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GeneScan Analysis Software Version 3.1

1. Preferences Page Results Displat Y Default Display Attributes Stacked Electropherogram Panels C Align By Size Show Offscale Regions C Use Common Vertical Scale C Show Peak Positions Panel Height Resize Limits X show Legends Minimum 25 em Plot Colors 2 Standard o Custom Masonic 4o oim Peak Highlighting Opaque Q Transparent o Page Break before Tabular Data Printing Preferences amp Height As Shown on Screen Q Fixed at x C There are three Results Display preferences Default Display Attributes Stacked Electropherogram Panels Printing Preferences Evaluating Analysis Results 7 13 7 14 Evaluating Analysis Results To change how results are displayed and printed continued Step Action 2 Set the Default Display Attributes to control the display attributes of new results displays as follows You can set For more information Show Peak Positions Displaying Peak Positions on page 7 29 Use opaque or transparent Highlighting Peaks on peak highlighting page 7 29 Show Legends Using Legends to Change the Display on page 7 30 Show Offscale Data Showing Off Scale Data on page 7 32 Align By Size Showing Data by Fragment Size on page 7 35 Use standard or custom plot Defining Custom Color for All colors Electropherogram on page 7 41 3 Configure Stacked Electrop
2. 002 6 13 Creating a GeneScan Injection List llle lees 6 14 Starting the Data Collection Software 04 6 15 If the Run was Cancelled llseeee eee eee 6 15 Run Me oie rhe e Olan atte ete Roce a eee aes 6 16 Assigning Matrix File 0 creeer eee ee 6 16 Loading and Running Dye Standards for the ABI PRISM 377 6 17 Introduction scele Be ea eb cael ee Shea m ed eure 6 17 Creating a GeneScan Sample Sheet 005 6 17 Loading Matrix Standards 0 0 00 0c eee ee eee eee 6 18 Running the Matrix Standards 00 0 00008 6 19 Loading and Running Matrix Samples on the ABI 373 non XL 6 20 Introduction seip aa whe Sa eee ER ee eS ee 6 20 Loading the Matrix Standards 0 0 0 0 eese 6 20 Completing the Sample Sheet in the 672 Software 6 21 Running the Gel ecole RR uere ee ha RAS 6 22 Completing the GeneScan Sample Sheet 6 23 Loading and Running Matrix Standards on the ABI 373 with XL Upgrade6 24 InttOdUCtlOD eere ela RES RR i e A C ewe 6 24 Completing the GeneScan Sample Sheet 6 24 Loading the Matrix Standards 0 00 00 00008 6 25 Running the Matrix Standard 00 0 less 6 26 Generating Matrix Sample Files for the ABI 373 and ABI PRISM 377 6 27 Who Should Use This Step 0 0 0 ce eee eee eee eee 6 27 Verily Trackin
3. 2 Select the name of the database Server that you want to use These Server names correspond to those listed in the interfaces file 3 Click Ping E 12 Using the BioLIMS Database Accessing the BioLIMS Database Step Action 4 If the connection is Then working the Ping window reports that the Ping was successful not working The Ping window reports that the Ping Failed or The Ping window gives no response Refer to Appendix F Troubleshooting the BioLIMS Database 5 Quit from the SybPing application To access the BioLIMS database Step Action 1 Choose Preferences from the Edit menu and BioLIMS Access from the submenu The Preferences dialog box appears Preferences Page BioLIMS Access t BioLIMS Access Q Sample Files BioL IMS Session Manager Username Password Bd Save Password Database Server Alias 3 O Open on Launch v Make Default ok Using the BioLIMS Database E 13 To access the BioLIMS database continued Step Action 2 In the BioLIMS Access section click the BioLIMS button The fields in the Session Manager subpanel are activated Note The following alert appears if there are unsaved sample files and fragments open when you switch to BioLIMS mode When you
4. 7 17 Introduction seccions toripe eee Ret dre rv 7 17 Important Considerations lee eee eee ee eee 7 17 Saving the Display Format 0 0 0 eese 7 17 Working with a Previously Saved Displays 7 18 Renaming the Current Results Display 7 19 About Electropherogram and Tabular Data Displays 7 20 Introductio pa ses stint tates PER awe Meda DES Sale bere 7 20 How the Window is Divided 0 0 0 0 0008 7 20 What Tabular Data Contains 0 0 e eee eee eee 7 20 How Electropherogram Panels are Sized 0 7 20 Peaks Visible in Electropherogram Not Tabular Data 7 21 For More Information 0 0 0 eee eee eee 7 21 Displaying Electropherogram and Tabular Data 7 22 Displaying Electropherogram and Tabular Data 7 22 Tabular Data and Electropherogram Example 7 22 Table Describing Columns 0 0 0 0 eee ee eee 7 23 Highlighting Information eee 7 23 Adjusting Window Size 20 2 eee eee ee eee 7 24 Hiding Selected Rows of Data 0 0 0 0 cece eee eee 7 24 Limited the Rows Displayed lsleeeeleeeeeeees 7 24 Displaying Electropherogram Data eslelelee eere 7 25 Definition o da rera egeat eer erem e 7 25 Base Pairs Versus Data Points 7 25 Procedures ood ER REREWREES A RU EMEN R
5. Analyzing Project Files 5 23 Using Analysis Parameter Files In This Section This section contains the following topics Topic See page Analyzing Samples Using the Same Analysis Parameters 5 24 Selecting Different Parameters for Analysis Samples 5 25 Displaying Default Parameters 5 26 Creating Custom Analysis Parameter Files 5 26 Changing an Existing Analysis Parameters File 5 27 Deleting Custom Analysis Parameters 5 28 Analyzing Samples To analyze samples using the same analysis parameters Using the Same Analysis Step Action Parameters 1 If the Analysis Control window is not displayed then choose Analysis Control 3 1 from the Windows menu 2 Click the arrow in the Parameters column heading and choose parameters from the pop up menu Your menu choice applies to all fields in the column Pop up menu SS Project Analysis Control C Print Results Print Setup Sample File Size Standard Parameters Analysis Parameters sAnalysis Parameters 5 24 Analyzing Project Files Step Action The pop up menu contains the following options Item Description Analysis Parameters Applies the parameters that are stored as preferences in the software Collection Settings Applies the analysis parameters file specified in the data collection program which is embedded in the Sample file Cust
6. Select criteria s to find Collections with Fragments Collection Creator contains t Collection Name contains t Collection Type is Creation Date ani Sequence Frag Name contains Sample Creator contains Sample Name contains Gel Path contains Offscale Data is present t Modified Creator 3 Use the pop up menus and text fields to define your search query Refer to Collection Search Criteria on page E 24 and Fragment Search Criteria on page E 25 for details about the search criteria When you are satisfied with the search click Search The results of the search appear in the lower portion of the window Note Collections returned by the Collection Browser window must match all of the collection criteria and contain at least one fragment that matches all of the fragment criteria Using the BioLIMS Database E 29 E 30 To search the BioLIMS database continued Step Action 4 To view the fragments contained in the collections click the small triangle to the left of the collection name Collection Browser Select criteria Cs to find Collections with Fragments Collection Creator Collection Name Name Modified Type p 373XL2 r6spec Gel File 6 7 May 21 1998 04 09 25 PM project 7 377 XL Gels May 15 1998 01 27 16 PM project Small 7 36 3 pod 01 GS0627 377XL May 15 1998 04 06 03 PM
7. l m MM Analysis Control The following table describes the above diagram Window Description No Description 1 Use these columns to choose the dye colors to analyze and to specify which is the size standard 2 Diamonds mark the standards For more information see What are Size Standards on page 5 29 3 click a dye sample field to specify that dye sample as the standard 4 From this pop up menu you can choose Oollection Settings A user defined size standard Define a new size standard for that sample For more information see Topic See page About Size Standards 5 29 Using Size Standards 5 36 5 From this pop up menu choose an analysis parameters file For more information see Topic See page About the Analysis Parameters 5 17 Using Analysis Parameter Files 5 24 6 Double click the size standard text field or the analysis parameters text field to edit the size standard or the analysis parameters For more information see Topic See page Editing an Existing Standard 5 37 Changing an Existing Analysis Parameters File 5 27 Analyzing Project Files 5 3 Customizing the Display Using the Analysis Control Window 5 4 Analyzing Project Files No Description 7 A notation appears in this Information Display field when you move the cursor over a Sample file name
8. tracked gel extract Sample file Extract Lane Extracting Data data without changing command each time Without Changing the the current line you change the tracker Current Tracker on tracking line positioning in a page 2 64 lane or the information in the Sample Sheet to incorporate the new information If the Run Folder Each time the GeneScan Analysis Software tracks and extracts a gel is Not Specified the software looks for the data collection Run folder that is specified in the gel file If the Then Software does not locate the the program creates a new one in folder or if the the folder that contains the gel file Gel file does not reference one The program places new Sample files in the Run folder How to Process and Edit the Gel File 2 59 Tracking the Gel Why Use this Procedure Without Use this procedure to Extracting Data View the results of auto tracking and if necessary to correct any tracking errors or tracker line interpolation before the software extracts the lanes and generates Sample files Edit tracker information and to redraw the lines based on original tracking information Procedure Choose Track Lanes from the Gel menu The Track Lanes dialog box appears Note Pressing 3 period cancels tracking Track Lanes A Proceeding with this command will over write current Lane Tracking f Cancel Reve
9. Troubleshooting the BioLIMS Database F 9 Troubleshooting the connection from the client Macintosh computer continued Step Action 6 If the Ping is unsuccessful Open the program NCSA Telnet The default installation places NCSA Telnet into the BioLIMS BioLIMS Extras folder Step Action a Select Open Connection from the File menu b Enter the host name from the interfaces file into the Host Session Name text field C Click Connect If the connection is Then successful a window is displayed with a UNIX login prompt Skip to step 2 of Troubleshooting the client connection from the Sybase SQL Server on page F 12 unsuccessful an error message is displayed The network is not working Call your network administrator Note If the host domain name does not work try the IP address of the host This number can be found in the etc hosts file on the UNIX Server F 10 Troubleshooting the BioLIMS Database Required Sybase The following Sybase library files are placed in the Extensions folder in Extension Files the System folder by the BioLIMS Client and instrument installers These files are required for connection to the BioLIMS database libblk libintl libcomn libsybdbl libcs libtcl libct libtcp libctb Required Oracle The following Oracle library files are placed in the Extensions folder in Exte
10. Unlabeled Native 36 928 946 677 695 674 692 539 557 421 439 299 317 275 293 244 262 118 136 108 126 81 99 75 93 56 85 64 82 37 55 33 51 29 47 B 8 GeneScan Size Standards Running Under The following diagram shows the peak pattern of fragments run under Native Conditions native conditions on an ABI PRISM 310 using 2 5 GeneScan Polymer Solution in a 30 cm Ld capillary IMPORTANT An asterisk for the 262 and 692 base pair peaks denotes peaks resulting from abnormal migration of double strands that did not completely separate under denaturing conditions when analyzed on the ABI PRISM 310 Do not use these peaks to size samples The peaks show smaller values than the actual size of the fragments Refer to the GeneScan Reference Guide Chemistry Reference for the ABI PRISM 310 Genetic Analyzer P N 4303189 rev A for further details Asterisks 3R GeneScan Size Standards B 9 GeneScan 2500 Standard How It Is Prepared The GeneScan 2500 standard is made from lambda phage DNA What To Use It For Running Under Native Conditions B 10 GeneScan Size Standards restriction digested with Pst I followed by ligation of either a TAMRA or ROX oligonucleotide It has 28 fragments ranging from 55 to 14 097 base pairs bp You can use the GeneScan 2500 standard for native applicatio
11. 1 From the project that contains your matrix standard Sample files open the Analysis Control window untitled Analysis Control O Print Results PrintSetup Sample File Size Standard Parameters sAnalysis Parameters sAnalysis Parameters sAnalysis Parameters Analysis Parameters Select the four matrix standard Sample files Choose Assign New Matrix from the Project menu Select the matrix file you just created Select numbers 1 2 3 and 4 on the left side of the window to highlight the colors for each row Choose Set Analysis Parameters from the Settings menu Enter the appropriate range for the Analysis Range and click OK to return to the Analysis Control window 8 Click Analyze Reviewing the results 1 Choose Results Control from the Windows menu The Results Control window appears see below Making a Matrix File 6 35 6 36 Making a Matrix File To assign a matrix file to Sample files continued Step Action Dye Samples of Panels pull down menu Quick Tile Qo eor 2 Select 4 from the of Panels pull down menu see above 3 Click 1 under Dye Samples see above 4 Click 1 on the Sample Files side of the Results control window 5 For matrices 2 3 and 4 click Analyze and repeat step 1 and step 2 Evaluat
12. 1 18 GeneScan Analysis Software Overview Sample File GeneScan Analysis Software Sample files can be written to and read Database Support from the BioLIMS database The BioLIMS Genetic Information Management System BioLIMS database provides a relational database for storage and retrieval of DNA fragment data If you have obtained the GeneScan Analysis Software as part of the BioLIMS Client package you can use the program in either of two modes Mode Description Sample File mode Fragment data extracted from gel files is written to individual sample files Extracted fragment data viewed and processed within the GeneScan Analysis Software is read from and saved to sample files BioLIMS mode Fragment data extracted from gel files is written directly to a BioLIMS database that resides on a UNIX Server Extracted fragment data viewed and processed within the GeneScan Analysis Software is read from and written back to the same BioLIMS database For more information on using the BioLIMS database see Appendix E Using the BioLIMS Database GeneScan Analysis Software Overview 1 19 Technical Support To Reach Us on the Web Hours for Telephone Technical Support To Reach Us by Telephone or Fax in North America Applied Biosystems web site address is http www appliedbiosystems com techsupport We strongly encourage you to visit our web site for answers to frequently asked questio
13. Printer The following printers are supported HP DeskJet 1200C PS HP DeskJet 1600C PS System software Macintosh computer system software version 8 0 Turning On To turn on Virtual Memory Virtual Memory Step Action 1 From the Apple menu amp choose Control Panels and from the list choose Memory The memory dialog box appears Disk Cache Cache Size Always On Select Hard Disk Virtual Memory c Hard Drive On Available on disk 999M Q Off Available built in memory 64M 65M Percent of available memory RAM Disk to use for a RAM disk Qon off 0 50 100 RAM Disk Size OK 2 Ensure that Virtual Memory is On and that the memory available is at least 48M GeneScan Analysis Software Overview 1 13 RAM Required to The system requires approximately 32 MB of RAM available to have Run the Software both the Data Collection software and the analysis programs open simultaneously This does not include the RAM required by the Macintosh computer operating system The RAM required by each program is as follows Program Approximate RAM required Data Collection software 5 5 MB GeneScan Analysis Software 32 MB How to Check the RAM Available Choose About this Macintosh from the Apple menu in the Finder Installing the The following procedure describes how to install the GeneScan Software Analysi
14. Printing Results 9 3 Printing the Gel Image About Printing the Gel Image Procedure 9 4 Printing Results You can print a gel image any time it is displayed Note For best results when printing adjust the gel contrast to increase the intensity of the gel colors Depending on your printer the printed image may be less intense than the screen image To print the gel image press Command 8 Shift 3 to get a screen capture of everything displayed on the screen Printing Selected Sample Files Introduction Setting Printing Options From the Results Control Window You can print selected Sample files using the Results Control window or by choosing the Sample file To set the printing options use the Results Display Preferences dialog box see Changing How the Results are Displayed and Printed on page 7 13 These options affect the electropherogram height and page breaks Depending on how you set these options the format that prints may be different from what is on the screen For information on printing saved Results Control formats see Working with a Previously Saved Displays on page 7 18 You print selected samples after analysis regardless of whether you choose automatic printing To print results for selected Sample files after analysis Step Action 1 In the Results Control window select the dye samples and format you wish to print Use the same technique as selecting the format and
15. electropherograms for the samples and dyes selected for analysis Select the appropriate radio button to specify whether the electropherograms for the four dyes appear Together in one panel overlaid or in Separate panels tiled tabular data 6 Click OK Displaying Size Use the Analysis Control window to open review or change Size Standards and Standard and analysis parameters Analysis Parameters To display these files You can Then For more information see double click the field containing the size standard or the analysis parameters file the Size Standard or Analysis Parameter window appears Using Analysis Parameter Files on page 5 24 Defining the Size Standard on page 5 31 Analyzing Project Files 5 11 Displaying Sample How to Display Sample and Dye Information 5 12 and Dye Analyzing Project Files i Move the cursor over a dye color field or over a Sample file name field Information to display information about the samples and dyes How to Specify the Information Displayed The following procedure describes how to specify the information displayed when moving the cursor over a dye color field or a Sample file name field To specify the information displayed Step Action 1 Choose Project Options from the Settings menu and Sample Info Display from the submenu The Sample
16. Black line Red line Green line EP Voltage umm wv 10 EP Current m mA EP Power mum i Gel Temp um C enga v Jo 00 0 0 elg Colored Lines The following table describes the lines in the above figure Described Line Description Blue Electric voltage in volts 10 Black Electric power in watts Red Gel temperature in C Green Electric current in mA mamp 3 18 Analyzing Sample Files Analyzing a Sample File Introduction The GeneScan Analysis Software analyzes raw data stored in Sample files according to parameters and standards that you select You can use the analyzed data to detect peaks associated with DNA fragments and identify those peaks with an established size standard Procedure To analyze a sample file Step Action 1 Choose Analyze Sample File Name from the Sample menu 3 Y The Analyze Sample file dialog box appears Analyze 01 50 Lane Demo Sample 5 Dyes To finalyze q M EB v Craze me Bs Rnalysis Parameters Collection Setting vj Size Standard lt gs350 gt vj Standard Dye R vj From the Analyze Sample file dialog box select one of the following options Choose To select Dyes to Analyze checkboxes any number of dyes to analyze Analyze Parameters pop up from the default parameters or menu any parameter files in the folder location specified in the application prefe
17. 00 0 0 02 ee eae 2 29 Using the Copy of the Sample Sheet 0 0 2 29 Displaying the Copied Sample Sheet 2 29 Displaying a New Sample Sheet 0 00 2 29 About the Sample Sheet 0 0 0 2 eee eee ee eee 2 31 Sample Sheet Example 0 0 0 0 ee eee eee eee 2 31 Sample Sheet Described 0 0 0 eee eee eee 2 31 Working with the Sample Sheet 00 0 0 ee eee eee 2 34 Editing Collection Information 00 00 0005 2 34 Changing Column Widths 0 0 0 0 02 eee ee eee ee 2 35 Editing the Sample Sheet 00 0 0 0202 2 35 Printing the Sample Sheet 00 0 0 000 2 eee eee 2 36 Installing a New Sample Sheet 0 0 00 2 36 Importing Data to Sample Sheets 0000 2 37 Exporting Data From Sample Sheets 005 2 37 About Displaying Regions of Off Scale Data 2 38 Why Displaying Off Scale Data is Important 2 38 Off Scale Detection and GeneScan 0005 2 38 Displaying Off Scale Data 0 0 0 eee eee eee 2 38 Gel File Window Example 2 0 0 0 c eee eee eee 2 40 Electropherogram Examples 0 00002 eee eee 2 41 Working with Lane Markers 0 0 0 e eee eee eee 2 43 Introduction 2 a RR RE RI S e eg eas 2 43 Topics in This Section 00 esaii meiras sn
18. These kinds of adjustments can make it easier to see the data in the gel and can improve the appearance of the gel for presentation Note While using the Adjust Gel Contrast dialog box to adjust the contrast the changes are shown in the background gel To adjust the contrast in the gel image Step Action 1 Select a lane in the gel image that contains the color you want to adjust Note Changes affect the entire gel not just the selected lane Choose Adjust Gel Contrast 36 J from the Gel menu The Adjust Gel Contrast dialog box appears see below 2 20 How to Process and Edit the Gel File To adjust the contrast in the gel image continued Step Action Adjust Gel Contrast i d ud Al 3 Place the cursor on the triangle for the color you want to adjust then hold down the mouse button and drag the triangle up or down to a new position Note The changes take effect in the gel image as you move the triangles How to Process and Edit the Gel File 2 21 To adjust the contrast in the gel image continued Step Action 4 You can take the following action Note It is best to adjust one color apply the change and view the effect in the gel image before you adjust another color If you want to Then increase the intensity of a color pull the top triangle for that
19. ud The following is an example of the Results Control window of Panels 4 v Dye Samples 08 P27 B 8 gePer a 89 7 IM ep27 11 7117 109927 939797 f 111 P27 11 7117 7 107 27 7 STS f OS ePa7 S 5 7 7 05 P27 B 5 05 p27 5 757 7 7 4 Evaluating Analysis Results Results Control Window Description Results Control window descriptions No Description 1 Select dye sample information by clicking one of the dye color fields 2 Each button corresponds to a panel in the display window 3 Click to display electropherograms for the selected samples 4 Choose the number of electropherogram panels available for display from the pop up menu 5 Click to display tabular data for the selected samples 6 Identifies the sample by row number and dye code Sample information is displayed as specified in the project options 7 Plot color indicator If you Then double click the plot color the Choose a Plot Color dialog indicator box appears Choose a Plot Color Il Black v aa For more information see Defining Individual Plot Colors on page 7 42 double click the dye color the plot color indicator returns indicator to the default color For more information see Setting Dye Indicator Preferences on page 5 15 Note If you change the plot color a vertical line appears beside
20. Choose Paste from the Edit menu Saving Archiving and Copying Files 8 7 Creating a Text To create a text file from tabular data File Step Action 1 Display the tabular data 2 Choose Export Table from the File Menu 3 Choose a name and file location in the dialog box and click Save 8 8 Saving Archiving and Copying Files Printing Results Introduction In this Chapter Topics in this chapter include the following Topics See page About Printing 9 2 Automatically Printing Run Results 9 3 Printing the Gel Image 9 4 Printing Selected Sample Files 9 5 Printing Supporting Information 9 6 Printing Results 9 1 About Printing Ways You Can Print If You Get Unexpected Results 9 2 Printing Results You can print the Results of your run automatically at the end of the run or interactively as selected Sample files or display combinations Gel file from the Gel File window and supplementary information from the Sample Info view You might initially get unexpected results from autoprinting or the Print One command if you switch printers The first time you print after changing the printer configuration do the following First Second Choose Print Setup from the File Choose Print from the File menu menu and click OK and use the standard Print dialog box Automatically Printing Run Results Introduction From the Dat
21. D 12 Troubleshooting the GeneScan Software Using the BioLIMS Database Introduction In This Appendix This appendix describes how to access the BioLIMS database how to set the preferences and how to open or process a fragment that is located in the BioLIMS database For information on troubleshooting the BioLIMS database see Appendix F Troubleshooting the BioLIMS Database Topics in this appendix include the following Topic See page About the GeneScan Analysis Software and the BioLIMS E 2 Database Configuring the BioLIMS Database Server E 4 Switching Between Sample File and BioLIMS Mode E 10 How to Access the BioLIMS Database E 12 About Server Names E 17 Using the Collection Browser Window E 20 Using the BioLIMS Database E 1 About the GeneScan Analysis Software and the BioLIMS Database What is the BioLIMS Database Accessing the BioLIMS Database Before Using the BioLIMS Database Modes in GeneScan Analysis Software E 2 Usingthe BioLIMS Database The GeneScan Analysis Software 3 1 introduced support for selecting viewing editing and processing fragments from the BioLIMS database The BioLIMS database provides a relational database for fragments created by ABI PRISM GeneScan Analysis Software This database accommodates multiple users and versions while preserving the original data The GeneScan Analysis Software user interface has changed to accommoda
22. Enter a Display Title tance Enter a name for the display and click OK Evaluating Analysis Results 7 17 Working with a Use the Previous Displays dialog box to display print remove or Previously Saved rename a saved display Displays To work with a previously saved displays Step Action 1 Choose Previous Displays from the Project menu The following is an example of the Previous Displays dialog box Previous Displays Previous Display List Project 01 03 98 Display 1 Project 03 04 98 Display 1 Display Print Remove Rename Select a display or multiple displays and take the following action Click an item in the list and To Click display the saved formats Display print the saved formats Print The standard print dialog box appears remove the saved formats Remove An alert appears rename Rename Note You can only rename one display at a time The Rename dialog box appears See Renaming the Current Results Display on page 7 19 7 18 Evaluating Analysis Results Renaming the To rename the Results display that is currently on the screen and to Current Results save the display under a different name Display Step Action 1 Ensure the display is the active window 2 Choose Rename Display from the Project menu The following is an example of the Ren
23. How to Display the Gel Info Window There are two ways to display the Gel Info window You can Result click the Gel Info button on the Gel The Gel Info window appears see File window Gel Info Window Example below choose Gel Info from the Gel menu 2 26 How to Process and Edit the Gel File Gel Info Window Example Gel file Gel Info Run Information User Name Kathy un Date Instrument 377 Start Time Data Coll Version 1 1 Run Duration Total Scans Tue Jul 2 1996 7 25 22 AM 2Hrs 15 Secs 4572 Gel Characteristics Gel Tupe Number of Channels 194 Gel Percent 0 00 96 GelThickness 0 00 mm Number of Lanes 34 Well To Read Distance 36 0 em Number of Dyes 4 Gel Image Information Gel Image Range O 4572 Estimated Maximum Peak Height 2000 Multicomponented Yes Matrix File matix filet Gel Image Review the gel image for a general measure of the quality of the run For more information see How to Adjust the Gel Image on page 2 18 IMPORTANT If you make any of the changes to the scan range after extracting the sample data from the gel file re extract the Sample files to include the new information For information see Regenerating the Gel Image on page 2 23 What to Review in the Gel Image Review these items Take this action Inspect th
24. Note Ifthe gel is already tracked choose Extract Lanes from the Gel menu The project Analysis Control window appears containing each of the Sample files Choosing a Scan Range for the Matrix Calculation Introduction Viewing the Raw Data Depending on how well your Matrix Standards run it may be necessary for you to choose a specific range of data points to be considered for your matrix calculation In order to choose appropriate values for the Scan range you must first view the Sample file raw data from each of the four matrix standard files so you can decide where to choose the start and stop points for the scan range When you have multiple Sample files raw data can be accessed more easily through a project s Analysis Control window Raw data can provide useful information about the Sample files you have created Note You can view Sample files without opening a project However this procedure is easier if you use a project to organize the Sample files see Using a Project to Manage Sample Files on page 4 8 To view raw data Step Action 1 Use the following steps to create a project for the Dye Matrix Standards Step Action a Choose New from the File menu The Create New dialog box appears b Click the Project icon An untitled Analysis Control window opens C Choose Add Sample Files from the Project menu d Find and open your matrix run folder e Sel
25. Represents SYBASE the name you chose to call the BioLIMS Server The server can have any name For more information see About Server Names on page E 17 query MacTCP the part of the entry that is always the same mac ether i Note The tab preceding this phrase is required neuron apldbio the host and domain name of the Sybase based com BioLIMS SQL Server machine In this example neuron is the host name and apldbio com is the domain name You can also use an IP address This information is available from your system administrator 2500 the port number that the Sybase based BioLIMS SQL Server is using to connect with the clients This number is assigned to the server when it is installed You can find the port number in the interfaces file which is located in the home directory of the Sybase based BioLIMS SQL Server This is an example of the server entry in the UNIX server interfaces file SYBASE on neuron Services query tcp 2500 The third line of this entry shows the port number 2500 of the BioLIMS Server Troubleshooting the BioLIMS Database F 7 Troubleshooting the connection from the client Macintosh computer continued Step Action 4 Confirm that the SybaseConfig control panel is set up correctly Step Action a Open the SybaseConfig control panel This control panel must be located in the Control Panels fold
26. pcrlab appliedbiosystems com Protein Sequencing Peptide and DNA Synthesis corelab appliedbiosystems com If you are outside the United States and Canada you should contact The Americas United States Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 Tel 650 570 6667 800 345 5224 Fax 650 572 2743 Latin America Del A Obregon Mexico Tel 305 670 4350 Fax 305 670 4349 Europe Austria Wien Hungary Budapest Tel 43 0 1 867 35 750 Tel 36 0 1 270 8398 Fax 43 0 1 867 35 75 11 Fax 36 0 1 270 8288 Belgium Tel 32 0 2 712 5555 Fax 32 0 2 712 5516 Italy Milano Tel 39 0 39 83891 Fax 39 0 39 838 9492 Czech Republic and Slovakia Praha Tel 420 2 61 222 164 Fax 420 2 61 222 168 The Netherlands Nieuwerkerk a d IJssel Tel 31 0 180 331400 Fax 31 0 180 331409 Denmark Naerum Tel 45 45 58 60 00 Fax 45 45 58 60 01 Norway Oslo Tel 47 23 12 06 05 Fax 47 23 12 05 75 124 GeneScan Analysis Software Overview Europe Finland Espoo Tel 358 0 9 251 24 250 Fax 358 0 9 251 24 243 Poland Lithuania Latvia and Estonia Warszawa Tel 48 22 866 40 10 Fax 48 22 866 40 20 France Paris Tel 33 0 1 69 59 85 85 Fax 33 0 1 69 59 85 00 Portugal Lisboa Tel 351 0 22 605 33 14 Fax 351 0 22 605 33 15 Germany Weiter
27. Doesthe tracker line seem to follow the brightest part of the lane usually the center for the full length of the lane Does the lane drift to the side while the tracker line does not Inspect the Slice View of the lane data Arethe raw data peaks high enough for the full length of the lane Are some peaks very low because the tracker line is not located correctly on the lane How to Process and Edit the Gel File 2 55 Moving Tracker Tracker lines can be moved in the following ways Lines To move Then the line over one channel a Click the lane marker for the tracker line you want to move The lane marker becomes outlined in red and the tracker lines for all other lanes become hidden or grayed out b Press the Left Arrow or Right Arrow key to move the line over one channel selected lines to the right choose Force Selected Lanes to the Right from the Gel menu the line in channel a Click the lane marker for the tracker line increments of 0 1 you want to move b Press the Option key and either the Left or Right Arrow Deselecting a Lane To de select an edited lane In the lane marker area at the top of the window Result click the background between the diamonds that mark the lanes to If Then deselect the lane marker all tracker lines all lines are displayed become grey all lines are the deselected hidden line becomes hi
28. E 03 P27 3 Sample File Information gt Run Information 7 Data Collection Settings Module File EP Voltage 3000 Volts Matrix File HS AMD MAR 4 12 EP Current 60 m mps Parameters HS STANDARD AP EP Power 200 Watts Size Standard GS 350 75 bp 350 bp Temperature 51 C Laser Power 4 mivatts gt Gel Information b Sample Information 7 Analysis Records gt B Analyzed 10 24 20 4M Thu Jun 20 1996 P G Analyzed 10 24 20AM Thu Jun 20 1996 b Y Analyzed 10 24 204M Thu Jun 20 1996 P R Analyzed 10 24 20 AM Thu Jun 20 1996 Description of The following tables list the information in the Sample Info View Information Run Information Information found under the version Run date and start time Run duration Total data points collected header Information inserted from Username ABI PRISM 310 data collection Instrument Run file and run information Data Collection software ABI 3 3 or ABUPRISM S27 el file data collection Run file and run information This information is embedded in the gel file Analyzing Sample Files 3 9 3 10 Analyzing Sample Files Data Collection Settings Information found under the header Information is inserted from Module File Matrix File Analysis Parameters Size Standard Electrophoresis voltage current and power ABI 373 and ABI PRISM 377 only Run Voltage Injection Voltage and Injection Dur
29. Gel File Window The following is an example of the Gel File window Diagram Buttons 2 Gel file EE EB i gt 4 4 gt x S 1 05 Chanel 43 00 Sen 4107 Lanesused 33 I TEE 9 10 n 12 13 RE 15 16718 19 20 21 22 23 d4 25 28 27 Z 3 GOOOQOOO0QO0O0QO0Q9 QOOQOOQOOQC 4550 4045p 4 3540p 4 3035p 2530p 2025p 15209 4 10159 510 p oii Curent Comb Type set in prefs Square Tooth 5 9 8 7 6 How to Process and Edit the Gel File 2 13 Gel File Window The following table describes the buttons on the Gel File window Buttons Window tool Description ra e v Egi Color buttons Turns on off the display of one or more colors in the gel image and slice view see also page 2 18 IER Sample Sheet button Displays a copy of the Data Collection Sample Sheet associated with the gel file You can use the sample sheet information to check that the lanes are correctly labeled for the gel image For more information see Displaying the Sample Sheet on page 2 29 Gel Info button Displays the Gel Info dialog box For more information see Gel Info Window on page 2 26 E Horizontal Shrink button Compresses the gel image horizontally in order to see all the gel lanes in a standard sized window Horizontal Expand button Expands the gel image horizontally in order to adjust the tracker lanes The four levels of horizo
30. Show Creation Date date and time the Sample file was created 4 Select the checkbox labeled Save as Defaults to have the options you choose saved as the default settings 5 Click OK Analyzing Project Files 5 13 Setting Sample File Sort Order 5 14 Analyzing Project Files Use this option to specify how Sample files are sorted using three criteria If no sorting option is specified then the program sorts by Sample number To set Sample file sort order Step Action 1 Choose Project Options from the Settings menu and Sample File Sorting from the submenu The Sample File Sorting dialog box appears Sample File Sorting Sort Sample Files in following order Precedence Item Sort Order Sample No v Ascending Descending Ascending Descending Ascending Descending X Save as Defaults Cancel OK 2 Choose from the following items from the pop up menus File Name Directory Gel Name Sample Number User Name Instrument Name Run Data Creation Date As Added sorts the files in the order that they were added The precedence indicates the sorting level 3 Select a radio button for each sort to indicate whether ascending or descending order 4 Select the checkbox labeled Save as Defaults to have the options you choose saved as the default settings 5 Click OK Setting Dye Indicator Preferences The following pr
31. The step numbers in the following procedure correspond to the steps marked in the flow chart above IMPORTANT The documentation for each BioLIMS Macintosh computer program includes a section about setting up the database connection Make sure you have followed that procedure carefully Troubleshooting the connection from the client Macintosh computer Step Action 1 Locate the interfaces file Confirm that the folders charsets and locales are located in the same folder as the interfaces file The default installation places interfaces charsets and locales in the BioLIMS BioLIMS Extras Sybase folder 2 Confirm that the Sybase library files are installed into the Extensions folder in the System Folder For more information see Required Sybase Extension Files on page F 11 In particular if the libtcp extension is missing or not turned on the Sample 2DB Software crashes when connection to the database is attempted 3 Open the interfaces file and confirm the server information is correct An example of the Sybase based BioLIMS SQL Server entry is shown below Sybase query MacTCP mac ether neuron apldbio com 2500 Note Refer to the table immediately below for an explanation of the BioLIMS Server entry F 6 Troubleshooting the BioLIMS Database Troubleshooting the connection from the client Macintosh computer continued Step Action The BioLIMS Server entry Where
32. collection The Collection Name Collection comment and Collection owner fields use this information edit a a Choose the Edit Collection Names pop up menu collection The Collection Name Editor dialog box appears Collection Name Editor Edit Collection Names lection Name J Colection tommen Collection Owner lt _ lt 7 b Edit the information in the Collection Name Collection Comment or Collection Owner fields and click OK 2 34 How to Process and Edit the Gel File Changing Column Widths Editing the Sample Sheet To use the pop up menu to edit the collection information continued If you want to Then adda a Choose the Edit Collection Names pop up menu collection The Collection Name Editor dialog box appears see edit a collection above b Click Add Row Enter a Collection Name Collection Comment and Collection Owner and click OK delete a a Choose the Edit Collection Names pop up menu collection The Collection Name Editor dialog box appears see edit a collection above b Selectthe row that contains the collection you want to delete c Click Delete row and click OK To change the width of columns in the Sample Sheet Step Action 1 Place the cursor on the divider line to the right of the column title 2 When the cursor changes to two arrows hold down the mouse button and drag the line to the
33. only one strand of the double stranded DNA is labeled whereas the other two standards have labels on both strands under denaturing conditions even if the two strands migrate at different rates only the one labeled strand is detected Because of this split peaks are avoided that result when two strands move through a denaturing polymer at different rates see the figure below 60 30 120 150 180 210 240 270 300 Asterisks 2 o m 2R 330 360 390 420 450 480 Double Stranded GeneScan 500 Fragments Conditions The following figure shows the sizes of double stranded GeneScan 500 fragments Use these values to size fragments run under native 2R GeneScan Size Standards B 7 GeneScan 1000 Standard How It Is Prepared The GeneScan 1000 standard is prepared by digesting pBR322 with the restriction enzyme Alu followed by the ligation of a ROX labeled oligonucleotide This standard has 17 fragments ranging from 47 to 946 base pairs GeneScan 1000 Fluorescently labeled native fragments are 18 nucleotides longer than Molecular Lengths denatured fragments see the table below You can use this standard to size fragments in the 100 to 900 base pair range GeneScan 1000 Fragment Molecular Lengths Base Pairs
34. 22 23 2 On the left side of the Results Control window deselect the dye color fields corresponding to the samples you want to remove The dye sample identifiers are removed from the Dye Sample Display list as you deselect samples Evaluating Analysis Results 7 11 Removing Samples To remove samples Step Action 1 Select the panel number on the buttons to the left of the Dye Sample Display list You can take the following action To remove Click all the samples you selected Clear Panel for display in a panel the samples you have selected Clear All for display in all panels Displaying the To display the results on the screen you can either Results 4 Click the Display button Press the Return or Enter key Printing the To print the results Results Step Action 1 You can either Click the Print button or Choose Print 3 P from the File menu Click Print in the dialog box that appears 7 12 Evaluating Analysis Results Changing How the Results are Displayed and Printed Procedure You can set certain display preferences that remain in effect each time you display or print results data To change how results are displayed and printed Step Action 1 Choose Preferences from the Settings menu and choose Results Display from the submenu The following dialog box appears
35. 5 40 Analyzing Project Files For information on using the pop up menu see step 3 above O Print Results Sample File Project Analysis Control Print Setup Size Standard Pop up menu Parameters IM Analusis Parameters al sAnalysis Parameters Analysis Parameters Defining Folder Locations Introduction Storing Matrix Files The GeneScan Analysis Software looks in the designated folders for the Size Standard file Analysis Parameter file Matrix file When saving one of these files for the first time the default folder locations for saving the files are those same designated folders Store matrix files that are intended for use by Data Collection software to assign to collection runs in the ABI Folder The ABI Folder is located in the System folder on the computer on which the Data Collection software is installed If Data Collection and Analysis are Performed on Different Computers Make a copy of a matrix and store as follows This is useful when data collection and analysis are performed on different computers Store a copy in the For use by the ABI Folder Data Collection software GS Matrix Folder GeneScan Analysis Software Note The ABI PRisM instrument Data Collection software uses the files installed by the GeneScan Analysis Software in the ABI folder When you run the analysis software the progr
36. AQ 9 771623E 01 Al 1 220838E 01 A2 4 856885E 06 R 2 1 000 Size Calling Curve Global Southern Method D D T T T 7 7 T 1000 1500 2000 2500 3000 3500 4000 4500 Data Point sa A000 isi The Equations The following table describes the equations work Equation Description L c m m0 LO Attempts to describe the reciprocal relationship between the mobility m and the length LO of the standard fragments Yi Li c mi m0 LO 2 The fitting constants LO mO and c are calculated by a least squares fit to minimize the following quantity Size Calling Methods C 7 How This Method All points in the standard are weighted equally and the curve is not Works constrained to go through any specific point The software can analyze a large range of fragment sizes with this method C 8 Size Calling Methods DNA fragments that are Are sized using not bracketed within the size standard curve a second order least squares curve extrapolation bracketed within the size standard curve the method that was chosen For best results use a standard that brackets all the fragments of interest Troubleshooting the GeneScan Software Introduction In This Appendix The tables in this section present information about problems you might experience with your GeneScan Analysis Software runs and suggest possible causes and corrections For informatio
37. How to Change the Horizontal Scale for Individual Electropherograms To change the horizontal scale for individual electropherograms Step Action 1 Display the electropherograms 2 Move the cursor over the horizontal axis of the panel that you want to change and double click The Horizontal Scale Parameters dialog box appears Enter the increments represented by the tick marks for the horizontal axis in the Tick Spacing box Enter a range in the entry fields labeled Display from and to Click OK The horizontal scale changes Changing the You can change the vertical scale for Vertical Scale 4 Ait electropherograms Individual electropherograms How to Change the Vertical Scale for All Electropherograms To change the vertical scale for all electropherograms Step Action 1 Display the electropherogram panel that you want to change 2 Choose Vertical Scale from the View menu The Vertical Scale dialog box appears Uertical Scale Parameters Vertical Scale Tick Spacing 600 units tick Display from 0 to 6000 ok Enter the increments represented by the tick marks for the vertical axis in the Tick Spacing box 7 38 Evaluating Analysis Results To change the vertical scale for all electropherograms continued Step Action 4 Enter a range in the entry fields labeled Display from and to
38. Name starts with 31characters fragment including letters This is the fil ends with numbers and bant unctuation name entered in contains p the Sample Cannot use Sheet colons Sample Creator is up to 255 Name of the starts with enaraciers p including letters responsible for ends with numbers and the run amp contains punctuation Sample Name is up to 255 Sample name f characters from the Sample h Stans wit including letters Sheet ends with numbers and contains punctuation Instrument is up to 255 Set in the Name starts with characters General Settings including letters Preferences of ends with numbers and the Data contains punctuation Collection software Instrumentation any NA Whether the sample was run gel on a gel or capillary capillary instrument Start Collect any date set with Date data Date E arrow buttons collection using format began before mm dd yy after between Using the BioLIMS Database E 25 Fragment Search Criteria continued Pop up Menu Criterion Choices Allowed Text Description End Collect any date set with Date data Date i arrow buttons collection is using format ended before mm dd yy after between Gel Path is up to 255 The full path starts with chalaciels acti to thie including letters original gel file ends with numbers and e g contains punctuation H
39. Results Dye Scales Dye Indicators Folder Locations Results Display BioLIMS Access This BioLIMS Access menu item will not appear if certain database support files are missing Be sure that all the Oracle or Sybase SQL Server database support files are installed correctly If files are missing reinstall the BioLIMS Client or Instrument package from the original CD ROM disc F 2 Troubleshooting the BioLIMS Database About Troubleshooting the Client to Sybase Connection Introduction A common source of difficulty using the BioLIMS System is establishing connection between the BioLIMS programs running on Macintosh client computers and the BioLIMS database on the Sybase Server Note For information about troubleshooting an Oracle Server connection see About Troubleshooting the Client to Oracle Connection on page F 14 SybPing and Two programs have been provided to help with troubleshooting Telnet Program Description SybPing A Sybase tool for testing network connections Look for this application in the BioLIMS BioLIMS Extras Sybase bin folder Telnet NCSA Telnet 2 6 is a program used for interactive access from a Macintosh client to a telnet host on TCP IP networks NCSA Telnet was developed by the National Center for Supercomputing Applications at the University of Illinois in Urbana Champaign NCSA Telnet 2 6 has been provided in the BioLIMS Extras folder If you need help their web sit
40. Saving Sample Files 0 0 0 ce eee eee eee ee eee 8 3 saving Gel Piles i oae Rete cete 8 4 Saving Results Displays lleleeeeeeeeeeeeeee 8 5 How to Archive Sample and Gel Files 00 narn 8 6 Introduction i cx ROREM Kei ees 8 6 Archiving Sample and Gel Files 00 8 6 Transferring Data to Other Applications llle 8 7 Other Applications societies ea te oe ded RUNE RR RES 8 7 Cutting and Pasting Tabular Data 00 8 7 Creating aText Filet sic ecneentser br oer n es 8 8 9 Printing Results cceeeeeees 9 0 Introduction ec mr hemp C teer e ete bite 9 1 In this Chapters ac cx cepe ERI UE EE EIU 9 1 About Printing uuo qe iets DURER Ere Nee ode Reha RR X RS RS 9 2 Ways You Can Print 2 0 0 eee ee ee 9 2 If You Get Unexpected Results eese 9 2 Automatically Printing Run Results 00 0 0 0000002 eee 9 3 Introduction eoe hee eese REA REREY RM YE CI 9 3 From the Data Collection Software 000 9 3 From the GeneScan Analysis Software 000 9 3 Printing the Gel Images oui ectiezueDie e du hoe Ah Dep Bey 9 4 About Printing the Gel Image 9 4 Proc d te eriin ri gb tates gle a ees ener ee EE 9 4 Printing Selected Sample Files 2 0 00 0 0 e eee eee eee 9 5 Introduction ioi ev tes Baek edhe dey NY C UNSER 9 5 Setting Printing Options 00 0 cece eee ee 9 5 Fr
41. Using the BioLIMS Database E 23 Collection Search The table below shows the collection search criteria The collections returned by the Collection Browser window must match all of the collection criteria and contain at least one fragment that matches all of the fragment criteria Criteria Allowed Collection Search Criteria Pop up Menu Criterion Choices Allowed Text Description Collection is up to 255 Name of the Creator s anewiih characters creator owner of the collection ends with contains Collection is up to 31 Name of the Name starts with characters collection ends with contains Collection Type any NA Collection type run Default is any project menu item other Creation Date any date set with Date the i arrow buttons collection was is using the format created before mm dd yy after between Modification any date set with Date the Date e is arrow buttons collection was using the format last modified before mm dd yy after between E 24 Using the BioLIMS Database Fragment Search Criteria The table below shows the fragment search criteria The collections returned by the Collection Browser window must contain at least one fragment that matches all of the specified fragment criteria Fragment Search Criteria Pop up Menu Criterion Choices Allowed Text Description Sequence Frag is up to Name of the
42. Works with 96 Lane Gels lese 1 2 Displaying Off Scale Data llle eee eee 1 3 Manual Tracker Interface Changes 0 0000005 1 3 Printing from Sample File Window 004 1 3 New Neural Net Tracker 1 3 Lane Extr ction o ila sees e eese Deed os a e etia Re 1 4 About This Manual and Other Instruments 00 1 5 ABL379 ut eRiRbI reed enone xD 1 5 ABI 373 XL Upgrade Users 0 0 0 cece eee eee 1 5 96 lane Upgrade Kit 0 cece een eee 1 5 Manual Text e mer agri Land Ue Sink Nig ee eR tere Ree 1 5 User Attention Words 0 0 00 cee eee eee eee 1 6 Using the Macintosh Computer 02 0 0 00 0 e eee eee eee eee 1 7 Macintosh Computer Vocabulary and Operations 1 7 Guidelines for Optimal Performance 000 1 7 Macintosh Computer Terms Used in This Manual 1 8 Registering the Software 0 0 cee eee eee eens 1 11 License and Warranty 0 0c cece eee eee eee 1 11 Registering Your Software 00 cece eee eee 1 11 Installing the Software 0 0 eect eh 1 12 System Specifications 0 0 0 cece cece 1 12 Turning On Virtual Memory 0 0 ee ee eee eee 1 13 RAM Required to Run the Software 005 1 14 Installing the Software 1 14 Program Files Installed Diagram 0005 1 15 Contents of Folders circs easet seini a E 1 1
43. b Determine the stop number by multiplying the applicable number from the above table by the number of hours that pass before the last peak is detected 2 4 How to Process and Edit the Gel File Image Generation options continued Item Description Multicomponent Gel Image Note This option affects only the gel image Select this checkbox to use the spectral separation algorithm in conjunction with a predefined matrix file to resolve the spectral overlap between dyes Matrix files are specific to a particular set of run conditions and to the instrument on which they are created In other words you must create a different matrix file for each dye set gel type and set of running conditions you use For more information see Chapter 6 Making a Matrix File IMPORTANT If the gel does not have an associated matrix you must attach a matrix to the gel or the software will not multicomponent the gel image even if you select this option The tracker relies upon multicomponent images for accurate tracking IMPORTANT To attach a matrix see Installing New Matrix Information on page 2 24 Note You must always attach a matrix to the ABI 373 Collection file in order to multicomponent the gel image ABI 672 Data Collection software is not capable of pre installing a matrix See Appendix A Using GeneScan with the ABI 373 How to Process and Edit the Gel File 2 5 Image Gene
44. data for a single lane or injection Size GS Standards GS Standards Identify peak sizes Standard folder or other folder or other for specified size Files specified folder specified folder standards run under certain conditions Define the standards after running them on the instrument Tracker files and extensions GS GelTracker folder in the GS 3 0 folder ABI PRISM 377 and ABI 373 XL GS Tracker folder in the GS 3 0 folder GeneScan Analysis Software Overview 1 17 GeneScan Analysis Software files continued Location on 373XL File type ABI PRISM 377 Description 377XL 377 96 Lanes or ABI Prism 310 ABI 373 Analysis GS Parameters GS Parameters Specify certain Parameters folder or other folder or other ranges and methods specified folder specified folder used during GeneScan analysis You can also create and save custom parameters files for future use Matrix files ABI folder GS ABI folder GS Contains Matrix folder or Matrix folder or mathematical other specified other specified matrices that correct folder folder for spectral overlap They are applied to data based on the chemistry run Analysis GeneScan GeneScan Contains a running Log folder folder record of analysis performed by the GeneScan Analysis Software Sample GeneScan or GeneScan or Sample information Sheet specified folder specified folder in spreadsheet form Used for attaching to gel file
45. lanes HEX labeled products Collected using Repeat electrophoresis appear green on gel Filterset A with display ABI 373 or Filter set B ABI 373 or Virtual filter A ABI PRISM 377 Virtual filter C ABI PRISM 377 Collection time was Gellmage a Regenerate gel sufficient but only a Processing image with new small portion of gel preferences did scan range displayed not include b Adjust to correct enough scans to settings repeat display entire gel electrophoresis Electrophoresis power too low D 6 Troubleshooting the GeneScan Software Troubleshooting gel data continued Problem Probable Cause Correction Improper tracking results Bad matrix Attach new matrix Sample Sheet not filled out properly Fill out Sample Sheet properly Comb types set improperly a Fixand type in gel preferences b Retrack gel Peak height or red signal too low Rerun gel with more size standard Troubleshooting the GeneScan Software D 7 Troubleshooting Genotyping Results Table Description The following table lists the problem probable cause and correction for troubleshooting the Genotyper software results Problem Probable Cause Correction Allele peaks seen in correct molecular weight range with additional peaks seen outside this range Bleed through from other colors because of off scale data Primers not f
46. number in the table corresponding peak in the electropherogram is highlighted b Type the value for the selected peak in the corresponding Size field in the table Refer to Appendix B GeneScan Size Standards for values and peaks patterns Note Leave a zero in the Size field to ignore a peak for the standard definition C Press Return to automatically move to the next size standard peak Analyzing Project Files 5 33 Using the Analysis Control Window 5 34 Analyzing Project Files To use the New command to define a new size standard continued Step Action 8 When you finish defining the peaks save the size standard by choosing Save As from the File menu Note You can also click the close box 9 Type in a descriptive name for the standard and click Save Note Run conditions are not stored in the Standard file Use a name that clearly defines the standard for future use This file is automatically saved in the Size Standards folder For information on how to define the folder location see page 5 41 To use the Analysis Control window to define a new size standard Step Action 1 Open an existing project or create a new project For information on See page opening an existing project 4 10 creating a new project The Analysis Control window should appear If it does not choose Analysis Control 3 1 from the Windows me
47. on page 2 29 2 28 How to Process and Edit the Gel File Displaying the Sample Sheet Whatis the The Sample Sheet contains sample information and is copied to the Sample Sheet Data Collection run file before you start the run Using the Copy of The Sample Sheet copy is automatically copied to the gel file You can the Sample Sheet view edit and print this Sample Sheet copy whenever the gel file is open Changes you make in this copy are stored in the gel file they do not affect the original Sample Sheet file Displaying the To display the copied Sample Sheet Copied Sample Sheet Step Action 1 Open the gel file 2 There are two ways to display the Sample Sheet You can Result click the Sample Sheet The Sample Sheet window button EB appears choose Gel Sample Sheet from See Sample Sheet Example the Gel menu on page 2 31 Displaying a New To display a new Sample Sheet Sample Sheet Step Action 1 Open the GeneScan Analysis Software program 2 Choose New from the File menu The Create New dialog box appears Create Neu i T 8 Project Sample Analysis Size Matrix Sheet Parameters Standard Cancel How to Process and Edit the Gel File 2 29 Step Action 3 Click the Sample Sheet icon The following dialog box appears Number Of Dyes Select the number 4 dyes of dyes Q5 dyes 4 Click the radio button t
48. rather than averaging three channels Pre Averaging Offscale Detection checkbox When you select this checkbox if any of the channels that are used for averaging has a value of 8191relative fluorescent units rfu then no averaging takes place and the value of 8191 rfu is written for that scan of that lane Note If you extracted a sample file with the Pre Averaging Offscale Detection checkbox selected then pre averaging has a value of zero in the Sample Info View For information about off scale data see About Displaying Regions of Off Scale Data on page 2 38 The default is checkbox is not selected How to Process and Edit the Gel File 2 9 Item Description Stop extraction If the Stop extraction when below confidence when below threshold box is checked the lane extraction will not confidence value be carried out and a warning dialog box will appear checkbox when the lane assignment confidence level is below that specified in the Confidence Threshold The dialog box gives you the option to cancel or continue the gel file extraction and analysis Confidence After a gel is auto tracked a lane assignment Threshold text confidence value is written to the Analysis Log entry field This value indicates the Tracker s confidence in how well the assigned lanes match the sample sheet This value is not an indication of how well the tracker lines follow the fluorescence int
49. the program automatically changes to the next panel so each selection is placed after the one containing your previous selection After you select a sample for the last panel the panel increments to the first panel again Example The following table describes two examples of how to use the Quick Tile feature If you Then have four samples and choose four click the column heading for the blue panels for display dye to select all four blue dye labeled samples The blue dye for each Sample file appears in a separate panel click the row index number for the each dye for that Sample file first Sample file to select all dye appears in a separate panel colors for one sample file 7 10 Evaluating Analysis Results Deselecting To deselect the samples that you have selected for display Samples Step Action 1 If you specified Electropherogram display click the panel number to the left of the Dye Samples display list to display the information for the panel containing the samples you want to deselect Dye color fields Panel numbers D oe lozep27 2 of Panels Dye Samples TB G P27 B 8 f FG O8 Pa7 8 3 4 FY O8 27 8 8 TR 0G PE7T B 8 m E a Clear Panel Quick Tile Clear All t uiu 24 CUT leizsezrs2 e 090P27 9 7 9 d EEE Ea 4 meu L6 L8 L9 18 Lit L12 L13 L14 EFE L16 18 L19 L21
50. 14 saving procedure 8 3 switching between Sample file and BioLIMS mode E 10 to E 11 unlocking sample files 4 12 using project to manage files 4 8 to 4 9 why save 8 2 Sample Info View 3 8 to 3 12 description of view 3 9 to 3 12 displaying the view 3 8 example 3 9 what it displays 3 8 Sample Results View 3 6 to 3 7 description of view 3 7 differences from Results Display 3 7 displaying the view 3 6 example 3 6 what it displays 3 6 sample sheet about 2 29 to 2 37 completing for the ABI 373 A 9 to A 10 saving after tracking 2 68 gelfiles 8 4 matrix file 6 34 projects 8 3 Results Control format 7 17 to 7 19 important considerations 7 17 renaming current display 7 19 renaming current Results Display 7 19 Index 6 saving 7 17 saving the format 7 17 using previously saved formats 7 18 working with previously saved displays 7 18 Results Displays 8 5 sample files 8 3 why save 8 2 scan numbers defined Glossary 3 separation distance defined Glossary 3 server names about E 17 to E 19 sharks tooth comb defined Glossary 3 size calling methods Cubic Spine method C 4 Global Southern method C 7 to C 8 Least Square method C 2 to C 3 Local Southern method C 5 to C 6 Size Curve View 3 13 to 3 14 displaying the view 3 13 size curve described 3 14 what is the size curve 3 13 what the size curve displays 3 14 size standard about 5 29 to 5 30 defining how to 5 31 to 5 35 GeneScan 1000 B 8 to B 9 GeneScan 2500 B 10 to B 11 Gen
51. 2 4 Note Setting a range for gel display processing is similar to the same function in the preprocessing parameters of the GeneScan 1 X software You can take the following action in the Lane Extraction section To Then use a weighted average select the weighted averaging checkbox specify the number of channels to be averaged enter a value in the Channel averaging text box stop extraction when the extraction process falls below a set confidence level selectthe Stop extraction when below confidence level checkbox Enter a value in the Confidence Threshold box For more information see Lane Extraction on page 2 7 Click OK A 6 Using GeneScan with the ABI 373 Opening the Collection File Introduction When first opening a Collection file you usually need to tell the GeneScan Analysis Software how many lanes were collected and what matrix file should be used to adjust for spectral overlap Procedure Opening the Collection file Step Action 1 Choose Open from the File menu The dialog box appears You can either Double click the file name or Selectthe desired Collection file and click Open The gel file verification dialog box appears Gel File This Gel File is an old Format Please verify that the Number of Lanes is 36 Cem When opening a Collection file specify
52. 310 Genetic Analyzer automatically processes collection data and generates Sample files when the run completes If you ran your Dye Matrix Standards on the ABI PRISM 310 go to Choosing a Scan Range for the Matrix Calculation on page 6 29 Verify Tracking Before generating Sample files verify that the lanes were set and tracked correctly The following table lists where the data appears for each instrument once you have successfully completed your run of Dye Matrix Standards On this instrument The data appears Next step ABI PRISM 377 in a Gel File window ABI 373 in a Data Collection file Verify that the lanes Convert the Data were set and tracked Collection file to correctly generate the gel image see Appendix A Generating Sample Files P To generate Sample files Step Action 1 Click the appropriate Lane Indicators at the top of the Gel window band in all lanes to make sure that the tracker line is optimally aligned over each If any of the lanes are not properly tracked use the tracker line editing tools to align the tracker lines in each lane See Working with Tracker Lines on page 2 53 Making a Matrix File 6 27 6 28 Making a Matrix File To generate Sample files continued Step Action 2 If you change any of the lane assignments in any way save the changes 3 From the Gel menu choose Track amp Extract Lanes
53. An untitled Sample Sheet appears Fill out the Sample Sheet for each of the colors Set the preprocessing parameters as follows Do And deselect Multicomponent select Use Sample Sheet choose the appropriate Sample Sheet select Auto Lane Track leave the Process amount as Baseline Data the default value Choose Install New Sample Sheet from the Gel menu Go to Generating Matrix Sample Files for the ABI 373 and ABI PRISM 377 on page 6 27 Making a Matrix File 6 23 Loading and Running Matrix Standards on the ABI 373 with XL Upgrade Introduction The ABI 373 with XL upgrade updates the Data Collection software of the ABI 373 to make it compatible with Power Macintosh computers and allows you to open gel files directly using the GeneScan Analysis Software in much the same way the ABI PRISM 377 does This section describes how to do the following tasks Topic See page Completing the GeneScan Sample Sheet 6 24 Loading the Matrix Standards 6 25 Running the Matrix Standard 6 26 Note The procedures in Appendix A are not necessary since the ABI 373 software operates on a Power Macintosh computer Completing the The GeneScan Sample Sheet assigns the sample and dye information GeneScan Sample to their appropriate lanes Sheet To create a GeneScan Sample Sheet Step Action 1 Open the Data Collection software 2 Choose
54. Analysis Results 7 25 Electropherogram The following is an example of an electropherogram Example ill UL A 11B 1347 02 P14 FAM LINE 11v 1347 02 P14 HEX GM 116 1347 02 P14 TET il 11R GS 360 Size Std Descriptions of the The following table lists the descriptions for the above figure Electropherogram 7 26 Evaluating Analysis Results No Description See 1 Cross hairs Displaying X and Y Axis Positions on page 7 27 2 Magnifying glass Zooming In and Out Use to zoom in a specific area or hold Bn page moi down while pressing the Option key to zoom out in successively smaller scale 3 Legend Using Legends to h he Display Text from the Sample file that appear E nid pos ISP beneath electropherogram panels in the i Results Display window 4 Dye color indicator Dye color indicator on page 7 6 5 Plot color indicator Plot color indicator on page 7 5 6 Scroll bar Scrolling the Display Use the scroll bar to scroll horizontally on page 7 30 Working with Electropherogram Data In This Section This section describes how to perform the following tasks Task See Displaying X and Y Axis Positions 7 27 Moving the Electropherogram 7 28 Changing the Dye Color 7 28 Displaying Peak Positions 7 29 Highlighting Peaks 7 29 Using Legends to Change the Display 7 30 Scrolling the Display 7 30 Zooming In an
55. Comb Py pet vn esc ene aoe a eor C WR RACES SOR aes ee 2 10 How to Display the Gel File 2 0 0 0 0 cece eee eee 2 11 Introduction scissa eset hee ep RI ea we id ae ee ES 2 11 Displaying the Gel File 0 0 cece ee eee ene 2 11 About the Gel File Window 0 0 0 0 00 cece eee eee eee ee 2 12 Differences Between Gel File Windows 4 2 12 Gel File Window Diagram 0 c cece eee eee nee 2 13 Gel File Window Buttons sse 2 14 Gel File Window Described 00 0000 0 eee eae 2 15 How to Adjust the Gel Image 0 0 00 eee eee eee eee 2 18 IntrOductlOn sra vpRUEPUR RT EU DICES oe Rares heen ar ae 2 18 Displaying Hiding a Dye Color 0 00 00 00000 2 18 Changing Dye Indicators 0 0 0 cece ee eee ee 2 19 Adjusting the Contrast 2 0 0 ee eee eee eee 2 20 Removing Gel Contrast Changes 00 000005 2 22 Regenerating the Gel Image 0 00 00 00005 2 23 Installing New Matrix Information 2 24 Verifying Gel Information 2 26 Where the Information is Archived 00 2 26 Data Collection Run Error Log 0 0 0 0 0 0 cece eee 2 26 Gel Info Window lesse 2 26 Gel Image vo ueste ure RC UR CE Ii ee ch n 2 27 Sample Sheets x aed ERR eR Gade te ee we 2 28 Displaying the Sample Sheet 0 0 0 0 eee eee eee 2 29 What is the Sample Sheet
56. Generated from Using lane information ABI 373 or ABI PRISM 377 instruments f fil ili iL After extracting lanes from a gel file Sample file data can be analyzed as follows Analyze Sample file data For information see directly from the Sample Analyzing a Sample file File on page 3 19 from the Analysis Analyzing Sample Control window in a Files Using the project Analysis Control Window on page 5 6 capillary ABI PRISM 310 instrument electrophoresis How GeneScan The GeneScan Analysis Software creates Sample files after extracting Generates Sample lanes The information from each lane in the gel file is tracked and Files extracted and the resulting Sample files are placed in their respective sample folder If you change tracking lane assignment or Sample Sheet information you have to regenerate the Sample files 3 2 Analyzing Sample Files The software consults Sample Sheet information to determine whether a lane is used contains sample The lane tracker uses this information to assign lane numbers to the tracker lines In addition the GeneScan Analysis Software only extracts those lanes identified as Used Ways to Generate There are two ways to generate sample files Sample Files Software Instrument Generates Data Collection ABI PRISM 310 Sample file for each software injection GeneScan Analysis ABI 373 or Sample file for each Softw
57. Info Display dialog box appears Sample Info Display d File Name 3 Sample Info Dye Sample Info amp Legend E Name L Comment Sample File Info Show User Name Show Gel File Show Instrument Name Show Run Date Q Show Path Name Show Creation Date 3 Save as Defaults Cancel Select the checkboxes in the Dye Sample Info amp Legend section to control what appears when you move the cursor over the dye color fields in the Control windows The following table describes the checkboxes If you select this checkbox This appears File Name Sample file name Sample Name Name of the sample file from the Sample file Sample Info Sample information from the Sample file Comment Comment from the Sample file To specify the information displayed continued Step Action 3 In the Sample File Info heading select the checkboxes for the information that you want to display when you move the cursor over the Sample File name field The following table describes the radio buttons Select this radio button To display Show User Name user name from the Sample file Show Instrument Name instrument name from the Sample file Show Folder Name path and name of the folder where the file is located Show Gel File name of gel file from the Sample file Show Run Date run date and start time from the Sample file
58. L lane markers working with 2 43 to 2 52 Least Square size calling method C 2 to C 3 legends defined Glossary 2 using to change display in electropherograms 7 30 license and warranty G 1 to G 3 Local Southern method C 5 to C 6 M Macintosh using the computer 1 7 to 1 10 manual text for specific instruments 1 5 matrix files about 6 2 to 6 6 assigning to sample files 6 35 to 6 36 bad matrix files causes 6 38 to 6 39 choosing a scan range 6 29 to 6 31 Index 4 defined Glossary 2 Dye Matrix Standard kits 6 8 evaluating the matrix file 6 37 generating files ABI 373 and ABI PRISM 377 6 27 to 6 28 installing new file gel file 2 24 to 2 25 sample file 3 20 loading and running dye standards ABI 373 6 20 to 6 23 ABI 373 XL 6 24 to 6 26 ABI PRISM 310 6 13 to 6 16 ABI PRISM 377 6 17 to 6 19 new matrix file generating 6 32 to 6 33 preparing matrix standards ABI PRISM 310 6 9 to 6 11 ABI Prism 377 and ABI 373 6 12 process of creating new file 6 7 to 6 8 saving and naming 6 34 matrix standards preparing standard samples GeneScan 6 10 module defined Glossary 2 multicomponent matrix defined Glossary 2 multicomponenting defined Glossary 2 N network using SybPing program E 12 note definition 1 6 0 off scale data showing in electropherograms 7 32 showing in gel files 2 38 to 2 41 optimizing tracker lines 2 54 Oracle server configuring the server E 4 to E 9 how server names are recognized E 17 to E 19 troublesh
59. Methods C 5 How This Method This method which is similar to the Cubic Spline method uses the four Works fragments closest in size to the unknown fragment to determine a best C 6 Size Calling Methods fit line value Using this method only the region of the size ladder near the fragment of unknown length is analyzed Note Size estimates may be off if any of the standard fragments run anomalously The following table lists how the Local Southern method works Step Action 1 The fitting constants of the curve are calculated for each group of three neighboring points on the standard A separate curve is created for each set of three points 2 A curve is then created by using three standard points two points below and one point above the fragment and a fragment size is determined 3 Another curve is created by looking at an additional set of three points one point below and two points above the fragment and another value is assigned 4 The two size values are averaged to determine the unknown fragment length Global Southern Method About This This method is similar to the Least Squares method in that it Method compensates for standard fragments that may run anomalously The method creates a best fit line through all the available points and then uses values found on that line to calculate the fragment values EL 11 P27 11 HE Best Fit 2nd Order Curve
60. Peak field Indicates peak matched to defined size standard HL P14 ctlst 2 13 plex 5ypL 7 Displa 80 100 120 140 150 180 200 220 240 260 280 300 320 340 GM 1R 65 350 Size Std MG K Dye Sample Minutes Peak Height Peak Area Data Point Peak If you find that the sizes were not calculated correctly you can Redefine the size standard or Change the analysis parameters and re analyze the affected samples see Defining Analysis Parameters on page 5 18 Evaluating Analysis Results 7 49 Evaluating for To evaluate size calculations for multiple internal size standards Multiple Internal Size Standards 7 50 Evaluating Analysis Results Step Action 1 In the Results Control window select the Electropherogram button Click the Clear All button to clear the panels Click the On radio button to turn on the Quick Tile option 2 3 4 Click the dye color indicators for the size standards you want to view When the Quick Tile option is on the GeneScan Analysis Software inserts each in a separate panel Note Click the header of the appropriate dye sample column to display all standards in the project that are the same color dye Click Display The standards appear in tiled electropherogram displays For information on setting the tiled electropherogram displays see Creating Tiled Electropherogram Displays on page 7 10 Choose A
61. SOFTWARE from the previous computer or network You may never have operational SOFTWARE on more than one computer or more than one network if a network version per original copy of the SOFTWARE at any time 2 You may make one copy of the SOFTWARE for backup purposes 3 You may transfer the SOFTWARE to another party but only if the other party agrees in writing with Applied Biosystems to accept the terms and conditions of this Agreement If you transfer the SOFTWARE to another party you must immediately transfer all copies to that party License and Warranty G 1 Restrictions Limited Warranty Limitation Of Liability G 2 License and Warranty or destroy those not transferred Any such transfer terminates your license 1 You may not copy transfer rent modify use or merge the SOFTWARE or the associated documentation in whole or in part except as expressly permitted in this Agreement 2 You may not reverse assemble decompile or otherwise reverse engineer the SOFTWARE For a period of 90 days after purchase of the SOFTWARE Applied Biosystems warrants that the SOFTWARE will function substantially as described in the documentation supplied by Applied Biosystems with the SOFTWARE If you discover an error which causes substantial deviation from that documentation send a written notification to Applied Biosystems Upon receiving such notification if Applied Biosystems is able to reliably reproduce that error at its
62. Save as Defaults 1 2 3 4 5 6 7 8 2 Choose new colors from the pop up menus beside the plot numbers Note The plot numbers indicate the order of the samples in the electropherogram legend Evaluating Analysis Results 7 41 To define custom colors for all electropherograms continued Step Action 3 Choose Other from the pop up menu to specify a color that does not appear in the pop up menu The following is an example of a color picker that appears Original E IYK Picker a Crayon Picker 4 al TD Hue Angle p28 HLS Picker Saturation s Value s J Cx 4 Select the checkbox labeled Save As Defaults to save the customized colors 5 Click OK Defining To define the individual plot colors Individual Plot Colors 7 42 Evaluating Analysis Results Step Action 1 Display the electropherograms with legends 2 Double click the plot color indicator next to the sample you want to change The Choose a Plot Color dialog box appears Choose a Plot Color Pop up menu Cancel OK 3 Choose a new color from the pop up menu If you choose Other then a color picker appears Step Action Click OK The color of the electropherogram for the individual sample changes and a vertical line appears beside the plot color indicator to signify that it is modified Note You can change the plot col
63. Skip to step 2 on page F 12 of Troubleshooting from the Unix Oracle Server unsuccessful an error message is displayed The network is not working Call your network administrator Note If you entered the host name in step b above and the connection fails try entering the host domain name or the IP address before calling your network administrator See step 2 mozart on page F 18 for more information Troubleshooting the BioLIMS Database F 21 Troubleshooting The step numbers in the following procedure correspond to the steps from the Unix marked in the flow chart on page F 16 Oracle Server You need to have the account name and password for the Oracle user on the UNIX system that runs the Oracle Server If you do not have access to the Oracle user account you should ask your database administrator to carry out the following procedure Troubleshooting the client connection from the Oracle Server Step Action 1 Log in to the Oracle user account on the UNIX Server Try to connect to the Oracle Server with SQL Plus Use the same client user name and password as the one entered in the BioLIMS access dialog box For example sqlplus george georgel SOL exit Where Represents the george client user name george1 client user password If the Login is Then successful skip to step 6 unsuccessful continue to step 2
64. Stored 04 5 30 Defining the Size Standard 2 0 0 eee eee 5 31 Two Ways to Define the Size Standard 5 31 Using the New Command 00 0 0c eee eee eee 5 31 Using the Analysis Control Window 005 5 34 Using Size Standards 0 cece cee eee erent eens 5 36 In This Section uod ou REPE Pk Sere ed Ba en 5 36 Changing the Number of Peaks Detected 5 36 Editing the Size Standard Definition 5 36 Editing an Existing Standard 0 00 00 00008 5 37 Deleting an Existing Standard 0 00 5 39 Analyzing Samples Using the Same Standard 5 39 Selecting Separate Standards for Samples 5 40 Defining Folder Locations 5 41 Introduction corner ee TERQUE Sn Le a es Een PE A 5 41 Storing Matrix Files 0 0 0 cece cece reece m 5 41 Specifying File Locations 0 0 0 e cece eee 5 42 6 Making a Matrix File 6 1 Introduction oe tete nis nerve tense rdv edes hie RM RES 6 1 In This Ch pter aile se em Sa Weg e ede reete 6 1 About Matrix Files cech ect wee pis age RR OEERHEN uer ENDE 6 2 Introduction ou cepere ebES RE e Ue URN 6 2 Matrix File Definition 0 0 00 0 eee eee eee eee 6 2 Multicomponent Definition 00 00 0202 e ee eee 6 2 Why is a Matrix File Necessary 0 0 00 002 eee
65. T 1500 2000 2500 3000 3500 4000 4500 Data Point Mobility of any DNA fragment can be affected by its sequence and by secondary and tertiary structure formation If any internal size standard fragment has anomalous mobility the Cubic Spline method may exhibit local sizing inaccuracy For example Assume that a standard fragment is close in molecular length to an unknown sample fragment Assume further that the standard fragment runs anomalously The Cubic Spline method assigns the official value to this standard fragment even though it may be slightly incorrect The size of the unknown fragment is then likely to be calculated incorrectly as well Note This method does not determine the amount of sizing accuracy error Local Southern Method About This The Local Southern method determines the sizes of fragments by using Method the reciprocal relationship between fragment length and mobility as described by E M Southern 1979 270 Best Fit 2nd Order Curve AO 1 990206E 02 Al 2 075795E 01 4 A2 1 545595E 05 R 2 1 000 Size Calling Curve Local Southern Method D D T T 7 7 T T T 1200 1400 1600 1800 2000 2200 2400 2600 2800 Data Point The Equation The following table describes how the equation works Equation Description L c m m0 LO Attempts to describe the reciprocal relationship between the mobility m and the length LO of the standard fragments Size Calling
66. This Range Define lower minimum and upper maximum limits Base Pairs radio of the peaks to include in the tabular data button About Size Calling Method Parameter Options Click a radio button to select the desired size calling method The GeneScan Analysis Software uses these methods to determine the molecular length of an unknown fragment Size Calling Method Parameter Options Described Item Description 2nd Order Least Both Least Squares methods use regression analysis Squares and 3rd to build a best fit size calling curve d beast For more information see Least Square Method on quares page C 2 Analyzing Project Files 5 21 Split Peak Correction Parameter Options 5 22 Analyzing Project Files Item Description Cubic Spline Interpolation Forces the sizing curve through all the known points of the selected GeneScan size standard For more information see Cubic Spline Interpolation Method on page C 4 Local Southern method Determines the sizes of fragments by using the reciprocal relationship between fragment length and mobility For more information see Local Southern Method on page C 5 Global Southern method Similar to the Least Squares method in that it compensates for standard fragments that may run anomalously For more information see Global Southern Method on page C 7 About the Split Peak Feature Under denaturing c
67. Topics See page GeneScan 350 Size Standard B 2 GeneScan 400HD Size Standard B 4 GeneScan 500 Size Standard B 6 GeneScan 1000 Standard B 8 GeneScan 2500 Standard B 10 GeneScan Size Standards B 1 GeneScan 350 Size Standard What To Use It For How It Is Prepared GeneScan 350 Molecular Lengths Running Under Denaturing Conditions B 2 GeneScan Size Standards GeneScan 350 is a size standard useful for sizing fragments between 35 and 350 base pairs The native fragments are uniformly spaced to provide accurate size calling GeneScan 350 is prepared by Pst 1 digestion of plasmid DNA followed by ligation of a TAMRA or ROX labeled 22 mer oligodeoxynucleotide to the cut ends A subsequent enzymatic digestion with BstU 1 yields DNA fragments containing a single TAMRA or ROX dye see GeneScan 350 Molecular Lengths below The following table lists the GeneScan 350 Denatured Fragment Molecular Lengths Nucleotides 250 139 35 300 150 50 340 160 75 350 200 100 The following table describes running the GeneScan 350 standard under denaturing conditions Like However Consequently the GeneScan 2500 and GeneScan 1000 standards the GeneScan 350 standard is made of double stranded DNA fragments like GeneScan 500 the GeneScan 350 standard has only one labeled strand under denaturing conditions even if the two strands migrate at different rates only the labeled st
68. When an Alert Appears 0 0 0 cece eee nee 4 14 Searching for Missing Sample Files 004 4 14 Re Establishing Links 2 2 0 00 0 0c cece eee ee eee 4 15 5 Analyzing Project Files 5 10 Iittoductioric cose A a t oe 8 amt EAT UE URBE REATO 5 1 In This Chaptet ek Ee T 5 1 Analyzing Project Files About the Analysis Control Window 5 2 Introduction ous ce uen het HERR Eel rq PR PER 5 2 Analysis Control Window Example 0005 5 2 Analysis Control Window Description 00 5 3 Customizing the Display 0 0 0 0 eee 5 4 Using the Analysis Control Window 2 00005 5 4 Analyzing Sample Files Using the Analysis Control Window 5 6 Introduction n e eter egt ep Ra e Ent he er bi tcs 5 6 Accessing Sample Files 2 0 0 0 0 eee eee eee eee ee 5 6 Analyzing Sample Files 2 20 00 0 0 e eee eee eee eee 5 7 Specifying the Format for Printed Results 5 9 Displaying Size Standards and Analysis Parameters 5 11 Displaying Sample and Dye Information 5 12 Setting Sample File Sort Order esee 5 14 Setting Dye Indicator Preferences 0004 5 15 About the Analysis Parameters 0 0 0 c ee eee eee eee 5 17 vii Introduction 2x t su oed Rog auta D e En ede es 5 17 Parameters Used During Automatic Analysis
69. When to Create New a Matrix File You can only assign a matrix file to Sample files generated on the same instrument under the same electrophoresis gel matrix and buffer conditions and using the same dye set Note If you are using a fifth dye then you need to create a new matrix file for that dye Create a new matrix file in the following conditions For each dye set NHS Esters Phosphoramidite set Fluorescent dNTPs Whenever you change the dye set you use to label sample fragments for example if you are using the fifth dye When you use gel materials or buffers with pH values that differ greatly from the pH value of the gel material or buffer on which the existing matrix files were generated When you use dyes other than those provided by Applied Biosystems When you run the same gel on a different instrument When you see multiple unexpected peaks of different colors under an expected peak When you recalibrate your CCD camera ABI PRISM 310 and ABI PRISM 377 and the change is greater than 3 pixels from the original pixel position When you replace your filter wheel ABI 373 or CCD camera ABI PRISM 310 and ABI PRISM 377 Making a Matrix File 6 5 Considerations The following table lists some of the considerations before making a Before Making a Matrix File matrix file Consideration Comment How much dye matrix standard to load With the ABI 373 or ABI PRISM 377 load
70. You can save the defined standard in a file and use it to automatically analyze other samples run with the same standard and under the same conditions Split peaks might appear in size standards in which both strands are labeled for example GeneScan 1000 see page B 8 and GeneScan 2500 see page B 10 For some peaks the two strands migrate at different rates when running under denaturing conditions and they appear as two peaks approximately half the height of normal non split peaks One peak of the two runs is true to size You should assign a size to that peak for the standard definition and assign zero to the other peak For more information see Split Peak Correction Parameter Options on page 5 22 Analyzing Project Files 5 29 Specifying Where Choose Preferences from the Settings menu and Folder Locations from Files are Stored the submenu to specify the folders in which the software automatically stores the analysis parameters and size standards files For more information see Defining Folder Locations on page 5 41 5 30 Analyzing Project Files Defining the Size Standard Two Ways to Define the Size Standard Using the New Command There are two ways to define a new size standard Topic See page Using the New Command 5 81 Using the Analysis Control Window 5 34 To use the New command to define a new size standard Step Action 1 Choose New from the File menu The Create Ne
71. a Find the new location b Highlight the target folder c Click the Select button at the bottom of the dialog box 4 Take the following action If you have Then other folder locations to choose another folder from the designate Folder Locations Preferences dialog box no other folder locations to click OK designate Analyzing Project Files 5 43 Making a Matrix File Introduction In This Chapter Topics in this chapter include the following Topics See page About Matrix Files 6 2 Process of Creating a New Matrix File 6 7 The Dye Matrix Standard Kits 6 8 Preparing Matrix Standards for the ABI PRISM 310 6 9 Preparing Matrix Standards for the ABI 373 and ABI PRISM 377 6 12 Loading and Running Dye Standards for the ABI PRISM 310 6 13 Loading and Running Dye Standards for the ABI PRISM 377 6 17 Loading and Running Matrix Samples on the ABI 373 non XL 6 20 Loading and Running Matrix Standards on the ABI 373 with XL 6 24 Upgrade Generating Matrix Sample Files for the ABI 373 and ABI PRISM 6 27 377 Choosing a Scan Range for the Matrix Calculation 6 29 Generating a New Matrix File 6 32 Saving and Naming the Matrix File 6 34 Assigning the Matrix File to Sample Files 6 35 Evaluating the Matrix File 6 37 Causes for Bad Matrix Files 6 38 Making a Matrix File 6 1 About Matrix Files Introduction Matrix File Definition Multico
72. activities such as electrophoresis current laser power running time and gel temperature If the Run was The following table lists what happens to the Sample file if the run was Cancelled cancelled If a run was cancelled and you are using Then the sample file is Data Collection software v 1 0 4 saved if you skip to the next sample or cancel a run Note Version 1 0 4 is a free upgrade To obtain the upgrade either access the ABI PRISM 310 Genetic Analyzer website at www appliedbiosystems com techsu pport or call technical support see page 1 20 For more information refer to ABI PRISM 310 Data Collection Software Version 1 0 4 User Bulletin P N 4305798 Making a Matrix File 6 15 Run Time Run time is approximately 30 minutes for the GS STR POP 4 module so the total run time will be about 120 minutes Assigning Matrix For information on Assigning the Matrix File to Sample Files see File page 6 35 6 16 Making a Matrix File Loading and Running Dye Standards for the ABI PRISM 377 Introduction This section describes how to do the following tasks using the ABI PRISM 377 and the ABI PRISM 377 with XL Upgrade Topic See page Creating a GeneScan Sample Sheet 6 17 Loading Matrix Standards 6 18 Running the Matrix Standards 6 19 Note When loading the matrix standards on an instrument note which colors you load in which lanes for gel based systems Cr
73. analyze locked files When you add locked files an alert appears To unlock locked files Step Action 1 Save and close the project 2 In the Finder select the applicable Sample files 3 Choose Get Info from the File menu 4 In the lower left hand corner of each Sample Info window deselect the checkbox labeled locked To unlock locked files Step Action 5 Close the window and reopen the project Note If you did not close the project before unlocking the files once the files are unlocked close and open the project so the GeneScan Analysis Software recognizes that the Sample files are unlocked Removing Samples Note A removed Sample file is not deleted from the hard disk The reference from a Project is removed from the project To remove Sample files from a project Step Action 1 Select the file or files in the Analysis Control window or the Results Control window that you want to remove Note X Shift click to select multiple consecutive file or 3 click to select multiple files that are not consecutive 2 Choose Remove Sample Files from the Project menu or press the Delete key A warning dialog box appears A 1 Sample Files are selected Are you sure you want to remove them Cancel Remove 3 Click Remove Creating a Project 4 13 Finding Missing Sample Files When are Files Considered Lost When an Aler
74. because the GeneScan 672 Software does not automatically embed a copy of the data collection Sample Sheet in the Collection file Procedure IMPORTANT When tracking and extracting the Collection file ensure the Sample Sheet associated with the gel file has the checkbox labeled Used selected for each sample you want to extract The GeneScan Analysis Software only extracts the samples with that checkbox selected To complete the Sample Sheet Step Action 1 Open the Sample Sheet by either Clicking the Sample Sheet button in the upper left corner of the Gel Window or by Choosing Gel Sample Sheet from the Gel menu 2 Enter the following information Sample file name Sample name for each sample Sample info Comment for each dye sample For more information see Displaying the Sample Sheet on page 2 29 3 Select checkboxes as applicable To Then select the indicate that the lane contains Used checkbox data automatically analyze Sample A checkbox files when generating them automatically print Sample files P checkbox when generating them Making a Matrix File 6 21 To complete the Sample Sheet continued Step Action 4 Identify the dye standard for each sample by clicking in the appropriate dye cells under the standard column Note Alternately you can create a stand alone Sample sheet and attach it to the gel file u
75. below showing the electropherogram and a table of peaks for the dye color and sample selected You should be able to recognize the peak pattern of the standard in the electropherogram Note You can only change the peak size value in the right column of the table You cannot change or rearrange the peak numbers Note If too many peaks appear in the electropherogram or the baseline is too high you might need to adjust the analysis parameters See Using Analysis Parameter Files on page 5 24 The software assigns a number to each peak found in the electropherogram in order from left to right To use the New command to define a new size standard continued Step Action GS 350 2400 36 STD 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 1 ERE i i i i 1 1 i 1 i ner qm Template File Info File O1el Run Date Wed Jul 31 1996 Run Time 3 45 39 PM Dye R Parameters 2400 36 STD AP Specify the peaks of the standards and their sizes as follows Step Action a Click the peak you want to define either in the electropherogram or in the table Use the Zoom In 38 Plus sign and Zoom Out 3 Minus sign commands from the View menu to zoom the electropherogram for easier viewing If you click a peak Then the in the electropherogram corresponding row in the table is highlighted
76. click OK the alert is dismissed and the Preferences dialog box appears with the Sample Files button selected The Sample Data Access mode cannot be changed while editing Sample Project Files Please make sure all open Sample Project Files have been saved In the Session Manager section in the Preferences dialog box enter Your user name on the server Your password for your server account Thename of the database on the server You may have access to more than one database on the server The server name The server name is contained in the interfaces file Sybase or in the tnsnames ora file Oracle IMPORTANT All these text boxes are case sensitive Click the check box labeled Save Password if you want to Save your password so that you do not have to enter it every time you open the connection Run AppleScripts that don t contain password information Automatically analyze the data after the Data Collection software has collected the data d Save Password E 14 Using the BioLIMS Database To access the BioLIMS database continued Step Action 5 If you want the database to open automatically when you launch the GeneScan Analysis Software click the check box labeled Open on Launch 4 Open on Launch Note You must also click the check box labeled Save Password if you want the database to open automatically in order to automatically analyze data If you int
77. color down For example pull the top blue triangle down until it is somewhat above the tallest blue data peaks not primer peaks in the displayed Slice View suppress the background noise of a color pull the bottom triangle for that color up For example to correct a red background haze because of signal noise or because the signal baseline is not flat pull the bottom red triangle up until itis just above the baseline and noise in the display Click OK to dismiss the dialog box when you are satisfied with the gel contrast Removing Gel To remove the changes you can Contrast Changes 4 Choose Undo Adjust Contrast 38 Z from the Edit menu Note Can only undo if you do not choose another command that is after adjusting the gel contrast if you choose Undo Adjust Contrast you cannot do segment tracking Redisplay the Adjust Gel Contrast dialog box and manually reset the settings Close the Gel File window without saving the changes 2 22 How to Process and Edit the Gel File Regenerating the Gel Image Regenerating the gel image is useful for example if there is unusable data near the beginning or the end of the run the maximum peak height is different from what you expected or when a new matrix is installed To regenerate the gel image Step Action 1 Choose Regenerate Gel Image from the Gel menu The Regenerate Gel Image dialog box appears The va
78. connection still fails go to step 7 7 Call Customer Support See Technical Support on page 1 20 Troubleshooting the BioLIMS Database F 13 About Troubleshooting the Client to Oracle Connection Introduction Telnet A common source of difficulty using the BioLIMS System is establishing connection between the BioLIMS programs running on Macintosh client computers and the BioLIMS database on an Oracle Server Note For information about troubleshooting a Sybase SQL Server connection see About Troubleshooting the Client to Sybase Connection on page F 3 To help with troubleshooting NCSA Telnet 2 6 has been provided in the BioLIMS Extras folder NCSA Telnet 2 6 is a program used for interactive access from a Macintosh client to a telnet host on TCP IP networks NCSA Telnet was developed by the National Center for Supercomputing Applications at the University of Illinois in Urbana Champaign If you need help their web site address is http www ncsa uiuc edu SDG Software Brochure Overview MacTelnet overview html You can also reach them at the address shown below Applied Biosystems does not support NCSA Telnet NCSA Telnet Jim Browne Scott Bulmahn Jim Logan auskopf Gaige Paulsen Aaron Contorer itt sil Duval Pascal Maes Mark Tamsky NCSA Telnet is in the public domain NCSA Software Development Comments and suggestions catalog 152 Computing Applications Bldg available may be dir
79. current tracker line locations to extract the data in this and other similarly marked files and puts the extracted data into new Sample files Unmarking a_ To unmark a lane that is marked for extraction Single Lane for Extraction Step Action 1 Click the lane marker for the lane that you want to unmark 2 There are two ways to unmark a single lane for extraction You can Result choose Unmark Lane for Extraction from the Gel menu press the Option key and click the lane marker The color of the lane marker changes to blue 2 52 How to Process and Edit the Gel File Working with Tracker Lines Topics in This This section includes the following topics Section Topics See page Hiding Showing Tracker Lines 2 53 Why Positioning Each Lane is Important 2 53 Causes to Misinterpreting Lane Positions 2 54 Verifying Optimal Channel Tracking 2 55 Reviewing Tracker Lines 2 55 Moving Tracker Lines 2 56 Deselecting a Lane 2 56 Hiding Showing The following table lists how to hide and show tracker lines The first Tracker Lines time the GeneScan Analysis Software opens a gel file tracker lines are added to the gel image To Then turn off the tracker lines choose Hide Tracker Lines from the Gel Menu All unselected tracker lines disappear If a lane is selected the white tracker line for that lane
80. defining size standards Error Message Comment Correction Refer To The affected Sample File is not available Locate the Sample File and try again If the Sample file name is dim in the Analysis Control window the GeneScan Analysis Software has not located the sample file You can instruct the program to search for the sample file Finding Missing Sample Files on page 4 14 A Dye Standard is not selected for the affected Sample File Select a Dye Standard and try again Select the dye sample that represents the standard by 3 clicking the appropriate dye sample field Callout 3 on page 5 3 Troubleshooting the GeneScan Software D 11 Error messages when defining size standards continued Error Message Comment Correction Refer To The affected Sample Select either Callout 5 on page 5 3 File does not have a amp The default valid Analysis program Parameters Selection parameters Select new Analysis Analysis Parameters and try Parameter gt or again Avalid analysis parameters file in the Analysis Control window No peaks were found Make sure the Defining Analysis within the Analysis Peak Amplitude Parameters on Range Threshold setting page 5 18 allows for Check your Analysis detection of the Parameters peaks in your sample If peaks in your data are narrow make sure the Minimum Peak Half Width is a small number
81. desired location To edit the Sample Sheet you can Type in new text Note Use the Fill Down command to apply a change to all the rows in a column by entering the text in one of the rows clicking the column header and then choosing Fill Down from the Edit menu Use the Edit menu commands Select and deselect checkboxes How to Process and Edit the Gel File 2 35 Printing the There are two ways to print the Sample Sheet Sample Sheet If you choose from the File menu Then Print 36 P the standard Print dialog box appears choose any options and click Print Print One when the Sample Sheetis one copy of the selected information active is printed bypassing the standard print dialog box Installing a New Use the following procedure to replace the original Sample Sheet Sample Sheet information by installing a new Sample Sheet IMPORTANT If you change the Sample Sheet after extracting Sample files from the gel file you must re extract the Sample files to include the new information Make sure that the checkbox labeled Used is selected for each sample that you want to re extract To install a new Sample Sheet Step Action 1 Choose Install New Sample Sheet from the Gel menu A directory dialog box appears 2 Select a new Sample Sheet from the directory dialog box The new Sample Sheet replaces the original Sample Sheet copied to the gel file
82. eee 6 3 When to Create a Matrix File sees 6 3 Sample Files Using Matrix File 0 0 00000 6 4 Applying a Matrix File l l ee ees 6 4 When to Assign a Matrix File 0 0 0 0 ee eee eee ee 6 4 Limitations to Matrix Files 0 0 00 e eee eee eee ee 6 5 When to Create New a Matrix File 000 6 5 Considerations Before Making a Matrix File 6 6 Where to Store Matrix Files 6 6 Process of Creating a New Matrix File 00 errr 6 7 Process Diagram ies reri yea Shed ee daha da esd 6 7 Steps to Creating a New Matrix 00 00 0002 eee ee eee 6 8 The Dye Matrix Standard Kits 6 8 Table of Kits SERERE Ree Eee eS 6 8 Preparing Matrix Standards for the ABI PRISM 310 6 9 About Formamide and Samples in Formamide 6 9 Deionizing Formamide 0 0 0 eee eee eee 6 9 Preparing the Formamide Size Standard Mix 6 10 Preparing Matrix Standard Samples 000 6 10 Preparing Matrix Standards for the ABI 373 and ABI PRISM 377 6 12 Introduction e ce re Rt ER rte steep alge shard bets 6 12 Procedute sore eR C uper pedis TUA e s a8 6 12 Table of Matrix Standards 0 00 0 2c eee ee eee eee 6 12 Loading and Running Dye Standards for the ABI PRISM 310 6 13 Introduction seit et Ree eee nae rer re 6 13 Creating a GeneScan Sample Sheet
83. eee eee 2 57 Procedure oleis ll eR a Ree ed Re ae ew N uer es 2 57 Tracking Lanes and Extracting Data 0 00 00 0000 2 59 Ways to Track and Extract Data eese 2 59 If the Run Folder is Not Specified 0 00 2 59 Tracking the Gel Without Extracting Data 2 60 Tracking and Extracting Data 00000000005 2 61 Extracting Data Without Changing the Current Tracker 2 64 How the GeneScan Analysis Software Names Sample Files 2 67 Naming Process 4248 pp REF ESTIS EIN RI ONE 2 67 Saving Gel Files After Editing Tracking 00 2 68 Introduction eue chm y EIUS Wee Pate ee cs 2 68 Saving Files After Editing Tracking 0 2 68 Introductiotr erea ee et test e Ere Ee E nem HR Roe 3 1 In This Chapter 7 cest ee ee ree ede eed precise eg 3 1 About Sample Files 2 222 RR deg eee Wee EYE M 3 2 What Sample Files Contain llsleee eese 3 2 How GeneScan Generates Sample Files 0 3 2 Ways to Generate Sample Files 0 00000 3 3 How GeneScan Analyzes Sample Files 3 3 Opening Sample Files 3 4 Introduction ee ods a eae aden EU tme a ee lem 3 4 Procedure sei st eus us erts ec af s eee de aati cig des 3 4 About The Sample File Window sese 3 5 Whatat Displays o ek m nt pe cette MRESPOBIS REN 3 5 Fiye VIEWS vtto eost bep ek pt ende E ob
84. eese 3 18 Analyzing a Sample File 0 0 0 0 0 0 eee eee eee eee 3 19 Inttod cti h orc e tte si ie et OS pee SE E 3 19 Procedures ied Lone e eH ener halts andi ue Wi eet od 3 19 Installing a New Matrix File 0 0 eee eee eee 3 20 Introd CtIon gsn hee tr eter rie ep p Ad e Cr defe d 4 1 In This Chapter ic Luo ie ee eere exe VARICES 4 1 Automatic Analysis and Project Creation Process 4 2 Introduction uoo RIO ake eee eae ee ete es 4 2 Process Using the ABI PRISM 310 0 0 cee eee eee eee 4 2 Process Using the ABI 373 and the ABI PRISM 377 4 3 Setting Up for Automatic Analysis 00 0 c eee ee eee ee 4 4 Introd ctiony uc eR ee a RE wae HER Wie 4 4 Procedure iuis meus e e RT vet bei ue Reads 4 4 Using a Project to Manage Sample Files 0004 4 8 What1s a Project i llueve bx EU EES da e EI 4 8 Why Createa Project iecore te RH a RR 4 8 When GeneScan Creates Projects 00 02 e eee 4 8 Where to Store Projects 0 0 eee eee eee 4 9 Working with Project Files 1 0 0 0 eee eee eee eee 4 10 Opening an Existing Project 0 00 0 0 ee eee ee 4 10 Creating a New Project llle nee 4 10 Unlocking Sample Files llle 4 12 Removing Samples from a Project 00 0000 4 13 Finding Missing Sample Files llle eese 4 14 When are Files Considered Lost 00000000005 4 14
85. folder Sample files 4 aoo00000000 O0D00000000 DODDPDDDDDDI 0000000000 Collecting eee Omm nion gel data Processing Analyzing f the gel file adds more Data generates data to Collection f tracking Sample files and i gt Analysis Extracting l completed lanes generates Sample files Create project and add Sample file references Project with all references Creating a Project 4 3 Setting Up for Automatic Analysis Introduction To set up the GeneScan Analysis Software for automatic analysis after data collection you must have previously defined analysis parameters and size standards Procedure 4 4 Creating a Project For more information see Defining Analysis Parameters on page 5 18 Defining the Size Standard on page 5 31 To set up for automatic analysis after data collection Step Action Complete the following steps in the data collection software 1 Set the GeneScan Run default preferences to auto analyze and use the pop up menu to locate and select the GeneScan Analysis Software In the GeneScan Sample Sheet for each sample to be analyzed do the following Enterthe sample name This field must be completed for the samples to be active in the Injection or Run Sheet Indicate which dye is the standard Select the checkbox labeled Pres Present for each dye sample you want auto analyzed Select any additiona
86. for the database server see About Server Names on page E 17 Replace neuron apldbio com with the IP address or host and domain name of the server machine 2500 is the default port number for the Sybase database If necessary replace 2500 with the port number recommended by your BioLIMS database administrator You can find this information in the interfaces file on the Sybase Server or your BioLIMS database administrator can provide the information 4 If you have access to more than one server duplicate the two lines and edit them for the other server s For example for two servers one called SYBASE and one called SERVER2 the interfaces file might look like this SYBASE query MacTCP mac ether neuron apldbio com 2500 SERVER2 query MacTCP mac ether 192 135 191 128 2025 5 Save and close the interfaces file 6 Open the SybaseConfig control panel This control panel is found in the Control Panels folder in the System folder Network Driver MacTCP Default Language LANG T Using the BioLIMS Database E 5 E 6 Using the BioLIMS Database To configure for Sybase SQL Server connection continued Step Action 7 The first time the SybaseConfig control panel is opened a file browser opens automatically If a file browser does not open immediately click the Interfaces Files button to open one Ej interfaces locales Where is the interfaces file 8 Use the f
87. gel image Multicomponenting is the adjusting for spectral overlap of the fluorescent dyes It also copies this information during lane extraction to each Sample file for use during Sample file analysis IMPORTANT If you install new matrix file information after extracting the sample data from the gel file the Sample files will not contain the new information You must either regenerate the Sample files or install the new matrix directly to the existing Sample files How to Install a New Matrix File If you specified the wrong matrix file or no matrix file in data collection you can install a new matrix file in the gel image You can also specify a different matrix file in order to test the results of using different matrices on the samples To install a new matrix file in a gel image Step Action 1 Open the gel image 2 Choose Install New Gel Matrix from the Gel menu A directory dialog box appears The Folder Preferences settings determine where the GeneScan Analysis Software looks for the matrix file For more information see Specifying File Locations on page 5 42 2 24 How to Process and Edit the Gel File To install a new matrix file in a gel image continued Step Action 3 Find and select the desired matrix file then choose Open The dialog box closes and the information is added to the gel file If the new matrix installs correctly the following dialog box appears
88. indicator colors see Setting Dye Indicator Preferences on page 5 15 To change the dye indicator colors Step Action 1 There are two ways to display the Dye Indicators dialog box You can Result Choose Preferences from the Settings menu and Dye Indicators from the submenu The Dye Indicators dialog box Hold the Option key and click appears one of the colored boxes in the upper corner of the gel display Note Use the scroll bar to view additional dyes Scroll bar Preferences Page Due Indicators Y Dye Color Plot Color Blue vj ES Blue vj ES Green vj ES Green v O Yellow vj E Black v EH Red vj EH Red v Reset to Factory Settings C How to Process and Edit the Gel File 2 19 To change the dye indicator colors continued Step Action 2 Select a color from the pop up menu to change the Dye color or the Plot color Changing dye and plot colors has the following affects Choosing a color from Shows the colors Dye Color column that represent the dyes in the Analysis Control and the Results Control windows and in the gel image Plot Color column used for displaying the data in electropherograms Adjusting the Use the Adjust Gel Contrast dialog box to increase or reduce the Contrast intensity of individual colors in the gel image
89. on page 5 20 At the position of one strong peak additional colors appear Off scale data not multicomponented correctly a Repeat electrophoresis load less sample underneath the peak Poor incorrect b Attach a new gel matrix matrix and regenerate Sample files or assign a new matrix to the Sample file and re analyze D 2 Troubleshooting the GeneScan Software Troubleshooting projects and results continued Problem Probable Cause Correction Peaks appearing in a Bleed through from Repeat dye color that should other colors because of electrophoresis load not be present off scale data less sample A sample run on two different gels does not give the same molecular weights Using GS 350 standard size peaks gt 350 bp cannot be sized Size standard peaks called incorrectly by software Different sizing method used for each gel Inaccuracies associated with off scale data do not allow for identification of top of true peak Sizing requires at least one standard fragment larger than peak to be sized For more information see Appendix B GeneScan Size a Checksize standard and sizing curve Re analyze the Sample file with a different size standard or create a new one b Make sure same sizing method has been used for both gels c Repeat electrophoresis load less sample Repeat electrophoresis using GS 500 Stand
90. the dye samples to display the data For more information see Using the Results Control Window on page 7 7 2 Take the following action Click Print Choose Print or Print One from the File menu Note If you choose Print One the Print dialog box does not appear Printing Results 9 5 From the File To print a Sample file from the File menu Menu Step Action 1 Choose Open from the File menu The Open Existing dialog box appears Note You can also double click the sample name in the Finder If the GeneScan Analysis Software is not running the software starts and opens the Sample file 2 Click the Sample file icon 3 In the dialog box that appears find and select the Sample file that you want to open 4 Click Open The Sample File window appears 5 Select a display mode and choose Print or Print One from the File menu Printing Supporting Information Introduction The following procedure describes how to print supporting information about a Sample file such as the Sample Info view or to print the sample sheet Note You can use the same procedure for ABI 373 and ABI PRISM 377 runs to print the Sample Sheet and an image of the Gel File window Procedure To print supporting information Step Action 1 Display the information you want to show 2 Choose Print 3 P or Print One from the File menu Note If you choose Print One the Print
91. to display information about the peaks detected and matched For more information see Description of Information on page 3 9 Evaluating Analysis Results 7 53 Saving Archiving and Copying Files Introduction In This Section Topics in this chapter include the following Topics See page Saving GeneScan File 8 2 How to Archive Sample and Gel Files 8 6 Transferring Data to Other Applications 8 7 Saving Archiving and Copying Files 8 1 Saving GeneScan File WhySave The following table lists why to save projects Sample files and gel files For information on archiving files see How to Archive Sample and Gel Files on page 8 6 Save Because See GeneScan projects Protects the links to Sample files and their preferences Projects contain links to Sample files and preferences regarding display and analysis Saving Projects on page 8 3 Sample files Protects the links to projects and their preferences Sample files also contain raw data and critical information about the run settings and analysis control Saving Sample Files on page 8 3 Gel files Preserves the integrity of the data Note If you require long term storage of multiple gel files saving selected information from the files reduces their size considerably Saving Gel Files on page 8 4 Results Displays Saves the Results Display settings in proj
92. track curved lanes IMPORTANT Before auto tracking the first time you need to verify that the comb type is set correctly The default comb type is Square The Neural Net Tracker program exists as a separate program within the GeneScan Analysis Software folder Also associated with the Neural Net Tracker program are a set of Tracker settings files that have been optimized for number of lanes and comb types GeneScan Analysis Software Overview 1 3 Number of Number of Number of Size of Gel The Neural Net Tracker program is headless This means that although it stands as a separate program file it does not have a user interface The Tracker program is opened automatically from within the GeneScan Analysis Software IMPORTANT The gel file must be multicomponented using the correct matrix in order to be auto tracked Table of Tracking Times Tracking times depend upon the number of lanes channels and scans in the gel file Consult the table below to estimate gel tracking times for your GeneScan Analysis Software Time min for CPU Speed Lanes Channels Scans MB 7200 90 4400 2002 9500 2005 G3 266 36 194 9152 21 6 15 9 c 5 36 194 11424 26 8 20 11 c 6 48 388 4516 21 5 26 10 c 5 48 388 7768 36 1 38 17 c 8 64 388 4565 21 7 26 10 c 6 64 388 5708 26 8 30 13 g 6 96 480 6840 39 5 46 24 c 13 a 7200 4400 G3 32MB 10MB VM b 9500 64MB 10MB VM c Could not test this system Lane Extraction In version 3 1 you
93. uses the parameters set in the Auto Analysis Defaults see step 2 on page 4 6 Choose Preferences from the Settings menu and Folder Locations from the submenu to specify the folders in which the software automatically stores the analysis parameters and size standards files For more information see Defining Folder Locations on page 5 41 Analyzing Project Files 5 17 Defining Analysis Choose Analysis Parameters from the Settings menu to define the Parameters Analysis Parameters The Analysis Parameters dialog box appears Analysis Parameters Analysis Range Full Range This Range Data Points start stop r Size Call Range All Sizes This Range Base Pairs E 1900 Data Processing x Baseline x MultiComponent Smooth Options None amp Light Heavy Size Calling Method 2nd Order Least Squares 3rd Order Least Squares Cubic Spline Interpolation Local Southern Method Global Southern Method Peak Detection Peak Amplitude Thresholds E0 Min Peak Half Width r Split Peak Correction None amp GENESCAN 2500 LeftMost Peak RightMost Peak Correction Limit 50_ Data Pts o0 Six Analysis There are six Analysis parameters Parameters 5 18 Analyzing Project Files Topic See page Analysis Range Parameter Options 5 19 Data Processing Parameter Options 5 19 Peak Detection Parameter Opt
94. verify that the comb type is set correctly by choosing Preferences from the Settings menu and Gel Preferences from the submenu For more information see How to Set Gel Processing Preferences on page 2 3 Creating a Project 4 5 4 6 Creating a Project To set up for automatic analysis after data collection continued Step Action 2 If you are using the Auto Analysis defaults choose Auto Analysis Defaults from the Settings menu The Auto Analysis Defaults dialog box appears Ruto Rnalysis Defaults Auto Analysis O Always Override Collection Settings Standard Ly lt None gt ov anew Parameters w lt Anal ysis Parameters Ruto Print X Show Electropherograms amp Overlaid O Tiled X Show Tabular Data The parameters set in the Auto Analysis Defaults dialog box apply when you have Specified Analysis Defaults gt in the data collection software Selected the checkbox labeled Always Override Collection Settings Select the checkbox labeled Always Override Collection Settings to have the parameters set take precedence over the following files specified in the Data Collection software Size standard Analysis parameters Dye standard Choose a new Size Standard if Analysis Defaults is specified for the size standard in the data collection settings or Tooverride the specified size standard Choose the
95. 027 107107 7 LINE 3v 109027 10 4104 BE SR 1090273107107 Highlighting Peaks Use the Peak Highlighting command to highlight a selected peak with the dye sample s plot color To highlight selected peaks Step Action 1 Choose Peak Highlight from the Views menus and either Opaque or Transparent from the submenu Use this option To Opaque fill the peak with a solid color that can obscure peaks behind the selected peak Transparent use a slightly diffused plot color that allows you to view overlapping peaks 2 Click a detected peak in an electropherogram The peak is highlighted Evaluating Analysis Results 7 29 Using Legends to The following table lists how to use legends to change how Change the electropherograms are displayed Display If you want to Then show or hide legends choose Show Legends from the View menu open Sample file windows double click the corresponding legend text reorganize overlaid a Display the electropherograms electropherograms with legends b Click either the dye color indicator plot color indicator or the text for the sample you want to move to the front Scrolling the The following table lists ways to scroll the display Display 7 30 Evaluating Analysis Results Use the Description scroll bar If you want to Then shift the electropherogr
96. 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 E n 1 What to Evaluate Use the Raw Data view to evaluate Problems or noise in the baseline that could result in poor size calling Areas with a lower signal may indicate bad tracking Start and stop points for analysis For information on changing the horizontal page 7 37 or vertical scale of the data see page 7 38 3 16 Analyzing Sample Files EPT Data View What it Displays Displaying the View Displays the electrophoresis power and temperature information collected for a sample This information is stored in the Sample file The gel file does not need to be available The following table lists ways to display the EPT Data View To display the view Do this from a Sample file Clickthe button for the EPT Data view at the window bottom left of the Sample file window or Choose EPT Data 36 M from the Sample menu from a project window a Select a sample or multiple samples in the Analysis Control or the Results Control window b Select EPT Data from the Sample menu or click the EPT data button Analyzing Sample Files 3 17 EPT Data The following is an example of the Sample File window in EPT Data Example View E 15 P27 18 E 1200 1500 1800 2100 2400 2700 3000 3300 3600 3900 4200 4500 4800 5100 5400 Blue line
97. 2 36 How to Process and Edit the Gel File Importing Data to To import data to a Sample Sheet Sample Sheets Step Action 1 With the Sample Sheet open choose Import from the File menu A directory dialog box appears Note Ensure that the text file you are importing is complete and properly formatted for a tab delimited text file for example a spread sheet program For more information refer to the AB PRISM 377 Instrument User s Manual 2 Select a text file from the directory dialog box and click Open The data in the Sample Sheet is replaced by the imported text file Exporting Data To export data from Sample Sheets From Sample Sheets Step Action 1 With the Sample Sheet open choose Export from the File menu A directory dialog box appears 2 Take the following action a Enter a name for the text file b Choose a folder location to save the file and click Save How to Process and Edit the Gel File 2 37 About Displaying Regions of Off Scale Data Why Displaying Off scale data refers to raw fluorescent signals that exceed the linear Off Scale Data is dynamic range of the CCD camera detection system A data point is Important Off scale when the raw fluorescent signal is 8 191 rfu Peak quantitation of off scale data is not accurate this includes both peak area and peak height Additionally the matrix file may not be accurately applied often resulting in pull up peaks and po
98. 5 GeneScan Analysis Software Files 0 0 0 0 eee eee eee eee 1 16 Table ob Elles ios ie uro MEETUEREEITS A EDI PR SEE px 1 16 Sample File Database Support 2 0 e cece ee eee 1 19 Technical SUpport e deci ee babes Pee peeled us 1 20 To Reach Us On the Web sls 1 20 Hours for Telephone Technical Support 1 20 To Reach Us by Telephone or Fax in North America 1 20 Documents on Demand leleeeeeeeeeeeeee 1 23 To Reach Us by E Mail 0 0 eee eee eee 1 24 Regional Offices Sales and Service 0005 1 24 2 How to Process and Edit the Gel File 2 1 Introduction i ee babs Ges baer awes a E ERE Ra RE bed 2 1 In This Chapter cx EPUERPER AEN E e pink 2 1 Topics in This Chapter 0 0 0 0 cece cee eee eee 2 1 Abo t Gel Files res peu ener D Racer ete aes 2 2 Instruments Producing Gel File 2 0 0 0 0 eee eee eee 2 2 96 Lane Gels Capability 0 0 cece eee eee 2 2 Gel File Contents e wert hes Set ead etna wae es 2 2 How GeneScan Tracks a Gel File 0 0 0 0 eee eee eee 2 2 How to Set Gel Processing Preferences 0 00 00 000 eee 2 3 Inttod ctioh rr hex EE Xp Ree Se eee aye ER 2 3 Displaying the Gel Preferences Dialog Box 2 3 Auto Launch Processing 0 0 cee eee eee eens 2 4 Image Generation Defaults 0 0 0 cee eee eee 2 4 Lane Extraction uie Ee 9 RSEN RR eS 2 7
99. 5 17 Specifying Where Files are Stored 05 5 17 Defining Analysis Parameters lllleleeeeleleeeel 5 18 Six Analysis Parameters 2 00 0 0 eee eee eee eee 5 18 Analysis Range Parameter Options 005 5 19 Data Processing Parameter Options 005 5 19 Peak Detection Parameter Options 005 5 20 Size Call Range Parameter Options 04 5 21 Size Calling Method Parameter Options 5 21 Split Peak Correction Parameter Options 5 22 Using Analysis Parameter Files 00 0 0 eee eee ee eee 5 24 Tn ERIS Section ries rt ere ke koh esi and bees 5 24 Analyzing Samples Using the Same Analysis Parameters 5 24 Selecting Different Parameters for Analysis Samples 5 25 Displaying Default Parameters 0 00008 5 26 Creating Custom Analysis Parameter Files 5 26 Changing an Existing Analysis Parameters File 5 27 Deleting Custom Analysis Parameters lesse eese 5 28 About Size Standards 0 0 2 eee ee ee ee eens 5 29 What are Size Standards 0 0 00 cece eee eee 5 29 Advantages of Using Size Standard 00 5 29 Size Standards Provided 0 0 0 0 eee eee eee eee 5 29 When to Define Size Standards 00 000 00008 5 29 If Split Peaks Appear 5 29 Specifying Where Files are
100. 5 Click OK How to Change the Vertical Scale for Individual Electropherograms To change the vertical scale for individual electropherograms Step Action 1 Display the electropherograms 2 Move the cursor over the vertical axis of the panel that you want to change and double click The following dialog box appears Vertical Scale Parameters Vertical Scale Tick Spacing 600 units tick Display from 0 to 6000 CO Apply to all Electropherogram panels ok 3 Enter tick mark increments and a range 4 Ensure that the Apply to all Electropherogram panels checkbox is not selected Select the checkbox only to apply the changes to all displayed electropherogram panels 5 Click OK The vertical scale changes only for the electropherogram panel that you selected Assigning To assign standard or custom colors choose the Plot Color command Standard or from the View menu and either Standard or Custom from the submenu Custom Colors For information on defining custom colors see How to Define Custom Colors on page 7 40 Evaluating Analysis Results 7 39 How to Define Custom Colors Introduction The GeneScan Analysis Software assigns a plot color to each dye sample added to an electropherogram Normally it is the color associated with the individual dye sample by the Dye Indicators Preferences Note To change the default dye colors in the Analysis Control windo
101. 7 50 Index 2 working with electropherogram data 7 27 to 7 39 assigning standard colors 7 39 changing horizontal scale 7 37 changing the dye color 7 28 changing vertical scale 7 38 to 7 39 for all electropherograms 7 38 for single electropherograms 7 39 displaying peak positions 7 29 displaying x and y axis positions 7 27 highlighting peaks 7 29 making active window 7 28 scrolling the display 7 30 showing data by fragment size 7 35 to 7 36 about 7 35 procedure 7 36 showing off scale data 7 32 using legends to change the display 7 30 zooming in and out 7 31 e mail address for technical support 1 24 EPT Data View 3 17 to 3 18 displaying the view 3 17 example 3 18 what it displays 3 17 error messages D 9 to D 12 extracting data and tracking 2 59 to 2 66 extracting without changing tracking 2 64 to 2 66 saving after tracking 2 68 specifying Run folder 2 59 tracking and extracting 2 61 to 2 63 tracking without extracting 2 60 to 2 61 ways to track and extract 2 59 F files software files table of 1 16 to 1 18 folders ABI folder files installed in 6 6 defining locations 5 41 to 5 43 formamide how to deionize procedure 6 9 size standard mix preparing 6 10 G Gel File window diagram 2 13 optimizing tracker lines 2 54 window callouts call outs described 2 15 to 2 17 window buttons 2 14 to 2 15 gel files about the gel files 2 2 archiving 8 6 displaying the gel file 2 11 Gel File window diagram 2 13 differences betw
102. 8 T from the View menu When the data is aligned by size the menu changes to Align by Data point Choose the command again to show the data aligned by data point setthe default peak choose Preferences from the Settings menu and alignment choose Results Display from the submenu You can use the Results Display Preferences dialog box to set certain preferences that remain in effect each time you display or print results data For more information see Changing How the Results are Displayed and Printed on page 7 13 Changing the You can change the vertical scale Horizontal Scale 4 All electropherograms Individual electropherograms How to Change the Horizontal Scale for All Electropherograms To change the scale of the horizontal axis for all electropherograms 1 Display the electropherogram panels you want to change 2 Choose Horizontal Scale from the View menu The Horizontal Scale Parameters dialog box appears Note You can also move the cursor over the horizontal axis of a displayed electropherogram and double click Horizontal Scale Parameters Horizontal Scale Tick Spacing 300 units tick Display from 0 to 4572 Co Enter the increments represented by the tick marks for the horizontal axis in the Tick Spacing box Enter a range in the entry fields labeled Display from and to Click OK Evaluating Analysis Results 7 37
103. 886 2 2698 3405 Korea Seoul Thailand Bangkok Tel 82 2 593 6470 6471 Tel 66 2 719 6405 Fax 82 2 593 6472 Fax 66 2 319 9788 1 26 GeneScan Analysis Software Overview How to Process and Edit the Gel File Introduction In This Chapter This chapter contains information about how to view and edit the gel file and how to generate Sample files or BioLIMS database records Topics in This Chapter after editing the gel file Note This section does not apply if you are analyzing Sample files generated on the ABI PRISM 310 instrument IMPORTANT Refer to Appendix A Using GeneScan with the ABI 373 before using the GeneScan program to analyze data collected on the ABI 373 Topics in this chapter include the following Topic See page About Gel Files 2 2 How to Set Gel Processing Preferences 2 3 How to Display the Gel File 2 11 About the Gel File Window 2 12 How to Adjust the Gel Image 2 18 Verifying Gel Information 2 26 Displaying the Sample Sheet 2 29 About the Sample Sheet 2 31 Working with the Sample Sheet 2 34 About Displaying Regions of Off Scale Data 2 38 Working with Lane Markers 2 43 Marking and Unmarking Lanes for Extraction 2 50 Working with Tracker Lines 2 53 Interpolating Tracker Lines 2 57 Tracking Lanes and Extracting Data 2 59 How to Process and Edit the Gel File 2 1 About Gel Files Instruments Producing Gel File 96 Lane G
104. 9 Marking and Unmarking Lanes for Extraction Lane Marker The GeneScan Analysis Software marks lanes according to the Rules following rules About Marking Lanes for Extraction If Then Using the lane is identified with sample names in the sample sheet of the Data Collection software it is automatically marked as Used blue markers the lane is unidentified itis marked as Unused gray markers you are opening a gel file for the first time all Used lanes are marked for extraction white markers you have extracted the lanes each lane is unmarked for extraction blue markers you have edited a lane it is automatically marked for extraction white markers the lane was inferred by the Tracker software the lane is marked orange markers a lane is modified and then extracted and its edited tracking information is not saved to a gel file this serves as a warning that the latest generated sample does not reflect the saved tracking information yellow markers During the extraction process GeneScan Analysis Software only extracts data from gel lanes that are marked for extraction This allows you to control which lanes to extract when using the Extract Lanes command For more information see Extracting Data Without Changing the 2 50 How to Process and Edit the Gel File Current Tracker on page 2 64 Marking All Lanes T
105. 96 Lane Gels GeneScan Analysis Software version 3 1 supports the BioLIMS Genetic Information Management System v 2 0 In the BioLIMS mode the GeneScan Analysis Software extracts sample data from gel files and writes it to the database The contents of the gel file are not saved to the database The GeneScan Analysis Software can read and write sequence fragment to the database just as it writes sample files to the Macintosh hard disks Both Oracle and Sybase databases are supported For information about using the GeneScan Analysis Software in BioLIMS mode see Using the BioLIMS Database on page E 1 The GeneScan Analysis Software version 3 1 can display and analyze raw data from up to five dye colors This feature has been implemented in the software to support future developments in the dye chemistries The current maximum number of dyes available is four The availability of a fifth dye for the GeneScan Analysis Software is dependent on the progress made in the dye development chemistry and can not be accurately projected at this point You can receive more information on the availability of additional dyes for the GeneScan Analysis Software when they become available through our web site see To Reach Us on the Web on page 1 20 The GeneScan Analysis Software version 3 1 can open and analyze 96 lane gels For more information refer to the AB PRISM 377 DNA Sequencer 96 Lane Upgrade User s Manual P N 4305423 1 2 GeneSc
106. BASE server name sfdb name of the BioLIMS database If the Login is Then successful skip to step 6 unsuccessful continue to step 2 Use the show server script to find out if the Sybase SQL Server is running If the server is Then running skip to step 4 not running continue to step 3 F 12 Troubleshooting the BioLIMS Database Troubleshooting the client connection from the Sybase SQL Step Action 3 Ask your database administrator to restart the Sybase SQL Server After restarting the server try to connect to the database using isql as in step 1 on page F 12 If the connection still fails go to step 7 4 Check the Sybase error log and make a note of any error messages The error log can be found in the install directory of the Sybase home directory and error messages are preceded by the string Msg 5 Refer to the Sybooks documentation for an explanation of the SQL Server Error Messages Attempt to fix the problem following the Sybook instructions Try to connect to the database using isql as in step 1 on page F 12 If the connection is Then successful continue to step 6 unsuccessful Skip to step 7 6 Try once more to connect to the BioLIMS database from the BioLIMS Macintosh program Make sure that the user name password database and server names are all typed correctly and are in the correct case If the
107. BI PRISM 377 and ABI PRISM 377XL data Differences From The Sample Results view displays the same electropherogram and the Results Display tabular data as the Results Display with the following differences One Sample file displayed Show or hide dye sample data by clicking the buttons below the electropherogram Cannot display legends Cannot use custom plot colors Analyzing Sample Files 3 7 Sample Info View What it Displays Displays the following Sample file information 9 Run and data collection information Gel information Sample information this information can be edited Analysis records Displaying the The following table lists ways to display the Sample Info View View 3 8 Analyzing Sample Files To display the view Do this from the Sample file window Click the button for the Sample Info view at the bottom left of the Sample file window or Choose Sample Info 36 I from the Sample menu from a project window a Select a sample or multiple samples in the Analysis Control or the Results Control window b Choose Sample Info from the Sample menu The information is organized in five panels Click the triangles to expand or collapse the panels to display specific information Sample Info View The following is an example of the Sample File window in Sample Info Example View
108. Chapters deoe acteies chp hoes pad sal eee gies 7 1 Process of Evaluating Analysis Results 00 00 00 0005 7 2 Steps to Evaluating Analysis Results 0 7 2 Ways to Display Analysis Results 00 0 ee eee eee eee 7 3 Two Ways to Display Analysis Results 000 7 3 The Results Display Window 0 0c eee eee eee eee 7 4 Introduction ipla gc Lee alee Guha bs tod huis OS 7 4 Displaying the Window leseleeeeeeeee ee 7 4 Results Control Window Description 000 7 5 Using the Results Control Window 0 0 ce eee eee eee 7 7 Selecting Display Format 0 00 eee eee eee eee 7 7 Selecting Electropherogram Panels 005 7 7 Selecting Samples to Display 7 8 Creating Tiled Electropherogram Displays 7 10 Deselecting Samples 00 0 0 eee eee eee eee 7 11 Removing Samples 7 12 Displaying the Results 7 12 Printing th Results 4 vp Eme RENE CONDES REY a 7 12 Changing How the Results are Displayed and Printed 7 13 Procedure etis eto ctv esr REL ber eed 7 13 The Sample Results View leeeeee eA 7 16 About the VIeWs cnrs eaaa RE nh poh EN RE E NS DECIR DS 7 16 For More Information 0 0 0 eee eee eee 7 16 Updating the Results 0 0 cee eee ee eee 7 16 Re Analyzing the Data 7 16 Saving and Renaming the Results Control Format
109. Choose Zoom Out 36 from after zooming in the View menu or Click the magnifying glass cursor hold down the Option key and click the electropherogram The data appears in successively smaller scale views quickly scale the data so that the Choose Zoom Out Full Range from entire length fits within the window the View menu again Evaluating Analysis Results 7 31 Showing Off Scale This section contains the following information 7 32 Data Evaluating Analysis Results Topic See page Procedure 7 32 For ABI PRISM 377 Runs 7 33 About Flat Topped Peaks 7 33 Electropherogram Displaying Off Scale Data 7 34 Electropherogram Displaying the Flat Topped Effect 7 35 Procedure To show off scale data Step Action 1 Choose Preferences from the Settings menu and Results Display from the submenu The Results Display Preferences dialog box appears 2 Choose the Show Offscale Regions checkbox to highlight with a red bar regions in the electropherogram that contain off scale see Electropherogram Displaying Off Scale Data on page 7 34 Note Choose the Zoom In command from the View menu to more clearly show the areas of off scale data The Analysis Log lists the numbers of off scale regions in the analysis range for each Sample file if the sample was sized Note You can toggle this command for individual electropherograms by choosing Hide Show Offscale reg
110. Defining the Size Standard 5 31 Using Size Standards 5 36 Defining Folder Locations 5 41 Analyzing Project Files 5 1 Analyzing Project Files About the Analysis Control Window Introduction Analysis Control Window Example When a project is opened the Analysis Control window appears The Analysis Control window is the main window of a project You can use this window to specify the following for each sample in the project Dye that represents the size standard you ran with the sample Size standard Analysis parameters Specific dyes to be analyzed Optionally format the document for printing and print the results automatically 9 9 9 For more information see Analyzing Sample Files Using the Analysis Control Window on page 5 6 The following is an example of the Analysis Control window HS P27 1 copy r Analysis Control L4 5 2 Analyzing Project Files Rndluze Created Thu Jul 12 1226 2 24 PM CJ Print Results Print Setup Size Standard P Parameters HS P27 Standard GS 350 ansiusis Parameters gt HS F27 Standard GS 350 gt lt analusis Parameters al HS P27 Standard 6S 350 gt lt analusis Parameters al HS F27 Standard 65 350 lt Analysis Parameters gt a Sample File tt tt ozep27 2 1 O 0P27 3 1 049P27 4 1 E 1 E 9 oserz7 5
111. Dye pop up menu to specify the dye that represents the internal size standard you are running with the samples Choose the analysis parameters you want to use from the Parameters pop up menu To use the default parameters choose Analysis Parameters To set up for automatic analysis after data collection continued Step Action 7 In the Auto Print section choose you have the following options Choose To print Show Electropherograms electropherograms Select the appropriate radio button to specify whether the electropherograms for the four dyes appear Together in one panel overlaid or in Separate panels tiled Show Tabular data tabular data 8 Click OK The GeneScan Analysis Software is now prepared to perform automatic analysis Note The settings that you specify are initial settings Make two or three trial runs fine tuning the parameters with each run to determine which parameters work the best for a particular protocol Creating a Project 4 7 Using a Project to Manage Sample Files What is a Project Why Create a Project When GeneScan Creates Projects 4 8 Creating a Project A project is a file containing references to a set of Sample files that you want to analyze and display together The project contains Analysis Control and Results Control windows that allow you to analyze specific dye samples and display the results of analysis Yo
112. F 22 Troubleshooting the BioLIMS Database Troubleshooting the client connection from the Oracle Server continued Step Action 2 Find out if the Listener Process is running As the Oracle user type lsnrctl status If the Listener Process is Then not running continue to step 3 running check that the server is running Type ps ef grep ora_ If server processes are Then displayed server is running Skip to step 4 not displayed continue to step 3 3 See your system administrator for help restarting the Listener Process or Server After restarting try to connect to the database using SQL Plus as in step 1 on page F 22 If the connection still fails go to step 7 4 Check the Oracle error log and make a note of any error messages The error log can be found in the install directory of the Oracle home directory and error messages are preceded by the string Msg Note Refer to the Oracle documentation for an explanation of the error messages 5 Attempt to fix the problem following the documentation instructions Try to connect to the database using SQL Plus as in step 1 on page F 22 If the connection still fails go to step 7 Troubleshooting the BioLIMS Database F 23 Troubleshooting the client connection from the Oracle Server continued Step Action 6 Try once more to connect to the BioLIM
113. File and BioLIMS Mode on page E 10 Procedure To open a Sample file as a separate file 3 4 Analyzing Sample Files Step Action 1 Choose Open from the File menu The Open Existing dialog box appears Note You can also double click the Sample file name in the Finder If the GeneScan Analysis Software is not running the software starts and opens the Sample file Open Existing t9 4 t9 TO Ul ful Collection Project Sample Sample Analysis Size Matrix Gel Sheet Parameters Standard Cancel Click the Sample icon A directory dialog box appears In the dialog box find and select the Sample file that you want to open Click Open The Sample File window appears See About The Sample File Window on page 3 5 About The Sample File Window What it Displays You can use the different display modes in the Sample File window to review the analyzed and raw data and all pertinent data collection sizing and Sample description information from a single window The Sample Results view appears as the default Five Views The five views of the Sample File window are View See page Sample Results view 3 6 Sample Info view 3 8 Size Curve view 3 13 Raw Data view 3 15 EPT Data view 3 17 Analyzing Sample Files 3 5 Sample Results View What it Displays The Sample Results View displays the Sample file s analyzed data in both electropherogram and tabul
114. GeneScarn Analysis Software Version 3 1 User s Manual Applied E Biosystems Copyright 2000 Applied Biosystems For Research Use Only Not for use in diagnostic procedures Notice to Purchaser License Disclaimer Purchase of this software product alone does not imply any license to any process instrument or other apparatus system composition reagent or kit rights covered under patent claims owned or otherwise controlled by PE Corporation either expressly impliedly or by estoppel ABI PRISM and the ABI PRISM design GeneScan Genotyper and Applied Biosystems are registered trademarks of PE Corporation or its subsidiaries in the U S and certain other countries BioLIMS is a trademark of Molecular Informatics Inc ABI Applied Biosystems and GenoPedigree are trademarks of PE Corporation or its subsidiaries in the U S and certain other countries Sybase is a registered trademark and Sybase SQL Server are trademarks of Sybase Inc Oracle is a trademark of Oracle Corporation All other trademarks are the sole property of their respective owners Contents 1 GeneScan Analysis Software Overview 1 1 Inttoduetion ose Sects dee eter ns tieu SG ave We SS UN dha ANTS FE NOI RE d 1 1 In This Chapter eee trm eee Re RE esce 1 1 What s New in the GeneScan Analysis Software 0 1 2 BioLIMS Support otic reru ru RR ER Meda EGG RON CRUS S 1 2 Fifth Dyes cso nre MER Rude De NE qu p ERR 1 2
115. In This Chapter Topics in this chapter include the following Topic See page Process of Evaluating Analysis Results 7 2 Ways to Display Analysis Results 7 3 The Results Display Window 7 4 Using the Results Control Window 7 7 Changing How the Results are Displayed and Printed 7 13 The Sample Results View 7 16 Updating the Results 7 16 Saving and Renaming the Results Control Format 7 17 About Electropherogram and Tabular Data Displays 7 20 Displaying Electropherogram and Tabular Data 7 22 Displaying Electropherogram Data 7 25 Working with Electropherogram Data 7 27 How to Define Custom Colors 7 40 How to Change the Dye Scale 7 44 Process of Verifying Results 7 46 How to Verify Size Calculations 7 48 Using the Analysis Log 7 51 How to Verify Peak Detection 7 53 Evaluating Analysis Results 7 1 Process of Evaluating Analysis Results Stepsto The following table lists the steps to evaluating the analysis results Evaluating Analysis Results Step Action For information see 1 Displaying Analysis Results Ways to Display Analysis Results on page 7 3 Using Electropherogram and Tabular Data Displays About Electropherogram and Tabular Data Displays on page 7 20 Viewing Electropherograms Displaying Electropherogram Data on page 7 25 Verifying analysis results Process of Verifying Results on page 7 46 Displa
116. Ld ul 2 a connection T successful Fi uet Try Macintosh connection step 6 on gt B page F 13 p meta Y fails Call Customer Support step 7 on page F 13 im zc fails Check libraries in the Extensions folder step 2 on page F 6 Open and check interfaces file step 3 on page F 6 a a Try connection connection step 6 on fails e m MN a it S EC DU a iu P e i a E a a PM server running Check theSybase SQL Server error log step 4 on page F 13 T Try Macintosh 2 s __ page F 13 NSCA Telnet step 6 s on page F 10 connection i successful i connection Is the server running step 2 on page F 12 7 M connection The network is not working step 6 on page F 10 Start the server step 3 on page F 13 Look up errors in theSybase error log step 4 on page F 13 Attempt to fix problems found in error log step 5 on page F 13 Troubleshooting the BioLIMS Database F 5 Procedures for Troubleshooting the Client to Sybase Connection Introduction Troubleshooting from the Macintosh Client The procedure for troubleshooting the Client Server connection is divided into two parts Troubleshooting from the Macintosh Client below Troubleshooting from the Unix Sybase Server page F 12
117. MS Extras Y Oracle ra Select Oracle 10 Use the file browser to find the Oracle folder in the BioLIMS Extras folder Press Select Oracle button Using the BioLIMS Database E 9 Switching Between Sample File and BioLIMS Mode Introduction If the BioLIMS database is installed on your system then the BioLIMS Access command is available from the Settings menu when you start the GeneScan Analysis Software Switching Modes To switch modes in the GeneScan Analysis Software E 10 Step Action 1 Choose Preferences from the Settings menu and BioLIMS Access from the submenu The Preferences dialog box appears Preferences Page BioLIMS Access BioLIMS Access o Sample Files BioL IMS Session Manager Username Password O Save Password Database Server Alias I3 O Open on Launch O Make Default ok To switch modes take the following action To change to Then Result Sample File mode click the Sample The Session Files radio button Manager text is greyed out BioLIMS mode click the BioLIMS radio button The fields in the Session Manager subpanel are activated Using the BioLIMS Database Step Action 3 You can take the following action Click To OK affect th
118. New from the File menu 3 Click the GeneScan Sample Sheet icon The GeneScan Sample Sheet appears 6 24 Making a Matrix File To create a GeneScan Sample Sheet continued Step Action Sample Sheet GS matrix GeneScan Sample Sheet Comments Jj Ju u Ji c H EX EJ DI EJI EJ E DIDI ET EJ 9 ET DT ETE 4 Enter the individual colors in the appropriate lanes where the matrix standards are loaded 5 Enter any additional information about the sample in the Comments column 6 Use the Save As command and save the Sample Sheet to the Sample Sheet folder Loading the To load matrix standards Matrix Standards Step Action 1 For denaturing and native gels load 1 5 pL of matrix standard per lane depending on comb configuration see instrument user s manual 8lanes with different colors leaving an empty lane between each lane of matrix standard 2 Select the appropriate filter set on the ABI 373 sequencer For this matrix Choose Dye Primer matrix Filter set A Fluorescent Amidite matrix Filter set B Making a Matrix File 6 25 Step Action Electrophorese samples according to conditions specified in your instrument manual Running the Run the matrix standar
119. ONNECT DATA SID WG733 Note Refer to the table immediately below for an explanation of the BioLIMS Server entry Troubleshooting the BioLIMS Database F 17 Troubleshooting the connection from the client Macintosh continued Step Action The BioLIMS Server entry Where Represents Oramozart the name you chose to call the Oracle based BioLIMS database connection This can have any name See About Server Names on page E 17 mozart the host of the Oracle based BioLIMS Server In this example mozart is the host name You can also use a host and domain name such as mozart apldbio com or an IP address such as 167 116 100 61 This information is available from your system administrator The IP address can also be found in the etc hosts file on the UNIX Server 1521 the port number that the Oracle based BioLIMS Server is using to connect with the clients This number is assigned to the server when it is installed You can find the port number in the tnsnames ora file that is located in the ORACLE HOME network admin directory on the Oracle based BioLIMS Server WG733 the Oracle database system identifier sid defined at server installation You can find the port number in the tnsnames ora file that is located in the ORACLE HOME network admin directory on the Oracle based BioLIMS Server a 3 Confirm that the Oracle Home is s
120. R 7 25 Electropherogram Example 0 0000 e eee eee 7 26 Descriptions of the Electropherogram 4 7 26 Working with Electropherogram Data 0 000 000 0000 7 27 In This Sections 2u enc Seid wa dg oh ea RR 7 27 Displaying X and Y Axis Positions 00 7 21 Moving the Electropherogram 0 000000 e ee eae 7 28 Changing the Dye Color 7 28 Displaying Peak Positions 0 00 0 ce eee eee eee 7 29 Highlighting Peaks 0 0 eee eee ee ee 7 29 Using Legends to Change the Display 7 30 Scrolling the Display 7 30 Zooming In and Out 00 0 ee ee eee 7 31 Showing Off Scale Data llle 7 32 Electropherogram Displaying Off Scale Data 7 34 Electropherogram Displaying the Flat Topped Effect 7 35 Showing Data by Fragment Size llle 7 35 Changing the Horizontal Scale sleleeeeeeese 7 37 Changing the Vertical Scale 0 0 eee eee eee 7 38 Assigning Standard or Custom Colors 0 7 39 How to Define Custom Colors lese 7 40 Introduction seee eess bee ee eec eek ok oa ee ee a 7 40 Why Change Colors in the Electropherogram 7 40 Saving the Display Format 0 00 00 eese 7 40 Defining Custom Color for All Electropherogram 7 41 Defining Individual Plot Colors 0 00 00 00000 7 42 H
121. S database from the BioLIMS Macintosh program Ensure that the user name password database and server names are all typed correctly and are in the correct case If the connection still fails go to step 7 7 Call Customer Support See Technical Support on page 1 20 F 24 Troubleshooting the BioLIMS Database LicenseandWarranty Software License and Warranty Applied PURCHASER CAREFULLY READ THE FOLLOWING TERMS AND Biosystems CONDITIONS THE AGREEMENT WHICH APPLY TO THE Software License SOFTWARE ENCLOSED THE SOFTWARE and Limited YOUR OPENING OF THIS PACKAGE INDICATES YOUR Product Warranty ACCEPTANCE OF THESE TERMS AND CONDITIONS IF YOU DO NOT ACCEPT THEM PROMPTLY RETURN THE COMPLETE PACKAGE AND YOUR MONEY WILL BE RETURNED THE LAW PROVIDES FOR CIVIL AND CRIMINAL PENALTIES FOR ANYONE WHO VIOLATES THE LAWS OF COPYRIGHT Copyright The SOFTWARE including its structure organization code user interface and associated documentation is a proprietary product of The PE Corporation and is protected by international laws of copyright Title to the SOFTWARE and to any and all portion s of the SOFTWARE shall at all times remain with Applied Biosystems License 1 You may use the SOFTWARE on a single computer or on a single network if your software is designated as a network version You may transfer the SOFTWARE to another single computer or network if a network version so long as you first delete the
122. TE Installation of matig file1 within the Gel File was successful NOTE Previously extracted sample files DO NOT contain the newly installed matrix Do you want to regenerate the Gel Image 4 Choose whether or not to regenerate the gel image Note If you do not regenerate the gel image now the matrix is included in the data when Sample files are generated For more information on matrix files see Chapter 6 Making a Matrix File How to Process and Edit the Gel File 2 25 Verifying Gel Information Where the Information is Archived Data Collection Run Error Log Gel Info Window Gel and run information is archived in the following Data Collection run error log Gel Info window Gel image Gel Sample Sheet The Data Collection Run Error log can provide information about Macintosh computer or instrument errors that occurred during the run What the Gel Info Window Displays The window displays information about the run conditions and parameters and the gel image You can change some of this information for your record keeping purposes and other information such as the date of the run cannot be changed The information in this window is saved with each Sample file generated from the gel file IMPORTANT Use the Matrix file section in the Gel Info window to verify what matrix is attached to the Gel file and use the Multicomponented section to verify whether or not the Gel file is multicomponented
123. Troubleshooting the connection from the client Macintosh continued Step Action If the login is Then successful the Result Explanation dialog box displays the following Result field ORA 00000 normal successful completion Connected To field Oracle Server version information Comments field Your SQL Net setup appears to be in working order Skip to Troubleshooting from the Unix Oracle Server on page F 22 unsuccessful the Result Explanation dialog box displays the following Result field the Oracle error encountered Comments field A detailed explanation of the login failure Continue to step 6 F 20 Troubleshooting the BioLIMS Database Troubleshooting the connection from the client Macintosh continued Step Action 6 If the NetTest login is unsuccessful Open the program NCSA Telnet The default installer places NCSA Telnet into the BioLIMS 2 0 BioLIMS Extras folder Step Action a Select Open Connection from the File menu b Enter the host name from the tnsnames ora file into the Host Session Name text field Note You can enter the host host and domain name or IP address here See step 2 mozart on page F 18 for more information C Click Connect If the connection is Then successful a window is displayed with a Unix login prompt
124. Used If you change the setting for this checkbox the corresponding lane setting in the gel file is automatically changed Name of the Sample file created for the data in this lane The Sample file is saved to the hard disk into the Run folder next to the gel file If the data is extracted into a BioLIMS database then this is the name assigned to the database record for that sample file Name of the sample which is used by the Data Collection software to generate the name for the Sample file The following table lists how the names correspond between the Data Collection software and the GeneScan Analysis Software Data Collection software GeneScan Analysis Software Sample Name Sample Name Sample Name and lane Sample number While this information is often the same as the sample info field it is used by the software for different purposes Indicates which dye contains the standard Information about the sample usually patient or sample ID Note This is a required field if the data is to be analyzed using the Genotyper software and stored in GenBase Comments about the sample Select this checkbox for the GeneScan Analysis Software to automatically analyze the Sample files after generating the gel image 2 32 How to Process and Edit the Gel File Sample Sheet described continued No Description 9 When this checkbox and the checkbox labeled A a
125. a Collection Software From the GeneScan Analysis Software You can specify that the results are printed in the Data Collection software or in the GeneScan Analysis Software When you choose automatic printing from the Data Collection software the GeneScan Analysis Software prints a separate page for each designated Sample file showing electropherograms and tabular data as specified in the Auto Analysis Defaults see step 2 on page 4 6 Choose automatic printing in the Data Collection software as follows On this instrument Choose ABI PRISM 310 Auto Print in the Injection list ABI PRISM 377 Auto Print in the data collection Run window For information about setting up your run refer to your instrument user manual When you analyze a project separately from the Data Collection software you can print results automatically as the samples are analyzed To print the results automatically as the samples are analyzed Step Action 1 In the Analysis Control window select the samples you wish to analyze For more information see Analyzing a Sample File on page 3 19 2 Select the checkbox labeled Print Results in the Analysis Control window 3 Click the Print Setup button to specify the samples and the format For more information see Specifying the Format for Printed Results on page 5 9 4 Click OK The results are printed after the results are analyzed
126. a bs Le eee ea ee tek E B 8 GeneScan 1000 Molecular Lengths 0 000000 e B 8 Running Under Native Conditions 0 0000 B 9 GeneScan 2500 Standard 1 0 sss B 10 How It Is Prepared o scie keg oe hie eee B 10 What To Use It For ceccar inia a III B 10 Running Under Native Conditions 000 B 10 GeneScan 2500 Molecular Lengths 0 B 11 Size Calling Methods LLL CI Introduction x ee ce Deep enl epe n Sob lene m recs C 1 In This Appendix icu eELLCR e Tier hee Rn C 1 Least Square Method 2 0 0 eee eee eA C 2 About This Method 0 0 0 0 0 eee eee eee ee C 2 Advantages eset pete reise eere ee e e Ee C 2 Cubic Spline Interpolation Method 0 00 0 eee ee eee C 4 About This Method 0 0 0 0 cee eee ee C 4 Possible Local Sizing Inaccuracy lseeeeeeeeeeeee C 4 Local Southern Method 0 0 cece ec eee C 5 About This Method 0 0 0 00 cece eee eee eee ee C 5 The BQuatOn si orenga mRCp m ESPERE RE EE C 5 How This Method Works 0 0 0 ee eee eee e C 6 Global Southern Method 2 00 0 cece eene C 7 About This Method C 7 The Equations dace e prete te ate e i euin C 7 How This Method Works eseseeeeeee e C 8 Troubleshooting the GeneScan Software D 1 Introduction 4 5 iets deve ve CERE E te Hooded UG E EN RECS D 1 xvii xviii In This A
127. aa n ee 2 43 Moving Between Lanes and Channels 4 2 44 Using Control Points 0 0 0 eee ee ee ee 2 45 Adding and Deleting Control Rows 0005 2 46 What Marking Lanes as Used Unused Means 2 47 Marking Lanes as Used or Unused 005 2 47 Moving Misplaced Lane Markers 0005 2 48 Marking and Unmarking Lanes for Extraction 2 50 lane Marker Rules eeu pL eR feeds Re 2 50 About Marking Lanes for Extraction 005 2 50 Marking All Lanes for Extraction 0005 2 51 Unmarking All Lanes for Extraction 005 2 51 Marking a Single Lane for Extraction 0 2 52 Unmarking a Single Lane for Extraction 2 52 Working with Tracker Lines 0 0 00 cee eee eee eee 2 53 Topics in This Section 0 0 0 eee eee eee eee 2 53 Hiding Showing Tracker Lines 00 0 0 00008 2 53 Why Positioning Each Lane is Important 2 54 Causes to Misinterpreting Lane Positions 2 54 Verifying Optimal Channel Tracking lesse esee 2 55 Reviewing Tracker Lines 0 00 esee 2 55 Moving Tracker Lines 0 0 eae eee ee 2 56 Deselecting a Eane iue RR FREE ERE es 2 56 Interpolating Tracker Lines 0 0 0 eee eee eee 2 57 Why Use Interpolation Mode 00 00
128. alyzing Sample Files Note X Sizing errors due to anomalous mobilities are displayed as non linear This curve Represents Red curve The size calling curve based on the size calling method used to analyze the data Black curve The best fit least squares curve which the GeneScan Analysis Software calculates for all samples regardless of the size calling method This curve is provided to help evaluate the linearity of the sizing curve When the sizing curve and best fit curve match they overlap so you see only the size curve Raw Data View What it Displays Displaying the View Displays the raw data collected for a sample Raw data is non baselined and non multicomponented This information is stored in the Sample file The gel file does not need to be available The following table lists ways to display the Raw Data View To display the view Do this from the Sample file window Click the button for the Raw Data view at the bottom left of the Sample file window Or Choose Raw Data 3 R from the Sample menu from a project window a Select a sample or multiple samples in the Analysis Control or the Results Control window b Choose Raw Data 3 R from the Sample menu Analyzing Sample Files 3 15 Raw Data The following is an example of the Sample File window in Raw Data Example View Relative peak amplitude signal intensity Data points TENTE
129. am click in the gray region of to the right or the left the scroll bar to the right or the left of the scroll box scroll across the click an arrow at the end of electropherogram the scroll bar control the amount of drag the scroll bar to the scroll right or the left scroller a Hold the mouse cursor over either the vertical or symbols horizontal scale of the electropherogram Either a vertical scroller symbol A or a horizontal scroller symbol zy appears b Hold down the mouse button and move the mouse in the direction of the information you want to view Zooming In and About Zooming In and Out Out By default the GeneScan Analysis Software scales each electropherogram horizontally to show all peaks detected during the run While this provides a good overview of the run some peaks may be quite compressed To improve visibility you can change the horizontal scale of the electropherograms by zooming Zooming affects Only the horizontal scale and zooms the middle portion of the window All displayed electropherogram panels How to Change the View Scale To change the view scale If you want to Then see views with greater detail Choose Zoom In 3 from the View menu or Clickthe magnifying glass in the upper left corner of the window and select a specific area on which to zoom in by drawing a box on the electropherogram see a smaller scale view of the data
130. am also creates several files such as a Preference file and an Analysis Log Analyzing Project Files 5 41 Specifying File Use the Folder Location preference in the Settings menu to specify Locations where the software saves size standards analysis parameters and 5 42 Analyzing Project Files matrices These preferences are saved as defaults for subsequent projects and Sample files To specify file locations Step Action 1 Choose Preferences from the Settings menu and Folder Locations from the submenu The Preferences window appears see below Note It is advisable that you use the appropriate folders that is the GS Standards Folder the GS Parameters Folder and the GS Matrix Folder Note If the Preference window is displayed choose Folder Locations from the Page pop up menu Preferences Page Folder Locations Size Standards Folder Hard Drive GeneScan Analysis 3 0 65 Standards Folder J Analysis Parameters Folder Hard Drive GeneScan Analysis 3 0 65 Parameters Folder J Matrix Folder Em Hard Drive GeneScan Analysis 3 0 65 Matrix Folder J ok To specify file locations continued Step Action 2 Click a button that shows the folder s path name A dialog box appears Select a Standards Folder c Hard Drive 36S Parameters Folder COGS Standards Folder 3 Take the following action
131. ame Display dialog box Rename Displa Enter a Display Title Ces CC 3 Enter a new name for the display and click OK Evaluating Analysis Results 7 19 About Electropherogram and Tabular Data Displays Introduction After analyzing the data you can display the results for each sample in electropherograms and tabular data You can also customize the electropherograms and tabular data display Note Altering the appearance of the electropherograms and the tabular data displays does not change the analyzed data contained in the Sample file on which they are based How the Window When electropherograms and tabular data are displayed together the is Divided window is divided into upper and lower windows Window Contains Upper window electropherograms Lower window tabular data You can customize the window s appearance by adjusting the relative size of each window see Adjusting Window Size on page 7 24 What Tabular f you analyze samples with an internal size standard and perform size Data Contains calling the tabular data contains the estimated sizes in base pairs of all detected fragments Use this information for detailed data analysis and further calculations The peaks matched to the defined size standard are identified by dots next to the Dye Sample Peak field Sample peaks that are larger in base pairs than the largest defined peak in the selected standard ar
132. amed 373 SampleFiles lt date gt where date is the original date on which the GeneScan 672 Software file was generated Using GeneScan with the ABI 373 A 3 GeneScan Prior to Version 2 0 GeneScan 672 Software on ABI 373 instrument Collection file GS Analysis preprocesses z Collection file to create gel file Gel file GS Analysis analyzes gel file to create Results file AA Analyzed data and BOE analysis information ee stored in a single Results file Results file A 4 Using GeneScan with the ABI 373 Using GeneScan 2 0 or Later GeneScan Analysis Software on ABI 373 instrument gs sinum Collection file Sample files after analysis Setting Gel Processing Parameters Introduction Before you open a Collection file set the gel processing parameters The GeneScan Analysis Software takes the information in the processing parameters into account when it opens the Collection file Procedure To set the gel processing parameters Step Action 1 Double click the GeneScan Analysis Software icon to start the program Choose Gel Preferences from the Settings menu The Gel Preferences dialog box appears Note The checkboxes under Auto Launch processing only apply when the Data Collection software automatically starts the GeneScan Analysis Software The GeneScan 672 Software does not automatically start GeneScan 2 1 IMPORTANT Befor
133. ample file Search a Folder Specify a folder in which to search for missing Sample files The GeneScan Analysis Software then immediately locates the Sample files and re establishes links to the project Searching for missing sample files continued Choose If you Description Exhaustive Search do not know where The GeneScan any of the specified Analysis Software missing Sample files searches all mounted are located disk drives and available servers When the files are found the software re establishes links to the project Re Establishing To re establish the links with Sample files Links Step Action 1 Click the dimmed file name to select it When the missing file is selected the Find file name command becomes active The name of the missing file appears inside the quotation marks 2 Choose Find file name A file dialog box appears 3 In the file dialog box locate the proper folder and file 4 Click Open or double click the file name Creating a Project 4 15 Analyzing Project Files Introduction In This Chapter Topics in this chapter include the following Topics See page Analyzing Project Files About the Analysis Control Window 5 2 Analyzing Sample Files Using the Analysis Control Window 5 6 About the Analysis Parameters 5 17 Using Analysis Parameter Files 5 24 About Size Standards 5 29
134. an Analysis Software Overview Displaying Off Scale Data Manual Tracker Interface Changes Printing from Sample File Window New Neural Net Tracker A new feature has been added in version 3 1 to display offscale regions By clicking the OS button on the tool bar when the gel image is displayed if the gel contains offscale data then this data appears as white bands on the gel image Off scale data can also be viewed while in Results Display by selecting Show Offscale Regions from the View menu Note To see off scale portions in the gel image click the Vertical Expand button The resolution of the full display is low and does not show the off scale data In version 3 1 the new tracker lines consist of control points The new interface allows the selection and movement of multiple control points simultaneously interpolation of tracker line shapes between two lanes multiple lane selection and movement In version 2 1 only the electropherogram display is printed when printing from the Sample File window The following displays can now be printed also Sample Info View Size Curve View Raw Data View EPT Data View About the Neural Net Tracker This was the most significant improvement in the version 3 1 release of GeneScan Analysis Software The Neural Net Tracker program uses a neural net based algorithm to automatically track gel lanes The Neural Net Tracker has been taught how to recognize bands and how to
135. an take the following action To You can use the control points to move Click the control point and the tracker lanes drag or Use the Arrow keys Note Pressing the Option key while using the Arrow keys moves the control point in 0 1 channel increments For more information see Using Control Points on page 2 45 track and extract lanes choose Extract Lanes from the Gel menu For more information see Tracking Lanes and Extracting Data on page 2 59 2 58 How to Process and Edit the Gel File Tracking Lanes and Extracting Data Ways to Track and Note The only action that changes the tracking is choosing the correct comb Extract Data type see How to Set Gel Processing Preferences on page 2 3 Changes to the Sample Sheet do not effect the tracking There are three ways to track and extract data in the gel file Choose from the Gel To Menu Comment See track the gel without Track Lanes command Tracking the Gel extracting Sample file to track the gel file Without Extracting data based on the current Data on page 2 60 Sample Sheet information Both these procedures both track the lanes Track and Extract generate the same Tracking and and extract data into Lanes command to tracking lines Extracting Data on the Sample files or the calculate the tracking page 2 61 BioLIMS database records and then extract the Sample file from the
136. anes This ensures that when the Sample files are generated they contain the correct information The Sample Sheet that is part of the Collection file is blank when it is opened This is because the GeneScan 672 Software does not automatically embed a copy of the data collection Sample Sheet in the Collection file IMPORTANT When tracking and extracting the Collection file ensure the Sample Sheet associated with the gel file has the checkbox labeled Used selected for each sample you want to extract The GeneScan Analysis Software only extracts the samples with that checkbox selected To complete the Sample Sheet Step Action 1 Open the Sample Sheet by either Clicking the Sample Sheet button in the upper left corner of the Gel Window or by Choosing Gel Sample Sheet from the Gel menu 2 Enter the following information Sample file name Sample name for each sample Sample info Comment for each dye sample For more information see Displaying the Sample Sheet on page 2 29 3 Select checkboxes as applicable To Then select the indicate that the lane contains Used checkbox data automatically analyze Sample A checkbox files when generating them automatically print Sample files P checkbox when generating them Using GeneScan with the ABI 373 A 9 To complete the Sample Sheet continued Step Action 4 Identify the dye stand
137. appropriate will enable the Save command in the File menu You may save all the information or selected parts of the gel file Saving Files After IMPORTANT If you save changes to the gel file the original tracking Editing Tracking information is overwritten You can retrieve the originally calculated tracking only by choosing Track or Track amp Extract Gel from the Gel menu to retrack the gel See Tracking Lanes and Extracting Data on page 2 59 You have the following options If you Then choose the Save command The contents of the Gel File window are saved to the gel file select the checkbox labeled Save It is not necessary to use the Save Gel after Extraction in the Extract command to save the gel file Lane dialog box For information regarding saving selected portions of gel files and archiving gel files see Saving Gel Files on page 8 4 2 68 How to Process and Edit the Gel File Analyzing Sample Files Introduction In This Chapter Topics in this chapter include the following Topics See page About Sample Files 3 2 Opening Sample Files 3 4 About The Sample File Window 3 5 Sample Results View 3 6 Sample Info View 3 8 Size Curve View 3 13 Raw Data View 3 15 EPT Data View 3 17 Analyzing a Sample File 3 19 Analyzing SampleFiles 3 1 About Sample Files What Sample Files Sample files contain data generated from the following Contain
138. ar data form Displaying the The following table lists ways to display the Sample Results View View To display the view Do this from the Sample file Click the button for the Sample Results view window at the bottom left of the Sample file window Choose Sample Results 3 E from the Sample menu from a project window a Select a sample or multiple samples in the Analysis Control or the Results Control window b Choose Sample Results from the Sample menu Sample Results The following is an example of the Sample Results view View Example EIE 119P27 11 Y al Dye Sample Minutes Size Peak Height Peak Area Data Point Peak B 1 40 59 76 81 53 Z m 3 6 Analyzing Sample Files Description of The following table describes the columns in the above figure Columns This column Identifies Dye Sample Peak Dye color Peak number Minutes The time in minutes from the start of the run to the time the fragment was detected Size The number of base pairs in the fragment This value is calculated automatically only if you Run the size standard in the same lane or injection as the sample and Perform size calling Peak Height Signal size Peak Area Area of the detected peak Data point Data point of the fragment at its maximum peak height Scan number for ABI 373 ABI 373XL A
139. ar data panel information below the icons is dimmed Tabular data appears in a single table so electropherogram panel configuration is not relevant Selecting In electropherograms the data appears in panels You can overlay up to Electropherogram Sixteen samples within up to eight panels Select the number of Panels available panels up to eight from the pop up menu labeled of Panels Project 1 Results Control of Panels Pop up menu Dye Samples GM 38 0292 GM 36 0242 LIN 3y 0z 2 MM 3k 029e2 oO Note Set the number of panels when using the Quick Tile option Using this option causes the GeneScan Analysis Software to automatically change panels as you select samples for display see Creating Tiled Electropherogram Displays on page 7 10 Evaluating Analysis Results 7 7 Selecting Samples Use the dye color field on the left side of the Results Control window to to Display select the dye for display 7 8 Evaluating Analysis Results You can select any or all dye colors including the standard for each Sample file To select samples for display Step Action 1 Click the Electropherogram icon The panel below the icon is enabled 2 Click a panel number in the list to the left of the Dye Sample Display list If fewer than eight panels are available and you want to use more choose a larger number up to eight from the of Pa
140. ard Disk Data GelRuns L28t Sample Info is up to 255 Sample characters information from h i Starts wit including letters the Sample ends with numbers and Sheet contains punctuation Sample is up to 255 Comment from Comment characters the Sample h Startsiwit including letters Sheet ends with numbers and contains punctuation Size Data ispresent NA is present e i means that one is not dues present Or Tore Cy contain sizing does not information apply is not present means none of the dye sample contain sizing information E 26 Using the BioLIMS Database Fragment Search Criteria continued Pop up Menu Criterion Choices Allowed Text Description Size Calling done NA done means notdo e sample file has completed size does not calling indicated apply by a size curve not done indicated by a missing size curve Matched any 0 100 Percentage Peaks based on size V SNA standard lessthan matched peaks divided by size reater than i standard between defined peaks Offscale Data present NA present means does not ine analyzed apply range contains off scale dye sample peaks Using the BioLIMS Database E 27 Searching the BioLIMS Database Follow these steps to use the Collection Browser window to search the BioLIMS database for specific collections and fragments To search the BioLIMS database Step Ac
141. ard for each sample by clicking in the appropriate dye cells under the standard column Note Alternately you can create a stand alone Sample sheet and attach it to the gel file using the Install New Sample Sheet command from the Gel menu see Installing a New Sample Sheet on page 2 36 5 Choose Save 36 S or Save As from the File menu If you click the close box before saving a dialog box appears with the message whether or not to save the entries A 10 Using GeneScan with the ABI 373 Tracking Lanes Introduction When opening a Collection file the GeneScan Analysis Software places an appropriate number of straight tracker lines on the image based on the number of lanes chosen using the Gel file verification dialog box see Opening the Collection File on page A 7 The software does not automatically track the lanes You must do so before generating Sample files Process The normal process is as follows Step Action 1 Track the lanes 2 Review and edit the tracking if necessary 3 Generate the Sample files Note However if you wish you can automatically extract sample information and create Sample files immediately after tracking Procedure To track the lanes and review the tracking before generating Sample files Step Action 1 Choose Track Lanes from the Gel menu IMPORTANT Before choosing Auto Track Lane verify that the comb type is set
142. ards Peak centers seem to Resolution of the Repeat electrophoresis be incorrect in gel might be at lower power or with electropherogram inadequate ABI 373 or ABI Prism 377 Signal to noise ratio might be too low longer gel Troubleshooting the GeneScan Software D 3 Troubleshooting projects and results continued Problem Probable Cause Correction Software cannot Sample s in lane Re analyze the display the sizing curve size standard does Sample file with a for a sample not match defined different size size standard standard or Sample file was create a new one not size called In the Analysis Control window d click dye sample that represents Size Standard for the Sample file e Choose a size standard definition file and re analyze Peaks disappear in the Included the primer electropherogram peak in the analysis D 4 Troubleshooting the GeneScan Software Troubleshooting Gel Data Table Description The following table lists the problem probable cause and correction for troubleshooting gel data Troubleshooting gel data Problem Probable Cause Correction At the position of one strong peak additional colors appear underneath the peak Off scale data not multicomponented correctly Poor incorrect matrix Gel Image not a Repeat electrophoresis load less sample b Attach a new gel matrix regenerate
143. are ABI PRISM 377 lane How GeneScan The GeneScan Analysis Software performs the following steps in Analyzes Sample analyzing Sample files Files Step Action 1 Processes the raw data signals to generate analyzed data signal and then uses the analyzed signals to detect the signal peaks associated with DNA fragments 2 Performs size calling by identifying the peaks of the in lane size standard found in each sample 3 Determines the fragment size of each experimental peak within the sample based on the size calling curve generated using the size standard peaks the selected size calling method and by comparing it to the pre defined size standard file The algorithmic steps of the process from raw data to analyzed data are as follows Multicomponenting Baselining Smoothing if any Peak detection Analyzing Sample Files 3 3 Opening Sample Files Introduction Sample files can be opened as separate files outside of projects and display related information about each Sample file If you are interested in Then one or two Sample files it is often more convenient to open Sample files individually and analyze or view the data without or opening an entire project multiple Sample files use a project For information on opening Sample files from within Projects see Accessing Sample Files on page 5 6 BioLIMS see Switching Between Sample
144. are to lose track of a lane at extremely bent some point along the lane s path lanes Make identification of lane positions difficult Failure to complete The software uses the Used lane information to the sample sheet determine if what is found corresponds to what was loaded according to the Sample Sheet If too few or too many lanes are marked Used then the software must guess at which lanes to throw out and which to keep Wrong or bad The software needs a proper matrix to determine the matrix centers of the bands of the gel 2 54 How to Process and Edit the Gel File Verifying Optimal To verify optimal channel tracking examine peak heights in the Slice Channel Tracking View of the Gel File window These peaks represent the selected lane and show the peak intensity Reviewing Tracker Lines If the lane is Then correctly tracked the white tracker line should appear in the middle of each band optimally located you may need to adjust the line and re extract the affected lane For more information see Tracking and Extracting Data on page 2 61 To review tracker line placement Step Action 1 In the Gel File window click the lane marker for the lane you want to review Note From the currently selected lane you can also press the Tab key to move right or press Shift Tab to move left Inspect the placement of the tracker line on the lane
145. ata Collection software For analysis of the data after completing the matrix run transfer the data collection file to a Power Macintosh computer This section describes how to do the following tasks Topic See page Loading the Matrix Standards 6 20 Completing the Sample Sheet in the 672 Software 6 21 Running the Gel 6 22 Completing the GeneScan Sample Sheet 6 23 Note See Appendix A for converting ABI 373 data collection data into gel files Loading the To load matrix standards Matrix Standards Step Action 1 For denaturing and native gels load 1 5 pL of matrix standard per lane depending on comb configuration see instrument user s manual 8 lanes with different colors leaving an empty lane between each lane of matrix standard instrument manual 2 Select the appropriate filter set on the ABI 373 sequencer For this matrix Choose Dye Primer matrix Filter set A Fluorescent Amidite matrix Filter set B 3 Electrophorese samples according to conditions specified in your 6 20 Making a Matrix File Completing the Sample Sheet in the 672 Software Introduction After opening the Collection file complete the Sample Sheet embedded and track the lanes This ensures that when the Sample files are generated they contain the correct information The Sample Sheet that is part of the Collection file is blank when it is opened This is
146. ation ABI PRISM 310 only Temperature Laser power ABI 373 or ABI PRISM 377 gel file data collection Run File and run information This information is embedded in the gel file ABI PRISM 310 data collection Run file and run information Gel Information Polymer Information found under the header Information is inserted from ABI PRISM 310 Gel type Optional user entered Length to detector Lot and expiration date Data Collection information ABI 373 and ABI PRISM 377 Gel type Optional user entered Name of gel file Gel file data collection Run file This information is embedded in the gel file Gel percentage and thickness Well to read separation distance Optional user entered Number of channels Number of Lanes Gel file data collection Run file This information is embedded in the gel file Information found under the header Information is inserted from Channel Averaging Note Zero 0 indicates use of pre averaging offscale data The following is an example of how pre averaging offscale data appears Extraction method Displayed 3 channel averaging 3 3 channel averaging 30 with pre averaging offscale 3 channel 3 averaging weighted 3 channel 30 averaging weighted pre averaging offscale Range of data point
147. ay be seen when the data is analyzed with no or light smoothing the flat topped peaks may not be apparent with heavy smoothing Choose Analysis Parameters from the Settings Menu see Smooth Options on page 5 20 How to Process and Edit the Gel File 2 39 Gel File Window The following is an example of off scale data displayed in a Gel File Example window Each white line contains off scale data points GS Gel 38 377 36cm34w34L H 1 9 Off scale data Current Comb Type set in prefs Square Tooth 2 40 How to Process and Edit the Gel File Electropherogram The following are two examples of off scale data displayed in Examples electropherograms Each red bar region indicates an off scale data point Electropherogram Displaying Off Scale Data Note Choose the Zoom In command from the View menu to more clearly show the areas of off scale data Off scale data 6B 16 16 16 GM 6G 16 67167 LIE sy 16 1e 7167 BE 6ER 16 167167 6B 16 16 16 Hi 55 18 16716 LI sy 16 1e 7167 MM sR iseie rises How to Process and Edit the Gel File 2 41 Electropherogram Displaying the Flat Topped Effect The off scale peaks in the zoomed in electropherograms illustrate the flat topped effect The second electropherogram has the red bars removed Im Flat topped peaks 2 42 How to Process and Edit the Gel File Working with Lane Markers Introduction When the GeneS
148. b is used to form a well during gel polymerization The toothed edge is inserted to form wells into which samples are loaded For GeneScan Analysis Software applications a square tooth comb is recommended to lessen the chance of sample spillage from lane to lane size calling curve Curve created by the GeneScan Analysis Software for size calling The software calculates this curve based on the size calling method you specify for data analysis This curve is red in the Standard Sizing Curve window When it matches the align by size curve the two overlap so you see only this curve See also align by size curve and size standard spline interpolation curve size standard Specific DNA fragments of known sizes After you define the peaks of a size standard the GeneScan Analysis Software matches this definition to the internal lane or injection standard that you include with your run The software assigns the defined size values to the appropriate peaks of the internal lane or injection standard and uses this information with the selected size calling method to size all unknown fragments For more information see About Size Standards on page 5 29 and Using Size Standards on page 5 36 Glossary 3 size standard spline interpolation curve Curve created by the GeneScan Analysis Software for aligning data by size The software creates this curve if you use the Local Southern or Cubic Spline Interpolation size calling method and the size standar
149. bular data in many ways for Displays display and the GeneScan Analysis Software allows you to save display combinations and formats for future viewing For information on saving a display for future viewing see Saving and Renaming the Results Control Format on page 7 17 Saving Archiving and Copying Files 8 5 How to Archive Sample and Gel Files Introduction Since the gel file is so large you will probably want to back up only specific Sample files Archive Sample files when you feel confident that the channel selections tracking used to generate them was correct If you need to save a gel file you can save only selected information from it and greatly reduce the file s size see Saving Gel Files on page 8 4 Archiving Sample The following table lists how to archive Sample and gel files and Gel Files Item Description How to archive Sample files A sample file is 60 KB Drag the file icon or the to 150 KB in size Run folder containing depending on the the files to floppy disk length of the run icon or to an alternative storage device A 1 4 MB high density disks holds about twelve files Gel files Gel files contain the Make a backup copy of raw data acquired by the Data Collection software and take up a lot of space 10 MB to 60 MB is typical After the GeneScan Analysis Software generates Sample files from a correctly tracked gel file you do not need to save the gel file the g
150. c analysis the Gel File window opens and displays the newly created gel file For more information see Setting Up for Automatic Analysis on page 4 4 manually you can either Double click the Gel file or a Choose Open from the File menu The Open Existing dialog box appears b Click the Collection Gel icon A directory dialog box appears C Locate and select the desired gel file and choose Open How to Process and Edit the Gel File 2 11 About the Gel File Window Differences The following table explains the differences between the GeneScan Between Gel File Analysis Software Gel File window and the Gel File window displayed Windows by the Data Collection software during a run Application Shows New Data Gel Image Data Collection software Gel window Real time data as it is being collected New data appears at the bottom of the Screen as it is collected so the top of the screen shows the start of the run Gel image is not baselined or multicomponented GeneScan Gel File window An image of the gel after the Data Collection software is finished This image is inverted The bottom of the window displays the start of the run that is the smallest fragments appear at the bottom of the window just as they would on an autoradiogram Gel image is baselined and may or may not be multicomponented 2 12 How to Process and Edit the Gel File
151. can Analysis Software first opens a gel file it adds lane numbers lane markers and tracker lines to the gel image In most cases if you used a good gel for your run and the run settings are correct the gel file should be properly tracked If however the tracking is incorrect because the signal is weak and the tracker missed a lane Topics in This Section you might need to make some changes This section includes the following topics For this topic See page Moving Between Lanes and Channels 2 44 Using Control Points 2 45 Adding and Deleting Control Rows 2 46 What Marking Lanes as Used Unused Means 2 47 Marking Lanes as Used or Unused 2 47 Moving Misplaced Lane Markers 2 48 Lane Marker Rules 2 50 About Marking Lanes for Extraction 2 50 Marking All Lanes for Extraction 2 51 Unmarking All Lanes for Extraction 2 51 Marking a Single Lane for Extraction 2 52 Unmarking a Single Lane for Extraction 2 52 How to Process and Edit the Gel File 2 43 Moving Between The following table lists how to move between lanes and channels Lanes and Channels If you want to move Then quickly from one lane to the next lane You can take the following action To move Then the lane click the Lane marker of the lane you want to move one lane to the press the Tab key right one lane to the left press the Shift Tab key the location of a s
152. can extract lanes using weighted averaging or pre averaging offscale detection These options are available in the Lane Extraction section of the Gel Preferences dialog box For more information see Lane Extraction on page 2 7 1 4 GeneScan Analysis Software Overview About This Manual and Other Instruments ABI 373 ABI 373 XL Upgrade Users 96 lane Upgrade Kit Manual Text GeneScan Analysis Software works version 3 1 with ABI 373 data in the same way as the it works with ABI PRISM 377 data If you use the GeneScan 672 Software to collect data on the ABI 373 you must transfer your data to a Power Macintosh computer and perform certain extra steps These extra steps are described in Appendix A Using GeneScan with the ABI 373 The ABI 373 DNA Sequencer with XL upgrade updates the data collection interface to match the ABI PRISM 377 increases the maximum image resolution to 388 channels and increases the maximum number of lanes to 66 Note ABI 373 with XL upgrade users do not need to follow the procedures described in this Appendix A Using GeneScan with the ABI 373 The ABI PRISM 377 DNA Sequencer 96 lane upgrade kit enhances both the capabilities of the 377 DNA sequencer to support up to 96 lanes for using the GeneScan Analysis Software Manual Text Applies to other ABI PRISM Genetic Analysis Instruments The GeneScan Analysis Software and this manual apply to three different genetic analysis in
153. colors in the row one sample are selected change all the standards in the size standards column to the same setting a Click the arrow in the column heading b Choose a file from the pop up menu The same size standard is displayed for all the samples change a size standard in one of the rows a Click the arrow in the row b Choose a file from the pop up menu The size standard for the selected row changes change all the parameters in the parameters column to the same setting a Click the arrow in the row heading b Choose a file from the pop up menu The same parameter is displayed for all the samples change a parameter in one of the rows a Click the arrow in the row b Choose a file from the pop up menu The parameter for the selected row changes apply a choice to selected fields in the size standards or parameters column a Click the row in the column containing the information you want to apply and drag down b Choose Fill Down from the Edit menu The value in the selected rows changes to the value in the first row selected Analyzing Project Files 5 5 Analyzing Sample Files Using the Analysis Control Window Introduction This section describes using the Analysis Control window to perform the following tasks Topic See page Accessing Sample Files 5 6 Analyzing Sample Files 5 7 Specifying th
154. correctly by choosing Preferences from the Settings menu and Gel Preferences from the submenu The Track Lanes dialog box appears Track Lanes A Proceeding with this command will over write current Lane Tracking Revert to Straight Tracking Auto Track Lanes Click Auto Track Lanes Using GeneScan with the ABI 373 A 11 Step Action 3 Review the tracking and edit as necessary For information on Editing tracking see Working with Tracker Lines on page 2 53 Changing the appearance of the window for better visibility see How to Adjust the Gel Image on page 2 18 4 When you are satisfied with the tracking choose Save from the File menu Generating Sample Files When to Generate Generate new Sample Files from the lanes if you Sample Files 4 pid not automatically generate Sample files after tracking or you Edited the lane tracking For information on generating sample files see Extracting Data Without Changing the Current Tracker on page 2 64 A 12 Using GeneScan with the ABI 373 GeneScan Size Standards Introduction In This Appendix Applied Biosystems provides four different size standards that you can choose from to analyze fragments run on the ABI PRISM 310 the ABI 373 or the ABI PRISM 377 See also About Size Standards on page 5 29 Topics in this appendix include the following
155. d Out 7 31 Showing Off Scale Data 7 32 Showing Data by Fragment Size 7 35 Changing the Horizontal Scale 7 37 Changing the Vertical Scale 7 38 Assigning Standard or Custom Colors 7 39 Displaying X and Click the cross hairs and select an area in the Electropherogram to Y Axis Positions display the x and y axis positions The x and y axis values appear in the box in the lower left corner of the electropherogram and if tabular data is also displayed the row in the table is highlighted Evaluating Analysis Results 7 27 Moving the Electropherogram Changing the Dye Color 7 28 Evaluating Analysis Results Move the associated electropherogram to the front by clicking in the legend either the Dye color indicator Plot color indicator or Text il LINE 11Y 1347 02 P14 HEX Dye color Plot color Text indicator indicator To change the dye color double click the dye color indicator If you change the dye color or scale a vertical line appears beside the indicator showing that it has been modified Note 36 double click the dye color indicator to return it to the default Displaying Peak Choose Show Peak Positions from the View menu Use this command Positions to examine how the GeneScan Analysis Software defined peaks by displaying markers that identify the beginning center and end of each peak HS P27 1 7 Display 1 re 9B 10 P27 10 10 GM 36 109
156. d create Close it one for the new Sample files cancel the track and extract Cancel command not open The GeneScan Analysis Software creates a new project and adds the new Sample files to it How to Process and Edit the Gel File 2 63 Extracting Data Without Changing the Current To extract the data without changing current tracker lines Tracker Step Action 1 Choose Extract Lanes 36 L from the Gel menu and the Extract Lanes dialog box appears Note Press 36 period to cancel the Extract Lanes operation Extract Lanes Lane Extraction Extract From All Used Lanes O Lanes D Over Write Original Sample Files Project File K Add Sample Files to Project Create a New Project Y K Auto Analyze New Sample Files Analyze All Files Print Result Use Sample Sheet Settings K Save Gel after Extraction Ga 2 64 How to Process and Edit the Gel File To extract the data without changing current tracker lines continued Step Action 2 In the Lane Extraction portion of the dialog box you can take the following action Choose To All Used Lanes radio button generate a new Sample file for every lane marked Used Lanes marked for Extraction generate a new Sample file radio button white markers only for each lane with a white lane marker Over Write Original Sample replace the old files with the Files checkbox n
157. d data does not match the best fit curve which is normally used for aligning the data by size This curve is blue in the Sizing Curve window See also align by size curve and size calling curve square tooth comb Piece of mylar inserted into a gel to form wells for sample loading You remove a square tooth comb prior to sample loading For GeneScan Analysis Software applications a square tooth comb is recommended to minimize the chance of lane to lane leakage which can occur more often with a shark s tooth comb tiled Displayed so they do not overlap The GeneScan Analysis Software displays tiled electropherogram panels in the Results Display If you display more than one electropherogram in each panel all electropherograms in the panel are overlaid tracker line Marks the channel exhibiting the strongest fluorescent signal in a lane For each lane the raw data extracted for the sample file is an average of the data in several channels You set the number of channels averaged in the Gel Processing Parameters WTR well to read Length from the wells of the gel to the read region of the gel See also separation distance Glossary 4 Index A ABI 373 automatic analysis diagram 4 3 Collection file opening A 7 to A 8 gel processing parameters setting A 5 to A 6 generating sample files procedure 6 27 to 6 28 when to generate new files A 12 how GeneScan works with 1 5 loading and running dye standards 6 20 to 6 23 ma
158. d within the sample lane Each position on the gel image is defined by a scan number and a channel number Tracker line for the selected lane Current Comb Type Displays the current comb type that is set in the Gel preferences For more information see Comb Type on page 2 10 Control row For information on adding and deleting a control row see Adding and Deleting Control Rows on page 2 46 2 16 How to Process and Edit the Gel File Gel File window described continued No Description 10 Scan number Reflects a particular point in time The address of a particular data point on the Gel file image This run Collects 2400 approximately 2250 scans per hour 1200 approximately 1125 scans per hour 11 Slice view Graphical view of the data values in the tracked channel of the selected lane The display changes as you move the tracker line from one channel to another in the lane Each peak in the Slice view corresponds to a band in the gel image and indicates a DNA fragment Although these bands and peaks do not represent analyzed data they provide an overview of the relative signal intensity between the bands in that lane and allow you to make a qualitative evaluation of the run The Slice view is empty black when no lane is selected or when the lane has no data How to Process and Edit the Gel File 2 17 How to Adjust t
159. data collected using the ABI 373 the software creates a Collection file that contains the raw data When using previous versions of the GeneScan Analysis Software before 2 0 you preprocessed this Collection file to create a gel file Once you were satisfied with the tracking of the gel file the gel file could be analyzed to create a Results file The Results file contained all of the analyzed data Using the GeneScan Analysis Software version 2 0 or later it was not necessary to preprocess the Collection file You process the Collection file to create Sample files then analyze the Sample files Each Sample file contains information from one lane of the gel and after analysis the corresponding analyzed data A 2 Using GeneScan with the ABI 373 Processing Steps The steps to using the GeneScan Analysis Software with ABI 373 data are as follows Step Action See page 1 Setting Gel Processing Parameters A 5 2 Opening the Collection File A 7 3 Completing the Sample Sheet A 9 4 Tracking lanes A 11 5 Generating Sample files A 12 Comparison of The following figure is a comparison of using the GeneScan Analysis GeneScan Analysis Software before version 2 0 and how to use the software with version 2 0 or later Once you have generated Sample files use the GeneScan Analysis Software as described in this manual Note After analysis the GeneScan Analysis Software automatically creates a Run folder n
160. dden 2 56 How to Process and Edit the Gel File Interpolating Tracker Lines Why Use Interpolation Mode Procedure Use the interpolation mode to adjust the tracking lines between two vertical points in the gel image This mode is very useful to manually manipulate the tracker lines for a set of lanes than have anomalous but similar migration patterns Once the two points are chosen you can use the control points on the tracker lines to move the lines To use the interpolation mode Step Action 1 With the Gel File window displayed press the Interpolation button to enter interpolation mode I In the region being tracked click the left hand lane marker at the bottom of the tracker line and then click the right hand lane marker These two lanes are the interpolation guides and the lanes between them are interpolated Note X While in interpolation mode all the other lanes are hidden and no editing of the tracker lines is allowed Press the Interpolation button again to exit the interpolation mode How to Process and Edit the Gel File 2 57 Step Action Em Gel file Ee v Ell El oo Ex S BR 05 Cha 5 25 Sean 4515 Lares Used 33 9 10 11 12 13 14 15 16718 19 20 21 22 23 24 25 OOOOOOOOOOOOOOQO OOOOO000 4550 4045p 3540p 3035p 2530p 2025p 1520p 1015p Interpolation guides 4 You c
161. dialog box does not appear 9 6 Printing Results Using GeneScan with the ABI 373 Introduction In This Appendix Note This section does not apply if you are processing data collected on ABI 373 DNA Sequencer with XL upgrade instruments Topics in this appendix include the following Topics See page Processing Using the ABI 373 A 2 Setting Gel Processing Parameters A 5 Opening the Collection File A 7 Completing the Sample Sheet A 9 Tracking Lanes A 11 Generating Sample Files A 12 Using GeneScan with the ABI373 A 1 Processing Using the ABI 373 Introduction ABI 373 Versus ABI PRISM 377 Data Using the GeneScan Analysis Software Before Version 2 0 Using the GeneScan Analysis Software Version 2 0 or Later This section describes using the ABI 373 data with the GeneScan Analysis Software in the same way as you use ABI PRISM 377 data When the GeneScan 672 Software has been upgraded to work on a Power Macintosh computer running System 7 5 or higher this will be true The ABI 373 and ABI PRISM 377 run on different computers If you use the GeneScan 672 Software to collect data on the ABI 373 the data collection program runs on a 68K Macintosh computer Since the GeneScan Analysis Software 2 0 or later runs on a Power Macintosh computer transfer the data to a Power Macintosh compatible computer before analyzing the data Using the GeneScan 672 Software on
162. ds under the precise conditions you want to Matrix Standard generate a matrix file To run the matrix standards Step Action 1 Complete the information in the Run window Note modules Make sure to choose the appropriate PreRun and Run Run 7 28 98 12 18 PM Lx OA D Plate Check Module Plate Ch v PreRun Module Run Module O Collect time hours Sample sheet cone v D Gel s Matrix File Bogus Matrix v Operator Lanes prt cain a Lane Sample Sample Name Well to Read distance cm Filter Set Matrix File Auto ta Analysis Parameters Sample File Name LU FIFI Choose the appropriate filter set for the conditions you wish to duplicate For this matrix Choose Dye Primer or dNTP matrix Filter Set A Fluorescent Amidite matrix Filter Set B Start the electrophoresis run according to the conditions specified in your instruction manual 6 26 Making a Matrix File Generating Matrix Sample Files for the ABI 373 and ABI PRISM 377 Who Should Use This Step Who should use this step Who shouldn t use this step This step is only necessary if you are using an ABI373 ABI PRISM 377 XL upgraded instrument 96 lane upgrade The ABI PRISM
163. e file appears all dyes for all samples area above the row index numbers Identify the sample containing the standard as follows To click the identify each sample that contains a standard colored field that represents the standard select the same dye as the standard for all samples colored column heading for that dye A gray diamond appears in the field to identify the dye color as the standard Choose a defined size standard setting from the pop up menu in the Size Standard column as follows To See define a new size standard Using Size Standards on page 5 36 edit a size standard Editing an Existing Standard on page 5 37 change the available size standards in the pop up menu Defining Folder Locations on page 5 41 Analyzing Project Files 5 7 5 8 Analyzing Project Files To analyze Sample files continued Step Action 4 To install a new matrix select a set or all of the samples in the Sample File column and choose Install New Matrix from the Sample Menu 5 Choose a parameters setting from the pop up menu in the Parameters column as follows To See use the default analysis Defining Analysis Parameters parameters to specify different on page 5 18 parameters change the available Defining Folder Locations on parameters in the pop up m
164. e this Agreement if you fail to comply with any or all of its terms in which case you agree to return to Applied Biosystems all copies of the SOFTWARE and associated documentation 1 Failure to enforce any of the terms and conditions of this Agreement by either party shall not be deemed a waiver of any rights and privileges under this Agreement 2 In case any one or more of the provisions of this Agreement for any reason shall be held to be invalid illegal or unenforceable in any respect such invalidity illegality or unenforceability shall not affect any other provisions of this Agreement and this Agreement shall be construed as if such invalid illegal or unenforceable provisions had never been contained herein 3 This Agreement shall be construed and governed by the laws of the State of California 4 This Agreement and the Applied Biosystems Sales Quotation constitute the entire agreement between Applied Biosystems and you concerning the SOFTWARE License and Warranty G 3 Glossary This glossary defines special terminology used in the GeneScan Analysis Software User s Manual The terms are listed in alphabetical order Many terms are defined in the text of the manual so if you do not find a term here check the index to see if you can locate it in the manual align by size curve Curve created by the GeneScan Analysis Software for aligning data by size The software calculates a best fit least squares curve for all samp
165. e Format for Printed Results 5 9 Displaying Size Standards and Analysis Parameters 5 11 Displaying Sample and Dye Information 5 12 Setting Sample File Sort Order 5 14 Setting Dye Indicator Preferences 5 15 Accessing Sample There are two ways to access Sample files contained in a project from Files the Analysis Control window Sample files that are dimmed could not be found by the project You can Then double click a Sample file name If the Sample file is Then that open Sample file window becomes active not open Sample file opens to its Sample Results view select a Sample file and choose one of the five display modes from the Sample menu the Sample file window appears in the display mode selected 5 6 Analyzing Project Files Analyzing Sample The Analysis Control window allows you to analyze multiple samples Files easily You choose dyes to analyze dye standard size standard and analysis parameters for each Sample file and then analyze the Sample files using these settings You can also install a new matrix to the Sample files To analyze Sample files Step 1 Action Click the dye color fields for each sample you want to analyze as follows To Click the select a dye for all samples colored column header for that dye select all dyes for a single Sample file index number at the left end of the row in which the Sampl
166. e See Sample Results View on page 3 6 Information Updating the Results Re Analyzing the The Results Control and the Sample Results windows are dynamic The Data following table lists how the windows are updated if you re analyze the data If you re analyze Then the your data with either window active software updates the window 7 16 Evaluating Analysis Results Saving and Renaming the Results Control Format Introduction Important Considerations Saving the Display Format You can use the Results Control window to view multiple Sample files in electropherogram and tabular format The GeneScan Analysis Software allows you to save formats for future use You can then redisplay or print these formats without having to redefine them again The following are important considerations for saving a Results Control format You must save the project for the display to be available when you open the project again Remembering a display preserves the combination of windows panels data and customized color settings It does not preserve any zooming you have performed To save a display format for future viewing Step Action 1 With the Results Control window set for the display either Click the Display button or Press the Return key With the display on the screen choose Remember Display from the Project menu The Remember Display dialog box appears Rename Displa
167. e Sheet Example on page 2 31 Procedure To track and extract the gel file Step Action 1 Choose Track amp Extract Lanes from the Gel menu The Track amp Extract Lanes dialog box appears Track amp Extract Lanes WW Over Write Original Sample Files Ds Auto Analyze after Extraction Analyze All Files Use Sample Sheet Settings ane CC How to Process and Edit the Gel File 2 61 2 62 To track and extract the gel file continued Step Action 2 Use the Over Write Original Sample Files checkbox as follows If you Then this select the overwrites any existing Sample files that have the checkbox same name with the same sample data Tracking and extracting the gel file a second time without overwriting the original Sample files creates new Sample files with a dot and a number appended to the original names The number increments each time the program creates new files do not preserves Sample file names that already exist and select the create new Sample files for this data checkbox Note In BioLIMS mode the file name must be unique If the file name is not unique then the sample file will not be written to the database Note If a series of numbered files exist and you discard one of the files and then have the GeneScan Analysis Software re extract that sample the software adds the first available number t
168. e Standard column for the sample that you want to change A pop up menu appears Pop up menu Project Rnalysis Control O Print Results Print Setup Sample File Size Standard Parameters zi Analysis Parameters al Analysis Parameters Analysis Parameters 3 Select a size standard from the pop up menu Edit the peak size values by following step 7 on page 5 33 to step 9 on page 5 34 5 Choose Save or Save As from the File menu You can take the following action If you choose Then the Save existing file is replaced Save As changes are saved using a file name you specify Deleting an The following procedure describes how to delete a user defined Existing Standard standard from the GS Standards Folder so that it no longer appears in the Standard pop up menus The standard is permanently removed and you must re define to use it again To delete an existing standard Step Action 1 Switch to the Finder Locate and open the Current Standards folder Select the definition file that you want to delete 2 3 4 Drag the file to the trash and empty the trash Note You can also drag the standard to another folder for storage Analyzing Samples To select the same standard for analysis of all samples Using the Same Standard Step Action 1 If the Analysis Control window is not displayed choose Analysis Control 3 1 from the Windows men
169. e a control row a Option click once on the control row triangle that you want to delete The following dialog box appears A Are you sure you wish to remove a control row Spline accuracy can be lost Cancel ok b Click OK The GeneScan Analysis Software only tracks a lane and extracts sample data if the lane is marked Used in the Gel Sample Sheet When a lane is marked Used its lane marker 4 is colored blue white or yellow All unused lanes have gray lane markers By marking lanes as Used or Unused you specify which lanes contain sample data This allows the software to correctly number the Used lanes and put the extracted sample data from each lane into the correct Sample file It also ensures that Sample files are generated from only the Used lanes and not from empty lanes Note Using any of these three methods to mark a lane used or unused changes the setting in both the gel image and the Sample Sheet The three ways to mark a lane as used or unused are by Clicking the lane marker then choosing Mark Lane Used Unused from the Gel menu Holding down the 3 key while clicking the lane marker Clicking the Sample Sheet button to open the Sample Sheet that is attached to the gel file and then selecting or deselecting the Used checkbox for the lane How to Process and Edit the Gel File 2 47 Moving Misplaced The following procedure describes how to move misplaced lane Lane Markers
170. e address is http www ncsa uiuc edu SDG Software Brochure Overview M acTelnet overview html You can also reach them at the address shown below Applied Biosystems does not support NCSA Telnet NCSA Telnet Scott Bulmahn Tim K Jim Browne Jim Logan opf Gaige Paulsen Aaron Contorer Former Developers ascal Maes Mark Tamsky rs on macintosh and cal support team and NCSA Software Development 152 Computing Applications Bldg 605 E Springfield Ave Champaign I11 61820 macteinetencsa uiuc edu NCSA Telnet iz in the public domain Comments and suggestions catalog available may be directed to 2 6 Think C 6 0 March 1994 Troubleshooting the BioLIMS Database F 3 Troubleshooting Process Troubleshooting On the following page is a flow chart illustrating the process for Flow Chart troubleshooting database connection between the Macintosh Client and the Sybase Server The step numbers given in the flow chart refer to the tables on pages F 6 to F 13 where the troubleshooting procedures are described in detail F 4 Troubleshooting the BioLIMS Database Find interfaces file charsets and locales step 1 on page F 6 Check SybaseConfig panel step 4 on page F 8 l a E cn SybPing step 5 a fails F A n RIO page F 9 He o z d ping V successf ra bod d M Try to log in to 5 the server step 1 on page F 12 E c U
171. e assignment confidence value of OB is less than the threshold value of 70 11168 Gel 38 377 Z6cm4w24L H 1 9 confidence value 100 The lane assignment confidence value of 100 is equal to or above the threshold value q f a program error Gel file 2 24 98 3 47 34 PM Started A gt gt 0101 B G R Analysis completed I 1 Exceeded the detection limit of 250 peaks for dye B gt gt Size Calling completed Matched Size Range 75 300 bps 09 PM Started Analysis Session 01e1 B G Y R Analysis completed Exceeded the detection limit of 250 peaks for dye B gt gt Size Calling completed Matched Size Range 75 300 bps Evaluating Analysis Results 7 51 What to Evaluate Removing Information from the Analysis Log Closing the Analysis Log 7 52 Evaluating Analysis Results Evaluate the following What to evaluate What not to evaluate Potential problems that the GeneScan Analysis Software might have had during size calling It will alert you to any peaks defined in the size standard definition that the software failed to match to the size standard run with your sample The GeneScan Analysis Software will not alert you to any consecutive peaks at the end of the definition This is to avoid logging warnings when your sample was not run long enough to include all the defined size standard peaks This prevents you from having to create a new standa
172. e c b tom 3 5 Sample Results View 0 0 0 0 cece cece e 3 6 Whatit Displays iesu ue oo So See eee ae XV PRAES 3 6 Displaying the View 0 0 0 cee eee ee eee 3 6 Sample Results View Example 00 0 00000 eee eee 3 6 Description of Columns 0 0 0 e eee eee eee ee 3 7 Differences From the Results Display 0 3 7 sample Info VEW piee cases oe ene QE E EIUS GRE EN NU REN UA 3 8 What it Displays s s scadaie nis use Be Here eite preda 3 8 vi Displaying the View 1 0 20 0 eee eee eee e 3 8 Sample Info View Example 0 0 0 cee eee ee eee 3 9 Description of Information 3 9 Size Curve VIEW ovt e ee RR HR e CERE UR epee es 3 13 What ait Displays ne note alae eee eye see A eee a 3 13 Displaying the View lesen 3 13 What the Size Curve Displays 2 0 ce eee eee ee ee 3 14 Curves Described sits 4 aagice expe DER ne PEST EY xe 3 14 Raw Data View ires e ERR Ge eae S eR d ee ecce 3 15 What it Displays tenu eret epe Suet as 3 15 Displaying the View 0 00 0 eee eee eee e 3 15 Raw Data Example 0 cece eee eee eens 3 16 What to Evaluate cient hr ba eR Sed Gare ees 3 16 EPT Data VIEW esee deerat pe UR e e ege eia 3 17 What it Displays 5 doe ET BS EL RE UR hee Xu 3 17 Displaying the View 0 0 cece ee e 3 17 EPT Data Example 0 0 0 eee cee ee eee 3 18 Colored Lines Described 0 000
173. e changes to the Preferences dialog box and to close it Cancel close the Preferences dialog box without changing the mode Using the BioLIMS Database E 11 How to Access the BioLIMS Database Introduction Before Accessing a Sybase Database The following procedure describes how to access the BioLIMS database by completing the Preferences dialog box Before you can work with the GeneScan Analysis Software software you must establish a connection to the BioLIMS database This connection is made through the BioLIMS Access page of the Preferences dialog box IMPORTANT If you try to connect to a Sybase database through the GeneScan Analysis Software when the network is down the computer may hang If this happens you have to restart the Macintosh computer Force the computer to restart by pressing the key combination Command Control Power key Before accessing a Sybase database you need to ping the database using SybPing to ensure that the database connection is working Step Action 1 Open the SybPing program At installation this program is placed in the BioLIMS 2 0 BioLIMS Extras Sybase bin folder SybPing To use SybPing Select the server you wish to ping from the popup menu The server must be defined in you interfaces file Servers Execute by clicking on the Ping button selecting Ping from the File menu or pressing the return key
174. e choosing Auto Track Lane verify that the comb type is set correctly by choosing Preferences from the Settings menu and Gel Preferences from the submenu Gel Preferences Auto Launch Processing s Auto Track Gel K Extract Lanes after Auto Tracking Image Generation Defaults Scan Range Step 00000 Start Ds Multicomponent Gel Image Estimated Maximum Peak Height 2000 Lane Extraction L1 Use Weighted Averaging Use s Channel Averaging o Pre Averaging Offscale Detection K Stop extraction when below confidence threshold Confidence Threshold Z Comb Type Sharks Tooth Square Tooth Using GeneScan with the ABI 373 A 5 To set the gel processing parameters continued Step 3 Action You can take the following action in the Image Generation Defaults section To Then start and stop processing data Note All scans outside the specified scan range are ignored enter the scan numbers in the Scan Range Stop and Start text entry boxes compensate for the spectral overlap between dyes select the Multicomponent Gel Image checkbox set the upper limit of the scale of the peaks in the Slice view of the Gel window Note This value also affects the brightness of the fragment bands enter a value in the Estimated Maximum Peak Height text box For more information see Image Generation Defaults on page
175. e dialog box appears 3 Enter a new scale in the dialog box and click OK Note The dye scale for the individual sample changes and a vertical line appears beside the dye color indicator to signify that it is modified You can change the dye color in the same way from the Results Control window The dye is scaled each time you open the Results Display window Changing the Dye To change the dye scale preferences Scale Preferences Step Action 1 Choose Preferences from the Settings menu and choose Results Dye Scales from the submenu The Preferences dialog box appears with the Results Dye Scales pop up Preferences Page C 2 Enter a positive number between 0 1 and 100 for each sample relative to any other sample and click OK Note Dye scale values do not automatically revert to default values Change them back to the defaults before examining results of another run Evaluating Analysis Results 7 45 Process of Verifying Results Introduction You can use the electropherogram and tabular displays to verify the results of analysis by checking the GeneScan Analysis Software calculated sizes and peaks Note The size calling of the standard and of sample fragments depend on the size calling method you defined in the Analysis Parameters and the accuracy of the defined standard Steps to Verifying To verify size calc
176. e following Standard pop up menu in the Analysis Control window Auto Analysis settings dialog box You can specify in the Auto Analysis Settings dialog box or in the Data Collection software that the defined standard be used for automatic analysis in future runs See Setting Up for Automatic Analysis on page 4 4 Analyzing Project Files 5 35 Using Size Standards In This Section Changing the Number of Peaks Detected Editing the Size Standard Definition 5 36 Analyzing Project Files This section contains the following topics Topic See page Changing the Number of Peaks Detected 5 36 Editing the Size Standard Definition 5 36 Using the Open Command to Edit an Existing Standard 5 37 Using the Analysis Control Window to Edit an Existing 5 38 Standard Deleting an Existing Standard 5 39 Analyzing Samples Using the Same Standard 5 39 Selecting Separate Standards for Samples 5 40 Use the Analysis Parameters dialog box to change the number of peaks detected in the Define New Standard window For information see Peak Detection Parameter Options on page 5 20 The following procedure describes how to edit the size standard definition lt Collection Settings gt standard that is embedded in the Sample file Double clicking the size standard definition will not open the file To edit the size standard definition Step Action 1 If the file is on your ha
177. e general condition of the bands in the lanes in the gel image Are the fluorescent signals displayed as discreet horizontal bands This may indicate a poor gel Are any of the colors too bright or too dark Is there a green or red haze Is this something that you can fix by adjusting the gel image contrast Either Adjust gel contrast see page 2 18 Adjust Estimated Maximum Peak Height see page 2 6 or Rerun the gel Inspect the lane markers Verify that each lane marker corresponds to a sample as designated in the Sample Sheet Adjust the locations of the lane markers as necessary How to Process and Edit the Gel File 2 27 Review these items Take this action Inspectthe tracker lines Adjust the tracker line placement as necessary Each tracker line should be centered over the brightest part of the bands it is tracking through Inspect the Slice View for each Adjust the tracker line placement as lane necessary Verify that all fragments in the size standard are included in the current tracking Sample Sheet Review the Sample Sheet to confirm that the information in the fields is correct If necessary edit the Sample Sheet information instrument file sample names comments and so forth that is automatically transferred to the Sample files or to the BioLIMS database For more information see Displaying the Sample Sheet
178. e not sized The corresponding size fields are blank Note Tabular data displays only peaks that are detected based on the Dye Amplitude Thresholds and Minimum Peak Half Width setting of the analysis parameters How n the Results Display window the GeneScan Analysis Software sizes Electropherogram all electropherogram panels to fit within the electropherogram portion of Panels are Sized the window by using the largest size that fits them into the visible area You can scroll to see the portion of the display that is not visible 7 20 Evaluating Analysis Results Peaks Visible in Peaks may be visible in the electropherogram and not listed in the Electropherogram tabular data because Not Tabular Data Reason For more information see The software detects the peaks based on the Dye Amplitude Thresholds and Min Peak Half Width Peak Detection Parameter Options on page 5 20 Electropherograms display the peaks that fall within the range specified by the Size Call Range parameters that are defined in the Analysis Parameters dialog box Size Call Range Parameter Options on page 5 21 For More For more information see the following topics Information Topic See Displaying Electropherogram and Tabular Data 7 22 Displaying Electropherogram Data 7 25 Working with Electropherogram Data 7 27 Evaluating Analysis Results 7 21 Displaying Electropherogram and Tabular Data D
179. eScan 350 B 2 to B 3 GeneScan 400HD B 4 to B 5 GeneScan 500 B 6 to B 7 using 5 36 to 5 40 Slice view how to determine the scale of the peaks in view 2 6 software files table of 1 16 to 1 18 license G 1 to G 3 registering 1 11 SSCP Minimum Peak Half width setting 5 21 Sybase SQL server configuring the BioLIMS server E 4 to E 9 how server names are recognized E 17 to E 19 troubleshooting connection F 3 to F 13 SybPing using E 12 System specifications 1 12 T tabular data displays about 7 20 to 7 21 displaying electropherogram and tabular data 7 22 to 7 24 using to verify peak detection 7 53 using to verify results 7 46 to 7 47 technical support 1 20 to 1 26 e mail address 1 24 internet address 1 20 regional sales offices 1 24 to 1 26 telephone fax U S 1 20 text in manual noted for specific instruments 1 5 tracking and extracting data extracting without changing tracking 2 64 to 2 66 saving after tracking 2 68 specifying Run folder 2 59 tracker lines defined Glossary 4 working with 2 53 to 2 56 tracking and extracting 2 61 to 2 63 tracking without extracting 2 60 to 2 61 ways to track and extract 2 59 troubleshooting bad matrix files causes 6 38 to 6 39 BioLIMS client to Oracle connection F 14 to F 24 client to Sybase connection F 3 to F 13 GeneScan crashes with BioLIMS D 9 if BioLIMS Preference page does not appear F 2 error messages D 9 to D 12 gel data D 5 to D 6 Genotyping software results D 8 projects and r
180. eating a The GeneScan Sample Sheet assigns sample and dye information to GeneScan Sample their appropriate lane Sheet 1 create a GeneScan Sample Sheet Step Action 1 Open the ABI PRISM 377 Data Collection software Define or verify the data collection preferences 2 Choose New from the File menu 3 Click the GeneScan Sample Sheet icon The GeneScan Sample Sheet appears Sample Sheet GS matrix GeneScan Sample Sheet Making a Matrix File 6 17 To create a GeneScan Sample Sheet continued Step Action 4 Enter the individual colors in the appropriate lanes where the matrix standards are loaded Note Itis important to fill out the Sample Sheet completely 5 Enter any additional information about the sample in the Comments column 6 Use the Save As command and save the Sample Sheet to the Sample Sheet folder Loading Matrix To load matrix standards Standards 6 18 Making a Matrix File Step Action 1 For denaturing gels load 0 5 2 uL of matrix standard per lane 8lanes with different colors leaving an empty lane between each lane of matrix standard 2 Complete the information in the data collection Run sheet making sure to choose the appropriate PreRun and Run modules Take the follo
181. ect the four Sample files representing the blue green yellow and red dye labeled runs and then click Add f Click Done after the Sample files are transferred For more information see Creating a New Project on page 4 10 Making a Matrix File 6 29 What to Look For 6 30 in the Raw Data Display Choosing a Scan Range Making a Matrix File To view raw data continued Step Action 2 From the Analysis Control window select the four matrix standard Sample files by clicking on the first Sample file holding down the mouse button and releasing on the last Sample file 3 From the Project menu choose Raw Data 36 R Electropherograms displaying raw data from the four matrix standard Sample files appear Note For the ABI PRISM 377 or ABI 373 you can also view raw data from the gel display by selecting one of the four lanes containing Dye Matrix Standard and looking at the Slice View to the left of the gel image For more information about viewing the gel image see What to Review in the Gel Image on page 2 27 In the raw data display of the Sample files verify the following Data peaks are present in all four of the matrix standards There are no anomalies The baseline is stable Peaks should be on scale no more than 4 000 relative fluorescent units and the peaks of the dye of interest should have a value of at least 200 If peak data does not show t
182. ected to 605 E Springfield Ave Champaign I11 61820 2 6 Think C 6 0 March 1994 mactelnet ncsa uiuc edu F 14 Troubleshooting the BioLIMS Database Troubleshooting Process Troubleshooting On the following page is a flow chart illustrating the process for Flow Chart troubleshooting the database connection between the Macintosh Client and the Oracle Server The step numbers given in the flow chart refer to the tables on pages F 17 to F 22 where the troubleshooting procedures are described in detail Troubleshooting the BioLIMS Database F 15 tnsnames ora file step 1 on page F 17 libraries step 4 on page F 19 an ha d ne EN AM an NetTest login NetTest Spes page F we c On page F 21 7 fails s a ji NetTest Pd w bod ty to log in to ihe server step 1 on i n page F 22 p 7 fails TU m NE e a E RW connection poss qu ba ty alae Connection step 6 page dd ag connection fails Call Customer Support step 7 on page F 24 fails F 16 Troubleshooting the BioLIMS Database a Try NSCA Telnet step 6 connection connection tnsnames ora file step 2 on page F 17 Home setting step 3 on page F 18 AON connection i E i a Es connection i successful D he the hw _ tistener and Serv r peer running step 2 on Town page F 23 E j a E te server running Check the Oracle Ser
183. ects when you have found a display format that suits your needs Saving Results Displays on page 8 5 8 2 Saving Archiving and Copying Files Saving Projects Note You do not need to save a Sample file after analysis The analyzed data is written directly to the Sample file during analysis The following table lists the options for saving a project If you choose Then Save Project 3 S you can take the following action If you Then previously saved the it is automatically saved project using the same name had not saved the a dialog box appears project Select a location for the file enter a name and click Save Save Project As a dialog box appears Select a location for the file enter a name and click Save Saving Sample Choose Save Sample Info 36 S from the File menu Files Note If you choose Close from the File menu or click the close box when you have not saved the changes a dialog box asks if you want to save them Saving Archiving and Copying Files 8 3 Saving Gel Files Why Save Only Parts of the Gel File Information A gel file can be large normally 10 MB to 60 MB You do not need to keep a gel file once the tracking is verified and the corresponding Sample files are created The following procedure describes how to keep parts of the information while discarding the rest IMPORTANT Do not discard any gel file until you have v
184. edure for making a new matrix file Prepare Dye Matrix Standards Evaluate the Matrix File Assign the Matrix File to Select Sample Files Save and Name the Matrix File Load Dye Matrix Standards Generate Sample Files View Raw Data Choose a Scan Range Making a Matrix File 6 7 Steps to Creating a The following table lists the steps to creating a new matrix file New Matrix Step Process See page 1 Preparing Matrix Standards for the ABI PRISM 310 6 9 2 Preparing Matrix Standards for the ABI 373 and 6 12 ABI PRISM 377 3 Loading and Running Dye Standards for the 6 13 ABI PRISM 310 4 Loading and Running Dye Standards for the 6 17 ABI PRISM 377 5 Loading and Running Matrix Samples on the ABI 373 6 20 non XL 6 Generating Matrix Sample Files for the ABI 373 and 6 27 ABI PRISM 377 7 Choosing a Scan Range for the Matrix Calculation 6 29 8 Generating a New Matrix File 6 32 9 Saving and Naming the Matrix File 6 34 10 Assigning the Matrix File to Sample Files 6 35 11 Evaluating the Matrix File 6 37 The Dye Matrix Standard Kits Table of Kits Applied Biosystems provides Dye Matrix Standard kits that are designed to test and define the multicomponent matrices for specific dye sets Tubes containing matrix standards for each of the four different dye colors blue green red and yellow are available in the each of these kits 6 8 Ma
185. ee as eEPUECERUUCERTGRAG A CPI UFIO PPS ers F 2 SOMMON 035 5 Stag ex AERE EAE QE QE NEUES F 2 About Troubleshooting the Client to Sybase Connection F 3 Introduction prs entanas a eni p Ie A p EE pets F 3 SybPing and Telnet 2 20 eee eee ee ees F 3 Troubleshooting Process 0 cece eee eee ne F 4 Troubleshooting Flow Chart 0 0 c cece cece eee eee F 4 Procedures for Troubleshooting the Client to Sybase Connection F 6 Introduction eeen en E a Se LUSAEREEUINE Sele bie ak od F 6 Troubleshooting from the Macintosh Client F 6 Required Sybase Extension Files 0 0 00 F 11 Required Oracle Extension Files llle F 11 Troubleshooting from the Unix Sybase Server F 12 About Troubleshooting the Client to Oracle Connection F 14 Introduction Lev soe e ee C eee tetas F 14 Teltietuc as ever UU DEI ANUS At el or queque ye pd e ES F 14 Troubleshooting Process leen F 15 Troubleshooting Flow Chart 00 0 00 ce ee eee eee F 15 Procedures for Troubleshooting the Client to Oracle Connection F 17 Introduction 2 1 ais es reu ei eer ets F 17 Troubleshooting from the Macintosh Client F 17 Troubleshooting from the Unix Oracle Server F 22 G License and Warranty cesses G I Software License and Warranty 0 cece eee eee eee G 1 Applied Biosyst
186. een windows 2 12 window callouts call outs described 2 15 to 2 17 window buttons 2 14 to 2 15 gel image adjusting 2 18 to 2 25 adjusting the contrast 2 20 to 2 22 changing dye indicators 2 19 to 2 20 displaying hiding a dye color 2 18 displaying hiding dye color 2 18 installing new matrix file 2 24 to 2 25 regenerating the gel image 2 23 to 2 24 removing contrast changes 2 22 Processing preferences setting 2 3 to 2 10 Auto Launch processing 2 4 comb type 2 10 displaying the Gel preferences 2 3 Image Generation Defaults 2 4 to 2 6 lane extraction 2 7 to 2 10 interpolating data 2 57 to 2 58 lane markers working with 2 43 to 2 52 off scale data displaying 2 38 to 2 41 sample sheet about 2 29 to 2 37 saving after editing tracking 2 68 procedure 8 4 tracker lines working with 2 53 to 2 56 tracking and extracting data 2 59 to 2 66 extracting without changing tracking 2 64 to 2 66 saving after tracking 2 68 specifying Run folder 2 59 tracking and extracting 2 61 to 2 63 tracking without extracting 2 60 to 2 61 Ge ways to track and extract 2 59 troubleshooting gel data D 5 to D 6 verifying gel information 2 26 to 2 28 why save 8 2 gel image adjusting 2 18 to 2 25 adjusting the contrast 2 20 to 2 22 changing dye indicators 2 19 to 2 20 displaying hiding a dye color 2 18 displaying hiding gel color 2 18 regenerating the gel image 2 23 to 2 24 removing contrast changes 2 22 printing 9 4 GeneScan about this manual 1 5
187. el file and remove the file from the hard disk For best performance keep as few files on your hard disk as possible Use magnetic tapes removable cartridge drives or optical disks to archive gel files Gel files are too big for diskettes 8 6 Saving Archiving and Copying Files Transferring Data to Other Applications Other Applications GeneScan files can be read by the following version of the Genotyper software for further analysis Genotyper software version Can read 1 0 Sample files from the GeneScan Analysis Software 2 0 both Sample files and project files Both programs can convert GeneScan Analysis Software data into formats used by downstream applications like the GenBase software GenoPedigree software and Linkage among others Cutting and To cut and paste tabular data Action Display the tabular data you want to copy Select the rows you want to copy by taking the following action If you want to select Then all tabular data choose Select All 3 A from the File menu several consecutive rows shift click the first and last row in the group you want to select several rows that are not listed next to each other click the rows Choose Copy from the Edit menu Open the new application and click where you want to place the information Pasting Tabular Data Step 1 2 3 4 5
188. elected lane by one channel You can take the following action Click the control point and drag or Use the Arrow keys Note Pressing the Option key while using the Arrow keys moves the control point in O 1 channel increments a lane segment Use the arrows to move the segment from one tracker line segment to the next segment You can take the following action To move Use the up one segment Up Arrow key down one line Down Arrow key segment 2 44 How to Process and Edit the Gel File Using Control When a gel is tracked the tracker places a tracker line with spline Points control points over the strongest signals for its lane Use the control points to control shape and position the splines When a control point is selected it appears as a filled in square The following table lists how you can use the control points If you want to Then move a single control point Clickthe control point and drag or Control point PS Use the Arrow keys altel vigi gs i viii 5 1153 Note Pressing the Option key while using the Arrow keys moves the control point in 0 1 channel increments select an entire row click one of the red triangles at the sides of the gel view Red triangle Use the Right or Left Arrow keys to i o 1 05 ceca move all the control points in that direction select mult
189. els Capability Gel File Contents How GeneScan Tracks a Gel File Topic See page How the GeneScan Analysis Software Names Sample Files 2 67 Saving Gel Files After Editing Tracking 2 68 The gel file which is generated only by the ABI 373 DNA Sequencer instrument the ABI 373 with XL upgrade the ABI PRisM 377 DNA Sequencer instrument and the ABI PRISM 377 with XL upgrade stores the raw data collected during the entire run of the instrument A gel file can be 10 MB to 60 MB The GeneScan Analysis Software can open and analyze 96 lane gels Initially the file contains the following Raw data collected during the run Gel image which is similar to an autoradiogram but in color Copy of the Data Collection software sample sheet Copy of the matrix file After lane tracking and editing the file also contains the lane tracking information and any changes you make to the original information in the file When the GeneScan Analysis Software tracks the gel file it creates a tracker line for each lane in the gel Each tracker line is trying to find the center of the bands not the brightest part of the band The tracker will only track if a matrix is attached to the gel file During data extraction the software generates a Sample file for each tracked lane by averaging the data from the tracked channel and the number of channels you specify on either side of it The software also copies to eac
190. ems Software License and Limited Product Warranty now ede REG aah eee a ed ieee eS RS G 1 CODYEIght sinners tes CR eI EATER RA ees AC EUER UY G 1 U1Cerse ioco trs etate es obese t tes tics duce tent s G 1 REStHICHONS ois er eee rupeten p BEER E En eee ae G 2 Limited Warranty sse G 2 Limitation Of Liability 00 0 ee eee eee G 2 TOT eps Seen cy ct ine Spe ving dite br a AER Oh abe eye E A E E G 3 Miscellaneous nex ete eR EE rure ee G 3 Glossary Index GeneScan Analysis Software Overview Introduction In This Chapter This chapter provides a general introduction to the ABI PRISM GeneScan Analysis Software It provides information about the organization of this manual and instructions on how to get help from Applied Biosystems Topics in this chapter include the following Topic See page What s New in the GeneScan Analysis Software 1 2 About This Manual and Other Instruments 1 5 Using the Macintosh Computer 1 7 Registering the Software 1 11 Installing the Software 1 12 GeneScan Analysis Software Files 1 16 Technical Support 1 20 Note Already familiar with previous versions of the GeneScan Analysis Software and want to know what is new and different in this version Turn to What s New in the GeneScan Analysis Software on page 1 2 GeneScan Analysis Software Overview 1 1 What s New in the GeneScan Analysis Software BioLIMS Support Fifth Dye Works with
191. end to use more than one database or user account enter an alias for this BioLIMS session information You can use an alias to connect to the database if no database connection is open Once you enter an alias you can take the following action If you click Then Open you connect to the database OK the changes are saved and you are connected to the database if a channel is open Use the pop up menu to add change or remove aliases If you have more than one alias click the Make Default check box to choose which one appears when you first open the Edit Session dialog box W Make Default Note The default alias is the database that opens if you choose to automatically analyze data Note If both the Make Default and the Save Password boxes are checked no dialog box will appear when a connection to the server is requested Since all the information required of the user has been saved the software will connect to the database automatically Using the BioLIMS Database E 15 E 16 To access the BioLIMS database continued Step Action 9 Take one of the following actions If the login was Then successful a Choose Open from the File menu The Open Existing dialog box appears b Click the Sample icon and the Collection Browser appears For more information see Using the Collection Browser Window on page E 20 unsuccessful an alert dia
192. ends on the instrument and run mode used Scan Current cursor position on the vertical scale Note These numbers change as you move the cursor over the image Lane Used Number of lanes used in the gel 2 Lane numbers Numbers across the top of the gel image show the lane number currently assigned to each lane on the gel How to Process and Edit the Gel File 2 15 Gel File window described continued No Description 3 Lane markers Diamond shaped markers 4 show the current status of each lane The following table lists the status values Lane color Description White Lane used and marked for extraction Blue Lane is used but not marked for extraction Yellow Lane was edited and extracted but the gel file was not saved with new information Gray The tracker does not expect to find a lane here if it does the tracker will confuse it and lane assignment confidence will be low Orange Lane was inferred by the Tracker software If you move or reshape an inferred lane tracker line it ceases to be inferred and the orange border is lost Control points Use to control shape and position the splines For more information see Using Control Points on page 2 45 Scroll bars Use to scroll the gel view horizontally or vertically Gel Image An image representing a time history of all fluorescence detected during the run Each peak is shown as a brightly colored ban
193. ensity within the lanes Lane assignment confidence values tend to be extreme numbers very low or very high Although a value of 70 or more generally indicates that the lane assignment for the gel is correct it is recommended that you check the tracker lane assignment anytime the reported lane assignment confidence value is less than 100 Comb Type IMPORTANT Before auto tracking the first time you need to verify that the comb type is set correctly The default comb type is Square The Neural Net Tracker uses special tracker setting files that are optimized according to the number of channels and lanes in the gel file and the comb type It is important that you set the correct comb type for the gel so that the Tracker applies the correct tracker setting files This value appears at the bottom of the gel window see Gel File Window Diagram on page 2 13 IMPORTANT The proper comb type needs to be selected in order to ensure proper tracker performance 2 10 How to Process and Edit the Gel File How to Display the Gel File Introduction Use the gel image to observe sample migration lane tracking and signal strength in the gel image You can also use the window to adjust the image to improve visibility re align individual lane markers or edit the position of the tracker lines Displaying the Gel You can display a gel file as follows File To display a gel file Then automatically after automati
194. ents have been checked for migration that is true to size under a wide variety of run conditions on all ABI PRISM instruments There are no anomalous fragments e g the 250 bp fragment in GeneScan 350 on the ABI PRISM 310 Genetic Analyzer Note GeneScan 400HD is the recommended size standard for use with the ABI PRISM Linkage Mapping Set Version 2 All aspects of the preparation of the GeneScan 400HD size standard are proprietary Each fragment contains a single ROX fluorophore The following table lists the lengths of the 21 fragments comprising the GeneScan 400HD size standard 50 160 260 360 60 180 280 380 90 190 290 400 100 200 300 120 220 320 150 240 340 Denaturing Although the GeneScan 400HD size standard is made of Electropherogram double stranded DNA fragments only one of the strands is labeled Consequently even if the two strands migrate at different rates under denaturing conditions you will not need to worry about peak splitting The following figure shows the peak patterns of GeneScan 400HD fragments run under denaturing conditions Fragments were run using the POP 4 polymer at 60 C o o 8 o o o o S 3 o t o o o o o x S An QA 9V 9 re oo a o N A a oO ae o or N A ape o ceo re o o o A Paf wi henas scd ha E hd Said S Nat eeu News fait Se Mi nate x ini oa Mutin Non denaturing The following shows the peak patterns of GeneScan 400HD fragments Electroph
195. enu page 5 41 6 Select the Print Results checkbox to print the results automatically For information on print set up see Specifying the Format for Printed Results on page 5 9 7 Click Analyze If a field Then contains a small triangle see the figure below you have analyzed the samples using the currently selected size standard and analysis parameters does not contain a triangle the sample has not yet been analyzed with the current settings O Print Results Sample File 2950 Lane Demo Sample Size Standard 50 lane Demo 6el 7 Analysis Control i Print Setup Parameters s nalysis Parameters S50 Lane Demo Sample Analysis Parameters gt 4050 Lane Demo Sample Analysis Parameters 15950 Lane Demo Sample 16950 Lane Demo Sample Analysis Parameters 7 50 Lane Demo Sample lt nalysis Parameters a a a Analysis Parameters a a woes a ctnaiysis Parameters Created Sat Jun 1 1996 11 53 AM Triangles indicate samples were analyzed with the current setting To analyze Sample files continued Step Action 8 The triangle disappears if you Selecta different size standard or analysis parameters file for a dye sample Modify the currently selected size standard or analysis parameters file or the analysis parameters stored as program pre
196. eparing Matrix Standards for the ABI 373 and ABI PRISM 377 Introduction Use the following procedure to prepare matrix standards for the following instruments ABI 373 ABI PRISM 377 ABI 373 with XL Upgrade ABI Prism 377 with XL Upgrade Procedure To prepare the matrix standards for loading Step Action 1 Set up a run with the instrument conditions you want to duplicate Run conditions depend on the following Instrument system you are using ABI PRISM 377 or ABI 373 Kind of experiment that you are running Select the four Dye Matrix Standards one for each color from the Dye Matrix Standard kit that you are using If you are using Then each dye that you use ABI PRISM 310 or must be recognized as a different color by ABI PRISM 377 the single virtual filter ABI 373 must be recognized by a different filter on one filter wheel Depending on the instrument system use the table below to prepare dye matrix standards for loading Table of Matrix The following table lists the matrix standards to prepare for each Standards instrument ABI PRISM 377 and with ABI 373 with XL For XL Upgrade ABI 373 Upgrade Denaturing Combine 2 5 uL of each Combine 2 5 uL of each Combine 2 5 uL of gels standard with 1 5 uL standard with 2 5 uL standard with 2 5 uL of Loading buffera Loading buffer deionized formamide Heating Heat each sample at 95 C for fi
197. er PowerPC CPU You will benefit from using the fastest computer available CD ROM Drive Any Operating System Mac OS version 8 0 with Open Transport 1 1 or later Disk Space A minimum of 15 MB free disk space Storage requirements depend primarily on the quantity of data to be generated and stored Sample files are approximately 150 250 KB each and gel files are 20 70 MB each It is common to store many Sample files on the analysis computer Gel files are usually stored on the computer that is connected to the instrument and are removed or archived frequently Memory RAM The minimum memory requirement is 32 MB total with at least 20 MB of this available to run the GeneScan Analysis Software Virtual Memory Virtual memory MUST be turned on and set to 16 MB Set the Memory control panel so that the memory available after restart is at least 48M For information on how see Turning On Virtual Memory on page 1 13 If your computer has 48 MB of physical RAM virtual memory does not need to be turned on Monitor A 17 inch monitor or larger is recommended Although a monitor of 640 x 480 resolution can be used you will benefit from having a monitor of higher resolution disk drive Hard disk with a minimum of 250 MB storage 1 Call technical support for the latest system specifications 1 12 GeneScan Analysis Software Overview Item Specifications
198. er defined analysis parameters files if you create your own files GS Standards folder Use for storing size standard files when you define them GS GelTracker folder GS Gel Tracker program tracker settings file and extensions You may also see a set of 20 to 30 matlab text files You can ignore these files if you throw them away new ones are created when you next track a gel Note Advise keeping the files because they are useful for troubleshooting auto tracking problems Note Do not change the name of the folder the location of the folder or the files in the folder GS Matrix folder Use for storing the matrix file generated using the GeneScan Analysis Software For more information about matrix files see Chapter 6 Making a Matrix File GeneScan Analysis Software Overview 1 15 GeneScan Analysis Software Files Table of Files The following table lists the files that the GeneScan Analysis Software reads writes and in most cases creates The software does not create gel files and creates Sample files only through lane extraction from a gel For information on the files installed by the GeneScan Analysis Software see Program Files Installed Diagram on page 1 15 GeneScan Analysis Software files File type Location on 373XL ABI PRISM 377 377XL 377 96 Lanes or ABI PRISM 310 ABI 373 Description GeneScan Analysis Software GeneScan 3 0 folder GeneScan 3 0 folder The Ge
199. er in the System folder If itis missing or disabled the GeneScan Analysis Software crashes when connection to the database is attempted Click the Interfaces File button to open a browser box Use the browser box to locate and select the interfaces file that you located in step 1 above Confirm that the correct default server is chosen Use the Default Server pop up menu to select the default server Note This is the same server you named for SYBASE in the BioLIMS Server entry table immediately above Confirm that the Default Language pop up menu is set to us english F 8 Troubleshooting the BioLIMS Database Troubleshooting the connection from the client Macintosh computer continued Step Action 5 Use the program SybPing to confirm communication with the Sybase based BioLIMS SQL Server The default installation places SybPing into the BioLIMS BioLIMS Extras Sybase bin folder Step Action a Start the SybPing program b Select the server from the Servers pop up menu The servers shown in the pop up menu are the servers listed in the interfaces file C Click Ping The program responds with a message of whether or not the Ping was successful If the Ping is Then successful skip to Troubleshooting the client connection from the Sybase SQL Server on page F 12 unsuccessful continue to step 6
200. erified the tracking and taken any required corrective action Procedure To save selected information about a gel file Step Action 1 Select Save As from the File menu The Save As dialog box appears Cp GeneScan Software c Hard Drive Analysis Log m Eject FJ GELO1 980120 15 EJ GeneScan Application C3 GSGelTracker folder Run 2941 01 980120 15 Desktop Save Gel As GELO1 980120 15 copy File Format CSI Gel without Image PICT File 2 Select a location and type in a name for the file The default file name is the original file name with copy added 8 4 Saving Archiving and Copying Files To save selected information about a gel file continued Step Action 3 You have the following options from the pop up menu File format Description Gel file Saves the entire gel file into the specified folder This includes the image raw data and other collection information Gel without image Saves all of the information in the gel file excluding the image Storage requirements are about one third less than the entire gel file When this gel file is later opened again in the analysis software the image will be regenerated PICT file Creates a PICT file from the gel image This PICT file can not be opened in the analysis software Saving Results You can combine electropherograms and ta
201. erogram run under non denaturing conditions Fragments were run using 396 GeneScan Polymer GSP at 30 C EIE Native3 Beach 7 Display 6 3r L E 2000 2400 2800 3200 3600 m ha h mE 1R 01 Sample File ry x 2367_ 5393 e m gE GeneScan Size Standards B 5 GeneScan 500 Size Standard What To It Use For How It Is Prepared GeneScan 500 GeneScan 500 is a size standard useful for sizing fragments between 35 and 500 base pairs The native fragments are uniformly spaced to provide accurate base calling GeneScan 500 is prepared by Pst 1 digestion of plasmid DNA followed by ligation of a TAMRA or ROX labeled 22 mer oligodeoxynucleotide to the cut ends A subsequent enzymatic digestion with BstU 1 yields DNA fragments containing a single TAMRA or ROX dye see GeneScan 350 Molecular Lengths below The following table lists the GeneScan 500 Denatured Fragment Molecular Lengths Molecular Lengths Nucleotides 400 250 139 35 450 300 150 50 490 340 160 75 500 350 200 100 400 250 139 35 Running Under The following table describes running the GeneScan 500 standard Denaturing under denaturing conditions Conditions B 6 GeneScan Size Standards Like However Consequently the GeneScan 2500 and GeneScan 1000 standards the GeneScan 500 standard is made of double stranded DNA fragments with GeneScan 500
202. es Opening an To open an existing project Existing Project Step Action 1 Choose Open from the File menu The Open Existing dialog box appears For information on opening a Sample file from the BioLIMS database see Switching Between Sample File and BioLIMS Mode on page E 10 Open Existing of T ful Collection Project Sample Sample Analysis Size Gel Sheet Parameters Standard 2 Click the Project icon A directory dialog box appears 3 In the dialog box find and select the project you want to open 4 Click Open The project opens in an Analysis Control window Creating a New Proj oject To create a new project Step Action 1 Choose New from the File menu The Create New dialog box appears Create Neu 13 598 fw fu Project Sample Analysis Size Sheet Parameters Standard Cancel 4 10 Creating a Project To create a new project continued Step Action 2 Click the Project icon An untitled Analysis Control window appears For information on using the Analysis Control window see page 5 4 3 There are two ways to add files to a project To add Choose sample files you select to the open project Add Sample Files 38 B The Add Sample dialog box appears see Using the Add Sample Dialog Box below a sample currently open to the open project Add file name U
203. esults D 2 to D 4 U unlocking sample files 4 12 V vertical scale changing 7 38 to 7 39 for all electropherograms 7 38 for single electropherograms 7 39 viewing scale changing for electropherograms 7 31 W warranty G 1 to G 3 well to read WTR defined Glossary 4 www address Documents on Demand Applied Biosystems 1 20 1 23 X x axis positions displaying in electropherograms 7 27 Y y axis positions displaying in electropherograms 7 27 Z zooming electropherograms 7 31 Index 7 Worldwide Sales Offices Applied Biosystems vast distribution and service network composed of highly trained support and applications personnel reaches into 150 countries on six continents For international office locations please call our local office or refer to our web site at www appliedbiosystems com Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free 1 800 345 5224 Fax 1 650 638 5884 www appliedbiosystems com AR Applied NP Biosystems PE Corporation is committed to providing the world s leading technology and information for life scientists PE Corporation consists of the Applied Biosystems and Celera Genomics businesses Printed in the USA 10 2000 Part Number 4306157B
204. et correctly Open the Set Oracle Home program At installation this program is placed into the BioLIMS 2 0 BioLIMS Extras Oracle Applications folder Use the file browser to locate and select the Oracle folder F 18 Troubleshooting the BioLIMS Database Troubleshooting the connection from the client Macintosh continued Step Action 4 Confirm that the Oracle library files are installed See Required Oracle Extension Files on page F 11 5 Use the program NetTest to confirm communication with the Oracle Server At installation NetTest is placed into the BioLIMS 2 0 BioLIMS Extras Oracle Applications Networking folder Step Action a Open the NetTest program Note No window appears for the NetTest program but the menu bar changes b From the Database menu choose Logon The Connect To Database dialog box appears mM Connect To Database Username Password Database Save As Default C d Enter the Username and Password for the database Note These are the same Username and Password you use to log into BioLIMS e g george george1 e In the Database field enter the alias name of the Oracle Server from the tnsnames ora file e g Oramozart f Click Login The message Attempting Connection appears then the Result Explanation dialog box appears Troubleshooting the BioLIMS Database F 19
205. ewly generated files If you deselect this checkbox a number is appended to the name for each newly generated file and the original files are preserved In BioLIMS mode the file name must be unique If the file name is not unique then the sample file will not be written to the database How to Process and Edit the Gel File 2 65 To extract the data without changing current tracker lines continued Step Action 3 In the Project File portion of the dialog box you can take the following action Choose To Add Sample Files to Project checkbox Create a new project choose Create a New Project from the pop up menu Use an open project choose Use the Open Project from the pop up menu Auto Analyze New Sample Files checkbox automatically analyze the new Sample files after extraction is finished Deselect this it you want the Sample files extracted but not analyzed This option is only available if the Sample files are being added to a project Analyze All Files radio button analyze all the new Sample files created from the gel Print Results checkbox print the results for all new Sample files after analysis Use Sample Sheet Settings checkbox analyze and print the Sample files as designated in the Sample Sheet Save Gel After Extraction checkbox save tracker lines and other gel file settings to the gel file afte
206. facility then Applied Biosystems will do one of the following at its sole option i correct the error in a subsequent release of the SOFTWARE which shall be supplied to you free of charge or ii accept a return of the SOFTWARE from you and refund the purchase price received for the SOFTWARE Applied Biosystems does not warrant that the SOFTWARE will meet your requirements will be error free or will conform exactly to the documentation Any sample or model used in connection with this Agreement is for illustrative purposes only is not part of the basis of the bargain and is not to be construed as a warranty that the SOFTWARE will conform to the sample or model EXCEPT AS SPECIFICALLY STATED IN THIS AGREEMENT THE SOFTWARE IS PROVIDED AND LICENSED AS IS THE ABOVE WARRANTY IS GIVEN IN LIEU OF ALL OTHER WARRANTIES EXPRESSED OR IMPLIED INCLUDING THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE NOTWITHSTANDING ANY FAILURE OF THE CENTRAL PURPOSE OF ANY LIMITED REMEDY Applied Biosystems LIABILITY FOR BREACH OF WARRANTY SHALL BE LIMITED TO A REFUND OF THE PURCHASE PRICE FOR SUCH PRODUCT IN NO EVENT WILL Applied Biosystems BE LIABLE FOR ANY OTHER DAMAGES INCLUDING INCIDENTAL OR CONSEQUENTIAL DAMAGES EVEN IF Applied Biosystems HAS BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES Term Miscellaneous You may terminate this Agreement by destroying all copies of the SOFTWARE and documentation Applied Biosystems may terminat
207. ferences Specify a different dye color as the standard move the diamond indicator to another dye sample Assign a new matrix to the Sample file 9 To verify the results refer to Process of Verifying Results on page 7 45 Specifying the Format for Printed 1 primate To specify the format for printed results Results Step Action 1 In the Analysis Control window select the checkbox labeled Print Results When this checkbox is selected the Print Setup button becomes active 2 Click the Print Setup button The Auto Print Setup dialog box appears see below All the dyes selected for analysis are also selected for printing Analyzing Project Files 5 9 5 10 Analyzing Project Files To specify the format for printed results continued Step Action Sample Dye color fields Ruto Print Setup LH menm 0 Overlaid Tiled Sample Information Display field 3 Moving the cursor over a Sample file name or over a dye color field a notation appears in the Sample Information Display field 4 Click the sample dye color fields to specify any sample you do not want to print see the above figure To specify the format for printed results continued Step Action right of the window 5 Choose the format by clicking either or both of the buttons at the Click this button To print
208. g Topics See page Automatic Analysis and Project Creation Process 4 2 Setting Up for Automatic Analysis 4 4 Using a Project to Manage Sample Files 4 8 Working with Project Files 4 10 Finding Missing Sample Files 4 14 Creating a Project 4 1 Automatic Analysis and Project Creation Process Introduction Process Using the ABI PRISM 310 The following two diagrams illustrate the process of automatic analysis and project creation for data from the ABI PRISM 310 and from the ABI 373 and ABI PRISM 377 instruments The following diagram illustrates data analysis using the ABI PRISM 310 instrument Run folder Add Sample file Add Sample file with 1 to Run folder 2 to Run folder analyzed a Sample files Collecting F E ponpon wel injection 1 mn gt Add Sample Start NEW Project na file Analyze amp Data GeneScan Sample file Print i Analyze amp Print Collection f Idle and Analysis i gt completed IH m ue file 1 Create project and Add reference to Project with add reference to Sample file 2 all Sample file 1 to project references 4 2 Creating a Project Process Using the The following diagram illustrates data analysis using the ABI 373 and ABI 373 and the the ABI PRISM 377 instruments ABI PRISM 377 Add Add Run folder re i Sample file with Gel file is created in the Sample file Run folder during data gel 1 to Run N to Run analyzed collection file folder
209. g esce orate ce ti ew eee ETS we 6 27 Generating Sample Files 0 00 0c eee eee eee 6 27 Choosing a Scan Range for the Matrix Calculation 6 29 Introduction os edere epis e geni sese 6 29 What to Look For in the Raw Data Display 6 30 Choosing a Scan Range 00 cece cee eee eee 6 30 Eliminating Primer Peaks 6 31 Generating a New Matrix File 0 0 0 eee eee 6 32 Procedure vss ere EHE cee Sw Geese be wes ERR 6 32 Matrix File Example 0 0 02 eee eee eee eee 6 33 Saving and Naming the Matrix File eee 6 34 Introduction oves ect nage ER HUN E eRe He ORNS 6 34 Naming Considerations 0 0 0c eee eee eee eee ee 6 34 Saving the Matrix File 00 0 2 eee ee eee 6 34 Where to Store the Matrix File 0 00 6 34 When to Make Copies of a Matrix 00 00 00 005 6 34 Assigning the Matrix File to Sample Files 000 6 35 Introduction resur eer tyes Shed ee eme xe 6 35 lene UCM Em 6 35 Evaluating the Matrix File 0 0 eee ee ee eee ee 6 37 Introduction oder reU ERE ER 6 37 Procedure sis iuair rine ren t PS Bet ER ee hae tie 6 37 Causes for Bad Matrix Files 0 0 0 eee eee eee 6 38 If an Error Message Appears 00 0002 eene 6 38 Two Causes of Bad Matrix Files 00 00 0000 6 38 Introduction soaa e 2 laa ced waste ER Pe sent Cre mE wae dened 7 1 In This
210. g and dragging Choosing commands Using pull down and pop up menus dialog boxes radio buttons and checkboxes Working with windows Opening and closing resizing and repositioning scrolling understanding the active window Using the Macintosh computer Finding files and creating folders hierarchical file system If you do not understand some or all of these refer to the Macintosh System Software User Guide or the Owner s Guide for more information Computers require regular attention and maintenance to operate efficiently and consistently The ABI PRISM software works with large files and accesses the hard disk often so it is especially important that you follow maintenance procedures to minimize the occurrence of errors during operation Follow these general guidelines for optimal performance Install only one Macintosh computer system per hard disk Back up all programs and files regularly Remove nonessential files from the hard disk Use a hard disk maintenance program regularly 9 9 9 Use discretion when adding software programs especially Control Panel CDEV and Extension INIT files Refer to the AB PRISM Instrument User Manual for more detail GeneScan Analysis Software Overview 1 7 Macintosh The following Macintosh computer terms are used frequently in this Computer Terms manual to describe how to use GeneScan Analysis Software Used in This Macintosh computer terms used in
211. g between Sample file and BioLIMS mode E 10 to E 11 testing network connection using tool E 12 troubleshooting client to Oracle connection F 14 to F 24 client to Sybase connection F 3 to F 13 Index 1 GeneScan crashes with BioLIMS D 9 if BioLIMS Preference page does not appear F 2 using the BioLIMS Chooser E 20 to E 30 C channels defined Glossary 1 colors assigning to electropherograms 7 39 defining in electropherograms 7 40 to 7 43 computer using the Macintosh 1 7 to 1 10 configuring the BioLIMS server E 4 to E 9 copying data to other applications 8 7 to 8 8 crashes GeneScan crashes with BioLIMS D 9 Cubic Spline method C 4 customer support e mail address 1 24 help 1 20 to 1 26 internet address 1 20 regional sales offices 1 24 to 1 26 telephone fax U S 1 20 D Data Collection software automatically printing results from 9 3 Gel File window how differs from GeneScan Gel File window 2 12 data point defined Glossary 1 Deionizing formamide procedure 6 9 Documents on Demand 1 23 dye color changing in electropherograms 7 28 Dye Matrix Standards kits 6 8 dye scale changing 7 44 to 7 45 E electropherograms about 7 20 to 7 21 changing dye scale 7 44 to 7 45 defining custom colors 7 40 to 7 43 displaying electropherogram data 7 25 to 7 26 displaying electropherograms and tabular data 7 22 to 7 24 using to verify peak detection 7 53 using to verify results 7 46 to 7 47 verifying size calculations 7 48 to
212. h new Sample file all the information required to identify and analyze the sample 2 2 How to Process and Edit the Gel File How to Set Gel Processing Preferences Introduction Displaying the Gel Preferences Dialog Box Use the Gel Processing dialog box to define the parameter values to use for gel file processing The following procedure applies to the ABI 373 and the ABI PRISM 377 instruments To display the Gel Preferences dialog box Step Action 1 If the GeneScan Analysis Software is not running then double click the program icon 2 Choose Gel Preferences from the Settings menu The Gel Preferences dialog box appears Gel Preferences Auto Launch Processing K Auto Track Gel s Extract Lanes after Auto Tracking Image Generation Defaults Scan Range Stop 100000 Start DJ Multicomponent Gel Image Estimated Maximum Peak Height 2000 Lane Extraction L1 Use Weighted Averaging Use s Channel Averaging o Pre Averaging Offscale Detection K Stop extraction when below confidence threshold Confidence Threshold Z Comb Type Sharks Tooth Square Tooth Cancel There are three Gel Preferences parameters Auto Launch Processing Image Generation Defaults Lane Extraction How to Process and Edit the Gel File 2 3 Auto Launch Select or de select the checkboxes to specify whether or not the gel is Processing to be au
213. hat corresponds to the number of dyes contained in your gel file and click OK An untitled Sample Sheet appears See Sample Sheet Example on page 2 31 2 30 How to Process and Edit the Gel File About the Sample Sheet Sample Sheet The following is an example of a Sample Sheet for DNA fragments Example labeled with a blue dye and the GeneScan 350 Rox Internal Lane Size Standard 0 0 000000 0 EI HERE qel fite Sampte Shedt File Name f Sample Name Dye f Sample Info f Comment i i tollection Name f Collection Comment f Collection Owner Sample Sheet The following table describes the above diagram Described Sample Sheet described No Description 1 Lane number for the sample GeneScan Analysis Software assigns lane numbers to the gel file lanes based on the numbers in this column If a lane in the gel is empty a corresponding empty row must appear in the Sample Sheet How to Process and Edit the Gel File 2 31 Sample Sheet described continued No Description 2 Select this checkbox to mark the corresponding lane in the gel image as Used When the GeneScan Analysis Software extracts the sample information from the gel file it creates Sample files only from lanes marked Used Note Use the Fill Down command from the Edit menu to check all lanes as being
214. he curve is black in the Standard Sizing Curve window ELI 11 P27 11 Best Fit 2nd Order Curve AQ 9 771623E 01 j Al 1 220838E 01 A2 4 856885E 06 R 2 1 000 Size Calling Curve 2nd Order Least Squares T T T T T 2500 3000 3500 4000 4500 Data Point T T 1500 2000 T 1000 Figure C 1 2nd Order Least Squares size calling curve 119P27 11 Best Fit 3rd Order Curve aso AO 1 266506E 02 Al 1 608974E 01 A2 2 041442E 05 AZ 1 887158E 09 R 2 1 000 300 250 200 150 100 50 Size Calling Curve 3rd Order Least Squares T T T T T T T T 1000 1500 2000 2500 3000 3500 4000 4500 Data Point BULAN o Figure C 2 3rd Order Least Squares size calling curve Size Calling Methods C3 Cubic Spline Interpolation Method About This Method Possible Local Sizing Inaccuracy C 4 Size Calling Methods By definition the Cubic Spline method forces the sizing curve through all the known points of the selected GeneScan size standard Although this produces exact results for the values of the standards themselves it does not compensate for standard fragments that may run anomalously Em 11eP2 11 Best Fit 2nd Order Curve AO 9 771623E 01 Al 1 220838E 01 A2 4 856885E 06 R 2 1 000 Size Calling Curve Cubic Spline Interpolation T T T 7 7 T
215. he Gel Image Introduction Although the data shown in the gel image is not analyzed the displayed information allows you to evaluate the quality of the run You can adjust the appearance of the gel image as follows Displaying Hiding a Dye Color Regenerating the Gel Image using a different scan number range estimated maximum peak height and multicomponenting option Changing Dye Indicators Adjusting the Contrast Installing New Matrix Information Note change the raw data contained in the gel file Displaying Hiding Introduction a Dye Color Changing any of the above features in the Gel File window does not You can control the display of specific fluorescent dye colors For example if you want to display only the bands labeled with blue dye you can suppress the display of all green yellow or red bands How to Display or Hide a Dye Color Display or hide by a color by Default Comment clicking the colored boxes near the top left corner of the Gel File window All colors displayed Any change you make in the button settings is saved in the gel file and used the next time that the file is opened 2 18 How to Process and Edit the Gel File Changing Dye The following procedure describes how to change the dye indicator Indicators colors You may want to change the dye colors to make the colors easier to distinguish For information on changing the default dye
216. herogram Panels as follows Choose To Use Common Vertical Scale set all panels in a display so checkbox they have the same vertical scale The common scale is based on the electropherogram with the largest vertical scale Panel Height Resize Limits set minimum and maximum values for electropherogram panel height in the results display This allows you to limit how much the electropherogram panels stretch or shrink to fit the size of the window To change how results are displayed and printed continued Step Action 4 Set the Printing Preferences as follows If Then the height of the panels that choose the As Shown on appears on screen is Screen radio button acceptable you want to print the click the Fixed at radio button electropherogram at a and enter a value in the field specified height you want to force a page break select the Page Break before after the electropherograms Tabular data checkbox have been printed 5 Click OK Evaluating Analysis Results 7 15 The Sample Results View About the View The Sample Results view is displayed within a Sample file window You can access the window through a Sample file or through a project s Analysis Control or Results Control window If you are opening a Sample file as a stand alone file the Sample Results view is the default display within the Sample file window For Mor
217. hese characteristics see Causes for Bad Matrix Files on page 6 38 for possible interpretations of your peak data To choose a scan range Step Action 1 Move the cursor well away from the primer peak in a region at the beginning of the run and in a flat part of the baseline and record the scan numbers Note When choosing the start point do not include primer peaks in the scan range see Eliminating Primer Peaks on page 6 31 Also the region for both the start and stop points should be flat points at the baseline Step Action 2 Record the data point values for both the start and stop points of the scan range You will need to enter those values in the next step when generating the new matrix file see page 6 32 3 Close the raw data boxes and the project by clicking in the upper left hand corner of the window Note Holding down the Option key while clicking in the upper left hand corner of the window will close the windows simultaneously Eliminating Both the primer peaks and the data peaks are displayed when viewing Primer Peaks the raw data of your matrix standards Anytime you run dye labeled samples on a gel ABI 373 and ABI PRISM 377 or capillary ABI PRISM 310 you always have excess dye labeled primer in the reaction The primer peak displays as the first peak usually off scale because it is in molar excess Eliminate the primer peak when making a matrix by c
218. hing Between Sample File and BioLIMS Mode E 10 Int OdUuCtlOn eee qt rp eb e e E 10 Switching Modes sseeeeeeee ee E 10 How to Access the BioLIMS Database llle esee E 12 Introduction er ELS ERU S ER ERES PES IRE E 12 Before Accessing a Sybase Database lesse esse E 12 Accessing the BioLIMS Database E 13 About Server Names mo aieea ri enei e A nnn E 17 How Names are Recognized 0 02 cece eee eee eese E 17 Sybase SQL Server Examples 0 00 00 00 000 E 17 Oracle Server Examples eirioed enere iae o cece eee eee eee E 18 Using the Collection Browser Window 00 0000 2 eee E 20 Applications that Use the Collection Browser Window E 20 Ways to Search the Database 00 0 0 ee ee eee E 20 Tn This Sectii 4 i ees kRDEeHEO ER TION E SERO RESP OPER E 20 Displaying the Collection Browser Window E 21 Collection Browser Window Example 4 E 22 Parts of the Collection Browser Window E 22 Collection Search Criteria eese E 24 Fragment Search Criteria 0 0 0 eee eee eee eee E 25 Searching the BioLIMS Database llle eese E 28 F Troubleshooting the BioLIMS Database F 1 Inttoduction ost reset f ESPERE EIER ae aes Hn ele RES F 1 In This Appendix si ceu eere Lb AE EU E eR F 1 If the BioLIMS Preference Page Does Not Appear suus F 2 Problemas ve gh
219. hoosing the start point after the primer peak in a flat area with a stable baseline Making a Matrix File 6 31 Generating a New Matrix File Procedure To generate a new matrix file 6 32 Making a Matrix File Step Action 1 Choose New from the File menu The Create New dialog box appears Click the Matrix icon The Make New Matrix dialog box appears Make New Matrix Select the Matrix Standard Sample Files Number Of Dyes No File Selected for Data Start At No File Selected for G Data Start At No File Selected for Y Data Start At No File Selected for R Data Start At Points 100000 Choose the number of dyes from the Number of Dyes pop up menu If 5 dyes are selected a button is added to the bottom of the list The B G Y and R buttons represent dye colors a Click a button to display a pop up menu b Use the pop up menu to access a Sample file to link to each of the four dye labeled primers c Choose the Sample file that represents the dye color for that button Enter the start point that you determined when choosing a scan range in the Start at field See Choosing a Scan Range on page 6 30 Enter the total number of data points to include to calculate the matrix in the Points field Note You must have at least five peaks to make a matrix In most cases leave the default value unless you must exclude a
220. ide Selected Rows 3 H from the View menu Limited the Rows To limit the display to the selected rows of data Displayed 7 24 Evaluating Analysis Results Step Action 1 Select the rows you want to display 2 Choose Show ONLY selected Rows 36 G from the View menu Note Choose Show All Rows 36 G from the View menu to display all of the tabular data after limiting the display Displaying Electropherogram Data Definition Each electropherogram provides a profile of the selected dye samples it represents The y axis represents the relative fluorescence of the detected fragments as they occurred over time The x axis represents time and can be displayed by data points or base pairs Base Pairs Versus You can choose to have the horizontal tick marks on the x axis Data Points represent size in base pairs instead of data points only if you ran an internal size standard with the sample see Showing Data by Fragment Size on page 7 35 Procedure To display electropherogram data Step Action 1 Choose Results Control 3 2 from the Windows menu to open the Results Control window Click the electropherogram icon If applicable select the number of electropherogram panels Select the dye samples for display c1 5 OJIN Click Display The electropherogram is displayed in the Results Display window Evaluating
221. ifferent level Multicomponent checkbox Use to specify that the GeneScan Analysis Software apply a predefined matrix to adjust for spectral overlap when it performs analysis Although the dyes used to label DNA fluoresce at different wavelengths the spectra overlap to some extent Create a matrix file containing a mathematical matrix to correct for this overlap For a description of matrix files and how to create them see Chapter 6 Making a Matrix File Analyzing Project Files 5 19 Item Description Smooth Options Use to help reduce the number of false peaks detected by the GeneScan Analysis Software You have the following options Select To None apply no smoothing Select this option if the data has very sharp narrow peaks of interest Light provide the best results for normal data Heavy apply to data from slower runs that has very broad peaks Choosing this option might reduce peak size or eliminate narrow peaks Peak Detection About the Peak Detection Parameter Options Parameter Options Use the Peak Detection parameter options to specify the minimum peak 5 20 Analyzing Project Files height to be detected for analysis This in turn controls the number of peaks analyzed Peaks falling below the parameters specified appear in the electropherogram but are not analyzed and no values appear for them in the tabular data Peak Detectio
222. ile browser to locate and open the interfaces file edited in the steps above Set the Default Language pop up menu to us english 10 Close the SybaseConfig control panel Configuring for Oracle Server Connection Follow the steps below to configure your Macintosh computer for connection to the Oracle Server IMPORTANT Any time you change the BioLIMS database server name or its IP address or host and domain name you need to repeat this procedure To configure for Oracle Server connection Step Action 1 Use the program Easy Config to configure for the Oracle Server connection At installation Easy Config is placed into the BioLIMS 2 0 BioLIMS Extras Oracle Applications Networking folder Open the Easy Config program The Remote Databases window appears Remote Databases H Currently Defined Aliases Oramozart Use this tool to Oraneuron deo samplel SQL Net v2 sample2 product sample3 i 2 Click New The Protocol dialog box appears m Protocol What networking protocol do you want to use with this alias TCP IP Q AppleTalk Cx Using the BioLIMS Database E 7 E 8 Using the BioLIMS Database To configure for Oracle Server connection continued Step Action 3 Select TCP IP and click OK The TCP IP dialog box appears TCP IP Alias Name Server Name Or IP add
223. ing more than 3 uL produces too much signal Any amount that results in a signal over 4 000 FU s is too strong Which lanes to load with the dye matrix standards For gel electrophoresis load the matrix standards with an empty lane between each sample to avoid contamination of the individual dyes by residual material leaking adjacent samples After generating a gel image for ABI 373 and ABI PRISM 377 instruments check that the tracking of the gel file is adequate Where to Store Store matrix files intended for use by Data Collection software in the Matrix Files ABI folder The ABI folder is located in the System folder on which the 6 6 Making a Matrix File Data Collection software is installed If Data Collection and Analysis are Performed on Different Computers Make a copy of a matrix and store as follows This is useful when data collection and analysis are performed on different computers Store a copy in the For use by the ABI Folder Data Collection software GS Matrix Folder GeneScan Analysis Software Note The ABI PRISM instrument Data Collection software uses the files installed by the GeneScan Analysis Software in the ABI folder When you run the analysis software the program also creates several files such as a Preference file and an Analysis Log Process of Creating a New Matrix File Process Diagram The following diagram shows the proc
224. ing the Matrix File Introduction Procedure After creating a new matrix file and assigning it to select Sample files the next step is evaluating the quality of the matrix file The quality of the matrix files has a direct impact on the quality of the results data To evaluate the matrix file Step Action 1 Analyze the Sample files used to make the matrix 2 Display Results data for all four Dye Matrix Standard Sample files on one screen showing only electropherogram data 3 For each displayed Sample file You should see If not that the only visible peaks represent the color of the Dye Matrix Standard run in that lane or for that injection ABI PRISM 310 all other lines should be relatively flat you probably have a bad matrix file For instructions on how to This indicates that the matrix identify and correct problems properly compensated for the with bad matrix files see spectral overlap Causes for Bad Matrix Files For example for the blue on page 6 38 matrix standard Sample file you should only see blue sharp well defined singularly colored peak data Making a Matrix File 6 37 Causes for Bad Matrix Files If an Error If an error message appears the following table lists two possible Message Appears causes For this cause Take this action designated the wrong files reassign the matrix files See Assign
225. ing the Matrix File to Sample Files on page 6 35 signal is too weak to make a matrix rerun the matrix standards Loading and Running Matrix Standards on the ABI 373 with XL Upgrade on page 6 24 Two Causes of Bad The following table lists two common causes of bad matrix files Matrix Files Problem Cause What to do Artifact peaks of Loading too much dye Complete another run different colors under when running matrix and recreate the the true peaks standards resulting in matrix See the figure below dye bleed through Wrong Matrix 4B A11 Test 24 2 7 LINE 4 A11 Test2 2 Ye 46 A11 Test 24 2 7 4R Al1 Test 24 2 6 38 Making a Matrix File Problem Cause What to do Noisy baseline If the matrix subtracts Compete another too much of a matrix run and make particular color from sure that you do not the sample then the have any off scale baseline may become data too elevated resulting in false peaks See the figure below Gel File rg 80 100 120 140 150 180 200 220 240 260 280 300 320 340 360 380 BB 11 test 2 test 2 EH 6G 11etest 2 test 2 CUM SY 11etest2 test 2 BE GR 11etest2 test2 Ns Dye Sample Minutes Peak Height Peak Area Data Point Peak Making a Matrix File 6 39 Evaluating Analysis Results Introduction
226. ioLIMS Database Example 2 If the interfaces file contains this Offenbach query MacTCP mac ether mozart apldbio com 2500 The Session Manager would look like this Username jane Password O Save Password Database Server In order for Offenbach to be recognized as a Sybase SQL Server the name in the Server field is suffixed with S Example 1 If the tnsnames ora file contains this Oramozart DESCRIPTION ADDRESS PROTOCOL TCP host mozart port 1521 CONNECT DATA SID WG733 Oramozart is recognized as an Oracle Server because the server name begins with O The Session Manager would look like this Username Password O Save Password Database Server Example 2 If the tnsnames ora file contains this SIBELIUS DESCRIPTION ADDRESS PROTOCOL TCP host SIBELIUS port 1521 CONNECT DATA SID WG733 The Session Manager would look like this Username jane Password O Save Password Database Server SIBELIUS 0 In order for SIBELIUS to be recognized as a Oracle Server the name in the Server field is suffixed with O Using the BioLIMS Database E 19 Using the Collection Browser Window Applications that Use the Collection Browser Window Ways to Search the Database In This Section Collection Browser window is common to the following BioLIMS aware applications AutoAssembler DNA Sequence Assembly Software GeneScan Analysis Software Factura Fea
227. ions 5 20 Size Call Range Parameter Options 5 21 Size Calling Method Parameter Options 5 21 5 22 Split Peak Correction Parameter Options Analysis Range The following are the Analysis Range parameter options Parameter Options Item Description Full Range radio button Use to analyze all the data collected on the genetic analysis instrument for each sample This Range Data Points radio button Enter Start and Stop data point numbers in the entry fields in order to specify only a limited range analyzed for each sample This affects what is displayed in the results display Normally set the analysis range to start after the primer peak Note Sample files generated from ABI 373 or ABI PRISM 377 may have already removed the primer peak This was done by setting the scan range for gel image generation to exclude the primer peak Data Processing About the Data Processing Parameter Options Parameter Options The Data Processing parameter options specify how the raw data is processed before peak detection and size calling Data Processing Parameter Options Described Item Description Baseline checkbox Use to automatically adjust the baselines of all detected dye colors to the same level for a better comparison of relative signal intensity When the GeneScan Analysis Software data is sent to the Macintosh computer during data collection the baseline for each dye is at a d
228. ions from the View menu For ABI PRISM 377 Runs The Show Offscale Regions feature must be used in conjunction with For ABI PRISM 377 runs use Refer to Pre averaging offscale detection in the Gel Preferences menu Pre Averaging Offscale Detection checkbox on page 2 9 Suppress Left Right Averaging checkbox in the General Setting Preferences dialog box ABI PRISM 377 Data Collection software Refer to Chapter 4 in the ABI PRISM 377 Instrument User s Manual About Flat Topped Peaks An additional feature is that peaks that contain off scale data points are drawn in the electropherograms as flat topped that is the top section of the peak is flat rather than pointed see Electropherogram Displaying the Flat Topped Effect on page 7 35 This feature may be seen when the data is analyzed with no or light smoothing the flat topped peaks may not be apparent with heavy smoothing Choose Analysis Parameters from the Settings Menu see Smooth Options on page 5 20 Evaluating Analysis Results 7 33 Electropherogram The following is an example of an electropherogram displaying off scale Displaying data Off Scale Data Off scale data 6B 16916 16 Hi 6G 16 67 16 7 LI sy 16 1e 7167 Mi sR 16 e1e7i6 2400 1200 o 6B 16 16 18 2400 7 1200 BH scG iee1erviss 2400 1200 LI 5v 186916716 480 240 MM ER 16 6716 7 34 Evaluating Analysis Result
229. iple control points draw a box around a set of control points All the control points that you drew a box around become enabled Use the Arrow keys to move all the control points How to Process and Edit the Gel File 2 45 If you want to Then move up or down the control points on the spline use the Arrow keys move forward right a lane Note When moving forward the control point is deselected press the Tab key move left backwards a lane Note When moving backwards the control point is deselected press the Shift Tab key Adding and To add and delete control rows Deleting Control Rows If you want to Then add a control row a Place the arrow cursor outside the image left or right side and press the Option key Place arrow cursor on left or right side al aol uer m SC T EI 19 20 21 22 23 24 25 26 27 dee OO OC OP OPO OC OOD OO OC 0101010101 8 The cursor changes to a symbol and a horizontal line will be displayed across the gel image Click the mouse button to add a control row at the desired location 2 46 How to Process and Edit the Gel File What Marking Lanes as Used Unused Means Marking Lanes as Used or Unused Lane marker Gel file Scan 4107 Laret Use 33 10 11 12 13 15 167 18 LOLOL OLS E If you want to Then delet
230. isplaying To display electropherogram and tabular data Electropherogram and Tabular Data Step Action 1 Choose Results Control 3 2 from the Windows menu to open the Results Control window Click the electropherogram icon ERR and the tabular icon If applicable select the number of electropherogram panels Select the dye samples for display Click Display The electropherogram and tabular data are displayed in the Results Display window See also Working with Electropherogram Data on page 7 27 Tabular Data and The following is an example of tabular data with a corresponding Electropherogram electropherogram Example zi 8R 08ep27 8 3 Kel 7 22 Evaluating Analysis Results Minutes Table Describing The following table describes the columns in the above figure Columns This column Identifies Dye Sample Peak Sample index number Dye color Peak number Minutes The time in minutes from the start of the run to the time the fragment was detected Size The differences in fragment mobility This value is calculated automatically only if you Run the size standard in the same lane or injection as the sample and Perform size calling Peak Height Signal size Peak Area Area of the detected peak Data point Data point of the fragme
231. ixture at room temperature for 30 minutes 3 Check the pH It must be 7 0 9 0 4 If the pH Then is in the proper range filter the mixture through a 2 micron filter is not in the proper range a Decantthe formamide into a beaker with 5g of resin b Stir at room temperature for 30 minutes c Checkthe pH it must be 7 0 9 0 Making a Matrix File 6 9 Preparing the Formamide Size Standard Mix Preparing Matrix Standard Samples 6 10 Making a Matrix File Step Action 5 Make 500 pL aliquots and store them at minus 20 C for up to 3 months To ensure reproducibility of results for all samples prepare the formamide size standard mix using the 12 1 ratio of reagents stated in the procedure below IMPORTANT The formamide size standard mix for the ABI PRISM 310 differs from the mix prepared for the ABI 373 and ABI PRISM 377 and do not use on other instruments WARNING CHEMICAL HAZARD Formamide is a known teratogen It can cause birth defects Wash thoroughly after handling formamide Wear appropriate protective eyewear clothing and gloves Obtain a copy of the MSDS from the manufacturer Wash thoroughly after handling formamide Prepare the formamide size standard mix as follows Step Action 1 Mix in a sample vial a 0 5 uL GeneScan 350 TAMRA Size Standard for example b 12 0 pL deionized formamide 2 Label the vial 3 Ge
232. ize Calculations Introduction This section describes the following Verifying for the GeneScan 350 Standard Evaluating for Multiple Internal Size Standards Verifying for the About the Standard GeneScan 350 Standard 7 48 Evaluating Analysis Results In the GeneScan 350 standard the size of the first peak should be approximately 50 bp the second 75 bp and so on assuming that processing started after the 35 bp fragment passed the scan region If this appears to be correctly measured for your run measurement of the sample fragments that ran with the standard should also be correct Note For a complete list of fragment sizes refer to Appendix B GeneScan Size Standards Procedure To verify the size calculation for the GeneScan 350 standard Step Action 1 In the Results Control window select the Electropherogram and the Tabular Display icons 2 Click the Clear All button to clear the panels 3 Click the dye color indicator for the size standard you want to view 4 Click Display Note You could also open the Sample File window of the Sample file of interest to verify size calling To verify the size calculation for the GeneScan 350 standard continued Step Action 5 Click each peak in the electropherogram and check the tabular data to ensure it is the correct size Note The peaks matched to the defined size standard are identified by dots next to the Dye Sample
233. king a Matrix File Kit Part number Dye Primer Matrix Standard Kit 401114 Fluorescent Amidite Matrix Standards 401456 Fluorescent dNTP Matrix Standards 402792 Preparing Matrix Standards for the ABI PRISM 310 About Formamide WARNING CHEMICAL HAZARD Formamide is a known and Samples in teratogen It can cause birth defects Wash thoroughly after handling Formamide formamide Wear appropriate protective eyewear clothing and gloves Obtain a copy of the MSDS from the manufacturer Wash thoroughly after handling formamide IMPORTANT Use the same matrix standards that you used for your GeneScan Analysis Software run The protocol uses formamide as a sample preparation reagent Fresh formamide must be deionized and aliquotted into smaller volumes for storage Each aliquot should be adequate for about one week s work Store aliquots of formamide at 20 C for up to three months Formamide stored at 4 C is good for about one week At room temperature samples in formamide are stable for a maximum of 48 hours Although not recommended on a routine basis you can keep samples prepared in formamide frozen for no more than three days with no detectable loss in resolution before running on the ABI PRISMe 310 Genetic Analyzer Deionizing To deionize formamide Formamide Step Action 1 Mix 50 mL of formamide and 5 g of ion exchange resin AG501 X8 from BioRad is recommended 2 Stir the m
234. l checkboxes IMPORTANT If you plan to use the Genotyper software then you must complete the Sample Info box correctly For more information refer to the Genotyper User s Manual To set up for automatic analysis after data collection continued Step Action 3 In the Injection List or Run Sheet for each applicable sample select AutoAnalyze and choose from the appropriate pop up menus one of the following Matrix file Analysis parameters Size standard The following table lists considerations when choosing a size standard If you choose a Then GeneScan Size Standard definition file in the Collection Injection List or Run Sheet but you do not specify which dye is the standard in the Sample Sheet performs analysis using the dye specified in the Auto Analysis defaults dye as the standard in the Sample Sheet but do not specify a size standard definition file in the Collection Injection List or Run Sheet does not perform size calling 4 Select Auto Print to print automatically Complete the following steps in the GeneScan Analysis Software 1 Take the following action for the ABI 373 and the ABI PRISM 377 data a Startthe GeneScan Analysis Software b Fromthe Gel menu and the Auto Processing submenu ensure that Auto Track Gel and Extract Lanes after Auto Tracking are selected IMPORTANT Before choosing Auto Track Lane
235. lay window about 7 4to 7 6 using 7 7 to 7 12 Results Displays saving procedure 8 5 why save 8 2 S Sample File window about 3 5 EPT Data View 3 17 to 3 18 displaying the view 3 17 example 3 18 what it displays 3 17 Raw Data View 3 15 to 3 16 displaying the view 3 15 example 3 16 what it displays 3 15 what to evaluate 3 16 Sample Info View 3 8 to 3 12 description of view 3 9 to 3 12 displaying the view 3 8 example 3 9 what it displays 3 8 Sample Results View 3 6 to 3 7 description of view 3 7 differences from Results Display 3 7 displaying the view 3 6 example 3 6 what it displays 3 6 Size Curve View 3 13 to 3 14 displaying the view 3 13 size curve described 3 14 what is the size curve 3 13 what the size curve displays 3 14 sample files about 3 2 to 3 3 how GeneScan analyzes sample files 3 3 how they are generated 3 2 ways to generate sample files 3 3 what sample files contain 3 2 analyzing 3 19 to 3 20 installing new matrix file 3 20 procedure 3 19 to 3 20 Index 5 using Analysis Control window 5 6 to 5 16 archiving 8 6 defined Glossary 3 finding missing sample files 4 14 to 4 15 how GeneScan names files 2 67 matrix file assigning to sample files 6 35 to 6 36 opening procedure 3 4 printing 9 5 to 9 6 removing from a project 4 13 Sample File window about 3 5 EPT Data view 3 17 to 3 18 Raw Data View 3 15 to 3 16 Sample Info View 3 8 to 3 12 Sample Results View 3 6 to 3 7 Size Curve View 3 13 to 3
236. le files opened for viewing database Using the BioLIMS Database E 3 Configuring the BioLIMS Database Server Sybase or Oracle This section gives instructions on how to configure the client computer that runs the GeneScan Analysis Software for database access The BioLIMS database resides on either a Sybase SQL Server or an Oracle Server To configure for the See Sybase SQL Server Configuring for Sybase SQL Server Connection on page E 4 Oracle Server Configuring for Oracle Server Connection on page E 7 Configuring for Follow the steps below to configure a Macintosh computer for Sybase SQL connection to the Sybase SQL Server Server Connection IMPORTANT Any time the name port number IP address or host and domain name of the BioLIMS database server is changed you need to repeat this procedure To configure for Sybase SQL Server connection Step Action 1 Find the interfaces file in the Sybase folder in the BioLIMS Extras folder vy BioLIMS 2 0 O BioLIMS Extras 3 Subase O bin 3 charsets D interfaces gt O locales v4 Open the file with SimpleText or a similar text editing application E 4 Using the BioLIMS Database To configure for Sybase SQL Server connection continued Step Action 3 Find the lines SYBASE query MacTCP mac ether neuron apldbio com 2500 and edit them Replace SYBASE by an alias name
237. les This is a third order curve when you use the Third Order Least Squares size calling method for all other size calling methods it is a second order curve This curve is black in the Standard Sizing Curve window although when the sizing curve and this curve match they overlap so you see only the sizing curve See also size calling curve and size standard spline interpolation curve analysis parameters Options that specify certain ranges and methods used during analysis using the GeneScan Analysis Software The software has default analysis parameters that are stored in the project itself These parameters apply globally unless you create your own parameters files for use with specific protocols See Defining Analysis Parameters on page 5 18 baselining Adjusting the baselines of detected dye colors to the same level for a better comparison of relative signal intensity channels ABI 373 and ABI PnisM 377 instruments Theoretical divisions across the read region of a gel where the Data Collection software software samples the data The number of wells in the loading comb used determines the approximate number of channels assigned per lane of the gel for instance with a 36 well comb one lane is approximately five channels When the GeneScan Analysis Software tracks a gel it places the tracker line for each lane in the channel showing the strongest fluorescent signal The data from that channel and the adjacent channels on each
238. lign by Size 3 T from the View menu if the electropherograms are not already aligned by size The size standards should line up when aligned by size Note You can set preferences so that all new displays show data aligned by size For more information see Changing How the Results are Displayed and Printed on page 7 13 Using the Analysis Log What is the Analysis Log Displaying the Analysis Log The Analysis Log maintains a running record of analysis performed by the GeneScan Analysis Software If a problem occurs during analysis of a Sample file the Analysis Log automatically opens and appears in the foreground as an alert Choose Analysis Log from the Windows menu The following is an example of the Analysis Log Analysis Log 11GS Gel 1 9 98 confidence value 100 The lane assignment confidence value of 100 is equal to or above the threshold value o 11168 Gel 1 9 98 confidence value 100 The lane assignment confidence value of 100 is equal to or above the threshold value o The Gel file length is not the expected length 11168 Gel 38 377 36cm34w34L H 1 9 confidence value 0 The lane assignment confidence value of 096 is less than the threshold value of 70 11168 Gel 38 377 36cm34w34L H 1 9 confidence value 100 The lane assignment confidence value of 100 is equal to or above the threshold value o 11168 Gel 38 377 Z6cm4w24L H 1 9 confidence value 0 59 The lan
239. lly has a Close box in the upper left corner If many windows are open click one window to make it active When a window is active you can click the top border hold down the mouse button and drag the window to another location on the screen When you are finished working with a window click the Close box to remove the window from the screen or click another window Close box Window Gel file EB i r x 1 05 Chanel 43 00 Sen 4107 Lanesused 33 9 10 11 12 13 15 16718 19 20 21 22 23 24 2 CSSSSSSSSEESEBGE _ SSESSESS Current Comb Type set in prefs Square Tooth GeneScan Analysis Software Overview 1 9 Macintosh computer terms used in this manual continued Item Description Entry fields Image Generation Defaults Scan Range Step 100000 Start 0 Rectangular areas in which you can enter information Click in an entry field to display a cursor and use the keyboard to enter the information Checkboxes Auto Launch Processing s Auto Track Gel s Extract Lanes after Auto Tracking Boxes that you click to select certain options in a dialog box When you click an empty checkbox an x appears in it indicating that you have selected that option You can usually select multiple checkboxes Radio buttons Comb Type Sharks Tooth Square Tooth Small circles that appear in fr
240. log box appears Check that Allthe login information was entered correctly and in the correct case Your interfaces file is correctly configured page E 4 Ifthe connection is still not open consult Appendix F Troubleshooting the BioLIMS Database Using the BioLIMS Database About Server Names Sybase or Oracle The BioLIMS Session Manager decides whether you are connected to Sybase SQL Server or to an Oracle Server database by looking at the name in the Server field in the Session Manager dialog box How Names are The table below summarizes how names are recognized Recognized If the Session Manager sees a Server name It assumes a Example All in uppercase letters Sybase SQL Server database connection MOZART Suffixed by s or S Sybase SQL Server database connection Offenbach S Containing any lowercase letters Oracle Server database Oramozart Suffixed by o or O Oracle Server database SIBELIUS O Sybase SQL Example 1 Server Examples MOZART If the interfaces file contains this query MacTCP mac ether mozart apldbio com 2500 MOZART is recognized as a Sybase SQL Server because the server name is in all uppercase letters The Session Manager would look like this Username ine Password O Save Password Database Server MOZART Using the BioLIMS Database E 17 Oracle Server Examples E 18 Using the B
241. lts are Displayed and Printed on page 7 13 project File containing links to a set of Sample files that you want to analyze and display together A project can contain Sample files from multiple runs Adding a Sample file to a project creates a reference to the file It does not copy the file into the project For more information see Using a Project to Manage Sample Files on page 4 8 project options Formatting information you can set for the current project Project options are remembered by the project when you open it again For a brief description of each project option see Changing How the Results are Displayed and Printed on page 7 13 sample files Computer files that contain raw and analyzed data Sample files are created directly by the ABI PRISM 310 and by the GeneScan Analysis Software for the ABI 373 and ABI PRISM 377 Sample files contain data such as peak locations size calling values and a record of analysis settings scan number ABI 373 and ABI PRISM 377 instruments Data Collection software samples data 194 times 194 or 388 times for XL instruments as it scans across the gel Each sampling is stored as a data point The scan number describes the location of the data point separation distance Length from the wells of the gel to the read region of the gel Also called the WTR well to read shark s tooth comb Piece of mylar inserted into a gel typically used for a sequencing run The flat edge of the com
242. lues displayed are an example that were used to create the current gel image Regenerate Gel Image Scan Range Sto Ba Start 0 Max 4572 Ds Multicomponent Gel Image Estimated Maximum Peak Height 2000 tance Make any required changes in the dialog box values as follows Use the To Stop text entry field include the last scan value desired in the gel image The Max value indicates the total number of scans in the current gel file Start text entry field include the first scan value in the gel image Multicomponent Gel Image checkbox cause GeneScan Analysis Software to apply the assigned matrix file to the gel image For information on the matrix file see Chapter 6 Making a Matrix File Estimated Maximum Peak Height text entry field enter the maximum signal level expected from samples in the run How to Process and Edit the Gel File 2 23 To regenerate the gel image continued Step Action 3 Click OK to close the dialog box and start regenerating the gel image Note 36 period cancels the regeneration Installing New About the Matrix File Matrix The Data Collection software copies the matrix information in the Information specified matrix file to the gel file during data collection The GeneScan Analysis Software uses this matrix information to multicomponent the
243. markers so that the lane numbers are properly aligned with the actual sample lanes The lane data is written to the correct files with the Sample files are regenerated To move misplaced lane markers Step Action area to the right of the lanes E LC OCLL 0000 000000000000 1 Inspect the gel image for incorrectly labeled lanes For example in the following illustration the GeneScan Analysis Software missed the faint signals from lane 25 As a result lanes 3126 36 are mislabeled and the lane 36 marker is over an unused Missed lane 25 has no marker Lane 36 marker is not over a lane 2 48 How to Process and Edit the Gel File To move misplaced lane markers continued Step Action 2 Click the incorrectly placed marker to select it the selected marker has a red border and while holding down the mouse button drag the lane marker to the correct location All the affected markers are renumbered accordingly Lane markers always remain in numerical order from left to right and are attached to their respective tracker line within a few channels to either side For example if you drag the 436 marker in the preceding illustration to the real lane 25 lanes 25 36 all become correctly marked as shown in the figure below Lane 25 now has a marker Lane 36 marker Q000000 000000000 Sentit o g How to Process and Edit the Gel File 2 4
244. menu Displaying Default There are two ways to display the default parameters Parameters You can Then choose Analysis Parameters from the Settings menu Analysis Parameters dialog box appears with the default parameters double click Analysis Parameters from within the Analysis Control window For information on defining the Analysis Parameters see page 5 18 Creating Custom To create a custom analysis parameter file Analysis Parameter Files 5 26 Analyzing Project Files Step Action 1 Choose New from the File menu Create New The Create New dialog box appears Project Sample Sheet Analysis Parameters Standard 13 598 fw fu Size Matrix Cancel Step Action 2 Click the Analysis Parameters icon The Analysis Parameters dialog box appears 3 Change the parameters as necessary For information on the specific options see About the Analysis Parameters on page 5 17 4 Choose Save 3 S from the File menu A dialog box appears 5 Enter a descriptive name and click Save Note Unless you choose a different folder location the GeneScan Analysis Software stores the file in the folder specified as the folder location for Analysis Parameters For more information see Defining Folder Locations on page 5 41 The file now appears in the pop up menu for analysis parameters in the GeneScan Analysis Cont
245. mponent Definition 6 2 Making a Matrix File There are three dye labeling chemistries currently available to prepare nucleic acid samples for using the GeneScan Analysis Software on ABI PRISM instruments Fluorescent NHS Ester Fluorescent dNTP Fluorescent Phosphoramidite Each chemistry has a set of dye labels that fluoresce at different wavelengths when excited by a laser During data collection on the The wavelengths are separated ABI PRISM 310 or ABI PRISM 377 by a spectrograph into a known 377XL or 96 lane upgrade spectral pattern across a detection system with the sequencer ABI 373 and the ABI 373 with XL using a filter wheel upgrade Matrix files are mathematical matrices that correct for spectral overlap of fluorescent emission spectra data collected from ABI PRISMe instruments A matrix file allows you to account for spectral overlap when analyzing Sample files This process of eliminating the bleed through caused by spectral overlaps is called multicomponenting Applying a matrix file to raw data allows you to generate multicomponented data Why is a Matrix A matrix file is necessary because the four or five dyes used to label File Necessary your fragments fluoresce at different wavelengths and may have spectral overlaps as shown below EMISSION L 1 1 1 fi 1 450 500 550 600 650 WAVELENGTH nm FAM JOE TAMRA ROX When t
246. n Parameter Options Described Item Description For example Dye Amplitude Threshold Set the dye amplitude threshold at a level that allows the software to detect peaks but eliminate noise For each dye the GeneScan Analysis Software detects peaks above the threshold entered in the entry field If you leave the default value of 50 peaks with amplitude above 50 are analyzed and appear in the tabular data Lower amplitude peaks still appear in the electropherogram but are not analyzed and do not appear in the tabular data Size Call Range Parameter Options Size Calling Method Parameter Options Item Description For example Minimum Peak Half Defines what If this number is large Width constitutes a peak the software ignores i ikes Use to specify the qo E smallest half peak If the peaks in the data width for peak are narrow set the detection value to a low number The range is from Experiment with this 2 99 value to determine the for th A typical number might Beet nue rani data be 3 for microsatellites or 10 for SSCPs About the Size Call Range Parameter Options Use the Size Call Range parameter options to specify the range of size fragment in base pairs to be included in the peak tabular data Size Call Range Parameter Options Described Item Description All Sizes radio All detected fragments appear in the tabular data button
247. n on troubleshooting the BioLIMS database see Appendix F Troubleshooting the BioLIMS Database Topics in this appendix include the following Topics See page Troubleshooting Projects and Results D 2 Troubleshooting Gel Data D 5 Troubleshooting Genotyping Results D 8 GeneScan Error Messages D 9 Troubleshooting the GeneScan Software D 1 Troubleshooting Projects and Results Table Description The following table lists the problem probable cause and correction for troubleshooting projects and results Troubleshooting projects and results Problem Probable Cause Correction File name is dimmed in The file has been a Move the file back Size Standard or moved from the folder to its original Parameters column in in which it was located location Analysis Control when it was first b Resetpreferences window selected to specify the new folder location c Create or select a new file Peaks appear on Peak Amplitude a Adjust minimum display but the Threshold set too peak height to GeneScan Analysis high include smallest Fede does not Minimum Peak poa aud and etect them canno Half Width set too re analyze select them in high b Reduce minimum electropherogram peak half width display Electrophoresis setting and run too quickly re analyze resulting in poor resolution c Repeat electrophoresis at reduced power For more information see Peak Detection Parameter Options
248. neScan Analysis Software analyzes data sent from the ABI PRISM instrument after a run The gel file 373 or 377 only Run folder created by ABI PRISM 377 Data Collection during run Run folder created by ABI 373 Data Collection during run A large file created by the Data Collection software The gel file contains the raw data collected during the instrument run It also contains the Sample Sheet and run information For a typical run this file can be very large More than 20 MB for 194 channels more than 40 MB for 388 channels and more than 50 MB for 96 lanes up to 70 MB for long runs 1 16 GeneScan Analysis Software Overview GeneScan Analysis Software files continued Location on 373XL File type ABI PRISM 377 Description 377XL 377 96 Lanes or ABI PRISM 310 ABI 373 The Project Run folder Sample file Organizes displays File created by the folder created by and analyzes Data Collection GeneScan Sample files and software during during run results ran Projects contain references to Sample files Project files can contain references to Sample files from more than one run Sample Run folder Sample file Created by Files created by folder created by ABI PRISM 310 data collection GeneScan software during during run For ABI O5 and HB ABI PRISM 377 GeneScan extracts sample information from the gel file to create Sample files Each file contains
249. nels pop up menu 3 Click dye color fields to select or deselect the corresponding samples Take the following action To Click the select the entire column header deselect the entire column header again select four colors for a Sample index number to the left of the file color columns 4 If you specified Electropherogram display the samples corresponding to the selected dyes appear in the Dye Sample Display list to the right of the Sample File list as shown in the figure below To select samples for display continued Step Action Dye color Plot color of of sample electropherogram OO Quick Tile Om eor Row number and dye code followed by dye sample information 5 When you change to a new panel the dye colors of the samples you selected in other panels appear dark gray to indicate that they have been selected You can select them again in the current panel Evaluating Analysis Results 7 9 Creating Tiled Procedure Electropherogram To create tiled electropherogram displays Displays Step Action 1 Choose the number of panels you want to display from of Panels pop up menu Click the On radio button under Quick Tile Select samples by clicking color fields For information on Topic See page Selecting Samples to Display 7 8 Setting the tiled electropherogram preferences 7 13 Each time a sample is selected
250. ns and to learn more about our products You can also order technical documents and or an index of available documents and have them faxed or e mailed to you through our site see the Documents on Demand section below In the United States and Canada technical support is available at the following times Product Hours Chemiluminescence 9 00 a m to 5 00 p m Eastern Time LC MS 9 00 a m to 5 00 p m Pacific Time All Other Products 5 30 a m to 5 00 p m Pacific Time See the Regional Offices Sales and Service section below for how to contact local service representatives outside of the United States and Canada Call Technical Support at 1 800 831 6844 and select the appropriate option below for support on the product of your choice at any time during the call To open a service call for other support needs or in case of an emergency press 1 after dialing 1 800 831 6844 For Support On This Product Dial 1 800 831 6844 and ABI PRISM 3700 DNA Press FAX Analyzer 8 650 638 5981 ABI PRISM 3100 Genetic Analyzer Press FAX 26 650 638 5891 DNA Synthesis Press FAX 21 650 638 5981 1 20 GeneScan Analysis Software Overview For Support On This Product Dial 1 800 831 6844 and Fluorescent DNA Press FAX Sequencing 22 650 638 5891 Fluoresce
251. ns to size fragments in the 100 to 5 000 base pair range The following figure shows the peak pattern of fragments run under native conditions on the ABI PRISM 310 using 2 5 GeneScan Polymer Solution in a 30 cm Ld capillary IMPORTANT An asterisk for the 508 base pair peaks denotes peaks resulting from abnormal migration of double strands that did not completely Separate under denaturing conditions when analyzed on the ABI PRISM 310 Do not use these peaks to size samples The peaks show smaller values than the actual size of the fragments Refer to the GeneScan Reference Guide Chemistry Reference for the ABI PRISM 310 Genetic Analyzer P N 4303189 rev A for further details 800 2100 2400 2700 3000 Asterisk IR GeneScan 2500 Fluorescently labeled native fragments are 18 nucleotides longer than Molecular Lengths denatured fragments see table below GeneScan 2500 Molecular Lengths bp Denatured Native 18 14079 14097 5099 5117 4771 4789 4529 4547 2860 2878 2481 2499 2465 2483 2162 2180 2008 2026 1722 1740 1181 1199 1115 1133 827 845 536 554 490 508 470 488 361 379 286 304 269 287 238 256 233 251 222 240 186 204 172 190 116 134 109 127 94 112 37 55 GeneScan Size Standards B 11 Size Calling Methods Introduction In Thi
252. nsion Files the System folder by the BioLIMS Client and instrument installers These files are required for connection to the BioLIMS database OracleCore23Lib OracleOra71Lib OracleKernel71Lib OraclePlsql21 Lib OracleNetNLLib OraclePstd21Lib OracleNetTCPLib OracleRuntime13Lib OracleNetTNSLib Oracle Sql16Lib OracleNetTNSTCPLib OracleTNSATKLib OracleNLS23Lib OracleVsoci71Lib OracleOci71Lib Troubleshooting the BioLIMS Database F 11 Troubleshooting The step numbers in the following procedure correspond to the steps from the Unix marked in the flow chart on page F 5 Step1 below corresponds to the Sybase Server diamond Try to log in to the server on page F 5 You need to have the account name and password for the Sybase user on the UNIX system that runs the Sybase SQL Server If you do not have access to the Sybase user account you should ask your database administrator to carry out the following procedure Troubleshooting the client connection from the Sybase SQL Server Step Action 1 Log in to the Sybase user account on the UNIX Server Try to connect to the Sybase SQL Server with isgl Use the same client user name as the one entered in the BioLIMS access dialog box For example isql U george P georgel S SYBASE 1 use sfdb 2 go 1 quit Where Represents the george client user name george1 client user password SY
253. nt Fragment press FAX Analysis includes GeneScan applications 23 650 638 5891 Integrated Thermal Cyclers Press FAX 24 650 638 5891 Biolnformatics includes BioLIMS BioMerge and Press FAX SQL GT applications 25 505 982 7690 PCR and Sequence Press FAX Detection 5 or call 240 453 4613 1 800 762 4001 and press 1 for PCR or 2 for Sequence Detection icd Telephone FAX 1 800 899 5858 and press 1 then press 6 508 383 7855 Peptide and Organic Press FAX Synthesis 31 650 638 5981 Protein Sequencing Press FAX 32 650 638 5981 Chemiluminescence Telephone FAX 1 800 542 2369 781 275 8581 U S only or Tropix 1 781 271 0045 9 00 a m to Tropix 5 00 p m ET GeneScan Analysis Software Overview 1 21 For Support On This Product Dial 1 800 831 6844 and LC MS Telephone FAX 1 800 952 4716 650 638 6223 9 00 a m to 5 00 p m PT 1 22 GeneScan Analysis Software Overview Documents on Demand Free 24 hour access to Applied Biosystems technical documents including MSDSs is available by fax or e mail You can access Documents on Demand through the internet or by telephone If you want to order Then through the Use http www appliedbiosystems com techsupport internet You can search for documents to order using keywords Up to five documents can be faxed or e mailed to
254. nt at its maximum peak height Highlighting To highlight information for one peak in the electropherogram and Information tabular data Click Then the peak in the electropherogram the peak fills with color and the corresponding row in the tabular data window is highlighted the Dye Sample Peak number in the highlights the corresponding peak in tabular data window the electropherogram How to Change the Highlight Color Choose Peak Highlighting from the View menu and either Opaque or Transparent from the submenu For more information see Highlighting Peaks on page 7 29 Evaluating Analysis Results 7 23 Adjusting Window Size Hiding Selected Rows of Data To adjust the relative size of the electropherogram and tabular windows Step Action 1 Move the cursor to the window divider the double line between the two windows 2 When the cursor changes to a bidirectional arrow E click the window divider line and drag it up or down To hide selected rows of data Step Action 1 Take the following action If you want to Then select a row either Click the first field in the row or Click the corresponding peak in the electropherogram select several consecutive shift click the first and last row rows you want to select select several rows that are not 3 click the rows next to each other 2 Choose H
255. ntal zoom are 1x 2x 4x and 8x Vertical Shrink button Returns the vertical scale to normal after using the Vertical Expand button i bs Vertical Expand button Expands the gel image vertically to see the quality of the peaks in Slice View There are two levels of vertical zoom full scale and 600 scans Interpolation button Places the user in tracker line interpolation mode For more information see Interpolating Tracker Lines on page 2 57 2 14 How to Process and Edit the Gel File Window tool Description as Off Scale button Displays off scale data Turns off all the colors in the gel image and displays in white any data points that are off scale Off Scale data is outside the range of the detector 8191 relative fluorescent units and greater Note To see all off scale data points the Horizontal Expand and the Vertical Expand buttons must be used For more information see About Displaying Regions of Off Scale Data on page 2 38 Gel File Window The following table describes the diagram on page 2 13 Described T Gel File window described No Description 1 Channel Current cursor position on the horizontal scale and is displayed in sub channel increments Channels are the divisions across the read region of a gel where the Data Collection software samples the data The number of available channels dep
256. ntly vortex the mixture for 3 5 seconds 4 Spin down the mixture 5 Store the mix at 2 6 C until ready to use About Matrix Standards You must run matrix standards and create a matrix file the first time you use a new chemistry or change the run conditions Do not prepare matrix standards more than two hours in advance To ensure reproducibility of results for all samples prepare the matrix standard mix using the 12 1 ratio of reagents stated in the procedure below IMPORTANT Do not add size standard mix to the matrix standard samples IMPORTANT The matrix standard mix for the ABI PRISM 310 differs from the mix prepared for the ABI 373 and ABI PRISM 377 and should not be used on other instruments For Prepare the the Fluorescent Genotyping Demonstration Kit Fluorescent Amidite Matrix Standards P N 401546 fluorescent dNTPs Fluorescent dNTP Matrix Standards P N 402792 Note Remember that the matrix standards run must always match the sample run chemistry and conditions Preparing Matrix Standard Samples For each matrix standard Step Action 1 Mix in a sample vial a 1 0 uL of matrix standard b 12 0 pL of de ionized formamide Label each vial according to the dye Gently vortex the mixture for 3 5 seconds Spin down the mixture c1 25 0 N Store at 2 6 C until ready to use Making a Matrix File 6 11 Pr
257. nu In the Analysis Control window find the row that contains the sample for which you want to define the standard In that row 3 click the dye color cell that represents your standard A diamond symbol 4 appears in the cell identifying it as the standard To use the Analysis Control window to define a new size standard continued Step Action 4 Click the arrow in the Parameters field of the same row and select an option from the pop up menu that appears The pop up menu contains the following options Item Description lt Analysis Parameters gt Applies the parameters that are stored as preferences in the software lt filename gt Applies the settings specified in the data collection run file custom parameters that are These are files that you defined listed at the bottom of the menu and they are located in the parameters folder specified in the Folder Locations preferences For information on setting the Folder Locations preferences see page 5 41 5 In the same row click the arrow in the Size Standard field and choose Define New from the pop up menu A window appears showing the electropherogram and a table of peaks for the dye color and sample you selected You should be able to recognize the peak pattern of the standard in the electropherogram 6 Follow step 7 on page 5 33 to step 9 on page 5 34 The name of the standard appears in th
258. o Createa Create a matrix file for each dye set used from that particular instrument Matrix File before analyzing fragment data You may have to create new matrix files for different gel compositions or unusual run conditions Making a Matrix File 6 3 Sample Files Using Matrix File Applying a Matrix File When to Assign a Matrix File 6 4 Making a Matrix File The figure below shows an example of data analyzed with and without a matrix file You can see that peak data from a Sample file analyzed without a matrix file displays the expected peak along with extra peaks in other dye colors or bleed through from other dye colors 12 50 Lane Demo Sample 4 1700 1800 1900 2100 2200 1800 1900 2000 2100 2200 Sample file analyzed with a matrix file You can apply a matrix to a gel image or to the raw data within Sample files The matrix from a matrix file is installed within a gel file or Sample file automatically upon generation during or after a run or manually from within the GeneScan Analysis Software If a matrix is installed to the See page Gel image during image generation 2 24 Sample file s raw data during analysis 3 20 Before you can successfully analyze Sample files using the GeneScan Analysis Software you must make a new matrix file or assign an existing one to a set of Sample files Limitations to Matrix Files
259. o mark all lanes for extraction for Extraction From the Gel menu choose Result Mark All Lanes for Extraction The color of all markers for Used lanes changes to white If you choose the Extract Lanes command the GeneScan Analysis Software uses the current tracker line location to extract the data in all Used lanes and puts the extracted data in new sample files Unmarking All To unmark all lanes for extraction Lanes for Extraction Step Action 1 Select Unmark All Lanes For Extraction from the Gel menu The color of all the lane markers changes to blue 2 You can take the following action To See extract all lanes marked as Used Step 2 on page 2 65 remark selected lanes for extraction Marking a Single Lane for Extraction on page 2 52 How to Process and Edit the Gel File 2 51 Marking a Single To mark only selected lanes or to extract a lane that was not Lanefor automatically extracted Extraction Step Action 1 Click the lane marker you want to mark for extraction 2 There are two ways to mark a single lane for extraction You can either Result choose Mark Lane for Extraction from the Gel menu press the Option key and click the lane marker The color of the lane marker changes to white When you choose the Extract Lanes command the GeneScan Analysis Software uses the
260. o the new file For example if SAMPLE 1 SAMPLE 2 and SAMPLE 3 exist and you discard SAMPLE 2 GeneScan names the next file SAMPLE 2 not SAMPLE 4 Use the Auto Analyze after Extract checkbox to Analyze the new Sample files after extraction is finished Do not select this checkbox if you want the Sample files extracted but not analyzed Use the Analyze All Files radio button to analyze all the new Sample files created from the gel file Use the Print Results checkbox to print the results for all new Sample files after analysis Choose Use Sample Sheet Settings radio button to analyze and print only those files that are marked for analysis and printing in the Sample Sheet for the gel file Note Select this option if you want to process only some of the new Sample files created from the gel file How to Process and Edit the Gel File To track and extract the gel file continued Step Action 7 Click OK See the table below for the action you can take If a project is Then open The following dialog box appears T R Project File is presently open Do you want to add the new Sample Files to this Project or to close it and have a new one created Cancel Use Open Project Closeit And you can take the following action To Click add the new Sample files to Use Open Project the open project close the project an
261. ocedure describes how to change the defaults that determine what dye colors appear on the screen and on printed results Setting default dye and plot colors sets the colors used for both the Control windows and the Results displays To set default dye and plot colors Step Action 1 Choose Preferences from the Settings menu and Dye Indicators from the submenu The Preferences window appears Note If the Preferences window is already displayed choose Dye Indicators from the pop up menu Vertical scroll bar Preferences Page Dye Color Plot Color Eeue Yj msue v creen vj ereen 7 LJ Yellow v Nl Black v EH Red v EH Red v C 2 The following table describes the Dye Color and Plot Color columns Item Description Dye Color Shows the colors that represent the dyes in the column Control window lists and the gel display of ABI 373 and ABI PRISM 377 data The dye color is also identified in the left color legend in the Results display Plot Color Shows the colors used for plotting the data in column the electropherograms 3 Use the vertical scroll bar to change the dye color and plot color for a fifth dye Analyzing Project Files 5 15 To set default dye and plot colors continued Step Action 4 To change a code type a different character in the appropriate entry field in
262. of 8191 Note If you extracted a sample file with the Pre Averaging Offscale Detection checkbox selected then pre averaging has a value of zero in the Sample Info View 2 38 How to Process and Edit the Gel File How Pre Averaging Offscale Detection Works The following table lists how the Pre Averaging Offscale Detection feature identifies off scale data points If Then each data point used in averaging is less than 8191 rfu relative fluorescent units the peak in the electropherogram is not off scale and appears normal any one of the data points used in the averaging is 8191 rfu no averaging takes place and 8191 rfu is written for that scan number of that lane In the electropherogram the off scale regions are displayed by drawing red vertical regions where the off scale data is present How to Display the Red Regions a Choose Preferences from the Settings menu and Results Display from the submenu b Select the checkbox labeled Show Offscale Regions Note You can choose Hide Show Offscale Regions from the View menu if the checkbox labeled Show Offscale Regions is not selected About Flat Topped Peaks An additional feature is peaks that contain off scale data points are drawn in the electropherograms as flat topped That is the top section of the peak is flat rather than pointed see Electropherogram Displaying the Flat Topped Effect on page 2 42 This feature m
263. ollowing table lists ways to display the Size Curve View View To display the view Do this from the Sample file Click the Size Curve button on the lower left window of the Sample file window E or Select Size Curve 36 U from the Sample menu from a project window a Usethe following table to select a sample or multiple samples in the Analysis Control or the Results Control window If you want to Then select a sample click the sample you want to select shift click the first and several consecutive last sample in the samples group you want to select select several click the samples samples that are not next to each other b Choose Size Curve from the Sample menu For each selected sample a Sample file window opens and displays its Size Curve view Analyzing Sample Files 3 13 What the Size Curve Displays The Size Curve view displays two curves as shown in the figure below For a description of the curves see Curves Described below ELI 08 P27 8 Riz Best Fit 2nd Order Curve aso 40 9 795660E 01 Al 1 222550E 01 A2 2 4868944E 06 800 R 2 1 000 250 200 150 100 50 Size Calling Curve Local Southern Method D 1 1 D D 1000 1500 2000 2500 3000 3500 4000 4500 Data Point Curves Described The following table describes the curves in the above figure 3 14 An
264. om parametersthat These are files that you defined and are listed at the bottom they are located in the parameters of the menu folder specified in the Folder Locations preferences For information on setting the Folder Locations preferences see Specifying File Locations on page 5 42 Selecting Different To apply separate analysis parameters to selected samples Parameters for Analysis Samples Step Action 1 If the Analysis Control window is not displayed then choose Analysis Control 3 1 from the Windows menu 2 Click the arrow in the Parameters column for the sample that you want to change the parameter settings A pop up menu appears Pop up menu Project Rnalysis Control O Print Results Print Setup Parameters sAnalysis Parameters Analysis Parameters Analysis Parameters Sample File Size Standard Analyzing Project Files 5 25 Step Action 3 The pop up menu contains the following options Choose To Analysis Parameters apply the parameters that are stored as preferences in the software Define New display the Analysis Control dialog box For information on completing the fields see About the Analysis Parameters on page 5 17 4 Repeat step 2 and step 3 for each sample Note You can also use the Macintosh computer Cut Copy and Paste commands from the Edit
265. om the Results Control Window 0 0000005 9 5 From the File Menu 0 00 0 ec eee ee 9 6 Printing Supporting Information 0 00 00 e eee ee eee eee 9 6 Introduction ceret eee rete de gehe oa a 9 6 lene M MMC 9 6 Introd ctioti ie ee rec ee mec e e etes a e eo e Sine cn A 1 In This Appendix eeina ne a inae we T RIA eat A 1 Processing Using the ABI 373 0 0 cee cece eee eee A 2 Introduction 2v ovis exp aoe dk PER RN A 2 ABI 373 Versus ABI PRISM 377 Data 00 00 000 A 2 Using the GeneScan Analysis Software Before Version 2 0 A 2 Using the GeneScan Analysis Software Version 2 0 or Later A 2 Processimg Stepss uos seas EE ee a NR UESTRE Beal ne aes A 3 Comparison of GeneScan Analysis 0000 0005 A 3 Setting Gel Processing Parameters 0 00 00 c eee eee eee ee A 5 Xv Introduction 21 ye o et phos beoe qur Re ec ed e Rd A 5 Procedure e Uds ces eee gea Re a alates chee A 5 Opening the Collection File 0 0 0 0 eee eee eee eee A 7 Introduction cs ee eev alee dete ULUSM Der A 7 Procedure cit ds ced ec ed ee Aa a aca ee os A 7 Completing the Sample Sheet 0 0 0 0 e eee eee ee eee ee A 9 Introduction 42 Sess LEUTE PERRA ERG UR e E eet A 9 P ocedute an CE DXRREEU SAREE as Sas A ES A 9 Tracking Lan s un Sis eae ake NODE S e e S Mee A 11 Introdiction 54 ie ctu esr he ape et A 11 PrOCe 84 E ERR e CERE A 11 Piocedute
266. onditions certain fragments in the GeneScan 2500 standard appear as doublets or split peaks This standard has labels on both stands of the DNA Under poor denaturing conditions you see split peaks One of the two fragments typically has normal mobility while the other does not The Split Peak Correction feature allows the software to correctly call each of the splits Split Peak Parameter Options Described If you select split peak correction you will also need to verify or change the correction limits After you decide on a split peak correction method use the same method for all projects to keep size calling consistent Split Peak Correction parameter options Item Description None No correction for doublets GeneScan 2500 Makes the following peak size assignments for GeneScan 2500 Theright peak for all fragments 222 233 238 286 and 490 Theleft peak for all other splits LeftMost Peak Chooses the left peak for every doublet Split Peak Correction parameter options continued Item Description RightMost Peak Chooses the right peak for every doublet Correction Limit Set a correction limit if correcting for doublets Set this value slightly larger than the largest split observed This value set in scan lines or data points specifies the maximum width of split that should be corrected the difference in scan numbers or data points of the positions of the two peaks
267. ont of choices When you click a radio button with the cursor a black dot appears in the center of the circle to indicate your choice You can only select one at a time Buttons Rectangles with rounded corners that allow you to accept or cancel the contents of a dialog box or perform functions such as printing within the dialog box A button with a heavy outline is the default button that applies if you press the Return key 1 10 GeneScan Analysis Software Overview Registering the Software License and Before you begin please read Appendix G License and Warranty Warranty This appendix explains your rights and responsibilities regarding the software Registering Your To register your copy of the GeneScan Analysis Software complete the Software registration card included in this software package and return it to Applied Biosystems Registering the software enables us to send you notification of software updates and any other future information that may be specific to GeneScan Analysis Software owners IMPORTANT Your product registration number is located on the Registration card Be sure to record this number here before you return the Registration card Registration Number GeneScan Analysis Software Overview 1 11 Installing the Software System The following table lists the system specifications Specifications Item Specifications CPU A Power Macintosh comput
268. ooting connection F 14 to F 24 overlaid defined Glossary 2 P PCR Single stranded Conformation Polymorphism SSCP Minimum Peak Half width setting 5 21 peak detection verifying 7 53 peak positions displaying in electropherograms 7 29 peaks highlighting in electropherograms 7 29 preferences defined Glossary 3 printing about printing 9 2 automatically printing run results 9 3 gelimage 9 4 sample files 9 5 to 9 6 supporting information 9 6 program files installed diagram 1 15 project options defined Glossary 3 projects analyzing project files 5 2 to 5 5 creating finding missing sample files 4 14 to 4 15 process diagrams 4 2 to 4 3 using ABI 373 and ABI PRISM 377 4 3 using the ABI PRISM 310 4 2 using to manage sample files 4 8 to 4 9 working with 4 10 to 4 13 creating a new project 4 10 to 4 12 opening an existing project 4 10 removing samples from a project 4 13 defined Glossary 3 defining folder locations 5 41 to 5 43 saving procedure 8 3 troubleshooting D 2 to D 4 why save 8 2 R RAM required 1 14 Raw Data View 3 15 to 3 16 displaying the view 3 15 example 3 16 what it displays 3 15 what to evaluate 3 16 registering the software 1 11 restarting Macintosh key commands for E 12 Results Control window saving and renaming 7 17 to 7 19 important considerations 7 17 renaming current display 7 19 saving the format 7 17 using previously saved formats 7 18 working with previously saved displays 7 18 Results Disp
269. or baselining Off Scale The off scale detection features of the GeneScan Analysis Software Detection and identify and flag such data points in the bands of a gel image and the GeneScan peaks of an electropherogram However these features must be used in conjunction with suppress left right averaging in Data Collection refer to Setting Preferences General Settings of chapter 4 in the ABI PRISM 377 User s Manual in order to detect all instances of off scale data Displaying Displaying Off Scale Data In the Gel Image Off Scale Data Take this action Description Click the OS button in the Gel File Each pixel that is off scale is window to display where the data displayed as a white dot went off scale data in the gel image The other colors in the gel are Note To see off scale portions in turned off the gel image click the Vertical Expand button The resolution of the full display is low and does not show the off scale data When finished viewing the off scale data turn on the other colors by clicking each color button or by clicking the OS button again to deselect it Displaying Off Scale Data In the Electropherogram Pre Averaging Offscale Detection must be selected when extracting lanes for accurate displays of off scale data in electropherograms During the extraction process each data point used to generate an average value for a scan number i e channel averaging is compared to the off scale value
270. or in the same way from the Results Control window When you do so the dye sample is plotted with the set color each time you open the applicable Results Display window Note Press 3 and double click the plot color indicator to reset it to the original color Evaluating Analysis Results 7 43 How to Change the Dye Scale What the Dye Scale Defines Increasing the Dye Scale Example The dye scale defines how dyes in an electropherogram appear relative to each other You can compensate for peaks with different intensities by redefining the dye scale Note data Changing the dye scale affects only the display not the underlying The following table describes an example of increasing the dye scale If Then Action you loaded a smaller amount of green sample in relation to the red sample the peaks for the green sample might appear half as tall as those of the red sample To make it easier to view both samples on the same scale increase the dye scale value of the green sample to make the peaks appear similar Changing the Dye To change the dye scale of an individual electropherogram Scale of an Electropherogram 7 44 Evaluating Analysis Results Step Action 1 Display the electropherograms with legends 2 Double click the dye color indicator next to the sample you want to Choose a Dye Scale Cancel change The Choose a Dye Scal
271. or over a dye color field For information on how to customize this field see Displaying Sample and Dye Information on page 5 12 The following table lists how you can customize the display by changing the settings Note Chapter 7 Evaluating Analysis Results These preferences also apply to the Results Control window See To change Choose For more information information displayed in the information display field Project Options from the Settings menu and Sample Info Display from the submenu Displaying Sample and Dye Information on page 5 12 the sorting of sample files Project Options from the Settings menu and Sample File Sorting from the submenu Setting Sample File Sort Order on page 5 14 dye Indicator code Preferences from the Setting menu and Dye Setting Dye Indicator dye color Preferences on Indicators from the page 5 15 submenu To use the Analysis Control window To Then Result select all the samples click the upper left cell Click here All the columns in the Analysis Control window are selected select all of one dye color click the column heading for that dye color The column is highlighted for the color selected To use the Analysis Control window continued To Then Result select all four dyes for click the row number All the
272. ouo e ot rnE ie Pes p mt pU EID A 11 Generating Sample Files ceresna 0 0 eee eee ee eee A 12 When to Generate Sample Files 0 0 0 0 ce eee eee A 12 GeneScan Size Standards Bel Introduction sss xi ned REM Lee bei aaa as B 1 In This Apperdix 0 06 ee eere ee eedem ARS B 1 GeneScan 350 Size Standard 0 0 0 eee ee eee B 2 What To Use It For sve ep URP e oe ds aie EX RES B 2 How It Is Prepared 2 RD a EE E eee x AES B 2 GeneScan 350 Molecular Lengths 0 0 0 0 eee eee B 2 Running Under Denaturing Conditions B 2 Double Stranded GeneScan 500 Fragments B 3 GeneScan 400HD Size Standard 0 0 eee eee ee eee B 4 What To Use It For 2 0 eee ee eee B 4 Special USES cui chew UT HER eg Ss Ee dd OR i B 4 How It Is Prepared 2 ce ete eens B 4 Fragment Lengths 1 o iem menm ex SE B 4 Denaturing Electropherogram 0 0 0 0 0 eese B 5 Non denaturing Electropherogram 0000 5 B 5 GeneScan 500 Size Standard 0 0 ee eee eee B 6 What To lt Use Forss i ecce se ei Ie Mad gia od ees B 6 How It Is Prepared oii ee ite RR be eke eed aes B 6 GeneScan 500 Molecular Lengths 000 B 6 Running Under Denaturing Conditions B 6 Double Stranded GeneScan 500 Fragments 4 B 7 GeneScan 1000 Standard iles ee eee B 8 How It is Prepared ioc
273. ow to Change the Dye Scale 2 0 0 0 eee eee eee 7 44 What the Dye Scale Defines sels 7 44 Increasing the Dye Scale Example 0 0 7 44 Changing the Dye Scale of an Electropherogram 7 44 Changing the Dye Scale Preferences 7 45 xiii xiv Process of Verifying Results 00 0 00 eee eee eee eee 7 46 Introduction Seip cate reset eee Bedard Weed BBG 7 46 Steps to Verifying Size Calculation 04 7 46 How to Verify Size Calculations 7 48 Introd ction o tas ey Ln LI era 7 48 Verifying for the GeneScan 350 Standard 7 48 Evaluating for Multiple Internal Size Standards 7 50 Using the Analysis Log 0 sers eee ce eee eee 7 51 What is the Analysis Log 7 51 Displaying the Analysis Log 00 0 0 cece eee eee 7 51 What to Evaltiate 3 4 6ho 4s gk Gtk seared dae eee 7 52 Removing Information from the Analysis Log 7 52 Closing the Analysis Log 1 0 0 0 ees 7 52 How to Verify Peak Detection 7 53 Inttod cti h zio exer 3T eek eee te eee eee 7 53 Verifying Peak Detection 0 00 0 eee 7 53 Introductioti i e pere Ha CORR EA EE Eee ne HE ie ex E 8 1 In This Section e oc eC envie URxedR ee Ss i EO ROUEN 8 1 Saving GeneScan File lssleeeeeeeeeee e 8 2 Why SaV6 ciis eiie te aee e Ee br pde eue 8 2 Saving Projects io ssl e eR RU Se EE TRAE EY 8 3
274. peak See Weighted Averaging on page 2 7 The number of channels to be averaged for each lane when extracting data from the gel file is normally set to 3 Each tracker line in the Gel File window marks the channel where the GeneScan Analysis Software located the strongest fluorescent signal for that lane If you use the default three channel average without weighted averaging the raw data in each sample file is an average of the data in the channel marked by the tracker line and one channel on either side of it IMPORTANT When using multiple channel averaging ensure the tracker lines mark central channels of the bands If they mark channels at the right or left edge of the bands the empty channel between lanes is included in the average or the signal from an adjacent lane could be included causing an erroneous value 2 8 How to Process and Edit the Gel File Item Description Altering the Channel Averaging If you choose Then two channel averaging data is taken from the tracked channel and the channel to the right of it without weighted averaging You can include data from up to nine channels Three channel averaging is recommended for most applications one channel averaging no averaging if the gel bands are severely tilted For example if the left lane of the gel ran faster than the right a better result would be obtained by taking the center channel alone
275. portion of your data because of artifacts or bleed through Matrix File Example Step Action 7 Click OK This generates a new matrix file The following is an example of the Matrix Values window that appears showing the values used to calculate the overlap correction SS Run Matrix Reactions B 6 v R 1 0000 0 2304 0 0073 0 0019 0 5916 1 0000 0 2640 0 0028 B 6 y o5175 05916 1 0000 0 1445 R 0 1211 0 2663 0 5000 1 0000 For each dye the value where the dye fluorescence is read by the appropriate filter is 1 000 The adjacent colors show the amount of overlap for which the system must compensate The adjacent values in most cases should be less than 1 000 but equal to or greater than 0 0000 Note In some Filter Set C matrices the matrix value for green in the first column can vary from about 0 8 to 1 2 Making a Matrix File 6 33 Saving and Naming the Matrix File Introduction Naming Considerations Saving the Matrix File Where to Store the Matrix File When to Make Copies of a Matrix 6 34 Making a Matrix File The matrix file is instrument specific You cannot apply a matrix file you made on the ABI 373 to data you collected on a ABI PRISM 377 or ABI PRISM 310 nor can you apply a matrix file made on a ABI 373 to a Sample or gel file made on another ABI 373 Also you cannot apply matrix files created on one instrument to othe
276. ppendix sum Ree Rer eye D 1 Troubleshooting Projects and Results 0 0 0 ce eee ee eee D 2 Table Description 0 0 eee cece tee een eae D 2 Troubleshooting Gel Data D 5 Table Description e xD ale ede as Rh D 5 Troubleshooting Genotyping Results D 8 Table Desctiptioti 34 488 oh eh oe PERO a ees I ete PE D 8 GeneScan Error Messages 0 0 cece cece eee eee een en eens D 9 Introduction se sil eke UR wok a ee eee RP eR D 9 GeneScan Analysis Software Crashes with BIoLIMS D 9 Analysis Log Error Messages 0 0000 e eee eee eee D 10 Error Messages When Defining Size Standards D 11 Using the BioLIMS Database E l Introduction 4 5 cente ac et Se tame ete aan dieu Sealey A ID e acea E 1 In This Appendix nior p LU ee oaks SEED Ru E 1 About the GeneScan Analysis Software and the BioLIMS Database E 2 What is the BioLIMS Database 0000 E 2 Accessing the BioLIMS Database 00 0 eese E 2 Before Using the BioLIMS Database E 2 Modes in GeneScan Analysis Software 00 E 2 Comparing Modes 0 cee eect eee eens E 3 Configuring the BioLIMS Database Server 000 E 4 Sybase or Oracle ish os epe tes Ee eR EN lee ates E 4 Configuring for Sybase SQL Server Connection E 4 Configuring for Oracle Server Connection E 7 Switc
277. r extracting Sample file data If you do not select this option the setting used for the extraction are discarded when you close the gel file without saving When all of the information in the Extract Lanes dialog box is correct click OK to begin extracting data 2 66 How to Process and Edit the Gel File How the GeneScan Analysis Software Names Sample Files Naming Process The following table lists the process the GeneScan Analysis Software follows when naming generated Sample files If the generated Sample file Then GeneScan uses the has an associated file name in the Sample Sheet file name in the Sample Sheet does not have an associated file name but has a sample name lane number of the sample concatenated in front of the sample name for example 02 Test1 does not have an associated file name or a sample name lane number of the sample concatenated in front of the name Sample file for example 02 Sample File has the same name as a previously generated Sample file lane number of the sample in parentheses concatenated to the file name for example Test1 1 Test2 2 a These defaults can be edited in GS Collection Refer to the ABI PRISM 377 Instrument User s Manual How to Process and Edit the Gel File 2 67 Saving Gel Files After Editing Tracking Introduction The Gel File window keeps track of whether or not you have edited data and when
278. r or in the matrix folder in the GeneScan Analysis Software folder The values of the matrix are stored in the gel file ABI 373 and ABI PRISM 377 and in the Sample files For more information see Chapter 6 Making a Matrix File module A file that provides instructions about conditions of operation to the ABI PRISM genetic analysis instrument Refer to your instrument manual multicomponenting Adjusting for spectral overlap of the fluorescent dyes overlaid Displayed together so they overlap In the GeneScan Analysis Software Results Display window all electropherograms in a single panel are overlaid You can bring a specific one to the front by clicking the color box that represents it in the legend See Using Legends to Change the Display on page 7 30 plot color indicator Right color box in the Results Control window and the legend of the Results Display In the Results Display window click this box to move the associated electropherogram to the front In the Results Control and the Results Display windows double click this box to change the plot color or 3 double click it to reset the plot color to the default Glossary 2 preferences Defaults you can set so that certain parameters are automatically applied when you are working with a project The GeneScan Analysis Software remembers preferences and applies them globally to all new projects For a brief description of each preference you can set see Changing How the Resu
279. r instruments of the same model When naming a matrix file consider including the following information in the name Item For example Instrument type ABI 378 ABI PRISM 377 or ABI PRISM 310 Filter set used A B or C Gel conditions native or denaturing To save the matrix file Step Action 1 If a matrix folder does not already exist inside your GeneScan applications folder create one 2 Choose Save from the File menu A dialog box appears 3 Enter a descriptive name for the new matrix file and click Save Store matrix files that are intended for use by data collection to assign to collection runs in the ABI folder The ABI folder is located in the System folder of the computer on which Data Collection software is installed You can make a copy of a matrix and store one in the ABI Folder for use by the Data Collection software and the other in the GS Matrix Folder for use by the GeneScan Analysis Software This is useful when data collection and analysis are performed on different computers Assigning the Matrix File to Sample Files Introduction After generating the new matrix file assign it to all the Sample files that you want to analyze Procedure IMPORTANT After assigning your matrix file to Sample files see Evaluating the Matrix File on page 6 37 To assign a matrix file to Sample files Step Action Assigning a matrix file to Sample files
280. rand is detected Because of this split peaks are avoided that result when two strands move through a denaturing polymer at different rates see the figure below UP 115 Gs 350 Size Std Double Stranded The following figure shows the sizes of double stranded GeneScan 500 GeneScan 500 fragments Use these values to size fragments run under native Fragments conditions IMPORTANT An asterisk for the 250 and 340 base pair peaks denotes peaks resulting from abnormal migration of double strands that did not completely separate under denaturing conditions when analyzed on the ABI PRISM 310 Do not use these peaks to size samples The peaks show smaller values than the actual size of the fragments Refer to the GeneScan Reference Guide Chemistry Reference for the ABI PRISM 310 Genetic Analyzer P N 4303189 rev A for further details 30 180 270 360 150 160 200 250 300 340 350 75 100 50 2R GeneScan Size Standards B 3 GeneScan 400HD Size Standard What To Use It For Special Uses How It Is Prepared Fragment Lengths B 4 GeneScan Size Standards You can use the GeneScan 400HD High Density size standard to determine fragment lengths between 50 and 400 base pairs The high density of marker bands in this standard makes it particularly useful for microsatellite analysis All fragm
281. ration options continued Item Description Estimated Maximum Peak Height In this text box enter the maximum signal level you expect from samples in the run This can be an approximate number based on your typical run conditions and samples The default is 2000 Note This option affects only the appearance of the gel image not the raw data when the image is generated the first time the gel file is opened Use the Regenerate Gel Image command to change the appearance of an open gel window See page 2 23 The gel image can also be modified using the Adjust Gel Contrast command see Adjusting the Contrast on page 2 20 The Estimated Maximum Peak Height value Affects the color brightness of the DNA fragment bands All bands with a data value at or above the Estimated Maximum Peak Height value are assigned the brightest dye color The dye colors for bands with values below that level are dimmed proportionally unless multicomponented In general the lower you set this value the brighter the bands appear in the Gel File window A value of 1000 is satisfactory for most gel files If the gel image is very dim try 500 if it is too bright try 2000 Determines the scale of the peaks in the Slice view of the Gel File window For the highest quality gel image the highest sample peaks not the Primer peak should just reach the top of the scale in the Slice view If you find that many of
282. rd disk choose Open from the file menu The lt Collection Settings gt does not change 2 Make any changes to the file 3 Choose the edited file from the Standard pop up menu to apply the changes Editing an Existing The two ways to edit a previously defined standard are by Standard 4 Using the Open Command to Edit an Existing Standard Using the Analysis Control Window to Edit an Existing Standard Using the Open Command to Edit an Existing Standard Step Action 1 Choose Open 36 O from the File menu The Open Existing dialog box appears Open Existing Collection Project Sample Sample Analysis Size Gel Sheet Parameters Standard 2 Click the Size Standard icon A directory dialog box appears 3 Select the standard file that you want to modify 4 Edit the peak size values by following step 7 to step 9 on page 5 34 5 Choose Save or Save As from the File menu You can take the following action If you choose Then the Save existing file is replaced Save As changes are saved using a file name you specify Analyzing Project Files 5 37 5 38 Analyzing Project Files Using the Analysis Control Window to Edit an Existing Standard Step Action 1 If the Analysis Control window is not displayed choose Analysis Control 36 1 from the Windows menu 2 Click the arrow in th
283. rd for shorter runs The Analysis log will however alert you if less than 50 of the defined size standard peaks were not matched regardless of the peak locations in the definition To remove information from the Analysis Log Step Action 1 Select the information you want to remove Note Choose Select All 38 A from the Edit menu to select all the information 2 Choose Clear from the Edit menu You can either Click the close box in the upper left corner Choose Close 3 W from the File menu How to Verify Peak Detection Introduction Use the Show Peak Position command while the electropherogram and associated tabular data are displayed to verify results by examining how the GeneScan Analysis Software defined the total area that comprises each peak and the center of the peak Verifying Peak To verify peak detection Detection Step Action 1 Choose Show Peak Positions from the View menu Markers appear that identify the beginning center and end of each peak Note For a better view choose Zoom In from the View menu or use the Zoom tool see Zooming In and Out on page 7 31 HL P14 ctist 2 13 plex Sul 7 Display 8 Examine the display to ensure that each peak s center beginning and end points are correct Choose Hide Peak Positions from the View menu to suppress the display of the peak markers Note You can also use the Sample Info view
284. re selected the GeneScan Analysis Software automatically prints the Sample file information after it automatically analyzes the files 10 Collection Name lIf the samples are extracted into the BioLIMS 2 0 database this is the name of the collection that will contain them If a collection of that name does not exist in the database one is created See Editing Collection Information on page 2 34 Use the Collection Manager to group samples in any number of desired collections see Appendix E Using the BioLIMS Database 11 Collection Comment Comment text associated with BioLIMS 2 0 collection name specified in the Collection Name field described above 12 Collection Owner Collection Creator text associated with BioLIMS 2 0 collection name specified in the Collection Name field How to Process and Edit the Gel File 2 33 Working with the Sample Sheet Editing Collection How to Edit Collection Information Information co the pop up menu in the Collection Name column to edit collection information that corresponds to that sample Pop up menu GELO1 980120 15 Sample Sheet B Sample Info i Comment Collection Name f Collection Comment i Collection Owner Edit Collection Names none Using the Pop Up Menu To use the pop up menu to edit the collection information If you want to Then select a choose a collection from the pop up menu
285. remains visible turn on the tracker lines choose Show Tracker Lines from the Gel menu The following table lists how all the tracker lines are displayed If Then no lane is selected all lanes are grey a lane is selected that lane is white and all the other lanes are grey display one tracker line click the lane marker 4 for the tracker line you want to view The unselected tracker lines are greyed out How to Process and Edit the Gel File 2 53 Why Positioning The GeneScan Analysis Software normally calculates the data values Each Laneis for each lane by averaging the data from selected channels portans Unless each lane is positioned at the optimum channel of the fragment band the resulting calculations for peak height and peak area may be incorrect Size calling however is still correct Causes to The following table lists the possible conditions that may cause Misinterpreting automatic tracking to misinterpret lane positions or fail to follow the path Lane Positions of a lane completely Condition Result Weak signals Causes the software to completely miss or be unable to follow a lane especially if the gel ran abnormally Although the software creates a track for each Used lane on the gel the tracker lines might be incorrectly placed indicating lane positions that do not exist or that are located elsewhere Gels that contain Causes the softw
286. rences Size Standard pop up menu from the default standard or any standard files in the folder location that you specify in the application preferences Standard Dye pop up menu the inlane standard dye Analyzing Sample Files 3 19 Installing a New Matrix File 3 20 Analyzing Sample Files To analyze a sample file continued Step Action 3 After analysis evaluate the results For information on evaluating the results see Chapter 7 Evaluating Analysis Results Use the following procedure to install a new matrix file for the Sample file that you want to analyze For information on attaching the new matrix to an ABI 373 or ABI PRISM 377 gel file see Installing New Matrix Information on page 2 24 To install a new matrix file Step Action 1 Choose Install New Matrix from the Sample menu A directory dialog box appears The Folder Preferences settings determine where the GeneScan Analysis Software looks for the matrix file For more information see Defining Folder Locations on page 5 41 2 Select the new matrix file in the dialog box and click Open A message appears when the matrix is successfully assigned 3 Re Analyze the Sample file Applying a new matrix file clears previous analysis information so you must re analyze the file Creating a Project Introduction In This Chapter Topics in this chapter include the followin
287. ress eg 129 25 51 36 Oracle SID Identifies the Oracle database 4 Enter text in the fields as follows Alias Name Enter an alias name for the database server See About Server Names on page E 17 Server Name Enter the server name This may be an IP address or host and domain name of the server machine Note This field does not scroll horizontally for display even though it accepts characters typed past the end of the field If the server name is longer than 20 characters you may want to enter the end characters first and go back or just use the IP address Oracle SID Enter the value of the ORACLE SID environment variable You can find this information in the tnsnames ora file on the Oracle Server or your BioLIMS database administrator can provide you with the information 5 Click OK to close the TCP IP dialog box 6 From the File menu choose Save Configuration 7 From the File menu choose Quit to exit the Easy Config program To configure for Oracle Server connection continued Step Action 8 Find the application Set Oracle Home ORACLE Set Oracle Home The application is contained in your BioLIMS 2 0 BioLIMS Extras Oracle Applications folder v BioLIMS 2 0 Vv O BioLIMS Extras v O Oracle v 3 Applications Set Oracle Home 4 SQL Plus gt O Libraries gt O Network 9 Open the application Set Oracle Home Select a new Oracle Home Select BioLIMS Extras C3 BioLI
288. rnal size standard Also called internal lane or injection size standard DNA fragment of known size that you include with your run On the ABI 373 or ABI PRISM 377 you include the size standard with the samples in each lane On the ABI PRISM 310 you include the size standard with each injection Running an internal lane standard results in particularly accurate and precise molecular length determination because the internal lane standard and the unknown fragments undergo exactly the same electrophoretic forces The software can then compensate for band shift artifacts caused by variations in the gel and in the run See About Size Standards on page 5 29 and Using Size Standards on page 5 36 legend Informational text that appears beneath electropherogram panels in the results displays You can show or hide legends see Using Legends to Change the Display on page 7 30 and you can use the color boxes displayed in them to bring specified electropherograms to the front of the panel Moving the Electropherogram on page 7 28 or customize the colors How to Define Custom Colors on page 7 40 matrix file multicomponent matrix File used to adjust for the spectral overlap between the fluorescent dyes used on the ABI PRisM instruments A mathematical matrix of the spectral overlaps is created and the inverse matrix is used to correct the data during analysis Matrix files are stored in the ABI folder inside the Macintosh computer System folde
289. rol window You can also select the file in the Data Collection software for automatic analysis Changing an To change an existing analysis parameter file Existing Analysis Parameters File Step Action 1 Choose Open 36 O from the File menu Note You can also double click the file name in the Parameters column of the Analysis Control window The Open Existing dialog box appears Open Existing gamag Collection Project Sample Sample Analysis Size Gel Sheet Parameters Standard 2 Click the Analysis Parameters icon A directory dialog box appears 3 Select a file that you want to change and click Open Analyzing Project Files 5 27 Step Action Make the changes and close the window by clicking the close box Close box SS 2400 36 STD RP Rnalysis Range Size Call Range Full Range All Sizes This Range Data Points This Range Base Pairs Start 1300 Stop 6000 Deleting Custom To delete a custom analysis parameters file Analysis Parameters 5 28 Analyzing Project Files Step Action 1 Find the file in the folder you specified as the location for the analysis parameters files Note The custom analysis parameters file is in the folder that is normally called the GS Parameters Folder This folder is inside the ABI PRISM GeneScan folder 2 Drag the custom analysis parameters file to the tra
290. rt to Straight Tracking Ruto Track Lanes You can take the following actions If you choose Then this Cancel cancels the tracking operation if you do not want to lose the current tracking information Revert to Straight Tracking adds straight evenly spaced tracker lines to the gel For information about moving these lines to the region of the highest signal within the lane see Working with Tracker Lines on page 2 53 2 60 How to Process and Edit the Gel File If you choose Then this Auto Track Lane places a tracker line with spline control points over the strongest signals for its lane IMPORTANT Before choosing Auto Track Lane verify that the comb type is set correctly by For information about choosing Preferences from the Setting the lane assignment Settings menu and Gel Preferences confidence value see from the submenu Confidence Threshold text entry field on page 2 10 Moving these lines to the region of the highest signal within the lane see Working with Tracker Lines on page 2 53 Tracking and Why Use This Procedure Extracting Data Use this procedure to automatically track and extract lanes IMPORTANT When you Track and Extract a gel file ensure the Sample Sheet associated with the gel file has the checkbox labeled Used selected for each sample you want to extract The software only extracts data from used lanes see Sampl
291. s Electropherogram The off scale peaks in the electropherogram in the expanded view Displaying the Flat below illustrates the flat topped effect Topped Effect Flat topped peaks Showing Data by Introduction Fragment Size If you analyze your samples with an internal size standard you can use the Align by Size command to show the horizontal scale of the electropherograms by fragment size instead of by data point Note You can only display data by size if you analyzed your samples using an internal size standard Adjusts for Run Variations The Align by Size command adjusts for run to run and lane to lane variations and thereby eliminates any apparent differences and any possible confusion caused by run discrepancies For example if you run two identical samples in different runs aligning peaks by size value eliminates any apparent differences and any possible confusion caused by run discrepancies With ABI 373 and ABI PRISM 377 data the command lets you align two different samples run on the same gel using the internal size standard as a reference Evaluating Analysis Results 7 35 7 36 Evaluating Analysis Results Note You can display overlaid samples in the same dye in different colors See How to Define Custom Colors on page 7 40 How to Switch Between Size and Data Point Display To switch between size and data point display If you want to Then show data by size choose Align by Size 3
292. s Appendix Topics in this appendix includes the following Topic See Least Square Method C 2 Cubic Spline Interpolation Method C 4 Local Southern Method C 5 Global Southern Method C 7 Size Calling Methods C 1 Least Square Method About This Method Advantages C 2 Size Calling Methods Both Least Squares methods 2nd Order and 3rd Order use regression analysis to build a best fit size calling curve This curve compensates for any fragments that may run anomalously As a result this method normally results in the least amount of error for all the fragments including the size standards and the samples Depending on whether you choose the 2nd or 3rd Order Least Squares Method in the Analysis Parameters dialog box the resulting size curve is either a quadratic or a cubic function The software uses the known standard fragments and the associated scan number positions to produce a sizing curve based on Multiple Linear Regression In the figures Figure C 1 and Figure C 2 you can see that in nearly all instances the mobility of an individual DNA fragment is coincident with the best curve fit of the entire data set Stated differently the mobility of most DNA fragments is strictly length dependent This method automatically compensates for fragments that run anomalously GeneScan Analysis Software calculates a best fit least squares curve for all samples regardless of the size calling method you choose T
293. s Software Before beginning the installation backup the copies of the older versions of the GeneScan Analysis Software IMPORTANT After installation be sure to register your software To install the GeneScan Analysis Software Step Action 3 Quit all currently running applications 4 Hold down the Shift key and choose Restart from the Special menu to turn off all extensions 5 Continue to hold down the shift key until the message Extension Off appears 6 Insert the GeneScan Analysis Software install disk 7 Double click the Installer icon The GeneScan Analysis Software installer appears 8 Choose a folder in which to install the software 9 Choose either 310 GeneScan Analysis 3 0 or 373 377 XL GeneScan Analysis 3 0 depending on your instrument 10 Click Install Insert other disks as the Installer asks for them 11 Click Quit at the end of the installation 1 14 GeneScan Analysis Software Overview Program Files The following diagram shows the program files installed on the hard Installed Diagram disk ABI PRISM GeneScan 3 0 Folder Contents ABIA PRISM ABI PRISM aca 3 0 GS Gel Tracker GeneScan GS Parameters GS Standards GS Matrix folder folder older folder o gt Contents of The installation program creates an ABI PRISM GeneScan 3 0 folder on Folders the hard disk containing the following Folder Contents GS Parameters folder Use to store us
294. s extracted Gel processing parameters Sample Information Information found under header Information is inserted from Sample data and comment for each dye color ABI PRISM 310 Sample Sheet ABI 373 or ABI PRISM 377 gel file Sample Sheet This information is embedded in the gel file Analysis Records Information found under header Information is inserted from Date and time each color was analyzed and more panel display arrows Analysis information Analyzing Sample Files 3 11 Dyes Within Analysis Records Information found under header Information is inserted from Analysis parameters file and Analysis Settings range analyzed Whether baselined or multicomponented Data smoothing Peak detection threshold and minimum half width Size standard file Analysis Control window The dye color used for the standard Sizing method and range Standard peak detection Analysis Settings threshold Split Peak correction Total number of peaks found in Analysis results sample amp dye standard defined in standard matched with standard peaks 3 12 Analyzing Sample Files Size Curve View What it Displays Displays sizing curves for Sample files The size curve is a measure of how well the internal size standard matches the standard definition and whether or not it is linear Displaying the The f
295. sh and empty the trash About Size Standards What are Size Standards Advantages of Using Size Standard Size Standards Provided When to Define Size Standards If Split Peaks Appear Size standards are specific DNA fragments of known sizes After defining the peaks of a size standard the GeneScan Analysis Software matches this definition to the internal size standard included with the run The software assigns the defined size values to the appropriate peaks of the internal size standard and uses this information with the selected size calling method to size all unknown fragments Running an internal size standard results in accurate and precise molecular length determination This is because the internal size standard and the unknown fragments undergo exactly the same electrophoretic forces The GeneScan Analysis Software can then compensate for band shift artifacts caused by variations in the gel and in the sample from lane to lane or injection to injection Applied Biosystems provides several fluorescently labeled standards which are described in Appendix B You can also label and use other fragments if they better suit the fragment sizes with which you are working Normally a size standard is defined using the GeneScan Analysis Software after running the standard with samples on the instrument The software detects peaks for a selected dye color in a selected Sample file and allows you to define the peak sizes
296. side are averaged to determine the raw data for the Sample file You set the number of channels to be averaged in the Gel Processing Parameters data point The ABI 373 and ABI PRISM 377 Data Collection software samples data 194 times as it Scans across the gel 194 or 388 times for the XL upgrades The ABI Prism 310 Data Collection software samples data as it passes by the detector Each sampling is stored as a data point In ABI 373 and ABI PRISM 377 data the scan number describes the location of the data point dye sample Individual sample labeled with a single dye within a Sample file Sample files normally contain up to four dye samples depending on how many labeled samples you included in each lane or injection of your data collection run Glossary 1 dye color indicator Left color box in the Results Control window and the legend of the Results Display In the Results Display click this box to move the associated electropherogram to the front In the Results Control and the Results Display windows double click this box to change the dye scale or 3 double click it to reset the dye scale to the default electropherogram Four color picture of a sequence showing peaks that represent the bases The term is used interchangeably with chromatogram in this manual grid Spreadsheet like display used for entering data in tabular format The Analysis Control and Results Control windows display grids for entering sample information inte
297. sing the Add Sample Dialog Box To use the Add Sample dialog box Step Action 1 Find the folder containing the samples that you want to add from the top scroll box x Sample Files v 5 02 Sample File 5 03 Sample File 5 04eSample File 5 05eSample File 5 06eSample File 0 Sample File 5 08eSample File Hard Drive Eject Desktop Add All Sample Files Hemave Hone Cancel Creatinga Project 4 11 Unlocking Sample 4 12 Creating a Project To use the Add Sample dialog box continued Step Action 2 You can take the following action If Then you want to select a single double click the file or select Sample file the file and click Add you want to select all the click Add All Sample files you want to add a random double click each file name selection of Sample files 3 Click Done when you have added all the Sample files If the Then Sample files appear in the see Analyzing Sample Files Analysis Control window Using the Analysis Control Window on page 5 6 Locked files alert appears see go to Unlocking Sample Files below below E Some Sample Files are locked They will be shown in italics within the Project windows If the Sample files added to a project are locked the GeneScan Files Analysis Software does not allow changes to them You cannot
298. sing the Install New Sample Sheet command from the Gel menu see Installing a New Sample Sheet on page 2 36 Choose Save 36 S or Save As from the File menu If you click the close box before saving a dialog box appears with the message whether or not to save the entries Running the Gel To run the gel 6 22 Making a Matrix File Step Action 1 Open the Data Collection software 2 Set the run time and Data Collection File name 3 On the ABI 373 instrument choose the appropriate filter set for the conditions you wish to duplicate For this matrix Choose Dye Primer or dNTP matrix Filter Set A Fluorescent Amidite matrix Filter Set B Run the matrix standards Start the electrophoresis run according to the conditions specified in your instruction manual Completing the To complete the Sample Sheet in the GeneScan Analysis Software GeneScan Sample Sheet Step Action 1 Open the GeneScan Analysis Software program 2 Choose New from the File menu The Create New dialog box appears Create Neu 9 598 fw ful Project Sample Analysis Size Matrix Sheet Parameters Standard Cancel Click the Sample Sheet icon The following dialog box appears Number 0f Dyes Select the number 4d of dyes 9 ges Q5 dyes tance Click the radio button that corresponds to the number of dyes and click OK
299. stadt Tel 49 0 6150 1010 Fax 49 0 6150 101 101 Russia Moskva Tel 7 095 935 8888 Fax 7 095 564 8787 Spain Tres Cantos Tel 34 0 91 806 1210 Fax 34 0 91 806 1206 South Africa Johannesburg Tel 27 11 478 0411 Fax 27 11 478 0349 Sweden Stockholm Tel 46 0 8 619 4400 Fax 46 0 8 619 4401 United Kingdom Warrington Cheshire Tel 44 0 1925 825650 Fax 44 0 1925 282502 Switzerland Rotkreuz Tel 44 0 41 799 7777 Fax 41 0 41 790 0676 South East Europe Zagreb Croatia Tel 385 1 34 91 927 Fax 385 1 34 91 840 Middle Eastern Countries and North Africa Monza Italia Tel 39 0 39 8389 481 Fax 39 0 39 8389 493 Africa English Speaking and West Asia Fairlands South Africa Tel 27 11 478 0411 Fax 27 11 478 0349 All Other Countries Not Listed Warrington UK Tel 44 0 1925 282481 Fax 44 0 1925 282509 Japan Japan Hatchobori Chuo Ku Tokyo Tel 81 3 5566 6100 Fax 81 3 5566 6501 GeneScan Analysis Software Overview 1 25 Eastern Asia China Oceania Australia Scoresby Victoria Malaysia Petaling Jaya Tel 61 3 9730 8600 Tel 60 3 758 8268 Fax 61 3 9730 8799 Fax 60 3 754 9043 China Beijing Singapore Tel 86 10 6238 1156 Tel 65 896 2168 Fax 86 10 6238 1162 Fax 65 896 2147 Hong Kong Taiwan Taipei Hsien Tel 852 2756 6928 Tel 886 2 2698 3505 Fax 852 2756 6968 Fax
300. struments supported by Applied Biosystems Instrument Description ABI PRISM 310 Analyzes one sample at a time using capillary Genetic Analyzer electrophoresis technology This instrument provides automatic sample loading while using a minimal amount of sample ABI 373 DNA Performs slab gel electrophoresis allowing the user Sequencer to analyze multiple samples on a gel ABI PRISM 377 A high throughput slab gel electrophoresis DNA Sequencer instrument created to meet the needs of high volume DNA sequencing or genetic analysis laboratories Throughput is up to than four times that of the ABI 373 GeneScan Analysis Software Overview 1 5 1 6 User Attention The text of this manual includes the following user attention words to Words draw your attention to specific details of the information presented in this manual User Attention word Description Note Used to call attention to information IMPORTANT Indicates information that is necessary for proper operation of the software GeneScan Analysis Software Overview Using the Macintosh Computer Macintosh To use the GeneScan Analysis Software you should be familiar with the Computer following basic Macintosh computer vocabulary and operations Vocabulary and Operations Guidelines for Optimal Performance Vocabulary and operations Description Using the mouse Clicking and double clicking selectin
301. t Appears Searching for Missing Sample Files 4 14 Creating a Project The project and related Sample files are usually located in the Run folder created by the Data Collection software If the Sample files or the project are moved so they are no longer in the same relative position the GeneScan Analysis Software might not be able to locate the Sample files when the project is opened Usually this only occurs if the Sample files are moved to another disk drive another server on a network or to another disk partition on the hard drive If the GeneScan Analysis Software does not locate the Sample files associated with a project an alert box appears and the Sample file names appear dimmed when the project opens You can re establish the links between the Sample files and the project by choosing Find Missing Sample Files from the Project menu and choosing one of the following options from the submenu Note Version 2 1 and later versions of the software allows you to choose how you want to search for missing Sample files Searching for missing sample files Choose If you Description Once the volume is mounted or the diskette containing the files is inserted Fast Search finds them immediately Fast Search suspect that the GeneScan Analysis Software could not find the missing Sample files because they are located on an unmounted external storage device or diskette know what folder contains the missing S
302. t to the Sample Sheet folder Creating a The Sample Sheet is imported into the Injection List which defines the GeneScan sample names and the initial injection order Inject List To create a GeneScan Injection List Step Action 1 Choose New from the File menu 2 Click the GeneScan Injection List icon The GeneScan Injection List appears Injection List Sample Sheet B Length to Detector em Operator Inj Tube amp Sample Name 1 6S Short Denatured C P j r GS Short Denatured C f 10 7 0 13 0 30 18 Bogus matris P O P GS Short Denatured C P 10 7 0 13 0 so 18 Bogus matris P O GS Short Denatured C gt 3 Choose a Sample Sheet from the Sample Sheet pop up menu 4 Choose the appropriate module in the Module pop up menu for lines 1 through 4 for example A1 A3 A5 and A7 5 Deselect Auto Anlz analysis Deselect Auto Print 6 14 Making a Matrix File Starting the Data To start the Data Collection software Collection Software Step Action 1 Click the Run button The following windows open Window Description Raw Data window Shows the real time chromatogram of the run Log Window Shows the real time written record of run events 2 Choose Status from the Window menu The current run is in italics in the Injection List You can monitor
303. table lists the error message you might encounter if the Software Crashes with BioLIMS application crashes with BioLIMS installed Observation Possible Cause Recommended Action GeneScan Analysis Software with BioLIMS crashes on launch or gives an error message un Could not open Insufficient memory to load the Oracle or Sybase libraries Ensure that there is at least 2 MB of free memory in addition to the preferred memory requirements set on the GeneScan Analysis Software Info box To access the Info box select the GeneScan application and choose Get Info from the File menu GeneScan Analysis Software with BioLIMS crashes when attempting to connect to a Sybase database SybaseConfig control panel or libtcp extension file is missing or disabled Use the Extension Manager to check that these files are present and turned on If either file is missing then reinstall the BioLIMS Client software Troubleshooting the GeneScan Software D 9 Analysis Log The following table lists the error messages you might encounter in the Error Messages GeneScan Analysis Software Log Analysis Log error messages Analysis Log Error Message Comment Correction Refer To The Analysis Range Make sure the Analysis Defining Analysis does not include Range in your Analysis Parameters on enough data points Parameters contains at page 5 18 2 in
304. te accessing the BioLIMS database Once you have accessed the BioLIMS database you can display the Collection Browser window by choosing the Open command from the File menu Before you can connect to the BioLIMS database you must have installed the BioLIMS 2 0 software It is recommended that you install the BioLIMS software first then install the GeneScan Analysis Software into the BioLIMS 2 0 software folder Note This organization allows you to install the Sequencing Analysis software at a later time For more information see Installing the Software on page 1 12 If you are using the GeneScan Analysis Software with the BioLIMS database you can use the software in either of two modes Mode Descriptions Sample File mode In Sample File mode fragment data extracted from gel files is written out to individual sample files BioLIMS mode In BioLIMS mode fragment data extracted from gel files is written to a BioLIMS database that resides on a Oracle Server database or Sybase SQL Server Sample files that came from the ABI PRISM 310 Genetic Analyzer can be uploaded to the BioLIMS database using the Sample 2DB Software Comparing Modes The following table is a comparison of BioLIMS and Sample File modes and for analysis from Feature BioLIMS mode Sample File mode data extracted from the BioLIMS sample files a gel file is written database to fragment data is the BioLIMS samp
305. the BioLIMS Database E 21 Collection Browser The following is an example of the Collection Browser window Window Example Collection search and text boxes Fragment search criteria pop up menus and text boxes Criteria pop up menu Search button Collection Browser Select criteria 4 to find Collections with Fragments Collection Creator contains Creation Date Modification Date Collection Name contains s criteria pop up menus Collection Type i Sequence Frag Name contains sit Sample Creator contains Sample Name contains Split bar Search results Status line Name Modified Tupe Creator 2 collections found b GS0259 373xL 64wSQ S3L Cust May 29 1998 10 41 11 AM project b GS0260 273xL 64wSQ S2L Cust May 29 1998 10 36 28 AM project Parts of the The table below describes the parts of the Collection Browser window Collection Browser that were labeled in the figure above Window Item Description Criteria pop up menu Use this pop up menu to specify the search criteria visible on the Collection Browser window Note If you only intend to use a subset of criteria setting only that subset visible helps to reduce clutter in the window The search results are the same whether a criterion is invisible or blank and visible Search button Click this button to quer
306. the Code column 5 To change a color select a new color from the pop up menu If you choose Other from the pop up menu a color picker appears Original B New 180 Hue Angle e2 Saturation 97 T Value 95 50 100 C Using the color picker To use the Then scrolling fields click one of the up or down arrows and hold down the mouse button The new color appears in the New box and the Original box retains the original color until clicking OK to accept the new color color wheel a Click the desired color in the wheel The new color appears in the New box b Move the insertion point and click again until the desired color appears 6 Click OK when finished changing dye indicator preferences 5 16 Analyzing Project Files About the Analysis Parameters Introduction Parameters Used During Automatic Analysis Specifying Where Files are Stored The GeneScan Analysis Software has default analysis parameters that are stored as preferences You can set and use these default analysis parameters or create analysis parameters files to use with specific protocols During automatic analysis the GeneScan Analysis Software uses the information from the data collection Injection List or Run Sheet window to determine which parameters to use If you do not specify the information in the Data Collection software the GeneScan Analysis Software
307. the gel image c Regenerate the multicompo nented gel image with multicomponent ing selected Peaks appearing in a Bleed through from Repeat dye color that should other colors because of electrophoresis load not be present off scale data less sample TAMRA labeled size standard appears yellow on the gel display Collected using Filter set A ABI 373 or Virtual filter A ABI PRISM 377 Repeat electrophoresis with Filter set B ABI 373 or Virtual filter C ABI PRISM 377 TET labeled products not seen on gel display Collected using Filter set A ABI 373 or Virtual filter A ABI PRISM 377 Repeat electrophoresis with Filter set B ABI 373 or Virtual filter C ABI PRISM 377 Troubleshooting the GeneScan Software D 5 Troubleshooting gel data continued Problem Probable Cause Correction Signal showing up in Leaking wells of gel Consider using a neighboring lanes square tooth comb instead of a shark tooth comb If using 96 lanes then rerun gel using protocol in the ABI PRISM 377 DNA Sequencer 96 Lane Upgrade User s Manual P N 4305423 Signal intensity very Move tracker lane high and signal is position from center of being detected in band to edge of the neighboring lanes due band away from strong to closeness of signal and extract as spacing usual Use 1 or 2 lane averaging to extract
308. the indicator showing that it has been modified Evaluating Analysis Results 7 5 7 6 Evaluating Analysis Results Results Control window descriptions No Description 8 Dye color indicator If you Then double click the dye color the Choose a Dye Scale dialog indicator box appears Choose a Dye Scale CNN Cum For more information see Changing the Dye Scale of an Electropherogram on page 7 44 double click the dye color the dye color indicator returns indicator to the default scale For more information see Changing the Dye Scale Preferences on page 7 45 Note If you change the scale a vertical line appears beside the indicator showing that it has been modified 9 Clear panel button See Removing Samples on page 7 12 10 Quick Tile radio buttons See Creating Tiled Electropherogram Displays on page 7 10 11 Display button See Displaying the Results on page 7 12 12 Clear All button See Removing Samples on page 7 12 13 Print button See Printing the Results on page 7 12 Using the Results Control Window Selecting Display Choose the results display format by clicking either or both the Format electropherogram and tabular icons You must select one of the icons to display or print data Click this icon To display And the electropherogram data panel information below the icon is enabled tabul
309. the peaks are cut off you may want to readjust the Estimated Peak Height value to a higher number 2 6 How to Process and Edit the Gel File Lane Extraction The following table lists the options in the Lane Extraction section Item Description Use Weighted Weighted channel averaging is a new feature in Averaging GeneScan v 3 0 checkbox Weighted averaging is now possible because the new Tracker interface allows tracker line placement to within a tenth of a channel No Weighted Averaging If the Use Weighted Averaging box is not checked data averaging is done per channel the same as with earlier versions of the GeneScan Analysis Software For example if the tracker line falls within channel 10 and three channel averaging is set long len 10 len 41 channel average 3 Where is the intensity for a given channel and scan number Weighted Averaging If the Use Weighted Averaging box is selected the tracker line falls 20 into channel 10 see diagram below and two channel averaging is set channel lenox 07 Cen 10 X 1 Clon ii D Con 12x 9 3 average 3 I Tracker line l EJ gt e S l D l 9 10 11 12 Channel Number How to Process and Edit the Gel File 2 7 Item Description Use Channel Averaging text entry field Averaging reduces the amount of noise in the sample file and allows for more accurate representation of the band as a
310. this manual Manual Item Description Dialog Boxes Appear when you must make a decision or enter information Choose a Dye Scale All other actions on the monitor 1 0 Screen are suspended until you close the dialog box by clicking a EE button such as Cancel OK or Done Menus Provide access to various functions you can perform with the software A heavy arrow fe after a menu item indicates that a submenu appears when you click that choice and hold down the mouse button When you choose a menu command followed by an ellipsis a dialog box appears Settings Gel Processing Parameters Analysis Parameters Ruto finalysis Menu Project Options NA Sample Info Display Sample File Sorting Results Display Submenu Custom Plot Colors Pop up menus Display a heavy arrow 9 and are found in dialog boxes gt Choose a Plot Color When you click a pop up menu and hold down the mouse button a submenu appears These menus allow you to choose dialog box entries from specific lists of items 1 8 GeneScan Analysis Software Overview Macintosh computer terms used in this manual continued Item Description Windows Display information and in some cases allow you to edit or enter additional information The top border of an active window always has six horizontal lines and usua
311. tion 1 Note From the Select Criteria pop up menu select the criteria by which you want to search Note To list all of the items in the BioLIMS database perform the search with no criteria specified For large databases this process may be slow Collection Browser Select criteria Collection Creator h Fragments v Collection Name Collection Type Creation Date Modification Date Sequence Fra Sequence Frag Name Sample Creator Sample Name Instrument Name Instrumentation Start Collect Date End Collect Date Gel Path Sample Info Sample Comment Size Data Size Calling Matched Peaks Offscale Data Creator To use the pop up menu To define the search Choose menu items by See page above the horizontal line Collection Search E 24 Criteria below the horizontal line Fragment Search E 25 Criteria Note As you choose items from the pop up menu a black dot appears next to the item on the menu and the item is added to either the collection search criteria or the fragment name search criteria section of the window E 28 Using the BioLIMS Database To search the BioLIMS database continued Step Action The following is an example of the Collection Browser window showing four collection search criteria and five fragment search criteria Collection Browser
312. to 1 6 archiving 8 6 copying data to other applications 8 7 to 8 8 crashes with BioLIMS D 9 license and warranty G 1 to G 3 preparing matrix standard samples 6 10 registering 1 11 saving gel files 8 4 projects 8 3 Results Displays 8 5 sample files 8 3 why save 8 2 size standard GeneScan 1000 B 8 to B 9 GeneScan 2500 B 10 to B 11 GeneScan 350 B 2 to B 3 GeneScan 400HD B 4 to B 5 GeneScan 500 B 6 to B 7 software files table of 1 16 to 1 18 what new in this release 1 2 to 1 3 GeneScan 100 standard B 8 to B 9 GeneScan 2500 standard B 10 to B 11 GeneScan 350 size standard B 2 to B 3 GeneScan 500 size standard B 6 to B 7 GeneScan 400 HD size standard B 4 to B 5 Genotyper copying GeneScan data to 8 7 troubleshooting software results D 8 Global Southern method C 7 to C 8 glossary Glossary 1 to Glossary 4 grid defined Glossary 2 Index 3 H help e mail address 1 24 internet address 1 20 regional sales offices 1 24 to 1 26 telephone hours 1 20 telephone fax U S 1 20 horizontal scale changing in electropherograms 7 37 I IMPORTANT definition 1 6 installing 1 12 to 1 15 folder contents 1 15 procedure 1 14 program files installed diagram 1 15 RAM required 1 14 system specifications 1 12 instrument manual text related to 1 5 internal size standard compensating for band shift artifacts Glossary 2 defined Glossary 2 internet address Applied Biosystems 1 20 Documents on Demand 1 23 interpolating data 2 57 to 2 58
313. to the GeneScan Analysis Software the number of lanes collected by entering the appropriate number in the entry field and clicking OK Another dialog box appears if the Multicomponent Gel Image checkbox in the Gel Processing Parameters was selected Identify the matrix file to use for adjusting spectral overlap For more information see Chapter 7 Evaluating Analysis Results Note If multicomponent was not chosen but later you decide to regenerate the image with adjustment for spectral overlap choose Install New Gel Matrix from the Gel menu to attach a Gel matrix see Installing New Matrix Information on page 2 24 Using GeneScan with the ABI 373 A 7 Opening the Collection file continued Step Action 5 Find and select the matrix file you wish to attach and click Open Note This version of the GeneScan Analysis Software can read a matrix generated using GeneScan 1 X Move the matrix file from the 68K Macintosh computer to the Power Macintosh computer you are using for analysis The GeneScan Analysis Software Attaches the matrix to the Collection file Adjusts for spectral overlap and Generates a gel image which appears on the screen For more information on the gel image see page 2 27 A 8 Using GeneScan with the ABI 373 Completing the Sample Sheet Introduction Procedure After opening the Collection file complete the Sample Sheet embedded and track the l
314. tomatically tracked and extracted at the end of a data collection run IMPORTANT Before auto tracking the first time you need to verify that the comb type is set correctly The default comb type is Square Note Select both checkboxes to analyze samples automatically after data collection Image Generation In the Image Generation Defaults options enter values that determine Defaults how the GeneScan Analysis Software processes the gel file when it generates the initial gel image from data collected after electrophoresis Image Generation options Item Description Scan Range Enter the scan numbers at which you want to display the gel image Use a trial run to determine the scan range since the time frame in which fragments are detected may vary from run to run During analysis the GeneScan Analysis Software ignores all scans outside the specified scan range and only includes the scans with the designated ranges when it generates Sample files See Regenerating the Gel Image on page 2 23 to change the number of scans displayed Determining the Start and Stop Scan Numbers a Determine the start number by multiplying the applicable number from the table below by the number of hours it takes for the peaks to appear Scans per Instrument amp Type of Run hour ABI 373 600 ABI PRISM 377 and ABI 373 1 200 to 2 4002 with XL upgrade a Depending on which module is used during data collection
315. triangle 105 02 GS0627 377KL May 15 1998 01 21 18 PM 9205 02 650627 377 L May 15 1998 01 21 18 PM 9105 04 GS0627 377KL May 15 1998 01 21 19 PM 1205 05 650627 377 L May 15 1998 01 21 19 PM Q5 06 GS0627 377XL May 15 1998 01 21 19 PM 245 07 650627 277 L May 15 1998 01 21 19 PM 85 08 GS0627 377 L May 15 1998 01 21 20 PM L uu Collection 377 XL Gels contains 48 items Selec 5 Select fragments Using the BioLIMS Database Troubleshooting the BioLIMS Database Introduction In This Appendix Topics in this appendix include the following Topic See Page If the BioLIMS Preference Page Does Not Appear F 2 About Troubleshooting the Client to Sybase Connection F 3 Troubleshooting Process F 15 Procedures for Troubleshooting the Client to Sybase F 6 Connection About Troubleshooting the Client to Oracle Connection F 14 Troubleshooting Process F 15 Procedures for Troubleshooting the Client to Oracle F 17 Connection Troubleshooting the BioLIMS Database F 1 If the BioLIMS Preference Page Does Not Appear Problem Solution If the BioLIMS Access page is not present after Results Display in the Preferences submenu the GeneScan Analysis Software is unable to find all the database support files and system extensions required to access the BioLIMS database Settings Gel Preferences Analysis Parameters Auto Analysis Defaults Project Options b Preferences
316. trix standards preparing 6 12 processing using ABI 373 A 2 to A 4 sample sheet completing A 9 to A 10 tracking lanes A 11 ABI 373 XL loading and running dye standards 6 24 to 6 26 ABI folder files installed in 6 6 ABI PRISM 310 automatic analysis diagram 4 2 automatically printing run results 9 3 loading and running dye standards 6 13 to 6 16 matrix standards preparing 6 9 to 6 11 ABI PRISM 377 automatic analysis diagram 4 3 automatically printing run results 9 3 generating sample files 6 27 to 6 28 loading and running dye standards 6 17 to 6 19 matrix standards preparing 6 12 Analysis Control window using to analyze project files 5 2 to 5 5 using to analyze sample file 5 6 to 5 16 Analysis Log using 7 51 to 7 52 analysis parameters about 5 17 to 5 23 defined Glossary 1 using 5 24 to 5 28 analysis results changing how displayed and printed 7 13 to 7 15 Results Display window about 7 4to 7 6 using 7 7 to 7 12 steps to evaluating evaluating results 7 2 updating the results 7 16 using the Analysis Log 7 51 to 7 52 ways to display 7 3 See Also Sample Results View analyzing project files 5 2 to 5 5 sample files 5 6 to 5 16 archiving 8 6 automatic analysis setting up 4 4 to 4 7 B band shift artifacts compensating for Glossary 2 baselining defined Glossary 1 BioLIMS about server names E 17 to E 19 accessing the database E 13 to E 16 configuring the server E 4 to E 9 GeneScan and BioLIMS about E 2 to E 3 switchin
317. ts Check your Analysis Best 250 data points Parameters The Range of Data Specify a smaller Defining Analysis Points to analyze is too range in the Analysis Parameters on large Parameters page 5 18 Check your Analysis Parameters The Analysis Make sure the Analysis Defining Analysis Parameters could not Parameters file Parameters on be accessed specified in the page 5 18 Check your Analysis Parameters Setting Analysis Control window is valid and accessible The Sample File does not contain a valid matrix Assign a new matrix to the Sample File and try again Assign a new matrix to the Sample file and re analyze Installing a New Matrix File on page 3 20 D 10 Troubleshooting the GeneScan Software Analysis Log error messages continued Analysis Log Error Message Comment Correction Refer To The Sample File s Matrix was not found Assign a new matrix to the Sample File and try again Assign a new matrix to the Sample file and re analyze Choose Preferences from the Settings menu and Folder Locations from the submenu Check the location of the matrix folder For more information see Specifying File Locations on page 5 42 Installing a New Matrix File on page 3 20 Error Messages The following table list the error messages you might encounter while When Defining defining size standards Size Standards Error messages when
318. ture Identification Software Sample 2DB Software 9 9 9 Sequencing Analysis software Using the Collection Browser window from within GeneScan Analysis Software you can search the BioLIMS database for specific collections and fragments The following table lists ways you can search Search by See page up to 5 collection specific criteria E 24 up to 14 fragment specific criteria E 25 This section includes the following topics For this topic See page Displaying the Collection Browser Window E 21 Collection Browser Window Example E 22 Parts of the Collection Browser Window E 22 Collection Search Criteria E 24 Fragment Search Criteria E 25 Searching the BioLIMS Database E 28 E 20 Using the BioLIMS Database Displaying the There are two ways to display the Collection Browser window Collection Browser Window If you want to Then Result open a fragment to a Choose Open The Collection Browser view or edit from the File window appears menu The Open Existing dialog box opens Click the Sample icon create a new project by adding sample files Choose New from File menu The Create New dialog box appears Click the Project icon An untitled Analysis Control window appears Choose Add Samples Files from the Project menu For more information see Collection Browser Window Example on page E 22 Using
319. u Click the arrow in the Standard column heading and choose a standard file from the pop up menu Your menu choice applies to all fields in the column Note X Alternatively you can choose a value from the pop up menu click the header to select the entire column and choose Fill Down from the Edit menu Pop up menu Project Rnalysis Control O Print Results Print Setup Sample File Size Standard Parameters Analysis Parameters lt Analysis Parameters Analyzing Project Files 5 39 Step Action The pop up menu contains the following options Item Description None Apply no standard definition filename Apply the size standard specified in the Data Collection software which is embedded in the Sample file For information on editing this file see Editing the Size Standard Definition on page 5 36 custom standards that are listed at the bottom of the menu These are files that you defined and they are located in the parameters folder specified in the Folder Locations preferences For information on setting the Folder Locations preferences see Defining Folder Locations on page 5 41 Selecting Separate To apply separate size standards to selected samples click the arrow in Standards for the Standard column for the sample that you want to change see figure Samples below and a pop up menu appears
320. u can create a new project and add any combination of Sample files allowing you to analyze and display samples from different runs Adding a Sample file to the project sets up a link between the project and the Sample file The file itself is not imported into the project For more information see Creating a New Project on page 4 10 The GeneScan Analysis Software creates a project file when Sample files are created during automatic analysis Note When the GeneScan Analysis Software automatically creates a project the default file name is the same as the gel file The following table lists when the GeneScan Analysis Software creates a project file When using this instrument A project is created ABI PRISM 310 immediately after the first sample run Note The default project name is GeneScan Project date ABI 373 and ABI PRISM 377 when the software extracts the lanes of the gel file to create the Sample files Note The default project name is the same as the gel with the following extension added to the end x Where to Store Keep the Sample files in the same location on the hard disk relative to Projects the project file so the GeneScan Analysis Software can locate them when the project is opened If Sample files are moved and the program does not find them when you open the project see Finding Missing Sample Files on page 4 14 Creating a Project 4 9 Working with Project Fil
321. ulations Size Calculation 7 46 Evaluating Analysis Results Step Action See 1 Compare how well multiple size standard electropherograms line up within a Results Display window when aligned by size How to Verify Size Calculations on page 7 48 View the sizing curve calculated by the GeneScan Analysis Software Size Curve View on page 3 13 Determine how well the defined size standard matched the size standard run with your sample Use the Peak Total information in the Sample Info view of the Sample File window Sample Info View on page 3 8 View the Analysis Log which provides messages for each analyzed Sample file If there is a problem or a questionable condition during size calling a warning message is displayed in the Analysis Log Using the Analysis Log on page 7 51 Use the Raw Data view to display information about the raw data for a sample Analyzed Sample files contain both raw and analyzed data Raw Data View on page 3 15 Step Action See Use EPT Data to troubleshoot problems caused by poor run conditions such as the EP voltage for ABI PRISM 377 EP current EP power for the ABI 373 Laser power for the ABI PRISM 310 Geltemperature versus time EPT data can be displayed for each Sample file EPT Data View on page 3 17 Evaluating Analysis Results 7 47 How to Verify S
322. ully optimized a Repeat electrophoresis load inject less sample b Check optimization With allele peaks of high intensity the GeneScan Analysis Software calls many small peaks Background above minimum peak height Too much PCR product loaded a Adjust minimum peak height re analyze b Repeat electrophoresis load inject less sample For more information see Peak Detection Parameter Options on page 5 20 A homozygous individual shows a dip at the top of an allele peak which may be called as two separate peaks Truncated single peak because of off scale data can appear as two peaks Repeat electrophoresis load inject less sample Warning message Could not complete Run Macro command because the labeled peak could not be found The first allele peak for one or more loci in the allelic ladder is lower than the preset minimum peak height specification in the categories list a Adjust minimum peak height re analyze b Repeat electrophoresis load inject less sample For more information see Peak Detection Parameter Options on page 5 20 D 8 Troubleshooting the GeneScan Software GeneScan Error Messages Introduction This section includes three tables GeneScan Analysis Software Crashes with BioLIMS Analysis Log Error Messages Error Messages When Defining Size Standards GeneScan Analysis The following
323. ve minutes to denature before loading a Loading buffer is 1 part 25 mM EDTA 50 mg ml Blue Dextran with 5 parts deionized formamide 6 12 Making a Matrix File Loading and Running Dye Standards for the ABI PRISM 310 Introduction This section describes how to do the following tasks before running matrix standards on the ABI PRISM 310 Topic See page Creating a GeneScan Sample Sheet 6 13 Creating a GeneScan Injection List 6 14 Starting the Data Collection Software 6 15 Note When loading the matrix standards on an instrument note which colors you load in which autosampler positions Creating a GeneScan Sample Sheet To create a GeneScan Sample Sheet Step Action 1 Open the ABI PRISM 310 Data Collection software 2 Choose New from the File menu 3 Click the GeneScan Sample Sheet 48 or 96 tube icon The GeneScan Sample Sheet appears Sample Sheet GS matrix GeneScan Sample Sheet Comments 4 Enter the appropriate colors Blue Green Yellow or Red in the positions for each sample name Making a Matrix File 6 13 To create a GeneScan Sample Sheet continued Step Action 5 Enter any additional information about the sample in the Comments column 6 Use the Save As command and save the Sample Shee
324. ver error log step 4 on page F 23 a s Try Macintosh connection step 6 on page F 24 a he network is not orking step 6 on page F 21 estart the Listener Server step 3 on page F 23 on Look up errors in Oracle manuals step 5 on page F 23 Attempt to fix problems listed in error log step 5 on page F 23 Procedures for Troubleshooting the Client to Oracle Connection Introduction Troubleshooting from the Macintosh Client The procedure for troubleshooting the Client Server connection is divided into two parts Troubleshooting from the Macintosh Client below Troubleshooting from the Unix Oracle Server page F 22 The step numbers in the following procedure correspond to the steps marked in the flow chart above IMPORTANT The documentation for each BioLIMS Macintosh program includes a section about setting up the database connection Ensure that you have followed that procedure carefully Troubleshooting the connection from the client Macintosh Step Action 1 Locate the tnsnames ora file The default installation places the tnsnames ora file in the BioLIMS 2 0 BioLIMS Extras Oracle Network Admin folder 2 Open the tnsnames ora file and confirm that the server information is correct An example of the Oracle based BioLIMS Server entry is shown below Oramozart DESCRIPTION ADDRESS PROTOCOL TCP host mozart port 1521 C
325. w and the Results displays refer to Setting Dye Indicator Preferences on page 5 15 Note Custom plot colors are not available in the Sample Results view Why Change Change the colors in the electropherogram to Colors in the 4 Electropherogram dye Differentiate between different samples labeled with the same color Improve contrast between different dye colors Show data in a special color for a presentation Optimize plot colors for a particular printer Saving the Display The following table lists the options for saving the display format after Format customizing the display colors If you And then specify save the display format of a Results Display window after customizing the display open it at a later time the custom colors still appear For more information see Saving and Renaming the Results Control Format on page 7 17 do not save the display format after manually customizing the colors redisplay the same results again the electropherograms are redrawn with default colors 7 40 Evaluating Analysis Results Defining Custom Color for All To define custom colors for all electropherograms Electropherogram Step Action 1 Choose Project Options from the Settings menu and Choose Custom Plot Colors from the submenu The Custom Plot Colors dialog box appears Custom Plot Colors Plot No Color Plot No Color 3
326. w dialog box appears Create New T3 Te g Project Sample Analysis Size Matrix Sheet Parameters Standard Cancel Click the Size Standard icon A directory dialog box appears Select the Sample file that contains the dye standard you want to use as the template and click Open The Select Dye and Analysis Parameters dialog box appears Select Dye and Analysis Parameters Select the Dye and finalysis Parameters to use in creating the New Size Standard Definition Dye Analysis Parameters 2400 36 STD AP v Cancel o Choose from the Dye pop up menu the code that represents the dye label of the size standard in the selected Sample file Analyzing Project Files 5 31 5 32 Analyzing Project Files To use the New command to define a new size standard continued Step Action 5 Choose from the Analysis Parameters pop up menu the analysis parameters to use The pop up menu contains the following options Item Description lt Analysis Parameters gt Applies the parameters that are stored as preferences in the software custom parameters that are These are files that you defined listed at the bottom of the menu and they are located in the parameters folder specified in the Folder Locations preferences For information on setting the Folder Locations preferences see Defining Folder Locations on page 5 41 6 Click OK A window appears see
327. wing action Choose modules For this matrix that use Module file Dye Primer matrix Virtual Filter A GS 36A Fluorescent Virtual Filter C GS36C Amidite 3 Electrophorese samples according to conditions specified in your instrument manual Running the Run the matrix standards under the precise conditions you want to Matrix Standards generate a matrix file To run the matrix standards Step Action 1 modules Complete the information in the data collection Run sheet Refer to the figure below to choose the appropriate Prerun and Run Run 6 15 98 3 58 PM LXX Bre Pm L Plate Check Module Plate Check A t PreRun Module PR GS 368 2400 Run Module GS 364 2400 E Sample Sheet ne Comb 34 well comb Collect time hours Well to Read distance cm Operator Take the following action Choose modules For this matrix that use Module file Dye Primer or Virtual Filter A GS 36A dNTP matrix GS Amidite matrix Virtual Filter C GS36C Start the electrophoresis run according to the conditions specified in your instruction manual Go to Generating Matrix Sample Files for the ABI 373 and ABI PRISM 377 on page 6 27 Making a Matrix File 6 19 Loading and Running Matrix Samples on the ABI 373 non XL Introduction The ABI 373 uses the 672 D
328. y the BioLIMS database Note You can also press the Return key to begin a search E 22 Usingthe BioLIMS Database Item Description Collection search criteria pop up menus and text boxes Use these pop up menus and text boxes to define the collection criteria of the search IMPORTANT Only those fragments that match each and every criterion you specify are returned That is search criteria are combined using the logical AND operation For more information see Collection Search Criteria on page E 24 Fragment search criteria pop up menu and text boxes Use these pop up menus and text boxes to define the fragment criteria of the search IMPORTANT A collection is returned if one or more of the fragments contained in it fulfill all of the specified fragment criteria Note Only fragments meeting search criteria will be displayed in the Collection Browser window For more information see Fragment Search Criteria on page E 25 Split bar Drag this bar to alter the relative amount of space allocated to the top and bottom portions of the Collection Browser window Search results After a successful query found sample files are listed in this area as Name Modification date type and Creator Status line Search results error messages and other important information is reported here For example the Status Line lists how many collections were returned in a search
329. ying Other Sample File Data Sample Info View on page 3 8 Size Curve View on page 3 13 Raw Data View on page 3 15 EPT Data View on page 3 17 Using the Analysis Log Using the Analysis Log on page 7 51 Remembering and Renaming the Results Display Saving and Renaming the Results Control Format on page 7 17 7 2 Evaluating Analysis Results Ways to Display Analysis Results Two Waysto The following table lists two ways to display analysis results Display Analysis Results You can use Description See page The Results This window is created from the Results 7 4 Display Control window of a project Window Use to group and view multiple Sample files as electropherogram and tabular data The Sample This view displays one Sample file at a time 7 16 Results View just as the Results Display window but is more convenient for viewing analysis results from a single Sample file It also allows quick access to supporting information views Evaluating Analysis Results 7 3 The Results Display Window Introduction The Results Display window is created from the Results Control window of a project and allows you to group and view multiple Sample files as electropherogram or tabular data You can use the window to show up to eight panels with multiple dye samples per panel Displaying the Choose Results Control 38 2 from the Windows menu
330. you by title by phone fromthe a Call 1 800 487 6809 from a touch tone phone Have United States or Canada your fax number ready b Press 1 to order an index of available documents and have it faxed to you Each document in the index has an ID number Use this as your order number in step d below c Call 1 800 487 6809 from a touch tone phone a second time d Press 2 to order up to five documents and have them faxed to you by phone from outside the United States or Canada a Dial your international access code then 1 858 712 0317 from a touch tone phone Have your complete fax number and country code ready 011 precedes the country code b Press 1 to order an index of available documents and have it faxed to you Each document in the index has an ID number Use this as your order number in step d below c Call 1 858 712 0317 from a touch tone phone a second time d Press 2 to order up to five documents and have them faxed to you GeneScan Analysis Software Overview 1 23 Regional Offices Sales and Service your local Applied Biosystems service representative To Reach Us by Contact technical support by e mail for help in the following product E Mail areas For this product area Use this e mail address Chemiluminescence info appliedbiosystems com Genetic Analysis galab appliedbiosystems com LC MS apisupport sciex com PCR and Sequence Detection

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