Home

GenomeLab Genetic Analysis System User's Guide

1. 2 e m E d i d 5 6 3 d E amp amp 4 LE 901632L Al Figure 1 1 Instrument Hardware Overview 1 Sample Access Cover extended 7 Manifold Access Cover 2 Capillary Access Cover extended 8 Gel Waste Bottle 3 Status Indicators 9 Power Switch 4 Plate Holders Sample Transport 10 Gel Pump 5 Capillary Temperature Control Cover 11 Gel Pump Gel Cartridge Access Cover 6 Rubber Latches 2 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Getting Started System Overview Sample Access Cover Provides access to the buffer plate wetting tray and sample plate Capillary Access Cover Provides access to the capillary array through the capillary temperature control cover Capillary Temperature Control Cover Provides an enclosed environment for the capillary temperature control and allows access to the capillary array Capillary Array Used to produce raw data for sequencing and fragment analysis is 33 centimeters in length and has an i d of 75 micrometers The capillary array Figure 1 2 h
2. Figure 4 21 Base Sequence and Trace Views after Sequence based Trimming Enable Internal Matches GenomeLab Genetic Analysis System User s Guide 1 31 PN A29142 AB Sequence Analysis Using the Sequence Analysis Module After you apply sequence based trimming the system replaces the internal matches in the resulting base sequence and trace view with the letter x as shown in the following example Q9 CD044 G01_02091814NX Analyzed Data Fluorescence 500 750 1000 1250 1500 1750 2000 2250 2500 Data Points CTGATAACGACATCCTCAGTGATATCTACCAGCAXXXXXXXXXTTATGTGGTCAGTGGCCAGCACCCTACGCTTTAAGGTGCTA TGCTTGATCGGCAACCTAATTTAGGGGTTTAGCACGTGTTTCTTCGCTACGGCGATGTTGTCCTTAAAACTAGCTACAGGATTG AGGAGTTAAAATGAAATCGAACCGTCAGGCACGTCATATTCTTGGACTGGACCATAAAATTTCTAACCAGCGCAAAATAGTTAC CGAAGGTGACAAATCCAGCGTAGTAAATAACCCAACCGGCAGAAAACGCCCCGCTGAAAAGTAATTCATAACCATCAGTCCTCA ATGACGATTAAACACCATTGCCTGCGCAATGGTGTTTTTGTTTTTATCTGCTTTATACTTGAGGCCGACGCCC TGGCGGTAAAG CAAAGACGATAAAAGCCCCCCAGGGATGGATATTCAAAAAAGAGTGAGTGACATGGAACCAAXHXHXXHXXXXXAGCGTTCGCT TTATATCCCTTACGCTGGCCCTGTACTGCTGGAATTTCCGTTGTTGAATAAAGGCAGTGCCTTCAGCATGGAAGAACGCCGTAA CTTCAACCTGCTGGGGTTACTGCCGGAAGTGGTCGAAACCATCGAAGAACAAGCGGAACGAGCATGGATCCAGTATCAGGGATT CAAAACCCGAAATCGACAAACACATCTACCTGCGTAACAT Figure 4 22 Base Sequence and Trace Views after Sequence based Trimming Enable Internal Matches Batch Trimming Enable automatic trimming of a large number of results To perform batch trimming l
3. Open Sequence Analysis Parameters Optical Scan Data Sample Data Sequence Resulta Sample Flate Results 032206 CEQ TROUBLESHOOT Start Date 07 21 2005 B Enable Cancel End Date 2i 2005 E Refresh Help Database Filter Project Mame Figure 4 9 Open Dialog Box Sequence Tes A03 0603012 03 02706 0 Sequence Sequence Test A04_0E03020 03 0206 0 Sequence Sequence TestA05 0603020 03 02706 0 Sequence Sequence Test BO 0603012 03 02706 0 Sequence Sequence Test B04_0603020 03 02706 0 Sequence Sequence TestbO5 DBO3O020 03 02706 0 Sequence Sequence Test COS_O60S012 03 02706 0 Sequence Sequence Test C04 UBOS020 03 02706 0 Sequence Sequence Test C05 0603020 03 02706 0 Sequence Sequence Tes D03 0603012 03 02706 0 Sequence Sequence TestO04 0603020 03 02706 0 Sequence Sequence Test005 0603020 03 02706 0 Sequence Sequence TestEOS_O60S012 03 02706 0 Sequence Sequence TestEU4 UBUS020 03 02706 0 Sequence Sequence TestEO5 0603020 03 02706 0 Sequence Sequence TestFOS_OBOS012 03 02706 0 Sequence Sequence TestFU4 UBOS020 03 02 06 D a k 2 Select the appropriate tab Sample Data Sample Plate Results Sequence Results Sequence Analysis Parameters or Optical Scan Data 3 Select the desired item from the list box then click OK Use the Sample View toolbar i
4. Bulk Disposal Table 9 3 7 WARNING GenomeLab Genetic Analysis System User s Guide PN A29142 AB When performing this procedure use an exhaust ventilation unit that meets TLV requirements Aspirate 400 pL of buffer gel mixture from the 96 well plate Dispose the buffer gel mixture into a hazardous liquid organic waste container Rinse the plate wells with water and dispose of the rinse in the liquid waste container After all wells are clear allow the empty plate to sit in the ventilation unit for 24 hours Dispose of the plate in a solid waste container Dispose of buffer gel mixture in accordance with all applicable federal state and local environmental regulations concerning hazardous liquid waste When performing this procedure use an exhaust ventilation unit that meets TLV requirements l Using a 1L side arm flask as a trap connect the trap to a vacuum Attach a pipette to the trap using chemical resistant tubing Aspirate the formamide from each well and dispose of it in a hazardous liquid organic waste container After complete removal of all formamide from the plates allow the empty plate to sit in the ventilation unit for 24 hours Dispose of the plate in a solid waste container 3 9 Maintenance and Diagnostics Biological Waste Disposal Table 9 3 8 WARNING Dispose of buffer gel mixture in accordance with all applicable federal State and local environmental regulations
5. 5 Loosen the Manifold Access Cover captive screw Figure 3 36 then remove the cover and set it aside GenomeLab Genetic Analysis System User s Guide 81 PN A29142 AB Run Module Using Direct Control a o o o 2 E N D a u Figure 3 36 Manifold Access Cover 1 Capillary Temperature Control Cover 4 Captive Screw open 2 Plenum Assembly 5 Manifold Access Cover removed 3 Manifold Access Cover 6 Loosen the two Plenum Assembly captive screws Figure 3 37 then pull the Plenum Assembly straight back and away from the instrument and set it aside CAUTION Slowly remove the Plenum Assembly as the electrode block may disengage from its mounting posts and become damaged 82 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Run Module Using Direct Control 901713L Al Figure 3 37 Plenum Assembly Removal 1 Captive Screw 3 Plenum Assembly 2 Rubber Latch 4 Captive Screw Lift the Eject Lever Figure 3 38 to release the Array Fitting Grasp the Array Fitting tab Figure 3 38 and then a Pull the fitting approximately one inch out of the manifold b Touch the tip of the fitting to the bottom of the Optics Base Plate c Hold and wait five seconds for the gel strand to dry d Pull the fitting away from the instrument e Wipe gel strands off of the inst
6. 900697L Al Figure 9 32 Plenum Assembly Capillary Routing Hole NOTE When reinstalling the plenum assembly gently gather the capillaries between your thumb and forefinger to make sure they pass through the hole in the plenum assembly without any constriction 14 Replace the plenum assembly and tighten the two captive screws Figure 9 32 15 Replace the manifold access cover and tighten the captive screw Figure 9 27 16 Lower the capillary temperature control cover and secure the two rubber latches 17 Lower the capillary access cover and sample access cover to their locking positions e If you are installing a new capillary array in the Install Capillary Array dialog box Figure 9 33 select the correct part number enter the serial number click Set to New then click Done The number of runs and days on instrument will revert to 0 e If you are installing the previous capillary array do not change the serial number number of runs or the number of days on the instrument as they will be correct e If you are installing a capillary array that was previously used but not the last capillary array on the instrument enter its part number if applicable serial 366 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Maintenance and Diagnostics Direct Control and Replenishment number and adjust the number of runs and the previous cumulative days on the instrument Then click Done NOTE Note the number of runs
7. Data File Edit View Tools Window Help Manager BEIE a oja Hee amp E GENOMELAB DBASE IN Ready g T Default E CEQBAK1 E CEQBAK2 D1151984 D145599 D151679 D225683 D352387 0952157 D95938 Default DXS7132 GATA193AD7 Test project E USERTEMPLATE lg Name Typ Modifie Project ee e o ot 1 10 10 10 10 10 10 10 10 10 11 11 11 11 11 11 11 11 11 12 12 12 12 47 Figure 8 1 Main Window Data Manager Module D1181984 O 1pmol 0 D1181984 0 1pmol 0 0228683 0 1pmol AD D382387 0 25pmol A D952157 0 1pmol AD 098938 0 2pmol 0 2 DXS7132 2pmol AD3 GATA193AD7 0 25pm 1 D1S1679 at 0 25pmol AD 1 pull 0 25 primer 9 599 D1181984 0 1pmol D225683 0 1pmol B D352387 0 25pmol 095215 0 1pmol B GATA193AD 7 0 25p D151679 at 0 25pmol B pull 0 25 primer 9 599 D1181984 0 1pmol D225683 0 1pmol C D352387 0 25pmol D952157 0 1pmol C D95938 0 2pmol D DXS7132 2pmol C04 GATA193AD7 0 25p D151679 at 0 25pmol C pull 0 25 primer 9 589 D1151984 D0 1pmol D228583 0 1pmol D D352387 0 25pmol D982157 0 1pmol D Naca fn f D95936 0 2pmol D DXS7132 2pmol B04 sample Data Sample Data sample Data
8. Menu Bar Options The following example shows the Sequence Investigator module menu bar options File Edit View Window Help The following topics describe each of these menus and their options File Menu Click the File menu to display its drop down menu as shown in the following example File Mew Ctrl M Open Ctri o Close Save Chri4 5 Save As Chri A Export Preferences Ctrl F Properties Report Format Print Preview Print Ctrl F Print Desktop 0 CACEQ System Results Investigate cgc Exit Use the File menu to create open save export set preferences view properties and print a document The following table describes the Sequence Investigator module s File menu options Table 5 2 File Menu Sequence Investigator Module Option Description New Prompts for a reference file from a Windows folder or disk and then prompts for one or two sequence results from the current working database Open Opens an existing compared sequence result Close Closes the document Save Saves the active document to a Windows folder Save AS Save the current document to a new name and or folder Export Exports the current document as a text txt or a protein file pro GenomeLab Genetic Analysis System User s Guide 167 PN A29142 AB Sequence Investigator Module Sequence Investigator Module Overview 168 Table 5 2 File Menu Sequence Investigator Module Option Preferences Properties Report Format
9. Select Edit Trim Based on Sequence The analysis parameters created in the steps above should be listed If not click Use Stored Parameters and select the appropriate analysis parameters Click OK The following example shows the resulting base sequence and trace view after performing a sequence based trim CD04A G01 02091814NX 14285 52 4 03 Analyzed Data Fluorescence 13000 13250 13500 13750 14000 14250 14500 14750 15000 Data Points CTGATAACGACATCCTCAGTGATATCTACCAGCAAACGATCAATTATGTGGTCAGTGGCCAGCACCCTACGCTTTAAGGTGCTA TGCTTGATCGGCAACCTAATTTAGGGGTTTAGCACGTGTTTCTTCGCTACGGCGATGTTGTCCTTAAAACTAGCTACAGGATTG AGGAGTTAAAATGAAATCGAACCGTCAGGCACGTCA TATTCTTGGACTGGACCATAAAATTTCTAACCAGCGCAAAATAGTTAC CGAAGGTGACAAATCCAGCGTAGTAAATAACCCAACCGGCAGAAAACGCCCCGCTGAAAAGTAATTCATAACCATCAGTCCTCA ATGACGATTAAACACCATTGCCTGCGCAATGGTGTTTTTGTTTTTATCTGCTTTATACTTGAGGCCGACGCCCTGGCGGTAAAG CAAAGACGATAAAAGCCCCCCAGGGATGGATATTCAAAAAAGAGTGAGTGACATGGAACCAAAAACAAAAAAACAGCGTTCGCT TTATATCCCTTACGCTGGCCCTGTACTGCTGGAATTTCCGTTGTTGAATAAAGGCAGTGCCTTCAGCATGGAAGAACGCCGTAA CTTCAACCTGCTGGGGTTACTGCCGGAAGTGGTCGAAACCATCGAAGAACAAGCGGAACGAGCA TGGATCCAGTATCAGGGAT T CAAAACCCGAAATCGACAAACACATCTACCTGCGTAACAT Figure 4 19 Base Sequence and Trace Views after Sequence based Trimming GenomeLab Genetic Analysis System User s Guide 1 29 PN A29142 AB Sequence Analysis Using the Sequence Analysis Module Applying Sequence based Trimming If the trimming is acceptable select E
10. 2 Online Help Click this icon and then on a menu item to open the help topic related to the selection Direct Control Toolbar The following example shows the Sample Setup modules Direct Control toolbar For descriptions of the actions that occur when you click a toolbar icon see Table 3 4 EE ig heel B m Figure 3 3 Direct Control Toolbar Run Module 111 WO E J wj Figure 3 4 Direct Control Toolbar Run Module Icon Description Inject Injects sample prior to its separation and detection See Injecting a Sample on page 357 Separate Initiates the separation of injected samples See Performing a Separation on page 358 Fill Capillary with Gel Fills all eight of the capillaries with fresh gel See Replenishing the Capillaries with Gel on page 358 Capillary Temperature Specifies the desired temperature for the capillaries See Specifying Capillary Temperature on page 356 s Hi Gi Denature Denatures the samples residing in a specific sample set See Denaturing a Sample on page 357 Optical Alignment Performs the alignment of the lasers with the capillary windows See Performing an Optical Alignment on page 359 Plate Position Selects where you want to position and immerse the capillaries Load Unload Plates Selects the position where the capillaries are immersed in the plate or wetting tray and moves the plates to the Load position at the front of the instrument for loadi
11. Default Search Load List Save List Figure 4 23 Batch Trimming Result Selection Click OK Verify that the required analysis parameters are selected in the Working Parameters dialog box If not click Use Stored Parameters and select the appropriate analysis parameters 8 If necessary select the Export Results check box M1 to export the results to files The Edit Export Options button becomes available For details see Export Options on page 135 9 Click OK GenomeLab Genetic Analysis System User s Guide 1 33 PN A29142 AB Sequence Analysis Using the Sequence Analysis Module 134 Trimming Report Exporting results creates a Trimming Report which you can view and print To view the Trimming Report select View Trimming Report The new pane opens at the bottom of the screen displaying the following details Result Name e Original Sequence Length Length after Quality based Trimming e Vector Other Subsequence Name e First Base Trimmed ast Base Trimmed e Enabled Length after Sequence based Trimming e Exported From the Trimming Report pane you can format the grid print the grid data and export the data as comma separated csv or tab delimited txt text files To access these options right click over the Trimming Report then select the desired option from the pop up menu The following table describes these options Table 4 25 Trimming Report Right Click Menu Option
12. Figure 2 12 View by Subject ID Sample Name Toggle GenomeLab Genetic Analysis System User s Guide PN A29142 AB 32 Sample Setup Module Using the Sample Setup Module Naming Samples and Assigning Subject IDs The sample plate window appends the plate location to each Sample Name entered in a sample cell For example enter MySample as the name of the cell at position A3 in the plate The sample plate view identifies the sample as MySample A3 NOTE Position the mouse pointer over a named cell to display a tool tip that identifies both the sample name and Subject ID If you enter a Subject ID before entering a sample name the system automatically populates the Sample Name field which you can change at any time To name a single sample 1 Select the cell where the sample resides in the plate 2 Enter the name of the sample in the Sample Name text box and press the Enter key 3 Enter the Subject ID in the Subject ID text box and press Enter To name contiguous samples 1 Select the cells where the samples reside in the plate by clicking and dragging the mouse cursor Enter the name of the samples in the Sample Name text box and press the Enter key Enter the Subject ID in the Subject ID text box and press Enter Naming all Wells To name all wells in the plate l Click Select All 2 Enter the name of the samples in the Sample Name field and press Enter 3 Enter the Subject ID data and pres
13. The analysis parameters area allows you to set filtering parameters for including or excluding fragment results in your AFLP analysis Maximum Bin Width Sets the limiting value given to the width of the individual bins The default value is 1 0 nt Enter the value in nucleotides Within each dye label the system clusters the fragments with the sizes that fall within the set bin width and creates a bin with the size rounded to the next integer Y Threshold Serves as a exclusion filter defining the lowest acceptable value peak height for the y axis The default value is zero RFU Set the y threshold in relative fluorescence units RFU The system excludes fragments with heights less than this threshold value from the clustering process Exclude Fully Populated Bins Select this option to exclude bins represented in all samples such as non polymorphic fragments Exclude Samples without Qualifying Peaks Select this option to exclude samples that have no qualifying peaks ALFP Dyes The Dyes fields determine the dye colors the system uses during the cluster analysis If the results list contains samples with fragments that have more than one dye either multiple dye labeled fragments pooled in a sample or multiple samples with single dye labeled fragments in each sample you can select the appropriate dyes for export GenomeLab Genetic Analysis System User s Guide 243 PN A29142 AB Fragment Analysis Module Performing AFLP
14. Cancel Help Figure 6 7 Select Raw Data Window Selecting the Components of the New Study Use this window to select the raw data to be analyzed from a database of samples that have been run in a particular projects The system includes the selected raw data in your Study and analyzes it as described in the following steps NOTE To view an individual sample electropherogram and other collected parameters prior to adding the sample to your Study right click on a highlighted sample and select Show Single Sample View The List View and Plate View tabs provide you with two ways to select raw data Each selection method provides the same sample information presented in different ways Selecting Raw Data from the List View Tab The List View tab allows you to select from a list of samples contained in a specific database and or collected during a particular time frame l Select the List View tab in the Select Raw Data window 2 Select the project folder that contains the sample data that you wish to include in the Study from the Project drop down list NOTE If the desired project is not listed it may be located in a different database To enter a different database you must close all applications and set the working database See Setting the Working Database on page 328 or Set Working Database in the online help for more information GenomeLab Genetic Analysis System User s Guide 1 95 PN A29142 AB Fragment Analysis Mo
15. Date Analyzed DATS 102 D4 H01 1060518 Y 6 5 2001 8 24 03 PM 4 25 2006 12 59 07 PM DATS 101 D4 G01_010605180 6 5 2001 8 24 01 PM 4 25 2006 12 53 14 PM DATS 100 D4 F 1 1050518QY 6 5 2001 8 24 00 PM 4 25 2005 12 53 21 PM DATS 99 D4 E01_010605180 6 5 2001 8 23 59 PM 4 25 2006 12 59 30 PM DATS 37 D4 D01 10605180Y 6 5 2001 8 23 57 PM Unanalyzed Analyze Data Result Data DATS S6 D4 C 1 1060518 Y 6 5 2001 8 23 56 PM Unanalyzed DATS 34 D4 B 1 1050518 Y 5 5 2001 8 23 54 PM Unanalyzed DATS 33 D4 AQ1 1050518 Y 6 5 2001 8 23 53 PM Unanalyzed PreProcessing channel 3 raw data RMS noise RFU 31 617333 PreProcessing Estimating baseline for channel 4 PreProcessing channel 4 average baseline level RFU 2077 442146 PreProcessing channel 4 raw data RMS noise RFU 46 558699 PreProcessing Finished baseline estimation PreProcessing Beginning estimation of dye spectra PreProcessing Estimating spectra from data PreProcessing Initial estimated number of dyes 2 PreProcessing Beginning spectral estimation Saal Total Samples 8 Analyzed 4 Passed 4 Failed 0 Figure 6 10 Analyze Data Window analysis currently in progress When the analysis is complete the status column indicates if the analysis succeeded or failed for each sample included in the Study To review the analysis log highlight a sample in the upper samples list and scroll through the analysis information in
16. GenomeLab Genetic Analysis System User s Guide 233 PN A29142 AB Fragment Analysis Module Performing Bin Analysis NOTE As an alternative you can access Bin Analysis from the Analyses tab of the Study Explorer The New Binning Analysis window opens as shown in the following illustration New Binning Analysis PII E3 Steps Parameters Dye D3 gl Binning Fragment Range From 300 E To 420 ES Binni z innimn Locus Tag Maximum Bin width iz E Source Data Minimum Data Points Per Bir 2 E Repeat Unit Length 4 E Allele Maming Convention Nominal Size Alphabetic Start At Nerd M 7 rog BD em c c c 4000 S000 Dye Signal RFU 2000 1000 350 355 360 365 370 sg 360 385 390 Fragment Size nt Help x Previous Finish Cancel Figure 6 27 New Binning Analysis Window Bin Parameters NOTE Bin Analysis requires a minimum of 10 samples Defining Parameters The first step in creating a new bin analysis is to set the bin analysis parameters These parameters identify the fragment list data you want to include in the bin analysis By setting these parameters you define the appearance of the view generated After they are set the system uses the information from the selected result set to generate a scatter plot The plot consists of all of the analyzed fragment data displayed as a function of the peak height y axis and fragment length
17. Humber of Auns Days on Instrument 13 1 Figure 3 42 Install Capillary Array Dialog Box 18 The Confirm Capillary Array Selection dialog will appear Read it and make the selection Yes to continue or No to abort the capillary installation Removing and Replacing a Gel Cartridge Gel Pump Plug CAUTION Care must be exercised when installing removing a gel cartridge due to the viscosity of the gel mixture NOTE This procedure assumes that an expended gel cartridge is being replaced with a fresh gel cartridge or that a used gel cartridge is being removed for storage purposes and being replaced with the gel pump plug l Select Replenish Release Gel Cartridge from the Run Module menu bar When the system is ready to release the gel cartridge the Release Gel Cartridge dialog box will be displayed Wait until the lead screw is completely disengaged 2 Open the Gel Pump Gel Cartridge Access Cover Figure 3 43 by gently pushing in on the top of the cover The cover is spring loaded and will pop open 3 Pullon the Cartridge Locking Lever The barrel will swing outwards to approximately a 90 angle from its locked position GenomeLab Genetic Analysis System User s Guide 87 PN A29142 AB Run Module Using Direct Control 88 901635L Al Figure 3 43 Removing Replacing the Gel Cartridge 1 Gel Pump Gel Cartridge Access Cover 4 Cartridge Locking Lever 2 Cartridge Locking Lev
18. Log into the eXpress Profiler and click on eXpress Analysis in the Menu Enter your username and password then click OK to log into the software Click Open Analysis Locate the desired analysis in the Analyses List Select and highlight the name of the desired analysis and click OK The plates assigned to the analysis will be listed in the Plates field 6 Make any changes to the analysis and click Save to save the modified file Importing Fragment Analysis Data Import GeXP Fragment Analysis data into eXpress Analysis in order to normalize gene expression relative to a reference gene rA eXpress Analysis Analysis Analysis 123 File Help GexP Analysis Plate Setup GexP Analysis Normalization m gt c les GexP Analysis Setup E GexP Import Use Browse button to select filename Filenames Plate Duplicate LD Found ee v Include Sample Names Add Plates to Database Figure 7 28 GeXP Import Screen Table 7 11 GeXP Import Options Option Plate ID Duplicate Found 300 Function Identifies the Plate ID from the GeXP fragment export file The Plate ID must be unique This option is selected if a Plate ID is not unique the database already contains an entry with the same Plate ID If a plate with a duplicate ID is added a warning dialog will open to confirm the replacement of the existing plate in the database If it is not desirable to replace an existing plate in the database the Plate ID must
19. NOTE See the Raw Data Legend area of the window for information regarding sample well selection e To remove samples from your Study click on the well location of the sample you would like to remove The sample ID moves to the Current Plate Selected Wells area of the window indicating that the sample has been removed from the Study To select or remove entire rows or columns of wells click on the corresponding row letters or column numbers e To select the entire plate to be included in your Study click on the red box in the upper left corner of the plate NOTE You may select Study data from multiple projects and or multiple plates Continue making selections from the list of available projects and plates to add all of the desired samples to the Raw Data Selected area 4 Click Next to proceed to the Analysis Parameters window Selecting an Analysis Parameter Set The second step in creating a new Study from raw data involves defining the parameters to use when analyzing selected samples Analysis parameters are used to process the fragment data to estimated fragment sizes and to identify alleles Analysis Parameters Fa Steps a cies Select Analysis Parameter Set Analysis Parameters Analyze Data New Result Data Edit Parameter Set Summary Project Default Date Modified 3 27 2006 1 55 54 PM Dye Mobility Calibration NoCorrection Size Standard SizeStandard 600
20. Sequence Analysis Using the Sequence Analysis Module Quality based Trimming Tab Use quality based trimming to trim poor quality data from the ends of the sequence The resulting base sequence and trace views display the base letters of the poor quality data as strike through Sequence Analysis Parameters Editor DefaultSequenceAnalysisPar EA 3eneral Initial Data Detection Heterozygote Detection Quality based Trimming Sequence based Trimming Alignment Reference Ends To Be Trimmed None C BothEnds C Let 5 End Right 3 End on a Trimming Stiagency Low Medium Save As Prnt Cancel Help Figure 4 15 Quality based Trimming Tab The following table describes the options available on the Sequence Analysis Parameters Quality based Trimming tab Table 4 23 Quality based Trimming Tab Option Description Ends To Be Trimmed Click the radio button to select the ends to be trimmed e Select Both Ends to trim outside the 5 and 3 coding region e Select Left 5 End to trim to the left of the 5 end e Select Right 3 End to trim to the right of the 3 end e Select None if you do not want to perform quality based trimming Trimming Stringency Click and drag the slider control to set the quality values from low to high There are five possible selections ranging from Low to High that correspond to error rate of 0 07 0 06 0 05 0 03 0 01 The default value is Med
21. Steps Raw Data Analysis Parameters Analyze Data Result Data w Filter by date List View Project AFLP 06 TP2 20 Start 4 25 2001 z End 7 25 2001 Raw Data Raw Data Available DATS 103 D4 402_01060520G 5 5 2001 10 06 30 PM DATS 105 D4 B02_01060520G 6 5 2001 10 06 31 PM DATS 107 D4 C02_01060520G 6 5 2001 10 06 32 PM DATS 110 D4 D02_010605206 6 5 2001 10 06 34 PM DATS 111 D4 E02_01060520G 6 5 2001 10 06 35 PM DATS 113 D4 F02_01060520G 6 5 2001 10 06 37 PM DATS 114 D4 G02_01060520G 6 5 2001 10 06 38 PM DATS 115 D4 H02 1060520G 6 5 2001 10 06 39 PM Selected 8 DATS 102 D4 H01 010605180 DATS 101 D4 G01 t 60518QY DATS 100 D4 F 1 010605180 DATS 33 D4 EU01 1060518 DATS 37 D4 D 1 010605180 DATS 96 D4 C01_010605180 7 DATS 94 D4 B01_010605180 7 DATS 93 D4 401_010605180 7 6 5 2001 8 24 03 PM 6 5 2001 8 24 01 PM 6 5 2001 8 24 00 PM 6 5 2001 8 23 59 PM 6 5 2001 8 23 57 PM 6 5 2001 8 23 56 PM 6 5 2001 8 23 54 PM 6 5 2001 8 23 53 PM DATS 93 D2 403_010605226D 6 5 2001 11 43 08 PM DATS 94 D2 B03_010605226D 6 5 2001 11 43 10 PM DATS 96 D2 C03_010605226D 6 5 2001 11 43 11 PM DATS 9 D2 D03_010605226D 6 5 2001 11 49 13 PM DATS 99 D2 E03_010605226D 6 5 2001 11 43 14 PM DATS 100 D2 F03_010605226 6 5 2001 11 49 16 PM DATS 101 D2 G03_010605226 5 5 2001 11 49 17 PM DATS 102 D2 H03_010605226 6 5 2001 11 43 18 PM lt lt Previous Finish
22. Unzoom Unzoom All Display Options View Last Analysis 90 Description Toggles Autoscroll off and on When enabled v the system will scale all data to the last 10 minutes of the run on the X axis and confine the display of data to the pane on the Y axis Selecting a checked option removes the check mark and disables the feature Toggles Autoscale off and on When enabled v the system automatically scales the data displayed on the window to the fit into the pane If autoscaling is disabled the data is scaled to its true values Selecting a checked option removes the check mark and disables the feature Pauses or resumes data display When enabled v this option pauses data display Pausing data does not stop the stream of data just the display of data Select Unzoom to undo one zoom level Select Unzoom All to undo all zoom levels Opens the Display Options dialog box which you can use to modify the parameters for currently displayed data including its title X axis Y axis dye traces current traces and colors For details see Setting or Changing Display Options on page 70 Select View Last Analysis to open the Sequence or Fragment Analysis module and display the most recently analyzed sample set of a running sample plate For details see Viewing the Last Analysis Performed on page 72 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Run Module Run Module Overview Run Menu Click the Run m
23. l Enter the sequence into the Search field 2 Select the Mask Lower Case option green circle to mask repeat sequences in lowercase 3 Select the species from the from drop down list red circle 4 Click Execute 286 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Gene Expression eXpress Designer Module The eXpress Profiler software will connect to the NCBI BLAST website and enter the queue for BLAST results Job Title Nucleotide sequence 26 letters o Your search parameters were adjusted to search for a short input sequence Request ID FUWGRHPU012 Status Searching Submitted at Fri Sep 28 17 28 55 2007 Current time Fri Sep 28 17 28 58 2007 Time since submission This page will be automatically updated in 14 seconds Figure 7 19 BLAST Screen Searching The results of the BLAST search will be displayed For more information on BLAST searches and interpreting the results see http www ncbi nlm nih gov BLAST Evaluating Primer and Amplicon Sequences with BLAST Evaluate the multiplex primer and amplicon sequences from a preliminary multiplex design If any primer or amplicon sequence is determined to be unsatisfactory then redesign the primer s for the gene target using the Primer Design function of eXpress Designer Identifying Repeat Sequences Use the BLAST function with repeat masking described above to examine the amplicon sequence generated by each set of primers for repeat sequences
24. 85 an g5 100 105 110 115 120 125 13 135 Size nt Figure 6 21 Single Result View The top of the Single Result window displays the name of the individual data sample selected All of the information pertaining to the data sample is distributed under several different tabs within this window Table 6 15 provides a brief description of the components of each of the tabs in the Single Result View NOTE Refer to the online help for detailed descriptions of each of the available tabs in the Single Result View Table 6 15 Information Available in the Single Result View Tab Fragment Data Raw Data Fragment List Current Inject Current Voltage Description Displays a plot of the dye signal traces Displays a plot of the raw four channel electropherogram Displays a list of all fragments that meet the minimum relative height criterion set in the parameter set This view displays all available fragment properties Displays a graph of the separation current versus time for the duration of the separation event Displays a graph of the injection current versus time for the duration of the injection event Displays a graph of the separation voltage versus time for the duration of the separation event GenomeLab Genetic Analysis System User s Guide PN A29142 AB Fragment Analysis Module Using the SNP Locus Tag Editor Table 6 15 Information Available in the Single Result View Tab Analysis Log Run Log A
25. A allele product NOTE You can select more than one of these check boxes at a time Read through the following descriptions or refer to the tables below to see how these selections affect A Detection and the allele peak assignments e Apparent size includes A Selecting this check box indicates that the sizes entered into the Apparent Size column of the allele list were for the A fragments The parameter affects where the system looks for peaks when identifying alleles e Detect A Selecting or clearing this check box determines When Detect A is checked the system identifies in the fragment list either the A or A peaks The system then identifies one of them as the true allele based on your selection in the Use A peak to call allele check box It designates the other peak as either A or A accordingly e When Detect Ais not checked the system identifies one of the two alleles as the true allele based on the Use A peak to call allele check box It considers other peaks on the fragment list as unknown alleles e Use A peak to call Allele Select this check box if the A form is expected to be the higher peak and leave it unchecked if the A form is expected to be the higher peak GenomeLab Genetic Analysis System User s Guide 21 T PN A29142 AB Fragment Analysis Module Using the STR Locus Tag Editor e If the Use A peak to call allele box is checked the system identifies the A peak as the true allele in the fr
26. Align Compare Views Display Options Heterozygote Display Color Analysis Menu Description synchronizes scaling zooming and panning of two analyzed data sets while in the Compare mode Aligns selected points of each of the displayed analyzed data sets while in the Compare mode Opens the Display Options dialog box which provides tabs used to modify any or all of the following parameters Title X Axis Options Y Axis Options Dye Traces Current Traces Colors and Quality Parameters For details see Setting or Changing Display Options on page 145 or click Help when using the dialog box Opens the Colors dialog box which you can use to change the color of the heterozygotes in the base sequence pane For details see Changing Display Colors on page 147 Click the Analysis menu to display its drop down menu as shown in the following example Analysis Analvze Stop Working Analysis Parameters Restore Original Base Sequence Batch Analysis Reanalvze Batch View Selected Batch Sample Result Skip Gurrent Sample Analyze Skip Gurrent Sample Set Skip Gurrent Sample Plate Pause After Current Sample Resume Batch Processing Third Party Analysis Third Party Analysis Setup Service Alignment k Align with Template Batch Alignment Trim Based On Quality Trim Based On Sequence Batch Trimming The following table describes the Sequence Analysis module s Analysis menu
27. Ancillary Information Primer set name GenBank Accession Repeat unit sequence Plaidy 2 Primer Label Forward Reverse f None Primer Sequence Forward Fr bo 3 Reverse BY bo 3 Comment amp x Previous Einish Cancel Figure 6 29 New Binning Analysis Window Updating Locus Tag You can use this window to select an existing Locus Tag from the drop down list or add any Ancillary Information to the selected locus tag at this time Save the new data to the selected locus tag to update the allele list with Locus Information including Dye Size and Repeat Unit Length NOTE Refer to Locus Tab on page 212 or Using the SNP Locus Tag Editor on page 219 for more information about locus tag parameters The system saves the following information e Observed Size bin mean Bin Minimum amp Maximum Standard Deviation e of Data Points Minimum Peak Height e X and Y coordinates of all the data points in the plot stored as additional information GenomeLab Genetic Analysis System User s Guide PN A29142 AB 23 Fragment Analysis Module Performing Bin Analysis The system automatically updates the allele list with the new information obtained during the bin analysis Reviewing the Source Data After applying the Bin Analysis parameters to the data selecting the reference and trace points and updating the locus tag and allele list you can review the source data for all
28. C Buffer Plate Wetting Tray Figure 9 10 Replace Wetting Tray Dialog Box Direct Control and Replenishment The following procedures provide system replenishment procedures using the Run module s Direct Control functions To access these functions use the Run module s Direct Control menu the Replenish menu and the selectable areas of the Direct Control window NOTE Use Figure 9 1 to locate hardware components referenced in this section GenomeLab Genetic Analysis System User s Guide PN A29142 AB Maintenance and Diagnostics Direct Control and Replenishment Accessing the Direct Control Window Launch the Run module to open the Direct Control window then select the Direct Control tab Data Monitor Direct Control Log instrument Data MM 2L Hu OF Access Plates Inject Separate Figure 9 11 Direct Control Window Loading the Sample Plate and Buffer Plate To load the sample plate or buffer plate l Select Direct Control Access Plates Access Plates IF vau are going to load new plates please Stark prepare your plates before clicking on Start You have 15 minutes to change plates once vou click on Start This time limit is critical sa that the Help capillaries are not adversely affected by prolonged exposure to air Figure 9 12 Access Plates Dialog Box GenomeLab Genetic Analysis System User s Guide 353 PN A29142 AB Maintenance and Diagnostics Direct Control an
29. Click on FASTA 3 Enter one or more accession numbers in the text box provided WARNING Make sure to enter the accession number correctly The entry of invalid accession numbers will cause the return of incomplete results GenomeLab Genetic Analysis System User s Guide PN A29142 AB 4 Gene Expression eXpress Designer Module Click Execute The screen will return the single or multiple FASTA formatted sequences as shown in Figure 7 16 Gene llame NM 002916 Accession Fasta NM 002916 gi 31881681 ref NM 002916 3 Homo sapiens replication variant l Sequence ACCGGCGTAT GCCCGTTATT GCGGTGACCT GACCAAGGAT AAAATGTGTG TTACGGACCA TGAGTTAAAT AGATGGGAAG CATGGAGAAG ATTCCGCTTC AATAGCTTAT GATCACAGAG TGACAAACTA AAATAACTTA GCAACTCATC TGAAGTTGTA mRNA TCCCGCCCTG CTCAGATTAG GCACGAGAAG CGAGGAGTAG GATGAAGTTG CCTGGAACTG GCATCTGATG CCGTGTCCGC GAGTCGAAAA AAGCCTCTGT CTTGTTAAAG AAAGTGATTA GAAGCTGTGG TCTGATAAAC AGCCTTTGTG ATAAAAATAA CTTTTCGCCC GTGACGGAGC CCAGGCTAAC CTGCCAGTGC CTTTCCAGGA GAAAAACATC AACGTGGAAT CTTTTAAGAT CCACCCGATT CAGATAAAAT TGTCAGAAGG CAGACATTGC TCAAGGATTT AGAAGTCTAT CAACTGTGAT AATGACCAAA GCCGTTCCGT TAAGACTTCG TGGGTGAAGT GGGAAGTAGC AGAAGTGGTT CACTATTTTG ACAAGTAGTT TGTGATTCTG CTGTCTTATC TCAACAGCAG AGACTTAAGA CGGGGTAATA AATAGATGAG TATCACAGAA GCAGCAGTTA AGCACCTTTA GGCGGGAACT AGACCATCTC ACCATGCAAG GGAGAGAACA GCAGTGCTGA GCAGCAGCTA CGAGAGAAAG GATGAAGCAG TGTA
30. DATS 100 D4 txt E DATS 93 D4 txt E DATS 101 D4 txt Z DATS94 D4 txt E DATS 102 D4 txt E DATS 36 D4 txt LocusList Ext E DATS 97 Da txt E DATS 99 D4 tect IY Result Output Options J Remove CEG Tracking Suffix W Resolve Filename Conflicts File name DEFAULT Save as type Test Tab Delimited tet Cancel Figure 6 41 Fragment Analysis Export Dialog Box To export the results as a text file from the Export Results Options window 1 Click Save As Use the folder navigation options next to the Save in field to locate and select the folder in which you want to save the export file 2 Enter the desired file name in the File name field NOTE Do not use special characters in the file name 2 V 5 l 3 Select Text Tab Delimited txt in the Save as type text box 4 Click Save The system returns to the Export Results Options screen displaying the file path to the specified text file in the Export results as field 5 If desired modify the fields that apply to the text file a Select Header and or Result Data in the Elements section Select Remove CEQ Tracking Suffix to automatically remove the plate position coordinates A01 BO1 etc as well as the time date stamp from the sample name These are generated by the CE system software c Select Resolve Filename Conflicts to enable the system to increment the sample names on export if it encounters samples with the m
31. General Initial Data Detection Heterozygote Detection z of Average Peak Spacing ro a Height Ratio fao a Sensitiity 0 25 Range From so nt to faso nt or to fio nt before the last called base Save As Print Cancel Help Figure 4 12 Heterozygote Detection Tab Sequence Analysis Parameters Editor NOTE If you opened this dialog box by selecting View Parameters Used to Compute Sequence this dialog box displays the analysis values but disables the fields described below GenomeLab Genetic Analysis System User s Guide 1 1 9 PN A29142 AB Sequence Analysis Using the Sequence Analysis Module 120 The following table describes the options available on the Sequence Analysis Parameters Heterozygote Detection tab Table 4 19 Heterozygote Detection Tab Sequence Analysis Parameters Editor Option Description Detect Heterozygotes Select this check box 1 if you want heterozygotes detected When selected the N Threshold on the General tab is disabled and N s will not be called The system detects heterozygotes after it determines the final basecall It detects peaks that meet the sensitivity criterion and examines each called base to determine if another peak nearby meets the Spacing and Height Ratio criteria specified If one or more nearby peaks meet the criteria the system merges their identities with the called base to produce an ambiguity code NOTE If you specif
32. GenomeLab Genetic Analysis System User s Guide PN A29142 AB Database Management Data Manager Procedures 3 Click on the symbol next to the Local Users and Groups item to expand its contents 4 Right click Users and select New User The New User dialog box opens Figure 8 11 Mew User El Ea User name Te stlsert Full name Description Password Confirm password v User must change password at next logon User cannot change password Password neven expires Account is disabled xe Figure 8 11 New Users Dialog Box 5 Enter the new User name required and edit the other fields as necessary 6 Click Create Adding Groups To add a new group from the Windows desktop 1 Select Start Settings Control Panel Administrative Tools Computer Management The Computer Management console opens 2 In the left pane click on the symbol to next to the System Tools item to expand its contents 3 Click on the symbol next to the Local Users and Groups item to expand its contents Right click Groups and select New Group The New Group dialog box opens Figure 8 12 GenomeLab Genetic Analysis System User s Guide 339 PN A29142 AB Database Management Data Manager Procedures 5 Enter CEQUSERS in the new Group name field and if necessary enter a description in the Description field Mew Group El Ea Group name CEQUSERS Description Members Add Remo
33. LEVELS are fixed as CEQ and 99 respectively for the CE system The TIME stamp is the current time at the time of export The DNA section is composed of the base call always lower case PHRED Quality Value and the Peak Index data point of the analyzed data contained within the associated SCF file Values are separated by spaces Selecting the PHRED format exports the Result Data Result Output Base sequence and Quality Parameters automatically if the sample has been analyzed with a sequence analysis parameter set If the sample has been analyzed with a fragment analysis parameter set nothing is exported The ESD electropherogram sample data format is used to export raw data from the CE system to a third party software package for analysis Selecting this option exports only the raw data and no other information GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Analysis Using the Sequence Analysis Module Table 4 29 Sequence Export Options Option CEQ cq Remove CEQ Tracking Suffix Resolve Filename Conflicts Apply Trimming GenomeLab Genetic Analysis System User s Guide PN A29142 AB Description selecting the CEQ format the format native to our database exports sample data sequence or fragment results sample plate results sequence or fragment analysis parameters sample plates methods locus tags fragment data only standards fragment data only and optical scan data The sa
34. Left click the GENOMELAB DBASE node at the top of the left pane 3 Select the database to be backed up An example is shown in Figure 8 5 Data Manager IDE x File Edit View Tools Window Help E GENOMELAB DBASE HE CEQ Al D1151984 HHE CEQBAKI G 0148599 HHE CEQBAK2 l D151679 a GA Cj 0228683 B D1151984 Cj 0382387 Al 149599 l p9s2157 Cl D151679 D95938 a 0225683 Cl Default a CF 03823987 amp pxs7132 a CY D952157 Gi GATA193AD7 Ci D95938 Cf Test project m E Default at CF Dxs7132 a CY GATA193AD7 CT Test project HE USERTEMPLATE Filter OFF NUM p 11 object s Figure 8 5 Select Database to Back Up 4 Select Tools Backup Database 5 Browse to the location on the local disk where the file will be saved Backup Database H E Save ir jo Dbase f pgs E logs CEQ 2006 4 29 BAK Filename SEQTEST_2006_05_0 BAK Save as type Database Backup Files bak Cancel Figure 8 6 Backup Database Dialog Box E 330 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Database Management Data Manager Procedures 6 Accept the file name or rename it as necessary NOTE Since databases are backed up frequently you might want to add the date as part of the name to prevent overwriting valuable backups and to ensure accurate database recovery 7 Click Save Restoring the Database Recover a database that was previously saved using the backup
35. Properties Report Format Print Preview Print Report Ctrl F Print Selected Pane Ctrl F Print Desktop DATS 100 D2 F03 0106052760 139424sprot CD amp 998121315XJ MySample D1 0109241066 MySample A1 0109241066 jo Ph Je oO DATS 100 D2 F03_ 0106052760 139424sprot CD6 DDD526143 MySample A1 0109241066 MySample B1_0109241066 4 2 amp 1 Exit Use the File menu to open save import export and print data and to set preferences The following table describes the Sequence Analysis modules File menu options Table 4 2 File Menu Sequence Analysis Module Option Description Open Open Sample Data Sequence Analysis Parameters Optical Scan Data Sequence Results and Sample Plate Results Close Closes the open sample Close Tab Closes the active tab save saves existing data save As save data to a new name Import Locate and select data to import The system can import data in Standard Chromatogram Format scf or Electropherogram Sample Data esd format 98 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Analysis Sequence Analysis Module Overview Table 4 2 File Menu Sequence Analysis Module Option Export Export from Plate Preferences Properties Report Format Print Preview Print Report Print Selected Pane Print Desktop Recent Objects Exit GenomeLab Genetic Analysis System User s Guide PN A29142 AB Description Export data in one of the follow
36. Red is the default color assigned to the Adenine A nucleotide bases in the analyzed red data pane C Black is the default indicator assigned to the Cytosine C nucleotide bases in the black analyzed data pane G Green is the default color assigned to the Guanine G nucleotide bases in the green analyzed data pane T Blue is the default color assigned to the Thymine T nucleotide bases in the analyzed blue data pane Fragment Dye Colors Toolbar The following example shows the Sample Setup modules Fragment Dye Colors toolbar For descriptions of the actions that occur when you click a toolbar icon see Table 3 13 D m 02 m os m os m Figure 3 8 Fragment Dye Colors Toolbar Run Module GenomeLab Genetic Analysis System User s Guide PN A29142 AB Run Module Run Module Overview Table 3 13 Fragment Dye Colors Toolbar Run Module Icon Description D1 Red is the default color assigned to Dye 71 in the fragment data pane and D1 is the red default name D2 Black is the default color assigned to Dye 2 in the fragment data pane and D2 is the black default name D3 Green is the default color assigned to Dye 73 in the fragment data pane and D3 is the green default name D4 Blue is the default color assigned to Dye 4 in the fragment data pane and D4 is the blue default name Status Monitor The Status Monitor displays the state of the current run Figure 3 9 shows the status monitor and Table 3 14 describes the
37. Seg Test B05 06030120 Seq Test B05_06 03 02 06 1 Seg Test B05 06030120 Seq Test C0306 037 02 06 1 Seg Test C03 0603011 Seg Test C03_06 03 02 06 1 Seg Test C03 0603011 Seq Test C03_06 03 02706 1 Seq Test C03 0603011 k Project M ame Search Figure 5 6 Open Sequence Results Dialog Box If two results are in different projects select none as the Project Name to display all available results files for a given database A new window opens GenomeLab Genetic Analysis System User s Guide PN A29142 AB NOTE By default the standard navigator dye color and discrepancy map toolbars appear docked If they are not displayed select View Toolbars and enable the desired toolbar 175 Sequence Investigator Module Sequence Investigator Procedures Numbering Base and codon numbering start at the beginning of the reference sequence The system uses these numbers as a navigational reference point The column position numbering begins at the first base in the comparison This may be a reference base a sample sequence base or a consensus base depending on the alignment of the sequences being compared The selected column number appears in the lower right hand corner of the status bar The History Log records all edits made in the Sequence Investigator and their column number Viewing Reference Amino Acid Translations When the system generates the comparison it translates the nucleotid
38. Select this check box VT to perform an alignment automatically when running an analysis The alignment uses the alignment template and parameters specified below and in the Advanced Alignment Parameters dialog box Reference Select the desired alignment template by clicking on its corresponding radio button puc18dG puc 18 or m13 To select a different template click Reference File Click Browse and navigate to the folder that contains the reference file then select it and click OK The text box displays the name of the selected reference file Cutoff Accuracy Enter the desired Cutoff Accuracy The alignment detects the base number in the called sequence at which the accuracy falls below this Cutoff Accuracy value The range is 0 0 to 100 0 Allowed N s Enter the percentage of N s allowed to occur before they are counted as errors when determining the alignment accuracy The range is 0 0 to 100 0 Advanced Alignment Click this button to open the Advanced Alignment Parameters dialog box Parameters Advanced Alignment Parameters Use the Advanced Alignment Parameters dialog box to define the Advanced Alignment Parameters used to perform alignments with the active sequence Advanced Alignment Parameters Ed Alignment scoring Match fi Insert get 2 Insert extend f l H 0 4 0 3 0 Mismatch 20 Delete stark 40 E Delete extend 30 Alignment controls v Find matching substrings I Left Edge Free
39. Sequence Analysis Using the Sequence Analysis Module 146 Title Properties Tab To set or change the title of the displayed data panel l Inthe Display Options dialog box select the Title tab 2 Change any item as necessary 3 Click Apply to continue or OK to close the Display Options dialog box X Axis Options Tab To define the X Axis properties of the displayed data panels l Inthe Display Options dialog box select the X Axis Options tab 2 Change any item as necessary 3 Click Apply to continue or OK to close the Display Options dialog box Y Axis Options Tab To define the Y Axis properties of the displayed data panels l Inthe Display Options dialog box select the Y Axis Options tab 2 Change any item as necessary 3 Click Apply to continue or OK to close the Display Options dialog box Colors Tab To define the color options of the window the current traces the voltage trace or the optical scan trace l Inthe Display Options dialog box select the Colors tab 2 Change any item as desired 3 Click Apply to continue or OK to close the Display Options dialog box Dye Traces Tab To set or change the dye trace properties In the Display Options dialog box select the Dye Traces tab Select the traces to display or not display under Show Dye Traces To change a color of any dye trace click on the appropriate Dye Colors dye select a color from the Color dialog box then click OK 4 When f
40. modity them using right click commands These commands allow you to select specific columns to display in the fragment results customize the arrangement of fragment results information and modify several other parameters of the fragment results For details on using the Column Selector window the Column Sort window and other display parameters see Customizing the Results List on page 248 Viewing Electropherogram Data You can view individual sample traces at any time either individually or in selected groups along with the parameters used or collected during the separation and analysis steps Sample traces can also be stacked or overlaid on top of one another for comparison purposes All of these features are available through the right click menus whenever a single or collection of samples is highlighted single Sample View Single Sample View displays all of the available information for an individual data sample including the collected trace views fragment data separation and analysis parameters the calibration curve the run log and more To access this information right click on the highlighted sample or multiple samples from any of the data or fragment lists and select Show Single Result GenomeLab Genetic Analysis System User s Guide 223 PN A29142 AB Fragment Analysis Module Using the SNP Locus Tag Editor 224 DATS 101 D4 G01_06042512776 dr T8 82 UD UA I r7 8 81 06 81 53 Br
41. n j gt OE NV S A N Da 901713L Al Figure 9 28 Plenum Assembly Removal 1 Captive Screw 3 Plenum Assembly 2 Rubber Latch 4 Captive Screw Lift the plenum assembly Figure 9 28 to release the Array Fitting Grasp the Array Fitting tab Figure 9 28 and then a Pull the fitting approximately one inch out of the manifold b Touch the tip of the fitting to the bottom of the optics base plate c Hold and wait five seconds for the gel strand to dry d Pull the fitting away from the instrument e Wipe gel strands off of the instrument using a damp tissue NOTE Note the number of runs and days on the instrument for this capillary array for reference If this capillary array is to be re installed this information is necessary A label has been provided on the back of the array packaging container so that this information may be recorded GenomeLab Genetic Analysis System User s Guide 363 PN A29142 AB Maintenance and Diagnostics Direct Control and Replenishment 901597L Al Figure 9 29 Removing Replacing the Array Fitting 1 Guide Pins 4 Gel Strand 2 Eject Lever 5 Optics Base Plate bottom 3 Array Fitting 6 Array Fitting 9 Grasp the electrode block tab Figure 9 29 and pull the block out and away from the instrument If removal of the block tab is difficult use the eject lever to assist 10 From the Remove Capillary Array dialog box Figure 9 26 select the Replace Capillary Arr
42. renamed NOTE The Profile report statistics keeps the original Plate ID GenomeLab Genetic Analysis System User s Guide PN A29142 AB Gene Expression eXpress Analysis Module Option Function Add Plate This option must be selected to add the plate NOTE To remove plates you do not want to add to the database clear this option Include Sample Names Select this option to import the sample names from the RN column of the GeXP fragment export file This automates the plate setup process while sample names properties are pre associated with wells Log into the eXpress Profiler and click on eXpress Analysis in the Menu Enter your username and password then click OK to log into the software Click on the GeXP Analysis tab to create a new analysis or open a previously created analysis See Creating a New Analysis on page 299 and Opening a GeXP Analysis File on page 300 Click on GeXP Import The GeXP Import tab will open Click Browse to navigate and select the files CSV exported from the GenomeLab application for this analysis Select the CSV file then click Open Several plates can be imported for the same analysis at once These plates can be grouped in the same CSV file or in separate files To add more plates to the analysis open each CSV file and add it to the plate list before the next step Click Add Plates to Database to save the selected plates to the database under the present analysis A dialog box wil
43. x axis To set the bin analysis parameters l Select the dye used during the raw data analysis from the Dye drop down list NOTE You can examine only one dye at a time during each bin analysis 2 Enter the fragment range the minimum and maximum fragment size in the Fragment Range From and To fields 3 Enter the Maximum Bin Width maximum difference between the smallest and largest fragments within a bin in nucleotides 4 Enter the Minimum Data Points Per Bin the limiting number of points necessary to form a bin in the scatter plot 234 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Fragment Analysis Module Performing Bin Analysis Enter the length of the repeat unit of the allele in the Repeat Unit Length field Select the preferred allele naming convention see Naming Alleles on page 213 Click Next The system applies the new parameters to the scatter plot and assigns the bins as specified New Binning Analysis IBI XI Steps ETE 7 p j i Bin View Trace Views iin is Nominal Size vs Apparent Size Slope 0 98685 y intercept 4 9889 f 0 99987068 373 902 937 151 Binning 407 709 0 327 S 750 EGATAI93407 n 4n i Locus Tag SN 100 1 120 039 030 187 160 123 187 18 500 Source Data ia o 250 gt a n a ty a E E 025 Relative Signal Strength Dye Signal E 320 330 340 350 360 370 380 390 ann Fragment Size nt Allele List jp
44. 2 Selecta file name and destination folder for the data 3 Click Save Some third party software may require that you export the sample information in a specific format When choosing to display the sample names in rows you can display the alleles in separate columns or in one column You can include the Bin Labels in the file by checking the appropriate boxes in the Export File Format for file export The Dyes parameters determine the dye color the system uses during the cluster analysis If the results list contains samples with fragments that have more than one dye either multiple dye labeled fragments pooled in a sample or multiple samples with single dye labeled fragments in each sample you can select the appropriate dyes to be exported 6 11 Performing LOH Analysis The Fragment Analysis module comes equipped with a macro that is intended to detect Loss of Heterozygosity LOH between alleles from Normal reference samples and those from other samples In LOH Analysis pairs of samples to be compared must have the same root names followed by characters that allow the distinction between reference and test samples or loci All data to be analyzed reside in a temporary file created by the System Fragment Analysis application The macro scans the fragment results contained in the Study to determine the loci to include in the LOH analysis For each result in the Study a list of alleles is then generated at each locus The peak he
45. 25 2006 1 55 24 PM DATS 36 D4 C 1 10605180Y 6 5 2001 8 23 56 PM 4 25 2006 1 55 30 PM DATS 37 D4 D 1 10605180 Y 6 5 2001 8 23 57 PM 4 25 2006 1 55 37 PM DATS 99 D4 E01_010605180 6 5 2001 8 23 59PM Unanalyzed DATS 100 D4 F 1 1060518QY 6 5 2001 8 24 00 PM Unanalyzed DATS 101 D4 G 1 1050518QY 6 5 2001 8 24 01 PM Unanalyzed DATS 102 04 HO1_010605180 6 5 2001 8 24 03PM Unanalyzed Analyze Current Trace End STATUS Data dimensions 10790 points x 4 channels STATUS Data rate 2 0 Hz STATLIS Constant velocity migration offset 1 00 min STATUS Capillary lengths 30 0 33 0 cm STATUS Separation Voltage 4800 0 V PreProcessing Assessing peak widths trial rank map window 22 points PreProcessing Data analysis range 1814 to 7204 icici Final rank map window is 22 points i us t Total Samples 8 Analyzed 4 Passed 4 Failed 0 Figure 6 25 Re Analyzing the Data NOTE For details regarding the data analysis process see Analyzing the Data on page 198 7 Click Analyze During analysis the system activates a progress bar and displays an analysis log for each sample analyzed When finished the status column indicates if the re analysis passed or failed for each sample selected for re analysis NOTE To stop an analysis currently in progress click Stop The system stops the analysis after completing the sample in progress 8 Review the analysis log a Selecta sample in the upper sampl
46. 37 Install Gel Cartridge Dialog Box 12 Dispose of the used tissue and spent gel cartridge in accordance with the procedure See Biological Waste Disposal on page 373 Removing the Manifold Plug The Manifold Plug is installed during shipping and when the instrument is not in use to prevent drying of gel NOTE This procedure assumes that the Manifold Plug is being removed in preparation for Capillary Array installation l Select Replenish Release Capillary Array from the Run Module main menu 2 Wait for the Remove Manifold Plug dialog box to appear 3 Open the Sample Access Cover Figure 9 1 and lift it to the vertical locking position 4 Open the Capillary Access Cover and lift it to the vertical locking position 5 Unlatch the two rubber latches holding the Capillary Temperature Control Cover and lift it to the vertical locking position Figure 9 38 6 Loosen the Manifold Access Cover captive screw remove the cover and set it aside GenomeLab Genetic Analysis System User s Guide 371 PN A29142 AB Maintenance and Diagnostics Direct Control and Replenishment 3 2 901633L Al Figure 9 38 Manifold Access Cover 1 Capillary Temperature Control Cover 4 Captive Screw 2 Plenum Assembly 5 Manifold Access Cover removed 3 Manifold Access Cover Lift t
47. 4 Click OK to open the selected sample plate Find Barcode Use the Find Barcode option to search for a sample plate using its barcode 1 From the drop down list select the Project Name that contains the sample plate barcode 2 Click Find Barcode and type the sample plate barcode exactly as it was saved If the sample plate barcode exists it will open the plate You may also use the wildcards and in your search If the search finds more than one match the Barcode Selection dialog box opens allowing you to select the proper plate Find Barcode Ed Figure 2 6 Find Barcode Dialog Box NOTE To search all projects for the sample name select none from the Project Name drop down list before clicking Find Barcode NOTE Ifthe sample plate does not appear in the list of available items it may be residing in another project To select the project where the sample plate resides select the project from the Project Name drop down list NOTE If the sample plate exists in a different database you can reset the working database For details see setting the Working Database on page 328 Other Ways to Open a Sample Plate You can also open a new or existing sample plate at any time while using the Sample Setup module e To open anew sample plate from the File menu select File New e To open a new sample plate using the toolbar click j e To open an existing sample plate from the File menu select File Open Use t
48. 4 Select a location to save the file 5 Enter a filename or leave the name as it is 6 Click OK Locking a Sample Plate To prevent editing of the currently displayed plate select File Lock The Notes Method and Analysis tabs are disabled when the Sample Plate is locked Viewing the Summary To display a read only window showing the attributes of the currently selected plate select View View Summary 44 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Run Module Run Module Overview Run Module This chapter provides an overview of the Run module including its menu options toolbars and dialog boxes It also shows you how to run sample plates and set the display options using this module 3 1 Run Module Overview Launch pre programmed sample plates and control individual functions of the instrument Use the Run module for e Running a sample plate e Performing all instrument control and operational functions such as denaturing samples injecting and separating samples loading and unloading plates controlling the capillary chamber temperature filling the capillaries with gel GenomeLab Genetic Analysis System User s Guide 45 PN A29142 AB Run Module Run Module Overview Using the Run Control Main Window The following illustration identifies the areas on the Run module s main window that are described in Table 3 1 A B C D f XRun Control GenomeLab System Py x File View Di
49. APERTURE approximate location O E d zd K vj e 0000000 AA DODODDDL 680000000000 PRRBFEBEPEBE EODDDDDDDUDD UGE 8000 approximate location 901651L AI Figure 0 Laser Label Locations 1 Capillary Access Cover extended 2 Laser Assembly Cover GenomeLab Genetic Analysis System User s Guide XIII Safety Information XIV System Operation and Electromagnetic Interference NOTE The following information addresses the EMI effect on system performance and provides recommended mitigations Under the test conditions specified by the European normative electromagnetic compatibility standard EN 61326 1 the instrument may exhibit temporary degradations in performance in accordance with the table below Because the environmental circumstances contributing to the problem can vary several different mitigation techniques have been provided that should help eliminate or reduce the interference Troubleshooting Performance Test Condition Radio Frequency Field Interference RFI System exposed to electromagnetic field strengths of greater than or equal to 3 V mat multiple frequency bands Effect on Performance May cause temporary degradation of data accuracy In some cases Significant noise may appear in the measured results Separation data will return to normal performance once exposure is removed May cause temporary loss of capillary temp
50. Analysis 244 Starting a New AFLP Analysis After setting the analysis parameters click OK to generate the New AFLP Analysis W AFLP New AFLP 1 H E sa roniva l a Fel Hee d 5414 55 03 54 65 0 14 3824 I 60 63 61 04 60 83 0 04 61 99 6252 62 34 0 06 66 71 67 15 67 00 0 04 76 92 77 17 77 11 0 01 78 62 78 85 78 78 0 01 87 51 87 74 87 66 0 00 90 24 90 24 90 24 0 00 91 45 91 65 31 53 0 01 93 09 94 07 3351 0 20 9423 94 23 94 23 0 00 96 39 96 66 96 47 0 01 97 37 97 62 97 45 0 01 100 46 100 46 100 46 0 00 0 102 49 102 86 102 63 0 02 120 12 120 12 120 12 0 00 0 125 14 126 01 125 33 0 12 0 1 D 123 33 130 18 123 53 0 07 1 0 1 1 1 1 135 95 135 95 135 95 0 00 0 0 0 0 0 0 139 45 139 45 138 45 0 00 7170 o ON a0 m HE Se ae ESSE Ge 141 79 142 56 14217 0 15 3303 D 146 58 146 68 146 68 0 00 2008 148 08 148 69 148 20 0 04 7961 150 38 150 38 150 38 0 00 17729 156 03 156 57 156 21 0 02 21863 150 42 150 42 160 42 0 00 1546 161 31 161 80 161 43 0 03 10588 162 16 162 63 162 26 0 02 10067 17457 17457 174 57 0 00 3860 180 41 180 41 180 41 0 00 1610 190 41 130 41 190 41 0 00 1660 200 39 200 39 200 33 0 00 203 06 203 43 203 33 0 01 mim n3 d ce e eo 7 74 cCn NJOJ e ce a ee ee ee ee ee ee eee D O17 amp o Oo Ooo mo co cn n ap k mami co
51. Analysis click Save to update the data in the g p y p database or before going to the next tab or function e Perform Plate Setup and Peak Binning for all plates in the Analysis before proceeding with Normalization Normalizing Peaks Normalize gene expression data to a selected reference gene and obtain a final report Log into the eXpress Profiler and click on eXpress Analysis in the Menu Enter your username and password then click OK to log into the software Click on the GeXP Analysis Normalization tab Click Open Analysis Choose the analysis and click OK pe BU x 2e de d Select the check box next to a potential reference gene in the Genes field The graph will plot the signal for that gene in relative fluorescence units on the y axis Raw peak area values are displayed for data points within a treatment or sample type The mean value for each treatment is connected by a line This preview of the unnormalized gene expression can aid in choosing the proper reference gene for normalization Choose a gene that has relatively even signal intensities across all of the samples within the sample capillary NOTE A reference gene that has constant expression regardless of sample type treatment or tissue is best suited as a normalization gene 7 After determining the gene to use for normalization select that gene from the Normalization Gene drop down list 8 Click Save to save the normalized gene 9 Select the Display No
52. Analysis Module Using the SNP Locus Tag Editor Detect A Detect A Checked Not Checked All fragments assumed to be A The A fragment is identified as the allele in the allele table Fragments without A if any are identified as unknown alleles or spurious peaks All fragments assumed not to be A Any fragments without A are identified as the allele in the allele table Fragments with A are identified as unknown alleles or spurious peaks Figure 6 19 SNP Locus Tag Editor Window Creating a New Locus Tag GenomeLab Genetic Analysis System User s Guide PN A29142 AB 219 Fragment Analysis Module Using the SNP Locus Tag Editor The Locus Tag Editor provides the following fields e Locus Tag Enter or edit the name of the locus tag in this field e Project Select the project to which the new or edited locus should be saved e Locus Name Enter the identification of the locus in the field This is the locus tag name that appears in the fragment list for all fragments that belong to this locus e Apparent Fragment Size Enter a real number greater than 0 and less than or equal to 150 This represents the estimated size in nucleotides that the user expects for fragments belonging to this locus e Ploidy This field is inactive for this system release e Allele ID s for This area provides fields for Allele IDs D1 D2 D3 and D4 Each field accepts 8 character alphanumeric values used to lab
53. CAUTION NEVER allow the liquid level to rise into the eight cannula recesses of the wetting tray lid nor drop below the fill level indicator line The top surface of the wetting tray lid must remain clean and dry under any and all circumstances GenomeLab Genetic Analysis System User s Guide 15 PN A29142 AB Getting Started Operating the System Starting the Sample Plate Run l Select Run I Start Sample Plate The Sample Plate Run Confirmation dialog box is displayed Sample Plate Run Confirmation Left Plate Right Plate Operator Mame Operator M ame Save ta Project Default Save ta Froject Default Plate Loaded C Run this plate first Plate Loaded C Run this plate first Sample Plate Barcode Sample Plate B arcade Sample Plate Marne Sample Plate Mame Mite Edit this sample plate configuration Mew Edit thie sample plate contiguration Load Plates Replace Gel Cartridge Details Cancel Help Figure 1 13 Sample Plate Run Confirmation Dialog Box 2 Select the desired project on the appropriate side left or right for each prepared sample plate and click OK 3 Click Load Plates The Access Plates dialog box is displayed Access Plates Fa IF vau are going to load new plates please Chart prepare pour plates before clicking on Shark you have 15 minutes to change plates once you click on Start as This time limit i critical sa that the Help capillaries are n
54. Capillary Array dialog box Figure 9 6 Please enter the serial number Far the capillary array 1E you wish to reset Dee the number of runs Far the capillary or number af days it has been on the Instrument enter the new values Cancel Click on Done when you have installed the capillary array and have closed bath the capillary access cover and the sample access cover Get to New Capillaries exposed to air Time Remaining Heb p min 14 SEC n Capillary Array Part Number 608087 Total Length 33 00 cm serial Number Length to Detector 20 00 cm Date Installed 04 27 2006 Internal Diameter 75 00 pr Time Installed f 6 45 20 Number of Auns E Days an Instrument 13 1 Figure 9 6 Install Capillary Array Dialog Box CAUTION After cleaning the detection windows perform the optical alignment procedure and monitor the baseline If background levels are above 6000 counts repeat the cleaning process Replacing the Gel Waste Bottle Empty Waste Bottle x NOTE This procedure assumes that you are replacing a used full waste bottle with an empty waste bottle Use the Run module to ensure the instrument is ready and follow these procedures to change the bottle In the Run module select Replenish Empty Waste Bottle The following dialog box displays a warning as the instrument prepares for removal m l Help Capillaries exposed to air Time Remaining min SEC je 57 Figure 9 7
55. Compare Data Compare the active analyzed data set against another analyzed data set Quality Parameters Displays a graphical representation of the quality values for the active sample Dye Colors Toolbar The following example shows the Sequence Analysis modules Dye Colors toolbar For descriptions of the actions that occur when you click a toolbar icon see Table 4 12 AFTC HEGET EH E Figure 4 5 Sequence Analysis Module Dye Colors Toolbar GenomeLab Genetic Analysis System User s Guide 1 09 PN A29142 AB Sequence Analysis Sequence Analysis Module Overview 110 Table 4 12 Dye Colors Toolbar Sequence Analysis Module Icon Description A Red is the default color assigned to the Adenine A nucleotide bases in the analyzed data red pane C Black is the default indicator assigned to the Cytosine C nucleotide bases in the analyzed black data pane G Green is the default color assigned to the Guanine G nucleotide bases in the analyzed green data pane T Blue is the default color assigned to the Thymine T nucleotide bases in the analyzed data blue pane N Gray is the default color assigned to ambiguous nucleotide bases in the analyzed data gray pane Batch Control Toolbar The following example shows the Sequence Analysis module s Batch Control toolbar For descriptions of the actions that occur when you click a toolbar icon see Table 4 13 waji om Figure 4 6 Sequence Analy
56. Data Sequence Results Sample Plate Results or Standards Print Prints a report of the selected item Print Setup Define printer properties Print Screen Send an image of the computer desktop or application window to the printer Delete Deletes items projects and databases NOTE The working database cannot be deleted Rename Rename items projects and databases Properties Displays the current database and modification date as well as other sample data collection information concerning the selected item in the same format as Sequence Result Properties See Sequence Result Properties on page 138 Import Import a file in one of the following formats Standard Chromatogram Format v2 10 and v3 00 scf ESD ESD CEQ cq NOTE You must select a project in which to import the items GenomeLab Genetic Analysis System User s Guide 321 PN A29142 AB Database Management Data Manager Module Overview 322 Table 8 2 File Menu Data Manager Module Option Export Description Export a file in one of the following formats Standard Chromatogram Format v2 10 and v3 00 scf CEQ cq ESD esd Tab Delimited ASCII Text txt SEQ seq FASTA fasta PHRED scf phd 1 CRV crv set as Working Database Defines the currently selected database as the default database Closes the Data Manager module Click the Edit menu to display its drop down menu as shown in the followi
57. Displaying the selected sample results in one of these modes will automatically display the appropriate EgramBand components for the selected display mode Table 6 12 Y Height 52215 49 Width 96 90042 EgramBand Y Axis Scale the Y axis EgramBand X Axis Scale the X axis EgramBand Zoom tool Allows you to zoom in ona A selected area of the Overlay Graph electropherogram display This option is the default selection when in Overlay Graph mode EgramBand Subset Selection tool Allows you to select a subset of data to be displayed in Stacked Graph mode This option is only available when in Overlay Graph mode GenomeLab Genetic Analysis System User s Guide 1 91 PN A29142 AB Fragment Analysis Module Fragment Analysis Procedures 6 2 Fragment Analysis Procedures To open the Fragment Analysis module from the Main Menu click the Fragment Analysis icon Fragment Analysis New Study LL Exi Eile View Results Analysis Reports Window Help nc melos ov Bil o2 I os IE os NN Stuy Epo x i Study W Result Set View AFLPs Binnings Results Exclusion Filter Set Peak Ratios ult dat analysis New Result Filter Set 2 Apply ax os c outcome DATS 93 D4 401_060425122M 04 25 2006 12 53 56 i LID Name Operator Value s DATS 94 D4 B01_060425122K 04 25 2006 12 53 50 DATS S6 D4 CO1 0504251221 04 25 2006 12 59 44 DATS 37 D4 D 01 0604251 22F 1
58. Fragment Analysis Module Option Description Fragment List Displays the Fragment List The Fragment List contains information for all analyzed fragments included in the Study such as size height area locus and allele ID Results Set Displays the Results Set View The Result Set contains information for all of the analyzed samples included in the Study Locus List Opens a dialog box that create the LocusList txt file used for the Genotype Summary Editor View GSV Genotype Displays a Study s genotype information loci and alleles Summary Status Bar Toggles between displaying or not displaying the Status Bar study Explorer Displays the Study Explorer The Study Explorer provides access to all of the parameters available in the selected Study Toolbars Select which toolbars to display See Toolbar Icons on page 190 Results Menu Click the Results menu to display its drop down menu as shown in the following example Results iv Show Excluded Ctrl F IM Show Capillary Summary NOTE This menu is available only when displaying the Result Set View The following table describes the Fragment Analysis module s Results menu options Table 6 4 Results Menu Fragment Analysis Module Option Description show Excluded Shows fragment results that were excluded from the Fragment Results Grid Show Capillary Displays a table that identifies the number of samples that are included and excluded summary in the Result Set w
59. IUB Ambiguity Codes W Weak 2 H bonds B CGT Not A D AGT Not C H ACT Not G V ACG Not T i m The system reports the heterozygous bases in the analyzed data and in the base sequence data Alignment Reference Tab Use the Alignment Reference tab of the Sequence Analysis Parameters dialog box to enable automatic alignment and to specify the template against which you want to align the active sequence Sequence Analysis Parameters Editor DefaultSequenceAnalysisPar EA General Initial Data Detection Heterozygote Detection Quality based Trimming Sequence based Trimming Alignment Reference Reference pucl dG pucig C mig Reference File File Browse Alignment Accuracy Cutoff Accuracy 98 5 Allowed N 20 E Advanced Alignment Parameters Save s Print Cancel Help Figure 4 13 Alignment Reference Tab Sequence Analysis Parameters Editor NOTE If you opened this dialog box by selecting View Parameters Used to Compute Sequence this dialog box displays the analysis values but disables the fields described below GenomeLab Genetic Analysis System User s Guide 1 21 PN A29142 AB Sequence Analysis Using the Sequence Analysis Module 122 The following table describes the options available on the Sequence Analysis Parameters Alignment Reference tab Table 4 21 Alignment Reference Tab Sequence Analysis Parameters Editor Option Description Perform Alignment
60. Menu Click the Help menu to display its drop down menu as shown in the following example Help Help Topics Using Help About Genomelab System Beckman Coulter Home Page The following table describes the Help menu options Table 5 6 Help Menu Sequence Investigator Module Option Description Help Topics Select this option to display the CE system online help By default the help system displays the Contents in the navigation pane located on the left and the introductory topic in the topic pane located on the right You can use the Contents to select and print specific help topics The navigation pane also provides a Search tab and an Index tab These tabs let you search for information by topic index entry and or keyword Using Help Select this option to display the Contents window on how to use the Windows Help system 170 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Investigator Module Sequence Investigator Module Overview Table 5 6 Help Menu Sequence Investigator Module Option Description About GenomeLab Select this option to access software instrument and system information system Beckman Coulter Select this option to access the Beckman Coulter Home Page on the Internet Home Page Toolbar Icons The toolbars provide quick and easy access to commonly used menu items Each toolbar contains icons that correspond to commonly used menu items Click the i
61. Method Analysis Reporta Analysis Export vw Perform Analysis Print Report Export Data Parameter Set equencednalysisParameters Edit Print Format For Plate Edit Export Options For Plate Defaults equence amp nalusizParame DefaultFragment4nalysisParamet DefaultShPAnalsisParameters DefaultGexPAnalysisP arameters Figure 2 17 Analysis Tab Selecting a Parameter Set Printing the Plate Report or Specifying Sample Plate Print Options Define the print options and report format for the currently open sample plate With a cell highlighted select the Analysis Tab 2 Select the Print Report check box M to print a report immediately after completion of the run 3 Click Edit Print Format For Plate The Report Format dialog box opens Verify or select the Printer and Page Layout options e f desired click Colors and verify or modify the trace colors of the Raw Data and or Analyzed Data then click OK 5 Inthe Sample Elements area of the Report Format dialog box select each element to be printed e If desired click Options and verify or change the Raw Data Sequence Analysis Result Data Fragment Analysis Result Data Base Sequence Grouping and Current Trace Options selections in the Print Options dialog box then click OK 6 Click OK on the Report Format dialog box to save the changes and close the dialog box 40 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sample Setup M
62. Minimum substrin Find Local alignment v Right Edge Free length po E es dee Figure 4 14 Advanced Alignment Parameters Dialog Box NOTE If you opened this dialog box by selecting View Parameters Used to Compute Sequence this dialog box displays the analysis values but disables the fields described below The system uses a scoring matrix to perform the alignment and displays the alignment that produces the highest total score In general matches should be given higher scores than non matches GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Analysis Using the Sequence Analysis Module The following table describes the options available on the Advanced Alignment Parameters dialog box Table 4 22 Advanced Alignment Parameters Sequence Analysis Module Option Description Alignment Scoring Match Sets the value of a match between a base position of the sequence and the reference template The range is 1000 0 to 1000 0 A value of 1 is the default value e MisMatch Sets the value of a mismatch between a base position of the sequence and the alignment sequence The range is 1000 0 to 1000 0 A value of 2 0 is the default value e insert start Sets the value of an insertion of a base in the sequence that does not exist in the alignment sequence The range is 1000 0 to 1000 0 A value of 4 0 is the default value e Insert extend Sets the value of a consecutive insertion
63. Module Fragment Analysis Module Overview Main Window The following illustration identifies the areas on the Fragment Analysis module s main window that are described in Table 6 1 A B C 0 Fragment Analysis D1151984 test Result Set Viev e XI File View Results Analysis Reports Window Help la xj D D ue 4 Pr ict ict Tos l E IET x Study r Results Exclusion Filter Set Fragments List analysis New Result Filter Set 2 xe Results Set result date outcom ID Name Operator Value s 49 D1151984 0 1 pmol 0 25uIDNA A02 060327114 03 27 2006 11 17 36 8 D1151384 0 1pmol 0 25uIDNA HO1 060327114 03 27 2006 11 17 40 7 D1151984 0 1pmol 0 25uIDN GO1 06032711Al 03 27 2006 11 17 44 16 D1151984 0 1pmol 0 25ulDNA FO1 060327114 03 27 2006 11 17 46 15 01151984 0 1 pmol 0 25uDNA EO1 050327114 03 27 2006 11 17 50 4 D1151984 0 1 pmol 0 25uDNA DO1 060327114 03 27 2006 11 17 54 3 D11519384 0 1 pmol 0 25uDNA CO1 050327114 03 27 2006 11 17 56 2 D1151984 0 1 pmol 0 25uDNA BO1 050327114 03 27 2006 11 18 00 10 D1151984 0 1 pmol 0 25uIDNA B02 06032711 03 27 2006 11 17 32 1 D1151984 0 1 pmol 0 25uIDNA A01_060327114 03 27 2006 11 18 04 1 selecb pcoogoOm mnc gm F Summary v Show Excluded Data G Ready Figure 6 1 Main Window Fragment Analysis Module The following table describes t
64. Module Performing Bin Analysis 236 Minimum Relative Peak Height Enter a value to exclude any data points generated from small noise peaks The relative peak height is calculated in the color and size range specified in the initial binning parameters window e Show Phantom Bins Select Show Phantom Bins 1 to highlight the area of all system generated phantom bins The system generates phantom bins at all intervening positions for whole number nominal sizes not already assigned to a bin Phantom bins encompass expected fragment sizes based on other observed sizes that may or may not be present in the data set To convert phantom bins to real bins alleles right click the desired bin location and select Create Allele The system adds a line to the Allele List representing the new bin which you should check for consistency Trace Views At the bottom of the Trace Views area select Show Bins 1 to display the bins with each trace shown in the stacked trace views Investigating Points Position your mouse pointer over any point in the Bin View plot and left click to select it The Trace View displays a trace for the selected point You can use this trace as a visual comparison to other points The visual comparison of the two traces help you confirm assigning a data point to an already defined bin or to a new bin of its own The system assigns a Name and Bin ID using the naming and ID conventions selected in the bin analysi
65. Move the mouse over each block to see the normalized value for each sample gene combination The table represents the data in a heat map or Eisen plot Color changes are formulated on a per row basis where each row represents a specific gene and each column represents a sample set in the analysis Warmer colors represent higher normalized values higher expression while cooler colors represent lower normalized values decreasing expression Editing the Color Scheme Although the information in the Table cannot be modified the color representation can be changed for aesthetic purposes The color scheme choices are Rainbow blue green red yellow blue to white red to black and blue to yellow The levels of gene expression are represented by a blending of these colors 1 Click on one of the color scheme options in the toolbar The eXpress Map will change colors to reflect the selected option Click Display Configuration green check mark The Table Configuration dialog opens Use the Gamma and Threshold setting to adjust the color intensity GenomeLab Genetic Analysis System User s Guide PN A29142 AB Gene Expression eXpress Map Module Creating a Profile Graph Normalized expression values of selected genes within the same analysis are represented by a Profile graph This tool is similar to the graph that is created when using the Normalize Peaks option in eXpress Analysis 1 Log into the eXpress Map and load the analysis to
66. Multiplex Run Statistics Hame Adjusted max sequence size TestPlex 2 300 Universal Tags Minimum sequence size Default Tags 100 Product size range Separation size 143 325 7 Separation size range Excluded Primer Pairs Lan Ico tal cp none Humber of Primer Pairs 10 Primer Pair 1 Primer Pair 2 Hame Hame AF034577 106 1272 20 1377 20 NM 005915 114 290 20 403 20 PCR product size including tags PCR product size including tags 143 TSI Fasta Fasta gt gilZ 72937 qgb AFO34577 1 AF034577 Rattus gi 33469920 ref NM 005915 4 Homo sapiens MCM6 norvegicus pyruvate dehydrogenase kinase minichromosome maintenance deficient 6 MISS isoenzyme 4 PDE4 mRNA complete cds homolog S pombe S cerevisiae MCM6 mRNA Left Primer Left Primer Sequence Sequence TCTGAGGCTGATGACTGGTG AACCAGCAACTTTCCACCAC Primer start length Primer start length 1272 20 290 20 Melting Tm Melting Tm 59 980 60 012 Right Primer Right Primer Sequence Sequence TTTGATCCCGTAAAAGTGCC ATCCTTGGCAAGAGGGATCT Primer start length Primer start length 1377 20 403 20 Melting Tm Melting Tm 59 938 60 037 Figure 7 25 eXpress Designer Multiplex Execute Results 9 Review the results and confirm that all of the genes and primers have been included in the Multiplex e fall of the genes and primers are included click Save to Database to save the multiplex data e If one or more genes and or primers are not included then reduce the minimum separation
67. Open the Sequence Analysis module 2 Select Analysis Batch Trimming 3 Select the Sequence Results or Sample Plate Results tab 4 Select the results to be trimmed 132 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Analysis Using the Sequence Analysis Module 5 Click the right arrow to move the selected results to the selected list box Batch Trimming Selection sequence Results Sample Plate Results Database 032206 CEG TROLIBLESHOU Filter Start Date 07 21 2005 Enable End Date 72 2005 Refresh Mame D ate Time Sequence Test 403 0603012257 Sequence TesLAUS 03 02 06 03 52 514M sequence Test B03 060301 2252 Sequence Test B05_ 03 02 06 03 52 56AM o D ae Sequence Test CO5_ 03 02 06 03 53 024M iili Test E03 DED3DI 2067 Sequence Test D5 03 02 06 3 53 08AM mpe Test FOS Denn 2967 Sequence Test E05 03 0206 03 53 134M Sequence Test G03 060301 2257 Sequence Test FOS 03 02 06 03 52 794M Sequence Test HOF 0603012757 Sequence Test 03 02 06 03 53 244M Sequence TestAQd OB030200L2 Sequence Test H0S 03 02 06 3 53 304M Sequence Test 604 O8030200L7 Sequencing A02 Ob 04 24 05 15 19 30PM Sequence Test C04 O6030200L2 Sequencing 407 Db 04 24 16 15 13 42PM Sequence Test O04 O6030200L2 Sequence Test E04 O6US0200L2 Sequence Test FO4 O6US0200L4 Sequence Test G04 O6030200L2 Sequence Test HU4 O6030200L2 Cancel Help ddi 1 i E pe Project Mame
68. Plate on page 354 and Loading the Buffer Plate and Evaporation Cover on page 355 7 Click Start to begin the Run GenomeLab Genetic Analysis System User s Guide PN A29142 AB 17 Getting Started Operating the System 18 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sample Setup Module Overview Sample Setup Module 2 1 Overview Create save and modify methods and sample plates Methods consist of sequential events needed to perform a DNA separation Sample plates provide a way to name and organize samples The sample plate definition contains the 96 well plate locations of the samples as well as the method assigned to each sample The capillary array must run the same separation parameters across eight samples one column at a time Each column that contains samples is called a Sample Set The separation parameters applied to each sample set are specified in the Method Method parameters can be changed to create new methods Using the Main Window The portions of the Sample Setup module s main window are described in Table 2 1 A E C D Sample Setup DefaultSample late E File Edit View Run Window Help Diae Ei ala aelel dl a ae r2 amp Run Sample Plates E m MySamplePlate view by sy View by Sample Name MySampleAQ1 Subject ID Plate Saved ff A2 A3 LY AS AB AT AS Ag A10 A11 32 B3 4 BS BE 7 8 9 B10 B B B MySample B01 Fm c MyS
69. Print Preview Print Print Desktop Recent Objects Exit Edit Menu Description Sets the preferences for the Sequence Investigator Displays the properties of the current document Set the format options used to print reports and adjust the printer settings Displays a print preview of the active document using the format specified in the Report Format dialog box Prints the active document as specified in the Report Format dialog box Prints the active desktop area as specified in the Preferences dialog box Provides a short cut to open previously opened documents Exits the Sequence Investigator module Click the Edit menu to display its drop down menu as shown in the following example Edit Insert Delete Replace Trim Search Restore To Original Quality Threshold Flip Traces Insert Delete trl Er H R Ctra Use the Edit menu to edit displayed data The following table describes the Sequence Investigator module s Edit menu options Table 5 3 Edit Menu Sequence Investigator Module Option Insert Delete Replace Trim Search Restore To Original Quality Threshold Flip Traces Description Inserts a space before the currently selected position Deletes the selected position Replaces the highlighted position with a selected IUB code Removes contiguous selected bases that contain an end base in the sample sequence base call text Searches forward and backward for the attr
70. Profile by Gene Profile statistics Profile third party Compressed v NM 031144 ri Normalize Peaks i Report View Multiplex GexP Plex v Normalize Peaks IV Display normalized data set Report Ss EATA EEE Repo Rep Ren mean siape sev Collapse all profile tables X Export Data v 567722 24hourUntreated D 952 0 902 1 087 0 98 0 096 9 765 pMsoo4w L133 110 L118 119 0 014 1267 pMsoosw 054 1189 1336 1195 0136 11 384 pmsoaw fus 1232 1342 1229 113 9224 Positive Control RNA 2 3 0 317 n4 osa 07 664 Repo Rep 2 Reres wem Stier v 24hourUntreated D 126 0 119 0 195 0 146 0 042 28 608 pMsoniw 0 217 226 o oge joze fo o79 44 642 pMsoos 0 15 0156 0262 189 0 063 33 436 pMsoi s i24 145 244 p 71 064 37 304 Positive Conto RNA 0 315 0 297 0 425 p 346 0 069 19 997 Description Gene centric organization with basic statistics Mean CV and Standard Deviation GeXP Genetic Analysis System Traceability data available as a QC tool Not suggested for use with the GeXP software due to less than reliable results Sample centric organization offering different view based on sample to sample comparison GenomeLab Genetic Analysis System User s Guide PN A29142 AB Gene Expression eXpress Analysis Modu
71. RR 42 Exporting Sample Plate Data sssssssesn RR 44 LockKIng a sample Fli s ac oui om ward aU RUD ra o UE bsc urbe e e uc Pole euet 44 Viewing the Summary ae d deo dnd tito e elo aptae ar tn Be get dom portada doit ata dei 44 Section 3 Run Module 2a does ub ai deo d boe Dato aed 45 3 1 Run Module DVBIVIBW sd disease itis we ent ur SEE tbe tU e art E PE Me eM XE Pen 45 Using the Run Control Main Window lssee RR ee RII 46 Gp HOS sc acm pea Mec cM IL ELE pM MEL 47 ITODIDSI ICONS ceto itae arde Eta ere bt E EX OE cd aa bd ae a me a i oad eoe Bn te ee ee 53 lan ITINERE REOR e OT TD TET QT ECT ee QUELLE 5 Window Selection TaDS e do eundo ne boob pur edem dpi m bo tot bci m date aiia 59 3 2 USING de RUDJMOGOIUIB s s co cd ccs corde a derer e ien CR eiu Ueber s peer 64 e eU US RIP EORR ETT Aea aa TORT TIR tA eee Seal aha ates Sra eee a ee a 64 GenomeLab Genetic Analysis System User s Guide il PN A29142 AB Defining System Preferences 0 00 eee n 65 RUNM Sa MIDIC PIALCS rcu troie ura tem e eek eens head gard CONSUE bu led ace dae 65 setting or Changing Display Options 0 0 0 eee een 70 Viewing the Last Analysis Performed 20 0000s 12 Oe SING MOG kG ORM crescant actuate che ae a ved cana cee e aud aaa anies cone be oe ech anh ante ees 13 Loading the Sample Plate and Buffer Plate 0 RR 74 setting the Capillary Temperature l l RR RR RR 76 Performing an Optical Alignment 0 0
72. Result Data to Study Export Results Export Fragments Genotypes Transfer Fragments Far eP Preferences Print Screen 1 GATA193A07 Exit Use the File menu to create open manage and save Studies or export edit and transfer Fragment results The following table describes the Fragment Analysis module s File menu options Table 6 2 File Menu Fragment Analysis Module Option New Study Open Study Close Study Manage Studies Save Study Save Study As Add Raw Data to Study Add Result Data to otudy GenomeLab Genetic Analysis System User s Guide PN A29142 AB Description Starts a new Study from either raw data or analyzed results Opens an existing Study Closes the active Study View and select all Studies saved to the database saves the new or edited Study saves the current Study with a new name Select additional data raw data to add to the Study Select additional data previously analyzed result data to add to the Study 185 Fragment Analysis Module Fragment Analysis Module Overview 186 Table 6 2 File Menu Fragment Analysis Module Option Description Export Results Export data in one of the following formats e Text Tab Delimited txt e CEQ format cq e CRV CRV Export Fragments Export the fragment genotype data in one of the following formats Genotypes e CSV Comma Delimited csv e Linkage Pedin pre e Discovery Manager DMPopulation
73. Rosina Size nil Apperent Size mi S Dev ot Num Points 1 gt Dye Signal 0 0 0 i E INT 0 i PU 2 E 0 in 8 s 3 6 To xm cO un Size nt gt Phase Shift 0 m Min Rel Peak Height 0 01 m Show Phantom Bins v Show Bins lt lt Previous L Net Einish Cancel Figure 6 28 New Binning Analysis Window Bin Analysis View Viewing Bin Analysis After applying the bin analysis parameters to the selected data the system displays the results of the bin analysis This bin analysis view is composed of three areas representing the selected results Bin View The Bin View consists of a scatter plot diagram showing the Fragment Size x axis versus the Relative Signal Strength y axis The points plotted represent the results selected when you created the Study The bins are defined and visible with the majority of the result points within their borders Allele List The system develops the initial Allele List from information provided in the first window of the Bin parameters plus the data from the fragment list see previous After completing the analysis you can correct the allele list using the following options e Phase Shift Enter an integer value nucleotide in either direction to move all identified bins This overrides the system determined position of perfect repeat alleles GenomeLab Genetic Analysis System User s Guide 235 PN A29142 AB Fragment Analysis
74. Select Run Start Sample Plate 10 Select the new sample plate and click OK The system processes the plate and stores it in the new database NOTE It is recommended that you archive databases that are larger than about 1700 MB For details see Checking the Database Size on page 329 Gel Re estimation The system determines if the gel cartridge volume is sufficient to safely process the plate Ifthe gel volume is greater that 1 5 mL more than the minimum amount needed to process the plate the plate runs and the system does not re estimate the gel volume e If the system determines that the gel cartridge volume is less than the volume needed to safely process the plate the system displays the two re estimation message boxes before running the sample plate 66 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Run Module Using the Run Module In the first step the system homes the gel pump and displays the Gel Volume Re estimation message as shown in Figure 3 19 Gel Volume Re estimation Step 1 The gel volume needed to process this samole plate is close to the amount available Gel volume re estimation ts required Please wait while the system homes the gel pump I Figure 3 19 Gel Volume Re estimation Step 1 Message Box In the second step the system engages the gel pump plunger and accurately determines the gel volume See Figure 3 20 Gel Volume Re estimation Step Z Ea Re positioning t
75. Select the appropriate Event Type and edit the method as explained in Creating or Editing a Method on page 38 When finished click Save As and save the edited method to an appropriate Project with the same method name as specified in the plate Import file Using a Sequence Reaction Separation l 2 3 4 Launch the Sequence Analysis module In the Sequence Analysis module select Analysis Working Analysis Parameters From the Working Parameters dialog select Use Stored parameters Confirm the presence of the appropriate pre selected analysis parameters in the Select Analysis Parameters popup window e If not present cancel the Select Analysis Parameters and click Edit in the Working Parameters dialog Select the desired tab and edit the parameters as appropriate in the Sequence Analysis Parameters Editor dialog box See Editing Sequence Analysis Parameters on page 116 for a description of the Sequence Analysis Parameters Editor and creating Analysis Parameters When finished click Save As and save the edited parameters to an appropriate Project with the same analysis parameters name as specified in the plate Import file Performing a Fragment Analysis Separation ae Launch the Fragment Analysis Module In the Fragment Analysis Module select Analysis Analysis Parameters From the Analysis Parameters dialog select the appropriate analysis parameters in the Select Analysis Parameters Set field If not present
76. System User s Guide 355 PN A29142 AB Maintenance and Diagnostics Direct Control and Replenishment Figure 9 16 Loading the Buffer Plate and Buffer Evaporation Cover 1 Front Location 4 Buffer Evaporation Cover Alignment Notch 2 Buffer Evaporation Cover 5 Wetting Tray Retainers 3 Buffer Plate 4 When finished positioning the sample and buffer plates close the Sample Access Cover and then select the position from which the plates were loaded left or right 5 Click Load on the Capillaries Exposed dialog box Figure 9 15 Specifying Capillary Temperature l Select Direct Control Capillary Temperature The Capillary Temperature dialog box opens as shown in the following example Capillary Temperature Ea T ture z emperature 35 c TE wait for temperature ta be reached Help Temperature 356 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Maintenance and Diagnostics Direct Control and Replenishment Figure 9 17 Capillary Temperature Dialog Box 2 Enter a capillary holding temperature value in degrees centigrade 3 Select Wait for temperature to be reached 4 Click Start Denaturing a Sample l Select Direct Control Denature Samples The Denature Samples dialog box opens Cancel Help Sample Plate Position Sample Set B Plates Value Temperature 80 E Duration f z ZEC Figure 9 18 Denature Samples Dialog Box 2 Enter a time dura
77. User s Guide 09 PN A29142 AB Run Module Using Direct Control Do not open gel access door PLEASE WAIT Releasing gel cartridge H Time Elapse min ZEC Epo Figure 3 45 Remove Gel Cartridge Dialog Box Remove Gel Cartridge Ea Tou may now open gel access door and change gel cartridge IF You are nat going to install a working gel cartridge please insert the gel pump plug ar clean empty cartridge to prevent any remaining gel from drying in the system Then click on Install Plug Install Cartridge Ez NN LONE Install Plug Cancel Please check the waste bottle to Help ensure that the battle will not overflow in future runs 11 Ifinstall Plug was selected in step 10 proceed to step 12 If Install Cartridge was selected in step 10 use the Install Gel Cartridge dialog box Figure 3 46 to enter the following information a If you are installing a new gel cartridge click New The system will automatically update the date and time installed and set the Hours on Instrument to 0 b Next enter the lot number c If you are installing a used Gel Cartridge select Used e If you are installing the previous used gel cartridge do not change the lot number or the hours on the instrument as they will be correct If you are installing a previously used gel cartridge but it was not the last one on the instrument enter its part number and or lot number and adjust the hou
78. Width Sets the limiting value given to the width of the individual bins The default value is 1 0 nt Enter the value in nucleotides Within each dye label the system clusters the fragments with the sizes that fall within the set bin width and creates a bin with the size rounded to the next integer Y Threshold Serves as a exclusion filter defining the lowest acceptable value peak height for the y axis The default value is zero RFU Set the y threshold in relative fluorescence units RFU The system excludes fragments with heights less than this threshold value from the clustering process NOTE You can reduce the chance of finding multiple components in a bin by decreasing the bin width GenomeLab Genetic Analysis System User s Guide 245 PN A29142 AB Fragment Analysis Module Performing LOH Analysis Customizing the AFLP Table The components of the cluster analysis are user defined You can modify them using the right click menus These commands allow you to select specific columns for display in the fragment list customize the arrangement of fragment list information and modify several other parameters of the fragment list NOTE See Customizing the Results List on page 248 for details regarding how to use the Column Selector window the Column Sort window and other display parameters Exporting the AFLP Analysis Results To export the AFLP analysis results l Select File Export AFLP The Export AFLP window opens
79. Yes Duration 15 sec Figure 2 3 Sample Setup Method Sub Window GenomeLab Genetic Analysis System User s Guide 21 PN A29142 AB Sample Setup Module Overview Using the Analysis Tab Enable or disable the Perform Analysis mode When enabled M this mode causes the CE system to analyze the sample at the end of the run Set the Perform Analysis option for each sample separately row column or entire plate Select the desired Analysis Parameter Set for each sample from the drop down list Select each sample separately row column or entire plate Enable or disable automatic printing of reports and or edit the print report parameters 2 xm qe X Select Edit Print Format For Plate The Report Format dialog box appears showing the current report settings for the sample plate The Print Format applies to all samples Print Report applies to individual samples p Enable disable automatic exporting of data and or change export options Click Edit Export Options For Plate The Export dialog appears showing the current settings for the sample plate The Export Format applies to all samples Export Report applies to individual samples NOTE New Sequence or Fragment Analysis Parameters must be created in their respective analysis modules Note Method Analysis Analysis Reports Esport v Perform Analysis Parameter Set Print Report Export Data equence4nalysisParameters Edit Print Format For Plate Edit
80. a batch analysis click this icon to pass over the sample plate EI currently being analyzed This item is not available if Sample Data was selected from the Batch Analysis Selection dialog box View Results After performing a batch analysis click this icon to view the results of a selected sample in the upper pane of the Batch Analysis window Sample Plate Toolbar To display the Sample Plate toolbar select View Toolbars then select the Sample Plate check box as shown in the following example I Standard v Data Um B W Sample View ina v Sample Plate Sample Plate x 123465 6 7 8 10171 12 v Base Sequence Plate Ua 001 Test 030106_060301 Sample Result Refresh Export Help Figure 4 7 Sequence Analysis Module Sample Plate Toolbar Use this toolbar to specify the samples to open or export Select the desired plate from the Plate drop down list Valid samples appear as filled wells Wells not specified as samples appear dark or empty Click on the samples to open or export e To view the name of a sample and associated result position the cursor over the sample The sample and result names appear in the Sample and Result text boxes e To select contiguous samples click on the first sample in the series hold down the Shift key then click on the last sample in the series The selected samples are highlighted GenomeLab Genetic Analysis System User s Guide 1 1 1 PN A29142
81. and days on the instrument for this capillary array for reference Install Capillary Array x Please enter the serial number for the capillary array IF you wish to reset the number of runs for the capillary or number of days it has been on the Instrument enter the new values Cancel Click on Done when you have installed the capillary array and have closed both the capillary access cover and the sample access cover Get to New Capillaries exposed to air Time Remaining o Heb p min 14 ec E Capillary Array Part Number 608087 Total Length 33 00 cm serial Number Length to Detector 20 00 emi Date Installed 04 27 2006 Internal Diameter 75 00 um Time Installed f 7 11 18 Humber of Auns E mm Days on Instrument o o Figure 9 33 Install Capillary Array Dialog Box 18 The Confirm Capillary Array Selection dialog will appear Read it and make the selection Yes to continue or No to abort the capillary installation Removing and Replacing a Gel Cartridge Gel Pump Plug CAUTION Care must be exercised when installing removing a gel cartridge due to the viscosity of the gel mixture NOTE This procedure assumes that an expended gel cartridge is being replaced with a fresh gel cartridge or that a used gel cartridge is being removed for storage purposes and being replaced with the gel pump plug l Select Replenish Release Gel Cartridge When the system is ready to release the gel cartridge the Releas
82. and designates which peaks should be present by their size Designed gene peaks that align with the imported sample data are represented in green peaks Unmatched or undesignated gene peaks are black Positions in which a designed gene peak is expected but not detected will show in red The X axis represents the actual fragment size nucleotides and the Y axis represents Peak Height RFU The colors shown in the graph are explained in the color key Reading the Peak Binning Table Peak Binning is the process of manually matching data peaks to the designed gene fragments The peak binning table represents the data from the GeXP System in a tabular form A designed gene that is associated with a peak or peaks in the data is highlighed in green A designed gene that is not associated with a data peak is represented in pink An unmatched peak highlighed in white is either a spurious undesigned peak or a genuine gene product that must be matched with a designed peak 306 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Gene Expression eXpress Analysis Module Table 7 13 Peak Binning Table Parameters Category Function Gene Displays the name of the gene The gene accession number will display by default unless the display name is changed in one of two ways e Administrator function using Manage Genes option e Under the user login in the eXpress Designer FASTA function using the View Existing Data option Viewing Existing Gene
83. and results to other third party software packages E Fragment Analysis New Study a B x File View Analysis Reports Window Help Template Contest Type Contest Analyses Data Reports Ready Z Figure 6 37 New Report Dialog Box All of the reporting features available in the Fragment Analysis module are located under the Reports tab of the Study Explorer In addition you can generate a new report using the Reports menu located on the menu bar The New Report window allows you to select the type of report you would like to generate from a list of available report templates see Using Report Templates on page 252 and saved data You can access the New Report window from either the Report menu or the Study Explorer To create a New Report from the Report menu Select Report New Report to display the New Report window 2 Select the type of report you would like to generate from the Template drop down list The selections available depend on the data contained in the open Study 3 Select the saved data from which you want to generate a report from the Context drop down menu 4 Click OK to display the new report containing the selected information To create a New Report from the Study Explorer l Select the Reports tab available at the bottom of the Study Explorer frame of the window GenomeLab Genetic Analysis System User s Guide 251 PN A29142 AB Fragment Analysis Module Reporting Re
84. apparent fragment sizes between two selected points Linear Regression calculates the line of best fit for the selected alleles The system uses this line to determine the apparent sizes for all points not selected This option requires you to select more than two apparent fragment sizes GenomeLab Genetic Analysis System User s Guide PN A29142 AB Fragment Analysis Module Using the STR Locus Tag Editor To generate an allele list using one of these methods 1 Individually edit the apparent fragment sizes within the allele list from which you want to generate interpolation or a linear regression line 2 Select the alleles you want to use for generating the allele apparent sizes e To select the rows within the allele list click the row header of the allele list e To select multiple consecutive alleles press and hold the Shift key while dragging through the headers e To select non consecutive rows press and hold the Ctrl key while selecting each of the rows with the left mouse button 3 Right click your selection and select Interpolate or Regression The Interpolation or Linear Regression function replaces existing or missing apparent fragment sizes or supplies the generated values Allele ID Criteria The Allele ID Criteria tab contains the parameters used to accurately identify your alleles and to set these true alleles apart from various other anomalies identified in the fragment list A sample fragment repres
85. apply to the data filter out all unwanted results from your Study This process is known as result set filtering 220 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Fragment Analysis Module Using the SNP Locus Tag Editor Accessing the Result Set View You can view the Result Set View while working with a Study using one of these methods e Select View View Result Set e Click the Results Set 4 toolbar icon Fragment Analysis New Study Eile View Results Analysis Reports Window Help Result Set Yiew i Binnings j Peak Ratios Results Exclusion Filter Set New Result Filter Set 1 v Apply m DATS 93 D4 401_060425122M 04 25 2006 12 59 56 d Operator Value s DATS 94 D4 B01_060425122K 04 25 2006 12 59 50 i E n DATS 96 D4 C01_0604251 221 04 25 2006 12 59 44 DATS 97 D4 D01_060425122F METATA AE DATS 99 D4 E01_0604251 22C 04 25 2006 12 59 26 DATS 100 D4 F01_0604251229 04 25 2006 12 53 20 DATS 101 D4 G01 0504251276 04 25 2006 12 59 12 DATS 102 D4 H01 0604251224 04 25 2006 12 59 06 IV Show Excluded Analyses Data Reports Ready Figure 6 20 Result Set View Window Second Level Filtering The Result Set View window provides three key elements e Fragment Results table displays all analyzed samples both included and excluded that have been selected for the Study The displayed columns are user defined and can be modified to show a
86. are groups of information properties that apply to a sample 1 To access the sample property sets select View Sample Property Sets or select a sample cell in the sample plate screen and refer to the Note tab 2 Inthe property set table use the Tab or arrow keys to move from cell to cell Press the Tab key moves the focus from property to value and then to the next row and the arrow keys move in the direction selected e Press the Enter key moves the focus down the table one cell at a time within the same column Mote Method Analysis ol x el e ud PROPERTY VALUE Template Source Mo insert Figure 2 13 Property Set Editor 3 To select an entire column for sorting click on its header The selected column becomes highlighted 4 To select a row click on its number As with the column selection the selected row becomes highlighted GenomeLab Genetic Analysis System User s Guide 35 PN A29142 AB Sample Setup Module Using the Sample Setup Module 5 To select consecutive rows left click and hold the mouse button while dragging the cursor over table cells 6 To move a selected row up or down one cell at a time click the toolbar arrow icons 7 To delete the contents of the rows select the desired rows and click the Delete icon X A warning message opens prompting you to confirm deletion e Click No to return to the editor without deleting the property e Click Yes to delete the contents and r
87. as the result name or status always in view e To lock a column highlight the column in the Selected Columns area of the Column Selector window and click Lock A lock icon appears next to the selected column to indicate that the column position is locked NOTE This also locks all columns positioned above the lock location in the Selected Columns area and thus to the left of the locked position in the list If this includes columns that you do not want locked use the positioning arrows to move the columns out of the lock range e If the view is filled with locked columns you may not be able to scroll through the grid If this happens reduce the number or widths of the locked columns To move the lock position to a different column location click up arrow lock or down arrow lock in the Column Selector window To remove the column lock click Unlock in the Column Selector window e To lock and unlock columns directly from the fragment result table right click the column header and select Lock Column or Unlock Column Sorting the Results List You can sort the components of the results list in ascending or descending order for any of the column parameters displayed For example you can sort the results list by ascending bin number descending bin fragment count etc You can sort columns in the Result Set View the Fragment List AFLP Analysis and Peak Ratios tables Sorting an Individual Column e Right click the header of the
88. back as shown in Figure 1 3 NOTE Refer to the caution label on the front of the plenum assembly for positive identification Figure 1 3 Plenum Assembly Back View 1 Routing Notch 2 Conical Depression A GenomeLab Genetic Analysis System User s Guide PN A29142 AB Getting Started System Overview Sample Transport and Plate Holders The Sample Transport Figure 1 4 contains the plate holders used to hold the sample plate the buffer plate with buffer evaporation cover and the wetting tray 901594L Al Figure 1 4 Sample Transport and Plate Holders 1 Plate Holders 2 Part of Sample Transport Sample Plate The sample plate Figure 1 5 is used to hold the samples for separation The plate is a V bottom thermal cycler compatible polypropylene plate containing 96 wells 8 rows x 12 columns The wells have a 200 pL volume capacity Figure 1 5 Sample Plate GenomeLab Genetic Analysis System User s Guide 5 PN A29142 AB Getting Started System Overview Buffer Plate with Buffer Evaporation Cover The buffer plate Figure 1 6 holds the DNA separation buffer used during a sample run The plate is a flat bottom polystyrene non sterile plate containing 96 wells When placed over the buffer plate an evaporation cover maintains the proper buffer level 250 300 pL in the butter plate by preventing the buffer from evaporating The cover slips over the buffer plate position of the sample transport As the CE system
89. bottom buffer plate Each well being 3 4 full 4 pack e e e DNA Separation 608087 1 Eight capillaries 75 um i d 33 cm long 200 o d complete with Capillary Array electrode block and detector array fitting 33 5B Buffer Plates 609844 Box Package of 100 flat bottom polystyrene plates nonsterile without lids Required for use as the separation buffer plate sample Plate 609801 Box Package of 25 V bottom thermal cycler compatible polypropylene plates with 200 uL volume capacity Required for use as the CE system sample plate 25 plates box 378 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Maintenance and Diagnostics Consumable Items List a Contact your local Beckman Coulter representative for specific consumable material requirements based on your application and frequency of use Table 9 15 provides a list of the required consumable items for the Fragment Analysis system Table 9 15 Consumable Items Required for Fragment Analysis Item P N Description DNA Separation 608087 1 Eight capillaries 75 um i d 33 cm long 200 o d complete with Capillary Array electrode block and detector array fitting 33 75B separation Gel 391438 1 20 mL of gel in CE system compatible container Sufficient to run LPA 1 two 96 well plates Separation 608012 1 Each container has a screw top and pour tip The container has Buffer enough buffer 30 mL to fill a 96 well flat bo
90. box M 5 Select the Use Final Values as Initial Values check box lvl to use the final values for the initial values for the computation recommended if the peak resolution is good Exporting Sequence Result To export from the Sequence Analysis module l Launch the Sequence Analysis module and open a sequence result 2 Select File Export to open the Export dialog box 3 Select a target folder and file name NOTE Do not use special characters in the file name 2 V 5 l 4 Select the desired format in the Save as type drop down list as shown in the following example Export Sample Elements Hess Save in Export 3 ex FE M Raw Data Imi Result Data Sequence Test 403 6042415KM Result Output I Guality Parameters Alignment Results Alignment amp ecuracy Options Remove CEQ Tracking Suffix Resolve Filename Conflicts File name Sequence TestAD3 0604247 6KN i Apply Trimmin iid save as Ippe str ver 3 00 sct L ancel SEE er OU etl ME SCF Ver 2 10 sct Text Tab Delimited tet SEQ Sequence Text only seq FASTA FASTA and QUAL Fasta PHRED PHRED and SCF sch phd Figure 4 40 Sequence Analysis Export Dialog Box 5 Click Export GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Analysis Using the Sequence Analysis Module Sequence Export File Types The foll
91. box opens 2 Selecta new color 3 Click OK The color of the ambiguity codes changes in the base sequence text NOTE To search for heterozygotes use the Heterozygote Backward Search and the Heterozygote Forward Search H icons in the Standard toolbar GenomeLab Genetic Analysis System User s Guide PN A29142 AB 147 Sequence Analysis Using the Sequence Analysis Module Pinning Results Several analyses single or batch may be necessary before a given result is satisfactory Overwrite a previous result or produce additional result tabs using functions in the CE 148 System To overwrite a previous result click the pin in the left hand corner of the result tab to unpin lt g the result A reanalysis of that sample overwrites the existing result To produce a new results tab click the pin to pin the result Upon reanalysis the system creates a new tab containing the reanalyzed results Performing a Batch Analysis To analyze more than one sample simultaneously l Batch Analysis Selection E Select Analysis Batch Analysis The Batch Analysis Selection dialog box dialog box opens Sample Data Sample Flate Results Database Filter CEQ DATABASE Start Date 07 21 2005 E 7 Enable End Date 2 2005 E Refresh FA Test H02_06030121L0 Sequence Test H04_0603020 03 02 06 02 10 308M Sequence Test G04 0603020 03 02 06 02 10 28M Sequence Test FO4_ 0603020 03 02 0
92. call is correct Shows the parameters used in quality based and sequence based trimming It includes the original sequence length the length after quality based Sequencing the length after sequence based trimming the vectors trimmed and the base sequence in its original and trimmed forms For sequence based trims an x replaces the trimmed bases that are located further than that specified in the Distance from End setting or if they are an internal match Displays the text results of the alignment between a template sequence and a sequence result The top row of text is the base number the second line is the template sequence the third line is the sample sequence and the last line is the consensus Shows the statistical analysis of the accuracy of the sample sequence This section groups the errors in the alignment into 50 base regions and displays the number and type of error for each region To define the report format and content select File Report Format e To print the report without previewing or formatting prior to printing select File Print Report e To the view the report select File Print Preview 152 GenomeLab Genetic Analysis System User s Guide PN A29142 AB The following example shows a sample report Project Sequence Sample testseg 404_ 030515 18HG Result testseq 404 3 030518HG Sequence Results testseq A04 03030518NG Created Usi 05 03 20 33 42 Project System CEQ 8080 Sequ
93. can assign dye mobility correction values to compensate for the varying effects of different dyes on fragment mobility select a standard mobility reference and indicate whether to use the dye spectra from sample data or from saved system spectra Edit Fragment Analysis Parameters IDE x Parameter Set Name 3A T193ALU7 Project Default Analysis Method STA Locus Tags SNE Locus Tags Advanced Mobility Dye Mobility Calibration MoCorection Standard mobility reference None Dye Spectra f Use calculated dye spectra f Use system dye spectra Figure 6 16 Fragment Analysis Parameters Window Advanced Tab Mobility Set up the mobility parameters for the current analysis e Dye Mobility Calibration specifies the calibration from the options available in the list Based on your selection this option adjusts migration times or mobility to compensate for the varying effects of different dyes on fragment mobility To choose a value select any of the following options from the drop down list e PAver 1 Select this option when analyzing samples made from primers labeled with the phosphoramidite dyes D2 PA D3 PA or D4 PA e AE ver 2 Select this option to improve calibration and size estimates for primers labeled with the active ester dyes D1 D2 D3 or D4 e AE ver 1 Select this option for the Fragment Analysis test sample e SNP ver 1 Select this option when performing an SNP analysis using the GenomeLab
94. change capillary information 1 Select Replenish Capillary Information The Capillary Information dialog box opens Capillary Part Number poena Serial Humber oan 050128 Date Installed 04 27 2006 Cancel Print Time Installed 17 01 47 Advanced FREE Number of Runs b Help Dave on instrument 13 1 Figure 9 24 Capillary Information Dialog Box 2 View the following information e Part Number Displays the part number of the installed capillary array GenomeLab Genetic Analysis System User s Guide 359 PN A29142 AB Maintenance and Diagnostics Direct Control and Replenishment 360 e Serial Number Displays the serial number of the installed capillary array The serial number is used to track the capillary array along with the samples that have used the array e Date Installed Displays the date the capillary array was installed Time Installed Displays the time the capillary array was installed e Number of Runs Specify the desired number using the spin control Set more than the recommended number of separations to be performed before the system alerts you that the recommended number of runs has been exceeded e Days on instrument Specify the number of days on the instrument using the spin control This allows the capillary array to reside on the instrument longer than the recommended length of time before the system alerts you that the recommended number of days on the instrument ha
95. check box M to find the entered text exactly the way you type it into the field If you do not select Match case the system finds all occurrences of the text regardless of case GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sample Setup Module Using the Sample Setup Module Select the Use wildcards check box MI to include wildcard characters in your search The follow table describes the possible wildcards Table 2 10 Wildcard Characters Wildcard Description The asterisk specifies to search for any letters before or after the entered text The question mark represents any single letter The exclamation point within brackets makes the query negative Enter the letter following the within the brackets if you want the system to find anything that is not the entered text For example entering A finds any string that does not start with A NOTE The system will support the following wildcard combinations and The system does not support the combination e Enter the text you wish to replace with in the Replace with text field 3 Choose the desired Search and Replace actions e Click Find Next to find the next occurrence of the entered text e Click Replace to replace the highlighted text with the text in the Replace with field e Click Replace All to automatically replace all occurrences of the search text with the replace text Using Property Sets Property sets
96. click Edit and modify the appropriate analysis parameters in the Edit Fragment Analysis Parameters dialog See Defining Fragment Analysis Parameters on page 200 for a description of this dialog When finished click Save As and save the edited parameters to an appropriate Project with the same analysis parameters name as specified in the plate Import file After creating the Non Default Run Method and Analysis Parameters follow the same steps as in Importing a Sample Plate with Default Run Method and Analysis Parameters as listed above GenomeLab Genetic Analysis System User s Guide 43 PN A29142 AB Sample Setup Module Using the Sample Setup Module Exporting Sample Plate Data Export the sample plate data to use in compatible equipment or to manually edit the plate information IMPORTANT If a Sample Plate file is already open make sure it has been saved before proceeding To export the sample plate l Open the sample plate file Select File Export Select Resolve Filename Conflicts to prevent the file from overwriting a previous file with the same name If another file with the same name exists the export function increments the new file with a unique sequence number NOTE If you do not select Resolve Filename Conflicts and a file with the same name exists the system displays a warning message asking if you want to replace the old file with the new one Select Yes to overwrite the existing file or No to cancel the export
97. click Reset Form to clear the form Enter the information for the new gene The Cut and Paste keystrokes do not clear the form IMPORTANT All changes are GLOBAL All users can view and use the entries created under the Administrator function unless the Hide Gene option is selected Viewing and Editing Genes l From the Administration Menu click Manage Genes 2 Select the gene of interest from the drop down list and click Load 3 Edit or update any of the fields as necessary 4 Rename the gene or add a version to the name if desired 5 Click Save to save the modified gene to the database GenomeLab Genetic Analysis System User s Guide 213 PN A29142 AB Gene Expression Software Administration Managing Primers Add primers that were produced with third party software view and modify user defined primers and make any primer set available to all users as Reference Primers NOTE Genes must be previously entered using Managing Genes on page 272 or by FASTA in eXpress Designer See page 284 eXpress Profiler BECKMAN COULTER Manage Primers p NM 017000 207 30 20 236 20 E Load Save Account Reset Form Manage Genes The data has been loaded Manage Primers Universal Tags Name NM 017000 207 30 20 236 20 Import Multiplex Help Parent Gene Nh 017000 Left Sequence ATGTCCGATCCTG GTGATGT Left Primer start length 30 20 Left Melting Temperature Celsius 59 933 R
98. column you want to sort and select Sort Column The system sorts the column in either ascending or descending order depending on the order in which it was previously arranged e To sort the same column in the reverse order right click the header and select Sort Column again GenomeLab Genetic Analysis System User s Guide 249 PN A29142 AB Fragment Analysis Module Customizing the Results List Accessing the Column Sort Window To access the Column Sort window right click the column header area of the fragment list and select Sort Columns Column Sort Oy x Sort by Result Name f Ascending Descending Then by SampleName C Ascending f Descending Ascending f Descending Cancel Figure 6 36 Column Sort Window Sorting the Results List Data Columns To sort a column XM Eb E m Click OK to sort the list Select the column parameter you want to be sort from the drop down list Select the order in which you want the parameter sorted Ascending or Descending Repeat the first two steps for each additional columns that you want to sort NOTE See the online help for more information on these and other display modifications 290 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Fragment Analysis Module Reporting Results 6 13 Reporting Results The CE system software includes a powerful suite of utilities for creating reports It also allows you to export data
99. common group of sample results that are analyzed and exported together This chapter provides an overview of the Fragment Analysis module including its menu options toolbars and dialog boxes It shows how to use this module to work with Studies to run a fragment analysis to set up parameters to use result properties and to display options It also describes how to work with report and export fragment analysis functions 6 1 Fragment Analysis Module Overview The Fragment Analysis module processes fragment data from the CE system platform and provides size and allele information for detected peaks The system organizes parameters that identify alleles as locus tags that you can customize You can then view export and print the analysis results Analysis parameter sets provide parameters specific to an experiment allowing size calibration and locus tag assignment as necessary when processing data Data analysis can be a manual function or set to run automatically Edit and re analysis functions provide the capability to verify the accuracy of the data Graphically view analyze edit compare and print data of the following drop down listing types e Raw Data Fragment Data Fragment Lists e Information about locus tags and assigned alleles Also available are Current Data and Voltage Data These types of data cannot be edited GenomeLab Genetic Analysis System User s Guide 1 83 PN A29142 AB Fragment Analysis
100. concerning hazardous liquid waste Disposal of the Capillary Array Table 9 3 9 WARNING After removing the expended capillary array from the instrument dispose of it in a solid hazardous waste container Disposal of the Gel Cartridge Table 9 3 10 WARNING After removing the expended gel cartridge from the instrument use a lab spatula or wooden dowel to push any remaining gel into a liquid hazardous waste container Dispose of the empty cartridge in a solid hazardous waste container Disposal of D I Water Gel Mixture from the Wetting Tray Table 9 3 11 WARNING When performing this procedure use an exhaust ventilation unit that meets TLV requirements l Pour the D I water gel mixture into a hazardous liquid organic waste container 2 Rinse the Wetting Tray with water and dispose of the rinse in the liquid waste container 376 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Maintenance and Diagnostics Biological Waste Disposal Table 9 3 12 WARNING Dispose of D I water gel mixture in accordance with all applicable federal state and local environmental regulations concerning hazardous liquid waste Disposal of the Gel Waste Bottle Table 9 3 13 WARNING When performing this procedure use an exhaust ventilation unit that meets TLV requirements Dispose of buffer gel mixture in accordance with all applicable federal state and local environmental regulations concerning
101. current working database is greater than 1700ME To maintain system performance it is recommended that vou create a new database before proceeding System is aborting this sample plate operation Figure 6 5 Database Size Warning 6 3 Working with Studies in the Fragment Analysis Module A Study is a set of multiple sample results together with the results of analyses performed on the group of sample results You can use the Study Wizard to create a new Study easily The Study Wizard guides you through the process of creating a new Study starting from either raw data or previously analyzed fragment results The Study window opens automatically when you start the Fragment Analysis module You can open a new or existing Study immediately from this window or at any time while working within the Fragment Analysis module GenomeLab Genetic Analysis System User s Guide 1 93 PN A29142 AB Fragment Analysis Module Working with Studies in the Fragment Analysis Module To access the Study window select File New Study or File Open Study Study Mel E Create a new Study from AS Analyzed Results e GP C Open an existing Study Date Time D1151384 test af2 2006 11 32 33 AM IATATS3AU test 3 27 2005 11 40 28 AM GATATISAO test 3 27 2005 12 44 52 PM Don t show this dialog box again Cancel Figure 6 6 Study Window Creating or Opening a Study You can create a new Study from either Raw Data or pr
102. define several other factors that affect how your data is analyzed You can access the Fragment Analysis Parameters window either while developing a Study from within the Study Wizard see Selecting Raw Data for the New Study on page 195 You can also access it at any time while working within the Fragment Analysis module by selecting Analysis Analysis Parameters Using either method the Analysis Parameters window opens showing the currently saved parameter sets available for selection e To create a new parameter set click New e To edit an existing parameter set click Edit The following illustration shows the Edit Fragment Analysis Parameters window Edit Fragment Analysis Parameters Mel Parameter Set Mame ODefaultaePAnalysisParameters Project Default Analysis Method STA Locus Tage SAP Locus Tags Parameter Set Date created 12 20 2005 11 04 02 4M Date modified 12 20 2005 11 04 02 46 Peak Criteria Allele Identification Identify STR Alleles Slope threshold 10 SNP Analysis l Size Estimation and Allele ID Confidence Relative peak height threshold 1 4 Confidence level gs x Save As Cancel Figure 6 11 Edit Fragment Analysis Parameters Window Creating or Editing a Parameter Set 200 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Fragment Analysis Module Defining Fragment Analysis Parameters General Tab The General tab is the first tab displayed when you first ope
103. eatin BENE uU REP ERAS 219 PS SUM OU Ir WGC MIAO ec aoiose cans Asset cise e E LE AE EL E 220 Accessing the Result Set VieW 0 00 ee II 221 Applying Exclusion Filters to the Result Set Data 0 0 0 ccc eee 222 PIMC EXC IUSIODEFIMBIS orbe etae iei bde bai ee bte addidi ng tes eats 222 Viewing the SUMMA a ido eite espaol en peius danii 6 DOE a dee esa toes ate hates dob 223 Customizing the Result Set View ssssssee eee eee 223 Viewing Electropherogram Data lsisesssee eee IRI 223 jReEATIOIUZITO RESUS ere ee aa a DKonU E e de uc dre Qa e ee 220 64 USME EFAOBIETIELISIS s ird Sophos bro os Pere obo he Sang E CE root petites 231 Accessing the Fragment List VIEW 2e be Sex dE EW APERTE SERRE 231 Applying Exclusion Filters to the Fragment List 0 00 0 0c cece eee 232 Available Exclusion FIIIBIS 2 ia deed atu ette Gand Shad daw ER Dd ar RISE EE 232 Customizing the Fragment List eee eee eee 232 Manually Selecting Peaks for the Fragment List 00 0 c cee eee 232 Exporting a Fragment List to CSV File 0 eee 232 SNOWING a Stacked Graph c ashe0d a6 d axis Ain bade oe Fourteen dept b 233 GenomeLab Genetic Analysis System User s Guide V PN A29142 AB Including Excluding amp Resetting Peaks in the Fragment List 04 233 6 0 Perrormitg BI AMA SIS es ca succ cr ets Gord one b IER be E ates AR Eb b ias 239 Setting up a Bin Analysis acie reU Gad ura tiunt oe cach
104. ef 331 Converting Individual IDs to Subject IDs 00 0 eee 332 Creating New Size Standards 0 00 00 eee eee RI 332 Exporting Database Files from the Data Manager 0 0 ccc cece eee 333 Importing Database Files 000 RR RR RR IRR 39 Generating a Sample Run History 000 000 ccc RR 338 PACHA TAS UN GHI VO ONG coo s ete sai End etur dot inh rts fru opes Ree ecce Seer tbe Erie sut ud 338 GenomeLab Genetic Analysis System User s Guide Vii PN A29142 AB Section 9 Maintenance and Diagnostics lesse 343 viii 9 1 Routine Maintenance sc ks rk yu Se Sasseas Geese ede beact das Geadeeadas 343 Cleaning the Capillary Array 0 0 RR RRRRRR RR IRRRIRRRRM RE 344 Replacing the Gel Waste Bottle 0 000 ccc eens 349 FGPG IG tne Wettin TV asd oe ae tant doe oue e ri eto tee dal het ette HORN ed eee dea 390 9 2 Direct Control and Replenishment liliis RR 352 Accessing the Direct Control Window 0 0 0 0 ccc ccc eee ees 353 Loading the Sample Plate and Buffer Plate 0 0 RR 393 specifying Capillary Temperature 0 0 0 0 0c eee een 356 Jena PIT Geli dT AD Doce etae toss a sans pts cadena aria a aa gee bee ea ed ad aed ante 35 Mecid aoamMp O ORE ETE TERT ETE LITT TIR TIT 35 Performing a Separation 0 ee RIRRRRR RR IR rn 358 Replenishing the Capillaries with Gel 0 RR IR 358 PUORO ME Mano gsc ocd oos bee a Sen draco tiem etes dedo eod er o
105. example dinucleotides trinucleotides etc The Stutter Definition area of the window allows you to set the parameters for which the system can identify these peaks as stutter and not as true alleles e Search for Stutter Select this option if you want the system to search for stutter When selected you must also choose at least one of the stutter options below it e Stutter detection window width Select the number of repeats to specify the width of the window around each allele in which the system detects the stutter e Maximum relative stutter peak height Enter the height percent of the stutter relative to the true allele below which detected peaks are considered stutters Peaks above this threshold are considered unknown alleles e Detect stutter shorter than allele Select this option to detect stutters shorter than occurring prior to the allele 216 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Fragment Analysis Module Using the STR Locus Tag Editor e Detect stutter longer than allele Select this option to detect stutters longer than occurring after to the allele Spurious Peak Detection Spurious peaks are generally small trace artifacts detected as a result of sample contaminants These peaks tend to have a different shape than fragment peaks but often fall within the size range of a locus tag and are detected However they generally do not meet the criteria to be identified as known alleles a
106. hazardous liquid waste GenomeLab Genetic Analysis System User s Guide 3 1 PN A29142 AB Maintenance and Diagnostics Consumable Items List 9 4 Consumable Items List Table 9 14 provides a list of the required consumable items for the Sequence Analysis system Table 9 14 Consumable Items Required for Sequence Analysis ltem P N QTY Description Method 608000 1 Dye Terminator Cycle Sequencing Kit for 96 reactions Development Kit Includes DNA Sequencing e DNA polymerase Ani Dye Terminators ddUTP ddGTP ddCTP ddATP e dNTP I Mix Solution e dNTP G Mix Solution e Sequencing Reaction Buffer e pUC18 Control Template e 47 Sequencing Primer e Glycogen e Mineral Oil or e Sample Loading Solution SLS Dye Terminator Cycle Sequencing Kit for 96 reactions in a single tube containing e DNA polymerase e Dye Terminators ddUTP ddGTP ddCTP ddATP e dNTP Mix Solution e Sequencing Reaction Buffer pUC18 Control Template 47 Sequencing Primer DTCS Quick Start 608120 Kit Glycogen e Mineral Oil e Sample Loading Solution SLS Sequencing Test 608070 1 Prepared pUC18 sequencing control sample in sample loading Sample solution Sufficient to perform 24 separations Separation Gel 391438 1 20 mL of gel in CE system compatible container Sufficient to run LPA 1 two 96 well plates Separation Buffer 608012 1 Each container has a screw top and pour tip The container has enough buffer 30 mL to fill a 96 well flat
107. in Database or Other Loci list If the loci exist in the database moves the loci to the Loci in Database list Use to rearrange the list order Select an item and click the appropriate arrow to move it up or down the list GenomeLab Genetic Analysis System User s Guide 263 PN A29142 AB Fragment Analysis Module Exporting Results 264 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Gene Expression eXpress Profiler Overview Gene Expression 7 1 eXpress Profiler Overview The GenomeLab GeXP Genetic Analysis System GeXP System uses a patented highly multiplexed PCR approach to efficiently examine the expression of multiple genes with sensitivity and speed Perform a multiplex gene expression analysis by completing the following procedures Design a custom multiplex using the eXpress Profiler module e Perform the Reverse Transcription RT reactions e Perform the PCR reactions e Run the PCR products on the GeXP System e Analyze the data using the eXpress Profiler module This flowchart depicts the GeXP System workflow eXpress Profiler GenomeLab eXpress Designer GeXP Chemistry Use Gene Set Kit Multiplexes GenomeLab GeXP eApress Analysis T s bed rz n kJ I la FW Plate Setup and Peak Binning dum Perform Capillary Electrophoresis J and Analyze Data Normalize Data and L Generate Analysis Report Export GeXP data eXpress Map SNe exDresson lagde
108. included samples before completing the Bin Analysis The Fragment Results table and Summary area list all of the data samples included in the Bin Analysis You may view and compare individual samples manually to include or exclude individual samples or groups of samples from the analysis and re analyze selected samples New Binning Analysis Sheps lel Es result date analysis analysis parameters outcome y p 3 27 2006 11 41 35 HGATATSSAU Parameters Binning Locus Tag Source Data amp x Previous Hent gt gt Cancel Figure 6 30 New Binning Analysis window Reviewing the Source Data Click Finish to add the Bin Analysis to your Study 230 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Fragment Analysis Module Calculating Peak Ratios 6 9 Calculating Peak Ratios After creating a Study you can begin Peak Ratio calculations Peak Ratio calculations analyze a Study result to quantify the amount of DNA present in a fragment The system calculates the DNA quantity as a relative comparison to a user defined reference peak Beginning a Peak Ratio Calculation When viewing an exiting Study select Analysis New Peak Ratio to calculate peak ratios The Peak Ratios window opens Fragment Analysis GATA193A07_test1 Peak Ratios New Analysis 1 I2 File View Analysis Reports Window Help D ael s NE o2 MI os MB oa MM Y Height 2368 452 H x width pana 4j K Pe
109. load the wetting tray sample plate and buffer plate by following the instructions Click Load to finish loading plates Verify the sample position in the plate Click Start to initiate the run NOTE Ifthe working database exceeds 500 MB an error message appears If this happens follow then troubleshooting procedures described in Database Size on page 66 then return to these steps GenomeLab Genetic Analysis System User s Guide 65 PN A29142 AB Run Module Using the Run Module Database Size If you select a database that is greater than 1700 megabytes the following warning message appears when you create and run a sample plate Run Control Ea e The size of the current working database is greater than 1700MEB To maintain system performance iE is recommended that you create anew database before proceeding System is aborting this sample plate operation Figure 3 18 Run Database Size Warning Message To resolve this problem Click No to dismiss the warning dialog box Exit the Run module and the Sample Setup module Make sure all of the other modules are closed Open the Data Manager module Create a new database For details see Creating a Database on page 327 Copy the sample plate from the previous working database to the new database Click OK All to copy any objects referenced by the sample plate to the new database Exit the Data Manager module and launch the Run module SF iot n a v eec qe A
110. mark matches the character to its left 0 or 1 times Brackets and enclosing a set of characters indicates that any of the enclosed characters may match the target character To search for the text regardless of case select the Ignore Case check box M To search for the exact match for text not the IUB codes select the Exact Match check box 1 e To search for text within a range highlight the range of interest in the base sequence pane and select the Search Highlighted Range check box M 112 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Analysis Sequence Analysis Module Overview The following table identifies the IUB codes you can enter to find the corresponding bases when the Exact Match check box is clear Table 4 15 IUB Codes c C9 Bases G A T or C G A or C G T orC A T or C G A or T GorT G or C Aor T A or C CorT A or G Jd zmim io mxio cr joiz i iz GenomeLab Genetic Analysis System User s Guide 1 1 3 PN A29142 AB Sequence Analysis Using the Sequence Analysis Module 4 2 Using the Sequence Analysis Module Run a sequence analysis and set up its parameters result properties and display options then work with report and export sequence analysis results Click the Sequence Analysis icon Viewing Sample Data To view sample data 1 Click on Sequence Analysis then select File Open The Open dialog box opens as shown below
111. method in which the system labels the identified alleles in the fragment list For example you can label the alleles the same as their expected fragment size in nucleotides 163 164 167 etc in alphabetic order A B C etc or in simple numeric order 1 2 3 etc Select the method in which to name the alleles in the fragment list e Nominal size Select this option to label the alleles by their nominal fragment size e Alphabetic Select this option to label the alleles in lettered order then select the letter in the Start at field e Numeric Select this option to label the alleles in numeric order then select the number in the Start at field NOTE With the exception of Ploidy the following fields are not required for any part of the analysis They are used for supplementary reporting purposes only Ancillary Information The Ancillary Information area of the window allows you to select any supplementary reporting information to include with the locus tag parameters This area includes the following fields e Primer set name Enter the name of the primer set e GenBank Accession Enter the GenBank Accession Number e Repeat unit sequence Enter the sequence of the repeat unit associated with the locus tag e Ploidy Enter the number of alleles associated with the locus tag Primer Label Optional The Primer Label area of the window allows you to indicate for supplementary reporting purposes on which st
112. of a base in the sequence that does not exist in the alignment sequence The range is 1000 0 to 1000 0 A value of 3 0 is the default value e Delete start Sets the value of a deletion of a base in the sequence that does not correspond to the alignment sequence The range is 1000 0 to 1000 0 A value of 4 0 is the default value e Delete extend Sets the value of a consecutive deletion of a base in the sequence that does not exist in the alignment sequence The range is 1000 0 to 1000 0 A value of 3 0 is the default value Alignment Controls To optimize the alignment to find matching substrings first then run the alignment algorithm only on the bases remaining outside the substrings reducing execution time and storage space select Find matching substrings 1 This check box is selected by default e To use only substrings with the highest score and ignore the rest as opposed to using every character from both strings to compute the similarity score select Find Local alignment v1 e To specify that overhanging bases on the template or sequence will not be counted in the alignment score select Left Edge Free vT and or Right Edge Free 1 e To specify a minimum length for detected substrings enter the number of bases needed to constitute a substring in the Minimum substring length text box The range is 5 to 2000 The default value is 30 GenomeLab Genetic Analysis System User s Guide 1 23 PN A29142 AB
113. on the window repeat this procedure GenomeLab Genetic Analysis System User s Guide 347 PN A29142 AB Maintenance and Diagnostics Routine Maintenance 15 In the Remove Capillary dialog box Figure 9 5 select the Clean Capillaries radio button and click OK Remove Capillary Array Ea You may now open sample capillaries access doors to change capillaries To replace the capillary array immediately select Replace capillary aray To install a manifold plug select Install Cancel manifold plug l Help To clean the capillary windows select Clean capillaries Remove Capilar Array Options Replace capillary array C Install manifold plug Clean capillaries Capillaries exposed to air L Time Remaining min sec Ra fie Figure 9 5 Remove Capillary Array Dialog Box 16 While holding the array fitting tab align the array fitting with the manifold opening and guide pins Push the fitting into the manifold until it is completely seated against the bases of the guide pins Figure 9 3 17 Replace the manifold access cover and tighten the captive screw 18 Lower the capillary temperature control cover and secure the two rubber latches 19 Lower the capillary access cover and sample access cover to their closed locked positions 348 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Maintenance and Diagnostics Routine Maintenance 20 Click Done in the Install
114. online help By default the help system displays the Contents in the navigation pane located on the left and the introductory topic in the topic pane located on the right You can use the Contents to select and print specific help topics The navigation pane also provides a Search tab and an Index tab These tabs let you search for information by topic index entry and or keyword Using Help Select this option to display the Contents window on how to use the Windows Help system About GenomeLab Select this option to access software instrument and system information System Beckman Coulter Select this option to access the Beckman Coulter Home Page on the Internet Home Page GenomeLab Genetic Analysis System User s Guide 105 PN A29142 AB Sequence Analysis Sequence Analysis Module Overview Toolbar Icons The Sequence Analysis module provides seven toolbars 1 Standard 2 Data 3 Sample View 4 Sequence Dye Colors 5 Batch Control 6 Sample Plate and 7 Base Sequence Each icon on these toolbars corresponds to a commonly used menu item By default all toolbars except Sample Plate and Base Sequence are displayed when you first open the Sequence Analysis module The following tables describe the function of each of the toolbar icons Standard Toolbar The following example shows the Sequence Analysis module s Standard toolbar For descriptions of the actions that occur when you click a toolbar icon see Table 4 9
115. options Table 4 6 Analysis Menu Sequence Analysis Module Option Description Analyze active sample data or reanalyze existing sequence results Selecting this option opens the current Working Parameters dialog box Click OK to start the analysis Stop GenomeLab Genetic Analysis System User s Guide PN A29142 AB Terminates the analysis currently in progress 103 Sequence Analysis Sequence Analysis Module Overview Table 4 6 Analysis Menu Sequence Analysis Module 104 Option Description Working Analysis View and change the analysis parameters to be used for subsequent analyses For Parameters details see Editing Sequence Analysis Parameters on page 116 Restore Original Base Sequence Batch Analysis Reanalyze Batch View Selected Batch Sample Result Skip Current Sample Skip Current Sample Set Skip Current Sample Plate Pause After Current Sample Resume Batch Processing Third Party Analysis Third Party Analysis Setup service Alignment Align with Template Batch Alignment Trim Based on Quality Trim Based on Sequence Batch Trim Returns analyzed data displays and base sequence text back to their original states losing all edits Analyze multiple sample data or sample plate results sequentially Selecting this option opens the following dialog box For details see Performing a Batch Analysis on page 148 After performing a batch analysis use this option to perform th
116. results 7 Click OK The system processes all fifteen results and the original result and displays the results on the screen NOTE The originally opened sequence result appears in the top pane To enable Compare Synch select Tools Compare Synch Zooming Results Click on the Zoom Mode icon and zoom in on a region in the original sequence result and the other fifteen results zoom into the same region Select Tools Unzoom All and all sixteen results zoom out so you can view the entire result GenomeLab Genetic Analysis System User s Guide PN A29142 AB Panning Results To pan results click the Pan Mode Sequence Analysis Using the Sequence Analysis Module e Pan through the data in the original sequence and all sixteen results pan together e Pan through the data in a result other than the original one and all sixteen results pan together e Use the horizontal scroll bar to move the data along the X axis The data panes scroll synchronously Aligning Results To align results click the Align Mode AR icon e Select a point in the original result Select points in the other results you want to align with the point in the original result e Access the Display Options dialog box by double clicking in the original result pane If you choose the X axis or the Y axis range the range changes in all of the panes e Disable Compare Synch by selecting Tools Compare Synch e Check zoom align and pan and
117. sample and consensus bars corresponds to the selected area in the trace data view and the selected base in the trace text view You can use this toolbar to navigate to desired areas by clicking on any point of interest The alignment and trace data panes refresh and display the area GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Investigator Module Sequence Investigator Procedures For a global view of the desired attributes select the corresponding check boxes located on the right side of the map For each selected attribute the system adds a new bar to the consensus area showing the locations where selected attributes exist To change the color code of the attribute display l Click on the color well at the left of the attribute The color palette for the selected well opens Select a new color Click OK The discrepancy map uses the newly selected color Printing Reports You may print a report for the active compared sequence result To set up a report Select File Report Format Select the default printer from the Printer Name down down list Set your preferred page layout by selecting Portrait or Landscape oe te Select the option elements you want displayed in your report Table 5 9 Element Description Header Prints information such as the name of the compared sequence and the name of the sample sequences as well as the database and project where the sample resides Alignment Prints
118. selected dye 96 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Analysis Sequence Analysis Module Overview Table 4 1 Main Window Sequence Analysis Module Item Description Analyzed Data Displays the data that has been analyzed for the active sample Base SequenceDisplays a text view of the bases from the analyzed data for the active sample Raw Data Displays the raw data for the active sample Current Displays the current data for the active sample Voltage Displays voltage data for the active sample Compare Data Compare the active analyzed data set against another analyzed data set Quality Parameters Displays a graphical representation of the quality values for the active sample sample Plate Enable the Sample Plate toolbar using the View Toolbars option shown as in Undocked mode Use this dialog box to select the samples to open or export Menu Bar Options The following example shows the Sequence Analysis modules menu bar File Edit View Tools Analysis Window Help The following topics describe each of these menus and their options GenomeLab Genetic Analysis System User s Guide 97 PN A29142 AB Sequence Analysis Sequence Analysis Module Overview File Menu Click the File menu to display its drop down menu as shown in the following example Open Cro Close Close Tab Save Ctri 5 Save 45 Import Export Export from Plate Preferences
119. standards must meet the following two requirements Fragments in the larger half of the size range should be spaced uniformly For example the peaks in the range of 200 to 400 nucleotides of the SizeStandard 400 are 20 nucleotides apart Fragments in the smaller half of the size range should include a set of at least three fragments that are spaced approximately half the distance of those that are in the larger fragment size range For example in the SizeStandard 400 the peaks in the larger half of the size range are spaced 20 nucleotides apart while some in the smaller half of the size range are spaced 10 nucleotides apart NOTE Custom Size Standards can be created and defined in the Data Manager module See Creating New Size Standards on page 332 Setting the Analysis Method To set the analysis method l Select the standard to run with the sample from the Size Standard drop down list The size standards available are SizeStandard 400 SizeStandard 600 and SizeStandard 80 Your selection determines which size standards become available for selection in the Selected Peaks list to the right 2 Select the peaks you want to use in the Selected Peaks list To use all fragments in the selected standard click Select All e To select individual fragments in the selected standard select the check box next to each of the fragment sizes you want to run with the sample e Ifyou do not want to use any of the standard sizes click D
120. system is powered on The light is off when the CE system is powered off LASER Green light is on when the lasers are turned on during a run The light is off when the lasers are turned off HV Green light is on when the high voltage is applied during the injection or separation The light is off when the high voltage is turned off GenomeLab Genetic Analysis System User s Guide 9 PN A29142 AB Getting Started System Overview Software User Interface The software provides the interface for manual or automatic pre programmed control of the system and for data capture and basic data analysis Combine with sentence below The CE system provides a graphical user interface GUI that enables you to open programs and initiate commands by clicking buttons or selecting menu options To open the Main Menu double click the CE system icon on your computer s desktop GenomeLab System The Main Menu opens as shown in the following example To access a module click its icon on the Main Menu as described in Software Module Descriptions on page 11 After opening a software module the Main Menu collapses either to the Windows taskbar or as a shortcut toolbar as described in Main Menu Collapse Options on page 12 Main Menu Version 10 0 GenomeLab GeXP Genetic Analysis System SEQUENGING UN INVESTIGATOR FRAGMENTS b sod VISUALIZE Figure 1 11 GenomeLab Genetic Analysis System Main Menu 10 GenomeL
121. the introductory topic in the topic pane located on the right You can use the Contents to select and print specific help topics The navigation pane also provides a Search tab and an Index tab These tabs let you search for information by topic index entry and or keyword Using Help Select this option to display the Contents window on how to use the Windows Help system About GenomeLab Select this option to access software instrument and system information system Beckman Coulter Select this option to access the Beckman Coulter Home Page on the Internet Home Page GenomeLab Genetic Analysis System User s Guide PN A29142 AB Toolbar Icons Sample Setup Module Overview The Sample Setup module s toolbar contains icons that correspond to common menu options The following table describes the Sample Setup toolbar icons Table 2 8 Toolbar Icons Sample Setup Module Icon a e lac i Description New Opens an empty sample plate Open Select an existing sample plate to open Save Saves the current sample plate to the same directory with the same name and format as was used on the previous save Print Sample Plate Print data associated with the active sample plate Print Preview Displays a sample format of a hard copy printout of the active sample plate A toolbar at the top of the window provides options for navigation viewing and printing the display To restore the defaul
122. the multiplex as possible This parameter can be manually adjusted The maximum size is linked to the capillary electrophoresis size standards used and should not exceed 400 bp set the minimum PCR product size excluding Universal Tags It is recommended to leave the default setting of 100 bp as the lowest size to avoid the loss of sizing of smaller products Set in the software by default WARNING Do not change the default setting It will affect the chemistry Use this option for troubleshooting multiplex design such as when a gene is excluded from a multiplex Selecting this option provides more detailed information regarding all available PCR products for each gene 281 Gene Expression eXpress Designer Module 282 Table 7 7 eXpress Designer Module Parameters Parameter Description Reference Primers Allows primers to be accessed by multiple users for inclusion in a multiplex e g KAN or housekeeping genes Click on to highlight and include this primer set in a multiplex NOTE New reference primers can only be added in the Administrator mode See Managing Primers on page 274 for more information Accession Numbers Enter GenBank accession numbers for genes of interest Type in as needed or cut and paste NOTE Accession numbers are case sensitive Creating a Multiplex with Accession Numbers Enter Accession Numbers to create a first pass multiplex 1 Log into eXpress Designer then click eXpress The eXpres
123. the migration time specified in the Time field listed for each of the four dyes Calculate Using In the Calculate Using area of the window select the method the system uses to calculate the quantity of your reference peak GenomeLab Genetic Analysis System User s Guide 205 PN A29142 AB Fragment Analysis Module Defining Fragment Analysis Parameters Height Select this option to calculate the quantity of your reference peak based on its peak height e Area Select this option to calculate the quantity of your reference peak based on its peak area Quantitation Values Set the parameters for each dye Enter values for each dye selection e Size Time These fields depend on the selected ID Standard e If you selected Size as the ID Standard the first field next to the dye requires you to enter the size of the reference peak nucleotides for that dye e Ifyou have selected Time as the ID Standard the first field next to the dye requires you to enter the migration time of the reference peak minutes for that dye e Window Enter a variable quantity to consider when identifying the reference peak For example if you listed the size of your reference peak as 90 nucleotides you may enter a value of 1 in the Window field This value should compensate for any variability that might arise in identification of the reference peak NOTE The values entered in these fields are based on the ID Standard used either nucleotide
124. the module name the active document name and has the standard minimize maximize i and close icons at the top right of the window Menu Bar See Menu Bar Options on page 167 standard Toolbar See Toolbar Icons on page 171 Display Window Use this area to graphically display the specific window or windows associated with the current task This window displays the following panes e Alignment Panes Displays the text alignment results including the following elements Base and codon numbering reference and consensus amino acid translations reference sequence sample sequences consensus sequence and the difference line e Trace Data Panes Displays trace data and base sequence results for the selected sample sequences E Dye Colors Toolbar Use these user defined colors in the trace data view and the bases in the text view when the text coloring is activated F Navigator Toolbar Offers search and navigation tools within an alignment 166 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Investigator Module Sequence Investigator Module Overview Table 5 1 Main Window Sequence Investigator Module item Description G Discrepancy Map Toolbar Displays color coded locations of any type of attributes selected You may view the coverage of the reference and sample sequences H status Bar Displays information concerning the current selection including the position of the currently highlighted text
125. the panes function independently of each other Close the Compare tab by selecting File Close Tab Sequence Result Report The sequence result report includes additional information when certain Sample Elements are selected in the Report Format dialog box The following table describes what each sample element includes Table 4 27 Sequence Result Report Options Option Header Raw Data Result Data Result Output Current GenomeLab Genetic Analysis System User s Guide PN A29142 AB Description Contains summary information for the sample such as sample name position the method under which it was run the sample plate name the analysis parameter set name and capillary serial number In addition the sample note property set and consumables information associated with the sample will be printed NOTE This is the same information found on the General Note Property Set and Consumables tabs in the Properties dialog box Includes the unanalyzed data from the sample run Displays data that has undergone final processing to produce result data If the data has been analyzed using a sequence analysis parameter set the data with each peak assigned a base is printed Displays the final output in text form If the data has been analyzed using a sequence analysis parameter set the called sequence will be printed in text form This section of the report corresponds to the base sequence text Includes the trace associ
126. the plunger with your thumb until a small amount of gel is pushed out of the cartridge tip Insert the new cartridge or the gel pump plug into the barrel and lock it into position by aligning the cartridge wings with the cartridge holder and pushing in Push the cartridge locking lever towards the back of the instrument approximately a 90 angle into its locked position Close the Gel Pump Gel Cartridge Access Cover 10 In the Remove Gel Cartridge dialog box Figure 9 36 Click Install Cartridge to indicate that a gel cartridge was installed OR e Click Install Plug to indicate that the gel pump plug or empty cartridge was installed GenomeLab Genetic Analysis System User s Guide 369 PN A29142 AB Maintenance and Diagnostics Direct Control and Replenishment Remove Gel Cartridge Fa Do not open gel Remove Gel Cartridge x e You may now open gel access door PLEASE WAIT access door and change gel cartridge Releasing gel cartridge E Time Elapse IF you are not going to install a Install Cartridge working gel cartridge please insert the gel pump plug or clean empty cartridge to prevent any remaining gel from drying in the system Then click on Install Plug Install Plug min sec EG Cancel Please check the waste bottle to Help ensure that the battle will not averflove ini Future runs NEM EN eS Figure 9 36 Remove Gel Cartridge Dialog Box 11 If Install Plug was
127. the results you want to export from a list of available results located in various projects a Select the Project that contains the sample data that you want to export from the Project drop down list b Inthe Available list select the samples to export then click the right arrow 5 to move the selection to the Selected list NOTE The functionality of this window is very similar to that of the Select Raw Data window described in selecting the Components of the New Study on page 194 4 When you have finished making your selections click Next to proceed to the Export Options window 256 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Fragment Analysis Module Exporting Results 5 Define the export parameters and options including the file name and location using the Export Options window as shown in the following example Steps Results E Export results as S Saved m Elements m Options M Header v Remove CEQ Tracking Sufis Raw Data w Resolve Filename Conflicts v Result Data v Result Output Use the Save As button to change the export file name format or directory Specifying a file name of Result Name assigns the actual result name to the exported file Note Result Name is not a valid file name for CRY export lt lt Previous Cancel Figure 6 40 Export Options The Export Results Options window provides the following fields and options e E
128. the sample data Select one of these Dye Spectra options e Use calculated dye spectra This default setting specifies using the set of dye spectra obtained from the sample data e Use system dye spectra When you select this option you must also select each Dye check box for the dye traces you want generated This setting applies the system dye spectra to the data instead of estimating the dye spectra from the current sample being run NOTE The system dye spectra is established when running the Fragment Analysis Test Sample during installation when viewing the Dye Matrices window for a sample result in which spectra for all four dye labels were estimated After you run a Fragment Analysis Test Sample the system displays a Spectral Matrix of this sample in the single result view under the Dye Matrices tab which provides a Save as System Dye Spectra option GenomeLab Genetic Analysis System User s Guide 21 1 PN A29142 AB Fragment Analysis Module Using the STR Locus Tag Editor 6 5 Using the STR Locus Tag Editor The STR Locus Tag Editor allows you to create a new Short Tandem Repeat STR Locus Tag or edit an existing one to be used for STR analysis You can access this editor from the STR Locus Tag tab of the Analysis Parameters window see STR Locus Tags Tab on page 206 Locus Tag Editor Id xi Locus Tag GATAISS4A07 Save As Project GATAISSA0 Date last modified 4 19 2001 3 22 06 PM Allele ID Criteria Locus Inf
129. these toolbars Study Toolbar Dye Colors Toolbar gg Bde iov Ml o2 Mil os Ml os NNI View Toolbar EgramBand Toolbar IE vHeig 5221549 H x width peso HIC Figure 6 2 Toolbars Fragment Analysis Module The following table describes these toolbar icons Table 6 10 Toolbar Icons Fragment Analysis Module Icon Description E New Create a new Study from either raw data or result data zx Open Open an existing or recent Study save Saves the new or edited Study Study Explorer Displays the Study Explorer Fragment List Opens the Fragment List S 190 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Fragment Analysis Module Fragment Analysis Module Overview Table 6 10 Toolbar Icons Fragment Analysis Module Icon lal D1 red D2 black D3 green D4 blue Description Result Set Opens the Result Set View Red is the default color assigned to Dye 1 in the fragment data pane and D1 is the default name Black is the default color assigned to Dye 2 in the fragment data pane and D2 is the default name Green is the default color assigned to Dye 3 in the fragment data pane and D3 is the default name Blue is the default color assigned to Dye 4 in the fragment data pane and D4 is the default name Table 6 11 EgramBand Toolbar IMPORTANT Portions of this toolbar are only available when viewing Sample Results in Single Result Stacked Graph and Overlay Graph View modes
130. this instrument Safety interlocks disable high voltage output while the capillary access cover is open and remove the risk of shock while performing routine instrument functions However removal of any panel may expose an individual to the possibility of severe electrical shock and or mechanical injury For this reason any service requiring removal of a panel or otherwise overriding or disabling safety interlocks must be done by Beckman Coulter personnel only GenomeLab Genetic Analysis System User s Guide Xi Safety Information Xii Moving Parts Moving parts are limited to the sample handling system Plate movement is safety interlocked through the sample access cover To avoid injury due to moving parts observe the following e Keep loose clothing and hair away from the plate area e Never attempt to physically restrict movement of the plate assembly Laser Safety WARNING The GenomeLab Instrument uses a Class 3B laser The 3B classification means that direct intrabeam viewing of this type of laser is always hazardous to personnel The laser and several other integral components are housed in a sealed container that together comprise the laser assembly The laser assembly has no user serviceable parts Service of the laser assembly is restricted to certified Beckman Coulter field engineers During normal operation of the system laser light is not accessible to the user Therefore the overall laser classificati
131. to another location When given enough space the toolbar becomes a two column vertical toolbar as shown in the following example The toolbar resizes to fit in any location in the active application window To move the toolbar to amore convenient location click the title bar and hold the left mouse button while dragging the toolbar to one of the four edges of the application s window The Main Menu toolbar icons are miniature versions of the ones provided in the expanded Main Menu Clicking one of these icons opens the corresponding software module as described Software Module Descriptions on page 11 1 2 Operating the System Perform the tasks involved in analyzing or managing samples and sample data Although the following procedures are independent from setting up and starting the system perform the procedures below in the sequence provided Before you begin confirm that the Capillary is ready for use See Removing and Replacing the Capillary Array on page 361 Install a new Gel Waste Bottle See Replacing the Gel Waste Bottle on page 349 Preparing a Sample Prepare a sample for sequence or fragment analysis in accordance with using the instructions contained in either the CE system DTCS or the Quick Start Chemistry Kits or the PCR Reagent manufacturers kit for Fragment Analysis Starting Up the System l Turn on the PC and wait for Windows to start 2 Turn on the instrument 3 From the Windows desktop select Star
132. txt e Genotype Summary Report Tab Delimited gsr Transfer Fragments for Export fragment data in a csv file compatible with the GeXP Analyzer GeXP Preferences Set the following preferences e Select whether to display the Study window when the Fragment Analysis module is launched e Specify whether to graph all electropherograms by size or time e Set the number of egrams to be cached e Select the initial view when selecting the Single Result View of a sample e Specify whether to display a warning message before resetting all manually included or excluded bins during an AFLP analysis e Specify whether to display a warning message before reanalysis if old results in the study will be replaced by new results Print Screen send an image of the computer desktop application window or main window to the printer Recent Studies Lists the most recently opened Studies Exit Closes the Fragment Analysis module View Menu Click the View menu to display its drop down menu as shown in the following example View Fragment List F2 Results Sek F4 Lacus List Editor FE Genotype Summary Fe IM Status Bar IM Study Explorer Toolbars i NOTE A Y next to an option indicates that the option is enabled GenomeLab Genetic Analysis System User s Guide PN A29142 AB Fragment Analysis Module Fragment Analysis Module Overview The following table describes the Fragment Analysis module s View menu options Table 6 3 View Menu
133. voltage used during the separation e Life Displays the remaining gel capillary usage and laser hours C Capillary Status Indicates certain capillary usage gel cartridge life and on line status When the life of the gel or capillary array is exceeded the associated indicator turns red When the system is on line the indicator is green 58 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Run Module Run Module Overview Window Selection Tabs View data on the Data Monitor Direct Control Log and Instrument Data The following topics describe these windows Data Monitor Window The following example shows the Data Monitor window To open this window select the Data Monitor tab This window displays information associated with a sample s analysis A B Data Monitor Direct Control Log Instrument Data 24 25 25508 64 Figure 3 10 Data Monitor Window Run Module Table 3 15 Data Monitor Window Run Module ltem Description A Data Monitor Tab Displays the Data Monitor window Data Monitor Window Displays the data for the running sample plate for the selected capillary buttons C Capillary Buttons These buttons A through H represent the eight capillaries of the array GenomeLab Genetic Analysis System User s Guide 50 PN A29142 AB Run Module Run Module Overview 60 Direct Control Window This window contains selectable areas and function buttons that perform some of the distinc
134. was estimated those with asymmetric peaks stutter peaks etc This third level filtering keeps only the sample data with fragments that you are certain of which saves you the time of weeding out inaccurate fragment data later in the analysis Available Exclusion Filters You can find all of the exclusion filters using the drop down list available in the Name column of the Filter Set area After selecting the name of the filter to use you must select an operator usually lt gt etc and a value to apply to the filter NOTE For details on using the Column Selector window the Column Sort window and other display parameters see Customizing the Results List on page 248 Customizing the Fragment List You can define and modify the Fragment List components using the right click menus These commands allow you to select specific columns for display in the fragment list customize the arrangement of fragment list information and modify several other parameters of the fragment list For details regarding the Column Selector window the Column Sort window and other display parameters see Customizing the Fragment List sections of the online help Manually Selecting Peaks for the Fragment List Manually select peaks you want to add to or clear from a fragment list or building a new one from scratch To access this function right click within the fragment list and select Manually Select Peak When selected the syste
135. you do not specify either PaneCount or PaneWidth the system uses the default PaneCount of 1 PaneCount is an integer e If you specify PaneWidth it represents either the size or time per pane depending on the XScaleType The graph is broken into MaxX MinX PaneWidth full sized panes as well as one additional pane if required for the remaining data PaneWidth is a float In the DisplayOptions section of the Alternative text box enter the desired number of panes to distribute the X axis range of data over as follows 254 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Fragment Analysis Module Reporting Results lt DisplayOptions XScaleType BySize MinX 100 MaxX 300 MinYz O0 MaxY 10000 PaneCount 3 gt Dyes Displayed on the Graph SelectedDyes are dye selection options It allows the e gram graph to select which dye to report The Fragment Analysis view dye visibility does not affect this option The possible value for SelectedDyes are all 1 2 3 4 or combination of 1 2 3 4 All displays all four dyes By default the display for the four dyes is 1 D1 2 D2 etc In the DisplayOptions section of the alternative text box enter the desired dyes as follows lt DisplayOptions XScaleType BySize Minx 100 MaxX 300 MinYz O0 MaxY 10000 PaneCount 3 SelectedDyes 1 4 gt GenomeLab Genetic Analysis System User s Guide 255 PN A29142 AB Fragment Analysis Module
136. 0 Commits to the sequence based trimming Toggles between enabling or disabling audio If enabled while in Edit mode the system audibly announces each letter as you type a base letter Audibly announces a series of selected base sequence text GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Analysis Sequence Analysis Module Overview View Menu Click the View menu to display its drop down menu as shown in the following example View Toolbars w Status Bar w Analyzed Data w Base Sequence Raw Data Current Volkage Alignment Alignment Accuracy Quality Parameters Trimming Report Analysis Log Parameters Used to Compute Sequence Run Lag Working Database NOTE A Y next to an option indicates that the option is enabled The following table describes the Sequence Analysis module s View menu options Table 4 4 View Menu Sequence Analysis Module Option Description Toolbars Used to select which toolbars to display Select 1 the options that identify the toolbars you want displayed To hide a toolbar clear its check box See Toolbar Icons on page 106 Status Bar Toggles between displaying or not displaying the status bar Analyzed Data Base Sequence Displays the data that has been analyzed for the active sample Displays a text view of the bases from the analyzed data for the active sample Raw Data Displays the raw data for the active sample Current Displays the c
137. 0 0 Tf Monitoring the Baseline 0 lisse RR IRR RRRRRRR RI 18 Viewing Capillary Information xs ba us boat Deb ee eet eee Mie eee eed EP ds 19 Viewing Gel Information ado ob icgadrtra tuc te acra abs Qt o conce De dte a e d eo bad cu ette t 80 Viewing or Changing Buffer Information lisse RR 80 Removing and Replacing the Capillary Array 0 0 0 0 ccc RR 01 Removing and Replacing a Gel Cartridge Gel Pump Plug 0 00 00 eee 0 Removing the Manifold Plug 3d God eng dee Rea Rope La ER ei DOES ER 91 Section 4 sequence Analysis ern 95 4 1 Sequence Analysis Module OvervieW 0 0 ccc RR 95 VERBI T C E M 95 Monu Nee POOR Gas sce caster ae es TOTO TTA CT TOO see eae ee coe ee 97 TOOIDdE ICONS 2222 1029626 RE De 20 eere od doe d ede ditte tr ARCU BRE denda aes 106 4 2 Using the Sequence Analysis Module lssessse RR 114 Viewing odhdple Dalai cn ies ed nek dea v in rip bet E MORES bee eS Ee oa 114 Editing Sequence Analysis Parameters 0 0000 a 116 Performing Quality based Trimming 0 0 0 eee eens 127 Performing Sequence based Trimming 0 000 n 129 FAUNO DAS Satta aes xd e ea ipod aesti bt iue Gare Dunt etapa kn ipi ceci SALE Discs arbres 135 sequence Result Properties llle RR RR 138 setting or Changing Display Options sssssses RR RR 145 Changing Display GOES 9 bateue Der eatin ele aede d beca eate a 147 TURIN Bs esi eere DN
138. 00 30000 20000 10000 50 100 150 200 250 300 350 400 Dye Signal Figure 6 23 Trace with Labels Stacked Graph The Stacked Graph function aligns the electropherograms of selected results by size or time Zooming and scrolling on the x axis is synchronous between all plots selected while the y axis of each plot selected is independently scaled based on the available data To access this function right click the selected results from any of the results or fragment lists and select Show Stacked Graph Overlay Graph The Overlay Graph function superimposes the electropherograms of selected results on a single x y plot To access the Overlay Graph right click the selected results from any of the results or fragment lists and select Show Overlay Graph This function has two different modes The default mode Zoom allows you to draw a rectangular box around a selected area to zoom into The second mode Subset Selection allows you to select a group of peaks and add them to a Stacked Graph view To switch between the two modes select the appropriate icon button from the EgramBand toolbar Include Exclude Results You can exclude results automatically using filter sets or manually by selecting the Exclude Results command You can re include any excluded results at any time using the Include Results Peaks command available in the right click menu GenomeLab Genetic Analysis System User s Guide 22 PN A29142 AB Fragment Analysis
139. 04 25 2006 12 59 36 DATS 99 D4 E01_060425122C 04 25 2006 12 59 26 DATS 100 D4 F01_0604251229 04 25 2006 12 59 20 DATS 101 D4 G01 0504251275 04 25 2006 12 53 12 DATS 102 D4 H01 0504251274 04 25 2006 12 53 06 m Summary v Show Excluded Analyses Data Reports Ready Figure 6 3 Fragment Analysis New Study Window Performance Safeguards Controller Resources The system checks the available controller resources before it starts processing results If the system determines that the resources needed to complete the analysis are not available it displays a warning message see Figure 6 5 If this occurs close all applications re start the Fragment Analysis module and attempt the analysis again Memory Usage Warning E D The system available memory is getting law IF the next study is likely to contain more than 200 analyzed results then it is recommended to terminate any other applications that are currently running and restart the Fragment Analysis application Do vou want to continue opening the next study Figure 6 4 Controller Resources Warning 19 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Fragment Analysis Module Working with Studies in the Fragment Analysis Module Database Size Warning If a database is selected that is greater than 1700 megabytes the following warning message will appear if you try to perform additional data analysis steps Run Control e The size of the
140. 1 5 PN A29142 AB Gene Expression eXpress Backup and Restore 7 8 316 eXpress Backup and Restore It is critical to save information to disk and back up system data Use the eXpress Backup and Restore program to back up database files as well as recover the database and configuration files used in the GeXP applications Database files include eXpress Profiler user information multiplex TDF files as well as gene and primer information Configuration files are settings and parameters saved by the user during primer and multiplex design Saving the Backup Files l From the Windows Start menu select Start Programs GeXPAdmin eXpress Backup and Restore A dialog box will open on the desktop 2 Select Backup Restore Configuration Files and Backup Restore Database Files option 3 Click Backup to start the backup process A Database Backup dialog box opens 4 Enter a name for the backup file in the Filename field The default File Type is Backup Files IMPORTANT Do not change the default folder DBBackup 5 Click Save If the backup was successful a message box displays Database backup was successful Click OK Click Close to quit the program Restoring the Backup Files If a system failure occurs use the GeXP eXpress Backup and Restore to restore previously saved database and configuration files NOTE If you saved both the database and recovery information in the same backup file as described in saving the Bac
141. 20 0181 300 1769 20 2068 20 0389 226 1746 20 1971 20 NM_000436 121 651 20 771 20 UM_000599 191 4458 20 4648 20 0689 134 1021 20 1154 20 73 248 940 20 1187 20 TestPlex_1 Load multiplex NM 000788 177 796 20 972 20 w amm Figure 7 24 eXpress Designer Multiplex Screen Table 7 9 Multiplex Parameters Parameter Multiplex Name Minimum Separation Size Maximum PCR Product Size excluding Universal Tags Minimum PCR Product Size excluding Universal Tags Universal Tags GenomeLab Genetic Analysis System User s Guide PN A29142 AB Description The identifier of the multiplex recognized by eXpress Profiler The multiplex name must be unique or else it cannot be saved to the database When the TDF file is opened in Excel the multiplex name is found in cell A2 Set the minimum base pair separation size between PCR products The default is 7 bp Optimal separation size is 5 to 7 bp NOTE The minimum separation size for multiplex design is 3 base pairs set the maximum PCR product size excluding Universal Tags This parameter automatically adjusts as needed to fit as many products in the multiplex as possible This parameter can be manually adjusted The maximum size is linked to the capillary electrophoresis size standards used and should not exceed 400 Dp set the minimum PCR product size excluding Universal Tags It is recommended to leave the default setti
142. 253 1 0 0 98 al amp z l9 l32 No No B 5 z 0 z5z 0 99 aU az l4 154 No No a C 0 253 1 00 35 2 19 173 No No 10 C l 249 1 00 al aa a Lge No No ll ia 0 95 la8 aa od auy No No lz G 3 O 251 1 00 36 ed a did aed No No l3 254 1 00 36 22 46 250 No No 14 G l 253 1 00 36 aa a4 are No No 15 A 254 1 00 36 22 635 295 No No 16 249 l 1 00 2E amp z T0 314 No No 17 211 4 1 00 4 age 15 331 No No la A 251 0 0 1 00 2 aa Ul 347 No No 18 G Uu l 51 1 00 2 az H85 366 No No al A 252 1 00 a6 ade da 354 No No al 246 0 2 0 99 ae a3 00 403 No No d 233 4 uu 0 99 ae 23 06 dal No No as 244 l 0 95 l4 23 lz 436 No No 24 C O 246 4 O 92 le 24 14 451 No No aa T oO amp eae 0 27 17 24 26 473 No No ab G O 0 253 0 28 17 24 29 495 No No a 254 0 27 17 amp 3 37 313 No No au G l 253 0 29 3l amp 3 44 537 No No Figure 4 39 Quality Parameters Report The following table describes the categories on the quality parameters report Table 4 28 Quality Parameters Report Categories Number Number of the Called Base Base single letter designation of the called base A C G T or other IUB code A Base identity score assigned by the base caller ranging from 0 255 C Base identity score assigned by the base caller ranging from 0 255 G Base identity score assigned by the base caller ranging from 0 255 T Base identity score assigned by t
143. 6 02 10 264M Sequence TestEO4 0603020 03 02 06 02 10 24 4 Sequence Test DO4 0603020 03 02 06 02 10 224M Sequence Test CO4_ 0603020 03 02 06 02 10 214 amp M Sequence Test B04_0603020 03 02 06 02 10 194M Sequence Test 404 0603020 03 02 06 02 10 174M Sequence TestHOS_O603012 03 02 06 O0 32 394M Sequence Test G03 0603012 03 02 06 O0 32 394M Sequence Test FOS_O60S012 03 02 06 00 32 354M Sequence TeshEOS_O60S012 03 02 06 O0 32 354M Sequence Test DOS_O603012 03 02 06 00 32 374M Sequence Test COS_O603012 03 02 06 00 32 364M Sequence Test B03_0603012 03 02 06 00 32 364M Sequence TestA 3 0603012 03 02 06 00 32 354M 03 01 06 22 43 44PM m k Project M ame Default Search Sequence Test B b O60302028P Sequence Test CO5_O60302028P Sequence Test 005_ O60302028P Sequence Test E05 O60302028P Sequence Test FO5 O60302028P Sequence Test G05_O60302028P Sequence Test H05_O60302028P Load List Save List Figure 4 35 Batch Analysis Selection Dialog Box ju d xe Im us UK Cancel Help Select a project name from the Project drop down list at the bottom of the dialog box Select the appropriate tab Sample Data or Sample Plate Results Select Enable and enter a filtering start and stop date if desired Highlight a sample name and click the right arrow to move the sample name to the selected list box on the right Continue highl
144. A29142 AB BECKMAN on ne COULTER GenomeLab Genetic Analysis System User s Guide Beckman Coulter Inc 4300 North Harbor Boulevard Fullerton CA 92834 3100 O Copyright 2006 2007 Beckman Coulter Inc Copyright Licenses and Trademarks Copyright Beckman Coulter Inc 2006 2007 All rights reserved No part of this publication may be reproduced transcribed transmitted or translated into any language in any form by any means without the written permission of Beckman Coulter Inc Licenses and Trademarks Beckman Coulter is a registered trademark of Beckman Coulter Inc GenomeLab is a trademark of Beckman Coulter Inc All other trademarks are property of their respective owners li Table of Contents Section 1 Getting Started 1 0 ccc mm rm 1 1 1 System Overview METTUS 1 Purpose OMNIS SyS lel eos te cao toe UE EU snes oie a A ota eae 1 Functional Descriptio esi ett 5 cris scans xed ar er oth Pro e id re Dee iod diede at d ea 1 WC CI PRESENT PETI 2 Software User Inter 068 2 io aiios probi tuere e ERO bb et hederae bet 10 1 2 ODOFaHEO Ihe Sy SIC eee ie eh x etat ced don sure Ua UE Le he bere TAR UEAN EEk 13 Preparing a or MN TH ne ese cost antares Se a ee he 13 Sa MNO UP the OVS EO TIE eost dc rp dotted a enmt bein eg edo V aed ates neq 13 Creating a Database and Project Folder 0 0 ccc ccc eee eee IRR 14 CHING UD ad odes e cde dir Estar Ee 1 Y OPEP EE eee EMO einn Sm eb
145. AAAUCCTGTUCUGTGCCAGCTGUATTARATGAATUOGGCCAAUUGCU GU GGGGALmIAUDGC GUOTTITGCGTIATTGGGCGCTCTTCCGCTTOCTCGCTCAUCTGACTCGUCTUGUCGCTCGGTOGTTCGGC Tit oGC AGUGGTATUOCAGUTCACTUOARAGGCUGGTAATACGGTTATCCACAGAATUOAGGGGATAACGUAGGAAAUARA CATGTGAGUAAARGGUCAUGUARRAGGUUCAGUOGAACCGTAAARAGGCCGUG TTGCTGGUCGTTTTTCCATAULG CTCCOGCCCCCOCTRACGAGCATCACAAAAATCOACGCTCAAGTCAGAGGTROCGAAACCCGACAGGACTAT AAAGATAUCUCAGGCGTTTCCCCCTGGAAUGUTCCCTUGTGCGCTCTUUC TOTTCCGACUCTGCCGUTTACCGG ATACCTGTCCGCCTTTCTOCCCTTUGGtiARGCGIGGUGUTTTCTUCATAGCTCAUGUTGTAGGTATCTCAG Figure 4 18 Base Sequence and Trace Views after Quality based Trimming Quality based Trimming Applied NOTE To recover the original sequence after performing Apply Quality based Trimming select Edit Undo 128 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Analysis Using the Sequence Analysis Module Performing Sequence based Trimming Setting Up Sequence based Trimming To setup sequence based trimming po Fe oy SY aS eS dom 9 NOTE When performing quality based and sequence based trimming quality based trimming is always performed first Open the Sequence Analysis module Open a result and select Analysis Working Analysis Parameters Click the Edit button to open the Sequence Analysis Parameters Editor Select the Sequence based Trimming tab Select the Perform Sequence based Trimming check box M and select the desired options Click Save As and save the new parameters Click OK
146. AATLCOCTOSTOSTCTCATTOTTTATACTAGST ATGGT ASAT GLAS TATACTTCCTGAAGTCT CATCT AAGGEAACTOAAAAATATECATCACCOACATCOCAGTACTOTTALCTOATTTTT TC TTTTTTAACCCT CtOGGGATGTGGTATTCCTAATTGAACTTCCCAGABGOGTCTTGAGTTCTCTTATTAAGTTCTCT GAA ATCTACTAATTTTCTCCATTTAGTACTGTCTTTTTTCTTTATGGCAAAT ACTUGGAGTATTGTATGGATTTTCAG CU CAATTTTTGAAATTTTCCCTTCCTTTTCCATTTCTGTACAAATTTCTACTAATGCTTTTATTTTTTCTTCTGTCAAT a C ATTGTTTAACTTTTGaGaCCATCCATTCCTGGCTT TAATTTTACTGUT ACARTTTCAAT AGGACT AAT GGG KNOWN AA MUTATIONS Kl57EQx 612595 A160R Lle6l1v L1524 Al6sEGV Flo4y F165IL P1665 xXl6 IM Al amp 68G PLOSL Fl lAs L1 2P Dl 3L El 4daG Pl 5LOG KNOWN BASE MUTATIONS GJS CABON T492N T493N A501N GSLON COLIN Reference Sequence TGCTGTO Amino Acid Mutations 9 KNOWN AA MUTATIONS Known Amino Acid Notation 02K Base Mutations KNOWN BASE MUTATIONS Known Base Notation A306 Figure 5 4 Sample Reference NOTE The system creates a default Reference folder during software installation You may store your reference files in that location or in any other folder When you create a new comparison navigate to the appropriate folder to select your reference Selecting a Reference and Sequence Results l 2 Open the Sequence Investigator module Select File New An Open dialog box appears listing the contents of a reference folder GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Investigator Module Seq
147. AB Sequence Analysis Sequence Analysis Module Overview Base Sequence Toolbar To display the Base Sequence toolbar select View Toolbars then select the Base Sequence check box as shown in the following example Base Sequence z w Standard W Data m e JY Sample View ES v Sample Plate W Ignore Case ExactMatch Search Highlighted Range Selected Bases Quality Scores wv Base Sequence 1 00 Base as ee P E 0 Migration 22 44 min UNE E Bo Edited Inserted G 230 if Help Figure 4 8 Sequence Analysis Module Base Sequence Toolbar Use this dialog box to search for specific base sequences and to view quality parameters Enter the desired string of bases and click the arrow buttons to search backwards and forwards through the base sequence text To use regular expressions to locate the desired text use the characters described in Table 4 14 Table 4 14 Characters Used in Expressions Character Description The pipe character 1 allows either expression on its side to match the target string The expression alb will match a as well as b The dot will match any character The asterisk indicates that the character to the left of the asterisk in the expression should match 0 or more times The plus is similar to the asterisk but there should be at least one match of the character to the left of the sign in the expression The question
148. ACTATG CGATTACTAG AAAGCCATTA CCAGCTGAGA GGTCATGCAG AAACTTGCCG TCTCAGAATT AAAAAAAAAA factor C activator 1 4 GAGGCGACTG GTCCTTTTTG CATTTCTTAA AGAAAGCCAA AAAAATCTIT GAGAACTCTT TGAAAAATTT ATTCTATGAC TCAGTCGAAT ACATTGCCAA CATTTCTTCA AA ATTGATGG CAACTCAGCT AACTTGACAA GTTAACGTGA AAAAAAAAAA Figure 7 16 eXpress Designer FASTA Sequence Results J Click Save to Database to save the sequence in the database for future use Viewing Existing Genes To view a gene that was previously saved to the database click View Existing Data The drop down list will show on the screen Genes that were previously saved can be selected and viewed on the FASTA screen AF093267 Load Save Data has been loaded Gene llame AF093267 Accession Fasta AF093267 gi 3s934624 gb A F093267 1 F093267 Rattus norvegicus homer lb mRNA Sequence CTAGTGGATC TCAAGAAGCC AGGACGTTGG TAAGCATAGC GCCCAAAGGC GCTGGCTTTT TTTGAACGTT ATCTCGGGGG CAAGTTTTCA GGTACCCACC GGCAATAATA TGTTTATGGA GGAGAAGTCG TATCAATGGG CCCCGGGCTG CATGACAGTT TTTCGGTCCC TTTTGCTGTC CGAGTAACCT GCGTTTTTCT TTGGTGTCAG TGGAAAAGCA TTGGGCAAAA AGCAAGCATG AATAGCACCA CTGGGATTCT CAGGAGAAGA ACAGATGATG CAGGAATTCT CCAGAAGTGA GATAGCTTTC ATCCTGGGGT GGCTGCTTGA ACACGTACAC CGCGAGTGAA ACTGCAAAAT TGGGGGAACA CAGTTACTGT TCACTCCAAA CCTCTGAGCA TGGAACTGAC AGAGAACACC GCGGCCGCAA TCCAGATCCT TTGAACTTCA CTTAAATGTG GTGTCGTGGA TAATTTTATG ATCATTTTAC AACA
149. B Run Module Run Module Overview Table 3 1 Main Window Run Module Item Description G Capillary Buttons Letters A through H represent the eight capillaries in the array Selecting one of these buttons displays an associated pane shown in the display area H Status Bar Displays messages such as e Currently active database Displays the name of the active database e Active Plate Displays the side of the active plate left right e Pause To Load If you selected Pause to load this area displays the wait time e Configured 2nd Plate If you loaded a second plate this area displays the name of the plate e Display fields on the right of the status bar indicate which of the following keys are latched down e CAP Caps Lock key is enabled e NUM Num Lock key is enabled e SCRL Scroll Lock key is enabled Menu Bar Options The following example shows the Run Control menu bar options File View Direct Control Tools Run Log Options Replenish Help The following topics describe each of these menus and their options File Menu Click the File menu to display its drop down menu as shown in the following example File Restore Data Monitor Defaults Save Log System Preferences Frink Setup Print Preview Print Lag Print Selected Pane Ctri 5 Print Screen Exit Use the File menu to save the log file set system preferences and print the log or an item on the window The following table de
150. Base Sequence Pane To delete bases in the base sequence pane 1 Select Tools Edit 2 Highlight the bases in the base sequence text to delete 3 Press the Delete key or use the Cut A icon The screen removes the selected bases from both the base sequence and analyzed data panes GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Analysis Using the Sequence Analysis Module Specifying Base Grouping To specify base grouping in the base sequence pane 1 Select Tools Base Spacing to open the cascading menu 2 Select the desired group spacing Viewing the Analysis Log The system logs all the steps used in performing the base calling routine on the raw data in the analysis log For example the system logs a message when it is unable to find the end of PCR product data You may view the analysis log to see if the system was unable to find the end of PCR data The following message appears in the analysis log Analyzed Data Cannot compute stop time Using data stop time of xxx xx minutes as shown in the following example 09728700 10 28 34 03 28 00 10 28 34 09728700 10 28 34 09728700 10 28 34 09728700 10 28 34 09728700 10 28 34 03 28 00 10 28 34 09728700 10 28 34 03 28 00 10 28 34 09728700 10 28 34 09728700 10 28 34 09728700 10 28 35 Analyzed Data Analyzed Data Analyzed Data Analyzed Data Analyzed Data Analyzed Data Analyzed Data Analyzed Data Analyzed Data Analyze
151. CCCCGATATGGGAGTCCTCCACAAAGAGATCCAAACTGGAACGGTGA GCGGCTAAACAAGTCTCATCGCCACCAGGGTCTTGGGGGCACCCTACCACCAAGGACATTTATTA A CAGGAATGCTGCAGGTACCGGCCGTATGTCTGCACCCAGGAATTATTCTCGATCTGGGGGCTTCAA GGAAGGTCGTGCTGGTTTTAGGCCTGTGGAAGCTGGTGGGCAGCATGGTGGCCGGTCTGGTGAGAC TGTTAAGCATGAGATTAGTTACCGGTCACGGCGCCTAGAGCAGACTTCTGTGAGGGATCCATCTCC A GAAGCAGATGCTCCAGTGCTTGGCAGTCCTGAGAAGGAAGAGGCAGCCTCAGAGCCACCAGCTGC TGCTCCTGATGCTGCACCACCACCCCCTGATAGGCCCATTGAGAAGAAATCCTATTCCCGGGCAAG A A GAACTCGAACCAAAGTTGGAGATGCAGTCAAGCTTGCAGAGGAGGTGCCCCCTCCTCCTGAAGG ACTGATTCCAGCACCTCCAGTCCCAGAAACCACCCCAACTCCACCTACTAAGACTGGGACCTGGGA Sequence Hide Gene If checked gene is hidden from the Gene List in the eXpress Designer Figure 7 5 eXpress Profiler Manage Genes Screen Table 7 3 eXpress Profiler Manage Genes Parameters Parameter Description Display Name Displays the user defined name which appears in the Gene list box when using eXpress Designer or Analysis and in reports Accession Displays the user defined accession number FASTA Uses the GenBank FASTA header format gt II Examplel descriptor where Example is the Accession Number and descriptor is text that describes the gene sequence Shows the gene sequence Hide Gene Indicates whether the gene will appear in the eXpress Designer Genes lists 272 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Gene Expression Software Administration Entering Gene Sequences When entering gene sequences it is ess
152. Click OK when prompted to verify this action 3 Wait until the dialog box message displays GO with a green text message 4 Open the sample access cover Figure 9 1 and lift to the vertical locking position 5 Remove the sample plate and set it aside 6 Rotate the wetting tray retainers outwards to release the wetting tray 7 Lift the wetting tray vertically Cleaning the Wetting Tray l PA Remove the wetting tray lid assembly from the wetting tray reservoir Rinse the wetting tray lid and reservoir with deionized water and dispose of the rinse into a hazardous liquid organic waste container GenomeLab Genetic Analysis System User s Guide PN A29142 AB Maintenance and Diagnostics Routine Maintenance 3 Dry the lid assembly and reservoir To fill with D I water l Remove the lid of the wetting tray and fill it with D I water to the indicator line 2 Replace the wetting tray lid CAUTION You should process no more than one 96 well plate for each wetting tray without replenishing the wetting tray Periodically check the liquid level in the wetting tray The liquid level should NEVER be allowed to rise into the eight recesses of the wetting tray lid nor drop below the fill level indicator line 9 mL minimum The top surface of the wetting tray lid must remain clean and dry under any and all circumstances Installing the Wetting Tray 1 Select Replenish Replace Wetting Tray 2 Click OK when prompted to
153. Description Print Grid Data Select this option to print the entire grid as it is represented on the screen Export Grid Data Select this option to save the grid data as a comma separated csv or tab delimited txt text file Format Grid Select this option to open a cascading menu with formatting options as shown in the following example Print Grid Data Export Grid Data Freeze Rows Unfreeze Rows Freeze Golumns LInFreeze Columns Resize Rows Resize Columns Hide Rows Hide Cals Unhide all Styles Use this menu to customize the look of the data grid If you hide rows or columns using this menu the Print Grid Data option omits them however this does not affect the Exported Grid Data this exports all data GenomeLab Genetic Analysis System User s Guide PN A29142 AB Export Options Sequence Analysis Using the Sequence Analysis Module The Export dialog box provides two options you can choose when exporting sequence data as described in the following table Table 4 26 Export Options Option Apply Trimming Description If you selected the Export option during sample setup or batch analysis selecting the Apply Trimming option removes the trims from the sequence before it is exported The exported file replaces all internal trims with the letter x Export Only If The resulting sequence after trimming may be too small to be useful sequence gt X nt Use the Ex
154. Dye Colors Each icon of the various toolbars corresponds to a commonly used menu item The following tables describe the function of each of the toolbar icons Standard Toolbar The following example shows the Sample Setup module s Standard toolbar For descriptions of the actions that occur when you click a toolbar icon see Table 3 9 om 4 a x Figure 3 2 Standard Toolbar Run Module Table 3 9 Standard Toolbar Run Module Icon Description Restore Data Monitor Defaults Displays the default color selections at the time of installation Run Sample Plate Runs a sample plate Pause Pauses the currently running sample plate otop Stops the currently running sample plate or direct control process GenomeLab Genetic Analysis System User s Guide 53 PN A29142 AB Run Module Run Module Overview 94 Table 3 9 Standard Toolbar Run Module Icon Description E View Last Results Executes the Sequence or Fragment Analysis module with the analysis of the last sample set run Help Topics Displays the CE system online help By default the help system displays the Contents in the navigation pane located on the left and the introductory topic in the topic pane located on the right You can use the Contents to select and print specific help topics The navigation pane also provides a Search tab and an Index tab These tabs let you search for information by topic index entry and or keyword
155. E ke i I Description Audio Playback Audibly announces a series of selected base sequence text Working Analysis Parameters View and or change the analysis parameters to be used for subsequent analyses Analyze Analyze active sample data or reanalyze existing sequence results Selecting this option opens the current Working Parameters dialog box Click OK to start the analysis Stop Terminates the analysis currently in progress Trim Based on Quality Performs a trim based on quality Trim Based on Sequence Performs a trim based on a sequence Restore Original Base Sequence Returns analyzed data display and base sequence text back to its original state losing all edits Third Party Analysis Opens the third party analysis package specified in the Third Party Analysis setup dialog box Align Performs an alignment of the sequence against the current alignment settings Base Sequence Toolbar Displays the base sequence toolbar that allows you to search for text and ambiguity codes View Sample Plate Toolbar Displays the sample plate toolbar that allows you to select the samples to open or export Heterozygote Backward Search Search backwards for heterozygotes in the base sequence text pane Heterozygote Forward Search Search forward for heterozygotes in the base sequence text pane Help Topics Displays the CE system online help By default the help system displays the Contents in the naviga
156. Empty Waste Bottle Dialog Box Stop GenomeLab Genetic Analysis System User s Guide 349 PN A29142 AB Maintenance and Diagnostics Routine Maintenance 350 2 Empty Waste Bottle Ea Open the sample access door and empty the Waste Bottle Clase the sample access door and click on Done Help Capillaries exposed to air Time Remaining min SEC s 27 Wait until the dialog box displays the GO message in green text At this time it is safe to open the sample access door Leave this dialog box open You may now open the sample access door and empty the waste bottle Figure 9 8 Empty Waste Bottle Dialog Box Go po qu uio E 9 10 1 Change the waste bottle as described in the following steps Remove the cap from the new empty waste bottle Open the sample access cover Figure 9 1 and lift to the vertical locking position With the gel waste bottle 80 9096 full remove the cap and pull the bottle out of the instrument Place the cap from the new bottle over the full waste bottle and secure Thread the new bottle onto the cap attached to the instrument and set the bottle into position Close the sample access cover Dispose of the full waste bottle according to procedures found in Disposal of the Gel Waste Bottle on page 377 In the Empty Waste Bottle dialog box click Done Replacing the Wetting Tray Removing the Wetting Tray 1 Select Replenish Replace Wetting Tray 2
157. Enter a name for the database and if desired select the Set As Working Database option Figure 8 3 Data Manager File Edit view Tools Window Help New Database xi Enter Name for New Database M yNewD atabase 150 MB HE CEQBAK2 5 MB FAII B a D1151984 HC 0149599 a4 D151679 a GF 0229683 a GF 0352387 a 0982157 H D95938 Cj Default Gf pxs7132 Gf GATA193A07 GF Test project HHE USERTEMPLATE Figure 8 3 Setting Up New Working Database in the Data Manager Module 4 Click OK GenomeLab Genetic Analysis System User s Guide 327 PN A29142 AB Database Management Data Manager Procedures Setting the Working Database Program the database that contains all of the projects and the location where the data are stored until another database is set as the working database Close all modules except for Data Manager module If necessary open the Data Manager module How do they know Select the database you want to use as the working database The system highlights the selected database in blue 4 Select File Set as Working Database The name of the working database appears in bold to indicate that it is the working database NOTE The database size is limited to approximately 1700 megabytes However we recommend that you work with smaller databases to improve performance Creating and Naming a Project To create and name a project 1 Inthe D
158. Export Options For Plate Figure 2 4 Sample Setup Analysis Tab Menu Bar Options The following example shows the Sample Setup menu bar options File Edit View Run Window Help 22 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sample Setup Module Overview The following topics describe each of these menus and their options File Menu Click the File menu to display its drop down menu as shown in the following example File Mew Ctrl M Open Close Chri o Save Girls Save 45 Import Export Properties Default Report Format Print Preview Frink Setup Print Print Screen Lock 0 DefaultSamplePlate 1 AFLP 06 Exit Use the File menu to create open save import export view properties and print sample plates The following table describes the Sample Setup module s File menu options Table 2 2 File Menu Sample Setup Module Option Description New Opens an empty sample plate Open select an existing sample plate to open Close Closes the selected sample plate save saves the current sample plate save AS save the sample plate with a new name Import Import a sample plate tab delimited ASCII text TXT format Export Export a sample plate tab delimited ASCII text TXT format Properties Provides an information window concerning the active sample plate Default Report Format Print Preview Print Setup Print Print Screen GenomeLab Genetic An
159. Exporting Results 6 14 Exporting Results When exporting a fragment result in text format you can include all properties You can then open the exported file in any text editor or MS Excel to preview the file prior to printing To export results l Open the Fragment Analysis module 2 Select File Export Results The Export Results window opens as shown in the following example Export Results E3 Steps Results Options Project AFLP 06 TP2 20 Filter by date Fragment Results Result Available Selected 8 DATS 93 D4 401_06042513WU 4 25 2006 1 55 16 PM DATS 93 D4 401_060425122M 4 25 2006 12 59 57 PM DATS 3 DATS 34 D4 BO1 05042513wW w 4 25 2006 1 55 23 PM DATS 94 04 BO 050425122K 4 25 2006 12 59 51 PM DATS 3 DATS 96 D4 CO1_06042513w2 4 25 2006 1 55 29 PM DATS 96 D4 CO1_0604251221 4 25 2006 12 59 45 PM DATS 3 DATS 37D4D01 06042513X1 4 25 2006 1 55 36 PM rz DATS 97 D4 001_060425122F 4 25 2006 12 59 37 PM DATS 3 DATS 99 D4 E01_06042513X3 4 25 2006 1 55 44 PM ud DATS 99 D4 01_060425122C 4 25 2006 12 59 27 PM DATS 3 DATS 100 D4FOT 05042513 6 4 25 2006 1 55 51 PM 4 25 2006 12 59 21 PM DATS 1 DATS 101 D4GO1 06042513 3 4 25 2006 1 55 58 PM DATS 101 D4 G01_ 0604251226 4 25 2006 12 59 13 PM DATS 1 DATS 102 D4HO1 06042513 B 4 25 2006 1 56 04 PM DATS 102 D4 H01 0604251224 4 25 2006 12 59 06 PM DATS 1 Help lt lt Previous TN Figure 6 39 Export Results 3 Select
160. GACGGA ACCTATCTTC GTCTTATTTC CATGACATTT TCATCTCTCA CAGTACCCCT CGATGTGACA Figure 7 17 eXpress Designer FASTA results GenomeLab Genetic Analysis System User s Guide PN A29142 AB CACCGCACTG CCAAGATCTT GAGGCCTTCA TGGAACCCCC AGACGTGAGC GAATAGTTAA CGACAAGAAC GAAAATTCCT AGCACTCGAG TATGACAGCA ACTAAAACAT AAATTTGCAG TCACAGGAAT CAGAACTCAG TGGTGGACAG CAGCTTTGGA GGTCCTTCCC CCAGGGACCT GAAATGATCA AGTGCTATAT TAACTGAATT TGGAAGTTAT CTCATGTCTT CAAGGAATGT CTCAAAAGTT AAAAGTTTCA CAGCAGGAGG AGCCAAGGGC TGGGGACATC TATCGCGGAA AGGTACATCC ACCCGTTCCC AGAAGGAGCA TGGGCCTGAA TGCTCAATTA CTCAGCTGCT AATTGAACCC GAAGGAAAAT AARGCGCTACT AGTATTTGCT CGTCAATCAA ATGCCTAGCA ATATATCTGG AA AAAAAAAA complete TGAGGGCCGG TGATCCCTTT ACCCCCTCCC GGTGTGGTGA GCGAACTCAT TCTCCGCGCA GTCTGCCTCG TTCTGTAGCA CCAGATCGAC GTATAGGATA TGGCCAATGG GGAATTTAAA AGATCTTCAG TGAGCCAGCT AGTGATCGTA ACCTGAGGAA ATCAGTACTA TGGGTGGAAA GATCTTCCTA CTTTTCCGAT ACTGTGTCAG CAGGCAGCTT CTGACCTCTA GTCAAAATTA CGATTAACAG GCCTGTCAGA CTCCATGATG GATGGTGCTG ATGGGGGGTT AA A AAAA li Export cds AGAGAGACCA TCCATCCTGG TCCCTGTTGC AATTTGAACC TTTTTATCAG ACCTTTTCAA TTGAGTTGCC TAAGAGCAGA CCAAACACAA ATCAGTCTAG GCTGATAGCC GAAGCTGCTC TCTCCTTTAA CAGAATGCAT AGTCTCCTGG CGAGCCGGCG AACCCCCGCT AATATCGCCC ATCTCTTGTT TAAGAGTTCT GAAGTCGCTC TAAGACGTAC GATGTTCAAA GTGATGAGGG GTGGAAAGGA GTGGCTCTTT TGGTTGTAGA ATGAACATTT TTGTAAATAA CAGTGACCCA ACCTGC
161. GATA193AD7 D1S1679 D148599 D1181984 D228583 D382387 D982157 naca2o xl Filter off NUM Menu Bar Provides access to all drop down menus Menu Bar Options on page 320 Toolbar Contains the icons that execute pre defined functions Toolbar Icons on page 325 Display Area Lists the files contained in the selected node otatus Bar Displays information concerning the current selection ptions The following example shows the Data Manager modules menu bar options File Edit 320 View Tools Window Help GenomeLab Genetic Analysis System User s Guide PN A29142 AB Database Management Data Manager Module Overview The following topics describe each of these menus and their options File Menu Click the File menu to display its drop down menu as shown in the following example File Mew Ctrl M Mew Database Ctrl B Open Ctri o Print Ctrl F Frink Setup Frink Screen b Delete Rename Properties Import Export Seb s working Database Exit Use the File menu to create a new project or database open a selected database delete a project or database rename database items view properties import export database items and set the working database Table 8 2 File Menu Data Manager Module Option Description New Create a new Project or Standard in a database New Database Create a new database Open Edit Fragment Results Optical Scan Data Sample
162. GCCOCGCTTTCCAGTCGUGGAAACCTUGTCGTGCCAGCTGCA T TAATGAATCGGUCAACUGCG CoGGUiSAGAGUIUGGTTTGCGTATTGGGUUGCTOCTTCCUGCTTOCCTCGOCTCACTGACTOCGCTGCGCTCGGTUCGT TOGGUTGCGUGCGAGUGGTATCAGCTCAUTCAAAGGUGGTAATACGGTTATCCAUCAGAATCAGGGGATAAC SCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCSGTAAAAAGGCCECETTSCTSGSCOT TITTCCATAGGCTUOCGCUCOCCUTGACGAGCATCARCARRAAATUGACGCUTCAARGTUAGAGUTGG GRAAUCC GACAGGACTATAAAGATACCAGGCGTTTCCCCCTGSGAAGCTCCCTCGTGCOCTCTCCTGTTCCGACCCTG CCGCTTACCGGATACCTUTCCGUOCTTTUCTCCUCTTCGGGAAGUGTGGCGOCTTTCTCATAGCTCOCACGCTUGTA GUOTATCTCA UNE x ae nA Figure 4 17 Base Sequence and Trace Views after Quality based Trimming GenomeLab Genetic Analysis System User s Guide 1 2 PN A29142 AB Sequence Analysis Using the Sequence Analysis Module Applying Quality Trimming If the trimming is acceptable select Edit Apply Quality based Trimming to apply the resulting base sequence and trace view test sequenceAl2 A11_02012519Al 7 New Analysis Untitled Analyzed Data i zu eu an a0 ru Bu poem GCG ETTE CAT GC CTE CAGGT CGACT CT AG OGG AT cc GGTACCGAGCTCOS amp ATTCGTAATCAT GT ATA G CT Huecrezceree iN Hehe lia fs N mM 4 SONDA Medal Jh i80 S00 rao 1 n 1240 1400 Wao 2000 Cata Polnti ACGGCCAUGTGCCAAGCTTGCATGCCTTGCAGGTCGAUCTCTAGAGGATCOUCCCGGGTAUCCGAGCTCGAATT COTAATCATGTCRATAGCTISTTTCCTGTIGTGAAATTGTTATCCGCTUOCACARATTCCAUCACRRACRATAUCUAUGCUC DA RAGCATAAACTGTAAAUGCUTGGGGTGCUCTARTGAGTGAGCTAAUCTOCACRATTAATTGCGTTGCGUTCAC GOCCGCTIICCAGTCGGG
163. Help system About GenomeLab Select this option to access software instrument and system information system Beckman Coulter Select this option to access the Beckman Coulter Home Page on the Internet Home Page Toolbar Icons The Data Manager module provides one toolbar with icons that correspond to common menu items Figure 8 2 shows this toolbar Pi a amp Ba i3 exp eres os r Figure 8 2 Toolbar Data Manager Module Table 8 8 Toolbar Data Manager Module Icon Description Up One Level Moves the directory structure tree up one level New Create a new project or database 4 Open Opens and edit standards a H Cut Deletes selected items projects and databases Copy Copies a selected item project or database to the clipboard e Paste Inserts one or more copied or cut items from the clipboard to selected project or database 8j Print Prints a report of the selected item GenomeLab Genetic Analysis System User s Guide 325 PN A29142 AB Database Management Data Manager Module Overview Table 8 8 Toolbar Data Manager Module Icon Description x Delete Deletes selected items projects and databases Properties Displays the current database and modification date and other information 3j concerning the selected data file a Backup Database Creates a backup of the database Restore Restores the database with the backup Help Topics Displays the CE In
164. JRE art san esie ve Ze ma H H v k 106 Figure 4 2 Standard Toolbar Sequence Analysis Module Table 4 9 Standard Toolbar Sequence Analysis Module Icon Description Open Opens data files including Sample Data Sequence Analysis Parameters Optical Scan Data Sequence Results and Sample Plate Results Save Saves existing data Print Prints the report Print Preview Displays a facsimile of a hardcopy printout of the selected sample plate Print Desktop Prints the desktop application or main window as defined in the Preferences dialog box Cut Cuts selected bases from the selected position and copies them to the clipboard Copy Copies selected bases to the clipboard Paste Inserts one or more copied or cut bases to the selected text position Undo Undoes the last action performed Redo Redoes the last undo action iol 15 leo e Apply Quality based Trimming Commits to the quality based trimming iL are Apply Sequence based Trimming Commits to the sequence based trimming ATTE E Audio Enable Toggles between enabling or disabling audio When this option is enabled in Edit I mode the system audibly announces each base letter typed GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Analysis Sequence Analysis Module Overview Table 4 9 Standard Toolbar Sequence Analysis Module Icon H m I 4 r1 ir Li
165. Kit A21019 1 Kit contents include e RT Buffer 5X 480 pL green e Reverse Transcriptase 120 uL at 20 units pL red e PCR Buffer 5X 480 uL amber e KANr RNA with RI 600 uL yellow e DNase RNase Free H20 1200 uL white e DNA Size Standard 400 55 uL e Mineral Oil 5 mL e GenomeLab Sample Loading Solution 6 mL GeXP Human A21055 1 e Kit contents include Reference Plex e RT Buffer 5X 480 uL green e Reverse Transcriptase 120 uL at 20 units pL red e PCR Buffer 5X 480 uL amber e KANr RNA with RI 600 pL yellow e DNase RNase Free H20 1200 uL white e DNA Size Standard 400 55 uL e Mineral Oil 5 mL e GenomeLab Sample Loading Solution 6 mL e RT Rev Primer Plex Human Reference Plex 240 uL orange e PCR Fwd Primer Plex Human Reference Plex 240 uL blue e Control RNA Templates Human Reference Plex 30 uL at 100 ng uL from human source gray sample Loading 608082 1 Sample Loading Solution SLS 6 0 mL Solution SLS Buffer Plates 609844 Box Package of 100 flat bottom polystyrene plates nonsterile without lids Required for use as the separation buffer plate Sample Plate 609801 Box Package of 25 V bottom thermal cycler compatible polypropylene plates with 200 uL volume capacity Required for use as the CE system sample plate 25 plates box a Contact your local Beckman Coulter representative for specific consumable material requirements based on your applicat
166. MLPLVPK Expanded KVSMLPLVLK KVSILPLVPK To export the active compared sequence 1 Select File Export 2 Choose the desired format as described in Table 5 9 3 Click OK to export the data NOTE Ifa file with the same name exists in the selected directory the system prompts you to rename the new file or confirm that you want to overwrite the existing file The system displays an hourglass while exporting the data Saving the Data To save a new document l Select File Save A Save As dialog box opens prompting you to enter a file name and folder location 2 Select the folder location and enter a file name 3 Click Save The system saves the data to the file for later retrieval NOTE Ifthe document has previously been saved select File Save to save the document immediately To change the name or location of an existing file l Select File Save As A Save As dialog box opens prompting you to enter a file name and folder location Select the folder location and enter a file name Click Save GenomeLab Genetic Analysis System User s Guide PN A29142 AB Fragment Analysis Module Fragment Analysis Module Overview Fragment Analysis Module The analysis of fragment or allele data is performed with the CE system software by using Studies A Study represents a collection of quality sample results used for locus tag generation genotyping quantitation or AFLP profile generation Ultimately a Study contains a
167. Model Quartic Locus Tags lt lt Previous Cancel GenomeLab Genetic Analysis System User s Guide 1 97 PN A29142 AB Fragment Analysis Module Working with Studies in the Fragment Analysis Module Figure 6 9 Analysis Parameters Window Selecting a Fragment Analysis Parameter Set Use the Analysis Parameters window to create select or edit the analysis parameter set for analyzing the data selected for the new Study Your choice of a particular set of analysis parameters depends on the particular fragments you are attempting to identify The CE system software has a set of default analysis parameters used to estimate unknown fragment lengths The calibration curve is based on the migration times of the size standard fragments The default set of analysis parameters cannot be edited The default parameters are as follows e DEFAULT FRAGMENT ANALYSIS PARAMETERS Used for applications such as STR AFLP etc DEFAULT GeXP ANALYSIS PARAMETERS Used for the Gene Expression application e DEFAULT SNP ANALYSIS PARAMETERS Used for the SNP application Selecting a Saved Analysis Parameter Set In the Select Analysis Parameter Set area of the window select the parameter set from the drop down list The Parameter Set Summary area of the window displays summary information about the selected analysis parameter set These include e Date and time the analysis parameter set was created or last modified e Selec
168. Module Using the SNP Locus Tag Editor To re include results that were excluded in the Study right click the selected samples from any of the data or fragment lists and select Include Results When displaying excluded results the screen uses a color code Teal shading identifies results excluded automatically using filter sets e Maroon shading identifies results excluded manually Light gray shading identifies previously excluded results that have been re included You can hide both filtered and manually excluded results from view by de selecting the Show Excluded command available in the right click menu NOTE Refer to the online help for detailed descriptions of each of these and other display parameters Re Analyzing Results The Fragment Analysis module contains a Reanalyze Results feature that allows you to select one or more samples from the Result Set view to be re analyzed This allows you to apply a different set of fragment analysis parameters to a previously analyzed data set to pick up missed peaks or to avoid picking up unwanted peaks The data resulting from the re analysis automatically replaces the older Result Set data allowing you to quickly continue with your Study To re analyze the results Select one or more results from the Result Set View 2 Right click the selected results and select Reanalyze Results Reanalyze presents the following options e From Sample Data replaces the previous analysis result wit
169. N N N EN NS Sequence Fragment Analysis Analysis VY NIS IS Part Number ABgene AB 0908 USB 71786 or Invitrogen 10977 015 USB 22638 608012 608087 20 mL 391438 10 mL 608010 or 381 Maintenance and Diagnostics Diagnostics Table 9 18 Equipment and Supplies ltem Part Number GenomeLab GeXP Genetic Analysis System A26572 or A28130 sample Microplates 96 Well 609081 8 Well Cap Stops BioRad TCS 0803 or Corning Costar 0556 Buffer Microplates 96 Well 609844 1 5 mL and 0 65 mL Microtubes Pipettors P10 P20 P100 P200 and P1000 Aerosol Resistant Tips for P10 P20 P100 P200 and P1000 Microtube Centrifuge 365603 Microplate Centrifuge Thermocycler with Heated Lid for 96 Well Plates Vortex Mixer Non Frost Free Freezers 80 C and 20 C 9 5 Diagnostics This section provides some common diagnostic procedures you may perform as needed on the GenomeLab GeXP system Re Initializing the System To re initialize the system select Run Reset in the Run module Homing the Plates and or Gel Pump To re establish the position of the plates and or gel pump Select Run Diagnostics in the Run module The Diagnostics dialog box opens Select the Home Plate and or Gel Pump check box Click OK Monitor the Tests dialog box and verify that the final results is Test passed jp cce uS usn dem Click the Close button Viewing PC Settings To view the computer settings Select Run Diag
170. O bre eed inda t her eae ae eat 383 MORIOTIRO NE BASEN a s sy aides bare dire a eee oa Ree e b Ur ee 383 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Safety Information Safety Information This section provides safety information and instructions for the hardware and accessories of the system It includes the following topics Safety Symbols on page x Chemical and Biological Safety on page xi Electrical Safety on page xi Moving Parts on page xii Laser Safety on page xii Electrical Safety on page xi Moving Parts on page xii Laser Safety on page xii Notes and Warnings The following information describes the notes and warnings used in this document NOTE Contains supplemental or explanatory information concerning the current topic or procedural step IMPORTANT Used whenever an action or circumstance may potentially cause personal injury Mechanical damage may also result Also can contain information about a possible software program failure draw attention to a specific software setting or point out that a loss of data may occur if information stated within the paragraph is not adhered to or if procedures are executed incorrectly CAUTION Used to prevent equipment damage To disregard that caution may cause mechanical damage however personal injury is not likely WARNING If the equipment is used in a manner not specified by Beckman Coulter Inc the protection provided by the equipment ma
171. Open Data Manager 2 Opena sequence result 3 Select File Export to open the Export dialog box 4 Browse to the target folder then select and highlight the file name 5 Select the desired format in the Save as type field as shown in Figure 8 9 Sample Elements Save in 3 Export a t v Header Raw Data Result Data Result Gutput F Guality Parameters o E Alignment Resulte Alignment Accuracy Options Remove CEG Tracking Suffix Resolve Filename Conflicts File name SEG TEST_2006_04_01 Appl Trimmin d save as Ippe scF ver 3 00 ct L ancel SCF ver 3 00 P scf SCF ver 2 10 ect Text Tab Delimited tet SEQ Sequence Text only seg FASTA FASTA and QUAL fasta FHRED FHRED and SCF scE phd 1 Figure 8 9 Sequence Analysis Export Dialog Box 6 Click Export GenomeLab Genetic Analysis System User s Guide 333 PN A29142 AB Database Management Data Manager Procedures 334 Data Manager Export File Types Table 8 9 describes the available export file types Table 8 9 Data Manager Export Options Format SCF scf Tab Delimited Text txt PHRED scf phd 1 Description The SCF Standard Chromatogram Format format stores analyzed DNA sequence or fragment data for a single sample The format describes both public and private data sections The well defined public data section contains trace data points the called s
172. Parameters Sequence Analysis Parameter Set determines the start and end times for the analysis of raw data the threshold above which data are considered peaks the data start criteria the heterozygote detection parameters and the alignment template and parameters You may also edit sequence analysis parameters under the Analysis tab in the Sample Setup module Optical Scan Data Lists the alignment optical scan data available in the selected project Optical scan data are the result of the two lasers scanning the capillaries to determine the position of the capillary windows where the lasers focus when collecting data If the scan is successful eight peaks appear for each laser denoting that all eight capillary windows were established See Table 4 11 Sample View Toolbar Sequence Analysis Module for more information on the Sample View Toolbar descriptions GenomeLab Genetic Analysis System User s Guide 1 1 5 PN A29142 AB Sequence Analysis Using the Sequence Analysis Module 116 Editing Sequence Analysis Parameters The Sequence Analysis Parameters Editor dialog box consists of six tabs General Initial Data Detection Heterozygote Detection Quality based Trimming Sequence based Trimming and Alignment Reference To edit the Sequence Analysis Parameters l Select Analysis Working Analysis Parameters to open the Working Parameters dialog box 2 Click Edit The Sequence Analysis Parameters Editor dialog box opens as show
173. SNP Primer Extension The SNP analysis can proceed if you do not select this mobility correction but the values obtained may vary slightly SNP ver 2 Select this option when performing a SNP analysis using the GenomeLab SNPStart Primer Extension Kit 210 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Fragment Analysis Module Defining Fragment Analysis Parameters e No Correction Keep this default value when you do not want the system to make any compensation for dye effects on fragment mobility NOTE When performing a Fragment Analysis Test Sample you must use the AE ver 1 dye mobility calibration for correct sizing of the fragments Dye Spectra Dye Spectra values are calculations of the relative emission intensities of each dye in each of the four detection channels The system calculates these values during each analysis and uses them to transform the four electropherogram channels to dye signal traces for each of the dye labels present in the sample When unable to calculate a dye spectrum from the sample the analysis process terminates unless system dye spectra are available The Dye Spectra options allow you to select an existing set of dye spectra to use instead of estimating the dye spectra from each sample NOTE This option can shorten the analysis time by up to 50 The system stores only one set of system spectra used automatically when the system cannot estimate one or more of the needed spectra from
174. Sample Data sample Data Sample Data sample Data Sample Data sample Data Sample Data sample Data Sample Data sample Data sample Data sample Data sample Data sample Data sample Data sample Data sample Data sample Data sample Data sample Data sample Data sample Data sample Data sample Data Sample Data sample Data sample Data sample Data sample Data Qarnnla Nata The following table describes the items called out in Figure 8 1 Table 8 1 Main Window Data Manager Module Opti A B Menu Bar 0 on Description Title Bar Shows the module name Data Manager 4 20 2001 11 35 2 4 20 2001 11 35 52 4 25 2001 17 23 53 4 18 2001 18 01 31 5 2 2001 09 33 22 5 17 2001 13 46 44 4 3 2001 18 44 22 4 3 2001 20 40 16 5 7 2001 10 25 34 4 26 2001 15 18 11 4 20 2001 12 36 30 4 25 2001 18 24 30 4 18 2001 19 00 02 5 2 2001 09 33 26 6 17 2001 15 20 04 4 3 2001 19 42 17 4 3 2001 21 38 12 6 7 2001 11 23 54 4 26 2001 17 16 37 4 20 2001 12 36 32 4 25 2001 18 24 31 4 18 2001 19 00 04 5 2 2001 09 33 27 6 17 2001 15 20 06 4 3 2001 19 42 16 4 3 2001 21 38 13 6 7 2001 11 23 56 4 26 2001 17 16 39 4 20 2001 12 36 34 4 25 2001 18 24 33 4 18 2001 19 00 05 5 2 2001 09 33 28 amp 17 9001 15 n nao D1181984 Default D228583 D382387 982157 D95938 DXS7132 GATA193AD7 D1S1679 D148599 D1181984 D228583 D382387 D982157 D95938 DXS7132 GATA193AD7 D1S1679 D148599 D1181984 D228583 D382387 D982157 D95938 DXS7132
175. Scale Type The possible values for XScaleType are Default BySize and ByTime XscaleType only applies to electropherogram view Default allows the graph type to determine whether to display by size or by time If you do not specify the XScaleType attribute it defaults to Default GenomeLab Genetic Analysis System User s Guide 253 PN A29142 AB Fragment Analysis Module Reporting Results In the DisplayOptions section of the alternative text box enter by size or time as follows lt DisplayOptions XScaleType BySize gt OR lt DisplayOptions XScaleType ByTime gt X Axis X axis values specify either the size or the time depending on the XScaleType If either or both are not specified they take on the minimum and maximum values from Unzoom all graph In the DisplayOptions section of the alternative text box enter minimum and maximum values as follows DisplayOptions XScaleType BySize MinX 100 MaxX 300 gt Y Axis In the DisplayOptions section of the alternative text box enter minimum and maximum values as follows lt DisplayOptions XScaleType BySize Minx 100 MaxX 300 MinYy 0 MaxY 10000 gt If you do not specify either or both MinY and MaxY they take on the minimum and maximum values from Unzoom all graph Pane Count or the Pane Width You may specify either PaneCount or PaneWidth e If you specify PaneCount it represents the number of panes in which to break the graph If
176. Select Tools Base Synch Peak Synch or click the Synch icon Select a base or range of bases in the base sequence pane The corresponding peaks appear between two hairline markers in sequence analysis Aligning Bases and Peaks To align the bases in the base sequence pane with the peaks in the analyzed data pane 156 Select File Open In the Open dialog box select the Results tab highlight the desired sequence results then click OK Click the Align Mode AR icon to visually align the peak with the base call Click on the apex of the peak for which you want to view the alignment A hairline cursor is displayed through the apex of the peak to its corresponding base Click the Zoom Pan or Edit button to exit from the Align mode GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Analysis Using the Sequence Analysis Module Specifying the Base Call Location To specify the base call location l Open a sequence results file 2 Select Tools Bases on Top to toggle the bases from the top to the bottom of the analyzed data pane or vice versa Using Audio Enable To enable the audio feature 1 With a sequence results file open select Edit Audio Enable 2 Enter text into the base sequence pane to hear the letter bases announced as they are typed NOTE To enter text in the base sequence pane Edit mode must be enabled Using Audio Playback To enable the audio playback feature l W
177. Status Ready UM Microsoft Office Document Image Writer Driver Type Where Microsoft Document Imaging Writer Part Comment Page Layout C Landscape Sample Elements I Header Analysis Log W Raw Data RunLag Result Data Method Summary Cancel Result Output Analysis Parameters Current M Quality Parameters Hep Voltage M Trimming Log Options Alignment Results Alignment Accuracy Figure 4 38 Report Format Dialog Box e To view the quality parameters of an analyzed data set prior to printing select File Print Preview To exit the Print Preview option click Close To print the quality parameters for that data set select File Print Report GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Analysis Using the Sequence Analysis Module The system prints the Quality Values as numerical values as shown in the following example ra aa Project Default System CEQ 2UDOIXL Operator Manual analysis gt Sample 4 3 F01 010061605 Instrument Unity fo Result 43 F01_011029090F Quality Parameters A 3 F01 011029090F Number Base T CallScore ualValue Migration DataPoint Insert Edit A E l G O 226 0 96 l l 73 iz No No 2 T O B O 250 0 86 l 21 64 47 No No 3 C 0 247 0 8z 10 al o4 68 No No 4 C O 244 8zZ 10 21 59 Gl No No 5 A 229 l 0 82 10 2l 96 g3 No No 5 246 4 0 27 15 2A Uz 110 No No T
178. TTCT CCATTGCCAA CACCGCCTTA ACTCGCTGAA ATTCCAAATG TCCGGAAAAG AACTTAAGAG AGAAGAACTG ACGGCTCAAA GGGCAAACAC GGCTGGCAAA CACCAGAAAG TGCCATTTTC 37kDa RFC4 transcript 285 Gene Expression eXpress Designer Module Evaluating Multiplex Primers Use the BLAST function to evaluate first pass multiplex primers and the resulting amplicons for undesirable sequences including polymorphisms repeats and homology and troubleshoot primer sequences The BLAST screen provides a direct interface with GenBank The parameters on the user interface are similar to those on the NCBI BLAST page ee Me oe Erasm A Esmit meinen ME dim CTTCTUAAATATCTACCTGGGATOGAGTUATAGTAAUC ACCTTC AGAAAAGGCTTATUACA AGCCACCACAGAAGCAAGOAAGGACAGGRAGAACGAOGACAATAC AGAAAGAACCACCTU AA Euch UCAGAGGAAGCAAGAAAGCATOGAAACTC AGGAGTUAC ATTCCCTTCACTCCTTTTCUCTAUC CCAAGOCOAARAUACTOGAGCCTAAGCTGCCTOCTACTUGCTTTAU ATGGT Options for advancad blasting Limit by aniraz guay Choose hie F Low complastiy Human ropeais Mirak frs iokup tab E Maik lower cass Expert ho E ar eslect ftom linor Y E Cther abana i Format Show EB Graphics Cee Linkout Sequence Hanez P BCBi gl Aigrenent in HTML Tunt Number of Dezzrpiang 10 Alignments Al en nee Paws l tasks ke amma mus br ameri tigr Hore coed ares ORIGIN STC TS ll re p Figure 7 18 eXpress Designer BLAST Screen
179. TTICI TR LITT IT IM 148 Performing d Batch Analysis sa ute cd ied ont e Poe io a once rt t ont end 148 USING Compare ModE a acera died cord ea src E AE eee dne FOR DE deb rates 150 Segue nce Resul REDON zu s ete tin ca ter Pat bd data bee 151 Section 5 sequence Investigator Module 165 5 1 Sequence Investigator Module Overview llis RR RR IR 165 VIE 165 Menu Bar ODHONS s s ce oases osten Sos Erica ient iue prete tee Seda Reich na aaa tee banca ards 167 MOO WR NCON eaaa og ced Jette noa dat iy unde chad retin dni ae tries a 171 5 2 Sequence Investigator Procedures 0 00 0 ccc ccc ee IRR IIR 173 Creating a Reference Fils a a a ina bebe aie ead hee avo a 173 selecting a Reference and Sequence Results 000 cc 174 UTO CMG 43 sean ote eevdentte rds Pt Rud eu nea eee ee eae eee cee re ie a ee 176 Viewing Reference Amino Acid Translations 0 0 0 cece RR 176 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Viewing Sequence Traces a auxscsceusuce Gree grece IND doe onera ada re nated meats hak mowed 176 Viewing Consensus Amino Acid Translations 0 0 cc cece eee RR 177 PRUNO ODO N neer at aeaa a aa a Oe cee uate eer EA 181 Exporting TNE Sequence scs esca heit pre ooo oU e Eu Aid Rn aA EE herd 181 Dd ING WNC dld ROUTE Uer 182 Section 6 Fragment Analysis Module 2 000 eee 183 6 1 Fragment Analysis Module Over
180. The PCR process is covered by patents owned by Roche Molecular Systems Inc and E Hoffman La Roche Ltd GenomeLab Genetic Analysis System User s Guide 265 PN A29142 AB Gene Expression eXpress Profiler Overview 266 eXpress Profiler Suite The eXpress Profiler controller runs the eXpress Profiler Suite of applications These programs can be used to perform multiplex primer design gene expression analysis and data visualization using the data that is collected by the GeXP controller Using the Controller Third Party Software IMPORTANT Do not install third party software on the GenomeLab GeXP controller or the eXpress Profiler controller Accessing the Internet An active internet connection must be established when the controller is turned on as well as maintained by the eXpress Profiler IMPORTANT Relaunch the software if the internet connection is lost at any time Microsoft Updates Although they are important to the Windows software security capabilities automatic Microsoft Updates should not be performed on these controllers This default setting is turned off Additionally updates to JAVA firewall settings or the SQL interface driver could be harmful to the eXpress Profiler controller GenomeLab Genetic Analysis System User s Guide PN A29142 AB Gene Expression Software Administration 1 2 Software Administration Prior to the initial use of this controller eXpress Profiler user accounts must be configu
181. To create a new locus tag click New Locus to display the STR Locus Tag Editor window NOTE See Using the STR Locus Tag Editor on page 212 for information about creating or editing locus tags GenomeLab Genetic Analysis System User s Guide 207 PN A29142 AB Fragment Analysis Module Defining Fragment Analysis Parameters 208 SNP Locus Tags Tab The SNP Locus Tags tab allows you to select the locus tags used for SNP analysis with the current parameter set Use SNP locus tags to detect the presence of specific Single Nucleotide Polymorphisms SNPs in the group of sample data selected for the Study SNP locus tags are much the same in many respects as STR locus tags Each SNP locus tag is configured with a specific set of parameters that tell the system what fragment size to look for and to assign an allele name based on the color dye label of the identified fragment The results produced from a SNP analysis looks the same as those produced by an STR analysis Fragments identified as alleles of a locus are labeled as such in the fragment list No more than one fragment labeled with a given dye can belong to the same locus up to four peaks can be labeled as alleles of the same locus but each must be labeled with a different dye You can choose SNP locus tags from a list of available SNP locus tags previously configured for analysis This tab also gives you access to the SNP Locus Tag Editor where you can edit existing or creat
182. User s Guide PN A29142 AB Fragment Analysis Module Reporting Results e Graph Placeholder pictures as shown in the following example Bin View 382 001 0 015 Ba 0025 Relative Signal Strength e D e an BB eo E c UM io SD nuo FOO 200 400 Fragment Size ri Figure 6 38 Placeholder Pictures You can edit or remove header text at will Use the header text provided in the Report Templates to identity the data objects that follow You can remove but should not edit the data object descriptions highlighted in gray because they are specifically coded to extract data from the database Do not overwrite the original report template doc files They contain a compilation of all available data objects for reporting Use loop delimiters or table iterations of similar data While the data object names may appear squashed in the report template if the data fits in the space provided the tables should look normal in the report Graph objects and graph placeholders must remain together for proper graph display Editing Graph Displays You can edit five features of the graph displays 1 X scale type 2 X axis scale 3 Y axis scale 4 Pane Count or Pane Width and 5 Dyes displayed To edit the graph l Right click the graph placeholder and select Format Object 2 Select the Web tab and create the following text DisplayOptions Your commands will go here gt X
183. VALUE Figure 2 2 Note Sub Window Editing Sample Properties Sort and remove sample properties To select a field in an existing property set left click the cell 2 Next use the Tab key to move your selection from cell to cell The Tab key moves the focus from property to value and then to the next row and the arrow keys to move in the direction selected 3 Press Enter to move the focus down the table one cell at a time within the same column To select an entire column for sorting click its header The selected column is highlighted e To select a row click its number As with the column selection the selected row is highlighted e To select consecutive rows left click and drag the cursor through the blocks Click the toolbar arrow buttons to move the row up or down one cell e To delete the contents of the rows select the desired rows and click Delete A warning message appears prompting you to confirm deletion Click Yes to delete the contents or No to return to the editor Viewing the Method Tab The Method sub window displays the parameters of the method for the selected samples Click the Edit button to modify one or all of the parameters of the existing method or to create a new method Note Method Analysis Denature Separate Method Name LFA b Temperature 90 C Voltage 6 0 kV Duration 120 sec Duration 60 0 min Capillary Inject Pause 0 min Temperature Br C Voltage 2 0 ky Wait for Temp
184. Working Database See Setting the Working Database on page 328 for more information The Subject ID attribute applies to the sequence and fragment records that were generated from the converted sample data record Performing this action removes the Individual ID column and adds a new Subject ID column to all existing studies within the database 1 Open Data Manager NOTE The working database cannot be changed while other modules are open 2 Select and highlight the database to convert and ensure that it is the Working Database NOTE This option is available only for the working database 3 Select Tools Convert Individual ID to Subject ID After the IDs are converted the following message is displayed in the status bar Conversion Completed Successfully Creating New Size Standards l Open Data Manager Select the Default or Project folder Open the Standards sub folder in the Default or Project folder Select File New to create a new standard item puc ae bs Double click on New Standard Item to begin defining the individual size standard fragments e Select the appropriate dye label attached to the size standard fragments Input the sizes of each standard fragment with one fragment per line Select Save As to save the new Size Standard with a new name 332 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Database Management Data Manager Procedures Exporting Database Files from the Data Manager l
185. a Monitor Date Time 10 12 01 10 12 01 10 12 01 10 12 01 10 12 01 10 12 01 10 12 01 10 12 01 10 12 01 10 12 01 10 12 01 10 12 01 10 12 01 10 12 01 10 12 01 10 12 01 10 12 01 10 12 01 10 12 01 10 12 01 10 12 01 10 12 01 10 12 01 10 12 01 10 12 01 10 12 01 12 20 27 12 20 27 12 37 20 12 38 01 12 38 26 12 38 37 12 38 53 12 33 13 12 33 13 12 33 25 12 33 35 12 33 33 12 33 48 12 33 55 12 33 58 12 40 08 12 40 10 12 40 15 12 40 18 12 40 51 12 42 13 12 42 45 12 43 08 12 43 28 12 43 37 12 43 46 Direct Control Logged Events A B C MM Inesrument Data Freeze Log System configured instrument detected CE quencer s ID Simulator 3 Gel life Capillary Array life exceeded Direct Control stage Load Gel Cartridge starts Capillary Array life exceeded Direct Control stage Load Capillaries starts Direct Control stage Detecting Array starts Sample Plate Default5 amplePlate begins Estimated sample plate end time 10 12 01 16 22 21 Begin Sample Set Startup Sample Set DefaultStartupM ethad begins Method stage Purge Manifold starts Method stage Fill Capillaries starts Sample Set DefaultStartupMethod ends Begin Sample Set 1 Sample Set LFH 1 begins Method stage Temperature Ramping starts Method stage Temperature Ramping starts Method stage Denature Sample starts Current capillary temperature is 34 degree C Met
186. a table of all analyzed fragments included in the Study You can modify the columns displayed to show different system variables and information You can use the Filter Set area to define and select exclusion filters you want applied to the fragment data Create modify and save filter sets Each Filter Set column provides a right click menu of parameters you can use to apply as exclusion filters NOTE The Filter Sets applied to the Result Set data second level filtering are different from the Filter Sets applied to the Fragment List data third level filtering You can apply filters to the Result Set to filter an entire sample that may contain numerous fragments You can also apply filters to the Fragment List to filter individual fragments identified within the various samples GenomeLab Genetic Analysis System User s Guide 231 PN A29142 AB Fragment Analysis Module Using Fragment Lists Applying Exclusion Filters to the Fragment List Use the exclusion filter set is to exclude any fragments that do not meet the criteria of the Study and to include fragments that do NOTE Refer to the online help for information and examples regarding the application of various filters to the Fragment List The system provides many filters which you can apply using an operator and a value You can apply any number of filters to a result set and save it for future use You can assign filter sets to filter out fragments in which no confidence interval
187. a unique mulitplex name in the Multiplex Name field 4 Enter PCR product specifications for the following e Minimum Separation size e Maximum PCR Product size Minimum PCR Product size NOTE If the multiplex contains more than 25 genes extend the Minimum Product Size Range to 100 and increase the Maximum Product Size range to 350 Since Verbose Mode is used for troubleshooting it is not necessary to select this option for the initial process 5 Select the Reference Primers that will be included in the multiplex IMPORTANT The KAN primers must be selected and highlighted for inclusion in the multiplex 6 Choose genes from the Genes list and or primers from the Primers list to be included in the multiplex NOTE The Genes option allows the algorithm to freely design new primers for that gene The Primers option forces the previously designed primers into the mulitplex For any particular gene only select one option Do not select both 7 Enter a setting name for this set of parameters in the Save Setting field and then click Save Setting By using the Load Settings drop down list the parameters can be selected opened and modified in the future GenomeLab Genetic Analysis System User s Guide PN A29142 AB Gene Expression Multiplex Designer 8 Click Execute to begin the multiplex formulation The results will be displayed on the Multiplex Design screen See Figure 7 25 for a sample of the represented Multiplex information
188. ab Genetic Analysis System User s Guide PN A29142 AB Run Module Using Direct Control Removing and Replacing the Capillary Array CAUTION Be careful when installing or removing the capillary array to prevent damage and maintain low background signals Clean all gel residue thoroughly NOTE This procedure assumes that an expended capillary array is being replaced with a new capillary array l Select Replenish Release Capillary Array from the Run menu After the system prepares for release of the capillary array the Remove Capillary Array dialog Figure 3 35 is displayed Remove Capillary Array Ea Tou may now open sample capillaries access doors to change capillaries To replace the capillary array immediately select Replace capillary array To install a manifold plug select Install Cancel manifold plug Help To clean the capillary windows select Clean capilares Remove Capillary Array Options Replace capillary array C Install manifold plug C Clean capillaries Capillaries exposed to air E Time Remaining min sec fia fie Figure 3 35 Remove Capillary Array Dialog Box Open the Sample Access Cover Figure 3 36 and lift it to the vertical locking position Open the Capillary Access Cover and lift it to the vertical locking position Disconnect the two rubber latches holding the Capillary Temperature Control cover and lift the cover to the vertical locking position
189. ab Genetic Analysis System User s Guide PN A29142 AB Getting Started System Overview Software Module Descriptions The following table shows the shortcut icons available on the Main Menu and describes the module that opens when you click the corresponding shortcut icon Table 1 3 Software Module Descriptions Icon Module Description Sample Setup Module Create save and modify sample plates Use sample plates to assign methods to control sample sets and determine the sequence of methods used to produce data Run Module Run sample plates and control individual functions of the instrument Sequence Analysis Module View analyze compare manipulate and print base sequence data produced by sample runs Sequence Investigator Module Compare sequences and create a consensus to compare against a reference Fragment Analysis Module View analyze compare manipulate and print fragment data produced by sample runs Data Manager Module Modify and print database files Exit Closes the CE system software GenomeLab Genetic Analysis System User s Guide 11 PN A29142 AB Getting Started System Overview Main Menu Collapse Options After opening the Main Menu you can set its collapse features using the pop up menu To do this right click the Main Menu s title bar and select your option from the pop up menu as shown in the following illustration Collapse to Toolbar Move Minimize x Close Alt F4 Al
190. able describes the Run module s Log menu options Table 3 7 Log Options Menu Option Description Errors Only Toggles this option off and on When enabled v the Log window lists only errors All Detail Toggles this option off and on When enabled v the Log window includes all messages and system activity sample Plate Toggles this option off and on When enabled v the Log window displays only the Only items related to a running sample plate Freeze Log Toggles this option off and on Select this option to freeze or resume the display in the Log window Freezing the log data does not stop the run just the display of log data Replenish Menu Click the Replenish menu to display its drop down menu as shown in the following example Replenish Capillary Information Install capillary Array Release Capillary Array el Cartridge Buffer Information Install Gel Cartridge Release Gel Cartridge Replace Wetting Trav Empty Waste Bottle The following table describes the Run module s Replenish menu options Table 3 8 Replenish Menu Option Description Capillary Opens the Capillary Information dialog box Use this dialog box to enter information Information that applies to your capillary array For details see Viewing Capillary Information on page 359 Install Capillary Opens the Install Capillary Array dialog box Use this dialog box to install the capillary Array array Verify or change the infor
191. ace capillary array C Install manifold plug C Clean capillaries Capillaries exposed to air n Time Remaining min sec ps fe Figure 9 26 Remove Capillary Array Dialog Box Open the Sample Access Cover Figure 9 1 and lift to the vertical locking position Open the Capillary Access Cover and lift to the vertical locking position Unlatch the two rubber latches holding the Capillary Temperature Control Cover and lift to the vertical locking position 5 Loosen the Manifold Access Cover captive screw Figure 9 27 remove the cover and set it aside GenomeLab Genetic Analysis System User s Guide 361 PN A29142 AB Maintenance and Diagnostics Direct Control and Replenishment 901633L Al Figure 9 27 Manifold Access Cover 1 Capillary Temperature Control Cover 4 Captive Screw open 2 Plenum Assembly 5 Manifold Access Cover removed 3 Manifold Access Cover 6 Loosen the two plenum assembly captive screws Figure 9 28 pull the plenum assembly straight back and away from the instrument and set it aside CAUTION Slowly remove the plenum assembly as the electrode block may disengage from its mounting posts and become damaged 362 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Maintenance and Diagnostics Direct Control and Replenishment
192. ad Setting The screen will display a drop down list that will include the Multiplexes that have been previously saved Click on and select the Multiplex of interest The screen displays the saved multiplex settings and the accession numbers Make any changes as necessary and click Save Setting Click Execute to generate the multiplex The screen displays the multiplex primer data See Modifying an Existing Multiplex on page 296 for instructions on modifying an existing multiplex with Multiplex Designer GenomeLab Genetic Analysis System User s Guide 283 PN A29142 AB Gene Expression eXpress Designer Module 284 Retrieving Gene Sequences with FASTA Enter GenBank accession numbers to retrieve gene sequences and save them to the eXpress Profiler database to be used for primer design This function can also be used for troubleshooting by checking to make sure accession numbers match the desired gene and have a valid sequence eXpress Designer FASTA Create New Data View Existing Data Review Current Run express m uu dcc to Load Setting f Save Setting x Reset Form FASTA Blast Primer Design Accession Numbers Multiplex Please enter the accession numbers of the data you wish to attain The data will be returned in FASTA format Account About NM 002916 Help eXpress Analysis express Map Figure 7 15 eXpress Designer FASTA Io retrieve one or more gene sequences l Loginto eXpress Designer 2
193. advances through the sample plate during a run the instrument pushes the evaporation cover back far enough to expose the next row of buffer wells to use in the next sample set run 901595L Al Figure 1 6 Buffer Evaporation Cover and Buffer Plate 1 Buffer Evaporation Cover 2 Buffer Plate Wetting Tray The wetting tray Figure 1 7 is used to immerse the ends of the capillaries in deionized water When properly filled the capillaries can be maintained for approximately seven days in the wetting tray without attention During continuous use of the CE system the wetting tray should be replenished with deionized water after a 96 well microplate has been run or prior to a sample plate run The wetting tray is also used to rinse capillary tips and provide a receptacle for separation medium that was purged from the capillaries CAUTION No more than one 96 well plate should be processed for each wetting tray without replenishing the Wetting Tray For details see Replacing the Wetting Tray on page 350 e d 900499e Al Figure 1 7 Wetting Tray 6 GenomeLab Genetic Analysis System User s Guide PN A29142 AB GenomeLab Genetic Analysis System User s Guide PN A29142 AB Getting Started System Overview Gel Waste Bottle The gel waste bottle Figure 1 8 is used to capture and store the waste gel that is pushed out of the manifold during the purge function The bottle can hold the equivalent of 25 gel cartridges To prevent ove
194. agment list when the Detect A box is also checked It designates the other peak as either A or A accordingly e If the Use A peak to call allele box is not checked the system identifies the A peak as the true allele in the fragment list when the Detect A box is also not checked The other peak will be included in the fragment list as an unknown allele e If the Detect Spurious Peaks check box is selected and the peak falls below the maximum height designation for spurious peaks the other peak will be included in the fragment list as a spurious peak NOTE The only time that Use A peak to call allele is checked while the Detect A is not checked is when there is complete conversion of the PCR product to A When neither Use A peak to call allele nor Detect A are checked no A labeling can occur In general the reproducibility of size estimates is better for higher peaks than for lower peaks Therefore it is good practice to direct the system to use the higher of the peaks associated with the allele to identify the allele Additionally it is generally a good idea to check the Detect A option so the system correctly categorizes peaks If the option is incorrectly configured the alleles are still identified but the A and A peaks are frequently categorized as unknown alleles and the system reports Too many alleles in the Comment column of the fragment data table Table 6 13 Apparent Size Adjustments A Detect
195. ak Ratios New Analysis 1 Reference Result Peaks Fragment Result Set Traces 10 GATA19340 0 25pmol BO amp 0603271100 4 328 13 94254 GATA1924207 715 57 571 29 378 01 116 601 2377 022 GATAIS3A07 Analysis Parameters 373 99 Use Help Peak Height Peak Area Help Reference Peak Known Quantity r Highlight Variation 2 BE 12 E v Highlight Variant Ratios E re GATAIS3A07 Max Bin Width vTheshod 5 122 Standard Deviation E 361 6 1 00 44 oH C 524 xEmo a EA di 362 46 C Result Table Show Excluded Result Name R356 T378 T378 R356 R356 H 4 10 GATA193407 25pmol B 6 060327110 0 771 10000 Ref 8 GATAT83AD7 0 25pmolA06_06032 110R N 8 GATATS3A07 0 25pmoL HOS 0603271105 N 2 GATAIS3A07 i 357 25 GATA193A07 0 25pmol G05_060327110T N 6 GATA19340 0 25pmol F 5 060327110U N 5 GATA193A07 0 25pmol EO5 060327110V OONA A SATA1Q3ADT7 N 2Enesol NOR 0802711 094 RIA Dye Signal 1287 Oe of Data Points 1 1 Standard Deviation N N A Ready Figure 6 31 New Peak Ratio Window The Peak Ratios window displays all of the result data and the selected parameters used to quantify the amount of DNA present in your sample Reference Result Peaks This area shows the selected r
196. al number etc To maintain exact copies of data including header information use the CEQ format and import and export these data using the Data Manager Tab Delimited Text txt The Tab delimited format is valid for raw data sequence results and fragment results Select the Raw Data check box L1 to include raw data as well as current and voltage data Select the Results Data and Results Output check boxes L1 to include the sequence results The data points are expressed as text values which can be viewed in any text editor or in Excel SEQ seq The Seq format only applies to the sequence results portion such as text bases of analyzed sequence data This format is used for instance to export data into Lasergene s SeqMan from DNASTAR Inc Selecting the SEQ format exports the Result Output Base Sequence automatically if the sample has been analyzed with a sequence analysis parameter set If the sample has been analyzed with a fragment analysis parameter set nothing is exported GenomeLab Genetic Analysis System User s Guide 1 59 PN A29142 AB Sequence Analysis Using the Sequence Analysis Module Table 4 29 Sequence Export Options Option FASTA fasta PHRED scf phd 1 ESD esd 160 Description The FASTA format only applies to the sequence results portion such as text bases of analyzed data and contains a comment line that is used to identify the data Along with the text file a quality values file is a
197. alue 95 is equal to one minus the significance level of the built in statistical tests used for allele identification and for computing apparent fragment size confidence intervals Analysis Method Tab Set the size standard and model The model selected should be the best fit for the size standard selected as it is used to generate the size calibration model which estimates sizes for the unknown fragments Edit Fragment Analysis Parameters Of xi Parameter Set Mame FABRI Project Default Size standard SizeStandard 600 m Dype D Size Standard Fit Coefficient 0 57 Model Quartic m Hequires a minimum of 5 selected peaks Migration Variable Migration time Mobility Select All Deselect All Save Ag Cancel Figure 6 12 Fragment Analysis Parameters Window Analysis Method Tab Size standards are sets of DNA fragments of known sizes run with a sample and used to create the size calibration model The system uses this model to relate the known fragment size to either migration time or mobility for estimating the size of the unknown fragments NOTE Refer to online help for information on creating size standards 202 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Fragment Analysis Module Defining Fragment Analysis Parameters Setting Size Standards To ensure the system finds the standards and makes correct associations between the fragment peaks and the fragment list size
198. alysis See Performing AFLP Analysis on page 242 New Binning Analysis Starts a new Binning Analysis See Performing Bin Analysis on page 233 New Peak Ratio Analysis Starts a new Peak Ratio Analysis See Calculating Peak Ratios on page 239 Run LOH Analysis Runs a Loss of Heterozygosity Analysis See Performing LOH Analysis on page 246 Reports Menu Click the Reports menu to display its drop down menu as shown in the following example Reports Mew Report Refresh F5 188 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Fragment Analysis Module Fragment Analysis Module Overview The following table describes the Fragment Analysis module s Reports menu options Table 6 7 Reports Menu Fragment Analysis Module Option Description New Report select a template and context type for the report Refresh Refreshes the display of the selected report Window Menu Click the Window menu to display its drop down menu as shown in the following example Window Cascade Tile Horizontally Tile Vertically Close All Arrange Icons IM 1 Fragment List The following table describes the Fragment Analysis module s Window menu options Table 6 8 Window Menu Fragment Analysis Module Option Description Cascade Cascades the open windows Tile Horizontally Tiles the windows in a horizontal orientation Tile Vertically Tiles the windows in a vertical orientation Close All Closes all currently acti
199. alysis System User s Guide Define the report formats for new and imported sample plates Displays a facsimile of a hardcopy printout of the active sample plate Define printer properties Displays a sub menu from which you can choose to print a detailed report of the active sample plate the contents of the sample plate the plate summary or the selected method Displays a sub menu from which you can choose to print an image of the computer desktop or application window to the printer 23 Sample Setup Module Overview 24 Table 2 2 File Menu Sample Setup Module Option Description Lock Use this option to prevent editing of the currently displayed plate Exit Closes the Sample Setup module Edit Menu Click the Edit menu to display its drop down menu as shown in the following example Edit Undo Ctrlt z Reda Clear All Ctrl L Select All Ctrl 4 Cut Ctrl x Copy Cri c Paste trl Find AIFS Replace Ctri F3 Method Selected Method 4uto Fill Method Mame Ctrl F Perform the standard editing operations cut copy paste find and replace It also provides an option for modifying previously defined parameters of a method contained in the active sample plate The following table describes the Sample Setup module s Edit menu options Table 2 3 Edit Menu Sample Setup Module Option Description Undo Use to undo the last action performed Redo Use to redo the last undo action Clear All Empties all cell p
200. ame If you select the Resolve Filename Conflicts check box 1 the system increments the sample names on export when it encounters duplicate names For example if the system encounters the duplicate Sample name of Fred on export it renames the file exported to Fred 1 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Database Management Data Manager Procedures Table 8 9 Data Manager Export Options Format Description Apply Trimming selecting this option removes the trims from the sequence before you export it and replaces internal trims with the letter x CRV crv The CRV export option from the Data Manager module exports the four colors dyes as four separate files When exporting batches the resulting four files contain separate dye colors for all the samples You can select CRV as an export file type for exporting fragment results from the Fragment Analysis module Individual files are created representing individual colors FileName _D1 crvD1 contains dye 1 red FileName _D2 crvD2 contains dye 2 black FileName _D3 crvD3 contains dye 3 green FileName _D4 crvD4 contains dye 4 blue Default dye colors You may reassign the dye colors at any time Batch CRV Exporting More than one result can be exported in a batch If for instance more than one result is exported in a batch the export generates five files one for each dye and one to assist in determining the trace order Se
201. amp X ee Enter Name for New Database MpNewDatabase 00000 E ebef E HHE CEQBAKI E CEG Cancel 15 MB HE CEQBAK2 E FAI 150 MB iE FAII B E USE 5 MB a D1151984 D148599 D181579 D228583 D382387 D9S2157 D95938 Default DXS7132 GATA193AD7 Test project i USERTEMPLATE E A Oe E Os Os Os D Os Os D C Figure 8 4 Checking the Database Size NOTE If you recently removed data from the database the size shown may not be accurate See Reducing the Database Size on page 329 for more information Reducing the Database Size Manually reduce the size of the database to its new smaller size After removing studies or results from the database the size of the database does not immediately reduce in size NOTE Always reduce the database size before backing it up The database size cannot be reduced during database back up l Open the Data Manager 2 Select and highlight the database that will be reduced 3 Select Tools Shrink Database GenomeLab Genetic Analysis System User s Guide 329 PN A29142 AB Database Management Data Manager Procedures Backing Up a Database Periodic backups are essential to reduce the risk of losing data Save backups to any folder on the local drive IMPORTANT If a database is deleted or damaged only the data created since the last backup will be lost NOTE Shrink the database before backing up any database For details see Reducing the Database Size on page 329 l Open Data Manager 2
202. ample C01 BEEN gt 7 D3 Cc D uysampie Dot ri gm 1 B E MySample EO1 aooo E FG Eii F F MySample F01 ee AE G fi 11 G MySample G01 ee c i T 2i Y Y H3 Hia H11 H1 H MySample HO1 Gm d d d d d d d d sd d d ds Note Method Analysis Legend O Saved OO Edited xl x hd PROPERTY Template Source Sample Prep 3 Forward Primer Hi Ready Working Database GEXP MODULE 2006 View by Sample Name NUM Figure 2 1 Main Window Sample Plate Module GenomeLab Genetic Analysis System User s Guide 19 PN A29142 AB Sample Setup Module Overview The following table describes the items called out in Figure 2 1 Table 2 1 Main Window Sample Plate Module ltem Description A Title Bar Shows the module name Sample Setup and the active sample plate DefaultSamplePlate Menu Bar See Menu Bar Options on page 22 Toolbar See Toolbar Icons on page 27 Legend Shows the color coding information Saved or Edited of the individual cells mI OO co WW Sample Name and Subject ID of the currently active cell The radio button toggles the sample plate cell labels between Sample Name and Subject ID Use the Barcode field to enter the barcode for the current plate F sample Plate Displays 96 cells that correspond to a 96 well sample plate Sample plate columns are numbered 1 through 12 and Rows are labeled A through H This naming conventi
203. an A C G or T matches any ambiguity codes containing the base For example searching for A results in any ambiguity code containing A specifically M R W U H D and N Editing the Consensus To edit the base selected by the search type the desired base or select Edit Replace then select the desired base or ambiguity code To automatically jump to the next base that matches your search criteria once you have completed the edit select Forward or Backward on the Edit tab of the Preferences dialog box Displaying the Discrepancy Map The discrepancy map toolbar allows you to look for areas of interest in relationship to the reference You may view the coverage of the consensus and sample sequences You may also view the color coded locations in the experiment of any type of attributes of interest that you specify v Mismatch M Ambiguity v Disagreement m r r2 VIIT VIIIF 12 2 txt Insertion Deletion Mutation Hotspot wv Low Quality m N E E r E E Manual Edit Figure 5 8 Discrepancy Map Toolbar e The top bar in the discrepancy map represents the reference sequence p pancy map rep q e The second and third bar if two sample sequences were selected displays the coverage of the sample sequences in relation to the reference The next area O represents the consensus and shows the color coded regions of interest associated with the specified attributes A red marker behind the reference
204. areas Status R un Sample Plate Event Type Temperature Ramping Progress Event 0 0 Sec Sample Set 1 37 Min N Sample Plate 2 228 Min sample u mp Device Life Active Plate LEFT Sample Plate SNP Method SNP 1 Project Default Sample Mame Position Al g1 A01_03062609LF Af f2g1 B01_03062609LF E1 r3 g1 C01_03062609LF C1 r4 g1 001_030626095LF D1 5 g1 E01_03062609LF El r6 g1 F01_03062609LF F1 r gli G 1 3 e2edsLF 4 amp 1 m g1 HO1_O3062609LF H1 Capillaries exposed to air Gel Cartridge lite exceeded Capillary usage exceeded On line Figure 3 9 Status Monitor Displays Run Module GenomeLab Genetic Analysis System User s Guide 57 PN A29142 AB Run Module Run Module Overview Table 3 14 Status Monitor Displays Run Module ltem Description A Progress Indicator This area of the Status Monitor shows the Status Event Type and Progress of a running sample plate or process Status F un Sample Plate Event Type Denature Sample Progress Event 20 20Min J Sample Set ll 2 128 Min Sample Plate 3 136 Min NOTE Times displayed are approximations B Information Tabs Select any of four tabs to display the corresponding status details e Sample Lists the sample plate sample names and the method being used e Device Displays the condition or value of devices or parameters e Amp Shows the approximate overall microamperes uA and
205. as three components electrode block inlet eight capillaries and the array fitting outlet The electrode block is the DNA sample inlet side of the array It holds eight hollow stainless steel electrodes Each stainless steel electrode holds a capillary in the center These electrodes are designed for immersion into an entire row 8 wells of a 96 well microplate 8 rows x 12 columns The capillaries pass through and exit the array fitting When the fitting is installed the ends of the capillaries are inserted into the gel buffer manifold reservoir The array fitting contains the detection window and is the outlet side of the array The detection window of the fitting exposes the eight capillaries to laser excitation The external polyimide coating of the capillaries has been removed for this purpose NOTE When the capillary array is not installed for example during shipping and storage the manifold plug is inserted into the manifold array fitting outlet to prevent any gel inside of the CE system from drying 901592 Al GenomeLab Genetic Analysis System User s Guide 3 PN A29142 AB Getting Started System Overview Figure 1 2 Capillary Array 1 Capillaries 3 Array Fitting outlet 2 Electrode Block inlet Plenum The CE system is supplied with a capillary heater plenum suitable for use with a 33 cm long capillary array This short plenum assembly has a capillary array routing notch and a conical depression on the
206. ata Manager window select and highlight the database where the project will reside then click File New 2 Highlight the new project and click File Rename 3 Enter a descriptive name for this project and press Enter NOTE Do not use special characters in the file name lt gt 1 4 Close the Data Manager module File Exit Deleting a Project or Database l Inthe Data Manager window select and highlight the folder or database to be deleted 2 Select File Delete A Data Manager dialog box opens prompting you to confirm deletion 3 Click Yes to delete the folder or database NOTE You cannot delete the working database CAUTION Deleting a folder deletes all data contained within that folder Renaming a Project or Database l Inthe Data Manager window select and highlight the project or database 2 Select File Rename 3 Enter the new name for the database and press the Enter key NOTE Do not use special characters in the file name lt gt 1 328 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Database Management Data Manager Procedures Checking the Database Size Check the size of each database Click Data Manager on the Main Menu 2 Left click the GENOMELAB DBASE node at the top of the left pane The size for each database is shown in the right pane as shown in Figure 8 4 EE Manager ed xi File Edit view Tools Window Help amp Bs im
207. atabase to save the primer pairs into the eXpress Profiler database These primers become available for use in the eXpress Designer module in the Primer list box The primers will only become available after they have been saved IMPORTANT The primers entered into the database in this manner are only available for the current user To make a primer available for all users see Managing Primers on page 274 Entering Previously Designed Primers Enter the previously designed primers into the eXpress Profiler database for use in multiplex design l Loginto eXpress Designer 2 Click FASTA 3 Enter the accession number for the gene of interest 4 Add the gene to the database by using FASTA See Retrieving Gene Sequences with FASTA on page 284 for instructions 5 Click on Primer Design Choose the newly entered gene from the Genes list Type the Left Primer and Right Primer sequences in the appropriate fields under Advanced Options GenomeLab Genetic Analysis System User s Guide 291 PN A29142 AB Gene Expression eXpress Designer Module 292 8 Select the option to use the left primer and right primer as shown below Advanced Options Use a Mispriming Library repeat library NONE IV Pick left primer or use left primer Pick hybridization probe internal oligo or use oligo Iv Pick right primer or use right primer below 5 3 on opposite below below strand CTCTCTTCCCAGCTCACAC RTGGCTTCCACCCTCTTCT Fig
208. atabase location and hardware information Displays a list of user defined property fields and the values associated with the selected result These parameters may be edited Displays a summary of the separation methods consumable parts and lot numbers that were used with the selected result 225 Fragment Analysis Module Using the SNP Locus Tag Editor Trace Copies To copy a trace as an image to the clipboard right click on the trace and select Copy see Figure 6 22 DATS 101 D4 GU1 06504251275 Fragment Data Haw Data Fragment List Current Inject Current Voltage Analysis Log Hun Log Analysis Parameters Quantital_4 t 100000 Dye Signal 60 7p sp 50 81 58 44 07 1791 53 70 83 57 g 102 54 amp r 1554 pg 97 41 62 51 _ amp 96 41 r8 83 54 78 93 13 f WLA ES d w Include Zoom to Peak Edit Annotation Show Sizes Show Allele IDs Show Locus Labels Show Comments Show Result Mames Show Y Axis Label Show Data Points zoom In zoom uk Unzoorm Full Screen View A ee e ae M IN LI IM Figure 6 22 Copying a Trace to the Clipboard 226 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Fragment Analysis Module Using the SNP Locus Tag Editor The system copies subtitles with the image as shown in Figure 6 23 To paste the image in other software use the Paste command DATS 101 D4 G01_0604251226 100000 90000 80000 70000 60000 50000 400
209. atching names Click Finish Navigate to the target folder and double click the exported file to open it in Excel Notepad or any other text editor You can view the properties at the beginning of the file 258 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Fragment Analysis Module Exporting Results CEQ File Format The CE system offers the CEQ export option from the Fragment Analysis module You can select cq as an export file type for exporting fragment results from the Fragment Analysis module Export Sample Elements Save in C3 Export amp es EB I Header EC TEST M Ray rata M Result Data M Result Output Options IY Remove CEQ Tracking Suffix w Resolve Filename Conflicts File name DEFAU LT save as type CEG eq L ancel Figure 6 42 Fragment Result Export Dialog Box To export fragment results in CEQ cq format from the Export Results Options window 1 Click Save As Use the folder navigation options next to the Save in field to locate and select the folder in which you want to save the export file 2 Enter the desired file name in the File name field NOTE Do not use special characters in the file name 2V 5 l 3 Select CEQ cq from the Save as type drop down list 4 Click Save The system returns to the Export Results Options screen displaying the file path to the specified CEQ file in the Export results as field 5 If d
210. ated with the current output of each capillary or the total current of the sample set run 151 Sequence Analysis Using the Sequence Analysis Module Table 4 27 Sequence Result Report Options Option Voltage Analysis Log Run Log Method Summary Analysis Parameters Quality Parameters Trimming Log Alignment Results Alignment Accuracy Description Includes the trace associated with the voltage output of the system during the Sample set run Contains all of the steps used to perform the base calling routine on the raw data Contains all of the actions performed and instrument messages for the sample run Contains the details of the method used to perform the sample run This is the information found on the Method tab of the Properties dialog box Shows the parameters used to produce result data and output for a given Sample including heterozygote detection and alignment If the data has been analyzed using a Sequence analysis parameter set the sequence analysis parameter set will be printed In addition the alignment template and the alignment parameters information will be included in this section when applicable This information is available on the Analysis and Alignment tabs of the Properties dialog box It includes alignment information only if an alignment was performed Includes the parameters that indicate that any given base is an A C G or T along with a value to indicate the likelihood that the
211. ay Options dialog box select the Dye Traces tab 2 Select the traces to display or not display under Show Dye Traces GenomeLab Genetic Analysis System User s Guide 11 PN A29142 AB Run Module Using the Run Module 3 To change a color of any dye trace click on the appropriate Dye Colors dye select a color from the Color dialog box then click OK 4 When finished with Dye Traces click Apply to continue or OK to close the Display Options dialog box Current Trace Tab Set or change the current trace options l Inthe Display Options dialog box select the Current Traces tab 2 Select the radio button for the current type to display under Data 3 Click OK Viewing the Last Analysis Performed View the last analysis performed on the CE system l With a sample plate running and with Automatic Analysis selected select Tools View Last Analysis The Sequence Analysis Module dialog box opens and displays the last row of sample analyzed along with the Sample Plate toolbar 2 To view additional samples select the appropriate sample positions in the sample plate toolbar and click Open If the last analysis was a sequence analysis result the Sequence Analysis module opens displaying the analyzed sequence data If a Fragment Analysis separation was performed the raw data will be displayed in the Sequence Analysis module 72 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Run Module Using Direct Contro
212. ay option and click OK 364 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Maintenance and Diagnostics Direct Control and Replenishment 901602 Al Figure 9 30 Removing Replacing the Electrode Block 2 Electrode Block 1 Guide Block Pins CAUTION Always grip the capillary array fitting near the end during removal or installation of the tip cap to prevent flexing and possible breakage of the capillary array as shown below 900654L Al 365 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Maintenance and Diagnostics Direct Control and Replenishment Figure 9 31 Array Fitting 11 While holding the Array Fitting tab of the new capillary array align the Array Fitting Figure 9 31 with the manifold opening and guide pins Push the fitting into the manifold until it is completely seated against the bases of the Guide Pins Make sure that the fitting tab is placed downward when inserting the capillary array into its slot 12 While holding the electrode block tab align the electrode block Figure 9 30 with the guide block pins and gently push it in until resistance is met The resistance is from the spring loaded contacts 13 When installing the capillary array carefully route the capillaries through the hole in the plenum assembly Figure 9 28 and straight across into the guide block pins Figure 9 30 CAUTION N USE THIS FRONT PLENUM ONLY WITH A 33 CM CAPILLARY ARRAY
213. ays the message Database backup restore Database restore was successful You must log off and back on or reboot in order to re establish database access Click OK then click Close to quit the program Exit all programs and shut down the computer Reboot the computer and restart Windows Go to the Windows Start menu and select Start Programs GeXPAdmin GeXP to start the eXpress Profiler software Login to the program using your Username and Password Select Multiplex from the menu The Multiplex drop down list has items listed from the recovered database Since all of the settings have been deleted the Load Settings drop down list will not have any items to select To recover this list proceed to Restoring the Configuration below Restoring the Configuration 2 From the Windows Start menu select Start Programs GeXPAdmin eXpress Backup and Restore A dialog box will open on the desktop Select the Backup Restore Configuration Files option ONLY IMPORTANT Make sure that the Backup Restore Database Files option is not selected 3 Click Restore The Database Restore dialog box opens 4 Selecta configuration file to restore from the file list 5 Click Open A dialog box displays the following message Are you sure This will destroy ALL data currently stored and revert to the data set existing when the selected backup was taken Do you wish to proceed 6 Click Yes to continue with the restore A dialog b
214. bal use as a Reference Primer Primer in a multiplex If it is not selected this primer will only be available to the Administrator and original primer designer NOTE Primers are stored in the database only as their native sequence Universal tags are only appended when a multiplex is created and account for an extra 37 base pairs The PCR Product Size is also native Creating a New Primer Set 1 From the Administrator Menu click Manage Primers 2 Select the Parent Gene from the drop down list and enter its description in the fields as shown in Figure 7 6 IMPORTANT The Primer Name Parent Gene Left and Right Sequence and PCR Product Size fields must be entered for the new Primer The Left and Right Primer start length and Melting Temperature field entries are optional IMPORTANT When naming the Primer do not use the parental gene Accession number Include a primer description to prevent confusion with other primers made from the same parental gene For example 3 Click Save Use Reset Form to clear the form and create another primer IMPORTANT Make sure to select the Set as Reference Primer option to allow Global access if the primer will be used by multiplier users Loading and Modifying an Existing Primer View or modify an existing primer including primers designed by users l Select the primer of interest from the drop down list 2 Click Load 3 Change the primer parameters as necessary 4 Select Set As Reference P
215. be viewed 2 Click Profile to view the profile graph 3 Select the genes to graph by selecting or clearing the Genes list options e X axis sample names e Y axis normalized data values The y axis scale varies based on the selected single or multiple genes eXpress Map 218i X le Tools Help 1 5 80 Select All Deselect All 20 wwop oxm o ac Treatment Time Figure 7 37 eXpress Map Profile Graph Renaming the X and Y Axes 1 Click Display Configuration green check mark The Chart Settings dialog opens 2 Enter anew name for the X axis label and Y axis label to better represent the data results GenomeLab Genetic Analysis System User s Guide 31 3 PN A29142 AB Gene Expression eXpress Map Module Creating a Correlation Graph Generate a graph that depicts the correlation between gene expression data for each gene in the analysis 1 Log into the eXpress Map and load the analysis to be viewed 2 Click Correlation to generate the graph 3 Click Display Configuration green check mark to choose whether to correlate genes or sample types treatments eXpressMap 81 x File Tools Help wH a olg E Table Profile a Correlation Figure 7 38 eXpress Map Correlation Graph by Genes 4 Move the mouse over each block to see the coefficient of correlation If the result is closer to 1 0 the correlation is stronger between
216. bove which the Signal must rise before the analysis starts detecting data The data must stay above this threshold for the minimum duration without gaps for more than 21 seconds The range is 0 to 100 The default is 40 Delay Enter a value for the delay between the detected start of data when the data that will be analyzed The range is 0 00 to 180 00 minutes e f 200 Bases is not checked on the General tab the default value is 0 75 minutes e f 200 Bases is checked the default value is 0 20 minutes Signal to Noise Enter the Signal to Noise value to specify the signal to noise ratio for significant data The range is 3 00 to 1000 00 The default value is 7 00 Minimum Duration Enter the value at which the signal to noise threshold must exceed with no gaps greater than 21 seconds The range is 2 00 to 50 00 minutes e f 200 Bases is not checked on the General tab the default value is 5 00 minutes e f 200 Bases is checked the default value is 2 00 minutes Heterozygote Detection Tab Use the Heterozygote Detection tab of the Sequence Analysis Parameters dialog box to enable Heterozygote detection and to specify the parameters for detecting heterozygotes If you enable this feature the next analysis run detects heterozygotes and displays ambiguity codes where appropriate Sequence Analysis Parameters Editor DefaultSequenceAnalysisPar E3 Quality based Trimming Sequence based Trimming Alignment Reference
217. ce Analysis Module Alignment Tab View the alignment template information and the alignment parameters used to create the alignment of the currently open result Select File Properties Alignment Properties E General Note Property Set Method Analysis j Alignment E Consumables Alignment Template Template Mama puc1 amp 8d Cutoff Accuracy 38 0 x Allowed N 202 Alignment Parameters Match Score 1 0 Mismatch Score 2 0 Insert Start 4 0 Insert Extend 3 0 Delete Start 4 0 Delete Extend 3 0 Find Match Substings Yes Find Local Alignment Ho Lett Edge Free es Right Edge Free es Min Substring Length 30 Figure 4 31 Analysis Tab Properties Dialog Box NOTE This tab is visible only if an alignment has been performed on the sequence result GenomeLab Genetic Analysis System User s Guide 1 43 PN A29142 AB Sequence Analysis Using the Sequence Analysis Module Consumables Tab View the status and identity of the capillary array the gel and the buffer information Select File Properties Consumables Properties Ea General Note Property Set Method Analysis Alignment Consumables Capillary Array Serial Number 1102050078 Capillary Array Part Number 60808 Total Lenath of Capillary 33 0 cm Length of Capillary ta Detector 30 0 em Internal Diameter of Capillary 75 0 pm Number of Runa 2 Days on Instrument 0 1 days Gel Part Number 608010 Gel Lot Numb
218. ce Analysis Module Performing Quality based Trimming Setting Up Quality based Trimming To setup quality based trimming 9 Peu v UR ee dos Open the Sequence Analysis module Open a result and select Analysis Working Analysis Parameters Click Edit to open the Sequence Analysis Parameters Editor Select the Quality based Trimming tab Select Ends To Be Trimmed and Trimming Stringency Click Save As and save the new parameters Click OK Select Analysis Trim Based on Quality The analysis parameters created in the steps above should be listed If not click Use Stored Parameters and select the appropriate analysis parameters Click OK The resulting base sequence and trace views display the base letters if poor quality as strike through after performing a quality based trim NOTE When performing quality based and sequence based trimming quality based trimming is always performed first Hueresceree test sequenceA12 A11 02012519AI Mew Analysis Untitled 1632 06 7 63 Analyzed Data i at an 3n at Gul rt ri a CG GCGHGT GOGLAGOCTTG CAT GO CTE MAGGT CGACT CT 45 AGG AT CCCG GGT ACCOA GETOS 44TTOGTAAT CAT GT CAT G CT a tc fa ER Ta fT rp hM T NN pel P sno TONTOTCAAGCGAUGGUCAGTUGCCAAUGCTTUGCATUGCCTTUGCAGUGTCGAUCTCTAGAGGATCCUCCGGUGTACC SAGUTCGAATTCGTAARTUCATGTORTAGUTGTTTCCTGTGTGRAATTGTTATCCOGUCTCACRATTOCAUCAUCA ACATACGAGUCCGISAAGUATAAAGTGTAAAGUCTGGGGTGUCTAATGAGTGAGUTAACTCACATTAATTGCU GITGUCOGCTCAC
219. cf Exporting Data to a Third Party Package CAUTION Verify that the third party analysis package accepts the data transferred using a command line and that it is compatible with the file types provided by the CE system software before attempting to perform this procedure To export sequence data to a third party analysis package Select File Open 2 Inthe Open dialog box select the appropriate tab highlight the desired data then click OK 3 Select Analysis Third Party Analysis Setup In the Third Party Analysis Setup dialog box a Specify the path and executable file of the third party analysis package b Select the appropriate Export Data Format radio button c Click OK GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Analysis Using the Sequence Analysis Module 5 Select Analysis Third Party Analysis 6 In the Export dialog box enter the filename then click OK The system exports the data and launches the third party package displaying the exported data GenomeLab Genetic Analysis System User s Guide 1 63 PN A29142 AB Sequence Analysis Using the Sequence Analysis Module 164 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Investigator Module Sequence Investigator Module Overview Sequence Investigator Module This chapter provides an overview of the Sequence Investigator module including its menu options toolbars and dialog boxes It also shows you h
220. ch alignment Opens the Current Working Parameters dialog box Click Edit to open the Quality based Trimming tab Opens the Current Working Parameters dialog box Click Edit to open the Sequence based Trimming tab in the Sequence Analysis Parameters Editor Opens the Batch Trimming dialog box GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Analysis Sequence Analysis Module Overview Window Menu Click the Window menu to display its drop down menu as shown in the following example Window Cascade Tile Horizontally Tile Vertically Close All Arrange Icons 1 row 402 Sn32908PE wf 2 fowl 401 D5032907Ml 3 results Az The following table describes the Sequence Analysis module s Window menu options Table 4 7 Window Menu Sequence Analysis Module Option Description Cascade Cascades the open windows Tile Horizontally Tiles the windows in a horizontal orientation Tile Vertically Tiles the windows in a vertical orientation Close All Closes any currently active windows Arrange Icons Automatically arranges the icons Help Menu Click the Help menu to display its drop down menu as shown in the following example Help Help Topics Using Help About Genomelab System Beckman Coulter Home Page The following table describes the Help menu options Table 4 8 Help Menu Sequence Analysis Module Option Description Help Topics select this option to display the CE system
221. ch sample plate or when cancelled See Specifying Capillary Temperature on page 356 Initiate the denaturing of samples See Denaturing a Sample on page 357 Align the lasers with the detection windows of the eight capillaries You can save and view the data in the Sequence Analysis module See Performing an Optical Alignment on page 359 Initiate the injection of the sample prior to separation See Injecting a Sample on page 357 Initiate the separation of injected samples The data generated is not saved See Performing a Separation on page 358 Fill all capillaries with fresh gel This forces any gel present in the capillary out to the specified waste position in the buffer plate or wetting tray See Replenishing the Capillaries with Gel on page 358 Clear the manifold of air bubbles and or remaining DNA fragments before the next separation process See Purging the Manifold on page 358 GenomeLab Genetic Analysis System User s Guide 49 Run Module Run Module Overview Tools Menu Click the Tools menu to display its drop down menu as shown in the following example Tools wv Autoscroll Autascale Pause Data Display Uneoon neocon dd Display Options View Last Analysis NOTE A v next to an option indicates that the option is enabled The following table describes the Run module s Tools menu options Table 3 5 Tools Menu Option Autoscroll Autoscale Pause Data Display
222. co 4 cn Sloan DF 25 EE DF ce oe a oo So oo ee ee Se ee Se ce oe ee Se a Pe ee ee my te ai gi ey ty tipi tt on te ti Son ss St on me oi i N 203 PIN 2n 4l mA Dat n n4 En 204 20 nn T n Sample Bin Fragment Count v Exclude Fully Populated Bins Maximum Bin Width C Sample Bin Binary Presence M Exclude Samples w o Qualifying Peaks C Sample Bin Fragment s Max Y Show Excluded Elements Y Threshold ogm Figure 6 33 New AFLP Analysis Window Viewing the Cluster Analysis After completing the clustering operation the system displays a table showing the results of the AFLP analysis You can format this table as either rows or columns of data for easy export to a third party software program for further analysis see sample axis below This table presents the data in rows or columns as follows e Bin Displays the bin number It assigns bins with A and B suffixes if two adjacent bins fall in the same integer after rounding the Bin X Mean e Dye Displays the dye included e Samples Displays the sample count within the bin e Fragments Displays the fragment count within the bin e X Min Displays the minimum fragment size within the bin e X Max Displays the maximum fragment size within the bin e X Mean Displays the mean fragment size within the bin e X Var Displays the fragment size variation within the bin e Y Mean Displays the mean peak height within the bin e Numbered headings Show the numb
223. con to execute the function By default all the toolbars appear when you first open the module Standard Toolbar Icons The Sequence Investigator module s standard toolbar contains icons that correspond to common menu options x ele aj m e z arian Figure 5 2 Standard Toolbar Sequence Investigator Module The following table describes the Sequence Investigator standard toolbar icons Table 5 7 Toolbar Icons Sequence Investigator Module Icon Description New Prompts for a reference file from a Windows folder or disk then prompts for one or two sequence results from the current working database Open Open an existing compared sequence result Save Saves the active document to a Windows folder Print Prints the active document as specified in the Report Format dialog box Print Preview Displays a print preview of the active document using the format specified in the Report Format dialog box y igo RD ee Print Desktop Prints the active desktop area as specified in the Preferences dialog box Insert Inserts a space before the currently selected position Delete Deletes the selected position Trim Removes contiguous selected bases that contain an end base in the sample sequence base call text Restore to Original Restores the compared sequence result to its original state removing all changes m GenomeLab Genetic Analysis System User s Guide 1 71 PN A29142 AB Sequence Inv
224. cons on page 53 Status Bar Toggles between showing and hiding the status bar Select this option to show the status bar To hide the status bar select this option again to clear the check mark otatus Monitor Select this option to activate or deactivate the Status Monitor window displays The default setting is activated See Status Monitor on page 57 Working Displays the name of the database currently in use Database GenomeLab Genetic Analysis System User s Guide PN A29142 AB Run Module Run Module Overview Direct Control Menu Manage separation functions conditions or system components These capabilities serve in both method development and diagnostic tools Direct Contral Access Plates Plate Position Capillary Temperature Denature Samples Optical Alignment Inject Separate el Capillary Fill Manifold Purge The table lists the Direct Control menu options Table 3 4 Direct Control Menu Option Unload Plates Plate Position Capillary Temperature Denature samples Optical Alignment Inject Separate Gel Capillary Fill Manifold Purge Description Move the plate holder to the position at the front of the instrument to load or unload plates See Loading the Sample Plate on page 354 Graphically select the well location where the capillaries will be immersed set the capillary holding temperature This temperature will be over ridden at the end of ea
225. cons to display the desired data GenomeLab Genetic Analysis System User s Guide PN A29142 AB 114 Sequence Analysis Using the Sequence Analysis Module The following table describes each tab in the Open dialog box Table 4 16 Open Dialog Box Tabs Sequence Analysis Module Tab Description sample Data Displays the list of raw data and baseline data stored in the selected project On the right hand side of the list box you may view the date and time the sample was run You may want to open a raw data set to analyze it Sequence Results Displays the list of analyzed data that resides in the selected project On the right hand side of the list box you may view the date and time the sample was run as well as the raw data from which the sequence result was derived Sequence data consists of analyzed data and its base sequence text When you Open sequence data you may also view the associated raw data current and voltage This option is available for sequence analysis only To filter the list select the Enable check box 1 and enter the Start and End dates for the items you wish to view Click Refresh to view the filtered results sample Plate Results Displays the list of sample plates run in the selected project Selecting a Sample plate result displays the Sample Plate toolbar Click on the desired Samples then click Open The desired samples open Sequence Analysis Displays the Sequence Analysis Parameters saved in the project The
226. csimile of a hardcopy printout of the selected sample plate Prints the report Prints the selected pane Prints the desktop application or main window as defined in the Preferences dialog box Lists the most recently opened objects The first set of four is sample data and the second set of four is sequence results Closes the Sequence Analysis module 99 Sequence Analysis Sequence Analysis Module Overview Edit Menu Click the Edit menu to display its drop down menu as shown in the following example Edit Undo Reda faut Copy Paste Apply Quality based Trimming Apply Sequence based Trimming w Audio Enable Audio Playback Ctrl Z Ctrl Er Ckrl2 br The following table describes the Sequence Analysis module s Edit menu options Table 4 3 Edit Menu Sequence Analysis Module Option Undo Redo Cut Copy Paste Description Cancels the last action performed Reverses the last undo action Cuts selected bases and copies them to the clipboard To activate inactivate this function select Tools Edit See Tools Menu on page 102 Copies selected bases to the clipboard Inserts one or more copied or cut bases from the clipboard to the selected text position To activate inactivate this function select Tools Edit See Tools Menu on page 102 Apply Quality based Trimming Commits to the quality based trimming Apply Sequence based Trimming Audio Enable Audio Playback 10
227. d 312 Creating a Profile Graph usos ere PIRE EURO RACER mE AUPAE peces 313 Creating a Correlation Graph isssssesese ee eee Ire 314 Creating K Means Clusters isses RR RRIRRRRRRR Re 315 7 0 eXpress Backup and Restore 0 0 0 0 cc RR RR IRR IRR IIR 316 SAVING the DACKUO ICS 4o do soe eet e he Vo thnk baie Diae 028 tO ere EE at Cb D deben 316 Restoring the Backup Files 0 0 0 0 ccc cee RR RR 316 Section 8 Database Management 00 00 cee eee 319 8 1 Data Manager Module Overview 0 0000 ee 319 VEL uim EPI 320 Menu Bar DHONS are cus tir on Re eo p DR n e OE re pe Dor iae Soc ind Ple be td 320 TOODA ICON Sassena teg au lh tite tea pna Dent eter alain id M oco iacs p Tp s Edessa aes 325 0 2 Data Manager ProcedureS 0 0 c ccc eee IRR IRR IIIS 327 Storing Methods and Parameters 0 00 c ccc eR IRR 327 Creating a Database sect agreste oS neeaaea 327 Setting the Working Database 0 0 0 0 aaa 328 Creating and Naming a Project nuunuu auaa 328 Deleting a Project or Database ss 4cicseadd cic ED vavasa chase the ieee ERE Reed 328 Renaming a Project or Database 0 0000 ccc ce ee eee ees 328 Checking the Database Size eee II 329 Reducing the Database Size 2 eR RRRRRRRRRRRRIRR RR ees 329 backing UD a DalabdS8s u a aq aret epu t Ro reor REDE dE vibe sS d 390 FCS UOTE MG AAAS Oe sess acy h apes sata ch cavers Nice t Oron ee uc Enel aed oa buds E a
228. d Data Analyzed Data Analyzed Data Estimate Noise Variance Warning Excessive noise in channel 4 at time 2228 392857 DeSpike Deadtime Correct Correct for Data Truncation Align Time Base Find Start Find End Cannot compute stop time Using data stop time of 103 38 minutag Spike Analysis omooth Compute Color Calibration Figure 4 25 Analysis Log Occasionally the system cannot find the end of PCR product data even if the PCR Product option is selected To determine if this is the case review the analysis log and use this information in future analysis of the same sample To view the analysis log l Open the sequence results you would like to review NOTE Ifthe sample data has not yet been analyzed analyze the sample data set using the desired analysis parameters Select the PCR Product check box 1 and enter 0 0 for both the Analysis Start and Stop times 2 Select View Analysis Log to view the analysis log for the sequence result The analysis log appears in a pane at the bottom of the window GenomeLab Genetic Analysis System User s Guide PN A29142 AB 137 Sequence Analysis Using the Sequence Analysis Module 138 Sequence Result Properties View method analysis alignment consumable general notes and property set information To view these properties l Select File Properties when a sequence result is open The Properties dialog box opens NOTE The Alignment tab appears in this dialog bo
229. d Replenishment 2 Click Start The Capillaries Exposed dialog Figure 9 13 opens INIM PLEASE WAIT Stor nin open sample e oe m haria the sample door Capillaries exposed to air Capillaries exposed to air Load p Eancel B Cancel Alarm Off Alarm Off Time Remaining Time Remaining 4l Tn min sec mir Sec E 53 Help EER 5a Help Lett Plate Right Plate Plate Loaded Plate Loaded Left Plate Right Plate Plate Loaded Plate Loaded Immerse Capillaries Immerse Capillaries Immerse Capillaries Immerse Capillaries requires wetting tray C requires wetting tray on the left side on the right side requires wetting tray C requires wetting tray on the left side on the right side Figure 9 13 Capillaries Exposed Dialog Box 3 Wait until the dialog box message displays GO with a green text message 4 Open the sample access cover Figure 9 1 and lift to the vertical locking position Loading the Sample Plate l Make sure the Wetting Station is installed or perform the procedure Installing the Wetting Tray on page 351 then return here 2 Align the sample plate guide pin with the notched corner of the sample plate and gently lower the plate into position Figure 9 14 901600L Al Figure 9 14 Loading the Sample Plate 1 Sample Plate 4 Sample Plate Notched Edge 354 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Maintenance and Diagnostics Direct Cont
230. dard Deviation SD Measures the spread of sample data around the mean CV Measures the variation among replicates by dividing the Mean by the Standard Deviation for a particular sample Analyzing Data from Third Party Software 1 To use third party software for gene expression analysis first perform eXpress Profiler data analysis through the peak binning step for each plate 2 Click on the Report View tab and select the desired report format 3 To export the non normalized data clear the Display normalized data set option 4 Click Export Data 5 Click Save to save the report file GenomeLab Genetic Analysis System User s Guide 311 PN A29142 AB Gene Expression eXpress Map Module T 1 312 eXpress Map Module View gene expression data as a table correlated to the expression of other genes or clustered in similar expression patterns NOTE The information represented in the eXpress Map module cannot be saved exported or printed Viewing Table Data Log into the eXpress Profiler and click on eXpress Map in the Menu 2 Enter your username and password then click OK to log into the software The eXpress Map Desktop will be displayed 3 Click Open Analysis from the database and select an analysis This loads the data from all plates within an analysis 4 Click Table to view the Table tab y eXpress Map BEE File Tools Help imo Profile Ral Correlation Figure 7 36 eXpress Map Table 5
231. date stamp from the exported files GenomeLab Genetic Analysis System User s Guide 41 PN A29142 AB Sample Setup Module Using the Sample Setup Module 42 Resolving Filename Conflicts Select this option to automatically increment the exported files of the same name with a unique sequence number Export Options Sequencing Results The following options are specific to sequencing results only Apply Trimming Select this option to remove the trimmed data from a sequence before it is exported The ec 2 export replaces all internal trims with the letter x Export Only If Sequence gt X nt After performing the trimming operation the resulting sequence may be too small to be useful Select the Export Only If Sequence gt X nt option to specify a minimum size for the sequence output when exporting This exports only those sequences greater than the set number of nucleotides See the Online Help for additional Export Sample Elements Importing Sample Plate Information from TXT Files Importing a Sample Plate with Default Run Method and Analysis Parameters Import the sample plate txt file s to the CE System The run default separation method s and analysis parameters that were pre selected in the imported sample plate data sets will be used for running and analyzing the plate Select an appropriate Working Database and Project in the Data Manager module Open the Sample Setup module From the Menu Bar select F
232. de PN A29142 AB Sample Setup Module Using the Sample Setup Module a Enter atime duration between 0 and 10 minutes b Click OK to exit the Method dialog box or continue with step 6 to make additional changes to the method 6 Select Inject from the Event list a Enter an injection voltage between 0 1 and 12 0 kV Enter a time duration in seconds c Click OK to exit the Method dialog box or continue with step 7 to make additional changes to the method 7 Select Separate from the Event list a Enter the separation voltage between 0 1 and 12 0 kV b Entera time duration in minutes c Click Advanced to specify the two separation phases for the method d Click OK to return to the Method dialog box The first phase is to coax the sample into the capillaries The second phase is to perform the normal separation of the samples 8 Click OK to exit the Method dialog box or continue to make additional changes to the method 9 Inthe Save As dialog box a Selecta Project Name from the drop down list b Enter a name in the name field c Click OK Defining Data Processing Conditions Analysis parameter sets define the conditions specific to an experiment used in data processing Modification of the Sequence or Fragment Analysis Parameters can only be performed in their respective analysis modules Sequence Analysis Parameter Sets define the start and end times for the analysis of raw data the threshold below which ba
233. described below The following table describes the options available on the Sequence Analysis Parameters Sequence based Trimming tab Table 4 24 Sequence based Trimming Tab Option Perform Sequence based Trimming Vector Other Sequence Files Max of Vector Other Sequences Min Substring Length Distance from End Enable Internal Matches Description Select this check box 1 to enable sequence based trimming i to select a reference sequence It must be in the fasta format and stored in an accessible folder e Click X to remove a previously selected reference sequence NOTE There is no practical limit on the number of fasta reference sequences used however larger files greater than 200 Kbase increase processing time set the number of Sequences to appear in the result This number is determined by the goodness of alignment by scoring the found Subsequences and displaying only the top number selected The trimming log details all vectors other sequences found regardless of the score set the minimum substring length Only the subsequences that meet or exceed this length will be trimmed Set the distance from the end of the sequence and contaminant for automatic removal You may set the Left 5 independently from the Right 3 end Select this check box 1 to allow trimming within the sequence GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Analysis Using the Sequen
234. dit Apply Sequence based Trimming to remove the trims from the sequence The following example shows the resulting base sequence and trace view after applying sequence based trimming NOTE When applying sequence based trimming quality based trimming is automatically applied first if quality based trimming was performed CD04A4 G01_02091814NX 15046 40 4 42 Analyzed Data 40 TT TGCCGGA AGT GG TC GAA ACC A TC GA AGAA CA A GCG GA AC GAGC A TGG A TCC AG TA TCA GGG A TT CA A AACCCG A AATC GACA AACACA TC T Fluorescence h o A cw I JM funi Winall 13000 13250 13500 13750 4000 14250 14500 14750 15000 Data Points CTGATAACGACATCCTCAGTGATATCTACCAGCAAACGATCAATTATGTGGTCAGTGGCCAGCACCCTACGCTTTAAGGTGCTA TGCTTGATCGGCAACCTAATTTAGGGGTTTAGCACGTGTTTCTTCGCTACGGCGATGTTGTCCTTAAAACTAGCTACAGGATTG AGGAGTTAAAATGAAATCGAACCGTCAGGCACGTCATATTCTTGGACTGGACCATAAAATTTCTAACCAGCGCAAAATAGT TAC CGAAGGTGACAAATCCAGCGTAGTAAATAACCCAACCGGCAGAAAACGCCCCGCTGAAAAGTAATTCATAACCATCAGTCCTCA A TGACGATTAAACACCATTGCCTGCGCAATGGTGTTTTTGTTTTTATCTGCTTTATACTTGAGGCCGACGCCCTGGCGGTAAAG CAAAGACGATAAAAGCCCCCCAGGGA TGGA TATTCAAAAAAGAGTGAGTGACATGGAACCAAAAACAAAAAAACAGCGTTCGCT TTATATCCCTTACGCTGGCCCTGTACTGCTGGAATTTCCGTTGTTGAATAAAGGCAGTGCCTTCAGCATGGAAGAACGCCGTAA CTTCAACCTGCTGGGGTTAC TGCCGGAAGTGGTCGAAACCATCGAAGAACAAGCGGAACGAGCATGGATCCAGTATCAGGGATT CAAAACCCGAAATCGACAAACACATCT Figure 4 20 Base Sequence and Trace Views after Sequence based Trimming NOTE To recover the original s
235. dule Working with Studies in the Fragment Analysis Module 196 3 Filter data If the data of interest was run during a particular time period you can filter the data by selecting the Filter by date check box M and choosing a range of dates to be searched When enabled the system displays only those samples that were run during the specified time period 4 Select samples from the Raw Data Available area To select the samples to be included in your Study click to highlight the sample then click the right arrow button to move the selection to the Raw Data Selected area e To select all of the displayed samples to be included in your Study click the double right arrow button to move them to the Raw Data Selected area e To remove samples from your Study click to highlight the samples in the Raw Data Selected area and click either of the left arrow buttons to remove them from your selected samples NOTE You may select data from multiple projects to be included in your Study as long as they are located in the same database Multiple results from multiple databases may not be combined into the same Study Continue making selections from the list of available projects and data until all of the desired samples have been added to the Raw Data Selected area 5 Click Next to proceed to the Analysis Parameters window Selecting Raw Data from the Plate View Tab The Plate View tab allows you to make your selections fro
236. dures for database activities such as setting up and managing databases exporting importing data and managing user accounts Maintenance and Diagnostics provides routine maintenance replenishment diagnostic and biological waste disposal procedures It also lists the consumable materials used in the system GenomeLab Genetic Analysis System User s Guide 1 PN A29142 AB Foreword Technical Support Technical Support If you encounter a problem that is not discussed in this guide and you need technical support contact your local dealer the provider of this product or contact Beckman Coulter directly using the information below NOTE Whenever you call your local dealer or Beckman Coulter be sure to have your registration material instrument serial number and software version number available For future reference record this information here Instrument Serial Number Software Version Firmware Version Dealer Name Dealer Phone Number Mail Beckman Coulter Inc 4300 North Harbor Boulevard Fullerton CA 92834 3100 Product Support 1 800 854 8067 Sales 1 800 742 2345 Service 1 800 551 1150 Telex 678413 FAX 1 800 643 4366 Internet http www beckmancoulter com 2 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Getting Started System Overview Getting Started This chapter discusses the purpose and functional description of the system provides an overview of the three ma
237. e 90 0 C Denature Duration 120 sec Pause Duration 1 sec Inject Voltage 2 0 EV Inject Duration 15 sec Separate Stage 1 Primary Voltage 4 2 kv Stage 1 Ramp Duration 5 l min Stage 2 Separation V altage 4 5 EV Stage 2 Start Time 5 0 min Stage 2 Ramp Duration 0 0 min Total Separation Curation 85 0 min Figure 4 29 Method Tab Properties Dialog Box GenomeLab Genetic Analysis System User s Guide 1 41 PN A29142 AB Sequence Analysis Using the Sequence Analysis Module Analysis Tab View the analysis parameters used to produce the open sequence result View the name of the parameter set used as well as all of the parameters and color calibration used to perform the analysis Select File Properties Analysis Properties General Note Property Set Method NS Analyste Alignment Consumables Analysis Parameter Set OC Method for Sequence Analysis M Threshold 0 00 Analysis Start Time 0 0 minutes Analysis End Time 0 0 minutes PCR Product Ma Color Calibratiar DefaultColorCalibration Threshold 40 00 Delay 0 8 minutes Signal to Noize 00 Minimum Duration 5 minutes Pre peak Reduction No Detect Heterozygotes No Base Range Before Last Called Base Average Peak Spacing Height R atia Sensitivity Automatic Alignment Yes Figure 4 30 Analysis Tab Properties Dialog Box 142 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Analysis Using the Sequen
238. e Gel Cartridge dialog box will be displayed Wait until the lead screw is completely disengaged 2 Open the Gel Pump Gel Cartridge Access Cover by gently pushing in on the top of the cover The cover is spring loaded and will pop open 3 Pull on the Cartridge Locking Lever The barrel will swing outwards to approximately a 90 angle from its locked position GenomeLab Genetic Analysis System User s Guide 367 PN A29142 AB Maintenance and Diagnostics Direct Control and Replenishment 1 901635L Al Figure 9 34 Removing Replacing the Gel Cartridge 1 Gel Pump Gel Cartridge Access Cover 4 Cartridge Locking Lever 2 Cartridge Locking Lever 5 Cartridge Barrel 3 Cartridge Barrel 4 Grasp the wings of the gel cartridge gel pump plug and pull it out of the barrel NOTE Always note the lot number and the hours on the instrument for a gel cartridge if you are planning to use it for more than one session 5 If necessary use a tissue to wipe gel strands off of the instrument 368 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Maintenance and Diagnostics Direct Control and Replenishment 901603L Al Figure 9 35 Removing Replacing the Gel Cartridge Continued 9 1 Cartridge Wings 3 Cartridge Locking Lever open 2 Gel Cartridge or Gel Pump Plug 4 Cartridge Barrel Remove any air pockets from the gel cartridge by grasping the cartridge wings between your first two fingers and pressing
239. e Peak Binning tab The first view does not show any data NOTE To view all of the fields on the Peak Binning tab click on the Maximize button 7 Click on a colored well in the 96 well sample plate to view the data associated with a particular well NOTE If the range of data is more than three genes it will be necessary to scroll down in the binning table to view all of the fields associated with peak binning GeXP Analysis Plate Setup GeXP Analysis Normalization 3 mj 6 sample Layout 2 Data Files WwW Peak Binning k Non Designed Peak NENNEN Designed Gene Peak Not Detected EEE Figure 7 32 Peak Binning First View Table 7 12 Peak Binning Tab Reference Item 1 Sample Plate 2 Peaks Graph 3 Peaks Color Key 4 Binning Table 5 Binning Range GenomeLab Genetic Analysis System User s Guide PN A29142 AB Function Select a well from the plate and view the data associated with the well This data is displayed in the Peaks Graph and Binning Table Displays peaks that were detected in the Sample The Y axis represents the peak height The X axis represents the fragment size Shows the status of each peak using a color representation Identifies each designed and detected peak according to its size Defines a size range in which to identify a designed peak 305 Gene Expression eXpress Analysis Module Viewing Data for a Selected Well Click on a gene from the Binning Table to show the bi
240. e Save As dialog box a Selecta Project Name from the drop down list b Enter a name for this plate in the name field c Click OK to save the plate Selecting Samples Select individual samples positions In addition select the entire plate row or column using a single click Use the Shift or Ctrl keys to select contiguous and non contiguous cells Selecting the Entire Sample Plate Select the entire sample plate by left clicking the grid box on the upper left corner of the plate grid To remove the selection left click any sample in the grid Figure 2 7 Select the Entire Plate 30 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sample Setup Module Using the Sample Setup Module Selecting the Entire Row Place the mouse pointer over the row label on the left hand side of the row to be selected A small black arrow will replace the mouse pointer Click once to select the row To remove the selection click any sample in the grid Figure 2 8 Select an Entire Row Selecting the Entire Column Place the mouse pointer over the column label at the top of the column to be selected A small black arrow replaces the mouse pointer see Figure 2 9 Click once to select the column To remove the selection click any sample in the grid Figure 2 9 Select the Entire Column Selecting Contiguous Cells To select contiguous cells 1 Left click the first cell in the column or row 2 Press and hold the Shift
241. e ahead irt ts ean a 233 DelnirgParamlelS eerren sarc ide erie corse de sdb ge Aly epu p pedi tp SE pup 234 MIBWIDU DIRUTA SIG Es acri docs etii an cose anced cata hee Aad ues ub aed Enden tent Un UP receta Rie Ea 235 I vVesttadtm FOIN cue needs Ub Ro pm by ee he Shake heed Bd inicie t EE 236 Updating the Locus Tag and Allele List 0 0 0 0 ccc cece 231 Reviewing the Source Data 0 ee eee eee eens 238 6 9 Calculating Peak BalloS ecebenpisebkbbfersesrekreb9embpvcerueeebesiveebbbesrgs 239 Beginning a Peak Ratio Calculation lisse ee ees 239 selecting a Reference Trace lssssessee RR RR Rene 241 Selecting a Reference PeakK isses RRRRRRRRRRR a 241 ogleellg a ToS Peak auae seriei qs br iet cea Oneal ite dea one tede sd ue d doit ten 242 b TOLPOEHIOFIBIFIG AER ANASI e s ico Soa uat eeu tre unt ca ose E a CR eek a Ens 242 USMO INCAFCF FedlU Bi or te a armo cot rad eme desea tenta state de dub feu e 242 setting the AFLP Analysis Parameters 0 0 0 ccc ccc eR RR 243 Starung a NeW AAFP AnaIVSIS xus cui mette bete tema eo e Aw d us a eed 244 Viewing the Cluster Analysis slseeee ee eee RII 244 setting Cluster Analysis Parameters 00000 c cc ccc RR RI Ie 245 Customizing the AFLP TOD Gs s raten as vie aet ah tion orco oe Bede un tu Rica oU heo eU Cette 246 Exporting the AFLP Analysis ResultS 0 00 00 ccc cece n nmn 246 6 11 Performing LOH Analysis naana eee eee eee
242. e analysis again For details see Reanalyzing a Batch on page 149 After performing a batch analysis use this option to view the results of a selected Sample in the upper pane of the batch analysis window While performing a batch analysis use this option to pass over the sample currently being analyzed This item is not available if Sample Plate Results was selected from the Batch Analysis Selection dialog box While performing a batch analysis use this option to pass over the sample set currently being analyzed This item is not available if Sample Data was selected from the Batch Analysis Selection dialog box While performing a batch analysis use this option to pass over the sample plate currently being analyzed This item is not available if Sample Data was selected from the Batch Analysis Selection dialog box Momentarily stops a batch analysis after the system analyzes the currently running sample Use Resume Batch Processing to continue the analysis Resumes a batch analysis after it has been paused Launches the third party analysis package specified in the Third Party Analysis setup dialog box specifies the path third party analysis package loaded on your PC and the export format of the data Used to e Align the sequence against puc18dG e Print the alignment report e Export the alignment data as a text file Performs an alignment of the sequence against the current alignment settings Performs a bat
243. e desired location Modifying an Existing Multiplex Modify a previously designed multiplex that was saved in the database to replace primers or include new genes 8 Log into eXpress Designer Click Multiplex Click on the Load Multiplex drop down list then select one of the multiplexes stored in the database Click Open Analysis to load primers from the selected multiplex The Primers list will show the selected primers that are contained in the loaded multiplex Use the CTRL Click function to select or clear a primer from the collection of primers in the Primers list Use the CTRL Click function to select the genes from the Genes list for inclusion in the multiplex Click to select the reference primers from Reference Primers list for inclusion in the multiplex Set the Minimum Separation Size to 25 Optimal separation is 5 to 7 nucleotides NOTE When modifying an existing multiplex it may be necessary to adjust the default parameters such as the Minimum Separation Size in order to successfully generate a new multiplex particularly if more gene products are being added to the multiplex 9 10 ll 12 13 Enter a unique multiplex name in the Multiplex Name field Name the new settings in the Save Setting field and click Save Setting to save the parameter changes By using the Load Settings drop down list the parameters can be selected opened and modified in the future This step is optional Click Execu
244. e for import Browse Manage Primers Universal Tags Import Import Multiplex Help Figure 7 8 eXpress Profiler Import Multiplex Screen 216 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Gene Expression Software Administration From the Administrator Menu click Import Multiplex Click Browse to locate the multiplex TDF file Click on the TDF file to select it Click Import once and wait for a message window that will confirm the process was successful p oS JS ues 5 Click OK to close the message window IMPORTANT All imported multiplexes are GLOBAL All users can view and use the entries created under this Administrator function IMPORTANT Once a specific TDF file has been imported another TDF file with the same multiplex name cannot be imported The multiplex names must be unique 6 To confirm that the TDF file has been imported log out of Administrator mode IMPORTANT An Administrator must have a User Account in order to access the eXpress Analysis module of the software 7 Log into eXpress Profiler as a User 8 Click eXpress Analysis and log in with your username and password 9 Click on the GeXP Analysis tab The multiplexes that have been saved in the database will be shown in the Multiplexes list rA eXpress Analysis Analysis Analysis 123 File Help GexP Analysis Plate Setup GeXP Analysis Normalization im gt m El kg GexP Analysis Setup Property 2 Pr
245. e new SNP locus tags to meet the specific requirements of your sample Edit Fragment Analysis Parameters Miel Ea Parameter Set Mame GATAISSAO Project Default Edit Locus Her Locus Save As Cancel Figure 6 15 Fragment Analysis Parameters Window Selecting SNP Locus Tags GenomeLab Genetic Analysis System User s Guide PN A29142 AB Fragment Analysis Module Defining Fragment Analysis Parameters Selecting Locus Tags Select one or more locus tags you want included in your parameter set e To select an individual locus select it in the Available list then click the right arrow gt to move the selection to the Selected list e To select all available locus tags click the double right arrow gt gt to move all of them to the Selected list e To remove locus tags from your parameter set select each locus tag in the Selected list and click either of the left arrow buttons to remove them from your parameter set To edit a locus tag select it and click Edit Locus to display the SNP Locus Tag Editor window e To create a new locus tag click New Locus to display the SNP Locus Tag Editor window NOTE See Using the SNP Locus Tag Editor on page 219 for information about creating or editing locus tags Running a SNP Analysis The following list identifies the three tasks you must complete before running an SNP analysis 1 Select the SNP Locus Tags as part of the analysis in the SNP Locus Tags tab of
246. e part number of the installed capillary array Serial Number Displays the serial number of the installed capillary array The serial number is used to track the capillary array along with the samples that have used the array Date Installed Displays the date the capillary array was installed Time Installed Displays the time the capillary array was installed Number of Runs Specity the desired number using the spin control Set more than the recommended number of separations to be performed before the system alerts you that the recommended number of runs has been exceeded Days on instrument Specify the number of days on the instrument using the spin control This allows the capillary array to reside on the instrument longer than the recommended length of time before the system alerts you that the recommended number of days on the instrument has been exceeded Advanced Displays the Capillary Advanced Information dialog box This dialog box displays the length and diameter of the installed capillary array based on the part number entered in the New Capillary Array dialog box The fields are read only and cannot be edited Click OK to enter the information and close the dialog box When finished click OK to close the dialog box GenomeLab Genetic Analysis System User s Guide 79 PN A29142 AB Run Module Using Direct Control Viewing Gel Information l Select Replenish Gel Cartridge Buffer Information from the Run Modu
247. e sequence of the reference file into an amino acid sequence and displays it above the reference base sequence line From the beginning of the reference the bases are separated into groups of three nucleotides codons Each group is translated into an amino acid which is displayed above the codon Each codon is shaded to show its boundaries If an inserted space exists signified by a dash the space is included in the codon The amino acid designation is left justified above the codon If two amino acids are possible the letters designating the possible amino acids appear If the system detects more than two possible amino acids at any location it displays an asterisk instead To show or hide the reference amino acid sequence select View Reference Amino Acid Translation The screen displays or hides the reference amino acid translation depending on whether the item is checked or unchecked Viewing Sequence Traces When you first perform a comparison select one or two sequences to form the consensus After performing the comparison the system displays the traces for these sequences When you click on any position in alignment view the corresponding peaks in the traces move to the center of the trace view framed by two vertical lines When you first open or create a comparison the system scales the traces to improve readability To zoom each sequence pane individually and repeatedly left click and hold the mouse button whi
248. e the table below FileName _D1 crvD1 contains all dye 1 results in the batch FileName _D2 crvD2 contains all dye 2 results in the batch FileName _D3 crvD3 contains all dye 3 results in the batch FileName _D4 crvD4 contains all dye 4 results in the batch FileName CRV TRACE ORDER txtcreated for batch export files to assist in determining trace order in the CRV files oee Sequence Export File Types on page 159 for additional information on sample elements or refer to the Online Help Importing Database Files To import files into the database l pv doc M e Open Data Manager In the Data Manager window browse and select the project where the imported file will reside Select File Import The Import dialog box opens Browse to locate the folder where the file resides Select the import format from the Files of type drop down list Select the filename Click Import GenomeLab Genetic Analysis System User s Guide 337 PN A29142 AB Database Management Data Manager Procedures Generating a Sample Run History l Select View Sample Run History The Sample Run History dialog box opens Select a Project name from the drop down list Click Compute pt se e p Run field Select Enable and enter a filtering start and end date View the total number of runs during the specified dates in the Total Sum of Samples 6 If desired print a report of all samples run during the specified dates by clicking the Pr
249. ect Separate NOTE Data will not be saved Replenishing the Capillaries with Gel 1 Select Direct Control Gel Capillary Fill from the menu 2 From the Gel Capillary Fill dialog box select the Buffer Plate or Wetting Tray option to identify the position where waste will be expelled from the capillaries e If Buffer Plate was selected use the Buffer Plate spin controls to identify the waste position in the buffer plate and then select Fill e If Wetting Tray was selected click Fill Purging the Manifold l Select Direct Control Manifold Purge from the menu 2 Inthe Manifold Purge dialog box enter a volume in milliliters mL 3 Enter the number of cycles and then select Purge Performing an Optical Alignment To align the lasers with the detection windows of the eight capillaries Select Direct Control Optical Alignment from the Run menu To save the alignment data select the Autosave check box Enter a name in the Name field Select a Work Folder from the drop down menu eS qw pg Select Align IMPORTANT Prior to performing an Optical Alignment it is advisable to purge the Array Manifold and fill the capillaries with fresh gel in that order See Purging the Manifold and Replenishing the Capillaries with Gel GenomeLab Genetic Analysis System User s Guide TI PN A29142 AB Run Module Using Direct Control Monitoring the Baseline 78 To monitor the system baseline NOTE An optical alignme
250. ees 246 6 12 Customizing the Results List 0 0 eee eee eee ees 248 selecting Columns for Display 0 00 0 0 ccc eee eee 248 SOMME BesultS LISE uia poasit to a Capo aet ER Rr Pob oot ae Sotto n 249 O21 o ReDOPTHIDO SUIS E cca tius at o TRE ah te DO E lcd ed Ehe ecce 251 Using Report Templates zac aur gato toe E XX best io ecd doe xo mb t rots edo ehe Ss 252 Edi ng Grap M DISDlaVS s icoiius its bot rere aeeae ky nd eee hook eae REESE Eu 203 D P4 EXDOMING RESUS s 5s mae belch othe oe d e RUE T Cea oer RU d Opes 256 iTeXU RI FOM a heraa do eae Ent dor aad RAE ice ba ae ees Lee rcs 200 GEO FIIs FOM dls yu wees atest cars pasa TC I OTT eee bers Meal tats ta ae 259 Exporting Fragment Lists or Genotypes froma Study 0 0 0 cc cee een 209 Transferring Fragment Data to eXpress Profiler lille 260 Section 7 Gene Expression foie ot te es ee oe wae ee i er ee 265 7 1 eXpress Profiler Overview lessen ce RR RRIRRRRIRRRRR RI 265 BADFeSS Profiler OUI ostium xn wks ee snes ee xim e dh td Mb n enti dod s Ed ue 266 Using iNe CONTONOT gerne mre a eo on oe 266 7 2 SOM Wale AGIMIMISUCALION perrin d Gch ty fading oh acmuhed Shand ratis Sedona domed Mand td oath windee elas 26 Managing User ACCOUNTS i a d redo he e ror BC Dx edet imt to e e t e ded e eios 269 Mandgnd Gell od coat dteusca ir EAE E ere cea LR DI Pu REDE 2 2 ManadinoFIIMCI S 22 Bote iot fraude tet cae mee estet attenta sax dL Pu dot dre 2 4 Managing Un
251. eference and test peak information You select the reference result which contains the best reference and test peaks for the system to use for a relative analytical comparison of trace peaks You may select any number of test peaks but only one reference peak for each Peak Ratio Analysis Analysis Parameters e Use Peak Height Select this option to perform a quantitation based on peak height Relative Fluorescence Units RFU GenomeLab Genetic Analysis System User s Guide 239 PN A29142 AB Fragment Analysis Module Calculating Peak Ratios 240 Use Peak Area Select this option to perform a quantitation based on peak area mm x RFU Reference Peak Known Quantity Optional If desired enter the known quantity of your reference peak in the field You must enter this value in scientific notation Select the units for the quantity from the drop down list The options are moles grams molarity and g L The system uses this information to calculate the quantity of DNA in the other results Highlight Variation Highlight Variant Ratios Select this option to enable the system to highlight any result ratios that deviate from average values The criteria for variant ratios are specified by either the Standard Deviation about the mean or the Error e Standard Deviation Select this option to base the system criteria for identifying any variant ratios on the number of standard deviations from the mean Enter the standard deviation
252. el the Allele ID s in the fragment list The default values are A T G amp C These fields cannot be blank e Accession and Primer Sequence fields are not used for analysis and are for supplementary reporting purposes only These fields are optional Apparent sizes of SNP probe fragments as estimated with SNP reference fragments often differ more from actual sizes than the STR allele fragments do This is due to the effects of primary structure on fragment mobilities Therefore it is important that the apparent size entered into the SNP locus tag definition is a known value This is best determined in preliminary runs as the average apparent size of all of the possible variants that might occur in a Study Because the effects of different dye labels are relatively greater on shorter fragments it is essential that in include the Dye Mobility Calibration in the parameter set for an SNP analysis Result Set Filtering The first steps in creating a Study involve applying a set of fragment analysis parameters to analyze the raw sample data These parameters are specifically configured to process the fragment data for a particular sample or set of samples The outcome of this analysis is a result set of data consisting of characterized fragments and identified alleles You can then use specific filters to scrutinize and selectively isolate results based on the parameters of the applied filters In other words the types of exclusions that you
253. emove the row from the property set Entering a New Property l i A new row appears into the property table 2 Enter the new property name and value into the cells of the new row e To assign a pre defined property set click and select from the defined property sets as shown below Sample Property Sets Detined Sets Properties PROPERTY 1 Template Source Create Delete Figure 2 14 Sample Property Sets Dialog Box 36 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sample Setup Module Using the Sample Setup Module Creating and Storing a Custom Property set 1 Click the Create button in the Defined Sets portion of the Sample Property Sets dialog 2 Enter a name in the Create Property Set dialog box Figure 2 15 3 If desired use the Copy settings from drop down list to choose an existing property set to copy into the new one Create Property Set Ea Set Name Copy setings fom ES Cancel Help Figure 2 15 Create Property Set Dialog Box 4 Click OK to save the new property set 5 Use the button to manually add properties to the new property set Using Methods A method is a program that includes a sequence of events that the system will use to collect the data The method controls the hardware the temperatures voltages and times which work together to gather data The system comes with several methods that are optimized for the different soft
254. ence Analysis Using the Sequence Analysis Module Operator Instrument CEQ S060 Ver 7 0 26 Madifred OSOS 03 20 33 42 Sample Mame Sample Plate Sample Plate Barcode Sample Position Method Instrument Analysis Parameters Mote Properties Consumables Capillary Array Serial Number Capillary Array Part Murnber Total Lenath of Capillary Length af Capillary ta Detector Intemal Diameter af Capillary PLurmber af Runs Days an Instrument Gel Part Number Gel Lot Plumber Gel Algorithm Type Hours an Instrument Buffer Part Number Buffer Lot Murnber Thu 06 26 03 10 27 43 Sy ster CEG 8080 Rev 0 Project Default Sy ster CEG 8080 Rev 0 testseq A04 0303051 8NG testSeaLong 234567891000 A04 LFF a CEQ 8080 ver 7 0 26 Default amp equence amp nalysisP arameters B 8087 33 0 em 30 0 em 75 0 um g 2 0 days 381438 CEG Sequencing Gel 48 hours BD 8012 CEQ Sequencing Separation Buffer Figure 4 37 Sequence Results Report GenomeLab Genetic Analysis System User s Guide PN A29142 AB 153 Sequence Analysis Using the Sequence Analysis Module 154 Displaying Quality Parameters in a Report To display the quality parameters in a report l Select File Report Format 2 Inthe Sample Elements section select the Quality Parameters option 3 Click OK Report Format Printer Name Microsoft Office Document Image Writer Properties
255. ensus Attribute Search radio button Select the attributes that you want to define as the search criteria Click on the forward gt gt or reverse lt lt button to search the consensus in the specified direction NOTE You may also press the Ctrl and the arrow lt keys to perform the search When the search finds consensus bases that conform to the search criteria it highlights them Searching the Consensus for Specific Bases To search for a specific base or contiguous multiple bases 1 Select the Consensus Text Search radio button 2 Enter the bases in the text box 3 Specify the search criteria GenomeLab Genetic Analysis System User s Guide 1 19 PN A29142 AB Sequence Investigator Module Sequence Investigator Procedures 180 e To search for the exact match for text select the Exact Match check box M1 Use the forward and back arrow buttons or press the Ctrl and arrow lt keys to search backwards and forwards through the base sequence for more text searches e To search for a base without requiring an exact match clear the Exact Match check box The search result includes all exact matches as well as all IUB codes with an overlapping base For example a search for the ambiguity code K not only results in the bases G or T but also result in any ambiguity code that contains G or T The search stops on all ambiguity codes other than M The bases represented by M are A and C Also searching for
256. ent lengths as the initial integer values You can modify these values to represent the allele sizes that appear when the data is analyzed The nominal sizes are also displayed in the apparent size column but these will usually be edited or regenerated Apparent Size Displays the fragment lengths which are either entered using the locus tag editor generated by interpolation or regression from an incomplete list or generated from the automatic binning analysis feature see Performing Bin Analysis on page A33 When apparent fragment lengths are user entered or generated by interpolation or linear regression you must enter the nominal sizes When the apparent fragment lengths are generated by binning analysis the nominal sizes are automatically generated Refer to the following pages for more information regarding generation of the allele list using interpolation or linear regression Std Dev and Num Points Describe the number of points included in each bin and the sample standard deviation Both of these columns are generated using the automatic binning feature see Performing Bin Analysis on page 233 and are empty for allele lists not based on a bin analysis Comment Displays any user comments associated with the allele Interpolation and Linear Regression You can generate an allele list from a list of nominal sizes and a partial list of apparent sizes using Interpolation and Linear Regression Interpolation calculates the
257. ent sample during a batch analysis select Analysis Skip Current Sample Skipping the Current Sample Set Analysis in a Batch To skip the sample set during a batch analysis select Analysis Skip Current Sample Set GenomeLab Genetic Analysis System User s Guide 1 40 PN A29142 AB Sequence Analysis Using the Sequence Analysis Module 150 Skipping the Current Sample Plate Analysis in a Batch To skip the current sample plate during a batch analysis select Analysis Skip Current Sample Plate Restoring the Original Sequence Data With a Results file open select Analysis Restore Original Base Sequence to return the result data to its original state losing all edits Using Compare Mode Select up to 15 results to compare against the open result enabling the comparison of a maximum of 16 sequence results at one time while within the Sequence Analysis mode IMPORTANT If you select more than 16 results from the Compare dialog box a message box opens displaying the following warning The maximum number of results allowed for comparison with the open result is 15 Please select again Comparing Sequence Results To compare sequence results Launch the Sequence Analysis application Select File Open Select the Sequence Results tab and select one sequence result Click OK to open the result Mi xb e qu Click on the Compare button on the Sample View toolbar The Compare dialog box opens 6 Select up to fifteen
258. ential that the gene sequence format is correct IMPORTANT Sequences containing IUPAC nucleic acid codes other than A C G T or N cannot be saved into the system Any sequence that contains unrecognized nucleic acid code will be rejected l From the menu options click Manage Genes 2 Inthe Display Name field enter the name to be displayed for this gene in the eXpress Designer Analysis and in reports IMPORTANT Do not use spaces or any special characters amp _ in the Display Name Use an underscore _ instead of a space NOTE To avoid potential confusion do not simulate the NCBI accession number format 3 Enter the same name used for the Display Name in the Accession field IMPORTANT The display name MUST be the same name as the Accession number 4 In the FASTA field enter the primer description using this format gt Example descriptor Where Example is the unique accession number and descriptor is text that describes the gene NOTE Before you enter the sequence strip the base pair numbers alignment characters and other notations from the sequence before it is submitted 5 Enter the sequence in the Sequence field 6 Determine whether this gene will be displayed in the Genes list for the eXpress Designer Primer Design and Multiplex functions To hide a gene or remove it from being viewed by other users select the Hide Gene option 7 Click Save to save the gene to the database To add more genes
259. ents a particular allele if its apparent size is similar to the size of a particular allele fragment in the allele list However because both the sequence and size of a fragment affects its mobility the nominal allele size will not likely be the same as its apparent size Therefore the system identifies the alleles based on their closeness to an apparent size using a user defined confidence level Confidence Interval You can either enter the confidence interval manually or generate it automatically using the bin analysis When automatically generated the value displayed depends on the overall standard deviation computed in the bin analysis and the user specified confidence level default 2 9596 When you manually enter the confidence interval the system uses the entered value and the confidence level to estimate an overall standard deviation Increasing the confidence interval increases the width of the bins You should set the confidence interval to a value less than 0 5 nt If you enter a value of zero in the Confidence Interval field the system uses only the standard deviation of the estimated sample fragment size in the statistical test used to identify a fragment as corresponding to an allele When you enter the confidence interval the allele identification will not correspond to an error probability estimated from actual data but is still a useful relative indicator of the goodness of the match e If would like to overwri
260. ents are necessary Use Reset Form to clear the form and create another multiplex GenomeLab Genetic Analysis System User s Guide PN A29142 AB Gene Expression eXpress Designer Module Multiplex Run Statistics Hame Adjusted max sequence size TestPlex 1 380 Universal Tags Minimum sequence size Default Tags 100 Product size range Separation size 137 365 7 Separation size range Excluded Primer Pairs tee ra none Humber of Primer Pairs 29 Primer Pair 1 Primer Pair 2 Hame Hame AI817737 100 111 20 210 20 AI377418 107 377 19 483 21 PCR product size including tags PCR product size including tags 137 144 Fasta Fasta gi l5436816 gb A I817737 1 AI817737 wk25f07 xl 4gi l4187271 gblA I377418 1 AI377418 tc35cll xl NCI CGAP Brn25 Homo sapiens cDNA clone Soares total fetus Nb2HF8 9w Homo sapiens cDNA IMAGE 2413381 3 mRNA sequence clone IMAGE 2066612 3 mRNA sequence Left Primer Left Primer Sequence Sequence TCCTGGGTGTGGATTCTGTT TTCCCCTTTTGGCAAGTTC Primer start length Primer start length 111220 377 19 Melting Tm Melting Tm 60 363 60 037 Right Primer Right Primer Sequence Sequence AGCCACAAAAATGTGTCACG CCCCAGCTATTGTCTGTTGTG Primer start length Primer start length 210 20 483 21 Melting Tm Melting Tm 59 615 60 556 Figure 7 14 eXpress Designer Multiplex Results Loading Existing Multiplex Settings 2 p 6 Log into eXpress Designer Click eXpress The eXpress screen will open Click Lo
261. enu to display its drop down menu as shown in the following example Run Start Sample Flate Pause Pause to load Stop System Monitor Baseline Diagnostics Reset The following table describes the Run module s Run menu options Table 3 6 Run Menu Option Description Start Sample Plate Opens the Sample Plate Run Confirmation dialog box Pause Pauses a running sample plate Pause to Load Load a second plate while a plate is running For details see Pause to Load on page 68 stop System Stop a running sample plate or direct control process For details see Stopping a Sample Plate Run on page 69 Monitor Baseline Set the desired parameters For details see Monitoring the Baseline on page 383 Diagnostics View the instrument version laser power PC Settings monitor status and to home the plates and or the gel pump For details see Diagnostics on page 382 Reset Re synchronizes system hardware with the firmware and to reset the system log GenomeLab Genetic Analysis System User s Guide 51 PN A29142 AB Run Module Run Module Overview 02 Log Options Menu Click the Log Options menu to display its drop down menu as shown in the following example Log Options Errors Only w All Detail Sample Plate Only Freeze Log NOTE A 7 next to an option indicates that the option is enabled The Log Options menu options are available when displaying the Log tab The following t
262. equence or identified fragments and the positions of each called base or fragment within the trace data A public comment section is also defined The private section stores other information that most software packages ignore The CE system uses the private data section to store sample data including raw data along with current and voltage data This may store sample data from either Sequencing or fragment analysis samples can be stored The notes and property Sets are stored in the comment section The CE system software supports SCF versions 2 1 and 3 0 You would use this format for instance to export analyzed sequence data into Lasergene s SeqMan from DNASTAR Inc Third party packages cannot currently view raw data current or voltage However custom applications can be written to view the data Use the SCF format to transfer data from instrument to instrument or to import data from third party software into the Sequence Analysis module If you use SCF to exchange data between instruments you will lose informational data such as sample plate name capillary array serial number etc If you want to maintain exact copies of data including header information use the CEQ format and import and export these data using the Data Manager The tab delimited format is valid for raw data sequence results and fragment results Select the Raw Data check box to include raw data as well as current and voltage data Select the Results Data and Re
263. equence after performing Apply Sequence based Trimming select Edit Undo Internal Trimming Select the Enable Internal Matches check box M on the Sequence based Trimming tab to trim subsequences from within the sequence To exclude a subsequence from final removal right click on the subsequence and select Vector Other Subsequences subsequence where subsequence is the subsequence name 130 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Analysis Using the Sequence Analysis Module NOTE The system removes only the selected subsequences from the sequence when you apply trimming Q9 CD04A G01 02091814NX Analyzed Data Fluorescence 1500 Data Points CTGATAACGACATCT Exp eT 007777 CAAACGATCAATTATGTGGTCAGTGGCCAGCACCCTACGCTTTAAGGTGCTA TGCTTGATCGGCAA Import Data ACGTGTTTCTTCGCTACGGCGATGTTGTCCTTAAAACTAGCTACAGGAT TG AGGAGTTAAAATGAJ ea TCATATTCTTGGAC TGGACCATAAAATTTC TAACCAGCGCAAAATAGTTAC CGAAGGTGACAAAT LAACCGGCAGAAAACGCCCCGCTGAAAAGTAATTCATAACCATCAGTCCTCA ATGACGATTAAACA Y Zoom Mode GTTTTTGTTTTTATCTGCTTTATACTTGAGGCCGACGCCCTGGCGGTAAAG CAAAGACGATAAAAL i id TCAAAAAAGAGTGAGTGACATGGAACCAAAAACAAAAAAACAGCGTTCGCT TTATATCCCTTACG Base Synch TTTCCGTTGTTGAATAAAGGCAGTGCCTTCAGCATGGAAGAACGCCGTAA CTTCAACCTGCTGG Base Spacing CGAAACCATCGAAGAACAAGCGGAACGAGCATGGATCCAGTATCAGGGATT CAAAACCCGAABAT C OS EE LEE Lure dig aes v RC pucig sct 2686 0 2686 SCF Unzoom Unzoom d Analyze Stop Properties
264. er S601091 Gel Algorithm Type j Hours on Instrument 2 hours Buffer Part Number 08012 Buffer Lat Number CEQ Sequencing Separation Buffer Figure 4 32 Consumables Tab Properties Dialog Box 144 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Analysis Using the Sequence Analysis Module Setting or Changing Display Options To set or change the graph data display options 1 Open the Display Options dialog box using one of these methods e Select Tools Display Options While viewing a graph display of sequence data right click the graph and select Display Options The Display Options screen opens as shown in Figure 4 33 Display Options Lx Dye Traces Current Traces Quality Parameters Title Aus Options Y Axis Options Colors Title Property Title Title Color MI Title Font Arial m i Save Customized Setting Save Setting Now Cancel Apply Help Figure 4 33 Display Options Dialog Box 2 Select the tab that identifies the type of display options you want to change For details see the following topics Title Properties Tab on page 146 X Axis Options Tab on page 146 e Y Axis Options Tab on page 146 Colors Tab on page 146 Dye Traces Tab on page 146 e Current Trace Tab on page 146 NOTE To view detailed descriptions while using this dialog box click Help GenomeLab Genetic Analysis System User s Guide 1 45 PN A29142 AB
265. er 5 Cartridge Barrel 3 Cartridge Barrel 4 Grasp the wings of the gel cartridge gel pump plug and pull it out of the barrel Figure 3 44 NOTE Always note the lot number and the hours on the instrument for a gel cartridge if you are planning to use it for more than one session 5 If necessary use a tissue to wipe gel strands off of the instrument GenomeLab Genetic Analysis System User s Guide PN A29142 AB Run Module Using Direct Control 901603L Al Figure 3 44 Removing Replacing the Gel Cartridge Continued 9 1 Cartridge Wings 3 Cartridge Locking Lever open 2 Gel Cartridge or Gel Pump Plug 4 Cartridge Barrel Remove any air pockets from the gel cartridge by grasping the cartridge wings between your first two fingers and pressing the plunger with your thumb until a small amount of gel is pushed out of the cartridge tip Insert the new cartridge or the gel pump plug into the barrel and lock it into position by aligning the cartridge wings with the cartridge holder and pushing in Push the cartridge locking lever towards the back of the instrument approximately a 90 angle into its locked position Close the Gel Pump Gel Cartridge Access Cover 10 In the Remove Gel Cartridge dialog box Figure 3 45 Click Install Cartridge to indicate that a gel cartridge was installed OR e Click Install Plug to indicate that the gel pump plug or empty cartridge was installed GenomeLab Genetic Analysis System
266. er of sample components peaks fragments within each bin for each sample GenomeLab Genetic Analysis System User s Guide Fragment Analysis Module Performing AFLP Analysis Setting Cluster Analysis Parameters The selections available at the bottom of the AFLP Cluster Analysis window provides additional settings that affect the values and output format of the fields displayed You can select one of the following Sample Bin options Fragment Count Displays the number of components within each sample bin as either zero components one component or multiple components Binary Presence Indicates the binary presence or absence within each sample bin Fragments Max Y Displays the absolute peak height of the fragments within each sample bin Select one or more of the following options to modify the displayed details Exclude Fully Populated Bins Excludes bins that are represented in all samples such as non polymorphic fragments Exclude Samples without Qualifying Peaks Excludes samples that have no qualifying peaks Show Excluded Elements Highlights all fragment data that was excluded from the analysis Sample Axis Arranges the table presentation by either Rows or Columns As an alternative you can switch between these views using the right click menu To do this select the row or column heading then select Transpose The following fields let you make additional adjustments to both the width and threshold Maximum Bin
267. erature control resulting in lower than expected capillary temperature The system logs the effect in the run log It will return to normal performance once exposure is removed May cause temporary loss of capillary temperature control resulting in run not being initiated because temperature cannot reach desired level Mitigation Always run duplicates and controls Evaluate peaks carefully for anomalous results Re run sample Move location of the product by several meters Change the orientation of the equipment by 90 degrees Avoid using transmitters or cellular phones within 1 meter of the equipment Review the temperatures in the run log for any anomalies and re run as necessary GenomeLab Genetic Analysis System User s Guide Safety Information Disposal and Recycling A A016608L EPS A28219 AA GenomeLab Genetic Analysis System User s Guide It is important to understand and follow all laws regarding the safe and proper disposal of electrical instrumentation The symbol of a crossed out wheeled bin on the product is required in accordance with the Waste Electrical and Electronic Equipment WEEE Directive of the European Union The presence of this marking on the product indicates that the device e was put on the European Market after August 13 2005 e is not to be disposed via the municipal waste collection system of any member state of the European Union For products under the req
268. es list b Scroll through the analysis information in the lower analysis log area of the window 9 Click Finish to return to the Result Set View When finished re analyzing the data the system removes the initially selected results from the Study and replaces it with the re analyzed results The screen refreshes the Result Set View including the Summary area with the new data and reapplies the selected filters to the entire set of data NOTE Refer to the online help for detailed descriptions of the Re Analysis process 230 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Fragment Analysis Module Using Fragment Lists 6 7 Using Fragment Lists The Fragment List window allows you to view and modify the results of the fragment data identified in the Study You may perform a third level of filtering on this fragment list by applying additional exclusion filters Accessing the Fragment List View To access the Fragment List view use one of these methods Select View View Fragment List e Click the Fragment List 5 toolbar icon The Fragment List window opens as shown in the following example Fragment List Fragments Exclusion Filter Set size std std frag estfrag pkarea pk height New Fragment Fiter Set 1 v Apply s size nt size nt rfuxmmo rfu Name perator Valu D AB 15383 e E E Show Excluded Figure 6 26 Fragment List Window The Fragment List displays
269. eselect All NOTE If you do not use a size standard when analyzing the data the unknown fragments will not be sized 3 Note the parameters provided in the following fields for the selected size standard e Dye displays the dye label on the size standard fragments e Size Standard Fit Coefficient displays the standard deviation of the differences between the actual fragment sizes and the apparent sizes of the standard fragments determined from a large number of repeated runs This parameter is sometimes called the lack of fit 4 Select the empirical model you want to use for generating the curve that best fits the identified standard fragments from the Model drop down list NOTE The relationship between fragment size and mobility or migration time in the CE system with replaceable gels is non linear and is not described with sufficient accuracy with any theoretical model to yield reliable fragment size estimates However polynomials of varying degrees serve as practical empirical models that will accurately account for this non linearity Based on the selected standard you can use one of these following models e Linear Fit is a straight line model which is useful only for a set of size standards spanning a very small range GenomeLab Genetic Analysis System User s Guide 203 PN A29142 AB Fragment Analysis Module Defining Fragment Analysis Parameters 204 Quadratic Fit adequately accounts for the non linear dependence
270. esired modify the fields that apply to the CEQ file a Select Remove CEQ Tracking Suffix to automatically remove the plate position coordinates A01 BO1 etc as well as the time date stamp from the sample name These are generated by the CE system software b Select Resolve Filename Conflicts to enable the system to increment the sample names on export if it encounters samples with the matching names 6 Click Finish to export the Result Data and the Result Output Exporting Fragment Lists or Genotypes from a Study This feature allows you to export the opened Study fragment list or genotype data The pedigree and genotype data included in the export come from the results included in the opened Study To export the properties with a fragment result l Open the Fragment Analysis module and open a Study GenomeLab Genetic Analysis System User s Guide 250 PN A29142 AB Fragment Analysis Module Exporting Results 2 Select File Export Fragment Genotypes The Export Fragments Genotypes As window opens as shown in the following example Export Fragments Genotypes As 27x Save in lo Export ex E3 Fe EC TEST My Recent Documents Desktop My Documents My Computer DESEE Filename Yew Study Save as type csv Comma delimited csv Cancel Figure 6 43 Exporting Fragment Lists and Genotypes 3 Use the folder navigation options next to the Save in field to locate and select
271. estigator Module Sequence Investigator Module Overview 172 Table 5 7 Toolbar Icons Sequence Investigator Module Icon Description Unzoom all Displays both sequence traces in their entirety Toggle Sequences Displays or hides the sequences in the text view Toggle Reference AA Translation Displays or hides the reference amino acid translation line in the text view Toggle Consensus AA Translation Displays or hides the consensus amino acid translation line in the text view Toggle Differences Displays or hides the differences line in the text view Dye Color Coded Text Displays base text in the specified dye colors when selected Displays the base text in black and white when not selected Dye Color Coded Text block Displays the base text background in the specified dye colors when selected Help Topics Displays the CE system online help By default the help system displays the _ Contents in the navigation pane located on the left and the introductory topic in the topic pane located on the right You can use the Contents to select and print specific help topics The navigation pane also provides a Search tab and an Index tab These tabs let you search for information by topic index entry and or keyword Context Sensitive Help Click this icon and then on a menu item to open the Help file related to the selection Dye Colors Toolbar The Sequence Investigator module s Dye Colors toolbar identif
272. eviously Analyzed Results Make your selection using one of the option buttons and click OK to continue The Study Wizard guides you through the steps of creating a Study After the system analyzes the samples you may include additional results in the new Study To open an existing Study select it from the list of Study names available under either the Existing Studies tab or the Recent Studies tab The Date Time column indicates when the Study was created or last modified When you have made your selection click OK to open the existing Study Selecting the Components of the New Study The first step in creating a new Study involves manually filtering data from the database by selecting the samples you would like to include in your Study This is first level filtering After the system analyzes these samples you may further filter the sample results second level filtering in the Result Set View 194 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Fragment Analysis Module Working with Studies in the Fragment Analysis Module Finally you may filter the generated fragment and allele data third level filtering in the Fragment List This leaves a collection of identified fragments and alleles that exactly match the requirements of your Study Selecting Raw Data for the New Study When you select the Raw Data option and click OK the Study Wizard opens displaying the Select Raw Data window Select Raw Data xi
273. g box displays the analysis values but disables the fields described below GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Analysis Using the Sequence Analysis Module The following table describes the options available on the Sequence Analysis Parameters General tab Table 4 17 General Tab Sequence Analysis Parameters Editor Option N Threshold Analysis Start Stop PCR Product lt 200 Bases Use Default Mobility Calib Pre peak Reduction GenomeLab Genetic Analysis System User s Guide PN A29142 AB Description Specifies the value for the threshold below which N s are substituted for called bases Enter the desired value in the N Threshold text box The range is 0 00 to 1 00 For example entering e 1 00 calls all bases as indeterminate N s e 0 50 calls all bases with an estimated error of probability of greater than 50 as N s e 0 00 does not call any N s e 0 60 is recommended called bases with an estimated error probability of greater than 40 will be called N s Enter the time in minutes from the start of the raw data where the analysis is to begin and the time in minutes from the start of the raw data where the analysis is to end e Start The default value of 0 for the start time uses the system algorithm to find the start of valid data and the values entered under the Initial Data Detection tab e Stop The default value of 0 for the stop time analyzes to t
274. gin 2 8 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Gene Expression Logging in as an eXpress Profiler User Table 7 5 eXpress Profiler User Menu Options Option Function eXpress Designer Design custom multiplexes See eXpress Designer Module on page 280 eXpress Analysis Visualize and analyze GeXP Multiplex Genetic Analysis data See eXpress Analysis Module on page 298 eXpress Map View gene expression data using visualization tools See eXpress Map Module on page 312 Changing a User Password NOTE The User password can also be changed by the Administrator See Managing User Accounts on page 269 for more information l To change your user password click eXpress Designer 2 Click Account 3 Next Reset Form to clear the information 4 Enter the new password in the password field 5 Click Save User NOTE The password is case sensitive and can be any combination of letters numbers spaces and special characters GenomeLab Genetic Analysis System User s Guide 2 9 PN A29142 AB Gene Expression eXpress Designer Module 7 4 280 eXpress Designer Module Design a multiplex from accession numbers of target and reference genes and evaluate the primer and amplicon sequences Next redesign individual primers and assemble a final multiplex for Gene Expression profiling l From the eXpress Profiler menu click eXpress Designer The main screen of the eXpress Designer will open eXpress Desi
275. gin Only members of the CeqUsers Group to SQL Login CEQUSERS group are given access the CEQ databases Window Menu Click the Window menu to display its drop down menu as shown in the following example Window Hew Window Cascade Tile Arrange Icons Close w 2 The following table describes the Window menu options for Data Manager Table 8 6 Window Menu Data Manager Module Option Description New Window Opens a new display window Cascade Cascades the open windows Tile Tiles the windows in a horizontal orientation Arrange Icons Automatically arranges the icons Close All Closes all currently active windows v1 Brings the selected window 1 to the foreground 324 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Database Management Data Manager Module Overview Help Menu The following table describes the Help menu options Table 8 7 Help Menu Data Manager Module Option Description Help Topics Select this option to display the CE system online help By default the help system displays the Contents in the navigation pane located on the left and the introductory topic in the topic pane located on the right You can use the Contents to select and print specific help topics The navigation pane also provides a Search tab and an Index tab These tabs let you search for information by topic index entry and or keyword Using Help Select this option to display the Contents window on how to use the Windows
276. gner eXpress 7 Maximum PCR Product Size excluding Universal Tags 300 Minimum PCR Product Size excluding Universal Tags fi 00 Default Tags r Figure 7 12 eXpress Designer Main Screen Table 7 6 eXpress Designer Menu Options Option eXpress FASTA BLAST Primer Design Multiplex Account eXpress Analysis eXpress Map Function Design a first pass multiplex by entering gene accession numbers See eXpress Designer Module on page 280 Enter and save accession numbers into the database as well as retrieve their corresponding sequences from the database for Primer Design or for troubleshooting purposes See Retrieving Gene Sequences with FASTA on page 284 Confirm primer sequence quality and perform troubleshooting See Evaluating Multiplex Primers on page 286 Provides advanced primer design tools See Creating a New Primer on page 290 Create custom multiplexes and modify existing multiplexes See Importing a Multiplex on page 276 Opens the Account screen to change the password associated with a user name See Changing a User Password on page 279 Normalize and report gene expression data See eXpress Analysis Module on page 208 View gene expression data in diagram form See eXpress Map Module on page 312 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Gene Expression eXpress Designer Module Designing a Multiplex with the eXpress Designer Desig
277. h a complete new analysis from raw data e Using New Parameters allows you to modify the analysis parameters previously used e Using Additional Edited Locus Tags does not change the basis analysis but allows you to modify allele identification 3 When selected a message box opens prompting you to verify that you want to replace the selected results with re analyzed results 228 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Fragment Analysis Module Using the SNP Locus Tag Editor 4 Click Yes to proceed with the re analysis The appropriate Fragment Analysis Parameters dialog window opens allowing you to select a new or edited set of parameters to use for re analyzing the data Analysis Parameters L X IETEVIGUS Inm Figure 6 24 Selecting a Parameter Set to be used for Re Analysis NOTE For details on selecting creating editing and saving Fragment Analysis Parameter Sets see Defining Fragment Analysis Parameters on page 200 5 Select an Analysis Parameter Set 6 Click Next to proceed to the Analyze Data window GenomeLab Genetic Analysis System User s Guide 229 PN A29142 AB Fragment Analysis Module Using the SNP Locus Tag Editor Steps Analysis Parameters Analysis Parameter FA600 Time started 4 25 2006 1 55 10 PM Analyze Data Status Date Analyzed DATS 33 D4 AQ1 10605180 5 5 2001 8 23 53 PM 4 25 2006 1 55 17 PM DATS 34 D4 B 1 10605180 6 5 2001 8 23 54 PM 4
278. h it The codes have a hierarchy the system displays the code with the highest precedence To view all difference codes associated with any position display the navigator toolbar by selecting View Toolbars Navigator The differences text box in the navigator toolbar displays the difference codes in order of precedence Navigation and Editing The navigator toolbar allows you to search for locations of interest in the consensus based on attributes as well as search for specific bases and view their associated quality values and difference codes If the navigator toolbar is hidden from view select View Toolbars Navigator The system displays the navigator toolbar Figure 5 7 When docked the toolbar appears to the left of the screen display GenomeLab Genetic Analysis System User s Guide 1 TI PN A29142 AB Sequence Investigator Module Sequence Investigator Procedures 178 If you need more space for your screen display you may move the navigator toolbar so it floats over the screen display e To reposition a docked toolbar select any background area of the dialog box press and hold the left mouse button while dragging the toolbar to a convenient position on the screen then release the mouse button e To re dock a floating toolbar left click the toolbars title bar press and hold the left mouse button while dragging the toolbar to the lower left corner of the screen then release the mouse button NOTE For details on h
279. he Eject Lever to release the Manifold Plug Grasp the Manifold Plug tab Figure 9 39 and then a bi C e Pull the plug approximately one inch out of the manifold Touch the tip of the plug to the bottom of the Optics Base Plate Hold and wait five seconds for the gel strand to dry Pull the plug out and set it aside for future use Wipe gel strands off of the instrument using a damp tissue GenomeLab Genetic Analysis System User s Guide PN A29142 AB Maintenance and Diagnostics Biological Waste Disposal 901597L Al Figure 9 39 Removing the Manifold Plug 1 Guide Pins 4 Gel Strand 2 Eject Lever 5 Optics Base Plate 3 Array Fitting 6 Array Fitting 9 Click OK on the Remove Manifold Plug dialog box 10 To install the capillary array see Removing and Replacing the Capillary Array on page 361 9 3 Biological Waste Disposal WARNING The GenomeLab Genetic Analysis system has been designed to minimize exposure to hazardous chemicals and biological waste However care must still be exercised when removing used chemicals and biological samples from the instrument The information below provides the minimum protocols to use when handling hazardous chemicals and biological waste GenomeLab Genetic Analysis System User s Guide 3 3 PN A29142 AB Maintenance and Diagnostics Biological Waste Disposal Disposal of Formamide from the Sample Plate Multi Channel Pipettor Table 9 3 1 WARNING Tab
280. he Sample Plate Selection dialog box to select your sample plate e To open an existing sample plate using the toolbar click Bl Use the Sample Plate Selection dialog box to select your sample plate GenomeLab Genetic Analysis System User s Guide 29 PN A29142 AB Sample Setup Module Using the Sample Setup Module Creating a New Sample Plate To create a new sample plate 6 4 iS E n Click nl A blank sample plate opens Select and highlight the cell or cells where the samples will reside Enter the name of the samples in the Sample Name field and then press Enter Enter the barcode for the plate in the Barcode field Apply sample specific information in the Note window of the Note tab and apply property sets and values Assign a method to each sample set To edit the method or create a new method see Creating or Editing a Method on page 38 re e To automatically analyze data after a sample set run select the Analysis tab then select the Automatic Analysis check box The system will analyze the sample data using the parameter set selected from the drop down list For details see Defining Data Processing Conditions on page 39 e To automatically print a report after a sample set run see Printing the Plate Report or Specifying Sample Plate Print Options on page 40 e To automatically export data after a sample set run see Specifying Sample Plate Export Options on page 41 Select File Save As In th
281. he base caller ranging from 0 255 Call Score Calculated probability of correctness indicated on a linear 0 00 1 00 scale Quality Value Probability of correctness presented on a logarithmic 1 100 scale Migration Actual migration time in minutes in the raw data corresponding to the called bases Data Point Actual number of the data points in the analyzed data corresponding to the called bases Insert Indicates whether a base has been manually added to the base sequence Edit Indicates whether the base call has been manually changed GenomeLab Genetic Analysis System User s Guide 155 PN A29142 AB Sequence Analysis Using the Sequence Analysis Module Magnifying Zooming Data To magnify data 3 Click on the upper left hand point of the area to be magnified Drag the mouse cursor down to the lower right hand point of the area and then release the mouse button Repeat this procedure to increase magnification NOTE Use the Unzoom or Unzoom All icons to zoom out Panning Data To pan back and forth in the data pane Select Tools Pan Click anywhere in the raw or analyzed data panes and drag the mouse cursor left or right to move the display Deactivate the pan mode by selecting Tools Zoom or by clicking on the Autoscale button Synchronizing Result Data with Result Output To synchronize the analyzed data with the base sequence Access the Sequence Analysis module Open the desired analyzed data
282. he end of collected raw data Selecting this check box 1 causes analysis to end when a sudden drop off of signal lasting at least two minutes is encountered This prevents the analysis of baseline data Signifies that the sequences generated from PCR are less than 200 nucleotides in length Selecting this check box 1 changes the default values for the Delay and Minimum Duration on the Initial Data Detection tab The default value for Delay is 0 20 minutes and the default value for Minimum Duration is 2 00 minutes NOTE This option is available only when PCR Product is selected Select this check box LJ to optimize the analysis results of most short PCR products This stabilizes the data adjustment that accounts for the difference in mobility of fragments due to their respective dye labels This differential mobility has the greatest effect on the shorter fragments and has to be accounted for in the analysis of data that includes short fragments NOTE This option is automatically selected when 200 Bases is selected which is recommended Select this check box 1 if you want the software to identify and reduce the inclusion of pre peaks in the analyzed data Pre peaks are artifacts that may appear one base prior to the significant peak They are usually caused by including primers that are not full length in the sequencing reaction Sometimes they are caused by slippage of the enzyme during the extension reaction in which case the
283. he gel cartridge plunger for gel volume re estimation Please wait BERNE Figure 3 20 Gel Volume Re estimation Step 2 Message Box If the actual volume re estimated by the system is less than 1 0 mL more than the total volume to run the plate the Sample Plate Gel Usage dialog box opens as shown in Figure 3 21 Sample Plate Gel Usage Confirmation Ea The total available gel is sufficient to run 3 sample sets Lett Flate Right Flate Total sample sets 4 Total sample sets 4 Run the first jo sample sets Replace Gel Cartridge Cancel Run the first 3 sample sets Figure 3 21 Gel Usage Confirmation Dialog Box 11 Click Accept to proceed with the run 12 Specify the sample sets to run 13 Select Replace Gel Cartridge to replace the gel cartridge before the run if necessary GenomeLab Genetic Analysis System User s Guide 67 PN A29142 AB Run Module Using the Run Module 68 Gel Volume Notification If you attempt any operation that requires gel and there is not enough gel in the cartridge to perform the operation a warning message appears For example if you attempt to perform a capillary gel fill when the cartridge does not have enough gel the system displays the message shown in Figure 3 22 Run Control Fa e There is nok enough gel to perform capillary Fill The gel cartridge reserves 1 0 ml of gel For system operation margin Figure 3 22 Gel Warning Dialog Box If you attempt
284. he items called out in Figure 6 1 Table 6 1 Main Window Fragment Analysis Module Option Description A Title Bar Shows the module name Fragment Analysis and the name of the currently open Study B Menu Bar See Menu Bar Options on page 185 C Display Area Graphically displays the opened data D Toolbar See Toolbar Icons on page 190 E Dye Colors Double clicking on a dye color opens the Color dialog box Change the dye color displayed for that particular dye Clicking on the dye name D1 D2 D3 D4 removes that dye trace from all traces F Study Explorer Displays and provides access to all of the parameters available in the selected Study These are divided into the tabbed sections Analyses Data and Reports which you can select at the bottom of the Study Explorer frame G status Bar Displays information concerning the current selection 184 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Menu Bar Options Fragment Analysis Module Fragment Analysis Module Overview The following example shows the Fragment Analysis module s menu bar options File wiew Results Analysis Reports Window Help The following topics describe each of these menus and their options File Menu Click the File menu to display its drop down menu as shown in the following example Eile Mew Study Open Study Close Study Manage Studies Save Study Save Study As Add Raw Data to Study Add
285. he operator name GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Analysis Using the Sequence Analysis Module Notes Tab Use the Notes tab to view the note that was entered in the Sample Setup when the sample ID was defined Select File Properties Notes Figure 4 27 Note Tab Properties Dialog Box GenomeLab Genetic Analysis System User s Guide 139 PN A29142 AB Sequence Analysis Using the Sequence Analysis Module Property Set Tab Use the Property Set tab to view the selected property set that was entered in the Sample Setup when the sample ID was defined Select File Properties Property Set Properties Ea Analysis Alignment Consumables General Hote Property Set Method Property Sek Static Property Value Figure 4 28 Property Set Tab Properties Dialog 140 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Analysis Using the Sequence Analysis Module Method Tab View the method associated with the result and the steps used to perform the separation as well as their associated values In addition view the parameters for the first part and the second part of the separation as well as the total separation time Select File Properties Method Properties Analysis Alignment Consumables General Note Property Set Method Method Seq T est Capillary Temperature Fe 0 C Walt for Temperature Yes Denature Temperatur
286. hod stage Optical Alignment starts Method stage Optical Alignment starts Method stage Sample Set Pause starts Method stage Inject Sample starts Scan Data MySample A01 01101212NS stored Method stage Separate starts Figure 3 12 Log Window Run Module Table 3 17 Log Window Run Module Item Description Window Selection Tab Select this tab to access the Log window Freeze Log Freezes the display in the Log window Freezing the log data does not stop the collection of data just the display of data Display Area Provides the list of logged events GenomeLab Genetic Analysis System User s Guide PN A29142 AB Run Module Run Module Overview Instrument Data Window View the current for the eight capillaries and the voltage level of the instrument for the current run To open this window select the Instrument Data tab A B 2 b Dat ponitor Direct Control Log Instrument Data BN A 97 e CS OL NE NA a EO m ES COLE AEN POY PET SE DAENDA i NESA Me AAE Nr AAN ai pct te REA POO ERT EN S ERSA rta es OA NE EN E S N IE Curent microA 20 30 40 50 60 70 80 Time Minutes 40 50 60 70 80 Time Minutes Figure 3 13 Instrument Data Window Run Module Table 3 18 Instrument Data Window Run Module Item Description A Current and Voltage Buttons Select panes for display Window Selection Tab Select this tab to access this window C Display Area Displays the cu
287. i eiles desde deese 390 Performing an Optical Alignment 0 0 0 0 ccc RR RR RII 359 Viewing Capillary Information liiis eee ens 399 Viewing ael nrormatiON uices ica tttm Er dE POE Goeth baba dm avd edis indes ques 360 Viewing or Changing Buffer Information 0 lisse ees 361 Removing and Replacing the Capillary Array 0 0 0 0 e RR 361 Removing and Replacing a Gel Cartridge Gel Pump Plug 0 00 00 eeu 367 Removing the Manifold Plug 0 000 eee IR 371 9 3 Biological Waste Disposal iuste us x eng C os CES Pe Ee we Era ees 3 3 Disposal of Formamide from the Sample Plate 0 0 0 ccc es 374 Disposal of Buffer Gel Mixture from the Buffer Plate 0 0 0 0 cee 375 Disbosal or the Gapillahy ATE aes coc d D Red mse ay ete oet estes AE tw ws GO rade eee dates int 3 6 Disposal of the Gel Cartridge 0 0 eee eens 3 6 Disposal of D I Water Gel Mixture from the Wetting Tray enn 376 Disposal of the Gel Waste Bottle nonna nannan 377 94 C nsumadle JTems EISE 2s tre e o e A E E OE DE 378 TAINO SLL O E Ain EEE E oy it te a E EE 382 Reminding TNE SyS OM eeen ers tre eee a EA REGE Den A AENA 382 Homing the Plates and or Gel Pump aonana 302 SETA ES 21 0 0 S e sere Eie taro EAE CURED SUIS ORC UND p recep dedu DNO NU E Uc Od 302 Viewing Instrument Status sss esae hd ect eto ce QT e UE AL GU UR T od aen inne 383 Viewing Optical Scam Dati cud o qu cute iod R
288. ibutes or text specified in the navigator toolbar Restores the compared sequence result to its original state removing all changes Changes the quality threshold below which bases in the consensus are identified as having low quality Moves the sequence trace shown in the top trace pane and exchanges its position with the bottom trace GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Investigator Module Sequence Investigator Module Overview View Menu Click the View menu to display its drop down menu as shown in the following example View Toolbars w Status Bar Unzoor All History Ckri H Base Numbering Ctrl ShiFk 6 w Codon Numbering CEri 5hifE2 O w Sequences CEri 5hifE4 5 v Reference Amino Acid Translation Ctrl Shift R w Consensus Amina Acid Translation CErla ShifE C wv Differences Ckri ShifE D Show Colors Working Database krel NOTE A Y next to an option indicates that the option is enabled Use the View menu to display data as desired The following table describes the Sequence Investigator module s View menu options Table 5 4 View Menu Sequence Investigator Module Option Toolbars otatus Bar Unzoom All History Base Numbering Codon Numbering Sequences Reference Amino Acid Translation Consensus Amino Acid Translation Differences Show Colors Working Database GenomeLab Genetic Analysis System User s Guide Description Select the toolbars
289. ics base plate Hold this position and wait five seconds for the gel strand to dry Pull the fitting away from the instrument Use a tissue to wipe gel strands off of the instrument GenomeLab Genetic Analysis System User s Guide PN A29142 AB 345 Maintenance and Diagnostics Routine Maintenance 901597L Al Figure 9 3 Removing Replacing the Array Fitting 1 Guide Pins 4 Gel Strand 2 Eject Lever 5 Optics Base Plate bottom 3 Array Fitting 6 Array Fitting 9 With the array fitting in hand blow dust and debris off of the windows with compressed gas Texwipe Microduster III PN TX2511 10 Using a water moistened swab Texwipe Swab PN TX754B gently wipe the detection window by stroking in one direction only as shown in Figure 9 4 346 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Maintenance and Diagnostics Routine Maintenance Figure 9 4 Cleaning the Detection Window 1 Detection Window 3 Array Fitting 2 Stroke in one direction ONLY 11 With a new water moistened swab repeat wiping on the other side of the window 12 With a dry swab gently wipe the windows to remove excess water again repeating on the backside with a new dry swab 13 Blow compressed gas on the windows to remove all excess water CAUTION Make sure not to invert the compressed gas bottle otherwise propellant will contaminate the capillary windows 14 If dried gel or other debris remains
290. ies the colors assigned to dyes displayed in the trace data view and for the bases in the text view To change any of these color assignments double click the desired icon and use the Colors dialog box to choose another color Dye Colors Ea A C HONT EN E Figure 5 3 Dye Colors Toolbar The following table describes the Sequence Investigator Dye Colors toolbar icons Table 5 8 Dye Colors Toolbar Icons Sequence Investigator Module Icon Description A Red is the default color assigned to the Adenine A nucleotide in the data view red C Black is the default color assigned to the Cytosine C nucleotide in the data view black G Green is the default color assigned to the Guanine G nucleotide in the data view green GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Investigator Module Sequence Investigator Procedures Table 5 8 Dye Colors Toolbar Icons Sequence Investigator Module Icon Description T Blue is the default color assigned to the Thymine T nucleotide in the data view blue N Gray is the default color assigned to ambiguous nucleotides other than A C G or T gray 5 2 Sequence Investigator Procedures This section shows you how to use this module to investigate and compare sequence analysis results To open the Sequence Investigator module click the Investigator icon from the Main Menu D Creating a Reference File The reference file is a text file you can create using N
291. ight Sequence ACACG CTCCCAGACGTAGTT Right Primer start length 236 20 Right Melting Temperature Celsius 53 84 PCR Product Size excluding Universal Tags 207 Set As Reference Primer Figure 7 6 Express Profiler Manage Primers Screen Table 7 4 eXpress Profiler Options Parameter Description Name Enter the accession number and primer description Use this format Accession number native PCR product size native left primer start length native right primer start length Parent Gene Choose the gene from which primers are derived This list is populated by the Manage Genes option Multiplex import or by FASTA in eXpress Designer Left Sequence The forward gene specific primer sequence Left Primer start The starting nucleotide position followed by the primer length length 214 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Gene Expression Software Administration Parameter Description Left Melting The melting temperature of the forward primer Temperatures Celsius Right Sequence The reverse gene specific primer sequence Right Primer start The ending nucleotide position followed by the primer length length Right Melting The melting temperature of the reverse primer Temperature Celsius PCR Product Size The PCR amplicon size produced with these primers without Universal Tags excluding Universal Tags set As Reference This option indicates whether the primer is available for Glo
292. ighting sample names and moving them to the selected list box until you have selected all the samples for analysis Click OK The Analysis Parameters Editor dialog box opens GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Analysis Using the Sequence Analysis Module 7 Select the appropriate analysis parameters or edit an existing parameter set and click OK The batch processing begins and a window opens Batch Analysis3 oj x f Batch 04 24 06 16 34 41 Start Analysis 04 24 06 16 34 45 Sequence Test A03 06042416KN Completed Result stored 04 24 06 16 34 49 Sequence Test B03 065042416K0 Completed Result stored 04 24 06 16 34 53 Sequence Test C03_06042416KQ Completed Result stored 04 24 06 16 34 57 Sequence Test D03_06042416KR Completed Result stored 04 24 06 16 35 01 Sequence Test EO3 06042416KS Completed Result stored 04 24 06 16 35 05 Sequence Test F03_06042416KU Completed Result stored 04 24 06 16 35 09 Sequence Test GO03 06042416KV Completed Result stored 04 24 06 16 35 12 Sequence Test H03 060424165KX Completed Result stored 04 24 06 16 35 12 Analysis Completed 04 24 06 16 35 11 Analyzed Data Computation Completed 04 24 06 16 35 11 Base Sequence Computation Start 04 24 06 16 35 11 Base Sequence Compute Base Number Curve 04 24 06 16 35 12 Base Sequence Convert X Axis to Base Numbers 04 24 06 16 35 12 Base Sequence Compute Initial Base Sequence 04 24 06 16 35 12 Base Sequence C
293. ights of alleles identified in similarly named samples are then compared and their status determined LOH AI MI Homozygous NoNorm Alleles 246 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Fragment Analysis Module Performing LOH Analysis To start LOH Analysis 1 Select Analysis Run LOH Analysis to launch Microsoft ExcelTM The LOH xml file opens displaying the START sheet File Edit View Insert Format Tools Data Window Help Type a question for help 2 X D gH J aria 1o Ji B z U lEz BIS E H OA B Y fe Minimum difference in Allele Peak Height Ratios for Detecting LOH 1 100 a LOH Threshold Minimum difference in Allele Peak Height Ratios for Detecting Allelic Imbalance p Al Threshold Al 1 to LOH threshold 0 means don t detect Characters embedded in sample names of Normal samples or loci L r Check if Tumor and Normal in same well Start m M 4 gt MN Instructions START jal Ele Ready Figure 6 34 Run LOH Analysis NOTE You must select Enable Macros to run the LOH Analysis otherwise a prompt reminds you to enable this feature 2 From the LOH xls START sheet set the LOH Threshold and the Al Threshold if desired 3 Specify the unique character combination used to identify Normal samples or loci i e Norm 4 Specify whether Normal and Tumor samples are in the same well e If you are running Normal and Tumor samples a
294. ile Import In the Import dialog navigate to the folder containing the Sample Plate TXT file Highlight the selected sample plate TXT file and click Open in the Import dialog The imported sample plate grid window will appear VE M E B M ee Re confirm the values and properties for all Sample Plate objects the barcode the sample names the separation method s the note the analysis parameters report and export options From the Menu Bar select File Save As 9 Save the Sample Plate to the desired project with a new name 10 Repeat steps 1 through 8 for the second Sample Plate if one is present Importing a Sample Plate Using Non Default Run Method or Analysis Parameters If using non default methods the appropriate run method s and analysis parameters that were pre selected in the Data Sets must be set up in the CE System software before importing the sample plate TXT file s GenomeLab Genetic Analysis System User s Guide PN A29142 AB po SUP xe PP des TN Sample Setup Module Using the Sample Setup Module Launch the Data Manager Setup an appropriate Working Database or create a new Working Database and Project Launch the Sample Setup module Cancel the Sample Plate Selection popup window From the Menu Bar select Edit Method From the Choose Method to Edit dialog confirm the presence of the appropriate or pre selected run method s If not present highlight any existing method and click OK
295. ime thereafter To activate the CEQUSERS group l Open the Data Manager module 2 Select Tools Administrative Tools Add CeqUsers Group to SQL Login All users added to the group CEQUSERS now have access to the database GenomeLab Genetic Analysis System User s Guide 341 PN A29142 AB Database Management Data Manager Procedures 342 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Maintenance and Diagnostics Routine Maintenance Maintenance and Diagnostics This chapter provides routine maintenance replenishment and biological waste disposal procedures It provides a list of the consumable materials used in the system It also includes some diagnostic procedures performed as needed 9 1 Routine Maintenance NOTE Use Figure 9 1 to locate hardware components referenced in this section 901632L Al GenomeLab Genetic Analysis System User s Guide 343 PN A29142 AB Maintenance and Diagnostics Routine Maintenance Figure 9 1 User Accessible Hardware Components 1 Sample Access Cover extended 7 Manifold Access Cover 2 Capillary Access Cover extended 8 Gel Waste Bottle 3 Status Indicators 9 Power Switch 4 Plate Holders Sample Transport 10 Gel Pump 5 Capillary Temperature Control Cover 11 Gel Pump Gel Cartridge Access Cover 6 Rubber Latches Cleaning the Capillary Array CAUTION The capillary array windows must kept free of any contaminants Otherwise high backgr
296. in components chemistry hardware and software comprising the system and details the safety features relevant to the system It also provides step by step procedures for operating the system 1 1 System Overview This section describes the purpose and function of the CE system provides an overview of the main components comprising the system and details the safety features relevant to the system Purpose of this System The purpose of the Capillary Electrophoretic CE Genetic Analysis System is two fold e To determine the nucleotide sequence of any given DNA sample To estimate sizes of DNA fragments Functional Description The GenomeLab Genetic Analysis System is fully automated and capable of determining the base sequence and fragment length of DNA samples that have been prepared with Beckman Coulter Inc dye labeled reagents Four color dye labeled terminator chemistry kits are used to process samples for base sequence analysis Generation of samples for fragment length analysis is performed using one of two techniques e dye labeled primers for micro satellite AFLP and other similar applications e four color dye labeled terminator chemistry for the single nucleotide polymorphism application The CE system incorporates two plate holders and accepts two 96 well plates at one time Each row of eight samples sample set containing labeled DNA fragments is automatically denatured and then separated by capillary electrophore
297. in eXpress Profiler Importing Fragment Analysis Data on page 300 Format Descriptions and Requirements for Exporting Fragment List and Genotypes Export data along with their export requirements by using one of the following file formats CSV Comma csv Contains the export parameters of a Study s fragment list as selected sorted and ordered Each line in the output file represents a fragment The data in this export are comma delimited suitable for viewing in most third party spreadsheet applications The export format has the csv extension Linkage Pedin pre Contains the export parameters of a Study s pedigrees and genotypes The Pedin file can be imported into a Linkage applications MakePed utility program This file includes the pedigree and genotype information from each result in the Study It supports up to two alleles per locus The export produces three files e pre the Pedin file e log the list of processing steps and errors encountered during the export e debug the text file with column headings and comma separated data to aid in the correction of errors in the pre file The Pedin export requires a list of locus names in a file named LocusList txt You must store this file in the CE system export or root directory This text file must have only one locus name per line The order of the loci included in this file determines the order of the loci in the Pedin file The Pedin export al
298. ing formats e Standard Chromatogram Format Ver 3 00 scf e Standard Chromatogram Format Ver 2 10 scf e Tab Delimited ASCII Text txt e SEQ Sequence Text seq e FASTA and Quality fasta e PHRED and SCF scf phd 1 e ESD esd e CEQ cq Opens or exports samples from a specific sample plate To select multiple samples press the Shift key while selecting samples The Sample Plate toolbar must be open to use this menu option Used to e View or change the panes displayed when you first open a sample e Show the Report Format dialog box selecting Filel Print Report e Open all associated results that belong to a sample when a samples is opened e Open export the most recent results from a sample plate e Show the Analysis Log during analysis e Print the entire desktop e Print the application window including all toolbars e Print only the displayed panes e Select audio playback speed View the general properties of the currently selected item You can view the item type database and project where the item resides as well as the date and time the item was last modified You may also view the sample name sample plate sample position in the sample plate the instrument and the operator name You can also view the Property Set run Method Analysis Alignment Consumables and Note of the currently selected sample opecify the format of the report to be generated for the active sample Displays a fa
299. inished with Dye Traces click Apply to continue or OK to close the Display Options dialog box Current Trace Tab To set or change the current trace options l Inthe Display Options dialog box select the Current Traces tab 2 Select the radio button for the current type to display under Data 3 Click OK GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Analysis Using the Sequence Analysis Module Changing Display Colors In addition to using the Display Options dialog box Colors tab page 146 to set display colors you can change colors used in raw data and heterozygote base displays as described in the following topics Raw Data Colors To display the Raw Data Colors toolbar 1 Select View Toolbars 2 Select the desired toolbar options 3 Click Close To change the colors of the raw data traces 1 Onthe Raw Data Colors toolbar click on the trace color to change A C G or T The Color palette dialog box opens as shown below Color El x Basic colors EI en ee g en es E E NEN UL BEE eee ee HNENSEN M Custom colors JIB BBE BE EEE ee Define Custom Colors gt gt TUN Figure 4 34 Color Dialog Box 2 Select the new color 3 Click OK Heterozygote Display Color The heterozygous bases are bold and red by default To change the color of the heterozygotes in the base sequence pane l Select Tools Heterozygotes Display Color The Color palette dialog
300. int button Administrative Tools The System Administrator can access the database without logging onto the computer as an Administrator Before using the Administrative Tools an Administrator must first set up a group and assign to it the users to have access to the CE system databases Users and Groups Adding Users and Groups is done outside of the CE system After creating the CEQUSERS group and adding users to it you must activate the group within the Data Manager module Adding Users l Select Start Settings Control Panel Administrative Tools Computer Management The Computer Management console opens see Figure 8 10 m Computer Management ITI File ction View Window SEEN Computer Management Local Eis System Tools gg Event Viewer F Shared Folders aon Device Manager EB Storage H A Removable Storage l Disk Defragmenter i Disk Management ee Services and Applications Figure 8 10 Computer Management Console Selecting Users Help Full Name OFF Administrator ASP NET Machine Account BE Helpassistant Remote Desktop Help ssi GESUPPORT_38 4 CN Microsort Corporation PII E Description Built in account For administering EF Account used For running the 45P Built in account for quest access bo Account Far Providing Remote Assi This is a vendor s account Far the F 2 In the left pane click on the symbol next to the System Tools item to expand its contents 338
301. ion Use A Peak to Call Allele Apparent Size Includes A Not Checked Checked Use A Peak to Call Allele No Size Adjustment One nucleotide is subtracted from Checked the apparent size of the allele in the allele list Apparent Size Includes A One nucleotide is added to the No Size Adjustment Not Checked apparent size of the allele in the allele list 218 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Table 6 14 Truth Table for Setting A Allele Identification A Detection Use A Peak to Call Allele Checked Use A Peak to Call Allele Not Checked All fragments assumed to be A The A fragment is identified as the allele in the allele table Fragments without A if any are identified as A peaks or Spurious peaks All fragments assumed not to be A Any fragments without A are identified as the allele in the allele table Fragments with A are identified as A peaks or spurious peaks 6 6 Using the SNP Locus Tag Editor Use the SNP Locus Tag Editor to create a new SNP locus tag or edit an existing one to be used for SNP analysis You can access this editor from the SNP Locus Tag tab of the Analysis Parameters window see SNP Locus Tags Tab on page 208 SNP Locus Tag Editor Locus Tag SMF1 Save As Project ddSNP Allele ID Lacus Mame SNF IZE Ploidy Allele ID s For Accession H Primer Sequence Apparent Fragment 19 47 Si Biel Es Cancel Fragment
302. ion and frequency of use 380 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Maintenance and Diagnostics Consumable Items List The following table identifies the materials that are required for either Sequence Analysis or Fragment Analysis but do not come with the product Table 9 16 Materials Required but not Supplied Item Refrigerated microfuge Molecular Biology Grade sterile dH O 95 v v ethanol dH 0 70 v v ethanol dH O Sterile tubes 0 5 mL microfuge 0 2 mL thin wall thermal cycling tubes or plates with caps Thermal cycler with heated lid sample Loading Solution SLS Mini Alpha Swab from Texwipe P N TX754B VWR Microduster III AccTech P N 58019 538 VWR 20 mg mL Glycogen Boehringer Mannheim Cat 901 393 3M Sodium Acetate pH5 2 Sigma Cat 7 7899 100mM Na EDTA pH8 0 500 mM Sigma Cat 7889 PCR enzyme and buffer Labeled primers contact your local Beckman Coulter representative for information on vendors carrying the appropriate primers Thermal cycling plates and caps Table 9 17 Materials Required but not Supplied with this Kit Reagents Item Thermo Start amp DNA Polymerase with separate 25 mM MgCl2 Nuclease Free H20 non DEPC Treated 1M Tris HCl pH 8 0 GenomeLab Separation Buffer GenomeLab Separation Capillary Array GenomeLab Separation Gel for single plate systems such as CEQ 8000 GenomeLab Genetic Analysis System User s Guide PN A29142 AB CPN
303. ioni uses 14 siTe IRI IO SCRI NET ELS TC TOT ene ce Bas one cee asd Maen ate ae 15 Section 2 sample Setup Module 0 cee eee eee 19 ZA OM CIVIC L Eres Be 19 USING THE Wid WINDOW 240 2 cere oe a e d a a date d a ea a i ai e boty amas cinteee 19 Men Bar ODTONS ec cuceisccdnait ga retein PE MNebeEbberQebeutsm4 add e iai Ra cis 22 OOM A NCC lg esane tay tee eet cess ea ets TEE ton eto aaa tate eee nt ee 21 2 2 Using the Sample Setup Module issssssseee RR 20 DDenipgasdimbie Pal cus atto rid eni ddp opener deus dido enn ee kw lube tpe 20 Creating a New Sample Plates eu zone 3 tea worse Em toe met DEDE BO acs Ub ee ate Eee 28 Opening a Sample Plate as sc oca d ad ee et V bae ratu et aah ai 29 Creating a New Sample Plate 0 RR IRR 30 Se lCCUNG SAIN OS o 5 2gcta tot qtia Pd ste este ede Ure yon beac tee ee dae teri dad 30 USING PEODOEB SOUS ose oo arts a te e DEREN oie eae a we ee 35 USING IMGINOOS asco icti tL odio et aco Daeg etico p area heme ORDER 3 Defining Data Processing Conditions 0 0 0c eee eee 39 Printing the Plate Report or Specifying Sample Plate Print Options 40 specifying Sample Plate Export Options 0 0 ees 41 EXD OL ODIO Sts os eanted aration a anes aaa aur ace meee en eee are oak 41 Export Options Sequencing ResultS 2 0 0 0 ccc RR RR 42 Importing Sample Plate Information from TXT Files lisse RR
304. irect Control Loading the Sample Plate and Buffer Plate 1 Select Direct Control Access Plates from the menu Access Plates x Stark Help IF vau are going to load new plates please prepare your plates before clicking on Start You have 15 minutes to change plates once wou click on Start This time limit is critical so that the capillaries are nat adversely affected by prolonged exposure to alr Figure 3 28 Access Plates Dialog Box 2 Click Start The Capillaries Exposed dialog opens as shown below Capillaries Exposed xi Capillaries Exposed xi PLEASE WAIT an Do not open sample e You may now open the sample door and load plates door Capillaries exposed to air Capillaries exposed to air Load Cancel Cancel Time Remaining Time Remaining Alarm Off min sec min sec ga p3 Help fiz 53 Help m Left Plate Right Plate m Left Plate Right Plate Plate Loaded Immerse Capillaries requires wetting tray on the left side Plate Loaded Immerse Capillaries requires wetting tray on the right side Plate Loaded Immerse Capillaries requires wetting tray on the left side Plate Loaded Immerse Capillaries C requires wetting tray on the right side Figure 3 29 Capillaries Exposed Dialogs 3 Wait until the dialog warning turns green 4 Open the Sample Access Cover Figure 3 27 a
305. is part of another word Select the Match case option to find the entered text exactly as typed into the field If Match case is not selected the system will find all occurrences of the text regardless of the case Select the Use wildcards check box M to include wildcard characters in your search The follow table describes the possible wildcards Table 2 9 Wildcard Characters Wildcard Description The asterisk specifies to search for any letters before or after the entered text The question mark represents any single letter The exclamation point within brackets makes the query negative Enter the letter following the within the brackets if you want the system to find anything that is not the entered text For example entering A finds any string that does not start with A NOTE The system supports the following wildcard combinations and The system does not support the combination 3 Click Find Next to find the next occurrence of the entered text Search for and Replacing Text in the Sample Plate 2 Select Edit Replace Use the Replace options to specify your search and replace settings Enter the desired text in the Find what text field Select the Match whole word only check box M to locate text that is flanked by spaces If Match whole word only is not selected the system will find the text even if it is part of another word Select the Match case
306. is option delays loading of plates until after immediate sample run After completion of sample separation the system enters the Access Plates condition After loading the new plate the system runs the next sample set from the original sample plate NOTE Ifthe plate has not been loaded by the time set in Modify Wait Time max 30 minutes the run resumes with the next sample set e Stop current sample set now to load plate This option immediately stops the run to allow for plate loading If selected the Save Collected Data option becomes available allowing the previously collected data to be saved e Stop current sample plate now to load plate This option breaks the plate run before the next sample If selected the Save Collected Data option become available allowing the previously collected data to be saved 3 Click OK Stopping a Sample Plate Run To stop the currently executing sample plate 1 Select Run I Stop System or click the Stop System al toolbar icon The the Stop System dialog box opens as shown in the following example Stop System Ea Stop pti ais Stop sample plate All Plates mand Skip current sample set Help Stop after current sample set completes W Save Collected Data I Perform Shutdown Method Figure 3 25 Stop System Dialog Box 2 Select the desired Stop Options radio button GenomeLab Genetic Analysis System User s Guide 69 PN A29142 AB Run Module Using the Run Modu
307. iteria of the Study and to include results that do For example suppose that your result set contains multiple samples collected under two different sets of fragment analysis parameters One of these parameter sets used the SizeStandard 400 to determine the fragment sizes while the second parameter set used the SizeStandard 600 to make the determination To exclude any data that were analyzed with the SizeStandard 600 1 Open the drop down list in the Name column of the Filter Set area and select SS Size Standard Open the drop down list in the Operator column of the Filter Set area and select Open the drop down list in the Values column of the Filter Set area and type in SizeStandard 600 4 Click Apply at the top of the Filter Set area All samples in the fragment results table that used the SizeStandard 600 are highlighted in teal visible by selecting this parameter for display This indicates that they have been excluded from the Study The Excluded column in the Summary area has increased by the number of samples using the SizeStandard 600 There are many filters available You must apply each one using an operator and a value You can apply any number of filters to a result set and save the filter sets for future use You can assign filter sets to filter out samples with excessive baseline noise excessively broad peaks no size standard out of calibration range etc This second level filtering leaves only the results
308. ith a sequence results file open highlight a series of base sequence text 2 Select Edit Audio Playback to hear the announcement of the highlighted base sequence text NOTE To use this feature Edit mode must be enabled Viewing Color Calibrations To view a color calibration for a sequence result l Select File Open 2 Inthe Open dialog box select the Sequence Results tab highlight the desired result name then click OK 3 Select View Parameters Used to Compute Sequence In the Parameters Used dialog box click Color e To view the color calibration prior to the run select the Initial Values radio button e To view the color calibration after the run select the Final Values radio button NOTE This is a read only dialog box To make changes to the color calibrations select Analysis Working Analysis Parameters Edit Color e To add a successful color calibration to the list of available color calibrations click Save As in the Color Calibration Editor dialog box Enter the desired name then click OK Computing a New Color Calibration To compute a new color calibration l Select Analysis Working Analysis Parameters 2 Inthe Working Parameters dialog box click Edit 3 Click Color in the Sequence Analysis Parameters Editor dialog box GenomeLab Genetic Analysis System User s Guide 1 57 PN A29142 AB Sequence Analysis Using the Sequence Analysis Module 158 4 Select the Compute new color matrix check
309. ithin each capillary Fragments Menu Click the Fragments menu to display its drop down menu as shown in the following example Fragments Show Excluded Manually Select Peaks Save Fragment Grid 45 CSV GenomeLab Genetic Analysis System User s Guide 1 8 PN A29142 AB Fragment Analysis Module Fragment Analysis Module Overview The following table describes the Fragment Analysis module s Fragments menu options Table 6 5 Fragments Menu Fragment Analysis Module Option Description Show Excluded Shows fragments that were excluded from the Fragment List Manually Add Provides an option to manually select peaks to be included in the fragment list Peaks save Fragment Save the Fragment Grid as a comma delimited file csv Grid As CSV Analysis Menu Click the Analysis menu to display its drop down menu as shown in the following example Analysis Analysis Parameters Mew AFLP Analysis Mew Binning Analvsis Mew Peak Ratio Analysis Run LOH Analysis The following table describes the Fragment Analysis module s Analysis menu options Table 6 6 Analysis Menu Fragment Analysis Module Option Description Analysis Parameters e View the current working analysis parameters e Edit the working analysis parameters maintained in memory for the duration of the application session e Select a previously saved parameter set to be used in the analysis New AFLP Analysis starts a new AFLP An
310. itioning the sample and buffer plates close the Sample Access Cover and then select the position from which the plates were loaded left or right 4 The Capillaries Exposed dialog will display and click Load CAUTION The separation gel within the capillaries will dry out if the capillaries are left exposed to the air for more than 15 minutes Setting the Capillary Temperature 76 l Select Direct Control Capillary Temperature from the menu 2 Inthe Capillary Temperature dialog box enter a capillary holding temperature value in degrees centigrade 3 Select Wait for temperature to be reached and then click Start Denaturing a Sample l Select Direct Control Denature from the menu 2 Inthe Denature Samples dialog box enter a time duration in seconds 3 Identify the position of the Sample Set and click Denature GenomeLab Genetic Analysis System User s Guide PN A29142 AB Run Module Using Direct Control Injecting a Sample 1 Select Direct Control Inject from the menu 2 Inthe Inject dialog box enter the Voltage in kV and enter a Time Duration in seconds 3 Use the Sample Set option to set the position of the samples and then click Inject Performing a Separation l Select Direct Control Separate from the menu 2 Inthe Separate dialog box a Enter a value for the voltage in kV b Enter a time duration in minutes c Identify the position of the buffer using the Buffer Set spin controls and then sel
311. ium 5 124 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Analysis Using the Sequence Analysis Module Using Sequence based Trimming Apply contaminant sequence based trimming This can be done in batch analysis mode or in sequence analysis mode The resulting base sequence and trace views display the base letters of the vector and other subsequences as underlined If multiple vector or subsequences matched they appear as multiple underlined in the Trace View Sequence Analysis Parameters Editor DefaultSequenceAnalysisPar General Initial Data Detection Heterozygote Detection Qualty baged Trimming sequence based Trimming Alignment Reference VectorOther Sequence Files RR t Distance from E nd Lett 8 25 E Right 37 25 blas H of Vector Other Sequences 2 E Min Substring Length E E Enable Internal Matches ma Save Ag Print Cancel Help Figure 4 16 Sequence based Trimming Tab GenomeLab Genetic Analysis System User s Guide 1 25 PN A29142 AB Sequence Analysis Using the Sequence Analysis Module 126 Contaminant trimming can be based on any combination of the following e Vector sequences such as plasmid BAC PAC and cosmids e Primer sequences e Restriction enzyme sites NOTE If you opened this dialog box by selecting View Parameters Used to Compute Sequence this dialog box displays the analysis values but disables the fields
312. iversal Tags 0 0 00 c ccc eee eee a 2 6 3 Logging in as an eXpress Profiler User 0 00 0 ccc ee ees 2 0 Changing a User Password 0 RR RR RR ER 2 9 7 4 eXpress Designer Module 0 0 0 0c RR IRR 280 Vi GenomeLab Genetic Analysis System User s Guide PN A29142 AB Designing a Multiplex with the eXpress Designer 0 0 00 cece ee 201 Retrieving Gene Sequences with FASTA 0 0 0 0 0 ccc cece RR RR 284 Evaluating Multiplex Primers issues eee eee eee eens 206 Evaluating Primer and Amplicon Sequences with BLAST 00 00 00 c eee eee 28 Ws e RUNS PrIM RO soe dic cera aaa cia cada mate ah aaa ara od beh oa ge eed ah 209 1 9 MUMIDIOX DeSIOFIBE aa Scd eed end anne S OE eee Behr mto E ACE e bot x thes 293 Creating a Multiplex with Genes and Primers 00 0000 RR RR 294 Performing a Gene Expression Analysis with the Multiplex Design 05 29 7 6 eXpress Analysis Module 0 0 00 cc ccc ee eee ee ee een ees 298 Creating a New ANAIVSIS dct teo xe epe x EE eek Meee eee eee hele sae 299 SOBRE d Pdl a odere d aoe a ee er ee Ede ques 302 Seting Up Peak BINNING zs seed and dex eon gebe Mais oerte deding dare Wand ih ack hears ere id 305 Viewing Data for a Selected Well 0 0 0 0 0c BB RI 306 NOMAA te AKG tance matte a tosta anui ees i uer A eet ae ee cedi Lg A dE E Stet I 308 Ef eXpress Map Module 432 2 giv eared draco dne dMe tet ue bd ete ak il Gee cain o
313. key 3 Left click the last cell in the column or row to be selected 4 Release the Shift key a eo R1 g1 amp 01 r9 g1 A02 r8 g2 A03 r ga Ang r2 q1 B01 r1 q2 B02 r9 g2 z r8 q3 B04 Bgc r292002 rig3c03 MERHED rdg DD 392 002 r2g3003 rai po4 i E el ad Coa ed a oo T 3 En2 ead Cod Figure 2 10 Select Contiguous Cells GenomeLab Genetic Analysis System User s Guide 31 PN A29142 AB Sample Setup Module Using the Sample Setup Module Selecting Non Contiguous Cells 1 Click the first cell 2 Use the Ctrl Click function to select each additional cell EIDEM HT EE HERI roto 892 403 723208 Dorcaneccaneneatenenconeecenen a a a enoonene ce ence paa Seeeneeeeeuaeneeeceauareeuseon cenereccseeersecserssertserrneengeanes r2 g1 B01 rl g2 602 r1 g3 c03 preme eed r3 qi r2 g2 C02 raqg2 002 Seems rl g4 D04 masussasuuossnssusuuosenscosussss usas r4 gf DOT LICUIT O1 A OOA O TO E Oe Figure 2 11 Selecting Non contiguous Cells Viewing a Sample Plate by Sample Name Subject ID Use the View View by Subject ID option to toggle the sample plate view between Sample Name and Subject ID This works in conjunction with the options found next to the Sample Name and Subject ID fields as shown in the following example View wv Toolbar w Status Bar w Cell Coordinates Sample Property Sets Summary View by Subject ID Working Database 9 Sample Name M Subiect ID 7
314. kup Files above then use this same backup file for the database recovery and configuration recovery NOTE You may recover both the Database and Configuration files at the same time by selecting the options for Backup Restore Database Files and Backup Restore Configuration Files This requires that the database and configuration files be backed up in the same backup file as described in eXpress Backup and Restore Restoring the Database l From the Windows Start menu select Start Programs GeXPAdmin eXpress Backup and Restore A dialog box will open on the desktop 2 Select Backup Restore Database Files ONLY IMPORTANT Make sure that the Backup Restore Configuration Files option is not selected 3 Click Restore The Database Restore dialog box opens 4 Select a backup file to restore from the file list 5 Click Open A dialog box displays the message Are you sure This will destroy ALL data currently stored and revert to the data set existing when the selected backup was taken Do you wish to proceed GenomeLab Genetic Analysis System User s Guide PN A29142 AB 10 ll 12 13 Gene Expression eXpress Backup and Restore Click Yes to proceed A Windows command dialog box displays the message Shut down message has been posted to the server Server shut down may take a while check logfiles for completion Press any key to continue Press any key on the keyboard to continue with the restore A dialog box displ
315. l 3 3 Using Direct Control Click on the Direct Control icon NOTE Use Figure 3 27 User Accessible Hardware Components to locate hardware components referenced in this section A al amp amp 901632L Al Figure 3 27 User Accessible Hardware Components 1 Sample Access Cover extended 7 Manifold Access Cover 2 Capillary Access Cover extended 8 Gel Waste Bottle 3 Status Indicators 9 Power Switch 4 Plate Holders Sample Transport 10 Gel Pump 5 Capillary Temperature Control Cover 11 Gel Pump Gel Cartridge Access Cover 6 Rubber Latches GenomeLab Genetic Analysis System User s Guide 13 PN A29142 AB Run Module Using D
316. l be expelled from the capillaries e Ifyou select Buffer Plate use the Buffer Plate spin controls to identify the waste position in the buffer plate then click Fill f you select Wetting Tray click Fill Purging the Manifold l Select Direct Control Manifold Purge The Manifold Purge dialog box opens FP Volume 0 40 ml Cancel Cycles f 7 Help 358 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Maintenance and Diagnostics Direct Control and Replenishment Figure 9 22 Manifold Purge Dialog Box 2 Enter a volume in milliliters mL 0 1mL default 3 Enter the number of cycles 4 Click Purge Performing an Optical Alignment To align the lasers with the detection windows of the eight capillaries l Select Direct Control Optical Alignment The Optical Alignment dialog box opens Optical Alignment Ea Scan Data m Autazawve im Mame SCAN Cancel Froject Default hi Help Figure 9 23 Optical Alignment Dialog Box 2 Save the alignment data a Select the Autosave check box b Enter a name in the Name field c Select a Project from the drop down menu 3 Select Align NOTE Prior to performing an Optical Alignment it is advisable to purge the Array Manifold and fill the capillaries with fresh gel in that order See Purging the Manifold on page 358 and Replenishing the Capillaries with Gel on page 358 Viewing Capillary Information To view or
317. l confirm that the import is complete and that the number of plates were added to the database IMPORTANT Make sure to import all of the plates to be included in a study in the first pass If more plates are added in the second round import the first set of plates will be deleted from the study Click OK to continue Click Save to save the changes to the Analysis Setup The data is now ready for Plate Setup GenomeLab Genetic Analysis System User s Guide 301 PN A29142 AB Gene Expression eXpress Analysis Module Setting up a Plate Assign an analysis multiplex and sample names for grouping purposes Log into the eXpress Profiler and click on eXpress Analysis in the Menu Enter your username and password then click OK to log into the software Click on the Plate Setup tab The Plate Setup screen will open jo ee qe Click on Open Analysis The dialog box opens 5 Select the analysis and plate to view NOTE Ifthe analysis includes multiple plates click one plate to view Follow the steps for plate setup and binning for each plate Save this information after each plate is set up Then open the next plate Once all of the plates are completed the analysis can continue to GeXP Analysis Normalization Normalizing Peaks on page 308 Using the Sample Layout 6 Click Sample Layout The Sample Layout tab opens with a sample plate display The name of this specific plate will show at the top of the screen The yellow shaded well
318. le 8 Click Export Data to export the report file The data will be shown on the report view screen 9 Rename the file as desired then browse and save the CSV file to a user designated folder This file can be viewed with Excel To view or hide the data for a particular gene on the Report View select or clear the option next to the gene name NOTE While a gene is cleared to prevent the gene from showing in the reports display an exported report includes all gene information regardless of check box status Column Heading Description Technical Replicates Two or more wells containing reaction products that were independently derived from the same starting material sample Technical replicates demonstrate the variability due to the GeXP process within a sample Expression Value Given for each technical replicate within each sample for each gene within the mulitplex The raw gene expression value peak area is displayed by default If the Display Normalized Data Set option is selected the normalized expression values are displayed The normalized expression value for each gene is calculated by dividing the peak area of a gene fragment by the peak area of the selected reference gene within the same reaction well sample Treatment or tissue type from a particular organism Three or more technical replicates are recommended for each sample Mean Relative The average of all the replicates for a particular sample type Expression stan
319. le e Stop sample plate Stops the currently running sample plate immediately e All Plates Check this box to make the Stop sample plate selection apply to both sample plates e Skip current sample set Skips the currently running sample set e Stop after current sample set completes Stops the run after the currently running sample set has been completed e Save Collected Data Saves data already collected for the currently executing sample set e Perform Shutdown Method Purges the system of any remaining DNA fragments and refills the capillaries with fresh gel after the sample plate has been cancelled The capillary temperature will be set to 35 C 3 Click OK Setting or Changing Display Options To set or change the graph data display options l Open the Display Options dialog box using one of these methods e Select Tools Display Options While viewing a graph display of sequence data right click the graph and select Display Options The Display Options screen opens as shown in Figure 3 26 Colors Dye Traces Current Traces Title 2 Axis Options Y Axis Options Title Property Title I Title Color MI Tithe Font Arial aa M Save Customized Setting Save Setting Now ceca Help Figure 3 26 Display Options Dialog Box 70 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Run Module Using the Run Module 2 Select the tab that identifies the type of dis
320. le 9 3 2 WARNING When performing this procedure use an exhaust ventilation unit that meets TLV requirements Aspirate 40 pL of formamide from the thermo cycling plate Dispense the formamide into a hazardous liquid organic waste container After all wells are clear allow the empty plate to sit in the ventilation unit for 24 hours Dispose of formamide in accordance with all applicable federal state and local environmental regulations concerning hazardous liquid waste Bulk Disposal Table 9 3 3 WARNING When performing this procedure use an exhaust ventilation unit that meets TLV requirements 1 Using a IL side arm flask as a trap connect the trap to a vacuum 2 Attach a pipette to the trap using chemical resistant tubing 3 Aspirate the formamide from each well and dispose of it in a hazardous liquid organic waste container 4 After complete removal of all formamide from the plates allow the empty plate to sit in the ventilation unit for 24 hours 5 Dispose of the plate in a solid waste container Table 9 3 4 WARNING Dispose of formamide in accordance with all applicable federal state 3 4 and local environmental regulations concerning hazardous liquid waste GenomeLab Genetic Analysis System User s Guide PN A29142 AB Maintenance and Diagnostics Biological Waste Disposal Disposal of Buffer Gel Mixture from the Buffer Plate Multi Channel Pipettor Table 9 3 5 WARNING Table 9 3 6 WARNING
321. le dragging the pointer to form a box around the desired area then release the mouse button e To unzoom both traces click the Unzoom All icon The traces in both panes revert to a completely unzoomed state displaying the entire range of the sequence results e To unzoom one of the two open data traces right click in the desired trace pane and select Unzoom Pane The selected trace displays the entire range of the result You cannot edit sequence traces in the Sequence Investigator However you may edit sequence traces using the Sequence Analysis module If you use an edited result for your comparison your edited bases appear in lower case 176 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Investigator Module Sequence Investigator Procedures Viewing Consensus Amino Acid Translations When the system generates the comparison it translates the consensus nucleotide sequence into an amino acid sequence and displays it below the consensus sequence The amino acid translation starts at the first base relative to a reference codon The consensus is also segmented into codons which are translated into their amino acids If edits occur in the consensus a re translation of the consensus amino acid translation occurs automatically and the system automatically updates the discrepancy map and the differences line Each codon is shaded to show its boundaries If a heterozygote occurs the system notes up to two po
322. le menu bar 2 Use the Gel Cartridge Buffer Information dialog box Figure 3 34 to view the following information e Part Number Displays the part number of the installed gel cartridge e Lot Number Displays the lot number of the installed gel cartridge The lot number is used to track the gel lot along with the separations that have used the gel e Gel Name The name of the gel e Date Installed Displays the date the gel cartridge was installed e Time Installed Displays the time the gel cartridge was installed e Hours on Instrument Displays the number of hours that the gel cartridge has remained on the instrument 3 When finished select OK to close the dialog box Gel Cartridge Buffer Information Gel Part Number 38 138 Lat Number SE020E6 Gel Name r Date Installed jover2008 Time Installed mons Haurs an Instrument 50 7 Buffer Part Number e0801 2 m OF Cancel Print Help dide Lat Humber CEG Sequencing Separation Buffer Figure 3 34 Gel Cartridge Buffer Information Dialog Box Viewing or Changing Buffer Information 1 Select Replenish Gel Cartridge Buffer Information from the Run Module menu bar 2 Use the Gel Cartridge Buffer Information dialog box see the figure above to view or change the buffer lot number NOTE The buffer lot number can only be changed when the instrument is not performing a separation 80 GenomeL
323. le users can be selected in the Users field to allow their access to the Analysis Setup and Results The Goal Comments and Property field entries are for ancillary information and are optional They serve to provide user entered information that is specific to the analysis To better represent the information entered into these fields click on the Property text above the field to rename the Property 6 After entering the information for this multiplex analysis click Save IMPORTANT When entering data in the eXpress Analysis module click Save to update the data in the database before going to the next tab or function rA eXpress Analysis Analysis Analysis 123 File Help GexP Analysis Plate Setup m gt j mj ley GexP Analysis Setup Goal Comments GexP Analysis Setup GexP Import GexP Analysis Normalization Sample Name Property 2 Property 3 Property 4 Multiplexes Users Human Reference Rat Multitox Rat Reference Figure 7 27 GeXP Analysis Setup Screen NOTE Do not attempt to delete properties from any of the fields on the GeXP Analysis Setup tab Though the properties can be deleted from the view they will reappear when the application is restarted GenomeLab Genetic Analysis System User s Guide 299 PN A29142 AB Gene Expression eXpress Analysis Module Opening a GeXP Analysis File Work with or modify a previously created analysis file while in eXpress Analysis pose S qe
324. lick on an area between two bases The Insert Base dialog box opens 135 Sequence Analysis Using the Sequence Analysis Module 136 The screen inserts the base as a lower case letter in both the analyzed data and the base sequence panes Inserting Bases in the Base Sequence Pane To insert bases in the base sequence pane 1 Select Tools Edit 2 Place the mouse cursor between the two bases where you want to insert the base 3 Type in the new base The screen inserts the base as a lower case letter in both the analyzed data and the base sequence panes Changing Bases in the Analyzed Data Pane To change bases in the analyzed data pane 1 Select Tools Edit 2 Click on a base in the analyzed data pane The Change Base dialog box opens 3 Select the desired base 4 Click OK The screen shows the changed base as a lower case letter in both the analyzed data and the base sequence panes Changing Bases in the Base Sequence Pane To change bases in the base sequence pane l Select Tools Edit 2 Highlight the bases to change in the base sequence text 3 Type in the new bases The screen shows the changed base as a lower case letter in both the analyzed data and the base sequence panes NOTE The left and right arrow keys move the cursor through the data in the base sequence pane To select bases hold the Shift key down while using the left and right arrow keys to select the desired bases Deleting Bases in the
325. list to associate it with the selected wells When the multiplex is selected the wells will display numerical values These values represent the number of genes in the selected multiplex and aid as a visual cue that the multiplex has been assigned to the well in case a well was missed y 7eXpress Analysis Plate Plate 1 File Help GeXP Analysis Plate Setup GexP Analysis Normalization B m E Sample Layout Well Specific Settings Analysis GexP_Analysis 1 Y a Multiplex Sample Name Cal Data Files JA 220009098 L d E J I cT 0mnmmooUuoofm Peak Binning Figure 7 31 Multiplex Selection of Multiple wells 9 Select each well individually to view the sample name information that was assigned to each well in the Plate Setup module of the GeXP Genetic Analysis System The sample name can be changed to another sample name in the same analysis by selecting the alternative name from the Sample Name drop down list NOTE The feature of associating selected wells to an analysis is important when technical replicates of the same sample are given different Sample Names Unless the replicates are all given the same name each well will be treated as an individual sample For example Sample 1 1 Sample 1 2 Sample 1 3 and Sample 1 4 are four technical replicates of the same sample Sample 1 To combine these replicates select each well that contains Sample 1 2 Sample 1 3 and Sample 1 4 and con
326. llary Array on page 81 GenomeLab Genetic Analysis System User s Guide 93 PN A29142 AB Run Module Using Direct Control 94 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Analysis Sequence Analysis Module Overview sequence Analysis This chapter provides an overview of the Sequence Analysis module including its menu options toolbars and dialog boxes It shows you how to use this module to run a sequence analysis and to set up its parameters result properties and display options It also describes how to work with report and export sequence analysis results 4 1 Sequence Analysis Module Overview View analyze compare edit and print data of the following types Raw Data Current Data Voltage Data Analyzed Data Base Sequences Optical Scan Data Baseline Data Quality Parameters These types of data cannot be edited This module accepts raw data and analyzed data Editing and re analysis functions enable you to verify the accuracy of the base calls You can also export these data to third party packages for further analysis Main Window The following illustration identifies the areas on the Sequence Analysis module main window that are described in Table 4 1 GenomeLab Genetic Analysis System User s Guide 95 PN A29142 AB Sequence Analysis Sequence Analysis Module Overview A B 4 Sequence Analysis Sequencing A01_06037313BS File Edit View Tools Analysis Window He
327. lp BE E e elle x Orley Qlals se E A M C GL 53 e Sequencing A01 06042415BP acer 2191 87 3 87 Cel F BP kV se l GGC CAG TGC CAA GCT TGC ATG 76 TGT CAT AGC TGT TTC CTG TOGT lal 151 GTA AAG CCT GGG GTG CCT AAT AGC TGC G CTG ACT 7 8 9 10 11 12 E E E EN gt Plate U33 001 Test 030106_0603012 Sample Result CAG AAT TEC Befresh Export Help GC 76 131 226 301 376 451 TCA AAA AAC GcT ATA CGT TAG GCC CTG TCC CGG AAA CTG TGG TCG TCG TTA AAG TIT GGT TGC CTC TEG GEC CCT GCC CAG ACT ACA GCG For Help press F1 CTG GAC GAA TTE CTG iTi Sequencing A01_06032313BS IDE x GTC ATT TGA AGG GTT GCT GAT TTC TTG GEG CAC CGT CGG ACA TGC GTA ACA GET TCG GCA TEG Figure 4 1 Main Window Sequence Analysis Module The following table describes the items called out in Figure 4 1 Table 4 1 Main Window Sequence Analysis Module Item m ocoi c u23 a Description Title Bar Shows the module name Sequence Analysis and the currently active sample Menu Bar See Menu Bar Options on page 97 Toolbar See Toolbar Icons on page 106 Display Area Graphically displays the opened data Double clicking on a base sequence text letter A C G T in the Dye Colors toolbar opens the Color dialog box which you can use to change the dye color displayed for the
328. lso generated FASTA QUAL which lists the quality value for each base Quality values correspond to 10 log10 error rate The range for the quality values is 1 to 99 Edited and inserted bases have a value of 99 You would use this format for instance to export data into PHRAP Selecting the FASTA format exports the Result Output Base Sequence and the Quality Parameters automatically if the sample has been analyzed with a sequence analysis parameter set If the sample has been analyzed with a fragment analysis parameter set nothing is exported NOTE For instance in PHRED a quality value of 10 means 1 error in 10 A quality value of 20 means an error rate of 1 error in 100 A quality value of 30 means an error rate of 1 error in 1 000 A quality value of 40 means an error rate of 1 error in 10 000 etc Normally PHRED would export these quality values into PHRAP In the case of exporting from the CE system we bypass PHRED and export directly into PHRAP The PHRED format produces two files a PHRED file and an SCF file A PHRED file is a text file composed of a SEQUENCE section containing a COMMENT and DNA section The SEQUENCE section also contains the name of the Analyzed Result that was stored The COMMENT section contains the file name of the associated SCF file CHROMAT FILE that was produced along with the PHRED file The values for ABI THUMBPRINT and PHRED VERSION are fixed text N A The values for CALL METHOD and QUALITY
329. lt Untitled gt a x Qu File Edit View Window Help 81 x osal e tale Axela aj fer Terz alee enel E UA BCHGNT NNE P cameras ADDS Geant Codon Number 5 76 77 78 79 80 81 82 83 84 5 86 87 88 89 90 391 KZ Mismatch 1 Reference AA Translation N L L T A P T Q F S E L W L W D Iv Ambiguity 7 VII VIIIF 12 2 txt GG AAT CTC CTT ACT GCT CCC ACT CAG TTT CT TTT CTC TGG CTT TGG GA 15 16 612 06042419BG GG AAT CTC CTT ACT GCT CCC ACT CAG TTT amp CT TTT CTC TGG CTT TGG GA 15 16b G01 06042419BB GG AAT CTC CTT ACT GCT CCC ACT CAG TTT CT TTT CTC TGG CTT TGG GA Differences Consensus GG AAT CTC CTT ACT GCT CCC ACT CAG TTT CT TTT CTC TGG CTT TGG GA Consensus AA Translation G I 5 L L L P L S F L F 5 G F G T 4 gt C Consensus Text Search n zl a Exact Match 15 16 612_06042419BG Reverse meum r Consensus 34 Called Base C Quality Value fi 3 3500 8250 2500 2550 8000 S050 5100 8150 5200 8250 8500 5550 Differences B lt Cats Points Help 4 a 2 15 16b G01_06042419BB Reverse Rucrescerce Rucrescence S700 S750 S800 5850 5500 55 Mismatch Ambiguity Disagreement Insertion Deletion Mutation Hotspot Low Quality Manual E dit e Figure 5 1 Main Window Sequence Investigator Module The following table describes the items called out in Figure 5 1 Table 5 1 Main Window Sequence Investigator Module item Description A Title Bar Shows
330. ly represents the equipment main power push button switch when it is in the on position GenomeLab Genetic Analysis System User s Guide Safety Information Chemical and Biological Safety WARNING Normal operation of the system can involve the use of solvents and reagents that are toxic flammable or biologically harmful WARNING e Observe all precautionary information printed on the original solution containers Operate the system in the appropriate environment Take all necessary precautions when using pathology or toxic materials to prevent the generation of aerosols Observe all applicable precautionary procedures when using flammable solvents in or near the instrument Wear appropriate laboratory attire for example safety glasses gloves lab coat and breathing apparatus when working with hazardous materials Dispose of all waste solutions in a proper manner Electrical Safety To reduce risk of electrical shock all devices employ a three wire electrical cable and plug to connect the equipment to earth ground e Ensure that the wall outlet receptacle is properly wired and earth grounded DO NOT use a three to two wire plug adapter DO NOT use a two wire extension cord or a two wire multiple outlet power strip e Disconnect power to the system before performing maintenance DO NOT remove any panels panels should be removed only by qualified service personnel WARNING A high voltage power supply is used with
331. m a visual representation of the sample plate l Select the Plate View tab in the Select Raw Data window Steps Raw Data Analysis Parameters Project AFLP 06 TP2 20 Analyze Data Result Data 123452678 910 11 12 A B C D E F G H Figure 6 8 Selecting Raw Data from the Plate View Tab Plate View Raw Data Legend o No Data Available o Selected For Analysis e Data Available e Selected For Re Analysis eo Data Already Analyzed Selected Wells lt lt Previous L Ne Einish Cancel GenomeLab Genetic Analysis System User s Guide PN A29142 AB Fragment Analysis Module Working with Studies in the Fragment Analysis Module 2 Select the project folder that contains the sample data that you want to include in the Study from the Project drop down list NOTE If the desired project is not listed it may be located in a different database To enter a different database you must close all applications and set the working database See Setting the Working Database on page 328 or Set Working Database in the online help for more information 3 Select the plate from the Plates drop down list A visual representation of the plate appears in the Current Plate area of the window e To select the samples for your Study click on the well location of the sample of interest The sample ID moves to the Current Plate Selected Wells area of the window indicating that the sample will be included in the Study
332. m asks if you want to clear the existing fragment list Choose one of the following Click No if you want to keep the list but add or remove peaks from it Click Yes if you want to discard the system generated list and start over Exporting a Fragment List to CSV File You can export the fragment list to other third party spreadsheet programs To export the fragment list l Right click the selected samples from any of the data or fragment lists 2 Select Export to CSV File The Export window opens 232 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Fragment Analysis Module Performing Bin Analysis 3 Select a name and location to save the file NOTE See Exporting Fragment Lists or Genotypes from a Study on page 259 for more information regarding data export Showing a Stacked Graph The Stacked Graph function aligns individual selected traces by size or time Zooming and scrolling on the x axis is synchronous between all plots selected while the y axis of each plot selected is independently scaled based on the available data To access this function right click the selected samples in the fragment list and select the Show Stacked Graph Including Excluding amp Resetting Peaks in the Fragment List You can individually include or exclude peaks from the existing fragment list e To include a peak in the fragment list right click the selected fragments and select the Include Peak e To exclude a peak i
333. mation then install the array GenomeLab Genetic Analysis System User s Guide PN A29142 AB Run Module Run Module Overview Table 3 8 Replenish Menu Option Release Capillary Array Gel Cartridge Buffer Information Install Gel Cartridge Release Gel Cartridge Replace Wetting Tray Empty Waste Bottle Toolbar Icons Description Displays the first in a series capillary array dialog boxes that let you remove the currently installed capillary array For details see Removing and Replacing the Capillary Array on page 361 Opens the Gel Cartridge Buffer Information dialog box which provides information concerning the gel cartridge and buffer For details see Viewing Gel Information on page 360 Opens the Install Gel Cartridge dialog box Use this option to remove the gel cartridge or if you are not going to install a working gel cartridge to insert a clean empty cartridge or gel pump plug into the cartridge chamber For details see Removing and Replacing a Gel Cartridge Gel Pump Plug on page 367 Displays the first in a series of dialog boxes that let you replace the wetting tray For details see Replacing the Wetting Tray on page 350 Displays the first in a series of dialog boxes that let you empty the waste bottle For details see Replacing the Gel Waste Bottle on page 349 The Run module provides six toolbars Standard Direct Control Data Monitor Log Sequence Dye Colors and Fragment
334. ment Result Set Traces area right click the reference peak and select Peak Reference to remove the check mark GenomeLab Genetic Analysis System User s Guide 241 PN A29142 AB Fragment Analysis Module Performing AFLP Analysis Selecting a Test Peak A test peak is a trace peak that you select from the reference result trace The system compares the test peak to the reference peak calculates a relative height and area then adds the results to the Peak Ratio Results Table e To select the test peak right click an individual peak from the selected Result Set Trace located at the top of the Result Set Traces area of the window and select Test Peak The system calculates the area and height by comparing it with the reference peak then enters this information into the Peak Ratio Result Table It uses each selected test peak to calculate ratios with the corresponding reference peak of the same trace e Select test peaks in the same manner until all desired peaks are identified e To de select a test peak from the Reference Result Peaks table right click the test peak and select Set Peak As Test to toggle the command to the off position unchecked To de select a test peak from the Fragment Result Set Traces area right click the test peak and select Peak Test to remove the check mark 6 10 Performing AFLP Analysis 242 Amplified Fragment Length Polymorphism AFLP Analysis is a highly sensitive DNA fingerprinting techni
335. mple elements are automatically selected in the Export dialog box such that all of the associated information listed above will be exported This format essentially takes a snapshot of the file and can retain all of the information belonging to that file This format can only be used when importing data into the Data Manager This format can also be used when exporting data from the Sequence and the Fragment Analysis modules and the Data Manager When exporting as CE system data the various data types are exported to files with the following extensions Sequence Analysis ParameterCQA Sequence InvestigatorCQC Fragment Size StandardCQD Fragment Analysis ParameterCQF Galvo Scan DataCQG Fragment Locus TagsCQL MethodsCQM SNP Locus TagsCQN Sequence ResultCQR Sample DataCQS Sample TableCQT Fragment ResultCQU Sample Table ResultCQX You may automatically remove the plate position coordinates A01 B02 etc and the time date stamp from the sample name which is normally generated by the CE system software on export To do so select the Remove CEQ Tracking Suffix check box v If the system encounters the same sample name at the destination it usually overwrites the name if you choose to remove the plate position coordinates and the time date stamp which ensures a unique name If you select the Resolve Filename Conflicts check box L the system increments the sample names on ex
336. n a first pass multiplex from accession numbers of target and reference genes Maximum PCR Product Size excluding Universal Tags Minimum PCR Product Size excluding Universal Tags p Load Setting G amp D Execut Save Setting Reset Form Multiplex Name TestPlex 1 Accession Numbers NM 002916 NM 020386 AI741117 A1817737 NM 000436 NM 004994 NM 015984 F073519 NM 005915 NM 006931 NM 003875 NM 014321 NM 014791 300 100 Default Tags r AFO52162 NM 016577 NM 000788 NM 003607 Figure 7 13 eXpress Designer eXpress View Table 7 7 eXpress Designer Module Parameters Parameter Multiplex Name Minimum Separation Size Maximum PCR Product Size excluding Universal Tags Minimum PCR Product Size excluding Universal Tags Universal Tags Verbose Mode GenomeLab Genetic Analysis System User s Guide Description The identifier of the multiplex recognized by eXpress Profiler The multiplex name must be unique or else it cannot be saved to the database When the TDF file is opened in Excel the multiplex name is found in cell A2 Set the minimum base pair separation size between PCR products The default is 7 bp Optimal separation size is 5 to 7 bp NOTE The minimum separation size for multiplex design is 3 base pairs set the maximum PCR product size excluding Universal Tags This parameter automatically adjusts as needed to fit as many products in
337. n below Sequence Analysis Parameters Editor DefaultSequenceAnalysisPar EA Quality based Trimming Sequence based Trimming Alignment Reference General Initial Data Detection Heterozygote Detection M Threshold x PCR Praduct 20 Bases S duss sto na Ex un Use Default Mobility Calib Analysis Stop 0 0 E min Pre peak Reduction Color DefauitColorCalbration Heterozygote Detection No Quality based Trimming None Sequence based Trimming Ma Automatic Alignment Ma Save As Print Cancel Help Figure 4 10 Sequence Analysis Parameters Editor Dialog Box 3 Use the tabs on this dialog box to make appropriate changes For parameter descriptions see the following topics e General Tab on page 116 Initial Data Detection Tab on page 118 Heterozygote Detection Tab on page 119 Alignment Reference Tab Sequence Analysis Parameters Editor on page 121 e Quality based Trimming Tab on page 124 Using Sequence based Trimming on page 125 4 Click Save As to create a new Sequence Analysis Parameter name or click OK to save the changes and exit the editor General Tab Use the General tab of the Sequence Analysis Parameters Editor dialog box to specify the parameters used to perform the basecalling operation on the desired samples whether or not you are performing an alignment NOTE If you opened this dialog box by selecting View Parameters Used to Compute Sequence this dialo
338. n junction or a single base polymorphism Enter single or multiple regions to be excluded from the gene sequence for the purpose of primer design This is useful for excluding regions that contain repetitive elements polymorphisms or a low quality sequence from being used as primers 289 Gene Expression eXpress Designer Module Creating a New Primer 1 Log into eXpress Designer 2 Click Primer Design The Primer Design screen will open eXpress Designer Primer Design Create New Data View Existing Data Review Current Run eXpress Save to Database FASTA Blast Primer Design Multiplex Primer Pair 1 Primer Pair 2 Name Name AFOO1898 203 186 20 388 20 AFO01898 202 500 20 701 20 Length without tags Length without tags Account 203 202 out Fasta Fasta gt gi Z183216 gb AFO01898 1 AF001898 Rattus gt gi Z183216 gb AFO01898 1 AF001898 Rattus Help norvegicus aldehyde dehydrogenase ALDH mRNA norvegicus aldehyde dehydrogenase ALDH mRNA complete cds complete cds Xpress Analysis Left Primer Left Primer Sequence Sequence eXpress Map JAGAAGGCGGACAAGGCAGAT GAACCTATTGGGGTGTGTGG Primer start length Primer start length 186 20 500 20 Melting Tm Melting Tm 60 074 60 088 Right Primer Right Primer Sequence Sequence CARGTACGCATTGGCAAAGA AA CCAGGGACAATGTTCACC Primer start length Primer start length 388 20 701 20 Melting Tm Melting Tm 59 872 59 679 Figure 7 20 eXpress Designer Primer Desig
339. n screen Main View 3 Click and select a gene from the Genes list Modify the primer design parameters or leave the default settings 5 Set the number of primer sets that are returned by entering a number in the Number to Return field 6 Click Execute 290 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Gene Expression eXpress Designer Module Figure 7 21 shows an example of the Primer Design results eXpress Designer Primer Design Create New Data View Existing Data Review Current Run eXpress Save to Database FASTA Blast Primer Design Multiplex Primer Pair 1 Primer Pair 2 Hame Hame NM 017000 180 1259 20 1438 20 NM 017000 170 351 20 520 20 Length without tags Length without tags Account 180 170 TAM Fasta Fasta gi l27501443 ref NM 017000 2 Rattus norvegicus gi l27501443 ref NM 017000 2 Rattus norvegicus Help NAD P H dehydrogenase quinone 1 Nqol mRNA NAD P H dehydrogenase quinone 1 Nqol mRNA Left Primer Left Primer Sequence Sequence CAAACAATGGCTGAGGGACT GAAGCTGCAGACCTGGTGAT eXpress Map Primer start length Primer start length 1252 20 351 20 Melting Tm Melting Tm GHI ITT 60 418 eXpress Analysis Right Primer Right Primer Sequence Sequence GGCGGAGACCTGTTTTTGTA GTGGTGATGGAAAGCAAGGT Primer start length Primer start length 1438 20 520 20 Melting Tm Melting Tm 60 110 59 973 Figure 7 21 eXpress Designer Primer Design Results 7 Click Save to D
340. n the Fragment Analysis Parameters window This tab contains general parameter set information and peak identification properties Parameter Set Information Parameter Set information depends on whether you are creating a new parameter set or editing an existing parameter set e If you are creating a new parameter set enter a descriptive name in the Parameter Set Name field and select the project where you want to save the parameter set e If you are editing an existing parameter set you may choose to save the edited parameters under a different name To do this click Save As and enter the new name in the Parameter Set Name field You may also choose to save the edited parameters under the original name The default set of fragment analysis parameters cannot be edited The General tab s Parameter Set area shows the date and time the parameter set was created and modified Peak Criteria You can edit the following Peak Criteria fields e Slope threshold Specify the minimum rate of the signal increase on the leading edge of peaks in order for a peak standard or unknown to be detected e Enter a value between 1 1000 The default value is 10 e The slope is proportional to the height of the peak and depends on the width of the peak You can set this parameter to detect peaks of interest while eliminating smaller or broader peaks that may not be of interest e The slope threshold is set relative to the baseline noise as the baseli
341. n the Peak Ratio Result Table Based on the reference and test peaks selected the system calculates the Mean the of Data Points and the Standard Deviation GenomeLab Genetic Analysis System User s Guide PN A29142 AB Fragment Analysis Module Calculating Peak Ratios Selecting a Reference Trace A reference trace is a user selected trace assigned as a reference by the system for comparative analysis of peak height and area You can select the reference trace from either the Peak Ratio Result Table or from the Fragment Result Set Traces areas of the window To select the reference trace from the Peak Ratio Result Table right click your selection and select Set Result As Reference The system highlights the result trace with a dark background and moves the trace to the top of the Result Set Traces area of the window to distinguish it as the reference result trace It marks the fragment result with an R in the left most column to distinguish it as the reference trace e To de select the reference result right click the reference result and select Set Result As Reference again to toggle the command to the off position unchecked The system removes the R from the de selected trace and returns it to its original highlight status and position in the Fragment Result Set Traces area of the window To select the reference peak from the Result Set Traces area right click your selection and select Reference The system highlights
342. n the fragment list right click the selected fragments and select the Exclude Peak An updated stacked trace view from which you can select the peaks of interest l Left click the peak of interest to select it 2 Right click to display the peak menu e To display all fragments excluded from the fragment list select Show Excluded Fragments e To reset peaks modified in the fragment list to their original configuration select Reset Peak 6 8 Performing Bin Analysis Binning is a one dimensional clustering by size of fragments compiled from multiple samples You can use bin analysis to generate an allele list that represents the population you are studying For example you might want to use this to establish a database of information on alleles and possible anomalies associated with them such as imperfect repeats Binning of alleles from multiple samples allow you to create a new allele list or update an existing allele list for a locus tag based on the collection of fragment results During bin analysis the system groups fragments by size to form bins characterized by an average apparent size and standard deviation of the fragments in the bin Setting up a Bin Analysis Bin analysis requires an exiting result set either from a previously saved Study or a new Study created exclusively for this purpose To set up a bin analysis l Open the result set you want to analyze 2 Select Analysis New Binning Analysis
343. nalysis Parameters Quantitation STR Locus Tags SNP Locus Tags size Calibration Dye Matrices General Notes Property Set Method Consumables GenomeLab Genetic Analysis System User s Guide PN A29142 AB Description Displays the list of messages generated by the primary analysis program during the analysis of the selected result The analysis log contains information that can help determine the cause of an analysis failure Displays the list of messages generated by the instrument during the pre separation and separation events of the eight samples that include the individual selected result Use the Run Log to troubleshoot the data in the case of a run failure Displays a summary of the fragment analysis parameters applied to the raw data in the selected result Displays a summary of the peak information used to estimate fragment amounts within the selected result Displays a list of STR locus tags that have been applied to the raw data in the selected result Displays a list of SNP locus tags that have been applied to the raw data in the selected result Displays a description of the size standards and calibration curve used to calculate the sizes of the unknown fragments in the selected result Displays the dye spectra and dye mobility calibration parameters used in generating the selected result Displays a list of system and user generated notes specific to the selected result for example sample name dates d
344. nd are therefore labeled as spurious peaks The Spurious Peak Detection area allows you to direct the system to look for spurious peaks in your analysis You can specify a height threshold under which the system identifies a peak as a contaminant and not include it in your allele list To enable this option 1 Select Detect spurious peaks if you want the system to remove any peaks that are below the maximum height threshold for spurious peaks 2 Inthe Maximum height for spurious peak field enter the percent of the spurious peak relative to the true allele below which detected peaks are considered spurious peaks A Detection A Detection is an allele calling criteria used to compensate for a common DNA polymerase phenomenon of adding a non template directed deoxyadenosine to the ends of PCR products The result is a mixture of PCR products Some of which are template true length called A products while others are one nucleotide longer called A products The ratio of these two types of products can vary greatly between different reactions and primers The result of having different fragment sizes from a single allele complicates the interpretation of fragment analysis data Instead of one peak for one allele there may be two that represent the same allele spaced one nucleotide apart The A Detection area of the window allows you to specify how the system will differentiate between the true allele and the
345. nd dialog boxes It also shows you how to run sample plates and set the display options using this module Sequence Analysis provides an overview of the module including its menu options toolbars and dialog boxes It shows you how to use this module to run a sequence analysis and to set up its parameters result properties and display options It also describes how to work with report and export sequence analysis results Sequence Investigator Module provides an overview of the module including its menu options toolbars and dialog boxes It also shows you how to use this module to investigate and compare sequence analysis results Fragment Analysis Module provides an overview of the module including its menu options toolbars and dialog boxes It shows you how to use this module to work with Studies to run a fragment analysis and to set up its parameters result properties and display options It also describes how to work with report and export fragment analysis results Gene Expression describes how the use the GenomeLab GeXP Genetic Analysis System This includes an overview of the system and describes how to use the GeXP applications that run independently from the GenomeLab Genetic Analysis System It also explains how to back up and restore files used in the GeXP applications Database Management provides an overview of the module including its menu options toolbars and dialog boxes It provides proce
346. nd lift it to the vertical locking position Loading the Sample Plate 1 Make sure the Wetting Station is installed or perform the following procedure Installing the Wetting Tray on page 351 and then return here 2 Align the Sample Plate Guide Pin with the notched corner of the sample plate and gently lower the plate into position Figure 3 30 74 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Run Module Using Direct Control 901600L AI Figure 3 30 Loading the Sample Plate 1 Sample Plate 4 Sample Plate Notched Edge 2 Wetting Tray Retainers 5 ALIGN together 3 Sample Plate Holder 6 Sample Plate Guide Pin Loading the Buffer Plate and Evaporation Cover l Align the notched corner of the Buffer Plate with the alignment line on the Buffer Plate Holder Figure 3 31 2 Gently push the Buffer Plate towards the front of the instrument and then set the plate into the transport 3 At the rear of the Buffer Plate Holder align the Buffer Evaporation Cover Guide Pin with the Buffer Evaporation Cover Alignment Notch and then gently lower the cover over the Buffer Plate GenomeLab Genetic Analysis System User s Guide 15 PN A29142 AB Run Module Using Direct Control 901599L Al Figure 3 31 Loading the Buffer Plate and Buffer Evaporation Cover 1 Front Location 4 Buffer Evaporation Cover Alignment Notch 2 Buffer Evaporation Cover 5 Wetting Tray Retainers 3 Buffer Plate When finished pos
347. ne noise varies for each dye trace Although you can define only one slope threshold value the system adjusts for each dye depending on the noise detected for that dye trace e Relative peak height threshold Set a minimum height criterion that unknown peaks must meet before being displayed in the fragment list or annotated in an electropherogram e Enter a value between 0 10096 The default value is 196 The unknown peaks above this set value 96 relative to the second highest peak in each dye trace will be displayed in the fragment list and the sizes will be displayed in the electropherogram e This value does not apply to standards e To size the peaks only clear the Allele Identification check boxes Allele Identification The Allele Identification check boxes let you add either of the following parameters e Identify STR Alleles Select this option MI to include allele identification as specified in the allele lists of the selected Short Tandem Repeat STR locus tags GenomeLab Genetic Analysis System User s Guide 201 PN A29142 AB Fragment Analysis Module Defining Fragment Analysis Parameters e SNP Analysis Select this option M to include Single Nucleotide Polymorphism SNP analysis in the parameter set NOTE You may include only one of these options in the same analysis Size Estimation and Allele ID Confidence Confidence level Set this level as required for your analysis The confidence level default v
348. ng example Exit Edit Menu Edit Cut Ckri x Copy Cri c Paste Er He Find Ctrl F Find MexE F3 Select All Ckrl A Use the Edit menu to cut copy paste and find items in the database Table 8 3 Edit Menu Data Manager Module Option Cut Copy Paste Find Find Next Select All Description Copies the selected item to the clipboard and removes it from its current location Copies the selected item to the clipboard without removing it from its current location Inserts one or more copied or cut items from the clipboard to other projects or databases Opens a Find dialog box which you can use to locate a specific item project or database Searches for the next occurrence of the item or folder defined in the Find dialog box Selects all items under the selected item GenomeLab Genetic Analysis System User s Guide PN A29142 AB Database Management Data Manager Module Overview View Menu Click the View menu to display its drop down menu as shown in the following example View w Toolbar wv Status Bar Refresh Filter Bv Database Sample Rum History NOTE A Y next to an option indicates that the option is enabled Use the View menu to toggle the toolbar and status bar on and off or to manage the database view Table 8 4 View Menu Data Manager Module Option Description Toolbar Toggles between displaying or not displaying the toolbar Status Bar Toggles bet
349. ng of 100 bp as the lowest size to avoid the loss of sizing of smaller products set in the software by default WARNING Do not change the default setting It will affect the chemistry 293 Gene Expression Multiplex Designer 294 Parameter Description Verbose Mode Use this option for troubleshooting multiplex design such as when a gene is excluded from a multiplex Selecting this option provides more detailed information regarding all available PCR products for each gene Reference Primers Allows multiuse primers to be included in the multiplex e g KAN or housekeeping genes Click to highlight and include this primer set in a multiplex NOTE New reference primers can only be added in the Administrator mode See Managing Primers on page 274 for more information Load Multiplex Select previously saved multiplex parameter settings from the drop down list Timeout in minutes Adjust the multiplexing algorithm run time The default setting is 5 If the multiplex has more fixed points for example previously created primers the software will take a longer time to bin the products together Enter 10 or more minutes if this is the case NOTE This is not an option in the eXpress function Genes The list of accession numbers available for use in a multiplex Primers The list of primers available for use in a multiplex Creating a Multiplex with Genes and Primers l Log into eXpress Designer 2 Click Multiplex 3 Enter
350. ng unloading plates B Release Gel Cartridge Removes the gel cartridge See Removing and Replacing a Gel Cartridge Gel Pump Plug on page 367 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Run Module Run Module Overview Figure 3 4 Direct Control Toolbar Run Module Icon Description Install Gel Cartridge Installs a gel cartridge m Release Capillary Array Removes the current capillary array See pa Removing and Replacing the Capillary Array on page 361 Install Capillary Array Installs the capillary array Replace Wetting Station Replaces the wetting tray See Replacing the Wetting Tray on page 350 4 jen Data Monitor Toolbar The following example shows the Sample Setup module Data Monitor toolbar For descriptions of the actions that occur when you click a toolbar icon see Table 3 10 a ss Figure 3 5 Data Monitor Toolbar Run Module Table 3 10 Data Monitor Toolbar Run Module Icon Description Unzoom Undoes one zoom level Unzoom All Undoes all zoom levels Autoscroll Scale all data to the last 10 minutes of the run on the X axis and confine the display of data to the pane on the Y axis Autoscale On Off By default data displayed on the screen is scaled automatically to the confines of the screen autoscale is on If autoscaling is disabled the data is scaled to its true values Pause Data Update Pause the display of data Pausing data doe
351. nning range and highlight the corresponding peak in the Peaks Graph In Figure 7 33 notice that gne M12516 with an expected size of 196 0 nt has been selected in the Binning Table 18 x PAS ier Analysis File Help GeXP Analysis Plate Setup GeXP Analysis Normalization Sample Layout w Peak Binning 80 000 70 000 P 60 000 e a k 50 000 r Ommoouomx H Peak Binning e 40 000 i B 30 000 t 20 000 Designed Gene Peak Detected 10 000 Non Designed Peak NENNNEN Designed Gene Peak Not Detected BEE Highlighted Peak Gene Expected Size Actual Size unmatched N 194 769 M12516 196 0 195 775 unmatched N 227 51 unmatched N 228 5061 Junmatched N 237 8324 U24174 239 0 238 8756 567722 2570 257 2817 Binning Range Height 884 1208 9276 697 2345 447 23154 03 1701 153 17209 56 8905 839 Area 405 8773 4480 658 1072 985 fi 1283 12 795 4359 8635 206 4443 652 NM 017101 307 0 306 0833 2694 031 1317673 NM 017101 307 0 307 1127 40620 3 20482 48 INM 031144 3140 3131245 3274 492 1558 01 Figure 7 33 Peak Binning Showing the Range for a Selected Peak Reading the Peak Binning Graph The goal of peak binning is to align all of the expected designed peaks with actual peaks found in the data The peak binning graph replicates the electropherogram data from the GeXP System The TDF file created during multiplex is assigned to the Analysis
352. normalized gene expression data l Loginto the eXpress Profiler and click eXpress Analysis in the Menu 2 Enter your username and password then click OK to log into the software The eXpress Analysis application opens as shown below y eXpress Analysis LL IBEX File Help Roamer GexP Analysis Normalization Plate Setup Figure 7 26 eXpress Analysis Desktop Table 7 10 eXpress Analysis Tabs Option Function GeXP Analysis Set up a new analysis or open a previously created analysis Import fragment data Plate Setup Connect Fragment Analysis data with existing multiplex information in the database GeXP Analysis Normalization Normalize peak data to a reference gene Export the results 298 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Gene Expression eXpress Analysis Module Creating a New Analysis Create a new analysis for gene expression Click on the GeXP Analysis tab Click New Analysis on the GeXP Analysis tab Name the analysis in the Analysis Name field and click OK pu qe qucm In the Multiplex list select and highlight a multiplex NOTE Ifthe multiplex of interest is not listed the eXpress Profiler Administrator must log in and import the missing multiplex TDF file See Importing a Multiplex on page 276 for more information 5 Inthe Users list select and highlight a user name IMPORTANT A user MUST be selected with a multiplex for Analysis to function properly NOTE Multip
353. nostics in the Run module The Diagnostics dialog box opens Click the PC Settings button The PC Communication Settings dialog box opens View the settings the system uses to communicate with the instrument then exit the dialog box 382 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Maintenance and Diagnostics Diagnostics Viewing Instrument Status To view the instrument status l Select Run Diagnostics in the Run module The Diagnostics dialog box opens 2 Click Status 3 View and or change the settings in the Run Control Monitor dialog box 4 Click OK Viewing Optical Scan Data To view the optical scan data in either the Sequence Analysis or Fragment Analysis module l Select File Open from the desired analysis module The Open dialog box opens 2 Select the Optical Scan Data tab highlight the desired sample name then click OK 3 Verify that the Optical Scan window is displayed Monitoring the Baseline To ensure that the optics are working correctly and that the capillaries are clean view the baseline after installing a new capillary array filling with gel for the first time or performing an optical alignment The baseline trace should be low and relatively flat NOTE You must perform the optical alignment procedures prior to monitoring the baseline Performing an Optical Alignment on page 359 To monitor the baseline 1 Select Run Monitor Baseline The Monitor Baseline dialog box
354. nstruct a size calibration GenomeLab Genetic Analysis System User s Guide PN A29142 AB Fragment Analysis Module Defining Fragment Analysis Parameters Quantitation Tab The Quantitation tab allows you to set the parameters for calculating the quantity of all peaks by comparing their heights or areas to a single peak of the same color whose quantity is known Edit Fragment Analysis Parameters D x Parameter Set Mame FAO Project Default Analysis Method STA Locus Tags SNE Locus Tags ID Standard Calculate Using Size Height v Time Area Quantitation V alues Time Window Amount mn omo Eo 4 mi 0 00 ep 4 pi n pa n pa fo min nn eo wap min foo ep i Save As Cancel Figure 6 13 Fragment Analysis Parameters Window Quantitation Tab Once you have entered the information for the known peak into this window the system looks for the reference peak in each trace using the specified size or time Based on this information the system can then calculate the quantity of each of the remaining peaks in the sample ID Standard In the ID Standard area of the window select one of the following options as the method the system uses to identify your reference peak Size Select this option to identify your reference peak based on the fragment size specified in the Size field listed for each of the four dyes Time Select this option to identify your reference peak based on
355. nt must be performed before enabling the Monitor Baseline function This will ensure accurate determination of the system baseline l Perform an optical alignment 2 Select Run Monitor Baseline The dialog will open Monitor Baseline x Enable Monitor Baseline LE Baseline Parameters Cancel Autosave v View Injection Instrument Data Help Mame BASELINE Project Default m dii Figure 3 32 Monitor Baseline Dialog Box 3 In the Monitor Baseline dialog select Enable Monitor Baseline then click OK 4 Select the Data Monitor tab to view the baseline trace After reviewing the baseline trace disable Monitor Baseline To do this 1 Select Run Monitor Baseline 2 Inthe Monitor Baseline dialog select the Enable Monitor Baseline option then click OK GenomeLab Genetic Analysis System User s Guide PN A29142 AB Run Module Using Direct Control Viewing Capillary Information l Select Replenish Capillary Information from the Run module menu bar 2 Use the Capillary Information dialog box Figure 3 33 to view the following information Capillary Part Number pngngz Serial Humber Dmt050128 Date Installed 0472772006 Time Installed f T AT Number of Runs EE Cancel Frint Advanced Help iket Days on instrument f 3 1 Figure 3 33 Capillary Information Dialog 3 View the following information Part Number Displays th
356. ntenance and Diagnostics Direct Control and Replenishment e Time Installed Displays the time the gel cartridge was installed e Hours on Instrument Displays the number of hours that the gel cartridge has remained on the instrument 3 When finished select OK to close the window Viewing or Changing Buffer Information 1 Select Replenish Gel Cartridge Buffer Information from the Run Module menu bar 2 Use the Gel Cartridge Buffer Information dialog box see the figure above to view or change the buffer lot number NOTE The buffer lot number can only be changed when the instrument is not performing a separation Removing and Replacing the Capillary Array CAUTION Take care when installing removing the capillary array to prevent damage and maintain low background signals Clean all gel residues thoroughly NOTE This procedure assumes that an expended capillary array is being replaced with a new capillary array 1 Select Replenish Release Capillary Array After the system prepares for release of the capillary array the Remove Capillary Array dialog box opens Remove Capillary Array Ea Tou may now open sample capillaries access doors to change capillaries To replace the capillary array immediately select Replace capillary array To install a manifold plug select Install Cancel manifold plug Help To clean the capillary windows select Clean capilares Remove Capillary Array Options Repl
357. number as the value a ratio must meet before the system considers the ratio a variant e Error Select this option to enable the system to base its criteria for identifying any variant ratios on the percentage of error Enter the percent value of error the system allows a ratio to reach before it considers the ratio to be variant Peak Ratio Result Table The system performs the analysis based on the selected reference and at least one test peak Show Excluded Select this option if you want the result table to display peaks excluded from the analysis The Peak Ratio Result Table displays details in the following columns Status Displays an R if the result has been selected as a reference Result Name Displays the result name Ratio R T Displays the ratio of the reference peak to the test peak Ratio T R Displays the ratio of the test peak to the reference peak Timestamp Displays the time the quantitation was performed sample Name Displays the sample name Reference Peak Height Displays the height of the selected reference peak Test Peak Height Displays the height of the selected test peak Test Peak Quantity Displays the calculated quantity of the test peak based on user supplied reference values Comments Displays any user comments associated with the result Mean Data Points amp Standard Deviation The lower left area of the Peak Ratio window displays system generated values for each of the calculated columns i
358. number of different system variables and information e Exclusion Filter Set area is where the user defined exclusion filters are selected and applied to the data Each column contains a menu of parameters to be applied as exclusion filters The system comes equipped with one pre defined sets of exclusion filters You can create modify and save additional filter sets e Summary area displays a table that identifies the number of analyses that have been affected by the application of exclusion filters As the result set is manipulated you can view the number of samples that have been excluded from your Study based on your filter set parameters GenomeLab Genetic Analysis System User s Guide 221 PN A29142 AB Fragment Analysis Module Using the SNP Locus Tag Editor 222 NOTE The Filter Sets applied to the Result Set data 2nd Level Filtering are different from the Filter Sets applied to the Fragment List data 3rd Level Filtering In the Result Set you apply filters to an entire sample that might contain numerous fragments In the Fragment List you apply filters to individual fragments that have been identified within the various samples See Using Fragment Lists on page 231 for more information Applying Exclusion Filters to the Result Set Data A Filter Set is a collection of any number of filters that are either pre defined by the system or user defined The purpose of the Filter Set is to exclude any results that do not meet the cr
359. o in NM_002467 NM_003234 r M NM_004702 M NM_004994 M NM 005228 MM 014883 cJ CS eni eni eni cajnip einjp eni E DE EE ine m u LH deWnH ze EH 1eWnH ce CHM 18WnH ca c LL 1e WnH CLA 38WnH ce u ELN 18WnH ce Treatments Figure 7 34 Normalized Gene Expression GenomeLab Genetic Analysis System User s Guide 309 PN A29142 AB Gene Expression eXpress Analysis Module 310 Viewing and Exporting a Report View the quantitative results of the normalized gene expression data The report includes data from all plates contained within the open analysis Export the data to Microsoft Excel or view the data on the screen eS ger c Click GeXP Analysis Normalization Report View Click Open Analysis The dialog box opens Log into the eXpress Profiler and click on eXpress Analysis in the Menu Enter your username and password then click OK to log into the software Select the analysis and click OK The default setting reports the non normalized data in relative fluorescence units Select the Display normalized data set option to view normalized data Choose a report format from the Report drop down list The recommended report is Profile By Gene since it contains the most pertinent statistics for the evaluation of changes in gene expression GexP Analysis Plate Setup GeXP Analysis Normalization MEME NI Report View Figure 7 35 Report View by Gene Option
360. odule Using the Sample Setup Module Report Format Printer Properties Status Ready Colors Type Microsoft Office Document Image Writer Driver Where Microsoft Document Imaging Writer Port Comment Page Layout Portrait C Landscape Sample Elements v Header Analysis Log Raw Data Run Log vw Result Data Method Summary Cancel w Result Output Analysis Parameters Current Quality Parameters o Help Voltage Trimming Log Options Alignment Results Alignment Accuracy Figure 2 18 Report Format Options Specifying Sample Plate Export Options Exporting Sample Results To export sample results after separation ze ee der des 6 Select the Export Data option in the Export section of the Analysis Tab Figure 2 17 Click on the Edit Export Options for Plate button Select the target folder Select the desired Sample Elements for the export format chosen Refer to the Online Help for a description of these parameters and the export formats Select the desired options for the chosen export format See Export Options on page 4 for descriptions of the export options used for exporting sample results Click OK NOTE The export formats selected will apply to all samples automatically exported during the plate run Export Options Removing CEQ Tracking Suffix Select this option to remove the appended sample plate position and time
361. of mobility on size under some conditions but usually over relatively short size ranges that are well separated from the size limits of the separation system Cubic Curve generally represents the curvature in a time or mobility versus size plot well enough that errors in estimated sizes are dominated by other factors such as effects of primary structure on mobility This is usually the best choice for routine use with the SizeStandard 400 set Quartic Curve is a fourth degree polynomial that provides more accurate size estimates than a cubic curve when used for calibrations that span a long size range or extend into size areas where the size selectivity of the separation system is very non linear This is usually the best choice for routine use with the SizeStandard 600 set Local Southern method estimates the size of a sample fragment from its mobility and that of the four standard fragments closest in size If you select this method the X Variable options are grayed out and mobility is automatically selected 5 Set the Migration Variable fields to either migration time or mobility as the independent variable for the Model Size is the dependent variable Migration time Select this option to use the average migration times and sizes of the standard fragments to construct a size calibration Mobility Select this option to use fragment velocity cm s divided by the separation potential V cm with the sizes of the standard fragments to co
362. og box opens 2 Select a method from the drop down list 3 Inthe Auto Fill area of the dialog box select All sample sets then click OK To view the selected method click the Method tab Conditioning the System It is recommended to purge the manifold with fresh gel followed by a capillary gel fill before each plate run If the system has not been used for a long period of time thoroughly purge the manifold and run the condition method before performing a sample separation Performing the condition method fills the capillaries with gel 6 times NOTE Thoroughly clean the wetting tray with D I water before every run to remove old or dried gel Creating or Editing a Method To create and or edit a method Select Edit Method from the Sample Setup main screen Highlight the desired method in the Choose Method to Edit dialog box and click OK When the Method Capillary Temperature dialog box opens enter a temperature between 35 and 60 C a Select the Wait for Cap Temp option if desired b Click OK to exit the Method dialog box or continue with step 4 to make additional changes to the method 4 Select Denature from the Event list a Enter a duration between 0 and 180 seconds if 0 is selected there will be no denaturation b Click OK to exit the Method dialog box or continue with step 5 to make additional changes to the method 5 Select Pause from the Event list GenomeLab Genetic Analysis System User s Gui
363. omeLab Genetic Analysis System User s Guide PN A29142 AB Fragment Analysis Module Exporting Results Creating a Genotype Summary Report The Locus List LocusList txt is required to create the Genotype Summary Report GSV and to perform some exports Use the Locus List Editor to customize the LocusList txt file Press the F7 key or select View Locus List Editor The Locus List Editor opens as shown in the following example Edit Locus List Copy af D 35 2387 2 eae ie Copy of 0952157 elected LOCI Copy of New Locus Tag GATATISADY 2 Other Loci cix Laocuslist Est File Date and Time 3 2 T 2006 1 43 26 PM GATATIS amp O 4 ba Cancel Figure 6 45 Edit Locus List Dialog Box Table 6 16 Locus List Editor Legend Loci in Database Other Loci Selected Loci 2 E D S Lists the loci from the database that have not yet been moved to the Selected Loci list Lists the loci to be removed from the LocusList txt file Lists the loci to be added to the LocusList txt file Moves all loci from the Loci in Database and Other Loci list to the Selected Loci list Moves only the selected loci from the Loci in Database and Other Loci list to the Selected Loci list Moves all loci from the Selected Loci list to the Loci in Database or Other Loci list If the loci exist in the database moves the loci to the Loci in Database list Moves the selected loci from the Selected Loci list to the Loci
364. ompare views Display Options Heterozygotes Display Color Option Zoom Pan Align Edit Unzoom Unzoom All Autoscale Base Spacing Base Synch Bases on Top Compare NOTE A v next to an option indicates that the option is enabled The following table describes the Sequence Analysis modules Tools menu options Table 4 5 Tools Menu Sequence Analysis Module Description Magnifies a specific area of data in the display Moves analyzed data in the X and Y direction Visually align bases and peaks in the analyzed data pane Insert change and or delete bases Undoes one zoom level Undoes all zoom levels scales data to the confines of the pane If autoscaling is disabled the system scales the data to their true values Use this item to turn autoscaling on or off Sets the space between base sequence text in groups of 0 3 5 or 10 With both the analyzed data and base sequence panes open synchronizes the analyzed data display with the highlighted base sequence text showing the corresponding peak or peaks between two hairlines for the selected base Toggles the base sequence text between displaying on the top or bottom in the analyzed data pane Selects an analyzed data set to compare to the current analyzed data set GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Analysis Sequence Analysis Module Overview Table 4 5 Tools Menu Sequence Analysis Module Option Compare Synch
365. ompute Final Base Sequence 04 24 06 16 35 12 Base Sequence Compute Initial Confidence Values 04 24 06 16 35 12 Base Sequence Refine Multiplets 04 24 06 16 35 12 Base Sequence Compute Final Confidence Values Bl 04 24 06 16 35 12 Base Sequence Perform Probability Analysis Sequence Test 403_06042416KN i2 Sequence Test B03 06042416KO 3 Sequence Test CO3_06042416KQ Sequence Test D03_06042416KR 5 Sequence Test E03 06042416KS Sequence Test F03_06042416KU Sequence Test G03_06042416K Y Sequence Test HO3_06042416kKx Figure 4 36 Sequence Analysis Batch Processing Reanalyzing a Batch To reanalyze a batch After performing a batch analysis select Analysis Reanalyze Batch 2 Select a new parameter set or edit the currently selected set from the Working Analysis Parameters dialog box to obtain different results 3 Click OK to start the reanalysis NOTE To keep the results from the previous analysis pin the results tab by clicking on the pin icon z Otherwise the system overwrites the results with any subsequent reanalyses Viewing the Selected Batch Result To view the selected batch result after performing a batch analysis l Click the desired sample in the upper pane of the batch analysis window 2 Select Analysis View Selected Batch Sample Result Skipping the Current Sample Analysis in a Batch To skip the curr
366. on of the CE Instrument is Class 1 defined as lasers which are safe under reasonably foreseeable conditions of operation To prevent users from potentially harmful laser light observe all safety warnings see Figure for label locations and NEVER REMOVE THE OUTER CASING OF THE LASER ASSEMBLY GenomeLab Genetic Analysis System User s Guide Safety Information 2 E a 5 CAUTION LASER LIGHT ACCESSIBLE WHEN COVER IS OPEN amp n OR REMOVED AVOID EXPOSURE TO BEAM i Ue S 8 1 fo O uA ua ic eal o m approximate location T EE 9 q eJ BECKMAN u o i A COUETEER AVOID EXPOSURE LASER LIGHT IS EMITTED FROM THIS
367. on uniquely identifies each sample location G Currently active Selected method Use this drop down list to select a pre defined method to the corresponding samples in the column above Selecting a method assigns it to all samples in a column sample set e Frag methods are used for all fragment analysis applications other than SNP primer extension e LFR method is used for long fast read sequencing on the 33 cm array e SNP method is used for the SNP primer extension application H e Note tab Displays the Note sub window that can be used to describe or apply a property set to single or multiple samples e Method tab Displays the Method sub window that shows the parameters defined for the selected method and allows you to edit or create separation methods e Analysis tab Display the Analysis sub window that can be used to Enable or disable the automatic analysis mode Select and edit a Sequence Analysis or Fragment Analysis parameter set Enable or disable automatic report printing and edit print report options Enable or disable automatic exporting of data and change export options 20 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sample Setup Module Overview Editing Property Values Attach information and a property set to the highlighted samples This informational note will accompany the sample throughout its lifetime modify the property sets Note Method Analysis x tl ie
368. opens Monitor Baseline Ea Enable Monitor Baseline OF Baseline Parameters Cancel Autosave v View Injection Instrument Data Name BASELINE Project Default b Figure 9 40 Monitor Baseline Dialog Box NOTE If you select Autosave on this dialog box the system stores baseline data under the Sample Data node for the selected project in the Data Manager If you do not manually end the Monitor Baseline it collects baseline data until you run a sample plate or until six hours have elapsed from the start of the baseline monitoring process 2 Inthe Monitor Baseline dialog box a Select the Enable Monitor Baseline check box b Click OK 3 Select the Data Monitor tab to view the baseline trace To view the baseline trace GenomeLab Genetic Analysis System User s Guide 383 PN A29142 AB Maintenance and Diagnostics Diagnostics Access the desired analysis module Select File Open Select the Sample Data tab and select the desired baseline data the baseline data is displayed After reviewing the baseline trace disable Monitor Baseline To do this 1 Select Run Monitor Baseline 2 In the Monitor Baseline dialog box a Select the Enable Monitor Baseline check box to clear the check mark b Click OK 384 GenomeLab Genetic Analysis System User s Guide PN A29142 AB
369. operty 3 Multiplexes Human Reference Rat Multitox Rat Reference Figure 7 9 Verification of the Multiplexes in GeXP Analysis Make sure that the multiplex of interest is shown in the Multiplexes list because gene expression analysis relies upon the associated multiplex TDF file GenomeLab Genetic Analysis System User s Guide 2 1 PN A29142 AB Gene Expression Logging in as an eXpress Profiler User 7 3 Logging in as an eXpress Profiler User IMPORTANT User Accounts must be set up by the Administrator prior to user login The eXpress Profiler Administrator creates accounts and can grant access to users to specific projects eXpress Profiler users log in under these non administrative accounts and have access to the eXpress Designer eXpress Analysis and eXpress Map tools 1 To log into the software double click the GeXP Launch desktop icon The GenomeLab eXpress Profiler login screen opens GenomeLab eXpress Profiler Please log in Username GexP_User Password Submit Figure 7 10 GenomeLab eXpress Profiler Login Screen 2 Enter your Username and Password then click Submit The eXpress Profiler Welcome screen will open and display the menu options available to eXpress Profiler users eXpress Profiler Welcome eXpress Designer eXpress Analysis eXpress Map Welcome Logout GeXP User Please make a selection from the menu on the left Figure 7 11 eXpress Profiler Welcome Screen User Lo
370. or see Managing Genes on page 272 for more information NOTE The following parameters are shown on the Primer Design screen but are not used with the GeXP System e General Primer Picking Conditions e Other Per Sequence Inputs e Sequence Quality e Objective Function Penalty Weights for Primers e Objective Function Penalty Weights for Primer Pairs Table 7 8 Primer Design Options Parameter Number to Return Genes Product Size excluding Universal Tags Primer Length Primer Tm User Left Primer Below Pick Hybridization Probe Use Right Primer Below Target Excluded Regions GenomeLab Genetic Analysis System User s Guide Description Specify the number of primer pairs to return Contains all global genes those entered by the Administrator as genes and multiplexes as well as all genes contained under the user s account specify the minimum maximum and optimal product size to define a range as needed specify the minimum maximum and optimal primer length WARNING Do not change the default setting optimal 20 nt It will affect the chemistry Specify the minimum maximum and optimal primer melting temperature WARNING Do not change the default setting optimal 60 C It will affect the chemistry opecify the forward primer sequence Not applicable for GeXP Chemistry Specify the reverse primer sequence Enter a region of sequence to be included in the amplicon sequence This can be an exon exo
371. ormation Ancillary Information Locus Mame GATA 9340 Primer set name GATA 4340 Dye iE M GenBank Accession Fragment length range between cal 3 15 a Repeat unit sequence gata Repeat unit length 4 nt Plaidy of repeats in shortest Allele 1 2 E Primer Label Primer Sequence Allele Naming Convention Shortest Allele size Forward Forward FT ta Nominal size Alphabetic Start at 340 d k id Reverse 5 to 3 Mone Numeric Start at l Generate Allele List a Allele List EINE Nominal Size nt Apparent Size nt Std Dev nt Num Points Comment 1 IREL EE EN ERR SINRAFEREESINENNTUERRRASSR RRSRSRSSREUEFFFENIYCER ESERNYNEERENEFEREENESRESREERSEFEENIKU EL QN EAN ES RSKSREERREEESRERESENEPPYCHFFURENS S eK ES KSNSEERSUSKERERRRRASSSESINEFINCSFNFNSASRFTRETESRSVERNEFRSRRSERRRSEREYKSRNINEPNYCUFSIFEN INE ERR RSKERNRRUSREKEEEENINSVETFSFNEVERYCUE SI ERREURREEdSRECEENARRSAM AE iR Ha eet eevee a eval vas essa fete EE I LEM T L LE CERT CT AEE E OLET E TID DLL IM TITBCIIBMIIGIIIIIIIIILDLBLICIL TM C IIIILIIIID TIBI LIT III EI NT Figure 6 17 Locus Tag Editor The Locus Tag Editor contains two tabs for creating or editing locus tags the Locus Tag tab and the Allele ID Criteria tab The top of the window displays the locus name and project You can save new or edited locus tags to a selected project for use in additional analyses Loc
372. ot adversely affected by prolonged exposure to air Figure 1 14 Access Plates Dialog 4 Click Start to continue 16 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Getting Started Operating the System 5 Install the plate and select the Plate Loaded option for the Left and or Right plate if applicable and then select the side to immerse the capillaries NOTE Prepare the new plates before proceeding Once the capillaries have been exposed to air you will have 15 minutes to load the plates Capillaries Exposed x PLEASE WAIT aif o not open sample Capillaries exposed to air p ance Time Remaining Alarm Off mir sec he 53 Lett Plate Plate Loaded Help ur Aight Plate Plate Loaded Immerse Capillanes C requires wetting tray on the right side Immerse Capillaries requires wetting tray an the left side Figure 1 15 Capillaries Exposed Dialog Box 6 Click Load to continue Capillaries Exposed You may now open the sample door and load plates Capillaries exposed to air Load Cancel Time Remaining Alarm Off min Sec 13 53 Left Plate Plate Loaded Help i Right Plate Plate Loaded Immerse Capillaries requires wetting tray on the right side Immerse Capillaries requires wetting tray on the left side NOTE Refer to Installing the Wetting Tray on page 351 Loading the Sample
373. otepad or any other text editor It may be composed of one or two sections the sequence and optionally specific nucleotide or amino acid mutations To create the file enter the text of the sequence starting in the first line of the text editor You may break the sequence text into several lines NOTE To create a reference file from the Sequence Analysis module export the data in seq format or choose txt with only Result Output checked To enter known protein or nucleotide mutations 1 On the line below the reference sequence type an open bracket followed by KNOWN AA MUTATIONS and or KNOWN BASE MUTATIONS then type a closed bracket 2 On the next line type the current reference amino acid or base the position number using the amino acid or base number then the expected amino acid or base change The following example shows a sample reference GenomeLab Genetic Analysis System User s Guide 1 13 PN A29142 AB Sequence Investigator Module Sequence Investigator Procedures 174 al referencel ExE Notepad Id xi File Edit Format wiew Help TCATTGACAGTCCAGCTGTCTTTTTCT6GaCAGC ACT AT AGGCTGTACTGTCCATTTATCAGUGAT GGAGTTICAT AAC CATCCABAC GAARTGGAGGTTCTTTCTGATGTTTTTTGTCTGGTGTGa T AASGTCCCCACCTORBACAG ATGTTGTCTCA GUCTCCTCTATTTTTGTTCTATGCTGCCCTATTTCTAAGTCAGATUCOCT ACAT AC AAATOATOCATOTATT GOAT AGATA ACTATGTCTGGATTTTGTTTTCT AAA AGGCTCTAAGATTTTTGTCATGCTACTTTGGAATATTGCTGGTGATCCTTT COATLCOCTOTOOAAGCACATTOTACTGOATATCT
374. ou specified a size standard see Analysis Method Tab on page 202 and calls alleles only if an allele list exists for the selected locus tag see Locus Tab on page 212 206 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Fragment Analysis Module Defining Fragment Analysis Parameters You can choose STR locus tags from a list of available STR locus tags previously configured for analysis This tab also gives you access to the STR Locus Tag Editor where you can edit existing or create new STR locus tags to meet the specific requirements of your samples Edit Fragment Analysis Parameters Miel Parameter Set Mame GATA193407 Froject Default IATATS3AU 2 1142001 10 07 40 A Edit Locus Hew Figure 6 14 Fragment Analysis Parameters Window Selecting STR Locus Tags Selecting Locus Tags Select one or more locus tags you want included in your parameter set e To select an individual locus select it in the Available list then click the right arrow gt to move the selection to the Selected list e To select all available locus tags click the double right arrow gt gt to move the selections to the Selected list e To remove locus tags from your parameter set select the locus tag in the Selected list and click either of the left arrow buttons to remove them from your parameter set e To edit a locus tag highlight the selection and click Edit Locus to display the STR Locus Tag Editor window e
375. ounds and or drifting baselines will occur All background levels should be below 6000 counts Water used during this procedure must be fresh distilled deionized water 18 Mohm cm water Select Replenish Release Capillary Array in the Run module Wait for the Remove Capillary Array dialog box to appear Open the sample access cover Figure 9 1 and lift it to the vertical locking position Open the capillary access cover and lift it to the vertical locking position pi ge ce YY d Unlatch the two rubber latches holding the capillary temperature control cover and lift it to the vertical locking position 6 Loosen the manifold access cover captive screw Figure 9 2 remove the cover and set it aside 344 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Maintenance and Diagnostics Routine Maintenance TBS AK Arr AN T L7 n Z 9016321 Al Figure 9 2 Manifold Access Cover 1 Capillary Temperature Control Cover 4 Captive Screw open 2 Plenum Assembly 5 Manifold Access Cover removed 3 Manifold Access Cover Lift the eject lever Figure 9 3 to release the array fitting Grasp the array fitting tab Figure 9 3 and then a b C d e Pull the fitting approximately one inch away from the manifold Touch the tip of the fitting to the bottom of the opt
376. ow to use the search and editing capabilities provided on the navigator toolbar click Help to open its context sensitive help topic Navigator x v Mismatch I ve Ambiguity 7 W Disagreement 7 Insertion Deletion Single Coverage 1 Mutation Hotspot M Low Quality lt Manual Edit C Consensus Text Search Exact Match LLonsensus Called B ase Quality Value Differences Figure 5 7 Navigator Toolbar Floating When editing the consensus follow these guidelines Complete all trim operations before performing other types of editing Using any other editing operation disables the trim feature e You may insert and delete spaces in the reference sequence but not change bases e Select a single consensus base if you want to perform base editing NOTE The system adds all editing operations to the history log along with the date time and position of the operation Complementing the Sequence When the system performs a comparison it determines which orientation forward or reverse to provide a better fit with the reference If the system determines that the sequences GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Investigator Module Sequence Investigator Procedures generate a better comparison in the opposite orientation it automatically reverse complements the sequences To manually reverse complement a sample right click the desi
377. ow to use this module to investigate and compare sequence analysis results 5 1 Sequence Investigator Module Overview Compare sequences to known reference sequences For each comparison you can use one or two new sequence results in either orientation to form a consensus for the comparison The software automatically determines and matches the orientation of the sequences forms the consensus aligns the consensus with the reference sequence and provides a detailed report of the differences The Sequence Investigator provides a number of tools you can use to inspect and edit the data before generating the final report The navigator toolbar allows you to jump to points of interest from specific base sequences to a variety of discrepancies in the aligned data set The discrepancy map provides an overview of the coverage of the reference sequence by the sequences and indicates the locations of the discrepancies Together these alignment inspection editing and reporting features make Sequence Investigator an ideal tool for identifying mutations or polymorphisms in DNA Main Window Figure 5 1 shows the Sequence Investigator screen layout This consists of a title bar menu bar several toolbars the display window and the status bar GenomeLab Genetic Analysis System User s Guide 1 65 PN A29142 AB Sequence Investigator Module Sequence Investigator Module Overview A B C D ne MM Sequence Investigator
378. owing table describes the available export file types and options Table 4 29 Sequence Export Options Option Description SCF scf Use the SCF Standard Chromatogram Format format to store analyzed DNA sequence data for a single sample The format describes both public and private data sections The well defined public data section contains trace data points the called sequence the positions of each called base within the trace data and the call scores for each called base It also includes a public comment section The private section may store other information that most software packages ignore The CE system uses the private data section to store sample data including raw data along with current and voltage data You can store sample data from either sequencing or fragment analysis samples The notes and property sets for the sample are stored in the comment section The CE system software supports SCF versions 2 1 and 3 0 You would use this format for instance to export analyzed sequence data into Lasergene s SeqMan from DNASTAR Inc Third party packages cannot currently view raw data current or voltage However custom applications can be written to view the data Use the SCF format to transfer data from instrument to instrument or to import data from third party software into the Sequence Analysis module Using SCF to exchange data between instruments removes information such as sample plate name capillary array seri
379. ox Figure 3 42 select the correct part number enter the serial number click Set to New then click Done The number of runs and days on instrument will revert to 0 e If you are installing the previous capillary array do not change the serial number number of runs or the number of days on the instrument as they will be correct e If you are installing a capillary array that was previously used but not the last capillary array on the instrument enter its part number if applicable serial GenomeLab Genetic Analysis System User s Guide PN A29142 AB Run Module Using Direct Control number and adjust the number of runs and the previous cumulative days on the instrument Then click Done NOTE Note the number of runs and days on the instrument for this capillary array for reference Install Capillary Array Ea Please enter the serial number for the capillary array IE you wish to reset the number of runs forthe capillary or number of days it has been on the Instrument enter the new values Cancel Click on Done when vou have installed the capillary array and have closed bath the capillary access cover and the sample access cover Cet ta New Capillaries exposed to air Time Remaining Help 8B h h mir li 4 sec a Capillary Array Part Humber e0087 Total Length 33 00 cm aerial Number Length to Detector 20 00 cm Date Installed iy zr 200E Internal Diameter 75 00 um Time Installed b 45 20
380. ox displays the message Database backup restore Database restore was successful You must log off and back on or reboot in order to re establish database access Click OK then click Close to quit the program Exit all programs and shut down the computer Reboot the computer and restart Windows 10 Go to the Windows Start menu and select Start Programs GeXPAdmin GeXP to start the eXpress Profiler software 11 Login to the program using your Username and Password 12 Select Multiplex from the menu and then click Load Settings The drop down list should now include the restored items GenomeLab Genetic Analysis System User s Guide 317 PN A29142 AB Gene Expression eXpress Backup and Restore 318 GenomeLab Genetic Analysis System User s Guide PN A29142 AB 8 1 Data Manager Module Overview Create save and modify databases These databases can contain any of the following data types Filter Sets Fragment Analysis Parameters Fragment Results Methods Optical Scan Data Sample Data Sample Plates Sample Plate Results Sequence Analysis Parameters Sequence Results SNP Locus Tags Standards STR Locus Tags GenomeLab Genetic Analysis System User s Guide PN A29142 AB Database Management Data Manager Module Overview Database Management 319 Database Management Data Manager Module Overview Main Window The main window of the Data Manager module is shown in Figure 8 1 and described in Table 8 1
381. play options you want to change For details see the following topics e Title Properties Tab on page 71 X Axis Options Tab on page 71 e Y Axis Options Tab on page 71 Colors Tab on page 71 Dye Traces Tab on page 71 e Current Trace Tab on page 72 NOTE For detailed descriptions while using this dialog box click Help Title Properties Tab Set or change the title of the displayed data panel l Inthe Display Options dialog box select the Title tab 2 Change any item as necessary 3 Click Apply to continue or OK to close the Display Options dialog box X Axis Options Tab Define the X Axis properties of the displayed data panels l Inthe Display Options dialog box select the X Axis Options tab 2 Change any item as necessary 3 Click Apply to continue or OK to close the Display Options dialog box Y Axis Options Tab Define the Y Axis properties of the displayed data panels l Inthe Display Options dialog box select the Y Axis Options tab 2 Change any item as necessary 3 Click Apply to continue or OK to close the Display Options dialog box Colors Tab Define the color options of the window the current traces the voltage trace or the optical scan trace l Inthe Display Options dialog box select the Colors tab 2 Change any item as desired 3 Click Apply to continue or OK to close the Display Options dialog box Dye Traces Tab Set or change the dye trace properties l Inthe Displ
382. port Only If Sequence gt X nt option to specify a minimum size that the sequence must be when exporting This exports only those sequences greater than the set number of nucleotides Export Sample Elements Header Raw Data Result Data y Result Output Guality Parameters Alignment Results Alignment Accuracy Options v Remove CEQ Tracking Suffix vw Resolve Filename Conflicts Figure 4 24 Export Options Editing Bases O m fal Sequence Test A03_060301225Z fasta E Sequence Tesk DO3 60301225z7 F Ej Sequence Test A04 O6030200L7 fasta Ej Sequence Test D04 06030200L7 Ff Ej Sequence Test BO3 O603012252 fasta Ej Sequence Test E03_060301225Z f E Sequence Test B04 O6030200L2 Fasta E Sequence Test E04_06030200LZ F Ej Sequence Test c03 0603012257 fasta Ej Sequence Test F03 0603012257 fF Ej Sequence Test c04 O6030200Lz Faska Ej Sequence Tesk FO4 O6030200L2 f Save as type Fas TA FASTA and QUAL fasta Cancel savein oEot Hege File name You can edit bases displayed in the analyzed and bases data panes using the Sequence Analysis module s Tools menu The system identifies edited bases by showing them in lower case letters Inserting Bases in the Analyzed Data Pane To insert bases in the analyzed data pane Select Tools Edit Select the desired base Click OK p e qw GenomeLab Genetic Analysis System User s Guide PN A29142 AB C
383. port when it encounters duplicate names For example if the system encounters the duplicate sample name of Fred on export it renames the file exported to Fred 1 Selecting this option removes the trims from the sequence before you export it and replaces internal trims with the letter x 161 Sequence Analysis Using the Sequence Analysis Module 162 Customizing Filename Extension To supply your own custom filename extension or none at all to exported files modify the CEQ ini file as follows NOTE If there is no Export section in the CEQ ini file export any sample file to generate the Export section then modify as needed Find the section EXPORT and add modify the appropriate entry TXT_EXT for Text files default is txt SEQ EXT for SEQ files default is seq FASTA EXT for FASTA files default is fasta SCF_EXT for SCF files default is scf PHD_EXT for PHRED files default is scf phd 1 To have no file extension added set the entry to nothing make sure there are no spaces after the SCF_EXT Example Output FileName SpaSample To use a unique file extension add or modify the entry with the desired file extension do not place the leading dot the system will add it SCF_EXT scf v3 Output FileName SpaSample scf v3 To hide an entry and make the system use the default either delete or place an before it SCF_EXT SCFV3 Output FileName SpaSample s
384. ppropriate analysis parameters as well as report and export parameters if needed Enter the barcode in the Barcode field Select File Save As Enter a name for the sample plate Select the project you just created from the Project Name drop down menu and click OK GenomeLab Genetic Analysis System User s Guide PN A29142 AB Getting Started Operating the System Running a Sample Starting the Run Module From the Main Menu click the Run icon y and verify that the Run window is displayed Checking the Capillary Array Check the capillary life and usage on the life tab and if necessary perform the procedure Removing and Replacing the Capillary Array on page 361 and then return here Installing a Gel Cartridge Check the gel life and usage on the life tab and if necessary perform the procedure Removing and Replacing a Gel Cartridge Gel Pump Plug on page 367 and then return here Installing the Gel Waste Bottle If necessary perform the procedure Replacing the Gel Waste Bottle on page 349 and then return here Preparing Plates for a Run l Load samples into the sample plate and cover with mineral oil 2 Load buffer 250 300 pL into the buffer plate 3 Fill the wetting tray with D I water to the level indicator marker IMPORTANT No more than one 96 well plate should be processed for each wetting tray without replenishing the wetting tray NOTE Periodically check the liquid level in the wetting tray
385. pre peaks are often found starting near the end of a series of the same base 117 Sequence Analysis Using the Sequence Analysis Module 118 Table 4 17 General Tab Sequence Analysis Parameters Editor Option Description Color Click this button to view the color calibration for the analysis parameters The Color Calibration dialog box opens The values in the matrix show the cross talk between the filters and the emissions for A C G and T If you want to select another color calibration click Select Stored Color Calibration select the new color calibration then click OK The name of the Selected color calibration appears in the field Informational The lower section of this dialog box displays specific features that are Display enabled disabled from other tabs To change these settings select the appropriate tab and edit the feature Initial Data Detection Tab Use the Initial Data Detection tab of the Sequence Analysis Parameters Editor dialog box to enable the system to detect the start of data to be analyzed The system attempts to detect valid data using the system algorithm and values entered on this tab only if the Analysis Start time on the General tab is set to 0 0 Sequence Analysis Parameters Editor DefaultSequenceAnalysisPar Qualty based Trimming Sequence based Trimming Alignment Reference General Initial Data Detection Heterozugote Detection Threshald Delay ozs eh min Signal to Noi
386. procedure WARNING If you are restoring a database that has the same name as one already residing in the Database Manager you must either restore it with a new name or delete the existing database before restoring it THIS Option IS NOT Recommended l Open Data Manager 2 Select Tools Restore Database 3 Browse to the location on the local disk where the backup file was saved Restore Database El XI Look irr Co D base e t CEQ 2006 4 29 BAK Filename CEQ_2006_4_29 BAK Files of type Database Backup Files bak Cancel Database To Restore CE uU 2n05 4 24 Figure 8 7 Restore Database Dialog Box 4 Select and highlight the file to restore and click Restore NOTE f a database with the same name exists a warning will appear Figure 8 8 Data Manager AN Database To Restore name already exists Please enter a new database name Figure 8 8 Database Exists Warning Message 5 If this occurs click OK and either e Delete the database in the Data Manager NOT recommended or e Rename the database being restored This is done by entering a new name in the Database To Restore field GenomeLab Genetic Analysis System User s Guide 331 PN A29142 AB Database Management Data Manager Procedures Converting Individual IDs to Subject IDs Converts all sample data records with Individual IDs to records that use Subject IDs Before you begin confirm that the present database is the
387. que that relies on the selective amplification of restriction fragments from a total digest of genomic DNA The CE system includes AFLP Fingerprint analysis which allows you to rapidly score AFLP fingerprints with output in a text format which is compatible with most statistical and pattern matching system The AFLP analysis tool is a cluster analysis module that scores the presence or absence of a fragment of a given size per sample Using the AFLP Feature The AFLP feature takes all results from the Study and creates bins from fragments with sizes that fall within a user defined bin width For each of the possible fragments in the Study the system assigns a 1 if it finds a fragment in that sample and assigns it a 0 if the fragment is absent It then creates a table of 1 s and Os You can export the text to most other statistical analysis and spreadsheet software GenomeLab Genetic Analysis System User s Guide PN A29142 AB Fragment Analysis Module Performing AFLP Analysis Setting the AFLP Analysis Parameters To start a new AFLP analysis select Analysis New AFLP Analysis The New AFLP window opens NOTE You can also access this from the Analyses tab of the Study Explorer E New AFLP PII ES Analysis Parameters E hd asimum Bin width 1 00 Y Threshold g Exclude Fully Populated Bins m Exclude Samples wro Qualitying Peaks Cancel Figure 6 32 New AFLP Window ALFP Analysis Parameters
388. rand the primer is labeled with the dye You may choose one of these options e Forward Select this option if the primer is dye labeled on the forward strand e Reverse Select this option if the primer is dye labeled on the reverse strand e None Select this option if you do not want to indicate which strand the primer is dye labeled GenomeLab Genetic Analysis System User s Guide 21 3 PN A29142 AB Fragment Analysis Module Using the STR Locus Tag Editor 214 Primer Sequence Optional The Primer Sequence area of the window allows you to indicate for supplementary reporting purposes the specific sequence of the primer used This can be up to 128 characters Forward If you labeled the primer on the forward strand enter the sequence of the primer in the 5 to 3 direction Reverse If you labeled the primer on the reverse strand enter the sequence of the primer in the 5 to 3 direction Generating an Allele List After setting the locus values click Generate Allele List to begin creating the allele list The system generates an allele list from the information provided The allele list starts at the low end of the fragment length range It creates an allele at each perfect repeat length until it reaches the end of the fragment length range The Allele List defines each allele under the following columns ID Uses the allele naming convention described in the section above Nominal Size Displays the generated fragm
389. re run in the same well select the corresponding check box f Normal and Tumor samples are in different wells clear this check box 5 Click Start NOTE Do not use the keyboard or the mouse during LOH analysis 6 If desired save the results when the processing is complete NOTE To save the data correctly you must save the LOH Analysis results as a Microsoft Excel Workbook xls file type You must also change the file name from the default name displayed in the Save As dialog box otherwise the system saves result as a temporary file which it will overwrite in the next LOH Analysis NOTE If more than two alleles are found for a locus the last allele listed for the locus will be followed by an asterisk GenomeLab Genetic Analysis System User s Guide 247 PN A29142 AB Fragment Analysis Module Customizing the Results List 6 12 Customizing the Results List The components of the results list are user defined You can modify them using the right click menus These commands allow you to select specific columns to display in the results list customize the arrangement of information add or remove columns and modify several other parameters of the results displayed Customized lists are available in the Result Set View the Fragment List AFLP Analysis and Peak Ratio tables Selecting Columns for Display The Column Selector window contains a list of all possible columns that you can display in the list You can selec
390. rect Control Tools Run Log Options Replenish Help An Cm GN T 4 2 Di BH D2 BH D E D4 E Staus Configuring Data Monitor Direct Control Log Instrument Data Event Type 0 14 0 78 nuu Progress Event 0 60 Min Sample Set 0 60 Min Sample Plate 0 60 Min e EN n Sample Amp Device Life e fn fa fm Jf fo fes fis F Active Plate Sample Plate Method T S Project Sample Name Position gt 5 x Capillaries exposed to air Gel Cartridge life exceeded 20 25 3 0 Capillary usage exceeded Time Minutes Off line H For Help press F1 Active Plate LEFT Pause To Load Inactive IConfiqured 2nd Plate Not Available INUM Figure 3 1 Main Window Run Module The following table describes the items called out in the image above Table 3 1 Main Window Run Module Item Description A Title Bar Displays the name of the module Run Control and the user defined system name Menu Bar See Menu Bar Options on page 47 Toolbars See Toolbar Icons on page 53 Window Selection Tabs Toggle between windows see Window Selection Tabs on page 59 E Display Area Graphically displays the raw data of the selected capillary or capillaries F Status Monitor Displays the current state of the run capillary and gel cartridge status and system on line off line status 46 GenomeLab Genetic Analysis System User s Guide PN A29142 A
391. red on the controller by the Administrator The eXpress Profiler Administrator has access to some administration tools that are not available to the eXpress Profiler users such as establishing user accounts importing multiplex data managing genes and making primers globally available See Table 7 1 on page 268 for more information IMPORTANT The Administrator does not have the ability to design or modify the multiplex as an eXpress Profiler user does 1 To access these administration tools double click the GeXP Launch desktop icon The GenomeLab eXpress Profiler login screen opens GenomeLab eXpress Profiler Please log in Username administrator Password ow Figure 7 1 GenomeLab eXpress Profiler Login Screen 2 Login as the Administrator by entering the username and password The default username and password is administrator To change the Administrator password see Changing the Administrator Password on page 271 NOTE All usernames and passwords are case sensitive 3 Click Submit The eXpress Profiler Welcome screen will open A menu on the left side of the screen will display the menu options available to the Administrator as shown in Figure 7 2 GenomeLab Genetic Analysis System User s Guide 26 PN A29142 AB Gene Expression Software Administration eXpress Profiler Welcome Users Account Manage Genes Manage Primers Welcome Universal Tags Import Multiplex Help System Administra
392. red trace data pane then select Reverse Complement The trace and associated bases are reverse complemented Flipping the Traces You may want to switch the order of sequence displayed To move the sequence trace shown in the top trace pane and exchange its position with the bottom trace select Edit Flip Traces or right click in one of the trace data panes and select Flip Traces This exchanges the position of the sequences in both the alignment pane and the trace panes Trimming the Bases When you first open or create a compared sequence result the sequences used in the comparison appear in the alignment pane You can then trim the ends of the sequences If sequences are not shown select View Sequences to display the sequences in the alignment pane To trim the sequence l Select a group of sequence bases at either end of the sequence 2 Click the Trim icon to remove unwanted bases from the alignment This deletes the bases from the text and trace data views NOTE You may only trim at either end of the sequence You may not trim within the sequence After each trim the system automatically aligns and redisplays the alignment text and traces NOTE The system adds trim operations to the history log along with bases trimmed with date and time of trim operation To access the history log select View History Searching the Consensus for Specific Attributes To search the consensus for specific attributes l Select the Cons
393. results sequence or fragment analysis parameters sample plates methods locus tags fragment data only standards fragment data only and optical scan data The sample elements are automatically selected in the Export dialog box which would export all of the associated information listed above This format essentially takes a snapshot of the file and can retain all of the information belonging to that file This format is available only when importing data into the Data Manager This format can also be used when exporting data from the Sequence and the Fragment Analysis modules and the Data Manager When exporting as CEQ data the various data types are exported to files with the following extensions sequence Analysis ParameterCQA Sequence InvestigatorCQC Fragment Size StandardCQD Fragment Analysis ParameterCQF Galvo Scan DataCQG Fragment Locus TagsCQL MethodCQM SNP Locus TagCQN Sequence ResultCQR sample DataCQS sample TableCQT Fragment ResultCQU sample Table ResultCQX You may automatically remove the plate position coordinates A01 B02 etc and the time date stamp from the sample name which is normally generated by the CE system software on export To do so select the Remove CEQ Tracking Suffix check box 1 If the system encounters the same sample name at the destination it usually overwrites the name if you choose to remove the plate position coordinates and the time date stamp which ensures a unique n
394. rflow observe the gel level in the waste bottle mW 901514L Al Figure 1 8 Gel Waste Bottle Gel Pump Gel Cartridge Access Cover Provides access to the gel pump and gel cartridge Table 1 1 Gel Pump Gel Cartridge and Gel Pump Plug Component Description Gel Pump Used to replenish the capillaries with fresh gel from the cartridge after each sample set and two 96 well plate runs Gel Cartridge Contains the fresh DNA separation gel The separation gel is used for both Sequence and Fragment Analysis When full the cartridge contains a sufficient amount of gel for 24 runs 96 templates Gel Pump Plug When the gel cartridge is not installed for example during shipping and storage the gel pump plug is inserted into the pump barrel to prevent gel from drying in the system Getting Started System Overview 901635L Al Figure 1 9 Gel Pump 1 Gel Pump Gel Cartridge Access Cover 4 Cartridge Locking Lever 2 Cartridge Locking Lever 5 Cartridge Barrel 3 Cartridge Barrel 8 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Getting Started System Overview Status Indicator Lights There are three status indicators PWR power LASER and HV high voltage as shown in the figure below 901652L Al Figure 1 10 CE System Location of Status Indicator Lights Table 1 2 Status Indicator Lights Indicator Description PWR Green light is on when the CE
395. rimer to allow Global access 5 Click Save to save the changes GenomeLab Genetic Analysis System User s Guide 975 PN A29142 AB Gene Expression Software Administration Managing Universal Tags Define the Universal Tag sequence that will be appended to the primer sequences that are created in the eXpress Designer IMPORTANT The current GeXP chemistry only works with the default sequences Do not modify the default sequences eXpress Profiler Manage Universal Tags Default Tags Account p Load Save Reset Form The data has been loaded Manage Genes Manage Primers Universal Tags Name Default Tags Import Multiplex Help Left Sequence JAGGTGACACTATAGAATA Logout Right Sequence GTACGACTCACTATAGGGA Set as Default Iv Figure 7 7 eXpress Profiler Manage Universal Tags Screen Importing a Multiplex Import gene set kit multiplexes as well as user defined multiplexes from another GeXP system into the database If a multiplex of interest was not created using eXpress Designer on the current eXpress Profiler controller or if a commercial gene set kit was used the multiplex TDF trace definition file file must be imported into the database before analysis can be performed NOTE Visit www beckmancoulter com GeXPplex for the multiplex TDF files to use with the GenomeLab multiplex gene set kits eXpress Profiler Import Multiplex Users Account Manage Genes Select Multiplex fil
396. rmalized Data check box to view normalized data for each gene IMPORTANT When entering data in eXpress Analysis click Save to update the data in the database or before going to the next tab or function Table 7 14 Graph Display Options Option Description Display as zero value All data points including zero value expression levels are displayed and expression levels taken into consideration when this option is selected Display normalized values Gene expression values that have been normalized to the selected Normalization Gene are displayed for the replicate data points within a treatment or sample type The mean normalized value represented by an X for each treatment is connected by a line NOTE When viewing normalized vs relative fluorescence units data the Y axis label changes accordingly 308 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Gene Expression eXpress Analysis Module 10 View each gene expression profile by selecting the gene display name from the Genes list GeP Analysis Plate Setup GexP Analysis Normalization i Normalize Peaks Multiplex HuMetastasis Normalize Peaks Normalization Gene NM 001 101 m 7 Display zero valued expression levels m o w in V Display normalized values Genes Select All Deselect All Kani NM_000346 NM_000389 DJ o h in N o in v NM 001101 NM 001158 Iv NM 002046 zonn xM apow v zZ oz 5v oz
397. rol and Replenishment 2 Wetting Tray Retainers 5 ALIGN together 3 Sample Plate Holder 6 Sample Plate Guide Pin 3 When finished positioning the sample and buffer plates close the Sample Access Cover and then select the position from which the plates were loaded left or right 4 Click the Load on the Capillaries Exposed dialog box Figure 9 15 CAUTION The separation gel within the capillaries will dry out if the capillaries are left exposed to the air for more than 15 minutes Capillaries Exposed Ed You may now open the sample door and load plates Capillaries exposed to air E Cancel Time Remaining Alarm Off min sec 4 26 Help Left Plate Jl Right Plate Plate Loaded Immerse Capillaries requires wetting tray on the left side Plate Loaded Immerse Capillaries C requires wetting tray on the right side Figure 9 15 Capillaries Exposed Dialog Box Loading the Buffer Plate and Evaporation Cover l Align the notched corner of the buffer plate with the alignment line on the buffer plate holder Figure 9 16 2 Gently push the buffer plate towards the front of the instrument and then set the plate into the transport 3 At the rear of the buffer plate holder align the buffer evaporation cover guide pin with the buffer evaporation cover alignment notch and then gently lower the cover over the buffer plate GenomeLab Genetic Analysis
398. roperties in the active sample plate Select All selects all cells in the sample plate Cut Deletes text in one or more cells of the sample plate and copies it to the clipboard You can then paste the cut text into different cells Copy Copies text or the applied properties selected from one or more cells of the sample plate You can then paste the cut text into different cells Paste Inserts copied or cut items at the cursor insertion point Find Opens the Find dialog box which you can use to search for text within the sample name entered in a sample plate Replace Opens the Replace dialog box which you can use to search and replace text within the Sample names entered in a sample plate GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sample Setup Module Overview View Menu Click the View menu to display its drop down menu as shown in the following example View v Toolbar w Status Bar w Cell Coordinates Sample Property Sets Summary View by Subject ID Working Database Activate or deactivate window display options The following table describes the Sample Setup module s View menu options Table 2 4 View Menu Sample Setup Module Option Description Toolbar Toggles between displaying not displaying the toolbar otatus Bar Toggles between displaying not displaying the status bar Cell Coordinates Toggles between displaying not displaying the coordinates of the plate cells Sample Propert
399. rrent and voltage levels of selected current run GenomeLab Genetic Analysis System User s Guide 63 PN A29142 AB Run Module Using the Run Module 3 2 Using the Run Module Run sample plates and set the display options To open the Run module from the Main Menu click the Run icon Gel Plug Warning 64 When you open the Run module the system initializes and checks the gel cartridge condition If the system detects an installed gel plug the Device Warning Message appears as shown in Figure 3 14 Device Warning Message Ea v Instrument status Gel Plug Installed Loading Gel Cartridge Please install a Gel Cartridge for proper operation Figure 3 14 Device Warning Message Gel Plug Installed In addition to this warning message the Device tab indicates the state of the Cartridge When a gel plug is present the Device tab indicates plug in the Cartridge field as shown in Figure 3 15 The plug must be removed and replaced with a gel cartridge before the next run When a cartridge is present the Device tab indicates Gel in the Cartridge field as shown in Figure 3 16 Sample wimp Device Lie Sample wimp Device Lie Plate Position sample 1 Plate Position Sample 1 Capillary Temperature 35 C Capillary Temperature 35 C Capillary Array Present Capillary Array Present Manifold Plug Mat Present Manifold Flug Mat Present Cartridge Barret Unlocked Cartridge Barret Locked sample Access Lover Clo
400. rs to properly reflect the hours this cartridge has been on the instrument d Click Done on the Install Gel Cartridge dialog NOTE Always note the lot number and the hours on the instrument for a gel cartridge if you are planning to use it for more than one session The lot number is an alphanumeric text box for your own identification purposes 90 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Run Module Using Direct Control Install Gel Cartridge Gel Cartridge Part Number 331 438 C Lot Number Elts Gel M ame LPA Help Date Installed D4 272006 Time Installed 75 00 New Used Hours on Instrument 27 Figure 3 46 Install Gel Cartridge Dialog Box 12 Dispose of the used tissue and spent gel cartridge in accordance with the procedure Maintenance and Diagnostics on page 343 Removing the Manifold Plug The Manifold Plug is installed during shipping and when the instrument is not in use to prevent the gel from drying NOTE This procedure assumes that the Manifold Plug is being removed in preparation for Capillary Array installation l Select Replenish Release Capillary Array from the Run Module main menu 2 Wait for the Remove Manifold Plug dialog box to appear 3 Open the Sample Access Cover Figure 3 27 and lift it to the vertical locking position 4 Open the Capillary Access Cover and lift it to the vertical locking position 5 Unlatch the two r
401. rument using a damp tissue NOTE Note the number of runs and days on the instrument for this capillary array for reference If this capillary array is to be re installed this information is necessary A label has been provided on the back of the array packaging container so that this information may be recorded GenomeLab Genetic Analysis System User s Guide 83 PN A29142 AB Run Module Using Direct Control 901597L Al Figure 3 38 Removing Replacing the Array Fitting 1 Guide Pins 4 Gel Strand 2 Eject Lever 5 Optics Base Plate bottom 3 Array Fitting 6 Array Fitting 9 Grasp the Electrode Block tab Figure 3 39 and pull the block out and away from the instrument 10 From the Remove Capillary Array dialog box Figure 3 35 select the Replace Capillary Array option and click OK 84 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Run Module Using Direct Control 901602 Al Figure 3 39 Removing Replacing the Electrode Block 2 Electrode Block 1 Guide Block Pins CAUTION Always grip the capillary array fitting near the end during removal or installation of the tip cap to prevent flexing and possible breakage of the capillary array as shown below Figure 3 40 900654L AI 85 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Run Module Using Direct Control 86 Figure 3 40 Array Fitting 11 12 13 While holding the Array Fitting
402. s for Size or minutes for Time Amount Enter the known quantity of your reference peak EEnter this value as the exponential notation Units Select the units for the quantity entered into the Amount field Values may be any of the following e Moles mol e Grams g e Molarity M e g L STR Locus Tags Tab The STR Locus Tags tab allows you to select the Short Tandem Repeat STR locus tags used for allele identification with the current parameter NOTE Although the STR Locus Tags tab is similar in appearance and functionality to the SNP Locus Tags tab the two types of Locus Tags are used for different purposes STR Locus Tags use size to define allele fragments having a single specified dye label in a specified size range SNP Locus Tags use multiple dye labels for fragments of a single specified size to define specific SNP variants The system uses locus tags to define a particular locus of interest Each available locus tag is configured with a specific set of parameters that define the manner in which the system identifies detected fragments as allele fragments These parameters include the fragment size range repeat unit length the dye used and an allele list associated with the locus tag When you select a locus tag and perform an analysis the system identifies the alleles present within the locus region using the estimated size of the fragments and the allele list NOTE The system estimates sizes only if y
403. s on page 285 Expected Size The PCR product size expected by eXpress Analysis for a designed gene based on the information stored in the multiplex TDF file Actual Size The PCR product size calculated by the GeXP Genetic Analysis System Minutes For non GeXP formatted files This is not applicable to the GeXP Genetic Analysis System Height Peak height obtained from GeXP fragment export file Area Peak area obtained from GeXP fragment export file NOTE t is helpful to use a printed or an electronic copy of the multiplex electropherogram from the GeXP Analysis System Fragment Data to better verify the designed peak sizes See Viewing Electropherogram Data on page 223 Binning Gene Peaks l Identify the peak that should align with the specific gene based on size and peak height 2 Click on the gene of interest in the Binning Table and perform one or both of the following steps to align the gene with the correct peak a Change the Binning Range and increase the bin width to include the peak The default binning range is 1 0 nucleotides 2 nt bin width to include a peak Changes to the Binning Range will affect all wells in the analysis for this gene peak size NOTE If multiple peaks reside in a particular bin the software will use the peak area associated with the maximum peak height for normalization calculations b Adjust the expected PCR product size within the Binning Range window to align the expected size of the gene
404. s Enter To name multiple non contiguous samples that are not within the same sample set Click on the first cell Press and hold the Ctrl key while selecting the additional samples Enter the name of the samples in the Sample Name text box and press the Enter key pu dude Enter the Subject ID in the Subject ID text box and press Enter The plate editor displays each edited cell with a green background shade The Plate Status indicator displays Plate Edited This indicates that the plate must be saved NOTE If you attempt to close the plate before saving it a Save Changes dialog box opens 5 Save the plate to retain the changes GenomeLab Genetic Analysis System User s Guide 33 PN A29142 AB Sample Setup Module Using the Sample Setup Module 34 Viewing Cell Coordinates To view or hide cell coordinates select View Cell Coordinates The option will toggle the display of the coordinates off and on Searching for Text in the Sample Plate l 2 Select Edit Find Use the options in the Find dialog to specify the search criteria Enter the desired text in the Search what field Select Down to search down each column beginning at the highlighted or selected cell Select Up to search up each column beginning at the highlighted or selected cell Select the Match whole word only option to locate text that is flanked by spaces If you do not select Match whole word only the system finds the text even if it
405. s been exceeded e Advanced Click this button to display the Capillary Advanced Information dialog box This dialog box displays the length and diameter of the installed capillary array based on the part number entered in the New Capillary Array dialog box The fields are read only and cannot be edited Click OK to enter the information and close the dialog box 3 When finished click OK to close the windows Viewing Gel Information 1 Select Replenish Gel Cartridge Buffer Information The Gel Cartridge Buffer Information dialog box opens Gel Cartridge Buffer Information Gel Part Number 38 430 Lot Number se02086 Gel M ame p Date Installed p 27 2008 Time Installed inops Hours on Instrument E Butter Part Number 608012 hd OF Cancel Print REE Help Lot Number CEG Sequencing Separation Buffer Figure 9 25 Gel Cartridge Buffer Information Dialog Box 2 Use the Gel Cartridge Buffer Information dialog box Figure 9 25 to view the following information e Part Number Displays the part number of the installed gel cartridge e Lot Number Displays the lot number of the installed gel cartridge The lot number is used to track the gel lot along with the separations that have used the gel e Gel Name The name of the gel e Date Installed Displays the date the gel cartridge was installed GenomeLab Genetic Analysis System User s Guide PN A29142 AB Mai
406. s not stop the stream of data just the display of data Monitor Baseline Displays the baseline data trace For details see Monitoring the Baseline on page 383 xml Display Options Modifies any or all display parameters See Setting or Changing GE Display Options on page 70 Log Toolbar The following example shows the Sample Setup module s Log toolbar For descriptions of the actions that occur when you click a toolbar icon see Table 3 11 zje Figure 3 6 Log Toolbar Run Module GenomeLab Genetic Analysis System User s Guide D5 PN A29142 AB Run Module Run Module Overview 06 Table 3 11 Log Toolbar Run Module Icon Description E Print Print the system log at any time the program is active System Logs contain all of the messages and activity from the time the program is opened to the time the program is closed Print Preview Display a print preview of the System Log Run Sample Toolbar The following example shows the Sample Setup module s Run Sample toolbar icon E Hun 5ample Platez Figure 3 7 Run Sample Toolbar Icon Click this toolbar button to open the Sample Plate Run Confirmation dialog box Sequence Dye Colors Toolbar The following example shows the Sample Setup modules Sequence Dye Colors toolbar For descriptions of the actions that occur when you click a toolbar icon see Table 3 12 AE CE GU rm Table 3 12 Sequence Dye Colors Toolbar Run Module Icon Description A
407. s parameters window The ID assigned by the system reflects the bin number with a decimal point and the number of bases over that of the bins For example ID 5 2 indicates a fragment with 5 repeats and 2 extra bases If you prefer a different allele ID you can click the system suggested ID and make changes The final classification is up to you Continue selecting points and viewing their traces until you are satisfied with all the points in the histogram The system stores the information for a new allele or the modified information of an existing one which you can apply in later records The system automatically recalculates the statistics based on the edited results GenomeLab Genetic Analysis System User s Guide PN A29142 AB Updating the Locus Tag and Allele List After completing the bin analysis the system generates an allele list based on the information gathered during the analysis It updates the list but at this point has saved it to the database The system prompts you for the locus tag to save to the generated allele list New Binning Analysis Sheps Parameters Binning Locus Tag Locus Tag Allele ID Criteria Locus Information Source Data Locus Mame Dye Fragment length range between 425 and AUD nt Repeat unit length 4 El nt of repeats in shortest Allele Locus Tag A Project Default m Fragment Analysis Module Performing Bin Analysis PII EI Date last modified
408. s screen will open 2 Enter the information into each of the parameter fields NOTE Multiplex files cannot be deleted from the database Make sure to name the multiplex so that it can be easily identified For example First pass Multiplex Name Final Multiplex Name HumanCancer_FirstPass HumanCancer_Final 3 Enter a multiplex name in the Save Setting field This name represents the parameter settings for this multiplex Click Save Setting The information will be saved to the database 5 Click Execute to generate the multiplex The screen displays the multiplex primer data See Figure 7 14 e Ifthe multiplex contains primers for every gene within the specified parameters click Save to Database e If one or more genes and or primers are not included then reduce the minimum separation size and click Execute to restart the multiplex formulation e Ifthe single or multiple genes and or primers are still not included select Verbose Mode and click Execute Verbose Mode provides information regarding the multiplex formulations and PCR products available for each gene This information can help to determine the reason why a particular gene and or primer was not included in the multiplex 6 Click Download to save the multiplex to a TDF file The primer sequences can be viewed by opening the TDF file with Microsoft Excel Use the Load Setting drop down list to load the multiplex settings at a later time or when parameter adjustm
409. s shown below indicate that data is associated with these wells To exclude a well from analysis RTminus No Template Control or unwanted sample click on and select the well and clear the Analysis option for the Analysis drop down list The well color will change to White y eXpress Analysis Plate Plate 1 File Help GeXP Analysis Plate Setup GexP Analysis Normalization B rj sample Layout Well Specific Settings Analysis Y Cal Peak Binning Figure 7 29 Sample Layout First View 7 Click on specific wells to select them for analysis The selected drop down lists under Well Specific Settings display the analysis specific information that has been applied to a specific well IMPORTANT If multiple wells are selected headers in red text indicate that the wells do not have the same settings The wells shown in Figure 7 31 have different sample names 302 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Gene Expression eXpress Analysis Module y eXpress Analysis Plate Plate 1 File Help GeXP Analysis Plate Setup GexP Analysis Normalization B x rj Sample Layout 5 Sample Layout well Specific Settings Analysis Multiplex Cal Sample Name Caf Data Files JA Peak Binning r Qum moomwxrx 222009990908 2 2909999 Figure 7 30 Sample Layout Well Selection 8 Select a multiplex from the Analysis drop down
410. s the 3 bases at 68 Click Execute Click Save to database to save the results to the eXpress Profiler database Tanels E g 50 2 requires primers to surround the 2 bases at positions 50 and 51 Or mark the source sequence with and e g ATCT ob i CCCC TCAT means that primers must flank the central CCCC Excluded E g 401 7 68 3 forbids selection of primers in the 7 bases starting at 401 and the 3 bases at 58 Or mark the source sequence with Regions and gt e g ATCT lt CCCC TCAT forbids primers in the central CCCC Max 3 Stability 2 0 Max Mispriming 12 00 Pair Max Mispriming 24 00 Figure 7 23 Advanced Options Targets Excluded Regions GenomeLab Genetic Analysis System User s Guide PN A29142 AB 7 5 Multiplex Designer Gene Expression Multiplex Designer Modify existing multiplexes or create new multiplexes with more control over primer selection This feature is particularly useful for incorporating redesigned primers into a previously designed multiplex express FASTA Blast Primer Design Multiplex eXpress Analysis eXpress Map Logout eXpress Designer Multiplex Create New Data p Load Setting Save Setting Multiplex Name TestPlex Minimum Separation Size Maximum PCR Product Size excluding Universal Tags 300 Minimum PCR Product Size excluding 1 00 niversal Tags Default Tags 0106 127 1448 20 1574 20 0181 240 1166 20 1 405
411. s the order of the loci in the DMPopulation file The DMPopulation export also requires that pedigree information at least the Individual ID be included in every result in the Study Additionally at least one result per individual must have a complete pedigree Population ID PopID Pedigree ID KinID Individual ID SubID Mother ID MID Father ID FID and Gender Sex Pedigree information is associated with samples through the sample plate editor using the sample property sets Pedigree template As an alternative you can associate the pedigree information with results using the single result view s property set tab Genotype Summary Report tab delimited gsr Accommodates Subject IDs the occurrence of more than two alleles and the absence of alleles as shown in the following illustration Use the Locus List Editor to customize the LocusList txt file before exporting a Genotype Summary Report For details see Creating a Genotype Summary Report on page 263 The report layout is described below e Column Row Individual headings are replaced with the heading of Subject ID e If the number of alleles is greater than two the cell will indicate Too many When an allele is not found the empty cell is replaced with Unknown Allele Subject 33 Subject 63 Subject 65 GATA193AD7 A1 GATA193A07 A2 D iei AT S E Tues a hee and Dein UU eee d D145588 A2 D151678A1 0151679 A Figure 6 44 Genotype Summary Report Gen
412. sal Tags Import Multiplex Help eXpress Profiler User Management GexP_User p Load User User has been loaded Reset Form First Name GexP Last Name User Username GexP_User Figure 7 3 eXpress Profiler User Management Screen Table 7 2 User Management Screen ltem Description First Name The user s first name Last Name The user s last name Username The name the user must enter each time they log into the GeXP application The username is case sensitive and can be any combination of letters numbers spaces up to 256 characters Password The password assigned to the user The password is case sensitive and can be any combination of letters numbers spaces up to 256 characters NOTE The user can change this password in the user account GenomeLab Genetic Analysis System User s Guide 269 PN A29142 AB Gene Expression Software Administration Viewing and Editing User Accounts View or edit the account of an existing user From the menu options click Users 2 Select the username from the drop down list then click Load User The user s current information appears in the fields 3 Edit any of the fields as needed Click Save User To display a new entry screen click Reset Form NOTE The Administrator can edit an existing user primarily to change the user s password NOTE To optimize database speed a generic user account can be set up for each project 210 GenomeLab Genetic Analysis S
413. scribes the File menu options within the Run Module Table 3 2 File Menu Run Module Option Description Restore Data Monitor Restores the colors of the data monitor to the original default shipped with the Defaults instrument Save Log Saves the system log in a folder you select The log is identified by the date and a unique identifier GenomeLab Genetic Analysis System User s Guide AT PN A29142 AB Run Module Run Module Overview 48 Table 3 2 File Menu Run Module Option Description system Preferences View and modify the System Name Operator Name Project Name and Dye Names for Fragment Analysis and or to activate or deactivate the alarms See Defining System Preferences on page 65 Print Setup Define printer properties Print Preview Displays a facsimile of a hardcopy printout of the system log prior to printing Print Log Prints the system log View Menu Click the View menu to display its drop down menu as shown in the following example view Toolbars wv Status Bar Status Monitor Working Database NOTE A v next to an option indicates that the option is enabled Activate or deactivate window display options The following table describes the Run module s View menu options Table 3 3 View Menu Option Description Toolbars Choose which toolbars to display Select vT the options that identify the toolbars you want displayed To hide a toolbar clear its check box See Toolbar I
414. se pooo g Minimum Duration 0 4 min a Save As Print Cancel Help Figure 4 11 Initial Data Detection Tab NOTE If you opened this dialog box by selecting View Parameters Used to Compute Sequence this dialog box displays the analysis values but disables the fields described below NOTE Set the Initial Data Detection parameters based on the sample cleanup method used If the cleanup method leaves large peaks due to unincorporated dye at the beginning of data set the parameters to avoid these blobs This makes it difficult to read to the end of the primer however the basecall accuracy improves since dye peaks do not interfere with early mobility correction If the cleanup method tends to leave dye peaks at the beginning of data set the Threshold to 30 or 35 and set the Delay to 0 75 If there are still fat peaks at the start of analyzed data make this a larger delay NOTE Ifthe cleanup eliminates dye peaks you can read closer to the end of the primer by setting the Threshold to 20 and the Delay to 0 6 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Analysis Using the Sequence Analysis Module The following table describes the options available on the Sequence Analysis Parameters Initial Data Detection tab Table 4 18 Initial Data Detection Tab Sequence Analysis Parameters Editor Option Description Threshold Enter the maximum ratio value of all the data in the energy profile a
415. sed Sample Access Lover Closed Capillary Access Lover Closed Capillary Access Cover Closed Voltage O 0 kv Voltage O 0 kv Total Current D p Total Current D u Figure 3 15 Device Tab Gel Plug installed Figure 3 16 Device Tab Gel Cartridge installed GenomeLab Genetic Analysis System User s Guide PN A29142 AB Run Module Using the Run Module Defining System Preferences To define the system preferences l System Preferences X Defaults Alarms Actve Inactive Select File System Preferences The System Preferences dialog box opens as shown in the following example System Name GenomeLab System Operator M ame Test Operator Project Mame Default Figure 3 17 System Preferences Dialog Box oS iiie uu ed Enter the System Name and Operator Name Select the Project Name Define Dye Names for the Fragment Analysis module if desired Enable or disable the alarms Click OK Running Sample Plates To run two sample plates once plate information has been created and saved for both plates 6 re 8 Select Run Start Sample Plate The Sample Plate Run Configuration dialog box opens Select the name of the sample plate from the Sample Plate Name drop down list for the Left and Right Plates Click Load Plates The Access Plates dialog box opens Click Start The Capillaries Exposed dialog box opens Follow the screen instructions in Capillaries Exposed dialog boxes to
416. selected in step 10 proceed to step 12 If Install Cartridge was selected in step 10 use the Install Gel Cartridge dialog box Figure 9 37 to enter the following information a If you are installing a new gel cartridge click New The system will automatically update the date and time installed and set the Hours on Instrument to 0 b Next enter the lot number c Ifyou are installing a used Gel Cartridge select Used e Ifyou are installing the previous gel cartridge do not change the lot number or the hours on the instrument as they will be correct e If you are installing a previously used gel cartridge but it was not the last one on the instrument enter its part number and or lot number and adjust the hours to properly reflect the hours this cartridge has been on the instrument d Click Done on the Install Gel Cartridge dialog NOTE Always note the lot number and the hours on the instrument for a gel cartridge if you are planning to use it for more than one session The lot number is an alphanumeric text box for your own identification purposes 370 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Maintenance and Diagnostics Direct Control and Replenishment Install Gel Cartridge Gel Cartridge Part Humber 331 438 C Lot Number eise Gel Name LRAT Help Date Installed 04 27 2006 Time Installed li 15 00 C Mew 6 Used Hours on Instrument 27 Figure 9
417. ses are called as N s and the information necessary to detect the start of data to be analyzed including the delay between the detected start of data and the start of data analysis the signal to noise ratio the minimum duration and the threshold above which data are considered peaks Fragment Analysis Parameter Sets including SNP analysis are used to process the fragment data to estimated fragment sizes and to identify alleles Fragment sizes are estimated by generating a calibration curve from a set of size standards run with the unknown samples The Fragment Analysis Parameter set defines the analysis model used to generate the calibration curve dye label mobility corrections allele identification criteria locus tags quantitation values and other factors that will affect how your data are analyzed GenomeLab Genetic Analysis System User s Guide 39 PN A29142 AB Sample Setup Module Using the Sample Setup Module Assigning Analysis Parameter Sets e Fora Sequence Analysis select a Sequence Analysis as the Parameter Set For Fragment Analysis select a Fragment Analysis as the Parameter Set Performing an Analysis on a Sample To perform an analysis on a sample 1 Select the cell cells as described in Selecting Samples on page 30 2 Select the Analysis tab at the bottom of the window 3 Select the Perform Analysis option 4 Select the parameter set from the Parameter Set drop down list see Figure 2 17 Note
418. sis The separation gel is automatically replaced in the eight capillaries after each separation The separation gel supply is an easily replaced cartridge with a capacity sufficient for two full microplates The instrument performs detection using laser induced fluorescence in four spectral channels It automatically processes the four channel raw data sets generated by each of the eight capillaries to produce high quality base sequences or fragment lists after separation Raw and analyzed data are stored in a database and may also be exported in file formats compatible with common analysis applications GenomeLab Genetic Analysis System User s Guide 1 PN A29142 AB Getting Started System Overview Hardware The hardware performs sample handling as well as tasks associated with the separation and detection phases of electrophoresis Figure 1 1 shows the GenomeLab instrument The following sections describe the GenomeLab instrument s user accessible hardware components
419. sis Module Batch Control Toolbar JAR Table 4 13 Batch Control Toolbar Sequence Analysis Module Icon Description Batch Analysis Analyze multiple sample data or sample plate results sequentially Batch Alignment Select sequence results or sample plate results to align Batch Trimming Trims multiple sample data or sample plate results sequentially Reanalyze Batch After performing a batch analysis use this option to perform a new analysis on the same data set stop Analysis Terminates the analysis currently in progress analyzed Use Resume Batch Processing to continue the analysis m Pause Momentarily stops a batch analysis after the currently running sample has been ire Resume Resumes a batch analysis after it has been paused okip Sample While performing a batch analysis click this icon to pass over the sample 7 currently being analyzed This item is not available if Sample Plate Results was selected from the Batch Analysis Selection dialog box GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Analysis Sequence Analysis Module Overview Table 4 13 Batch Control Toolbar Sequence Analysis Module Icon Description Skip Set While performing a batch analysis click this icon to pass over the sample set d currently being analyzed This item is not available if Sample Data was selected from the Batch Analysis Selection dialog box Skip Plate While performing
420. size and click Execute to restart the multiplex formulation e Ifthe single or multiple genes and or primers are still not included select the Verbose Mode option and click Execute Verbose Mode provides information regarding the multiplex formulations and PCR products available for each gene This information can help to determine the reason why a particular gene and or primer was not included in the multiplex 10 After the multiplex has been saved to the database click Export and browse to the desired location then save the new multiplex TDF file Open the TDF file in Excel to view the information The primer sequence data from this TDF file can be used to order oligonucleotides NOTE Do not order KAN primers They are included in the GeXP Start Kit chemistry Use the Reset Form option to restore the default settings and clear any selected genes or primers GenomeLab Genetic Analysis System User s Guide 295 PN A29142 AB Gene Expression Multiplex Designer 296 Viewing and Exporting Existing Multiplexes View existing multiplex primer data and export the sequences to order primers for multiplex formulation Log into eXpress Designer Click Multiplex Click View Existing Data to view the primer sets of multiplexes that are currently saved in the database Select the multiplex from the drop down list Click Load The multiplex primers list will open Click Export to export the multiplex TDF file and save the file to th
421. so requires that every result in the Study includes the pedigree information at least the individual ID It also requires that at least one result per individual has a complete pedigree Pedigree ID Individual ID Father ID Mother ID and Gender Pedigree information is associated with samples through the sample plate editor using the sample property sets Pedigree template As an alternative you can associate the pedigree information with results using the single result view s property sets tab Discovery Manager DMPopulation txt Contains the export parameters of a Study s pedigrees and genotypes The DMPopulation file is designed to be imported into Genomica s Discovery Manager application The DMPopulation file can also be imported into Discovery Manager as a DMFastPopulation file The pedigree and genotype information from each result in the Study is included in the DMPopulation file The DMPopulation export produces two files e txt the DMPopulation file log a list of processing steps and errors encountered during the export GenomeLab Genetic Analysis System User s Guide 261 PN A29142 AB Fragment Analysis Module Exporting Results 262 The DMPopulation export requires a list of locus names in a file named LocusList txt This file must be stored in the CE system s export or root directory This text file must have only one locus name per line The order of the loci included in this file determine
422. ssibilities if more possibilities exist an asterisk appears instead The consensus sequence is editable within the Sequence Investigator Edited bases appear in lower case letters Using Quality Values e If you are using a single sequence the quality value assigned to each base in the consensus match each base in the sequence e When using two sequences and the bases agree one of two calculations occur e If the orientation of the two sequences is opposing the system adds two quality values e If the orientation is the same the system uses the higher quality value e If the bases disagree the system performs a calculation to determine the probability of error for that position providing a quality value which is the difference between the higher and lower value To set the value at which bases in the consensus are considered low quality select Edit Quality Threshold Low quality bases are identified in the differences line the navigator toolbar and the discrepancy map Viewing Sequence Investigator Differences The system uses codes to denote the type of discrepancy encountered when performing the comparison These codes are shown in the differences section of the alignment pane To turn the differences line on or off select View Differences The highest level code appears in the differences line in the location where the discrepancy occurred NOTE Any consensus position may have multiple difference codes associated wit
423. strument System Help By default the help system displays the Contents in the navigation pane located on the left and the introductory topic in the topic pane located on the right You can use the Contents to select and print specific topics in CE Instrument System Help The navigation pane also provides a Search tab and an Index tab These tabs let you search for information by topic index entry and or keyword Context Sensitive Help Click this icon and then on a menu item to open the Help file related to the selection 326 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Database Management Data Manager Procedures 8 2 Data Manager Procedures Storing Methods and Parameters The USERTEMPLATE database stores newly created methods and parameters This database is added when the during software installation These methods and parameters are then included in all subsequently created databases Use the Cut Copy and Paste functions to add methods and parameters to the USERTEMPLATE Although you cannot set the USERTEMPLATE database as the working database you can delete it If this database is deleted no template will be used for subsequent databases If you need a new template create a new one named USERTEMPLATE Creating a Database 2 Click Data Manager on the Main Menu In the Data Manager window right click the GENOMELAB DBASE node and select New Database The New Database dialog box opens
424. sults 2 Right click to highlight the type of report you would like to generate from the list of those available in the Reports tab and select New The New Report window opens displaying the Context Type for your selection 3 Select the report template from the Template drop down menu The selections available depend on the Context Type 4 Click OK to display the new report containing the selected information Using Report Templates There are several types of elements in each report template e Header text for data tables or graphs For example Bin Scatter plot e Regular Data Objects For example Context Locus Name Locus Loop Delimiters For example BeginLoop Allele Context Locus Alleles ID Allele ID Nominal Size FormatNumber Allele TrueSize 0 Apparent Size FormatNumber Allele ApparentSize 2 Standard Deviation FormatNumber Sqr Allele FragmentSizeVariance Number of Data Points Allele FragmentCount Comment Allele Comment EndLoop Allele Context Locus Alleles e Table Delimiters TableLoop Allele Context Locus Alleles ID Nominal Size nt Apparent Size nt Standard Num Points Comments Deviation nt Allele D FormatNumber FormatNumber FormatNumber Allele Fragment Count Allele Comment Allele TrueSize Allele Apparent Sqr Allele Frag m 0 oize 2 entSizeVariance Graph Objects For example Graph Service BinGraph Context Site DisplayOptions 252 GenomeLab Genetic Analysis System
425. sults Output check boxes to include the sequence results The format expresses the data points as text values which can be viewed in any text editor or in Excel The PHRED format produces two files a PHRED file and an SCF file A PHRED file is a text file composed of a SEQUENCE section containing a COMMENT and DNA section The SEQUENCE section also contains the name of the Analyzed Result that was stored The COMMENT section contains the file name of the associated SCF file CHROMAT FILE that was produced along with the PHRED file The values for ABI THUMBPRINT and PHRED VERSION are fixed text N A The values for CALL METHOD and QUALITY LEVELS are fixed as CEQ SEQUENCING and 99 respectively for the CE system The TIME stamp is the current time at the time of export The DNA section is composed of the base call always lower case PHRED Quality Value and the Peak Index data point of the analyzed data contained within the associated SCF file Values are separated by spaces GenomeLab Genetic Analysis System User s Guide PN A29142 AB Database Management Data Manager Procedures Table 8 9 Data Manager Export Options Format Description ESD esd The ESD electropherogram sample data format exports raw data from the CE system to a third party software package for analysis This option exports only raw data and does not include any other information SEQ seq The SEQ format only applies to the sequence res
426. t Programs GenomeLab System Control Center The instrument initializes and after several seconds displays the Main Menu GenomeLab Genetic Analysis System User s Guide 13 PN A29142 AB Getting Started Operating the System 14 Creating a Database and Project Folder Opening the Data Manager Module From the Main Menu click on the DATABASE icon and verify that the Data Manager window is displayed Creating a Database To create a database l In the Data Manager window select File New Database The New Database dialog box opens Enter the name for this new database Select Set as Working Database Click OK Creating and Naming a Project Folder To create and name a project folder l In the Data Manager window highlight the database where the project will reside and select File New Highlight the new project and select File Rename Enter a descriptive name for this project and press Enter Select File Exit to close the Data Manager module Setting up a Sample To set up a sample Sn a ae N Se LIIIIIIITITI i i H H 4 a From the Main Menu click the SETUP icon and verify that the Sample Plate Selection dialog is displayed Select the Create a new sample plate radio button and click OK Set up the sample plates by naming the desired cells Add any associated notes or property information Select a method from the drop down menu at the bottom of the sample set Select the a
427. t tasks listed in the Direct Control and Replenish drop down menus For details see Direct Control Menu on page 49 and Replenish Menu on page 52 Data Monitor Access Plates Right click anywhere in the window to open a full menu Direct Control Log Instrument Data Access Plates Plate Position Capillary Temperature Denature Samples Optical Alignment Inject Separate Gel Capillary Fill Manifold Purge Capillary Information Install Gapillary Stray Release Capillary Array Gel Buffer Information Install Gel Gartridge Release Gel Cartridge Replace Wetting Tray 89995 73101 Senarate Figure 3 11 Direct Control Window Run Module The example above identifies the selectable areas on the Direct Control window When you click a selectable area the system initiates a unique Direct Control task Table 3 16 lists the tasks available in this window Table 3 16 Direct Control Window Selectable Areas Item A B Description Capillary Temperature Change the capillary temperature Gel Capillary Fill Fill all capillaries with fresh gel Any gel present in the capillary is forced out to the specified waste position in the buffer plate or wetting tray Optical Alignment Align the lasers with the detection windows of the eight capillaries Data can be saved and viewed in the Sequence Analysis module GenomeLab Genetic Analysis System User s G
428. t Sensitive Help Click this icon and then on a menu item to open the Help file related to the selection GenomeLab Genetic Analysis System User s Guide 27 PN A29142 AB Sample Setup Module Using the Sample Setup Module 2 2 Using the Sample Setup Module Open set up import and export sample plates To open the Sample Setup module click the Sample Setup icon on the Main Menu Opening a Sample Plate Open an existing sample plate or create a new one l Getto this location in the software 2 Step instructions The Sample Plate Selection dialog will open Sample Plate Selection Create a new sample plate Open an existing sample plate Sample Plates Database GEXP MODULE 2006 Filter Start Date 02 14 2006 E Enable Cancel End Date 0214 2008 Hefresh Help Project Hame Find Barcode Figure 2 5 Sample Plate Selection Dialog Box Creating a New Sample Plate l To create a new sample plate select the Create a new sample plate radio button 2 Click OK The Sample Plate window opens 28 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sample Setup Module Using the Sample Setup Module Opening a Sample Plate 1 To open an existing sample plate select the appropriate plate from the list 2 To help in the search filter the plates by their displayed date Selecting the Enable option 3 Use the Up and Down scroll arrows to change the Start Date and End Date
429. t screen press the Esc key or click the Close button Cut Deletes text selected in one or more cells of the sample plate which you can paste into a different cell or cells Copy Duplicates text selected in one or more cells of the sample plate which you can paste into a different cell or cells Paste Inserts into one or more cells the text copied to the buffer when using the Copy or Cut function Unlock Indicates that the active plate is available for editing Clicking this icon locks the plate to prevent editing and changes the icon to Run Opens the Run module and executes the active sample plate select All Highlights selects all cells in the sample plate Clear All Removes all cell properties in the sample plate View Summary Displays a summary of a specific sample plate To use this open the desired sample plate then click this icon The Sample Plate Summary spreadsheet appears with a summary of each sample in the plate This is a read only spreadsheet and cannot be edited Help Topics Displays the CE system online help By default the help system displays the Contents in the navigation pane located on the left and the introductory topic in the topic pane located on the right You can use the Contents to select and print specific help topics The navigation pane also provides a Search tab and an Index tab These tabs let you search for information by topic index entry and or keyword Contex
430. t these columns individually or in groups arrange them in any order and lock them so they always remain in view when scrolling through the fragment list Accessing the Column Selector Window To access the Column Selector window right click the column header area of the list to display then select Select Columns The Column Selector window opens as shown in the following example Column Selector P3 Possible Columns Result Name Data Exclusions i ms si Filter ID Siencand General Peak Classification Ctandard Eis Sour Ho fragment size estimate Estimated ie Separation Method Peak Area ae Date Method Modified Peak Height uU Quantitation Standard Locus i Length Attributes Edited gt Wo confidence interval es at Average Migration Time pm Allele ID Allele Match Quality Comment Locus Specific Information P gt No Alleles Found m Unknown allele ee Too many alleles Drusus si Stutter Peak settee gt Spurious Peak si Minus A Figure 6 35 Column Selector window Selecting Columns for Display in the Table NOTE The application area in which you select the Column Selector window such as Result Set View Fragment List or AFLP Cluster Analysis determines which columns are available for display NOTE Refer to the online help for detailed descriptions of the available columns and their use Selecting Col
431. tab of the new capillary array align the Array Fitting Figure 3 38 with the manifold opening and guide pins Push the fitting into the manifold until it is completely seated against the bases of the Guide Pins Make sure that the fitting tab is placed downward when inserting the capillary array into its slot While holding the Electrode Block tab align the Electrode Block Figure 3 39 with the guide block pins and gently push it in until resistance is met The resistance is from the spring loaded contacts When installing the capillary array carefully route the capillaries through the hole in the Plenum Assembly Figure 3 41 and straight across the guide block pins Figure 3 39 CAUTION N USE THIS FRONT PLENUM ONLY WITH A 38 CM CAPILLARY ARRAY 900697L Al Figure 3 41 Plenum Assembly Capillary Routing Hole NOTE When reinstalling the Plenum Assembly gently gather the capillaries between your thumb and forefinger to make sure they pass through the hole in the Plenum Assembly without any constriction 14 15 16 17 Replace the Plenum Assembly and tighten the two captive screws Figure 3 37 Replace the Manifold Access Cover and tighten the captive screw Figure 3 36 Lower the Capillary Temperature Control Cover and secure the two rubber latches Lower the Capillary Access Cover and Sample Access Cover to their locking positions e If you are installing a new capillary array in the Install Capillary Array dialog b
432. te the confidence interval value generated by automatic binning select Overwrite system interval value check box e Ifyou are overwriting the system generated value specify a confidence interval number of nucleotides in the field provided GenomeLab Genetic Analysis System User s Guide 21 5 PN A29142 AB Fragment Analysis Module Using the STR Locus Tag Editor Locus Tag Editor Locus Tag GSTAT33AU7 Project 3A T 183ALU Stutter Definition Search for Stutter Stutter detection window width hi asimum relative stutter peak height W 7 Detect stutter shorter than alelle Detect stutter longer than allele Confidence Interval System generated allele confidence interval Overurite system confidence interval PII Es Save As Date last modified 4 19 2001 3 22 05 PM Spurious Peak Detection Detect spurious peaks t asimum height for spurious peaks 20 xa A Detection Apparent size includes Detect Use 4 peak to call Allele Comment Cancel Figure 6 18 Locus Tag Editor Window Allele ID Criteria Tab Stutter Definition Stutter consists of small peaks generally DNA amplification artifacts that form as a result of halted polymerase activity When this happens allele fragments form that differ in length from the true allele by an integral number of repeats such as n 1 n 2 etc The absolute size of a fragment is a function of the variety of repeats for
433. te to generate a new multiplex See Figure 7 25 Click Save to Database to save the new multiplex to the database Click Export to export the new multiplex TDF file and save the file to the desired location The GeXP chimeric primers containing gene specific and universal tag sequences can be retrieved from the TDF file Use these sequences to order oligonucleotides NOTE Do not order KAN primers They are included in the GeXP Start Kit chemistry GenomeLab Genetic Analysis System User s Guide PN A29142 AB Gene Expression Multiplex Designer Performing a Gene Expression Analysis with the Multiplex Design At this point the individual multiplex primers are ready to be combined into forward and reverse multiplexes For instructions on how to build the primer multiplexes see the GenomeLab GeXP Chemistry Protocol A29143 Once the multiplexes are built and optimized gene expression assays are performed and analyzed with the GenomeLab GeXP Genetic Analysis System The data is analyzed using Fragment Analysis and the fragment data is transferred to eXpress Profiler Analysis module for normalization GenomeLab Genetic Analysis System User s Guide 297 PN A29142 AB Gene Expression eXpress Analysis Module 7 6 eXpress Analysis Module Import fragment data from the GenomeLab GeXP Genetic Analysis System into the eXpress Analysis module of eXpress Profiler for normalization and statistical analysis Create and export a report of the
434. ted size calibration standard calibration curve and optional dye mobility correction e Currently assigned locus tags that have been selected for this Study Click Next to proceed to the Analyze Data window NOTE If the available parameter sets do not apply to your Study you can create a new set of analysis parameters or edit an existing set to meet the requirements of your Study Refer to Defining Fragment Analysis Parameters on page 200 or the online help for more information Analyzing the Data With the new data selected and the analysis method defined the third step in creating the Study is to analyze the data The Analyze Data window displays all samples selected for analysis the date each was collected the status unanalyzed of each sample and the parameter set being applied to the Study see Figures 6 10 e To begin the analysis click Analyze As the system proceeds with the analysis the progress bar becomes active and the lower dialog box displays the analysis log for each sample as it is analyzed 198 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Fragment Analysis Module Working with Studies in the Fragment Analysis Module e To stop the analysis while it is in progress click Stop The system stops the analysis when it has finished with the sample in progress KY Analyze Data xi Steps Raw Data Analysis Parameter FABOD Time started 4 25 2006 12 58 58 PM Analysis Parameters Status
435. that can led to Taq polymerase slippage and result in stutter peaks If repeat sequence s are found redesign the primer s such that a region without repeat sequence is targeted Identifying SNPs Examine each primer for single nucleotide polymorphisms SNP especially at the 3 end that can lead to the preferential amplification of one allele over another If a polymorphism is found redesign the primer to a more conserved region Go to NCBI BLAST SNP website http www ncbi nlm nih gov SNP snp_blastByOrg cgi Confirm that the Program is blastn and that Megablast will be performed Choose a SNP BLAST Database based on the species of origin Enter the amplicon sequence in FASTA format into the Query Sequence text box Select the Human Repeats option to identify repeats in human gene sequences optional pc SP p xc e qe Click Submit Query The query is entered into the NCBI queue and a Format Request page appears 7 Under Format Display make selections for the Masking Character and Masking Color options to repeat mask the sequence optional 8 Click View report 10 Once the results page appears scroll down to Alignments to identify SNPs and repeats GenomeLab Genetic Analysis System User s Guide 28 7 PN A29142 AB Gene Expression eXpress Designer Module Identifying Homologous Sequences e Ifa gene is a member of a gene family has highly homologous regions or is an alternative transcript consider using the BLAST func
436. that will be displayed Selecting one of these options toggles between showing and hiding the corresponding toolbar See Toolbar Icons on page 171 Toggles between showing and hiding the status bar Shows each sequence in its entirety Opens the history log Toggles base numbering off or on Toggles codon numbering off or on Toggles between showing and hiding the sequences Toggles between showing and hiding the reference amino acid translation Toggles between showing and hiding the consensus amino acid translation Toggles the sequence differences off or on Toggles color used for text and background Displays the active database 169 Sequence Investigator Module Sequence Investigator Module Overview Window Menu Click the Window menu to display its drop down menu as shown in the following example Window Cascade Tile Horizontally Tile Vertically Close All Arrange Icons w 1 lt Untitled gt Use the Window menu to display data as desired The following table describes the Sequence Investigator module s Window menu options Table 5 5 Window Menu Sequence Investigator Module Option Description Cascade Cascades the open windows Tile Horizontally Tiles the windows in a horizontal orientation Tile Vertically Tiles the windows in a vertical orientation Close All Closes all open windows Arrange Icons Arranges all icons on the desktop Open file list Displays a list of all open documents Help
437. the Analysis Parameters window see SNP Locus Tags Tab on page 208 2 Select SNP Analysis in the General tab of the Analysis Parameters window see General Tab on page 201 3 Select an appropriate SNP Size Standard Size Standard 80 in the Analysis Method tab of the Analysis Parameters window see Analysis Method Tab on page 202 Asize standard with exactly two fragments must be selected for SNP analysis to succeed e The size standards must bracket all expected SNP fragments Both of these fragments must be selected The only valid model for a two fragment standard is the Linear model These three requirements enable you to successfully run the SNP analysis even if you do not select the mobility calibration but the values obtained might vary slightly If you want to correct for the mobility differences between the reference fragments and each of the four possible dye labeled SNP fragments use the Advanced tab of the Analysis Parameters window to select the SNP Dye Mobility Calibration see Advanced Tab on page 210 NOTE The SNP Ver 1 Dye Mobility Calibration is for use with the GenomeLab SNP Primer Extension Kit and the SNP Ver 2 calibration is for use with the GenomeLab SNPStart Primer Extension Kit GenomeLab Genetic Analysis System User s Guide 209 PN A29142 AB Fragment Analysis Module Defining Fragment Analysis Parameters Advanced Tab Set additional fragment analysis parameters From this window you
438. the folder in which you want to save the export file 4 Enter the desired file name in the File name field NOTE Do not use special characters in the file name lt gt 1 5 Select one of the four available export formats from the Save as type drop down list e CSV Comma csv e Linkage Pedin pre e Discovery Manager DMPopulation txt e Genotype Summary Report tab delimited gsr 6 Click Save to start the process and save the export file using your specified file parameters The export status along with a processing log appears in a status window This window indicates when the export procedure is complete and any export errors that may occur Transferring Fragment Data to eXpress Profiler Transfer fragment data from the GeXP System to eXpress Profiler for use with normalization of gene expression From the menu options click Fragment Analysis Open a set of analyzed GeXP data Select File Transfer Fragments for GeXP Click Transfer Fragments for GeXP as After the dialog box appears name the file and browse to a transfer location PV d ups ee ee i Click Save to save the exported file as a CSV file A message box will display confirming the export process 7 Click OK to confirm the completion of the process and close the message box 260 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Fragment Analysis Module Exporting Results For additional information on using Fragment Data
439. the lower analysis log area of the window Click Next to proceed to the Select Result Data window Selecting Additional Results to add to the Study Add result data to your new Study in this fourth step Additional result data may include samples that were previously analyzed under a different set of analysis parameters than those analyzed in the preceding steps of the Study currently being developed The Select Result Data window is identical to the Select Raw Data window displayed in the first step of the Study Wizard NOTE Refer to the Selecting Raw Data for the New Study on page 195 for an explanation of the features available in the Select Result Data window Select any additional results that you would like to add to the new Study and click Finish to complete the Study To save your new Study select File Save or File Save As GenomeLab Genetic Analysis System User s Guide 1 99 PN A29142 AB Fragment Analysis Module Defining Fragment Analysis Parameters 6 4 Defining Fragment Analysis Parameters The following sections describe how to edit a parameter set or create an entirely new set of analysis parameters to meet the specific requirements for your samples You can create or edit parameter sets using the Fragment Analysis Parameters window This window contains a number of tabs in which you define the general parameter set information set locus tag parameters select an analysis method assign quantitation values and
440. the reference amino acid translation the reference the sequences the difference codes the consensus and the consensus amino acid translation History Log Prints a history of changes you performed in the alignment Statistics Prints the statistics of the report such as the number of discrepancies before and after editing Mutations Prints all mutations including known and novel bases and amino acids Exporting the Sequence You may export the active compared sequence in either of three formats as described in the following table Table 5 9 Export Formats Format Description text txt Exports the base sequence of the consensus GenomeLab Genetic Analysis System User s Guide 181 PN A29142 AB Sequence Investigator Module Sequence Investigator Procedures 182 Table 5 9 Export Formats Format Description tab delimited Exports data as a single line and all carriage returns 1 are replaced text txt with tabs 2 protein pro Exports the amino acid sequence To set the protein format select File Preferences and choose one of the following format in the Preferences dialog box Condensed Provides all protein translations in the same text line and designates heterozygotes between brackets Expanded Includes all possible translations on different lines The following example compares the differences between condensed and expanded protein formats KVS IM LPLV LP K Condensed or KVSILPLVLK KVS
441. the result trace with a dark background and moves the trace to the top of the Result Set Traces area of the window to distinguish it as the reference result trace It marks the corresponding result with an R in the left most column to distinguish it as the reference peak To de select the reference result right click the reference result trace and select Reference again to toggle the command to the off position unchecked The system removes the R from the de selected trace returns it to its original highlight status and position in the Fragment Result Set Traces area of the window Selecting a Reference Peak A reference peak is a trace peak you select from the reference result trace The system compares the reference peak to the test peak calculates a relative length height and area then adds the results to the Peak Ratio Results Table To select the reference peak right click an individual peak from the selected Result Set Trace located at the top of the Fragment Result Set Traces area of the window and select Reference Peak The system then calculates the length area and height of the test peaks by comparison and enters this information into the Quantitation Result Table e To de select the reference peak from the Reference Result Peaks table right click the reference peak and select Set Peak As Reference again to toggle the command to the off position unchecked To de select the reference peak from the Frag
442. the two data sets Red datapoints indicate a significant correlation Grey data points indicate little to no correlation NOTE The data shown on these tabs is for viewing purposes only and cannot be saved modified or printed 314 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Gene Expression eXpress Map Module Creating K Means Clusters Group or classify data into a user specified number of clusters k based on similar expression profile attributes K means clustering is an algorithm used to classify or group genes based on patterns into k numbers of groups k is a positive integer number The grouping is done by minimizing the sum of squares of distances between the data and the corresponding cluster centroid Thus the purpose of K means clustering is to classify the data based on similar gene expression patterns Log into the eXpress Profiler and click on eXpress Map in the Menu Click K Means The parameters dialog box will open to set the parameters for clustering Set the number of clusters K Set the number of iterations Change the cluster initialization Select the distance measure means ee a m Click OK to generate data clusters File Tools Help H 8g Table Profile Ral Correlation K means B Table e Profile Correlation gag K Means LA Er Er Figure 7 39 eXpress Map K Means GenomeLab Genetic Analysis System User s Guide 3
443. tion above to verify that only the desired gene product will be amplified with the designed primers e If multiple different genes are retrieved from the BLAST search redesign the primer s to target a region of low homology especially at the 3 end of each primer Preventing Undesigned Peaks UDPs Any set of primers that has the potential to generate an undesigned peak UDP within the size range of the multiplex should be redesigned to prevent the production of a UDP in the multiplex profile Once the primer and amplicon sequences have been reviewed proceed with ordering and testing the multiplex primers Order the primers with the appropriate universal tag sequence IMPORTANT Do not order the KAN primers as they are included in the GeXP Start Kit Chemistry 288 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Gene Expression eXpress Designer Module Designing Primers Design individual sets of custom primers from difficult or proprietary gene sequences target or exclude a certain area of a sequence or change the parameters to more tightly define PCR product size for a selected gene The Primer Design feature is useful for redesigning primers that were rejected during primer evaluation or do not perform well within a multiplex In order to use Primer Design the gene sequence must be previously entered into the database individually or as part of a multiplex See Retrieving Gene Sequences with FASTA on page 284
444. tion in seconds 3 Identify the position of the Sample Set 4 Click Denature Injecting a Sample l Select Direct Control Inject The Inject dialog box opens Voltage 2 0 kM Cancel Duration 30 ZEC Help Sample Plate Position Sample Set fe Plates Figure 9 19 Inject Dialog Box 2 Enter a value for the voltage in kV 3 Enter atime duration in seconds 4 Identify the position of the sample using the Sample Set spin controls 5 Select Inject GenomeLab Genetic Analysis System User s Guide 357 PN A29142 AB Maintenance and Diagnostics Direct Control and Replenishment Performing a Separation l Select Direct Control Separate The Separate dialog box opens Values Voltage PAI Cancel Curation 35 0 min Help Buffer Plate Position Buther Set E Plates Figure 9 20 Separate Dialog Box Enter a value for the voltage in kV Enter a time duration in minutes Identify the position of the buffer using the Buffer Set spin controls pt od lem gie Select Separate NOTE Data will not be saved Replenishing the Capillaries with Gel l Select Direct Control Gel Capillary Fill The Gel Capillary Fill dialog box opens Waste Position C Buffer Plate fe Cancel Wetting Tray Plates Help Figure 9 21 Gel Capillary Fill Dialog Box 2 Select the Buffer Plate or Wetting Tray radio button to identify the position where waste wil
445. tion pane located on the left and the introductory topic in the topic pane located on the right You can use the Contents to select and print specific help topics The navigation pane also provides a Search tab and an Index tab These tabs let you search for information by topic index entry and or keyword Context Sensitive Help Click this icon and then on a menu item to open the Help file related to the selection GenomeLab Genetic Analysis System User s Guide 1 07 PN A29142 AB Sequence Analysis Sequence Analysis Module Overview Data Toolbar The following example shows the Sequence Analysis module s Data toolbar For descriptions of the actions that occur when you click a toolbar icon see Table 4 10 alale aale BALA ale ss Figure 4 3 Data Toolbar Sequence Analysis Module Table 4 10 Data Toolbar Sequence Analysis Module Icon 108 Description Zoom Mode Magnifies a specific area of data in the display Pan Mode Move analyzed data in the X and Y direction Align Mode Visually aligns bases and peaks in the analyzed data pane Edit Mode Insert change and or delete bases Unzoom Zoom out one level Unzoom All Zoom out all levels Autoscale Scales data to the confines of the pane If autoscaling is disabled the data is scaled to its true values Use this item to turn autoscaling on or off Base Spacing Sets the space between base sequence text in groups of 0 3 5 or 10 Base S
446. to run a sample plate and the gel cartridge does not have enough gel to process any sample sets in that plate the system displays the message shown in Figure 3 23 Sample Plate Gel Usage Confirmation There iz only enough gel in the system to fully process the first O of the 3 requested sample sets Do you want to continue Figure 3 23 Gel Usage Confirmation Dialog Box To end the run and return to the Run main window click Abort Pausing a Sample Plate Run e To pause the currently executing sample plate click the Pause ul icon The system pauses at the next executable step of the sample plate e To resume the run click the Pause u icon again Pause to Load To pause a current run so you can load another plate GenomeLab Genetic Analysis System User s Guide PN A29142 AB Run Module Using the Run Module 1 Select Run Pause to Load to pause the system and load another plate The Pause To Load Plates dialog box opens as shown in the following example Pause To Load Plates Ed Options i Load plate after current sample set Cancel Stop current sample set now to load plate Stop current sample plate now to load plate Help Save Collected Data Perform Shutdown Method I Audible Alarm The current mas wait time is 30 minutes Modify ait Time Figure 3 24 Pause to Load Plates Dialog Box 2 Select one of the following load options e Load plate after the current sample set Th
447. tor Logout Please make a selection from the menu an the left Figure 7 2 eXpress Profiler Welcome Screen Logged in as Administrator The Administrator can perform the software functions noted in Table 7 1 Table 7 1 eXpress Profiler Administrator Menu Options Option Function Users oet up user accounts See Managing User Accounts on page 269 Account Change the password assigned to the software Administrator account See Managing User Accounts on page 269 Manage Genes Enter sequences without GenBank accession numbers and edit all genes in the system see Managing Genes on page 272 Manage Primers Add primers produced in third party software and edit all primers in the system See Managing Primers on page 274 Universal Tags Define the Universal Tag sequences which are appended to primer sequences created in the eXpress Designer See Managing Universal Tags on page 276 Import Multiplex Import newly developed gene set kits as well as user defined multiplexes from other GeXP systems into the database See Importing a Multiplex on page 276 268 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Gene Expression Software Administration Managing User Accounts Set up or edit user accounts Creating a New User Account l From the menu options click Users 2 Enter the users first last name username and password 3 Click Save User Users Account Manage Genes Manage Primers Univer
448. ttom buffer plate Each well being 3 4 full 4 pack DNA Size 608395 Contains fragments of 13 and 80 nucleotides designed to Standard Kit accommodate a wide range of sizes for multiplexed and 80 poolplexed SNP fragments DNA Size 608098 1 DNA size standard for analysis of fragments up to 400 Standard Kit nucleotides Includes 400 e Mineral Oil e DNA fragments of the following sizes labeled with WellRED fluorescent dye 60 70 80 90 100 120 140 160 180 190 200 220 240 260 280 300 320 340 360 380 400 and 420 nucleotides e Sufficient for 96 fragment analysis separations DNA Size 608095 DNA size standard for analysis of fragments up to 600 Standard Kit nucleotides Includes 600 e Mineral Oil e DNA fragments of the following sizes labeled with WellRED fluorescent dye 60 70 80 90 100 120 140 160 180 190 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 and 640 nucleotides e Sufficient for 96 fragment analysis separations SNPStart Primer A23201 1 Dye Terminator SNP Primer Extension Kit for 96 reactions in a Extension Kit single tube containing e oNPStart Master Mix e Control e Mineral Oil e Sample Loading Solution SLS PN 608082 GenomeLab Genetic Analysis System User s Guide 3 9 PN A29142 AB Maintenance and Diagnostics Consumable Items List Table 9 15 Consumable Items Required for Fragment Analysis Item P N QTY Description GeXP Start
449. ubber latches holding the Capillary Temperature Control Cover and lift it to the vertical locking position Figure 3 47 6 Loosen the Manifold Access Cover captive screw remove the cover and set it aside GenomeLab Genetic Analysis System User s Guide 91 PN A29142 AB Run Module Using Direct Control 92 H o o 2 s Figure 3 47 Manifold Access Cover 1 Capillary Temperature Control Cover 4 Captive Screw 2 Plenum Assembly 5 Manifold Access Cover removed 3 Manifold Access Cover Lift the Eject Lever to release the Manifold Plug Grasp the Manifold Plug tab Figure 3 48 and then a bi C d e Pull the plug approximately one inch out of the manifold Touch the tip of the plug to the bottom of the Optics Base Plate Hold and wait five seconds for the gel strand to dry Pull the plug out and set it aside for future use Wipe gel strands off of the instrument using a damp tissue GenomeLab Genetic Analysis System User s Guide PN A29142 AB Run Module Using Direct Control 901597L Al Figure 3 48 Removing the Manifold Plug 1 Guide Pins 4 Gel Strand 2 Eject Lever 5 Optics Base Plate 3 Array Fitting 6 Array Fitting 9 Click OK on the Remove Manifold Plug dialog box 10 To install the capillary array see Removing and Replacing the Capi
450. uence Investigator Procedures 3 Select the sample reference and click Open The Open Sequence Results dialog box opens as shown in the following example Open Look in E Heterozygote and Alignment tutorial 4m E Y II VITIF 12 2 Ext pos genomic rc ExE o E File name PI VITIETI 2 2 Files of type Reference files bet Cancel Jl Figure 5 5 Open Reference Dialog Box 4 Select one or two results and click OK NOTE To select two results left click the first result press and hold the Ctrl key then left click the second result Select 1 or 2 Sequences to Compare Ea Sequence Results Database Filter CEQ OG TESTING_GENESIS Start Date 0117 2008 E Enable Cancel Ends 01 17 2006 Fated Help EAA 04 26 06 1 1516 612 001004033 ESAE 04 26 06 1 15 16b 601_00110221 Seg Test A03_06 03 02 06 1 Seg Test A03 0603011 Seq Test A03_06 03 02 06 1 Seg Test A03 00603011E Seq Tesha04 06 03 02 06 1 Seg Test A04 06030115 Seq Teshad4 06 03 02706 1 Seg TestA04 0603011 Seg Test A05_06 03 02 06 1 Seg TestA05 0603012 Seq Test A05_06 03 02 06 1 Seg Test A05 06030120 Seg Test B03_06 037 02 06 1 Seg Test B03 0603011 Seq Test B03_06 037 02 06 1 Seg Test BUS 0603011 Seq Test B04_06 03 02 06 1 Seg Test B04 0603011 Seq Test B04_06 03 02 06 1 Seg Test B04 06030115 Seq Test B05_06 03 02 06 1
451. uide PN A29142 AB Run Module Run Module Overview Table 3 16 Direct Control Window Selectable Areas Item Description D Manifold Purge Clears the manifold of air bubbles and or remaining DNA fragments before the next separation process E Install Release Gel Cartridge Releases and or installs a gel cartridge Denature Samples Initiates the denaturing of samples G Plate Position Select the plate or tray position where the capillaries will be immersed H Wetting Tray Replace or refill the wetting tray Install Release Capillary Installs the capillary array Verify or change the information requested and then install the array Access Plates Load Unload Plates Displays the Unload Plates dialog box and e Select the position for capillary immersion in the plate or wetting tray e Move the plates to the Load position at the front of the instrument to load or unload plates Inject Inject Initiates injection of the sample prior to separation Separate Separate Initiates the separation of injected samples For procedures on using the Direct Control window to replenish the system see Direct Control and Replenishment on page 352 GenomeLab Genetic Analysis System User s Guide 61 PN A29142 AB Run Module Run Module Overview 62 Log Window View all of the messages and activity for a sample plate run The following example shows the Log window To open this window select the Log tab Dat
452. uirement of WEEE directive please contact your dealer or local Beckman Coulter office for the proper decontamination information and take back program which will facilitate the proper collection treatment recovery recycling and safe disposal of the device XV Safety Information XVI GenomeLab Genetic Analysis System User s Guide Foreword About this Guide Foreword About this Guide This guide is intended for use with the GenomeLab Genetic Analysis System referred to as the CE system in this manual This manual is divided into the following sections Foreword this section describes the purpose of this guide provides a list of its contents discusses the use of the notes used in the document tells how to get online Help and provides service contact information Getting Started discusses the purpose and functional description of the system provides an overview of the three main components chemistry hardware and software comprising the system and details the safety features relevant to the system It also provides step by step procedures for operating the system Sample Setup Module provides an overview of the module including its menu options toolbars and dialog boxes It also shows you how to work with sample plates using this module such as opening setting up exporting and importing sample plates Run Module provides an overview of the Run module including its menu options toolbars a
453. ults portion such as text bases of analyzed data You would use this format for instance to export data into Lasergene s SeqMan from DNASTAR Inc FASTA fasta The FASTA format only applies to the sequence results portion such as text bases of analyzed data and contains a comment line that is used to identify the data Along with the text file a quality values file is also generated FASTA QUAL which lists the quality value for each base Quality values correspond to 10 log10 error rate The range for the quality values is 1 to 99 Edited and inserted bases have a value of 99 You would use this format for instance to export data into PHRAP For instance in PHRED a quality value of 10 means 1 error in 10 A quality value of 20 means an error rate of 1 error in 100 A quality value of 30 means an error rate of 1 error in 1 000 A quality value of 40 means an error rate of 1 error in 10 000 etc Normally PHRED would export these quality values into PHRAP In the case of exporting from the CE system we bypass PHRED and export directly into PHRAP GenomeLab Genetic Analysis System User s Guide 335 PN A29142 AB Database Management Data Manager Procedures Table 8 9 Data Manager Export Options Format CEQ cq Remove CEQ Tracking Suffix Resolve Filename Conflicts 336 Description Selecting the CEQ format the format native to our database exports sample data Sequence or fragment results sample plate
454. umns for Display Highlight the column selection in the Possible Columns area of the window and click the right arrow to move the selection to the Selected Columns area of the window e To select all columns for display in the list click the double right arrow 248 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Fragment Analysis Module Customizing the Results List To remove columns from the Selected Columns area highlight the column selection and click the left arrow to move the selection back to the Possible Columns area e To remove all columns from the list right click the header and select the Hide Column Arranging the Column Display You can arrange the left to right order of columns in the list by ordering the columns top to bottom in the Selected Columns area of the Column Selector window e Highlight the column selection in the Selected Columns area and click either the up arrow or down arrow buttons to move the column to the selected location Order the columns top to bottom in the Selected Columns area relative to the left to right order you want the columns to be displayed in the fragment result table Locking the Column Position You can lock selected columns into position so they remain in view as you scroll across the remaining columns in the list This helps when the results list contains multiple columns of data which require left to right scrolling and you want to keep a particular column such
455. ure 7 22 Primer Design Selecting Primer Sequences 9 Click Execute The primer design results will be displayed 10 Click Save to Database to save the results to the eXpress Profiler database Targeting Primer Design with Advanced Options Include or exclude regions of a gene for primer design to prevent an undesirable sequence from being included in the primer or amplicon sequence Log into eXpress Designer Click on Primer Design Choose the gene of interest from the Genes list Modify the primer design parameters or leave the default settings pice Xe coge x Set the number of primer sets that are returned by entering a number in the Number to Return field 6 Inthe Target field enter the region of sequence to be included in the amplicon sequence using the following format start length where start is the index of the first base of the target and 1ength is the length of the amplicon For example an entry of 50 2 requires primers to surround the 2 bases at positions 50 and 51 NOTE Ifa Target is specified then it must be flanked by a legal primer pair 7 In the Excluded Regions field enter a region of sequence to be excluded from primer design using the following format start length where start is the index of the first base of the excluded region and length is the length of the excluded region For example an entry of 401 7 68 3 excludes the selection of primers in the 7 bases starting at 401 as well a
456. urrent data for the active sample Voltage Displays voltage data for the active sample Alignment Displays the alignment data for the active sample Alignment Displays the alignment accuracy data for the active sample Accuracy Quality Shows a graphical representation of the quality values for the active sample Parameters Trimming Toggles the trimming report pane on or off Report Parameters Displays the sequence analysis parameters used to produce the analyzed data This Used to dialog box provides the same information displayed when editing these parameters Compute however the fields in this dialog box are read only and are not editable This item is Sequence not available for data that has not yet been analyzed For parameter descriptions see Editing Sequence Analysis Parameters on page 116 or click Help when using the dialog box GenomeLab Genetic Analysis System User s Guide 1 01 Sequence Analysis Sequence Analysis Module Overview 102 Table 4 4 View Menu Sequence Analysis Module Run Log Working Option Description Displays the messages received from the instrument during the sample run Displays the name of the database in use Database Tools Menu Click the Tools menu to display its drop down menu as shown in the following example Tools wf amp 4 x Zoom Pan Align Edit Unzoorn Unzoom All Autoscale Base Spacing d Base Synch Bases on Top Compare Gompare Synch lign G
457. us Tab Use the Locus Tag tab to define the general locus and supplementary reporting information These parameters include the fragment size range repeat unit length and the dye to be used This is the minimum amount of information necessary to generate an allele list Locus Information The Locus Information area of the window contains parameters used to generate a list of expected alleles in your analysis Locus Information includes the following fields e Locus Name Enter a locus identification in this field This is an optional field Dye Select the dye to be used for the locus from the drop down list 212 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Fragment Analysis Module Using the STR Locus Tag Editor e Fragment length range between Enter the fragment length range the minimum and maximum fragment size associated with the locus in these two fields This defines the range in which the system searches for the particular fragment e Repeat unit length Enter the length of the repeat unit that represents the allele e of repeats in shortest Allele Select the number of repeats in the shortest allele from the drop down list Variant alleles that contain a particular repeat are designated by a decimal followed by the number of bases in the partial repeat For example a variant allele with the designation 5 3 contains 5 complete repeat units and a partial repeat unit of 3 nucleotides Naming Alleles Select the
458. ve xe Figure 8 12 New Group Dialog Box 6 8 Click the Add button to add the users created as described in Adding Users on page 338 The Select Users or Groups dialog box opens The local computer appears as the default location in the Look in field If it isn t use the drop down list and select the local computer Scroll down the Name list and select the user names created If you created more than one user click on the first name press and hold the Ctrl key then select any additional names to add to the group Click Add and then click OK to add the selected users to the new group Select this object type users ar Built in security principals Object Types From this location EC WINXP TEST Locations Enter the object names to select examples Check Names EC WiIMeP TEST Testlserl ok cee n Figure 8 13 Select Users to the New Group 340 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Database Management Data Manager Procedures 9 Click Create to save the new group New Group Ei Ea Group name CEQUSERS Description FO Members Testllserl Remove Create Close Figure 8 14 New Group Dialog Box Activating the CEQUSERS Group After creating the CEQUSERS group and adding users to the group you must activate the group within the Data Manager module This is done only once and users can be added removed at any t
459. ve windows Arrange Icons Automatically arranges the icons v Brings the selected window 1 to the foreground Help Menu Click the Help menu to display its drop down menu as shown in the following example Help Help Topics Using Help About Genomelab System Beckman Coulter Home Page GenomeLab Genetic Analysis System User s Guide 1 89 PN A29142 AB Fragment Analysis Module Fragment Analysis Module Overview The following table describes the Help menu options Table 6 9 Help Menu Fragment Analysis Module Option Description Help Topics Select this option to display the CE system online help By default the help system displays the Contents in the navigation pane located on the left and the introductory topic in the topic pane located on the right You can use the Contents to select and print specific help topics The navigation pane also provides a Search tab and an Index tab These tabs let you search for information by topic index entry and or keyword Using Help Select this option to display the Contents window on how to use the Windows Help system About GenomeLab Select this option to access software instrument and system information system Beckman Coulter Select this option to access the Beckman Coulter Home Page on the Internet Home Page Toolbar Icons The Fragment Analysis module provides three toolbars that activate some of the most commonly used commands Figures 6 2 identifies
460. verify this action 3 Open the sample access cover Figure 9 1 and lift to the vertical locking position 4 Rotate the wetting tray retainers outward Insert the wetting tray into the receptacle between the Sample and Buffer plates Figure 9 9 and then gently press it down into the well 5 Rotate the wetting tray retainers inwards to lock the wetting tray in place NOTE If a sample plate is installed in the system you must remove it before installing the wetting tray to allow movement of the wetting tray retainers 901598L Al Figure 9 9 Replacing the Wetting Tray 1 Front Location 4 Wetting Tray 2 Wetting Tray Well 5 Wetting Tray Retainers 3 Wetting Tray Lid 6 Sample Plate Holder GenomeLab Genetic Analysis System User s Guide 351 PN A29142 AB Maintenance and Diagnostics Direct Control and Replenishment 9 2 352 6 T Close the sample access cover Click Done on the Replace Wetting Tray dialog box Figure 9 10 Replace Wetting Tray Ea Tou may now open the sample access door and replenish the wetting tray Open the sample access cover and remove the Wetting Tray Close the sample access cover and select the location where you wish the capillaries to be immersed after you click on Done Done Cancel Help ur Capillaries exposed to air L Time Remaining min ZEC pa n Capillary Array Location Position Set Plates Location C Sample Plate
461. vert these Sample Names to Sample 1 1 by selecting the new name from the Sample Name drop down list IMPORTANT When entering data in eXpress Analysis click Save to update the data in the database before going to the next tab or function GenomeLab Genetic Analysis System User s Guide 303 PN A29142 AB Gene Expression eXpress Analysis Module Well Color Code Description Yellow Gray C The Analysis name or Multiplex name is not associated with a well that contains data If the well will be included in the analysis click on the well to select it and use the Well Specific Settings to assign the well to an analysis and a multiplex Gray Purple The Analysis has been assigned Green Purple Bright The Analysis Multiplex and Sample Name have been assigned The Analysis and Multiplex have been assigned Green Gray White No data was associated with the well or the well was excluded from the analysis 304 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Gene Expression eXpress Analysis Module Setting up Peak Binning Align the expected PCR product sizes with actual peaks shown on the Peaks Graph DUR wWN E Log into the eXpress Profiler and click on eXpress Analysis in the menu Enter your username and password then click OK to log into the software Click on the Plate Setup tab Click Open Plate Select an Analysis and the desired plate from the Open Plate dialog box and click OK Click Peak Binning to view th
462. view RR 183 VIE BID MC r E 184 Menu Bar UID EC Pc od Eee ea as ey eke hale 185 MOON DAE NG ONS steeds etc ct tobe sp sods tht i mace md ob BAB rhetorum What ba apre qaae 190 6 2 Fragment Analysis Procedures 0 0 00 ccc ce eee RR RR III 192 Performance SaleQuards 44 2 vx tsacextee es Rege ore ETE Ice dhe ERE ER E qu 192 6 3 Working with Studies in the Fragment Analysis Module llslesesseesse 193 selecting the Components of the New Study 0 0 00 cc ccc RR 194 selecting Raw Data for the New Study 0 0 RR IRR 195 selecting an Analysis Parameter Set 0 0 0 cc RR 197 PATIO AA MICH UA os orae seer iat edicit ota Bae Bait alas ioe nataracaie R rami S dodo oan 198 selecting Additional Results to add to the Study 0 0 0 cece eee 199 6 4 Defining Fragment Analysis Parameters 0 0 0 0 0c cc ee eee 200 General LoT 201 Alay SISA MEMO MAD erena Grad Seance de comes Saad weaved a eee ae OAS eG ee ak aes 202 EE AE a TO Dis cd eee shed Men on hte a ices onerat ve Re tenue oh ees ed wanes dm ded 205 village ci ELS Cre 206 SNP LOCUS THUS TaD 628 it resides epee eater ene fed dd aod ema it pid UM 208 REIN MIRRORS PETUNT LL 210 5 5 Using the STR Locus Tag EGIEOE auro te er er pea pe be Dr bet bn sa 212 LOCUS DL o PERDERE TED Oe ce 212 PCIe ID OMON eos ox cy 61 are coc RE HUR ect d Rah d US doctr uo dob dnce acu go as 215 6 6 Using the SNP Locus Tag EGILOE oer ois ei
463. ware applications Use the Frag methods to collect data for fragment analysis Use the LFR methods to collect sequence data Use the SNP method to collect SNP primer extension data After creating a new sample plate the Method selection for each column is left blank Assigning Methods To assign a method 1 Click on and select a cell in the column 2 Click the down arrow to open the method selection list Figure 2 16 and select the method Condition LFR 1 Frag Test LFR a LFR B LFR c amp MP 1 Seq Test Figure 2 16 Method Selection Drop Down List GenomeLab Genetic Analysis System User s Guide 37 PN A29142 AB Sample Setup Module Using the Sample Setup Module 38 3 Edit the new method as desired NOTE You must assign a method to each named row before saving the sample plate If any named row is not assigned a method and a save is attempted a warning message appears listing each column that needs a method assigned to it Applying Methods to Multiple Sample Sets To apply a method to multiple sample sets 1 Highlight the desired sample sets as explained in Selecting Samples on page 30 2 Select Edit Auto Fill Method Name The Choose Method dialog box opens 3 Select a method from the drop down list 4 Inthe Auto Fill area of the dialog box select Selected sample sets only then click OK To apply a method to all sample sets l Select Edit Auto Fill Method Name The Choose Method dial
464. ways Collapse To Toolbar w Always Collapse To Taskbar Figure 1 12 Main Menu Window Pop Up Menu The following table describes the collapse options Table 1 4 Main Menu Window Collapse Options Option Collapse to Toolbar Always Collapse to Toolbar Always Collapse to Taskbar Main Menu Toolbar Description Select this option to immediately collapse the Main Menu to display the Main Menu toolbar Select this option v to set the Main Menu to collapse to the Main Menu toolbar every time you open a software module from the Main Menu Selecting Always Collapse to Toolbar makes it the default setting instead of Always Collapse to Taskhar Select this option Y to set the Main Menu to collapse to the Windows taskbar every time you open a software module from the Main Menu Selecting Always Collapse to Taskbar makes it the default setting instead of Always Collapse to Toolbar From any application you can access the Main Menu by left clicking the Main Menu button located on the Windows taskbar Provides access to all CE system modules from any active application window By default the Main Menu toolbar provides a miniature version of software module icons as a horizontal toolbar as shown in the following example 12 GenomeLab Genetic Analysis System User s Guide PN A29142 AB Getting Started Operating the System To move the toolbar click the title bar and hold the left mouse button while dragging the toolbar
465. ween displaying or not displaying the status bar Refresh Rebuilds the window display to reflect the most recent changes Filter By Filter the list in the right hand side window by the dates modified Database Displays all items in the selected database sample Run History Displays a list of items that were run in a project during specified dates Tools Menu Click the Tools menu to display its drop down menu as shown in the following example Tools Backup Database Restore Database Shrink Database Associate Default Dye Spectra Convert Individual ID to Subject ID Administrative Tools k Use the Tools menu to back up restore and shrink a database and to perform other database management tasks Table 8 5 Tools Menu Data Manager Module Option Description Backup Creates a backup copy of the selected database Restore Restores a previously backed up copy of a database to the Data Manager shrink Database Reduces the size of the currently selected database Associate Default Dye Spectra Associates previously saved data with a new default dye spectra GenomeLab Genetic Analysis System User s Guide 323 PN A29142 AB Database Management Data Manager Module Overview Table 8 5 Tools Menu Data Manager Module Option Description Convert Individual ID to Converts all sample data records that use Individual IDs to records that Subject ID use Subject IDs Administrative Tools Add Adds the CEQUSERS group to the SQL lo
466. with the actual size of the peak Important Considerations e Modifications to the expected PCR product size will change the place where the software will look for the designed peak e Modifications to the bin width will change the area surrounding the expected PCR product size where the software will look for the designed peak Changes to the PCR product size and bin width are made across the plate and throughout the analysis Changes are not made on a well by well basis Therefore peak binning in one well affects the peak binning of ALL of the wells in the analysis e Changes to the expected PCR product sizes or bin widths should not be performed arbitrarily but rather connected with experimental data to verify that the gene sizes and bins are accurate and reproducible GenomeLab Genetic Analysis System User s Guide 307 PN A29142 AB Gene Expression eXpress Analysis Module e Changes to the expected PCR product sizes and bin widths are owned by the user who is logged into eXpress Analysis These changes do not affect the multiplex TDF file that is available to all users The adjusted PCR product size and bin width parameters are applied to the current analysis and will be applied to future analyses by this user when the same multiplex is used e Changes to peak bins or PCR product sizes should be connected with experimental data to verify that the gene sizes and bins are accurate and reproducible e When entering data in eXpress
467. x only if you have performed an alignment on the open sequence result 2 Select the tab that displays the type of properties you want to view For details see the following topics General Tab on page 138 e Notes Tab on page 139 Property Set Tab on page 140 Method Tab on page 141 Analysis Tab on page 142 Alignment Tab on page 143 Consumables Tab on page 144 NOTE To view detailed descriptions while using this dialog box click Help 3 When finished reviewing the properties click Close General Tab Use the General tab to view the general properties of the currently selected sequence result Analysis Alignment Consumables General Note PropetySet Method Dg Sequence Test 403_060301 2252 Type Sequence Result Database CEQ DATABASE Project Default Modified Monday April 24 2006 16 08 36 Sample Name Sequence Test 403_060301 2252 Sample Plate U33 001 Test 030106 Sample Position A 3 Operator Name DC CEQ System Name CEQ 8000 Firmware Version 6 0 2 Instrument S N 3067033 Data Collection 8 0 52 Software Version Collection D ate Thursday March 02 2006 00 32 34 Sample Subject ID Figure 4 26 General Tab Properties Dialog Box View the item type database and project where the item resides as well as the date and time the item was last modified You may also view the sample name sample plate sample position in the sample plate the instrument and t
468. xport result as Displays the selected export file including its file path file name and extension which identifies the file format This is a read only field To change this selection click Save As and enter the required details to save the file using the application s format extension For details see Text File Format on page 258 or CEQ File Format on page 259 e Header Select this option to include general information regarding the sample and run conditions with the exported results e Raw Data Select this option to include raw data with the exported results e Result Data Select this option to include dye traces and data points with the exported results e Result Output Select this option to include a fragment list with the exported results e Remove CEQ Tracking Suffix Select this option to remove the sample plate and date from the exported file name e Resolve Filename Conflicts Select this option to automatically append a unique sequence number to exported files of the same name 6 Click Finish to complete the export application GenomeLab Genetic Analysis System User s Guide 25 PN A29142 AB Fragment Analysis Module Exporting Results Text File Format You can export the results file in a text file format The data in this file are tab delimited and the export format has the extension txt Export x Sample Elements ee Save in D Ew si t Ef EJ Raw Data v Result Data
469. y Sets Create delete or modify the property set summary Displays a summary of the currently selected plate View by Subject ID Toggles the sample plate cell labels between Sample Name and Subject ID Working Database Displays a dialog box showing the database in use Run Menu Click the View menu to display its drop down menu as shown in the following example EA Start This menu provides one option Table 2 5 Run Menu Sample Setup Module Option Description Start Invokes the Run module and loads the active sample plate GenomeLab Genetic Analysis System User s Guide 25 PN A29142 AB Sample Setup Module Overview 26 Window Menu Click the Window menu to display its drop down menu as shown in the following example Window Next Ctrl Tab Clase Ctrl F4 1 Untitled Plate w 2 Untitled Plate The following table describes the Sample Setup module s Window menu options Table 2 6 Window Menu Sample Setup Module Option Description Next Toggles between open sample plates Close Closes the active sample plate or sample plate summary Help Menu Click the Help menu to display its drop down menu as shown in the following example The following table describes the Help menu options Table 2 7 Help Menu Sample Setup Module Option Description Help Topics Select this option to display the CE system online help By default the help system displays the Contents in the navigation pane located on the left and
470. y be impaired GenomeLab Genetic Analysis System User s Guide IX Safety Information Safety Symbols The symbols displayed below and on the instrument should remind you to read and understand all safety instructions before attempting installation operation maintenance or repair to this instrument POPE P HIGH VOLTAGE Paragraphs marked by this symbol indicate that a potential hazard to your personal safety exists from a high voltage source BIOHAZARD Paragraphs marked by this symbol indicate that a potential hazard to your personal safety exists from a biological source LASER LIGHT Paragraphs marked by this symbol indicate that a potential hazard to your personal safety exists from a laser source SHARP OBJECTS Paragraphs marked by this symbol indicate that a potential hazard to your personal safety exists from unblunted corners or other appendages on the outside or inside of the equipment HOT SURFACE Paragraphs marked by this symbol indicate that a potential hazard to your personal safety exists from heated surfaces or other appendages on the outside or inside of the equipment PROTECTIVE EARTH OR GROUND TERMINAL This symbol identifies the location of the protective earth or ground terminal lug on the equipment OFF POSITION OF PRINCIPAL POWER SWITCH This symbol graphically represents the equipment main power push button switch when it is in the off position ON POSITION OF PRINCIPAL POWER SWITCH This symbol graphical
471. y the Detect Heterozygote option N s are not called The N Threshold text box is grayed out to disable calling N s Of Average Peak Enter a value to determine how far to look on either side of each called base to opacing look for other peaks This value is centered over the peak for the called base for instance a value of 50 looks 2596 on either side of the peak for secondary peaks The range is 0 100 Height Ratio Enter the height ratio of the secondary peak relative to called base peak The range is 0 1009 sensitivity Enter the value that specifies the minimum sharpness of detected secondary peaks The higher the value the higher the sensitivity used to detect smaller less visually defined peaks The range is 0 00 to 1 00 Range Enter the range in the called sequence where the system looks for heterozygotes within a called sequence Also provide the number of bases prior to the last called base you want heterozygote detection to end in the case of short PCR samples that may vary in length The range is O length of the sequence If a value is outside of an expected range a message box opens requesting you to enter a valid value The system uses the IUB ambiguity codes shown in the following table to represent heterozygous bases Table D 20 IUB Ambiguity Codes Code Mnemonic GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Analysis Using the Sequence Analysis Module Table D 20
472. ynch With both the analyzed data and base sequence panes open synchronizes the analyzed data display with the highlighted base sequence text showing the corresponding peak or peaks between two hairlines for the selected base Bases on Top Toggles the base sequence text between displaying on the top or bottom in the analyzed data pane Compare Synch Synchronizes scaling zooming and panning of two analyzed data sets while in the Compare mode Align Aligns selected points of each of the displayed analyzed data sets while in the Compare mode Display Options Opens the Display Options dialog box GenomeLab Genetic Analysis System User s Guide PN A29142 AB Sequence Analysis Sequence Analysis Module Overview Sample View Toolbar The following example shows the Sequence Analysis module s Sample View toolbar For descriptions of the actions that occur when you click a toolbar icon see Table 4 11 Figure 4 4 Sequence Analysis Module Sample View Toolbar Table 4 11 Sample View Toolbar Sequence Analysis Module Icon Description Analyzed Data Displays the data that has been analyzed for the active sample Base Sequence Displays a text view of the bases from the analyzed data for the active sample Raw Data Displays the raw data for the active sample Current Displays the current data from the capillary arrays for the active sample Voltage Displays the voltage data from the instrument for the active sample
473. you are sure of and saves you the time of removing unwanted fragment data later Finding Exclusion Filters You can find all of the exclusion filters in the drop down list available in the Name column of the Filter Set area Once you have selected the name of the filter to use you must select an operator usually lt gt etc and a value to be applied to the filter Refer to the online help for detailed descriptions of the available exclusion filters and their use GenomeLab Genetic Analysis System User s Guide PN A29142 AB Fragment Analysis Module Using the SNP Locus Tag Editor Viewing the Summary The Summary area displays a table that identifies the number of samples that have been affected by the application of exclusion filters As you manipulate the result set you can view the number of samples excluded from your Study based on your filter set parameters The Summary displays the following The number of results that are included in result set within each sample capillary A H As samples are filtered out of the result set by the exclusion filters they are moved from this row to the excluded row The number of results that have been excluded from the result set by the exclusion filters e The total number of all samples both included and excluded that are part of the result set Customizing the Result Set View You can define the fragment result components available in the Result Set View You can also
474. ystem User s Guide PN A29142 AB Gene Expression Software Administration Changing the Administrator Password l From the Administrator Menu click Account 2 Click Reset Form to clear the information 3 Enter the new password in the password field 4 Click Save User NOTE The password is case sensitive and can be any combination of letters numbers and spaces IMPORTANT Care must be taken when changing the default Administrator password If the new password is lost contact your local Beckman Coulter representative eXpress Profiler Account Users Account Manage Genes Manage Primers Universal Tags Import Multiplex Changing password for user Help System Administrator Logout m Figure 7 4 eXpress Profiler Account Screen for System Administrator GenomeLab Genetic Analysis System User s Guide 2 1 PN A29142 AB Gene Expression Software Administration Managing Genes Enter proprietary gene sequences without the use of GenBank accession numbers and edit the user defined genes that are stored in the system eXpress Profiler BECKMAN COULTER Manage Genes Users PO p Load Save Account Reset Form Manage Genes Manage Primers Universal Tags Display Name ProprietaryGene Import Multiplex Help Accession broprietaryGene FASTA P ProprietaryGene Description of the proprietary gene E CCGACAGGAGCTCATTGCTCTTTATGGTTATGACATTCGCTCAGCTCATAATCCTGATGACATCAA ACCTCGAAGAATCCGGAAA

GenomeLab Genetic Analysis System User's Guide

Contents

Download Pdf Manuals

Related Search

Related Contents