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GeneMarker® User Manual

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1. 5 d S Access Kette Lier Tias e entered twice to ensure accuracy Fae Ad Creer sf fnsert Alleles Access Rights Delete Aleles Lab Manager Undeseze Alo Aart Launches the Access Rights of User Types box where the Z Confer Alles different access rights available to each user type can be Corman As identified Clicking the Set Default button will return the TT Access Rights for the User Type selected back to factory defaults Fe ee a Laa NOTE Only the Administrator can change Access Rights for a z Enable Deable Samples Ceeemnt Sure Ulcer Type Add Saneled to Pipini Comment Pohi Save Project Change User erer Fer jou x Export Tree Update Scene 178 Z D s Qu farer May 2012 Chapter 9 User Management Prompts for a confirmation of action then launches the Login box password to login History The User Manager History tab monitors user activity associated with the user manager function Date Time Records the computer s date and time for the activity User Identifies the username of the person that performed the activity Events Records the user manager activity that was performed Comments Choose a new user and enter the user s fl User Manager Admin logged in lo e x User Manager History Settings Comments o Jonathan Events Delete user Log in Log out Log in Log out Log in Log out Log
2. 64 Save Del ae 24 Save Peak Table ooooccconcccnccccncccnnocnnaccnnnoss 32 41 SAVE Pl AEE 107 Save Pedigree File 108 112 115 162 SAVE e EE 35 74 SAVE RE POF dui idad 42 save Report Te 34 Save Synthetic Sample 85 SBE TECHNIQUE Vasari 123 ENN 176 SOE Ne 32 Seire vasre 29 Search OPTIONS sanne ini 29 Second Derivative Trace 20 SEP Nidia 103 US A A wees 109 Select Marker unas aa 108 SEVER COMPU vade 9 SOTOS CONTO eae eee cae odo 83 Set as Non Control 83 Set DNS eee 31 41 113 116 157 SFPE 74 SEL E 176 FUNN 74 Short tandem repeat markers ssessssessssessereseresseeen 145 SHOW Da 168 Show Chart Table aaaeeseeeessssssresss 32 41 Show Color eessen 41 113 116 157 162 Show Columns oocccococcncnnccnonncononocononocononacononacononarononos 32 Show Conflict arroen r 109 112 116 Show Difference Histogram cccccccssseeceeeeeeees 135 Show Disabled Samples in Report 36 SHOW EGIE HON SSN 33 SHOW Gel IMACS EEE 35 Show Individual Name 108 112 116 Show Intensity Rou 69 Show Last Event se 40 Show Loss in Histogram 133 SHOW Navigator ainia 35 Show Only Uncertain Alleles 68 69 71 Show PEAKATE Eege EENEG See Show Ratio in bercentage 89 Show Rejected Low Score Alleles 68 69 71 72 SHOW REDON RE 35 May 2012 Index Show Statistics Info
3. 53 May 2012 Chapter 5 Panel Editor 54 May 2012 Chapter 5 Panel Editor Chapter 5 Panel Editor Chapter 5 Panel Editor Overview Procedure Icons and Functions What to Expect 55 May 2012 Chapter 5 Panel Editor Overview The Panel Editor can be accessed from the Tools menu in the Main Analysis window OR via the Panel Editor icon in the Run Wizard Template Selection box The purpose of a Panel is to outline the position of expected alleles Loci or Markers give a range where a group of alleles is expected to appear and Bins indicate the specific basepair position of the expected allele In GeneMarker Markers are indicated by a horizontal gray bar across the top of the electropherogram Bins are indicated by the dye colored brackets at the top and bottom of the electropherogram Only in the Panel Editor do the vertical gray bars within the electropherogram indicate the center of the Bin For all other views in GeneMarker the vertical gray bars in the electropherogram indicate the center of the detected peak Panel Editor Panel List Panel Table J Sample List Overlay Trace p a RRRERRRRARAASRARARD gt gt s wl Fy r E gi 2 i A Hi H ale he Lal LUH ud H Fi bd i gt gt s vn e R AA AO ARA A SE Ze PES ra Sa OP j EPIA AAA AA HArLLNISGSE OSEN LL rar ross e o E oO Dee oO DS oO D ee SIK kk Gb bo Gb AHD RS gt T
4. 000nnooeenneeseeesseeeessreressseres 132 MSI Peak Normaltzation 133 Eeleren eeneg 132 133 134 MS MEET 95 96 MS MLPA Anahysis 38 MS MLPA Analysis Settigg s 97 MS MLPA Import Common Reference 96 MS MLPA Print Report 100 MS MLPA Report Content 99 MS MLPA Report Table 99 Multiplex Ligation dependent Probe Amplification MEPA save 80 N Naming Options occccocccccnccnnnnccnnnnncnnnnacnnnnacononanos 68 Navigatio DEE 30 NetDo8 E 11 K NT 11 NetDog Server Management coccoocccnccnnocncncnnonnnononononanoness 12 NetDos UPA aa 13 NetDog Upgrade sv GEA 13 Network Version Registration rrvrnnnnrrrnnnnnnvnnnnnnnvrnnnn 12 New Page for Each Sample ccccssssececeeseeeeeseeeeeeees 73 New Panel nro arrancada 63 New e E 107 New Pedigree File 108 112 115 162 New Size Standards vaare 47 Hew template JR 20 non disjunctiON occcccccnccnnncnnoconnnonaconnnonaronononanos 142 150 non synonymous SNPs nsSNPS ccooooccccnnnnccnonoconnnoss 122 Normalization Accurady enca 85 Nucleotide Repneat 57 58 Nucleotide RCDCATS rcn A 58 Number of Liability Classes 108 Number of Peaks ss 121 O Off Ladder OL serende 53 65 Offline Registration 8 10 12 ONE COlOl ANALYSIS Lanseres 21 Online Registration ccccocccncnnccncnncnnonacanonananons 7 10 12 Open Data ia nas ias 35 40 Open Data FICS as 16 Open Multiple Charts rrrrnnnnrrrnnnnnrrvnnnnnnvrnnnnnnrrnnnnnner
5. Automatically Save Run Wizard Parameters to INI file Apply International Date Format Check this option if you would like the dates of the Reports for Applications to be in the International format day month year Large Size Std Fitting Parameters Customizes the Large Size Call OK Cancel Algorithm by applying the user specified values to the Large Size Call See Chapter 2 General Procedure The icon links users to a spread sheet that assists in determining the best parameters for the custom large size standard Automatically Save Run Wizard Parameters Saves Run Wizard parameters in an ini file ProjectName_RunWizardParameters ini Channels Opens the Set Channels box and allows the user to choose from ABI MegaBACE and Beckman Coulter standard dye colors The user can also manually enter dye color and name The default channel color setup is ABI See Chapter 2 General Procedure Project Menu The Project menu contains options for how the data is processed and printed Run Activates the Run Wizard and begins the data processing setup This allows the Run user to select or adjust program settings in a sequential manner The same process gt Auto Run action can also be accomplished by clicking the Run icon in the toolbar Add Samples to Project Auto Run 5 Print Report GeneMarker will process data using the last set of parameters selected If one or Apply Sample Grouping more of the parameters require changing
6. 119 May 2012 Chapter 7 Special Applications Quantitative Analysis The Quantitative Analysis feature determines areas under the curve of complex peaks Two different analysis methods are included the Curtain Method and the Deconvolution Method The Curtain Method allows the user to define the start and end range for area calculation under a peak curve The Deconvolution Method compares a selected sample s peak areas to the peak areas of the other samples at the same position Quantitative Analysis ee Sample List Report Table a PS a al A 0154 PN fa d x 015 46 T tsa METIN fra DEBAT fsa On 7 55 N Les 0 7 56 T faa OES A fra 1 6 52 T fea 075 59 N fra 3607 tea 020 61 N faa 0 627 lea OT EN fa 0021 64 T les 2265 fra 0022657 Ie S LO 00 J OD LP de GA Sample List The Sample List displays all current samples in the project Single left click a sample name or use the Up Down Arrow keys to scroll through the list Electropherogram The Electropherogram frame displays the currently selected sample s trace with either Curtain shaded areas or Deconvolution dashed trace overlay Allele editing options are similar to the Main Analysis window Electropherogram frame See Chapter 3 Main Analysis Overview Report Table The Report Table shows the size height and area calculations for the peaks under analysis Adjusting the start and end range points with the Curtain Method wil
7. 62 May 2012 Chapter 5 Panel Editor Major and minor Auto panel adjust 1 Major panel adjustment icon should be used with a panel that was previously saved with signal information from the genetic analyzer if the data run is shifted 1 5 base pairs from alignment 2 Click on the auto major panel adjustment icon 3 The panel is automatically adjusted for this project 4 Minor panel adjustment icon aligns the center of the Bin to the center of the nearest peak within one basepair of the Bin sd o e de a o e a o e ee 23334 a i Icons and Functions The following are explanations of menu and icon options in Panel Editor Menu Options The Panel Editor contains three menu options File Tools and Help The File menu allows the user to create save and export Panels The Tools menu contains options for datasets with allelic ladder samples and exporting a Panel The Help menu contains navigation hints for Panel Editor File Menu Create New Panel Launches the Create New Panel dialog box with the options to create a new Panel Automatically or Manually Delete Current Panel Marker Deletes the Panel or Marker that is currently highlighted in the Panel List Save Changes Saves edits and changes to the Panel in the SoftGenetics GeneMarker Panel directory Hot Key CTRL S Save as New Panel Opens the Input Dialog box with a field to enter a new Panel name The Panel is added to the P
8. 8 S g D135628 og 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 2 000 Ki Auel ofeseje sky liado 110 do E 296 238 872 44 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 410 420 B 2 000 JA Corrected Height Ra Corrected Height Ratic Corrected Height Rat Correcte N gt E j gt bd marker Jame DEE ERIC Jo1ese7s__ O21S11 sma o lb b b b b h jo bh aee h en baw baw kaw k O b P 15219 02s 215218 06727 1135 1214 866 858 Trisomy Scores 013 880 ame rar jara e jor fom 12 om Report Header Contains information about the analysis project sample and parameters Signature Box Date and initial space for report reviewers Electropherogram Similar to the Trisomy analysis window displays all dye colors of the sample trace Corrected Ratio Plot Contains the entire dataset s plot points for all Markers in the dye color Symbol shapes represent different Markers and can be deciphered from the Symbol row in the Report Table Yellow filled symbols represent the current sample s data points Red outlined symbols represent trisomy calls Report Table Displays selected peak and Marker values for the current sample Trisomy calls are highlighted grey 148 May 2012 Chapter 7 Special Applications Best Practices Guideline BPG and Aneuploidy Print Reports When BPG or Aneuploidy is selected
9. Auto Range frame The range in camera frames will automatically find the processable data range If Auto Range is not selected manually enter the start and end frame numbers of the data set for analysis ata Intensity Correction S Intensity Coefficients Allows for manual correction of excessive bleed E ae through peaks best used for experiments with one color analysis Coefficients Smooth S J Smoothes the baseline by eliminating smaller noise peaks O Enhanced Smooth Dye e This feature is used only in cases where the data is extremely difficult to Dyes DR analyze and cannot be corrected with the Smooth function Peak Saturation The software will analyze saturated data points by creating a synthetic estimate of the peak shape based on the curves prior to saturation The results will be less accurate than that of non saturated peaks Pull up Correction This function removes peaks caused by wavelength bleed through to other wavelengths The function should be disabled if a Manual Pull up Correction was used in the Raw Data Analysis window Baseline Subtraction This function removes the baseline completely so that the Y axis will be raised above the noise level Enhanced Baseline Subtraction p May 2012 Chapter 2 General Procedure This function should be applied for samples that have a high baseline especially in the 40 80 bp mini STR range Spike Removal Removes peaks from voltage spikes caused by
10. Chapter 7 Special Applications Ratio Plot The Ratio Plot depicts data points representing Marker peak ratios on a peak ratio vs basepair size graph Markers with three peaks will display three data points one for each peak The three data points will typically appear within the user defined ratio threshold limits black lines and along the red fit line Markers with two peaks are represented by only one data point in the Ratio Plot If the data point is a blue triangle then the ratio of the two peaks is within the user defined ratio threshold limits If the data point is a red triangle then the ratio of the two peaks is outside the user defined ratio threshold limits and will be considered trisomy Data points that approach the black limit lines need to be considered carefully as they are not in perfect 1 2 0 5 or 2 1 2 0 ratio NOTE Markers which contain a single peak homozygote are not represented in the Ratio Plot Single click data points in the Ratio Plot to view the Marker in the Electropherogram and the Report Table values To zoom in hold down left click and drag a box from upper left to lower right To zoom out hold down left click and drag a box in the opposite direction from lower right to upper left Click the Settings icon in the Ratio Plot The Trisomy Marker Statistic Settings box appears Adjust the settings to display the Ratio Plot as a direct ratio calculation or as the corrected ratio calculation plot Change the co
11. D551982 D551982 338 Find Individual 161 May 2012 Chapter 7 Special Applications A Y k sa a SD H New Pedigree File Select New Pedigree File to create a new pedigree with multiple families OR select New Family to add a family to the pedigree file Enter the first family member s information into the New Family New Individual box and click OK to create a new Pedigree Tree Open Pedigree File Launches the Load Pedigree File box Select a PED or PRE file to upload the SMP file will automatically upload and click OK Save Pedigree File Launches the Save Pedigree File box Enter filename and change directory to save the Pedigree Files PRE SMP DAT Update Sample Data Select to refresh the Mendelian inheritance calculation after a node or allele is edited and after selection a different family when the show genotype display is used Haplotype Analysis Parameters Launches the Haplotype Anaylysis Settings box Options for selected samples or all samples Autosomal or X linked analysis and display options Show Genotype Toggle between Displaying and Not Displaying the Genotypes of the selected node Family 2 2 2 Family 2 Jones Family Select a family from the currently uploaded pedigree file to view and edit Marker 12 TPOM Marker Select a Marker or Locus to view in the Electropherogram Charts Show Color Allows the user to select all colors to view hide all colors or choose a si
12. Displays gain and loss information for individual peaks of a sample compared to a reference Samples designated as reference by the filename group text file are marked as blue rows in the report while samples being compared to the reference are in the white rows The Overlay Trace window will correspond to which ever peak cell is selected in the Report Table Report Settings Launches the Report Options dialog box Eeg Output Status Peaks Selecting this option displays the peak Ce see F intensity ratio values in the allele report ao Statistics T Equivalent EE Abide by Panel Displays allele information that is within the n Ee i EE selected panel Alleles outside of the selected panel will be EN represented by an OL Y Abide By Panel Show Ratio in Percentage i s A Y Vertical Forma Vertical Format Arranges the allele report in a vertical format Se T Display Quality Control Fragments Report Contents Selecting Status 1 0 1 will display the values mes of 1 0 and 1 for each detected allele to represent the presence absence and questionable presence of alleles respectively Statistics Displays the number of probes mean standard error and standard deviation for the control and sample probes at the bottom of the MLPA ratio report table Peak Differentiation Options to display peak differentiation in terms of peak height ratio peak area peak height and probability are available
13. Group Size Enter the number of groups for the analysis Standard MS MLPA analysis would use 2 groups an LOH analyses comparing one normal sample to three grades of tumor would use 4 groups Control Identifier Enter the character from the Control Identification section highlighted green that describes the control or reference sample Example N normal or R reference Select Case Sensitive if the Control Identifier needs to be identified by upper or lower case letters Control Match Mode Choose either Whole Words or Include Whole Words should be used if the characters entered into the Control Identifier field need to match exactly Include should be selected if the characters in the Control Identifier field only need to be identified in the filename i e not an exact match Macromolecules Tools gt Macromolecules The Macromolecule Tool aids with analysis of macromolecules without an internal lanes size standard Depending on the macromolecule size and configuration migration rates through CE vary greatly Internal size standards of the same macromolecule may not be readily available This tool enables researchers to physically identify reference peaks known to have the same size and uses the information to calibrate from one capillary to another Characteristics of the aligned data such as relative size peak height peak area can then be exported in an excel sheet or printed as an allele report Procedure Ha Select File
14. SNaPshot amp SNuPE One method to determine SNP genotypes is single base extension or SBE An unlabeled primer with its 3 end directly flanking the SNP is extended one nucleotide by Taq polymerase and fluorescently labeled ddNTPs complementary to the polymorphic base are added The resulting fragment is one nucleotide longer but the observed fragment size on a gel will be greater than expected due to the influence of the fluorescent dye on the electrophoretic mobility of these small fragments SNPs can be identified by the one or two color peaks associated with the incorporated labeled ddNTP and the length of the primer Primer extension method has the advantage of accurate genotyping using a low number of unlabeled user defined primers 122 May 2012 Chapter 7 Special Applications The SBE technique can quickly interrogate a small number of SNPs Two commonly used kits for SBE technique are the MegaBACE SNuPE Genotyping Kit Amersham Bioscience and the SNaPshot Genotyping System Applied Biosystems To fully utilize the investigative potential of SBE a robust genotyping and data analysis system should be employed GeneMarker genotyping software is designed for fast accurate and efficient analysis and reporting of primer extension data Overview The SNP analysis report window displays a synthetic gel image list of samples cluster plot to analyze peak information and assign SNP genotypes and sample electropherogram The informati
15. Special Applications Report Content Choose peak and Marker values to display in the Report Table The analysis type chosen in the Trisomy Analysis Settings box determines the Report Content initially chosen NOTE When selected Peak Ratio will correspond to the Quantification option Report Settings 28 Report Content Iw Allele Number Iw Trisomy Types chosen in the Trisomy Analysis Settings box Peak Height or Peak Area FZ Allele Name Y Peak Ratio Orientation 7 Peak Height When Horizontal is chosen Marker information will be displayed in columns Dr Peak Start and End along the top of the Report Table When Vertical is chosen Marker information will be displayed in rows Trisomy Score Orientation Show Only Trisomy When selected only trisomy calls will be displayed in the ee Report Table all other cells will be blank T Show Only Trisomy IW Hide Extra File Names Hide Extra File Names When selected sample filenames will appear only once in the first row of the sample s information Only available when Vertical Orientation is chosen Save Report Choose to save the Report Table as an Excel file xls or a tab delimited Text file txt Trisomy Print Report Settings Click the Print icon in the main toolbar to launch the Trisomy Print Settings box and preview print or save the Trisomy Print Report Cancel NOTE The analysis type chosen in the Trisomy Analysis Settings b
16. installing the NetDog key to correct this E Modus if 1 A EI Module 12 B Column B indicates the maximum number of El Module 13 15 E Module 14 concurrent users that are allowed to access the El Module 15 software C Column C indicates the number of current users running the software D The Module 0 row indicates programs being used in the network version of Mutation Surveyor Not applicable if you do not have Mutation Surveyor E The Module 1 row indicates programs being used in the network version of GeneMarker 5 After installing the NetDog Server Program please configure any firewalls on the server to allow the following default ports TCP IP 4587 11 May 2012 Chapter 2 General Procedure UDP IP 4587 IPX 17799 If other ports are preferred please launch the NetDog Server Management console and make the necessary changes Install the NetDog Key install NetDog In the toolbar there is an option to Install or Uninstall which allows you to numb delete or re install the NetDog ae 1 Delete any existing Mutation NetDog Key before beginning the Geen installation M 2 Click Install NetDog and the Install NetDog box will appear ee e Use configuration file NetDog NetDog Server NDoglnst Cid Browse Cancel i Install NetDog 3 Import the NDogINst cfg file which is located in the same directory as the NetDog Server setup package 4 The NetDog Server
17. v v H Y yyy 1 82 bp gt II 1 123 bp AP PG VM PL PL 1 280 bp R 1 439 bp 4 1 599 bp 1 680 bp 3 1 760 bp 1 801 bp ES 1 843 bp S 1 885 bp 37 1 927 bp 7 1 968 bp 37 1 1010 bp y 1 1050 bp 37 1 1092 bp y 1 1135 bp y 1 1178 bp 1 1212 bp 1 1250 bp Allele Call E Run Wizard n The Allele Call section allows the user to set allele calling range SENER detection thresholds and filters er Raw Data Analysis Allele Call Auto Range I Auto e frame tal I Auto Range bps The software will automatically display the processable data range me E Sur foo 2 ro 3 Y Smooth Enhanced Smooth Peak Detection Threshold for each lane I Peak Saturation V Baseline Subtraction Intensity gt 100 Percentage gt 25 Max Enhanced Baseline Subtraction ae i PutupCarecion FF Spke Removal a eee Manual selection of Range Nox Catintensy 500 Size Call ZC gt p a ocal Southern PERS wea Sie I Stutter Peak Filter IV Plus A Filter To select a specific analysis region de select Auto Range and input Cup C onesie ene TE mg the desired base pair range Additionally if automatic size call rd PEREZ fails due to high saturation de select Auto Range and manually TE LA input the required data range 23 May 2012 Chapter 2 General Procedure Peak Detection Threshold The recommended initial settings are different for each analysis type Please see the appropriate
18. 2 o Samples YA Chatts PAT_1_C fsa PAT_1_F fsa PAT_1_M fsa PAT_2 Cfsa PAT_2 F fsa PAT_2 M fsa PAT 3 Cfsa DAT 3 F fsa DAT 3 M fsa PAT 4 Cfsa DAT 4 F fsa DAT 4 M fsa DAT 5 Cfsa DAT 5 F fsa DAT 5 M fsa DAT 6 Cfsa DAT 6 F fsa DAT 6 M fsa PAT 7 Cfsa DAT 7 F fsa PAT Ladder 1 fsa PAT Ladder 2 fsa Sample File PAT 7 C fsa Icons and Functions New Pedigree File Select New Pedigree File to create a new pedigree with multiple families OR select New Family to add a family to the pedigree file Enter the first family member s information into the New Family New Individual box and click OK to create a new Pedigree Tree lar H H a Open Pedigree File Launches the Load Pedigree File box Select a PED or PRE file to upload the SMP file will automatically upload and click OK Save Pedigree File Launches the Save Pedigree File box Enter filename and change directory to save the Pedigree Files PRE SMP DAT Show Individual Name When selected the individual ID will be displayed in the nodes of the Pedigree Tree Update Sample Data Select to refresh the Mendelian inheritance calculation after a node or allele is edited Pedigree Parameters Launches the Loci Description box Enter the Affection Locus Description Gene Frequencies Select Markers Number of Liability Classes Penetrances Recombination Values and view the Allele Label and Frequencies 108 May 2012 Chapter 7 Special Applications Show
19. 2 The Input Dialog box appears Fes Sue Standard 3 Enter a Size Standard name and click OK Cancel 46 May 2012 Chapter 4 Fragment Sizing Standards me ot OY She 10 11 Right click at a known peak in the Sample ILS frame 5sm Select Insert Size CENE BR SES Hep The Insert Size box appears T aaa a ass 2 Enter the basepair size of the peak in the Size field SN SE and click OK S em S A green triangle will appear atop the peak in the Los j Sample ILS and a new peak will appear in the Srl ae Expected Size Standard frame meg T ince Sue Continue Insert Size operation for the rest of the oe men e peaks in the Sample ILS es A Se GeneMarker will interpolate Size values after two gi peaks are added to the Size Standard 5 on Sn NOTE It is recommended to use the interpolated Size EG values when creating a Size Standard due to the SZ differential migration patterns of each sample 12 13 14 The Expected Size Standard frame will be blank When the Size Standard is complete select File gt Save Changes OR click Save Changes icon Click OK in Size Template Editor Proceed with Run Wizard data analysis Icons and Functions The following are explanations of menu and icon options in Size Template Editor Menu Options The Size Template Editor contains three menu options File BestMatch and Help Th
20. 297 3390 5 69 Haplotype Analysis Familial DNA fragment data is used for haplotype analysis in areas such as genetic disorder research and pre implantation studies Commercially available kits with markers for both autosomal and X linked traits are available for diseases such as cystic fibrosis CF and Duchene Muscular Dystrophy DMD Traditionally researchers determine the genotype of each family member draw a pedigree diagram and assign the phase of the alleles where possible based on the familial data of parents and child ren Overview The Haplotype Analysis tool is directly linked to the main analysis screen in GeneMarker After confirming the allele calls for each family member the data is opened in Haplotype Analysis where the program uses the allele calls of children and parents to assign phase of the alleles with a first order approximation Whenever the alleles are informative for phase assignment a color pattern bar is assigned to indicate most probable phase Un informative markers receive a solid black phase bar Features 1 Follows Bennett et al nomenclature for pedigrees X linked and autosomal pedigree formats Edit family and individual information Displays markers allele calls personal information in pedigree Control ordering of markers in pedigree by customizing panels Automatically makes first order phase assignment based on parent child ren Edit capability for reassigning phase Edit capability for crossover Sa
21. Family selectton 109 Federal Bureau of Investigation 102 METON A en ter nee cree en ne eee 16 ETE EEE 34 FIC NOME GOUD srecne de era 172 File Name Group Tool ooennoosnnnessennnsseeresserressrrresse 39 Filename Egeter EE 172 Filename Group TOO ive as E 96 NEO AV ACO EE 176 PUK SIZE LA nave ead aero eas 51 Fed Bin Wid th vr 57 58 TOWN CVCOMELLY ve 93 tour color trade EE 176 CT VIE 21 frameshift MUtationskuasvearamasrsesaasnannand 131 FREQUENCIES LT 108 Funcional SNP een 122 G Gain Loss Histogram 132 135 EIERE 36 Gender segn 104 Gene e Une LE 108 GENRCIVIO DDE cs 60 63 GeneMarker HID Local version 6 7 9 GeneMarker HID Network Upgrade 13 GeneMarker HID Network version 9 General Settings siii a 35 BEIENEE a SG 103 PENECLIC relations aaa 103 genomic Mprinting sssessssssesserssessrrsserssessersseese 95 98 Genomic EEN serae een Bieta aa 97 98 Get Start POME ee 18 Gray for Single DV EC s scasceccasedcacs ccasversacedeasdiedavedsduedenss f der 36 Grey Scale Gel Image ooccccooocccnccnoconnncnanonononos 31 Group Feed vase 172 Group ldentUfNication orion 172 Grouped DD PG 73 Grouped by Marke lS cin Sege REENEN 71 183 H Haplotype Analys S iia 158 PO 32 Height ds Ju sansene 32 Help Meru 40 e EE 97 e IR WER 101 HID Panel Adjustment ccccooccncccnnccnnncnoncnnnonanonononanonnos 102 HID Run Wizard Settings cccccooccnnccnoncnnnonanonnnananenoss 101 Hide Extra Sample Name
22. Ii D 8 E0 se Ro am so EES ES Ebo Wo DC D a7 DIS scF Rap bes B MER Se B 552 FOSSCF B 553 GIES0F D i FIS SOF BES SESEEHEEREEEES 440 44040 Sample File Tree The Sample File Tree of the main analysis window contains two folders The ima first is the Raw Data folder which when expanded displays a list of all the 22 seca 564 B Group1_0 05 fsa dataset samples When a sample is double clicked its preprocessed Bonne F i o B 564 T Group1_0 07 fsa Select Page electropherogram trace will appear in the Raw Data Analysis window See amp 564 Groupl_102104_07488 Select Next Group B 7458 Group 0 09 Chapter 2 General Procedure D 7458 Groupt 102104 09fsa Select Max B 745 T Group1_0 11 fsa De select All B 745 T Group1_102104_11 fsa 5 The second folder Allele Call also contains a list of all the samples but Search File when the filename is double clicked the sample s electropherogram trace eer S appears in the Main Analysis window with all sizing information and allele i Enable Ctrl Del call filtering applied The Allele Call folder also flags each sample with a Edit Comments Fa green sheet yellow sheet or red strike through indicating size calling success See Chapter 4 Fragment Sizing Standards Right click the sample filename in the Raw Data or Allele Call folder to see additional options 28 May 2012 Chapter 3 Main Analysis Overview Sorting Options Select Page Open
23. Sample names are listed in rows in the left column and Markers are listed along the top C Alele List row in columns A Total Number column lists the number of peaks detected in the Ci Makee Tate F isinan sample EES NOTE Allele Count requires that a Panel is applied to the data See Chapter 5 Panel e Beran Editor C Sample Name File Name Orientation W 71 Horizontal C Venica CG Show Rejected Low Score Alleles May 2012 Iw Hide Extra Sample Names Chapter 6 Reports and Printing Features Orientation Horizontal Sample names appear on the left in rows and Markers appear at the top in columns Vertical Markers appear on the left in rows and sample names appear at the top in columns Show Rejected Low Score Alleles When selected the peaks with peak scores below the Run Wizard Additional Settings Allele Evaluation Peak Score Reject setting will be displayed in the table Hide Extra Sample Names This feature is not active for Allele Count Report Style Print Report 2 Faas a Faas a Faas Jus sos ser e oez cos ser e Fass a foes ros ser NM NM NM NI NIPI NINI NIN OINI Nin PIN I NI NT NM N PINI N N Oj FP er P NR RP NM NH NM RFP ba h N N MM NM ON W NM NM HM HM HM NM FP M amp M MM pp IN Pp The GeneMarker Print Report displays Electropherogram and or Peak Table information for all or selected samples in a dataset To access the Print Report go to Project Print Report OR c
24. The information in these fields cannot be edited This option is only available with Allele List Marker Table and Peak Table Report Styles Save Report Table To save all information currently displayed in the Report Table click the Save Report icon in the Report Table toolbar Choose a directory enter a filename _AlleleReport is the default and save as an Excel xls or tab delimited Text txt file To export only selected cells in the report table first select the cells by left mouse drag across the cell range or hold SHIFT key and select cells Right click on the highlighted cells and select Copy Hot Key CTRL C The information is saved to the Windows clipboard and can be pasted into any common word processor or spreadsheet program like Microsoft Excel The row and column headers for those cells will be copied with the highlighted cell information Menu Options The following menu options can be found in the menu bar of the Main Analysis window File Menu gt Open Data The File menu contains functions for opening and saving raw and processed data Open Project Reopen Project 34 f Save Project May 2012 Close All Exit Chapter 3 Main Analysis Overview Open Data Launches the Open Data Files window where the user can select raw data files for upload into GeneMarker Accepted file formats include fsa abl abi scf rsd esd smd smr See Chapter 2 General Procedure Open Project Opens a folder searc
25. These calculations will be applied to the peaks selected to display in the Output Status Peaks Show Ratio in Percentage Displays the peak height ratio in percentage if this option is selected Display Quality Control Fragments Displays the quality control fragments for panels that contain quality control fragments designated with a 1 negative 1 in the Control column in the panel editor Save Report Choose to save the Report Table as an Excel file xls or a tab delimited Text file txt MLPA Clinical Report Settings Click the Print Report icon to launch the MLPA Print dialog box and preview print or save the MLPA Clinical Report Samples Select All Samples or just the samples you wish to report To Select Samples click the button next to Selected Samples and place a check mark in the box next to the samples you wish to appear in the report Sort Probe MLPA Print Sort the probes in descending order By Size or By Name in the report table Samples Print Grayed Samples All Samples C Selected Samples When selected the report will include samples below the minimum score set in the MLPA Settings box Minimum Lane Score Threshold das C By Size Ce By Name List Samples Used for Synthetic Control When selected a maximum of ten samples will be displayed in the pe Ge sanser Babe Mien bes printed report If more than ten samples are used the symbol will 17 List Samples Used for Synthetic
26. an exponential function a e is used to fit to the square root of peak intensities where z is size and a and b are fitting constants Essentially both normalization methods take the square root of the intensity ratio and plot the ratios to model a linear regression Internal Control Probe Normalization The Internal Control Probe Normalization method corrects the trend of dropping intensities by setting the height ratio of the control probes to 1 00 and then calculating ratios for all other probes in the sample as compared to the controls The control probe peaks are identified in the selected Panel with a 1 in the Control column NOTE Some MLPA probe kits do not contain control probes Because the trend of control peak intensities varies greatly from one sample to another the Population Normalization method is the recommended method for most datasets 1Lampros A Mavrogiannia et al St James s University Hospital Leeds http leedsdna info science dosage REX MLPA REX MLPA_CMGS04 ppt http www eurogentest org uploads 1247475007479 MLPA validationreport TJ version 20090710 pdf Population Normalization The Population Normalization method uses all peaks in the sample to correct for preferential amplification effects Median peak intensities are derived from the first nine data points in a trace then sliding to data points 2 10 3 11 etc to ascertain the local median intensities This median filter reduces variation in
27. contain the Bin information will be deleted not the columns which contain the sample information Binning Options To adjust which Bins are displayed and to merge Bins in the Report Table click the Bin icon in the toolbar of the Report Table The Report Bin Columns box will appear Display Bins By default all Bins will be selected with a checkmark at the beginning of the row Individually deselect Bins for exclusion from ISI Bhe 11 Im me i the Report Table by single left clicking the checkmark box To SZ 11208 00 00 Bhet 134 Blue 128 9 0 0 0 0 Blue 135 Blue 129 0 0 0 0 0 Blue1 deselect all right click any cell in the Report Bin Columns box and 13 Bue 128100 a select Uncheck All To deselect only a few Bins left click a cell to Ke Er or Check highlight the row then hold CTRL or SHIFT key and select 140 Bue 1324 04 04 SE 141 Blue 133 4 0 0 0 0 142 Blue 133 6 0 0 0 0 Uncheck All additional rows Next right click and select Check or Uncheck to IS Bhe 185 00 00 include or exclude the Bins respectively Click OK in the Report we Ble 147 om aa as 146 Blue 135 2 04 04 Bluel Bin Columns box when finished and only the Bins with checkmarks will be displayed in the Report Table 0 5 0 5 Blue 0 4 0 4 Bue Merge Bins To make two or more Bins become one Bin single left click a row to highlight it Next hold down SHIFT key to select additional rows Right cli
28. gt Import Data from the main analysis window Select Tools gt Macromolecules gt Settings 3 Select Add and enter peak range for one group of fragments 4 Repeat for a second group of fragments at the opposite end of the size range 5 Select Ok and verify fragment selection white boxed 6 If needed select Tools gt Macromolecules Range to launch the Peak Range Editor and correct for any fragments outside of the range 7 Align the results Tools Macromolecules gt Show Results 8 Analyze data with Project gt Run gt Select Analysis type Animal gt Next gt Next gt OK N vj Dr Dr D 3 B Y E D DE Dr Dr 4 CR Dr Sr Dr Ei Dr Dr Gr mr Dr Dr D DE Sr Dr D Dr p a B Sr o Dr Sr e Dr Dr D Sr BEST 9 Review data Export Report Table xIs or txt or Print the Allele Report 10 If desired a panel may be made in the Panel Editor to allow application of local max filter in the Run Wizard 11 Project gt Run gt Select Panel from the drop down menu 12 Next gt Next gt OK Mutant 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 1 No Dye Size Height Area Marker Allele Difference Quality Score Comments 1 Blue 103 5 27077 108794 Blue_1 102 0 1 Pass 500 0 E Blue 109 9 7980 30021 Mutant 108 0 2 Pass 500 0 d 100 May 2012 Chapter 8 Additional Tools File Conversion The file conversion tool is used by in
29. inbreeding wild populations e Controlling rate of inbreeding wild life management and livestock e Quantifying natural population size e Identification of clones within a population e Identifying and ranking potential relatives e Verification of the number of successfully breeding individuals e Identification of lost stolen pets or livestock Procedure 1 Open data file or previously saved project 2 Run Wizard to call alleles 3 Select Relationship Testing from the Applications drop down menu 4 Select Allele Frequency of the appropriate population from the Tools drop down menu Individual sample 1 Tools gt family group tool gt OK to allow selection of each individual in the file as a separate node Use the Family dropdown menu to select the individual file Right click on the node and select find family from the drop down menu Left click on the Report icon in the tool bar to display the file name and any duplicates of that file found in the data base The samples with the highest LR for each relationship type are displayed in descending order in addition to the sex number of matched alleles and matched markers po lt lt Lei In this example there are seven samples with the identical microsatellite profile A Sr fil E vater mates ls D The random match probability indicates Samples LA Chans B Repor Bi E FileName iD Name x Matched Alleles Matched Markers that there is a 1 in 529 000 chance th
30. second position in a marker vice versa for a 2 1 ratio So the question arises is this ratio imbalance real or is it due to preferential amplification of the first allele Ratio Plot The GeneMarker Trisomy tool offers two answers to the 1 2 2 1 trisomy detection question First in the Ratio Plot in the bottom left corner of the analysis window the peak intensity ratio of all markers are plotted A linear regression line is run through the center of the data points and is used to correct for intensity drop due to fragment size increase The Ratio Plot can be viewed as a linear regression plot or corrected for slope This method of data correction aids in the detection of imbalanced ratio trisomy T Score The second aid in trisomy determination is the trisomy score First a t value is determined and defined as the difference between the sample and the expected value divided by the standard deviation There are two possible t values for every marker one is the t value for heterozygote and the second is for a trisomy T Score is the ratio of the heterozygote t value divided by the trisomy t value Therefore as the T Score increases the confidence of the trisomy call also increases A T Score greater than 5 0 is a confident trisomy call A T Score less than 0 6 indicates a confident heterozygous call 145 May 2012 Chapter 7 Special Applications Raw Data Plot vs Corrected Data Plot 2 2 T T Ka Ka ei D a T E V
31. 1 1 1 1 1 137 0 OO a e e OO OO ON e OO OO ON 139 141 0 0000000000000 2 DOI fsa K27774_D70908 2 K27774_D70906 2_ K27774 D70907 2 K27774_D70908 2 K27774 D70909 2 K27774 070910 K27774 D70911 K27774 D70916 2 HO h AU tea B02 tsa DU tsa DOG tse 2 E02 tsa 2 FOZ tea K27774_D70912 2_ K27774_D70913 2_ K27774_D7091 4 2 K27774_D70915 2 2 C03 tsa K27774_D70917 2 G02 fsa HO2 fse ANS r BO fra DO3fsa Match by Sections Match by Fired Position Group By Order Control slk 1 CaeSentive Group Identification Section Separators f p gt Corteol Mach Mode Compare by Section 2 c Wordt er Control Identification Match to identiiee by Section 1 7 f X Cancel 140 May 2012 Chapter 7 Special Applications What To Expect K31568_D70904_G0 K 31568 D70905 HO K31568 D70913 H K31568 D70906 Am K31568 D70908 CG K31568_ De BOF K 31568 Dao DO E563 070811_F02 E31568 070812 Go 31568 Damon EG K 315 8 070817_D00 E 562 Dame CO 31569 D 70914 403 31568 D70915 Du K27774 D70904 3 GO K27774 D709132 Hoe K27774_D709052_H01 K27774_D70905 2_402 K27774 D70908 2 CO2 K27774 D70907 2 BO2 K27774 070911 2 FO2 K27774_D70912 2_G02 K27774_D70909 2_D02 K 27774_D70910 2_E02 Kam D70914 2 em K27774_D70815 2_B03 K27774 D70916 2 D 27774 D70917 2 D The analysis in the upper diagram is based on just the results from the 4
32. 1 PAT 1 Cfsa 678 XY 26 26 13 13 3 93E 23 Father drop down menu 1 PAT Ftc 679 XY 16 26 13 13 4 386 09 3 Left click on the Report Select Node 1 EG 680 XX 15 28 13 13 1 116 10 icon in the tool bar to tn 1 ETT 727 XY 23 28 2076 13 Select Parents Io 8 ee 2 Mha 718 XY 23 28 207E 13 display the file name and Select Sibling 3 773 fsa 720 XY 14 26 7 24E 04 fsa 685 0 24E 0 any duplicates of that file Select Family PAT Fia e 18 EE 6 PAT5 Cfa 733 XX 10 28 5186 01 found in the data base The Edit Node 7 sa 724 XY 9 26 5136 01 Add Child 8 PAT_5 F sa 740 XY 10 26 3 996 01 samples with the highest Add Mate lo Aretha T 2 ee LR for each relationship Find Family Hal St GH L E ES type are displayed In FETE 3 PAI 1 Fifsa 728 XY 14 26 5 06 05 elete Node with Branchs 4 PAT 1 Mifsa 723 XX 12 26 1 856 05 descending order in Delete Side Ned 5 mia 72 XX 12 26 1 856 05 6 FAT 2 Mise 683 XX 10 26 2 226 04 addition to the sex number eee 7 PAT 5 Fiisa 631 XY 11 26 1216 04 d Pp P 8 2 fsa 722 XX 11 26 1 046 04 3 PAT7CI 635 XY 12 26 3 086 03 of matched alleles and E 10 PE Ca 690 XX 11 26 5 336 03 matched markers Sample File PAT_1_C fsa Icons and Functions d Relationship Testing Relationship Testing Main Drop down menus Ede gt DetsBase gt Tonlt Select from File DataBase or Tool options di Relationship Testing Relationship Testing Tools include File DataBase JB Family Group Toot Family Group Tool
33. 184 184 Mb cM MAP1B54 b 225 221 221 219 D552046 b 3 1 1 207 20 200 Sen SH Ce SE Bags D556 9 b 250 259 246 D5S637 a 297 EH Si 297 En MAP1B78 a 235 235 240 238 D55681 b 2 6 1 287 287 287 D5S351 0 SE oe a So D55435 b 2 1 1 182 170 170 D55112 a 304 304 304 300 D5S629 a 49 2 499 415 415 D5S1982 8 332 332 332 336 D55823 a 1 7 184 184 184 MAP1B54 b cl A 1 225 221 219 aa D551556 a 0 417 419 426 417 421 Mb b 2 3 2007 E D5S2046 b 31 207 207 E fi SE D55681 b 2 6 287 287 a SE 2 1 178 170 D55351 a 13 i 325 331 325 D55823 a 17 184 184 MAPIB54 b 1 3 221 219 D55112 a TIRS 304 304 300 D5S1556 8 0 415 419 421 S D5S610 b 0 7 218 216 D551982 a 3 1 332 336 332 SG Se s MAP1B78 a 1 3 235 238 D55351 a 13 325 325 D5S112 a 13 304 300 D5S1982 a 3 332 336 Automatically refreshes and re assigns parental phase with addition of child ren information 13 Review phase assignment and make edits right click and select swap or edit haplotype or cross over To edit crossover use the mouse to draw a box around the appropriate section of the haplobar ll Haplotype Analysis New File _Haplotype Analysis C Users SoftGenetics Desktop Haplotyping Data 07_08_2009 be 44 Haplotype Analysis C Users SoftGenetics Desktop Haplotyping Data 07_08_2009 haplotype File DataBase Tools File DataBase Tools File DataBase Tools F vz T BIr Family 141 1 Family 1 1 lyv a T E Q Q amp Family 1 1
34. 450 Sample Name 041915 JS P34 Machine 3100 Run Time 4 14 2005 13 27 40 gt 4 14 2005 14 17 43 Statistics Probes Mean StdDev Contro Sample 6 41 0 86 1 14 0 29 0 36 Peak Ratio Conclusion r jan arse o d 200 400 500 Size bps Electropherogram allele label options are set in the Main Analysis Window gt View Preferences gt Display Settings gt Chart Settings gt Max Allele Label Layers Select this option to display the allele labels in layers rather than the above view which maximizes the peak display If there are more than 46 samples a second page will be generated Page option B4 allows up to 52 probes to be included on the first page of the report table Highlighting selected probes in the Final MLPA Report In some chemistries such as those that may contain pseudo genes it is desirable to highlight certain alleles in the final report Designate these alleles with HL in the comments column of the panel editor The allele names that are designated HL will be highlighted in yellow in the final report table and represented as a fuchsia triangle in the ration plot MLPA Analysis Report General Metro Hospital TC Tree Size PO34 41115 Software GeneMarker V220 Analysis type m Tee er Project dmd practice SGF Compare Type MLPA Ratio Technician Mormalization By aisen Normalzation en a pe Report Time 10 26 2011 102943 Quantification By Peak Height er hor Panel HL test P034 DM P
35. CHAPTER 9 USER MANAGEMENT ss 177 A 178 PROCEDURE a lA A idas 178 SER IVI AINA GER cs od e a ln da a id o a nd rai eee eos 178 AO 179 EEIN A NNN 179 EDIT HISTORY AUDIT TRA an a 179 NEE 181 4 May 2012 Chapter 2 General Procedure Chapter 1 Installing GeneMarker Chapter 1 Installing GeneMarker Computer System Requirements Validation Version Local Version Network Version Questions 5 May 2012 Chapter 2 General Procedure Computer System Requirements GeneMarker software has been tested and validated for various computer systems The minimum system requirements are Windows PC OS Windows XP Vista Windows 7 Processor Pentium III 1 GHz CPU RAM 512MB Available hard disk space 20GB Intel Powered Macintosh Parallels desktop for Mac Mac OS virtual machine dependent or Apple Boot Camp or VMware Fusion Mac OS virtual machine dependent RAM 2GB Available hard disk space 20GB Installation of GeneMarker is not supported on Linux or UNIX based operating systems GeneMarker will only recognize PC file formats To convert Macintosh file formats to PC file formats please download the ABI PRISM 3100 Genetic Analyzer Conversion Utilities to convert Mac files to PC files at http www appliedbiosystems com support software 3100 conversion cfm Validation Version The validation or trial version of GeneMarker can be installed on as many computers as you wish The trial period expires 35 days afte
36. Control lt TO 3 be displayed after the tenth sample name p Print Statistics Information Print Summary Led leng Ven eeng Dn eg eg amp Ok X Cancel 89 May 2012 Chapter 7 Special Applications Print Statistics Information Displays the number of probes mean and standard deviation for the reference and sample in the bottom left corner of the report Print Summary Attaches an extra report to the end of the Clinical Reports that summarizes the ratio information for all the selected samples Display Quality Control Fragments Displays the quality control fragments in the print report MLPA Clinical Research Report Below is a description of the MLPA Clinical Report features For an explanation of functions within the Print Preview window see Chapter 6 Reports and Printing If there are more than 50 probes in the analysis a second page is generated with the remaining alleles in a table the header trace overlay conclusion box and ratio plot Report Header Contains information about the analysis project sample and parameters Overlay Trace Similar to the MLPA Analysis window displays the reference trace in light red behind the sample trace dye color The zoom setting in the MLPA Analysis window is applied in the Print Report Ratio Plot Similar to the MLPA Analysis window the plot points indicate the Peak Height Area Ratio of sample to reference Statistics Information When selected the statistics
37. Dimensional and a 3 Dimensional view of the selected samples See Chapter 8 Additional Tools Quantitative Analysis Offers two different analysis types for quantifying peak areas Curtain Method and De convolution Method SNPlex SNaPshot A SNP discovery application for use with SNPlex SNaPshot and SNuPe data analysis Includes two color comparison and a ratio plot of homozygotes and heterozygotes MSI Analysis Allows comparison of microsatellite markers in normal and tumor samples to detect instability Includes a trace comparison histogram and a patient report with positive and negative MSI marker calls Clustering Analysis Often used with AFLP data the Clustering Analysis module creates a dendrogram view of all samples in a project Users can choose from Euclidean Distance or Pearson Correlation and Single Complete and Linkage type analyses See Chapter 7 Special Applications AFLP Trisomy Analysis Detects additional alleles beyond the expected ploidy within a marker by peak intensity or peak area and calculates a confidence score A patient report is also available Relationship Testing Uses Identity by Descent methods to search a database for nearest relatives kinship analysis and parentage verification Pedigree drawing may also be helpful in reviewing uniparental disomy LOH Analysis The user defines limits for a ratio plot which then uses normalized peak intensities or peak areas to determine the presence or absence of allel
38. FORTH IN THIS SECTION GIVES YOU SPECIFIC LEGAL RIGHTS YOU MAY HAVE ADDITIONAL RIGHTS WHICH VARY FROM JURISDICTION TO JURISDICTION For further warranty information please see the jurisdiction specific information at the end of this Agreement if any or contact SoftGenetics LLC s Customer Support Department 7 DISCLAIMER THE FOREGOING LIMITED WARRANTY STATES THE SOLE AND EXCLUSIVE REMEDIES FOR SOFTGENETICS LLC S OR ITS SUPPLIER S BREACH OF WARRANTY SOFTGENETICS LLC AND ITS SUPPLIERS DO NOT AND CANNOT WARRANT THE PERFORMANCE MERCHANTABILITY OR RESULTS YOU MAY OBTAIN BY USING THE SOFTWARE EXCEPT FOR THE FOREGOING LIMITED WARRANTY AND FOR ANY WARRANTY CONDITION REPRESENTATION OR TERM TO THE EXTENT TO WHICH THE SAME CANNOT OR MAY NOT BE EXCLUDED OR LIMITED BY LAW APPLICABLE TO YOU IN YOUR JURISDICTION SOFTGENETICS LLC AND ITS SUPPLIERS MAKE NO WARRANTIES CONDITIONS REPRESENTATIONS OR TERMS EXPRESS OR IMPLIED WHETHER BY STATUTE COMMON LAW CUSTOM USAGE OR OTHERWISE AS TO ANY OTHER MATTERS INCLUDING BUT NOT LIMITED TO NON INFRINGEMENT OF THIRD PARTY RIGHTS INTEGRATION SATISFACTORY QUALITY OR FITNESS FOR ANY PARTICULAR PURPOSE The provisions of this Section 7 shall survive the termination of this Agreement howsoever caused but this shall not imply or create any continued right to Use the Software after termination of this Agreement 8 LIMITATION OF LIABILITY INNO EVENT WILL SOFTGENETICS LLC OR ITS SUPPLIERS BE LIABLE TO YOU FOR ANY DA
39. Fa Jee T RISI Family 141 1 Family 1 1 O A B SMA3 SMA3 8 8 D552046 E 198 203 D552046 E 198 198 D552046 E 198 203 D55679 250 288 D55679 288 250 D55679 288 250 D55681 d 293 293 D55681 293 293 D55681 293 293 D55435 170 170 D55435 5 170 182 D55435 170 170 D55629 417 415 D55629 415 419 D55629 415 417 D551556 419 421 417 4117 D551556 419 421 D55823 184 184 D55823 184 184 D58823 i 184 184 Select Node D551556 411 41 419 421 D55610 f 218 218 D55610 i 218 D55637 297 295 D58637 295 D55610 l 218 218 MAP1B54 S 219 223 MAP1B54 223 D55637 295 297 Select Parents MAP1B78 E 235 231 ek MARID G AE D58351 325 333 ct Sibling MAP1B78 S 235 231 D55112 306 304 D55351 E 325 333 D551982 332 338 Jeselect Node MAP1B78 235 D55351 325 D55112 e 306 D551982 332 Select Family D55112 S 306 304 Edit Node D551982 33288 En Add Child dd Mate ye Find Famil SI WM D552046 198 ETER D55679 288 D552046 D552046 198 D58681 293 D55679 Edit Haplotypes D58679 288 D55435 170 D55681 Cross Over D55681 i 293 D55629 415 PESEE 8 Delete Node with Branchs D55435 j 170 D55823 184 D55629 i D55629 j 415 D551556 411 417 D55823 E DO Node D55823 f 184 D55610 218 D551556 Export Image D551556 411 417 D55637 295 D55610 i D55610 E 218 MAP1B54 223 D55637 D55637 295 MAP1B78 231 MAP1B54 MAP1B54 S ye D55351 333 MAP1B78 MAP1B78 231 D55112 304 D55351 D58351 333 D551982 338 D55112 D55112 304
40. Free form text box to enter a name for the Edit Individual individual Display the in dividual s name in the Pedigree Tree by clicking the Show Individual ID icon in the toolbar Se Gender Select either Male Female or Unknown gender for the individual Male nodes are squares female nodes are circles and Unknown gender Father 2 ES nodes are displayed as a circle within a square Father Mother When more than one mate is Mother 1 El displayed for a Mother or Father a drop down menu allows the user to choose which possible parent to Sample Fie PAT_3_F fsa associate with the child NOTE The Gender and Father Mother options are not Cancel available when adding a mate Person Info Affect Status Joe O Unknown Gender Male Female C Unkown C Unaffected Affected Status Affected Status options are available to mark individual nodes for genetic linkage calculations Before marking individual nodes with Affected Status click the Pedigree Parameters icon in the toolbar and adjust settings accordingly Unknown The individual s phenotype is unknown The node is displayed as an empty square or circle Unaffected The individual does not show signs of the expected phenotype The node is filled in white Affected The individual expresses the phenotype The node is filled in with diagonal hashed lines Deceased If the person is deceased select deceased in the individual edit screen This extended information affect s
41. Gel Image displays the unprocessed data in a traditional gel format with larger fragments located on the right The Electropherograms display fluorescent signal intensities as a single line trace for each dye color The signal intensities recorded in Relative Fluorescent Units RFUs are plotted along a frame scale in the Raw Data Analysis window with fragment mobility from right to left The smallest size fragments are on the far left of the trace 16 May 2012 Chapter 2 General Procedure Raw Data Analysis Window Sample Tree Synthetic Gellmage Raw Data Trace d GeneMarker Prntied jo gt ev Sg Urtited amp Row Data 015 45 N tea Y 01546 T fr w TIESIN ha O16 54 T 118 017 55N tsa 017 56 T fra O18 57N iza 0M1958 7 1 a 019 59 N fra 013 60 T fos 00614 Ia 020 62 7 tee 021 63N ta O21 64 T fra 022 65 N tea 022 65 T fr 3091 7 19407 x Main Toolbar Icons a gt x s 8 2 H Spike Removal Removes peaks from voltage spikes caused by micro air bubbles or debris in the laser path This option is selected by default in the Run Wizard Saturation Correction A synthetic peak is created based on peak shape before and after saturation The results of these will be less accurate than that of non saturated peaks This option is selected by default in the Run Wizard Smooth This function smoothes the baseline by eliminating smaller noise peaks This option is selected by default in the Run Wizard B
42. MLPA technique for detecting genetic deletions and duplications in various diseases including cancer Recently the MLPA technique has been improved to detect methylation sites within promoter regions and for genomic imprinting applications Promoter Methylation kits from MRC Holland include ME001B Tumor Suppressor ME002 Tumor Suppressor and MEOL1 for Mismatch Repair genes Genomic Imprinting kits from MRC Holland include ME028 PWS AS and ME030 BWS RSS GeneMarker s new Methylation Specific MLPA MS MLPA module quickly and accurately detects methylation sites for researchers studying promoter methylation and genomic imprinting diseases GeneMarker s ease of use and professional reporting options are an excellent choice for MS MLPA applications MS MLPA Sample Group List Report Table Electropherogram Ratio Plot d MAER Aessen en El PA BE B QQ GE Blue D ss zl O Sagie A 2 Report QW O Gap See Patent Digened SCT eg Hie el Set ien fratidnel Digested a a e _ D E eg i D er VE Me t o pened 2 ge EN W 260 603 158 EZ 350 320 S s S 2 FEF Patere Urdges F e Potert _Dgenet SOF 4 096 fs se Ca Gag 4000 19001 in a i i 1 20 DI 1 000 LA A o gt w fe sem aan BOR e ra noansss SAD Benen Ar Atm RARO papaz pous juss Bras eer part A ED pma 17 mn WT sg PAR Rz TSC Con SR T RST Los uc Y NN 7 d d dh A SW WR TWRSF LA KLKX CASS fis
43. MSI Score Setting High MSI Score Setting Reports and Printing GeneMarker automates the analysis process and creates an easy to read report for fast MSI High or Low determinations Report Table Displays gain and loss information for individual peaks of a sample compared to a reference Samples designated as reference by the filename group text file are marked as blue rows in the report while samples 134 May 2012 Chapter 7 Special Applications being compared to the reference are in the white rows The Overlay Trace window will correspond to which ever peak cell is selected in the Report Table Report Settings Launches the MSI Report Options dialog box Output Status Peaks MSI Report Option KR Choose to display Loss Equivalent or Gain peaks Loss peaks are Er SE represented with a 1 Equivalent with a 0 and Gain with a1 Peaks that do Lose not meet the MSI Score thresholds for loss and gain will not be displayed in Equivalent the Report Table Gain Abide By Panel Iw Abide By Panel When selected all peaks regardless of their loss gain status will be displayed When deselected only those peaks positions with a loss gain Gan pray y H H 5 will be displayed OO Save Report Choose to save the Report Table as an Excel file xls or a tab delimited Text file txt MSI Clinical Report Settings Click the Print icon in the main tool bar to launch the MSI Print Settings box and preview
44. Manager in the Select Program window and click Next Click Next in the Destination Location window Next in the Select Program Manager Group window and Next in the Start Installation window to enter the LSM installation wizard Click the Next button in the Welcome window Read the SoftGenetics End User License Agreement and click the I Agree button in the Read Me File window Click Next in the Destination Location window to install LSM in the default folder Click the Browse button to choose a different Welcome to SoftGenetics License Server Setup program This program will install SoftGenetics License Server on your computer It is strongly recommended that you exit all Windows programs before running this Setup Program Click Cancel to quit Setup and close any programs you have running Click Next to continue with the Setup program WARNING This program is protected by copyright law and international treaties Unauthorized reproduction or distribution of this program or any portion of it may result in severe civil and criminal penalties and will be prosecuted to the maximum extent possible under law installation directory program is C ProgramFiles SoftGenetics License Server 11 Click Next in the Start Installation window to install License Server Manager 9 May 2012 EST en Chapter 2 General Procedure 12 Select the Launch License Server Manager option and click Finish 13 Click OK in th
45. Name DAVID DESKTOP Stopped has an internet connection select Online Registration If the computer does not have an internet connection or is connected to a proxy server select Offline Registration Online Registration A Locate the Account and Password on the SoftGenetics CD B Enter your Account Password and e mail address information in the appropriate fields C The Request Code information is automatically generated by License Server D Click Register E Your software will be registered automatically A confirmation e mail will be sent to you once registration is complete NOTE Some characters can commonly be misread If you get an error trying to register check for number 1 and lower case letter L or number 0 and upper case letter O confusion F Restart License Server to apply the registration information Offline Registration G Copy and paste the entire Request Code string and type your Account and Password information from the SoftGenetics CD into the body of an e mail H Send the email to tech_support softgenetics com I The Register ID will be sent to you via email within one business day J Copy and paste the Registration ID from the e mail into the Register ID field of the Offline Registration tab K Click Register 10 May 2012 Femme o o i Account Email Register Cancel Register Local Text based Key Option Ze Register Online Request Code M
46. Network Client No previous network configuration has been detected Run Validation v Details Cancel Choose Registration Method xj Register Local Hardware Key Register Network Hardware Key e Register Local Text based Key Register Network Text based Key iS Installing License Server Manager will require restarting the system to complete installation Please save all work and close all applications before installing LSM Install License Server Manager 1 St 19 10 NOTE The default Destination Location for the License Server Manager Insert the SoftGenetics CD into the optical or CD ROM drive If your computer is not set to automatically open a CD navigate to the optical or CD ROM drive on the computer and open the directory Double click the GeneMarker Setup executable file EXE The Installation Wizard will launch Click the Next button in the Welcome window Read the SoftGenetics End User License Agreement and click the I Agree button in the Read Me File window Select Program Important Network Configuration SoftGenetics GeneMarker 2 2 0 x Install GeneMarker Recommended Ze Install License Server Manager Install GeneMarker and License Server Manager The license Server Manager is needed for users running this product in a The License Server Manager must be installed on the Server It is not to be installed on Client Computers Select Install License Server
47. Output Trace Data The Output Trace Data tool exports raw or sized data of uploaded sample gt files as Text txt or SCF scf or SG1 sg1 files Reeg CSoftGeretcaVGene Marker Datasets MSI TraceD ata tet E Procedure EE a 1 Select whether to export the data as a Text or SCF file EH SC 2 Choose the directory and folder to save the exported data to in the Output File Name field 3 Select the samples to include in the output file from the Select Samples field 4 Select which dye color data to export from the Select Dyes field Select whether to export raw or sized data from the Data Type options 6 Click Export to export the data to the specified folder A Project Comparison Tools Project Comparison The Project Comparison tool can serve two functions First it can be used to compare two independent analysts analyses Second it can be used as a validation tool to determine differences in allele calls based on analysis parameters or instrument runs Procedure 1 After initial dataset analysis select Tools Project Comparison 2 The Project Comparison window appears 3 Click the Open Project to Compare icon 4 Use the file directory window to locate and select a previously saved SoftGenetics project file sgf sfp NOTE Projects with similar datasets and analysis types should be chosen 5 Click Open and the second project will be uploaded to the Project Comparison tool 6 The first
48. Overlay position Click the Save Report icon to save the Report Table as an Excel or tab delimited Text file Procedure 1 Import LOH raw data files and filter with Run Wizard Fragment Animal Analysis settings See Chapter 2 General Procedure Create a Panel for the data with the Panel Editor tool See Chapter 5 Panel Editor Apply the Panel Select Applications gt LOH Analysis The LOH Analysis Settings box appears Upload a tab delimited text file that identifies Reference samples first column and Experimental samples second column to the Group File field NOTE Use the File Name Group Tool to create a group file See Chapter 8 Additional Tools 7 Adjust LOH Analysis Settings as desired 8 Click OK 9 The LOH Analysis window appears 10 Click the Print icon to print the LOH Clinical Report ol te alt e Icons and Functions Load Group Information Choose a tab delimited Text txt file which contains information on how to group individual sample files For example pair Patient A s tumor sample to Patient A s normal sample by placing the normal sample filename in the first column and the tumor sample in the second column See Chapter 8 Additional Tools Analysis Settings LOH Analysis Settings X 1 Opens the LOH Analysis Settings box SE C SoftGenetics Gene MarkerDatasets LOHSLOH g Quantification by Group File Click Open File icon to upload a txt file that groups samples by filename Peak H
49. Pedigree tool select Applications gt Pedigree from the menu bar of the Main Analysis window File types accepted or generated by GeneMarker Pedigree module Pedigree File PRE PED Contains information about the Pedigree Tree display Individual Sample Accordance File SMP Contains Family and Individual ID information associated with specific sample file names Loci Description File DAT Contains gene frequency information generated in the Pedigree Parameters box There are two ways to begin a relationship test with the Pedigree tool upload a previously created pedigree file OR create an entirely new pedigree file The procedures are described below Upload Previously Created Pedigree A Load Pedigree File ele x 1 In Pedigree tool click the Open Pedigree File icon SE 2 The Load Pedi gree File box appears C Users SoftGenetics Desktop TestPed pre 3 Click the Open Files icon next to the Pedigree Files field I IndividualSample Accordance File SMP 4 Auto Load 4 Use the Windows Explorer window to locate the Pedigree File PED telen ol El PRE 5 ee the file and click Open E NOTE The Pedigree File selected must be associated with the samples currently uploaded to GeneMarker If the sample filenames are not the same the Pedigree Tree will appear grey and will not link with the Sample List or Electropherogram Chart 6 The directory path will appear in the Pedigree File field 7 The s
50. Process HID Analysis Set date process options Raw Daa Anshen Abele Cal Y Auto Fange rare fa Y Auto Range bos Si D Stat Je 3 end fem 3 Smooth l Erbunced Smooth Peak Detecton I hmesrold Y Pest Sauron V Baseine Subtraction Irene tp 50 Percentage gt 1 S Max M Enhanced Baseline Subtraction 5 oca Regon gt 5 SI Local Max V Pubup Conection Spike Removal Max Cad intensity 30000 3 Size Call i F x usd Fite Local Southern Cube Spire Lage See Y Ster Poak Firer X Pi ne Lek Li 3 ptn Load Dead Swe Desa Chapter 7 Special Applications Local Percentage 25 Max Call Intensity 30 000 Stutter Peak Filter Left 20 Right 20 Plus A Filter Selected Run Wizard Additional Settings Allelic Ladder Select an Allelic Ladder sample from the dataset drop down list Allele Evaluation Peak Score Reject lt 0 5 Check 7 lt Pass NOTE If the Reject value is set too high a false negative call could result If the Reject value is set too low then a false positive call may occur HID Panel Adjustment Addtional Settings HID Analysis Set addhonal aphore related to the dieser Abele Ladder After the data has been sized and filters have been applied go to Tools Panel Editor to adjust the selected Panel for the dataset For explanation of specific functions in Panel Editor see Chapter 5 Panel Editor Se OY St eS Ze In Panel Editor select the appropriate forensic Panel from the Pane
51. Run Wizard Then select Save on the Tepmalte Selection screen To create a new template click Select an existing template or create one A template can also be selected from the list of available templates in the left section of the window and then saved for future use by clicking the Save button If you do not want to use a template select the appropriate size standard standard color and type of analysis Use last template will automatically be selected Panel GeneMarker comes preloaded with many common kit Panels including Promega s MSI kit and MRC Holland s MLPA kits Additional Panels can be imported by selecting the Open Files icon next to the Panel field A custom Panel can be created in the Panel Editor tool See Chapter 5 Panel Editor NOTE It is recommended to size the data prior to selecting a Panel for comparison Select NONE in the Panel field for the first Run Wizard data process action 20 May 2012 Chapter 2 General Procedure Panel Editor A Panel can be selected from any available from the drop down menu or can be viewed UL and selected by clicking the Panel Editor icon Import a Panel If a Panel cannot be found in the Panel Editor tool it can be imported by clicking on the Lair Import a Panel icon Size Standard GeneMarker comes preloaded with many common size standards including GeneScan 500 and LIZ600 A custom Size Standard can be created by selecting the Size Template Editor icon next to the Size Sta
52. SHIFT and mouse over the vertical white line of the Bin edge When a double headed arrow appears hold down left click and drag the Bin edge to adjust the range Delete Bin Right click the vertical grey bar in the center of the Bin in the Overlay Trace Select Delete Allele The Bin will be deleted from the Panel To delete multiple Bins hold down CTRL key and left click and drag across peaks in the Overlay View A light blue hashed box will appear Right click in the hashed box and select Delete Alleles The Bins highlighted by the hashed box will be removed from the Panel Panel Table The Panel Table displays Marker and Bin information for the dye color displayed in the Overlay Trace frame All columns except Dye and Marker can be edited in the Panel Table Right click a highlighted cell and select Set Value to Column to make all values in the column equal to the value in the highlighted cell Dye Indicates the dye color of the Bin Marker Indicates which Marker the Bin is contained in Size Indicates the position of the Bin center in basepairs Left Right Range Indicates the range of the Bin on either side of the Bin center Allele Name Peaks that appear within the Left Right Range of the Bin will be labeled with the Allele Name Control Bins marked with a 1 are considered Control Bins Enter 0 in Control column of any allele that the contrasting color is needed example Abbott Cystic Fibrosis mutant alleles
53. Samples click the button next to Selected Samples and place a check Geer C Selected Samples mark in the box next to the samples you wish to appear in the report sane Sort Probe O By Size By Name Sort the probes in descending order By Size or By Name in the Methylation Report Table E Ok x Cancel Dem MS MLPA Print Report Below is a description of the MS MLPA Print Report features For an explanation of functions within the Print Preview window see Chapter 6 Reports and Printing Report Header Contains information about the analysis project sample and parameters Electropherogram Similar to the MS MLPA Analysis window displays the reference trace in light red behind the sample trace dye color The zoom setting in the MS MLPA Analysis window is applied in the Print Report Ratio Plot Similar to the MS MLPA Analysis window the plot points indicate the Peak Height Area Ratio of sample to reference Signature Box Date and initial space for report reviewers Methylation Report Table A list of all probe s peak ratio information Peak ratio cells which meet the methylation threshold criteria are highlighted grey Control probes are in bold font MS MLPA Print Report MS MLPA Analysis Report SoftGenetics E A Te du scF Software GeneMarker V1 70 Analysis Type Promoter Methylation Project Untitled Compare Type MLPA Ratio Technician Normalization By Population Normalization Sentai
54. amet 0 Een ee E 6 BRCA2 4159 Control Patient1_Undigested SCF Report Value Type Peak Ratio een 3008 1000 Patient1 Digested SCF a case 2670 0 000 z 8 ease 4760 oss ME0018 10 CD44 3201 0 000 750 m fcon eses ros GE Jen 278 CC ER 1611 0000 he conza rz Jam t el ee T327 se e emer 4705 fore 20 parkt 3484 ooo 21 Jesrt foros usa 22 fram fass jooo 24 mer 2209 00 25 ks 3883 0 982 28 mm fees Jooo 29 mns 2o05 st 30 Pan 12299 oss 31 PARK2 1533 De epn fae 6 000 ERC 34 RASSF1 Ge 5 Gees 000 135 RASSF1 3801 0 000 Machine EG 56 mues 1884 Joo 37 TNFRSFIA 1744 W9887 7 Run Time 00 00 00 gt 00 00 00 i i 138 TNFRSF7 4419 0699 ea 699 39 DEG 13973 0000 msez 2874 es ar var 3533 oo0 Peak Ratio 300 Size bps 100 May 2012 Chapter 7 Special Applications Human Identity HID The Human Identity HID Analysis Type in GeneMarker was designed for use with forensic short tandem repeat STR data The forensic community recognizes approximately 18 core loci by which humans can be tested and typed The core loci were selected for human typing because they follow the Mendelian laws of segregation and independent assortment they typically do not code for any particular protein and they are highly variable in th
55. ana 51 Call Allele oooooccconccnncccnocccnccnnnacinincnonos 25 41 Call Size ee WEE 25 ev nere EEEIEE ANERE 26 Call the ET TE 26 Cartesian POU rene eng 123 Change Morker 58 Change Password rrnnnrrrnnnnnnrvnnnnnnrvnnnnnnrrnnnnnrsrnnnnsssnennn 178 Change User 178 Cones DULEON suse 37 Chart Heie D EE 73 Chart Overlay usaras 73 Chart Sense 36 Ebart Sopelatronb neen 52 Check Range md 64 Sag NE EE EE 29 chronic lymphocytic leukemia CLL 146 Circularizing padlock ligation porobes 129 May 2012 Index ee araenaereaneuiess 35 Classic Trisomy Print Report 148 CentcomueruoassausansmussomnGtosane 9 CCM 35 Clustering Analysis cocooocccnccnoccnnnonononononanonononos 38 Clustering Analysis Settings 00 000eeeooseeeesseeeeen 137 Clustering Information ooncnncnnnnnonarononarononaronanarononass 126 Clustering Report ss 138 Clustering Rule ooocccccooccnncconccnnnconcconononononnnos 137 CODISRepO HS 102 Color Channels 16 37 COMMONS vana caras 71 Combined DNA Index System CODIS 102 Complete MK 137 Complex Peaks unio considerada did 122 Condense FINS td 176 CONMACNCE Eura 24 Confirm Peak pave 70 Confirm GC 34 Confirm Unconfirm A 33 Confirm Unconfirm Allele oo 33 Conflict with Parents 104 Conflict with Siblings coooooococccooccnnnnos 104 contact US a 13 Content ODTIONS aS as
56. and Allele ZE Gem Label of each called peak Samples are listed in rows in the far left column and CA E Feet Homozygous Panel Marker names indicate the columns at the top of the table a ER Marker Table Fragment is the default Report Style when Fragment Animal Fe Fragment Plant and HID Analysis Types are selected C Sample Name e File Name NOTE Marker Table Fragment requires that a Panel is applied to the data See rm Jae ele ea Chapter 5 Panel Editor Horizontal Verica Iw Show es Low Score Alleles Iw Hide Extra Sample Names Features z EA Options Extend Diploid Homozygous Repeats the same Allele Label in the second allele position of the marker when only one peak is detected in the marker Only active when the Edit Panel Ploidy option is set to 2 Diploid Show Allele Name Size 0 1bp Height Area Allele Name is displayed in the Report Table by default regardless of table Orientation Select to display Size Height and or Area of the peak all within the same cell Parentheses separate the peak statistics from the Allele Name Only enabled when Vertical Orientation is selected Orientation EEES III IO OE A n oa oa na na so oo aa aa oa na oa ag els OE g gt a gt OE gs g sg gt g gt g g gt OE g ss ow o w a w e oe o oe o w w 68 May 2012 Chapter 6 Reports and Printing Horizontal Sample names appear on the left in row
57. and Serbia and that you are not otherwise prohibited under the Export Laws from receiving the Software All rights to Use the Software are granted on condition that such rights are forfeited if you fail to comply with the terms of this Agreement 10 Governing Law This Agreement will be governed by and construed in accordance with the substantive laws in force in the State of Pennsylvania United States of America June 2012 Table of Contents GeneMarker v 2 4 0 TABLE OF CONTENTS hhv 1 CHAPTER 1 INSTALLING GENEMARKER scada ENS 5 COMPUTER SYSTEM REQUIREMENTS avse 6 VALIDATION VERSI N cosido diia T ET E T EOR 6 MENN 6 RENT NN 7 TVS COUN GIO EE o A 7 FN 7 TN 8 If you wish to exchange hardware based licensing for text based licensing please contact er Re CU 9 gH TENES CON EE EEE SATE IOP aia 8 ENV ORK LICENSING OPTION EE EEE EE SE cie opens 9 Mey 9 Install GeneMarker software on the client COMPUTED rrrrrrrrrrnnnnnrrrrnnnnnrrrrnnnenrrrrrnnenrrrrrnnenrrrrnnnesssrrsnnenssennnneee 11 Upgrade of Egeter 11 Upgrade of GeneMarker software on client computer 11 Install NetDog Server Mongogement 11 PO SEO EE 12 Pla o AR EEE EE 12 PCGITHONO User EE 13 A A o e 13 CHAPTER 2 GENERAL PROCEDURE ee 15 IMPORT DATA MES oie 16 Me 16 POO CUICS eee eee 16 RAW RE orto A A 16 VINN 17 MA 18 ANN 20 TV Template FN 20 MINTE E 21 Run Wizard Additional SCttinGS ccccseecccsseecccaneeccssseccsausecsauneccsausecsaaueeesaunses
58. as OL Select this option to label alleles that are outside of allele ranges as OL Use Size String for Label Select this option to label peaks in the electropherograms according to size instead of the allele label To display a rounded size string set the Decimal Precision to 0 Larger Font Doubles the font size of the allele label characters This increased font size will carry over to the Print Report Highlight abnormal allele Select this option to highlight the labels of abnormal alleles with a bright color Excellent for Abbott Cystic Fibrosis Kits or any chemistry that amplifies mutant and wild type alleles Note these alleles must be marked with a 1 in the Control Column of the Panel File See Chapter 5 Panel Editor Chart Settings Max of Open Charts Select the maximum number of samples you would like to display as an electropherogram at one time Max 96 Use the Sample File Tree right click option Select Max to open the number of samples specified Max Chart in Page Select the maximum number of sample electropherograms you would like displayed in the Main Analysis window at one time Max 8 Use the Sample File Tree PageUp Down option to select subsequent groups of samples Max Allele Label Layers Select the number of allele label layers to view at once Max 10 This determines how far you must zoom in to clearly read neighboring allele labels and affects how the print report will be displayed This setting al
59. been selected in the 7 PeakTabie Main Analysis window Sample File Tree Iw Electropherogram Y Dyed Mix Dyes Advance ER XK Lance Contents Electropherogram Prints the peak trace for each dye color and sample selected NOTE The zoom setting of the Electropherogram in the Main Analysis window will be represented in the Print Report Zoom out fully to include all peaks in the Print Report Peak Table Prints the Peak Table for each dye color below the dye color s electropherogram trace NOTE If neither Electropherogram nor Peak Table were selected the Print Report will contain a list of each dye color selected for each sample selected and the allele count within each dye color Dyes Dye 1 4 Click the checkbox to include the dye color in the Print Report Mix Dyes Prints all selected dye colors on one electropherogram Advanced Options Print Report Print Project Comments Includes the Project Comments at the top of the Welte GES Print Report Select Each Page option to display the Project Comments on each page in the report Select Word Wrap if comments require more oe Ze i C All Samples W Dyel P than one line on the page Selected Samples Y Dye2 Label Dyes amp Peak Numbers Labels dye color with number of peaks for Contents A EN electropherogram vw Electropherogram IV Dyed Iw PeakT able i Mix D N Implement Y Axis Settings Prints the report using the Y axis settings the r e
60. chapters for the required analysis type Intensity Minimum RFU threshold of peak height used for peak detection Peaks below this value will not be called Percentage Relative minimum intensity of allele peaks to the 5th highest peak in the dye color used for peak detection Local Region Percent Uses the percentage of the highest peak for that marker to define the minimum peak detection threshold For example if the threshold is set to 33 the height of all allele peaks must reach at least 33 of the height of the highest peak in that particular locus to be called Max Call Intensity Maximum RFU threshold of peak height used for peak detection Peaks above this value will not be called Stutter Peak Filter Forward and reverse stutter peaks commonly caused by PCR chemical reactions can be removed using the Stutter Peak Filter The settings are in percentage of the primary peak The default settings will remove stutter peaks within 2 5 base pairs of the primary peak Plus A Filter When selected the second peak of a split peak one basepair spacing between two peaks will not be called Deselect to call both peaks separately Load Default Recalls any settings previously saved by the user If there are no user saved settings the program loads the default settings for that particular analysis type Save Default Saves any settings defined by the user that is different from the default These settings can be recalled for consistency
61. continue with the Setup program WARNING This program is protected by copyright law and international treaties Unauthorized reproduction or distribution of this program or any portion of it may result in severe civil and criminal penalties and will be prosecuted to the maximum extent possible under law 4 SoftGenetics GeneMarker 2 2 0 x Select Program oS FAS e Install GeneMarker Recommended Install License Server Manager C Install GeneMarker and License Server Manager Important The license Server Manager is needed for users running this product in a Network Configuration The License Server Manager must be installed on the Server It is not to be installed on Client Computers lt Back i Cancel xi No security key has been detected If a local license was purchased click Register Now If a network license was purchased click Configure Network Client Configure Network Client No previous network configuration has been detected Run Validation Details Cancel Configure Network Client No previous network configuration has been detected Choose Registration Method xi Register Local Hardware Key Register Network Hardware Key Register Local Text based Key Register Network Text based Key Y 5 Register Local Text based Key xj C Register Offline Request Code Mk1UTXhFRFEWRwpROmRETXWRES4QUAN Accont TO S Register Op
62. directory to output the trace files See Chapter 8 Additional Tools Luminex MLPA Analysis For researchers and clinicians using Luminex colored bead method for MLPA analysis See Chapter 7 Special Applications MLPA 39 May 2012 Chapter 3 Main Analysis Overview Project Comparison Allows the user to compare the same data set two different projects and detect differences based on a number of parameters including peak size and height quality score and commented alleles See Chapter 8 Additional Tools Convert Text to Binary Files For customers developing their own instrumentation the Convert Text to Binary Files option allows users to upload four or five color Text files without headers for conversion into SCF four color data or SG1 five color data trace files for analysis with GeneMarker See Chapter 8 Additional Tools Export Electropherogram Allows the user to export the trace images to a specified folder png bmp or jpeg Magic Wizard Start your project Contains three option boxes Start Your Project Run and Report Start Your Project EE Allows the user to easily access the Open Data or Open Project Ee P upload windows The user can also re open the four previously opened projects by selecting the black arrow next to Open Project Run Selecting Run launches the Run Wizard Selecting AutoRun will process the data automatically with the process options currently selected See Chapter 2 General Procedure R
63. e 58 Bhs a 0 1 2 0 1462 04 as 145 0 0 00 0 0 0 1 0 0 She She 61 She 62 She 63 She 64 She 1472 03 1483 14 3 Gaass a 35 5 S S CO B h at ai a ah ab at EEE E ep oe moo oe la E EEE SS EEE Gla window rons gta a 5 eg 135 15 13 mane 08 mv cos ia o om 14 Select the newly created Panel from the Run Wizard is gn er rg 119 FOS fsa Panel Name AFLP Panel Ploidy 2 POTTI TITTET TT TT TT tt Y pent EC ee Template Selection Panel drop down menu 15 Proceed through Run Wizard and click OK in the Data Process box 16 The Panel has been applied What to Expect Three options are available for analyzing AFLP data Allele Detection with Bin Table Trace Comparison and Phylogeny Clustering Analysis The following sections describe these features in detail Allele Detection Bin Table The Main Analysis window of GeneMarker is used for AFLP data analysis The Report Style associated with the AFLP Analysis Type is Bin Table The Bin Table is useful for identifying allele positions common in the dataset and also readily displays outlier peaks The Bin Table can be exported as an Excel xls or tab delimited Text txt file See Chapter 3 Main Analysis Overview for Report Table operations and See Chapter 6 Reports and Printing for Bin Table options AFLP Analysis and Bin Table fl GeneMarker Untitled B File View Project Applicati
64. each individual sample was matched to the Size Standard selected access the Size Calibration Charts Within Size Calibration Charts the user can modify how each sample was sized and view the statistical information for disabled Size Standard peaks Sample List The Sample List includes filename Match Score and disabled peak information for each sample in the dataset Sort the list by single left clicking the column header The list will re sort in ascending or descending order based on the values in the column selected Single left click a sample to view its Sample ILS and Calibration Plots on the right OR use the Up Down Arrow keys Right click the sample row and select Mark as Failed to disable the sample select Unmark Failed to reverse the action Disabled samples will appear grayed out in the Sample List Score The Score column displays the sample s Match Score which corresponds to the degree of pattern match between the sample s ILS and the Size Standard selected Perfect matches receive a score of 100 no correlation receives a score of 0 and the sample is considered to have failed size calling Disabled Size Columns The Sizes that were disabled in the Size Standard see Size Template Editor section above will appear as column headers in the Sample List If no Sizes were disabled then only the Sample Name and Score columns will appear in 49 May 2012 Chapter 4 Fragment Sizing Standards the Sample List The basepair size p
65. edits will be lost when data is re analyzed Re analyze with Run Wizard To re analyze with the Run Wizard tool simply click the Run Project icon in the main toolbar The Run Wizard will launch and the most recently ss selected parameters will be displayed Adjust parameters as necessary and A E eT ae eee click OK in the Run Wizard Additional Settings box The Use Old Calibration You may check Call Size Again to recall te sizes box will appear with the option to Call Size Again Only select Call Size EE Again if the Run Wizard Template Selection Size Standard selection was changed or any of the Run Wizard Data Process Raw Data Analysis parameters were changed Click the Apply to All button The Data Processing box will appear again and the data will be re analyzed with the new parameters Use Old Calibration Being closed in 23 seconds ZS F Call size again Apply to All Apply to Current Re analyze with Auto Run To re analyze with Auto Run first select Project 0 Options The Project Options Settings box will appear This box offers all the same parameters settings as are available in the Run Wizard Use the tabs to view the Template Selection Data Process and Additional Settings boxes Click OK when finished Next select Project Auto Run The data will be re analyzed with the new parameters NOTE The Additional Settings Allele Evaluation Peak Score parameters can be changed in the Project Options
66. folder and SizeStd folder Upgrade Procedure Text based 1 Before proceeding prepare a backup copy of the previous version of GeneMarker recommended Double click the GeneMarker executable file EXE on the SoftGenetics Upgrade CD Proceed through the Installation Wizard as described in the Installation section above Once the Installation Wizard is complete launch GeneMarker by double clicking the new GeneMarker desktop icon OR open the Start menu and navigate to SoftGenetics gt GeneMarker the version that was just installed gt GeneMarker program The Configure Registration window appears Click Register Now to register the local license Click Register Local Text based Key from the Choose Registration Method dialog box Proceed through the Registration steps as described in the Registration section above Launch GeneMarker and begin analysis PS ve ECKE 8 May 2012 Chapter 2 General Procedure Upgrade Procedure Hardware based 1 EG ID 10 Before proceeding prepare a backup copy of the previous version of GeneMarker recommended Ensure the GeneMarker USB key is inserted into the computer s USB port Double click the GeneMarker executable file EXE on the SoftGenetics Upgrade CD Proceed through the Installation Wizard as described in the Installation section above Once the Installation Wizard is complete launch GeneMarker by double clicking the new GeneMarker desktop icon OR open the Start menu and navig
67. for automated pedigree trio drawing Allele Frequency Allele Frequency Import species specific allele frequency txt files Mutation Rate er een Mutation Rate Import species specific txt file Kinship Analysis Population Statistics for the file under analysis Genetics Analysis Settings Genetic Analysis Settings allows setting the above options at the same time New Pedigree File Select New Pedigree File to create a new pedigree with multiple families OR select New Family to add a family to the pedigree file Enter the first family member s information into the New Family New Individual box and click OK to create a new Pedigree Tree Open Pedigree File L gt Launches the Load Pedigree File box Select a PED or PRE file to upload the SMP file will automatically upload and click OK Save Pedigree File Launches the Save Pedigree File box Enter filename and change directory to save the Pedigree Files PRE SMP DAT 115 May 2012 Chapter 7 Special Applications Show Individual Name When selected the individual ID will be displayed in the nodes of the Pedigree Tree Update Sample Data Select to refresh the Mendelian inheritance calculation after a node or allele is edited and after selection a different family when the show genotype display is used Relationship Testing Parameters Launches the Relationship Testing Settings box Options for selected samples or all samples selecting the appropriate allele frequency
68. for the population mutation rate and prior probabilitiy Show Conflict Toggle between Show Conflict with Parents and Show Conflict with Sibling Conflicting and suspected Markers based on Mendelian inheritance are highlighted Show Genotype Toggle between Displaying and Not Displaying the Genotypes of the selected node 8 a H Family 2 2 2 Family 2 Jones Family Select a family from the currently uploaded pedigree file to view and edit Marker 12 TPOX Marker Select a Marker or Locus to view in the Electropherogram Charts Show Color Allows the user to select all colors to view hide all colors or choose a single dye layer Choose a single dye by single left mouse clicking on the icon Zoom In Use the icon to zoom in on the image or hold down the left mouse button and draw a box from the top left corner to bottom right corner around the area you wish to zoom in po Zoom Out Use the icon to zoom out on the image or hold down the left mouse button and draw a box from the bottom right corner to top left corner E Set Axis The default setting automatically sets the Y axis according to the maximum peak intensity of the samples Two other options are available auto fit the Y axis using peak intensities of the alleles or the user can select the ranges for the X and Y axis ES Browse by All Colors ES Displays a comparative view of sample electropherograms by dye color Individual samples can
69. height of the Reference trace is normalized to the Experimental sample traces in the Group To disable Reference trace normalization click the LOH Display Settings icon and deselect Peak Normalization The uncorrected Reference trace peak heights will be displayed in the Trace Overlay The allele editing options in the Trace Overlay frame are similar to those in the Main Analysis window Electropherogram See Chapter 3 Main Analysis Overview Ratio Plot The Ratio Plot depicts graphically peak ratio versus basepair position The peak ratio value is calculated by first determining the height or area ratio of peaks within a Marker in the Experimental and Reference sample separately The Experimental and Reference ratios are then compared and a final peak ratio is determined and plotted in the Ratio Plot Select Peak Height or Peak Area in the Quantification by section of the LOH Analysis Settings box Ratio thresholds are set in the LOH Analysis Settings box If a plot point occurs outside of the ratio thresholds then the point will appear red Double click plot points to view the trace in the Trace Overlay frame The number of Ratio Plots displayed at one time can be adjusted in the LOH Display Settings box Report Table The Report Table lists all samples in a column on the left Reference samples are marked with an R Click the Report Settings icon to select display options Double click values in the Report Table to link to the associated Trace
70. in Log out Log in Log out Log in Log out Log in Log out Log in Log out 3 Lasa e User Admin Admin David David Kevin Kevin Admin Admin Admin Admin Admin Admin Admin Admin Admin Admin Admin A denia Date Time 9 5 2007 3 14 45 PM 9 5 2007 3 14 40 PM 9 5 2007 3 14 32 PM 9 5 2007 3 14 10 PM 9 5 2007 3 13 31 PM 9 5 2007 3 13 21 PM 9 5 2007 3 13 14 PM 9 5 2007 3 11 08 PM 9 5 2007 3 10 33 PM 9 5 2007 3 10 14 PM 9 5 2007 1 15 05 PM 9 5 2007 9 52 27 AM 8 31 2007 11 10 40 8 31 2007 10 44 32 8 30 2007 11 36 30 8 30 2007 11 36 25 8 30 2007 11 28 34 QIN HINN 14 97 90 Forced to log out Forced to log out Forced to log out Iw Run User Protection Gives additional information for the event that was performed For example if a user is added then the username of the person that was added is recorded under Comments Settings The User Manager Settings tab contains additional options for the User Management function Overtime Protection When selected GeneMarker will logout the user after the specified time entered in the Wait field When the user is logged out the status of the analysis remains unchanged until the user logs back in with username and password Record Data Edit History When selected any changes made to the allele calls of the project will be saved in the Edit History log Please see Edit History section below for more information Edit Histo
71. in each individual SEET ES sample by right clicking in the Ratio Plot of the sample Cancel lt and selecting Adjust by Control Probes or Reset Save Parameters when Save Report Automatically generates an ini file of the MLPA Analysis Parameters when the MLPA report is saved Naming convention for the file is Report file name_MLPA_Settings ini Adjust by Control Probes and_Check Control Probe Quality When selected all samples with control probes that meet a certain quality criteria minimal amount of variation among control probe intensities will be adjusted by the sample s control probes All other samples will be fitted based on the population of control and test probes The method of adjustment can be changed in each individual sample by right clicking in the Ratio Plot of the sample and selecting Adjust by Control Probes or Reset Minimum Lane Score Threshold Sample files are given a signal to noise type score 0 to 100 that corresponds to the quality of the trace To view only the samples above a certain score threshold set the Minimum Lane Score Threshold to the desired value All samples below the selected value score will be deactivated and appear grey in the Sample List Quantification Select whether to calculate peak ratios by area or height To view the Quantification option click the double arrow button at the bottom of the dialog box MLPA Ratio to Copy Number When selected the software will convert pea
72. launch the upgrade tool NetDogUpdate exe that is located in the GeneMarker directory 1 Click the Produce Request String button 2 Copy the string in the text field and paste it into an email addressed to info softgenetics com for further order information Another encrypted string of characters will be emailed to you 3 Copy and paste the string into the second text field 4 Click Update and the number of licenses in NetDog will automatically be updated 5 Re install the NetDog Server following the Installation section above Questions If you have any questions during installation setup or program operation please contact us at 814 237 9340 OR 888 791 1270 OR email us at tech_support softgenetics com 13 May 2012 Chapter 2 General Procedure 14 May 2012 Chapter 2 General Procedure Chapter 2 General Procedure Chapter 2 General Procedure Import Data Files Raw Data Analysis Process Data Adjust Analysis Parameters 15 May 2012 Chapter 2 General Procedure Import Data Files After installing GeneMarker software you are ready to begin fragment analysis First raw data files must be uploaded to the program Below is the list of file types supported by GeneMarker ABI fsa abi ab1 hid MegaBACE rsd Beckman Coulter esd Procedure Spectrumedix smd Generic scf sgl f Open Data Files ER 1 Launch GeneMarker E ata File List 2 Click O en Data C S
73. marker multiplex The analysis in the lower diagram is from the combined allele reports 141 May 2012 Chapter 7 Special Applications Trisomy Detection Full trisomy of an individual occurs due to non disjunction during meiosis I or meiosis II of gametogenesis resulting in 24 vice 23 chromosomes in a reproductive cell sperm or egg Thus after fertilization the resulting fetus has 47 chromosomes vice the typical 46 The most common forms of autosomal trisomy are trisomy of chromosome 21 which results in Down Syndrome and trisomy of chromosome 18 which results in Edwards Syndrome In rare cases a fetus with trisomy of chromosome 13 can survive Trisomy 13 is called Patau Syndrome Autosomal trisomy is frequently associated with severe congenital abnormalities mental retardation and shortened life expectancy Aneuploidy of sex chromosomes can also occur The presence of extra X chromosome s causes Klinefelter syndrome in men and Triple X syndrome in women while monosomy X 45 X gives rise to women with Turner syndrome GeneMarker is a software tool used in clinical diagnostics and research laboratories across the world to analyze DNA fragments GeneMarker s new Trisomy detection module aids clinicians and researchers in analyzing QF PCR products to detect aneuploidy Overview In GeneMarker s Trisomy Analysis module the ratio of peaks within a marker is calculated Markers which contain three peaks are obviously trisomy however mar
74. match an allele from the father or mother it can be assumed that one of the parents is not related to the child Because all three individuals share Allele 8 it cannot be determined which parent is not related to the child Review additional loci to determine which parent is unrelated Mendelian Inheritance of Alleles il Pedigree Edit C SoftGenetics Forensics Frag Data DDC Paternity Data DDC Test TestPed pre ell amp P En T Eh P Family 2 2 2 Family 2 Jones v Marker 3 D75820 v Search d d lt lt Samples VA Charts m 2 Q bi G 279878 30 07 fsa 259 3 1749 x sa D75820 245 250 255 260 265 270 275 280 285 290 295 300 E 1 500 1 000 500 0 279878 10_06 fsa 262 6 5767 x 62 245 250 255 260 265 270 275 280 285 290 295 300 d 279879 30_11 fsa x 245 250 255 260 265 270 275 280 285 290 295 300 Person ID 3 Person Name Jon Sample File 279878 20_04 fsa 109 May 2012 Chapter 7 Special Applications Kinship Analysis Overview Identity by descent IBD uses the number of alleles shared and allele frequencies to calculate the probability that two individuals have a specific relationship versus the probability that two random individuals from that population have the given genotypes IBD excludes any individuals that are not potential relatives and allows ranking of potential relatives by probability of relationship level STR profiles of two in
75. micro air bubbles or debris in the laser path Spikes are typically less than a base pair wide Do not select Spike Removal when 214 Derivative Trace has been applied Size Call GeneMarker offers three sizing methods Local Southern Used in most genotyping software applications and is recommended for most analyses This method is based on the idea that smaller size fragments run faster Plot a size v time graph and overlay a size v 1 time graph to determine linear trace Southern E M Measurement of DNA Length by Gel Electrophoresis 1979 Analytical Biochemistry 100 319 323 Cubic Spline Method Cubic Spline is offered as an alternative method that may be more appropriate for some data This method uses a cubic equation to connect known points on the size v time graph An example of a cubic equation ax bx cx d The Astrophysical Journal December 1 1994 436 pages 787 794 Local Southern Method Cubic Spline Method Three sections used in cubic equation Region of Interest time Ttime Large Size AlgorithmGeneMarker s new algorithm provides accurate linear sizing of the data using a DNA derivative migration time correction to large DNA fragments Selecting Large Size algorithm enables accurate sizing from small to large 30 1400 base pair fragments The peak area in base space accurately determines the copy numbers because the peak area normalization is much less variable than that of the height normalization The
76. module will be discussed in the What to Expect section MLPA Run Wizard Settings For explanation of specific functions in Run Wizard see Chapter 2 General Procedure Run Wizard Template Selection Panel NONE OR Click the Open Files icon next to the Panel field and import the correct MLPA panel for analysis NOTE MRC Holland MLPA panels are stored in CA ProgramfFilesN SoftGeneticsN GeneMarkerV1 7VMLPA Panel folder Size Standard User defined Analysis Type MLPA Run Wizard Data Process MLPA Analysis Run Wizard Data Process slark Peak Detection Intensity Threshold 100 Global Percentage 1 Max Local Percentage 5 Max Call Intensity 30 000 Stutter Peak Filter Left 25 Right 25 Plus A Filter Selected NOTE The correct settings for MLPA Analysis should be a small global percentage plus a slightly larger or equal local percentage Run Wizard 4 Run Wizard Additional Settings pi atacan Peak Score Allele Evaluation Reject lt 1 Check lt 10 lt Pass NOTE If the user chooses all allele peaks with a score below 1 to be a ma rejected there is the possibility of a false negative whereas if scores P Unona below 0 are rejected there is the possibility of a false positive MLPA Hendin ted Population Nomakzalion Advanced MLPA Normalization Method Normalization adjusts the peak intensities within individual samples to the same scale for comparative purposes Internal Probe Norm
77. non controls in the electropherogram of the Main Analysis Screen Bins marked with a 0 are not controls and will not be used for data normalization in MLPA Analysis Bins marked with a 1 negative 1 are displayed as quality control fragments for MLPA analysis final report See Chapter 7 Special Applications MLPA 59 May 2012 Chapter 5 Panel Editor Distance kb Allows the user to input the distance in kb that each allele is from the beginning of the sequence For example 38 1 means that the allele is 38 1 kb from the beginning of the sequence for MLPA Analysis or in the case of Haplotype Analysis the Mb distance is used to order the markers in the pedigree diagram A 0 is used to indicate the marker to be located at the middle of the marker list positive numbers below the 0 marker and negative numbers are above the 0 marker in Haplotype Analysis NOTE Applicable for MLPA Analysis See Chapter 7 Special Applications MLPA NOTE Application for Haplotype analysis See Chapter 7 Special Applications Haplotype Analysis Recombination Frequency Used in Haplotype analysis pedigree diagram to report the Genetic distance between markers Comments Free form text field to associate a comment with the Bin Procedure As mentioned previously Panels are created to outline the position in basepairs of expected peaks In GeneMarker the Panels associated with several commercially distributed genotyping kits are included Ex
78. operate the program If you wish to exchange hardware based licensing for text based licensing please contact info softgenetics com Installing Over the Previous Version If you choose to install the new version of GeneMarker over the previous version you will need to choose the same directory for installation Several of the old files will be replaced with newer files Other files that are not present during installation but are created during analyses or by the user will remain in the folder and can easily be recognized by the new version of GeneMarker Please make a backup copy of the target directory before installing the newer software Installing into a New Directory If you choose to install the newer version in a different location be sure to specify a unique directory name or Program Manager Group for the upgrade to prevent overwriting any previous versions of GeneMarker Several files created by the users or created during analyses conducted by the previous version of GeneMarker will not be recognized by the new version of GeneMarker unless they can be found in the directory of installation If you intend for the new version to recognize these files then you will need to copy them from the older version s installation folder and paste them in the folder containing the new version of GeneMarker Some of the more common customized GeneMarker files are GeneDB mdb GeneMarker mdb codis ini CommentsTemplate ini ExpTemplates ini Panel
79. peak intensities among the probes All of the median intensities are then used to fit the exponential function which results in higher accuracy and lower false positive rates NOTE Because control probes can fail or may contain duplications or deletions Population Normalization is a more robust method of normalization as compared to Internal Control Probe Normalization 86 May 2012 Chapter 7 Special Applications Internal Control Probe vs Population Normalization P034 409 02fsa P034 409 02 fsa o T Ta ES T Di q cL cL Size has Size bp Advanced Population Normalization The Advanced Population Normalization method is designed to normalize data that contains a large number of test probes displaying copy number change Problematic data sets that are difficult to normalize using the other two methods can usually be successfully normalized using the Advanced function To activate Advanced Population Normalization select Population Normalization and check the Advanced option in the Run Wizard Additional Settings box Advanced Population Normalization solves problems when other normalization methods prove less than optimal It is a combination of Population and Internal Control Probe normalization methods Some samples that contain many probes with deletion or duplication copy number changes may be difficult to normalize When samples display an upward trend in peak ratios it is an indication of sub optimal normalization
80. peak width is less than 1 bps for all fragments less than 1k B 3 ji IMO 6D 80 IBN 40 EH SNS BINDE S RES OLE BEVEGES VE BL iT SV OS IO TROY OE 30 1160 120 10 000 i II II Ly Mir yr L Nb e HS Am St WE pat 501 x0 1 200 e x e 100 1100 100 Ca 1900 Ca 00 39 2 ee au 0 Sam 299 Em Zem Zen EDO ao Rem exe 400 m ace Lo 9 zo 100 100 100 o p 7 Sr SS bs St po 10 90 y e aan Fiare Frare Fiane AER 501 PE AER 501 200 1200 e x0 ef pr 100 1100 100 D 1900 a g mo S 700 Ram exo Zen Zem a 20 5 sm CEV 400 w 400 xo xo 100 100 100 Fiane Frare frm Chapter 2 General Procedure Custom Large Size Calling Some large custom size standards have unique size fragments that are not evenly spaced requiring the ability to change some of the parameters in the large size calling algorithm to provide a best fit to the fragments The large size calling algorithm above will use analyst supplied parameters to provide accurate linear sizing of the custom large size standard Use the spread sheet in the C ProgramFiles SoftGeneticsGeneMarkerSize Standard folder to assist in determining the appropriate parameters for custom large size standards Flanking Peak Size Calling With some non linear data it is not possible to make a best fit curve using the algorithms above The flanking peak method uses the fragments on either side of the data peak to interpolate the size This allows size call
81. ratio line and deletions will be below the line When you double click on any point the dosage histogram and the report will also display the selected point In MLPA Ratio Analysis it is possible to toggle between the Population and Control Probe normalization by right clicking in each sample graph For some samples the Control Probe normalization may be more accurate when the control probes and the test probes are in two distinct groups Adjust by Control Probes Adjust by Population Adjust Ratio 0 57 Peak Ratio Peak Ratio MLPA Regression Method MLPA Regression analysis relies upon a T Distribution to form the regression line between the square root of the sample and the square root of the control GeneMarker utilizes an automatic iteration based regression method Using this method the software forms a best fit line retaining 80 of peaks with smaller deviations and 20 are rejected GeneMarker then iterates multiple times to reject and retain peaks with a confidence of 99 This process is repeated until the regression line has reached the desired confidence Finally the removed data points are 3 placed back into the plot and will show up either on the regression line or RATE TIME TRI NV as outliers 88 May 2012 Chapter 7 Special Applications Reports and Printing GeneMarker automates the analysis process and creates professional clinical reports for accurate insertion deletion detection Report Table
82. sample 023 04 12 sample 024 ka 13 sample 15008 1d sample bea 15 sample DT ka TE sample 032 Ia L sample 033 Iza 18 sample 034 ten 18 2 H HH A 625 3 27 28 sample tra Li 127 May 2012 Chapter 7 Special Applications Procedure SNP analysis in GeneMarker requires the data to be sized and a Panel applied prior to launching the SNPlex SNaPshot module The ABI SNPlex Panel comes standard with GeneMarker and can be found in the pre defined Panel List SNPlex Run Wizard Settings For explanation of specific functions in Run Wizard see Chapter 2 General Procedure Run Wizard Template Selection Panel SNPlex_48Plex_v1 Size Standard SNPlex_48Plex_v1 Analysis Type SNPlex NOTE A Panel is required to analyze SNPlex data Run Wizard Data Process amp Additional Settings SNPlex analysis does not require Allele Call or Peak Score Threshold settings to be adjusted therefore the settings are inactive in the Run Wizard SNPlex Panel Adjustment Run Wizard Template Selection Set the Template of the Project Select an existing template or create one t 4 ABI MegaBACE C Use last template Template Name AB Panel SNPlex_48plex_v1 Size Standard SNPlex 48plex vi Standard Color Red Lal Led Le le Analysis Type SNPlex 0 wo X ei a Run Wizard Set data process Raw Data Analysis Iw Auto Range frame P 3 Smooth Y Pea
83. similar to the main analysis window displays a histogram based on MSI Score and can be adjusted through the MSI Analysis Settings box 135 May 2012 Chapter 7 Special Applications Marker Table Identifies the marker and whether it contains MSI POS or does not show MSI NEG The Comments column identifies the peak positions of the gains and losses as compared to the reference MSI Clinical Report por Report Type Peak Height Ratio BAT 25 Pos Gain 112111311142 POS Gain 99 9 100 8 101 8 102 7 MONO 27 POS Gain 138 6 139 6 140 7 141 7 142 7 NR 21 POS Gan882892902913923 POS Gain 121 8 122 8 123 8 124 8 Penta c NEG Penta D_ me gt gt gt NR 21 BAT 25 MONO 27 80 100 120 140 160 Phylogeny Clustering Analysis Biological applications of data clustering calculations include phylogeny analysis and community comparisons in ecology gene expression pattern enzymatic pathway mapping and functional gene family classification in the bioinformatics field It has been successfully paired with the AFLP analysis technique for a variety of applications There are two types of data clustering hierarchical and partitional Partitional clustering includes the K means and Self Organizing Map methods and will not be discussed here Hierarchical clustering treats each data point as a single cluster and successively merges clusters until all points have been merged into a single remaining cluster Hierarchic
84. the Sample List The sample s ILS appears in the Sample ILS frame Right click in the Sample ILS frame and chose Add Delete or Fix Size to correct size call Right click again and select Update Calibration The changes will be implemented for the sample and the Match Score will be updated When editing is finished close Size Calibration Charts The Size Match Score indicators in the Sample File Tree of the Main Analysis window will be updated eve Icons and Functions Toolbar Icons 7 Size Calibration Found in the main toolbar of the Main Analysis window View Mode Change the layout of the Calibration Plots frame Adjust the maximum number of rows and columns displayed Maximum number of rows and columns is 5 Chart Synchronize When selected both the Expected Size Standard and Sample ILS traces become synchronized This option is not selected by default Preprocess Raw Data Select Preprocess Raw Data to smooth the samples raw data ILS Auto Fit Y Ge Provides the option to automatically fit the Sample ILS s y axis by the maximum peak height in the trace OR by only the highest matched peaks sl Calibration Print Print e S 7 Print Icon Select Launches Print Preview and formatting options to F Calibration Charts Max Cot E El print the size calibration page s qe er Sort Samples Sort by Score C Sort by Filename Save Options to save print calibration page s jew I E Ok x Cancel
85. the hybridized probe and analysis of the resulting PCR products By labeling each set of hybridized probes with a unique microsphere up to 100 distinct reactions can be multiplexed in a single reaction volume Flow cytometry principles are used to allow microsphere tagged particles passing through a detection chamber to be measured discretely Multiple MLPA assays can be developed in a single volume for detection of gene deletions and copy number changes GeneMarker software has been developed to quickly and accurately analyze data from the Luminex flow cytometry instruments Luminex 100 IS and Luminex 200 for microsphere detection The software is therefore compatible with Luminex instrument data format csv With GeneMarker MLPA microsphere data analysis is a quick and accurate analysis with customizable features including reports and printing Overview The steps required to analyze Luminex instrument data after MLPA are different from other analyses done by GeneMarker Due to the nature of microsphere labeling peak intensities are artificial and represent the number of beads detected by the flow cytometer The software normalizes to an 93 May 2012 Chapter 7 Special Applications arbitrary 3000 using a combination method Because the Luminex data is different than standard capillary electrophoresis data a size standard and panel do not need to be applied Procedure Open GeneMarker Software Close Start Your Project box Go to T
86. the specified interval Cancel Auto Binning Fixed Bin Width Check this option to enter the number of basepairs on the right and left of the center of the Bins If 0 5 is selected as the Bin Width then the total Bin range will be 1 0 basepairs Auto Label When deselected the Bins are automatically labeled with the basepair size of the Bin position to the nearest tenth of a basepair If selected the basepair size is rounded up to a whole number value 57 May 2012 Chapter 5 Panel Editor Edit Marker Double click the Marker bar OR right click the Marker bar and select Edit Marker The Edit Marker box appears Adjust parameters and click OK NOTE The Edit Marker box can also be accessed by right clicking the Marker name in the Panel List and selecting Edit Marker Parameters Marker Name Edit the Marker Name field to change how the Marker will be labeled in the Panel rs Edit Marker Marker Parameters Nucleotide Repeats Use the Nucleotide Repeats drop down menu 1 6 or iaa bas enter a value into the field to set the number of basepairs expected Nucleotide Repeats 1 E between each allele in the Marker Boundary 97 0 ro 1163 Boundary To move a Marker left or right hold down SHIFT key and left click and drag the Marker bar To adjust the basepair range over which a Marker is located hold down SHIFT key and mouse over the edge of the Marker bar until a double headed arrow appears then left c
87. the specified ratio limits Blue Peak is a control probe Red Peak is outside the specified ratio limits Yellow Current selected probe Ratio Plot vs Regression Plot P034_C12_06 fsa ga a 2_06 fsa 2 w D bei E a 50 100 150 200 250 300 7 1 Size bps Control Normalization Succeeded E Normalization Succeeded Probe number Total 47 Normal 34 Control 6 Probe number Total 47 Normal 34 Control 6 Distribution of ratios Excluding zero and infinite Regression line Slope 0 982 Intercept 0 000 Std Error 0 025 Total Mean 1 079 Std Error 0 049 Distribution of the distances to regression line Excluding zero Normal Mean 0 958 Std Error 0 010 Total Mean 0 089 Std Error 0 022 Control Mean 0 894 Std Error 0 128 Normal Mean 0 021 Std Error 0 003 Control Mean 0 112 Std Error 0 078 Double click a point in the Ratio Plot to view the peak in the Trace Overlay and the ratio value in the Report Table Right click in the Ratio Plot and select Adjust by Control Probes to fit the data by only the control probes blue points in the sample Right click and select Reset to restore population fit calculation all control and sample probes in the sample are used to fit the data If the square representing a bin is not color coded empty this indicates that there was no data fragment in the sample Open square data points are the result of either a mis aligned panel during the initial analy
88. threshold intensity value may be estimated based on the peak with the maximum intensity from the negative control in the sample set As always these parameters can easily be adjusted to optimize the analysis for your specific requirements Addtional Settings AFLP Analysis The Run Wizard Additional Settings box displays the scoring threshold for sen ar i allele peak detection Reject lt 0 Check 1 lt Pass NOTE If the user chooses all allele peaks with a score below 1 to be rejected there is the possibility of a false negative whereas if scores below 0 are rejected there is the possibility of a false positive AFLP Unconfidence at Rightside Score is 1 NOTE This requires that two peaks to the right of the examined peak must have a score of at least 1 for the peak to be accepted UL Genetarker Untitled a hem Project Applicators Tools Mel gt HERO Raab DED We matali gt Ze What to Expect E eee Following size calling the Main Analysis window will activate The samples a listed on the far left are marked with green symbols indicating that size calling was successful Sample Electropherograms are displayed in the center of the window The Report Table on the right records the presence 1 or absence 0 of a fragment at specific positions for each sample 79 May 2012 Chapter 7 Special Applications Multiplex Ligation dependent Probe Amplification MLPA GeneMarker c
89. to improve analysis select Project Options Options change the desired setting s and re process the samples for analysis CE Project Comments Add Samples to Project The user can add samples to a project that has already been sized and analyzed When selected the Open Data Files box will appear Click Add to select individual files to the project and click OK The raw data file will be sized and processed with the same settings as the other files in the project and added to the bottom of the Sample File Tree Print Report Selecting Print Report launches the Print Report Settings box which allows the user to define display settings in the Print Report The software permits printing of the sample electropherograms You can choose to print all samples selected samples or print samples along with the allele table if desired See Chapter 6 Reports and Printing Options Allows you to access and change parameters in the Project Option Settings window This three tab window contains settings identical to the Run Wizard Adjust settings in the Project Options Settings box before selecting Auto Run See Chapter 2 General Procedure NOTE Auto Run does not need to be selected after adjusting the Additional Allele Evaluation Peak Score settings The changes will automatically be applied when the Project Option Settings window is closed Project Comments 37 May 2012 Chapter 3 Main Analysis Overview Allows the user to write
90. whether to scroll to alleles in the trace when selecting eee Dee Saree hpa the allele in the report Leave this feature on to have the software Open Mute Chats When Browsing Repor automatically call up alleles in the trace when you double click on them in the report Show Disabled Samples in Report GeneMarker identifies samples that failed during electrophoresis or size calling The default setting excludes the disabled samples from the report The Cancel option may be selected to have failed or user disabled samples to be identified in the report Open Multiple Charts When Browsing Report When selected this option with keep all open charts active while the analyst is editing individual charts Start up Settings Display Settings Report Settings Others Iw Automatically Scroll Chart to Alleles When Selected in Report 36 May 2012 Chapter 3 Main Analysis Overview Others Enable Sample Grouping When Project gt Apply Sample Grouping is a Preferences implemented the Enable Sample Grouping option will be automatically selected De select Enable Sample Grouping to Start up Settings Display Settings Report Settings Dier inactivate the Apply Sample Grouping option The Apply Sample Grouping information is saved and can be recalled by selecting Enable Sample Grouping See Chapter 8 Additional Tools Filename Group Tool se Enable Sample Grouping Apply International Date Format
91. will show only called alleles within Panel Marker ranges This option is only active when a Panel is applied to the data Grouped by Markers When selected alleles within the Marker will be listed one after the other in the columns at the top of the table When de selected each allele will be represented by a row so that the Marker name may be listed several times according to the number of alleles in the Marker This option is only active when a Panel is applied to the data Columns Click the Columns button to open the Set Peak Table Columns box All column options are listed in the All Columns field on the left The columns currently being displayed in the Report Table are listed in the Selected Columns field on the right Selecting Columns Single left click options in the All Columns field and click the Add button farman Ea to add the column option to the Selected Columns field Hold down CTRL RESI TENE or SHIFT key to select multiple options then click Add Click the Add Make O All button to move all the options in the All Columns field to the Selected SR Remme JS Columns field End Comments Quality Reasons De selecting Columns Single left click options in the Selected Columns field and click Remove to _ Adda o gt move the column option to the All Columns field Hold down CTRL or JE SHIFT key to select multiple options then click Remove Click the ok re Remove All button to move all the opti
92. x 5 D D a 5 oO 10 15 20 Distance Size 15 Distance Size Other Considerations Analysts trying to detect trisomy are challenged when aberrations caused by variation within an individual s own cells occur Examples of individual variation include mixed samples mosaicism and triallelic homozygotes Maternal Cell Contamination MCC Pregnant women over the age of 35 are screened for trisomy caused syndromes in the fetus because the risk of these syndromes increases as the mother ages During the procedure whereby cells are collected for analysis maternal cells can be mixed in with fetal cells This phenomenon is called Maternal Cell Contamination MCC MCC is recognized when extra alleles appear and when examining inconclusive dosage ratios When there are three alleles at a locus the intensity of all three should be essentially equal An imbalance is said to exist when the intensity ratio between the highest and lowest peak is greater than 60 40 The GeneMarker Trisomy tool identifies loci that contain imbalanced peaks The user can define within the settings parameters the imbalance ratio that is significant for the data Mosaicism Trisomy may not necessarily be present in all cells in an individual It may be detected in just a specific tissue or within different cells in a tissue When the presence of chromosomal abnormalities occurs differentially within an individual it is called chromosomal mosaicism In general as we wou
93. 2 181 8 827 86385 384 7 M262 262 0 1110 8277 276 8 1 653 82 JoL 457 7 636 800 559 37151 150 9 995 91325 325 1 482 35131 131 1 507 37g2e0 260 1 2041 1327 326 7 1269 18411 410 8 1171 1495 495 3 856 52 F141 140 6 1781 ise 185 8 1083 8277 276 7 906 90392 392 6 661 B9 JOL 461 6 5 659 47146 146 4 436 40313 312 4 1066 7 995 91 432 32 439 34 2041 1 1141 1 1039 1 788 56 1756 1 1051 8 658 76 92 808 612 392 If a peak is detected in at least one sample the Bin Table Report Style will report the presence or absence of a peak at that position for the rest of the samples in the dataset Bin Table is the default Report Style when AFLP MLPA or SNaPshot Analysis Types are selected Features Options Abide By Panel When selected the table will show only called alleles within Panel Marker ranges This option is only active when a Panel is applied to the data Show Type Symbol Enter values to indicate the presence of a peak at the position Positive the absence of a peak at the position Negative and a Check or Undetermined Quality rank at the position Suspected Show Intensity Raw Displays the peak intensity RFU value for all Positive and Suspected peak positions A 0 value is given to Negative positions Selecting Raw will show the peak intensity values for all positions including Negative positions Show Peak Area Displ
94. 2012 Chapter 8 Additional Tools Remove Files Removes any files selected in the Filename List Select multiple files to remove by holding down the SHIFT key and selecting additional samples Save Groups to File Saves the filenames of the samples paired in the Matched Groups field Samples identified as Controls will be in the first column of the Matched Groups tab delimited Text file File Name Group Editor MatchedGroups E Match by Sections Automatically separates the sample filenames into groups based on the specified Section Separators Group Identification Identifies how to match the filenames into eroups based on the section entered into the Compare by Section field The section of the filename specified will be highlighted red in the File Name List oo SCF SCF F F SCF SCF SCF ST SCF SCF SCF SCF F F oo Match by Sectors Match by Fived Postion Group By Order Control Identification Identifies which section of the filename s s See Sept contains the reference vs sample information based on the ssn section number entered in the Match to Identifier by Section field The section of the filename specified will be highlighted green in the File Name List Match by Fixed Position Allows the user to manually identify the characters of the filename for grouping the samples Section Separators like are counted as individual characters Group Identifi
95. 20_08 fsa 10 279879 10 01 6 7 B 279879 30_02 tsa m l d LD Ladder 1 fsa 0 11 1279879 20 08 f 53ff 3 f i 9 B Ladder_2 fsa 12 279879 30 02 fs4 MY 6 T 3 279878 20_03 fsa x P e 155 160 165 170 175 180 185 190 195 200 205 PageUp Page Down 4 Procedure HID analysis in GeneMarker is similar to Fragment Animal analysis with the exception that the Stutter Filter and Peak Score thresholds have been lowered to allow more peaks to be called In the Run Wizard Template Selection box be sure to choose the correct Size Standard and set the Analysis Type as HID Additionally forensic datasets generally contain 2 or more Allelic Ladder samples by which the forensic Panel can be adjusted HID Run Wizard Settings For explanation of specific functions in Run Wizard see Chapter 2 General Procedure The following are the default settings for HID Analysis Type Run Wizard Template Selection Panel Select the Panel that matches the Allelic Ladder samples Size Standard User defined Analysis Type HID Run Wizard Data Process Peak Detection Intensity Threshold 50 Global Percentage 1 Max 101 May 2012 Run Weard Template Selection Set the Template of the Project E Select an essing lengise of create ore J ABI Template Nane Promegs 4 MegaBACE Pant Powe 16 Ula DE SieSundent aen 3 J Standard Color Orange Analysis Type up Swe x Delete lun Wizard Data
96. 7 00 see 694 3 355 7 824 15453 18 54 22 pli 6937 3 839 5122 1914 23 pk 9192 1308 230 4143 510 ae d E 1621 0879 758 304 2243 26 pl 9483 10 49142 404 a Mutation Chart 02_plate1_1111_B01 fsa 19 285 3 7647 748 4264 2056 29 pk 100 150 200 250 300 350 400 450 500 550 600 g50 700 750 800 850 900 950 20 279 7 F 533 10114 17 42 Zen 2052 8448 279 1395 986 rae 3083 7417 412 2196 1457 Ge 35_ple 666 6 SL KE O 38 pl 387 9 6621 1006 6085 3557 39 pl Blue 937 3 1127 476 16730 4 93 ma Green 3834 6665 479 5157 1145 42 ple Y Sample List 155 May 2012 Chapter 7 Special Applications The Sample List displays all current samples in the project Single left click a sample name or use the Up Down Arrow keys to scroll through the list Right click on a sample name for options on selecting deselecting sample information sorting disabling samples or editing comments Reference Sample Subtracted sample mutation Traces The reference file will be subtracted from each individual sample trace center trace yielding a plot Mutation Chart highlighting the SNPs Peak Table The called peak size will be reported in bold type and complementary size cSize will be reported in grayed out font Mutation Report The information contained in the report is interlinked to the sample traces Double clicking on the cell in the report highlights the cell corresponding sample ID i
97. 74 EHNEN 59 94 Control Identification coocoooncnnnoonnonnnonononononanonoos 172 Control ldentitter 171 173 Control Match Mode 171 173 CONTO Probe E 81 96 CONTEO POD ia at ita 83 control probe normalization sosssssesseseesseeresseeresseeee 86 Control Sample Selection 000nnnooseneosseennssreeesseeee 85 convert Macintosh file tormats 6 Convert TEX tTO BINOIY ia 176 Convert Text to Binary Ples 40 Convert TXT GBNR 176 Coordinate Y see 144 Copy Current Calibration Data o 51 Corrected Ratio blot 149 Correlation Coefficient 137 Create BIN sa 58 Create Mae 57 Create New Range coincida gie 63 Create New Size Standard 48 Ve 93 Cubic Spline Method 22 CENT OIG ENEE EE 180 Cursor Locator EE 31 Curtain Metho EE 38 120 121 Custom Panel Creation EE 61 Customize Bin Column 42 CVSUIC FNS Sia 36 59 162 D DAT Me cuna A 106 Data Process 21 182 Data e e TEE 24 Database save combined genotypes ccccoocccnccconcnnncno 117 Database Search Locate Duplicate Samples and Nearest Fever 114 Decimal Precision E 35 Deconvolute Method occcocnccncnnccncnncononarononanononarononos 122 Deconvolution Method 38 120 Delete Blind ceda 59 Delete Bin Columns 70 Delete Current Size Standard 47 Delete Marker 58 Delete Palin iii 56 63 Delete Peak ies 70 Delete Peaks dido 34 Delete SIZ nag 45 Delete Size E 51 Delete Size Standard 45 48 Dele
98. BMP image file Show in Window Opens a separate window containing the Synthetic Gel Image The separate window can be maximized for closer gel image inspection 30 May 2012 Chapter 3 Main Analysis Overview Image Display Intensity Move the Intensity slide bar located in the upper left corner of a i IEE the Synthetic Gel Image up and down to adjust the intensity of the p m n fragments displayed Pa Grey Scale a Y 7 1 ni Go to View Preference Display Settings Gel Image Select ab pol Ai Gray for Single Dye to change the single dye Synthetic Gel Image to black and white when only a single dye color is selected when gt HI tl I multiple dye colors are selected the fragments will appear in their ml E H respective colors Click the Background in White option to sl di alt a A d d reverse the black and white exposure for single dye color gel L_ 5 ar o A images Electropherogram and Peak Table Features The Electropherogram displays fluorescent signal intensities from capillary electrophoresis instruments as a single line trace for each dye color The signal intensities are recorded in Relative Fluorescent Units RFUs which are plotted along the y axis Along the x axis are the basepair sizes of the fragments The frame units plotted along the x axis in the original Raw Data Analysis window are converted to basepair size units as defined by the Size
99. Bins as described in the previous section Panel Editor Overview 11 Follow steps 9 14 above Manual Panel Creation Er 1 In Panel Editor select File Create New Panel from the menu bar or click the Create New Panel icon 2 Enter a Panel name in the Name field 3 Choose the appropriate Analysis Type from the Type drop down menu 4 Select Manually Create When finished click OK 6 The Panel name will appear in the Panel List however no Markers or Bins will be associated with the Panel 7 Follow the steps in the previous section Panel Editor Overview to create Markers and Bins v LLI TETT EE EE EE ET LS ERASE TORES PARAR AAA A n 61 May 2012 R I SR sO OeEeRRePP x TE RSeheptoz SR Chapter 5 Panel Editor Adjusting and Calibrating Panels It is common for panel alignment to be shifted due to variations in genetic analyzers or run conditions such as templerature injection time Markers or bins can be manually aligned to the allele ladder using the shift and mouse key Once a panel has been adjusted to fit the output of a specific genetic analyzer the panel should be saved with the signal information The combination of the panel bins and signal information provides GeneMarker s pattern recognition algorithm with the data necessary to use major and minor auto panel adjust icons to align the panel for projects from future runs on that same analyzer If more than one genet
100. Conflict Toggle between Show Conflict with Parents and Show Conflict with Sibling Conflicting and suspected Markers based on Mendelian inheritance are highlighted Family 2 2 2 Family 2 Jones Family Select a family from the currently uploaded pedigree file to view and edit Marker akt DESE Y Select a Marker or Locus to view in the Electropherogram Charts What to Expect Mendelian inheritance is guided by Mendel s two basic laws Segregation and Independent Assortment The law of segregation describes how phenotypes are an expression of two alleles inherited independently one from each parent Independent assortment is the idea that the inheritance of one gene or allele does not affect the likelihood of inheritance of a different gene Today we have found that this is not true of all genes some genes are invariably linked and inherited together Linked genes do not follow Mendelian inheritance patterns With GeneMarker s Pedigree module Mendelian inheritance patterns can be analyzed That is alleles are inherited independently one from each parent and alleles are not influence by the inheritance of other alleles Forensic STR loci were selected because they follow the laws of Mendelian genetics In the example below the child s Marker D75820 is highlighted red The child s Allele 8 matches with the mother and father however the child s Allele 10 does not match with either the father or the mother Because Allele 10 does not
101. Edit Node allows editing of file or Eat Node Orr KK FEE electropherogram information Be er sure to use the refresh key after any Find individual changes A GE DE 8 Add Mate or child to expand the Es Export Bamap pedigree E Ca Relationship Testing File DataBase Toot JI E iTA BS TIT Ala Fany ug ET vate 1 065111 Sesch A D Sopes LA Charts D Report BB Catcudation Destad 59 2 642 Dez ug 120 125 a 145 150 155 16 e 170 130 135 1 Allele conflicts are listed and the node is highlighted in red Clicking on the marker in the list links to the section of the electropherogram where the conflict can be visualized 118 May 2012 Chapter 7 Special Applications Save Report Export Bitmap to save the diagram t DataBase Tools SE TAB r F AQ Fama 6 Fams Fomks v Maker Jm lte AR gt Samples VA Charts Ej Repot E Calculation Detads Wa of xie 4 PAT_6_F ise 13 12 12 fe 10 11 11 13 2 2 12 22 32 2 18 PAT 6 Mila O 190 200 on ID 1 on Nama 1 ple Fie PAT 6 C fsa Save pedigree drawings with the save icon Pedigrees may be re opened and edited as more information becomes available dl Relationship Testing File DataBase Tools Jil Save Pedigree File Pedigree File Pre Ped pre i Individual Sample Accordance File SMP Iw Auto Load PE h Loci Description File dat dat Cancel
102. Electropherogram or just the Electropherogram i Save Peak Table Exports the Peak Table as an Excel xls file or tab delimited Text txt file 3 Call Allele Call alleles by sample s by marker or by dyes Permits slight modifications to the samples without having to activate Run Wizard again Settings to change include Peak Detection Threshold Stutter Peak Filter and Peak Score Threshold Marker Drop down Menu Marker rellow 1 Zi Allows the selection of a marker to view This is available after the samples have been compared to a Panel Event Log Displays each lane s processing success or failure ati Magic Wizard Activates the Start Your Project Run and or Report dialog boxes D 41 May 2012 Chapter 3 Main Analysis Overview Report Table Icons The icons are located directly above the Report Table Report Settings Allows the user to customize Report Table display settings Save Report Exports the Report Table as an Excel xls file or tab delimited Text txt file i Customize Bin Column Bin Allows the user to select which bins to include exclude in the Report Table 42 May 2012 Chapter 4 Fragment Sizing Standards Chapter 4 Fragment Sizing Standards Chapter 4 Fragment Sizing Standards Size Template Editor Size Calibration Charts 43 May 2012 Chapter 4 Fragment Sizing Standards Size Template Editor The Size Template Editor is a tool in GeneMarker for creating and modifying
103. MAGES CLAIMS OR COSTS WHATSOEVER OR ANY CONSEQUENTIAL INDIRECT INCIDENTAL DAMAGES OR ANY LOST PROFITS OR LOST SAVINGS EVEN IF A SOFTGENETICS LLC REPRESENTATIVE HAS BEEN ADVISED OF THE POSSIBILITY OF SUCH LOSS DAMAGES CLAIMS OR COSTS OR FOR ANY CLAIM BY ANY THIRD PARTY THE FOREGOING LIMITATIONS AND EXCLUSIONS APPLY TO THE EXTENT PERMITTED BY APPLICABLE LAW IN YOUR JURISDICTION SOFTGENETICS LLC S AGGREGATE LIABILITY AND THAT OF ITS SUPPLIERS UNDER OR IN CONNECTION WITH THIS AGREEMENT SHALL BE LIMITED TO THE AMOUNT PAID FOR THE SOFTWARE IF ANY SoftGenetics LLC is acting on behalf of its suppliers for the purpose of disclaiming excluding and or limiting obligations warranties and liability as provided in this Agreement but in no other respects and for no other purpose For further information please see the jurisdiction specific information at the end of this Agreement if any or contact SoftGenetics LLC 9 Export Rules You agree that the Software will not be shipped transferred or exported into any country or used in any manner prohibited by the United States Export Administration Act or any other export laws restrictions or regulations collectively the Export Laws In addition if the Software is identified as export controlled items under the Export Laws you represent and warrant that you are not a citizen or otherwise located within an embargoed nation including without limitation Iran Iraq Syria Sudan Libya Cuba North Korea
104. Manual Calibration Provides the option of manually entering standard peak sizes if many peaks have been modified This window contains three columns Standard Size fragment sizes of standards used for size calling Peak Position in frames and Size sizes are automatically entered but easily edited 52 May 2012 Chapter 4 Fragment Sizing Standards What to Expect It is important to verify sizing accuracy prior to analyzing a dataset If a sample is not sized correctly peaks may be called Off Ladder OL if a panel is applied or will not be contained in the same Bin as other peaks in the case of AFLP analysis Incorrect sizing most dramatically affects larger size fragments Before amp After Editing Size Call dl Calibration Charts y g B x wo 8 Expected Size ILS600 50 100 150 200 300 350 400 450 500 550 600 650 015 45 N fsa 9 2 015 46 T fsa 38 100 3 016 53 N fsa 38 4 016 54 T fsa 38 50 5 017 55 N fsa 38 6 017 56 T fsa 38 H 7 018 57 N fsa 38 8 9 018 58 T fsa ES 22 65 N fsa 4100 2263 022 65 Nisa 43547 497 Ze MEN fea 3 140160180 200225250 275300325350375400425450475500 550 600 60 80 100120140150180200 225 250275300325350375400425450475500 550 600 bU Tea 11 020 61 N fsa 98 1 000 12 020 62 T fsa 98 13 021 63 N fsa 98 14 021 64 T fsa 98 0 SS 15 022 65 N fsa 65 022 66 T fsa 64 4 000 5 000 6 000 7 000 8 000 TESEI Size bps
105. Marker The Biologist Friendly Software A ae A R SS Wm S gt d ef p SOFTGENETIGS Software PowerTools for Genetic Analysis www Softgenetics com Copyright Licenses and Trademarks 2001 2012 SoftGenetics LLC All rights reserv ed No part of this publication may be reproduced transmitted transcribed or translated into any language in any form by any means without the written perm ission of SoftGenetics LLC The software is copyrighted and cannot be altered or given to a third party without the written authorization from Soft Genetics LLC Thes oftware may be licen sed from SoftGeneticsLLC Mutation Explorer Mutation Surveyor JelMarker ChimerMarker NextGENe and GeneMarker are trademarks of SoftGenetics LLC All other product names and or logos are trademarks of their respective owners Limited Liability of Using the Software In no event shall SoftGenetics LLC be liable for direct indirect incidental special exemplary or consequential dam ages including but not lim ited to procurem ent of substitute goods or services loss of use data or profit s or business interruption however caused and any theory of liability wh ether in contract strict lability or tort including negligence or otherwise arising in any way out of use of this software even if advised of the possibility of such damage SoftGenetics End User License Agreement GeneMarker GeneMarkerHID JelMarker and Mutati
106. Nore DH a Report DD Help The Report Table is linked to the other frames in the Main Analysis sm sses somone ass window Double click on the desired allele OR use the Arrow femmer E keys to move to the cell of interest and hit Enter key OR use Alt Arrow keys to move to different cells and zoom in on the peak in the Electropherogram Select multiple cells by holding down SHIFT key OR hold left mouse button and drag over desired cells IQA RA 33 May 2012 Chapter 3 Main Analysis Overview The rules by which the Report Table and other frames in the Main Analysis window are linked are controlled by options in the View Preferences Others tab Display Settings ser Click the Report Settings icon in the Report Table toolbar The Allele Report e Settings box will appear Select different Report Styles to see additional options A ee OG After selecting Report Style options click the Save as Default icon in the bottom C bnTse FLPMLPA left corner of the Allele Report Settings box Your options will be saved and will ee be recalled the next time you select that Report Style Additionally select View SE j Preferences Others Show Disabled Samples in Report to include samples C Sample Nano Deeg that are disabled in the Sample File Tree meer Hide Extra Sample Names Sort Options tl Carcel Sort by Marker Select Sort by Marker from the rig
107. OfftAllele IS NA ske 36 Marker Boundary cccccssscccccessececcaseceesaeseeeesaaesees 57 58 Marker Drop down Menu 41 Makker NAME a 57 58 Marker Parameters ccccccsssccccesseeeeeeeeeeeeeseeeeeees 58 Marker Table 136 Marker Table Fragment c cssssecccceeceesseeceeeeeeeeees 68 Marker Locus Specific Viewing 30 184 Marea ON 56 Match by Fixed Position 170 173 Match by Sections 170 173 Match Ladder avs TG 64 Mato SCOl ae 46 49 51 Maternal Cell Contamination IMC 146 Max of Open Charts i ia 36 ale Ce EE 57 64 Max Allele Label Layers oooccccccocccnnccnoccnnnonaronnnonanonoos 36 Max Call intensidad 24 Max Chart in Page Jane 36 Mendelian inheritance occcccconccnnccnoccnnnonanonnnnnanonoos 109 Mendelian Inheritance cccccoocccnccnnccnnnonanonnnananenoss 103 WIEN UO DLIONS sensies ieee Ee 34 Merge BINS gebaier dada 70 Meta casara 95 Methylation Report Table 000annooeeeneeseenesseeeesseen 100 methylation sensitive endonucleases seses 97 Methylation Specific MLPA ssnsesseenesssensssreresseressseeee 95 Microbiology Research Center MRC Holland 80 Microsatellite instability 00ennneoseeneeseennsseeressrersss 131 Microsatellite Instabiltw 131 172 leie Ee EE 131 microsphere data analysis 93 microsphere detection 80 Microsphere MLPA Analysis ccccssssccceessseceeeeeeeeeees 93 minim
108. Passau 123 Population Normalization 25 83 86 Prader Willi Syndrome oocccccccoccnnnccnoccnnnonaronnnonanonoos 98 PRE GE 106 Preprocess Raw Data 52 NE II EE 74 A SAA PP EA 73 Print Grayed Samples 89 Pritt Marker aars 73 Print Project COMMENTS ccooccccnccnncccnnoncnncnonannnnancnnonnnnos 73 Print Report rrarrnnnrrnnnrrnnnrrnnnrnnnrrnnnsrnner 37 40 72 168 Print Statistics Information oarnrnnrnnnnrnnnnrnnnnnnnnnnnnnr 90 Print Summary EE 90 Print Ke TEE 73 Probabili arva 130 May 2012 Index Project Comments ue 37 Project Compartson 40 174 Project Comparison Settings ccooocccococnnnnos 174 Project Option Settings ccooccccocnononcononnnocnnnancnnanonnns 37 Project Options SAettigge 25 Promesa Panels aaa 101 Promoter Methylation cccccoonncnnccnnnnnnnnnnnnnnnonanonnnnnos 97 Pullup Correction vaser 19 Pull up Correction ooccccccooccnncconccnnncnanonononanonoss 21 SJ EN EE 32 ale CN ER Quantification WEE 84 85 Quantitative Analysis aeeeeeeeeerresreen 38 120 Quantitative Analysis Settings oooooom o 121 quantitative trait loci OT 122 R ROMO IO tree 80 Raw Data Analysis 00 neennneeeeenesseeessereessreressreresse 21 raw data Tile tor Matic a 16 Raw Data folder unicidad 28 Raw Data Main Analysis ccoooocccnnccnonnnnncnanonnnonanononnnos 16 Re analyze Individual Samples 25
109. Reference sample The Poisson Difference Histogram appears below each selected sample s Electropherogram A positive bar in the histogram represents a peak gain at the position as compared to the Reference and a negative bar indicates the absence of a peak at the position as compared to the Reference AFLP Trace Comparison Ta GeneMarker Untitled l B X File View Project Applications Tools Help gt 0 BH amp H 9 Q MGE Maker f None x E Y H D Report e E Reference frag_001_H01 fsa 180 200 220 240 260 frag_002_G01 fsa x 12 180 200 220 240 260 2 500 2 000 1 500 1 000 Poisson Difference Histogram Report Table The Poisson Difference values are shown in the Report Table Peak gains are displayed in dye colored font and are positive numbers Peak losses or absence are displayed in grey font and are negative values The Poisson Difference classification for loss and gain can be modified in the Report Settings dialog box To access the Report Settings dialog box click the Report Settings icon above the Report Table Classification Poisson Difference Trace Compare Report Poisson Difference Set the Poisson Difference values at which a peak will be considered a Loss or a Gain Default Loss lt 0 20 lt Equivalent lt 0 20 lt Gain Output Status Peaks Choose to display Loss Equivalent or Gain values in the Report Table Report Contents When Status
110. Relationship Testing from the Applications drop down menu 4 Select Allele Frequency of the appropriate population from the Tools drop down menu The dropdown menu contains all samples from the current project Additional samples can be added to the dropdown list by using the icons to open a folder or select files from the database 5 Select Kinship Analysis Tool use drop down menus to select individuals for comparison 6 Select the desired relationship level and likelihood ratio probability or both at the kinship analysis settings 7 Probabilities for the occurrence of the genotypes within the population having a specific relationship or being unrelated for each locus and all loci combined are displayed in table form 8 Likelihood ratios for each locus and combined likelihood ratio of a related parent child sibling half sibling are presented in table form Icons and Functions Relationship Testing Main Drop down menus Select from File DataBase or Tool options d Relationship Testing File DataBase Tools d Relationship Testing Relationship Testing Tools include Family Group Tool Family Group Tool for automated pedigree trio drawing Allele Frequency En Allele Frequency Import species specific allele frequency txt files Tools gt Allele JUNE Frequency gt open folder icon gt select file gt save Genetics Analysis Settings Mutation Rate Import species specific txt file Population Statistics
111. S but will not be used to size fragments aa in the other dye colors Disable a Size if its position is variable from sample to sample NOTE If the Enabled value is changed in the Size Table you must click another cell in the Size Table before saving the Size Standard or the change will not take effect Insert Size Right click at the position in the Expected Size Standard frame or in the Sample ILS where the Size should be placed The Edit Size box will appear GeneMarker will automatically interpolate the value in the Size field if there are two or more Sizes present in the trace Adjust as necessary and click OK A green triangle will appear at the cursor position indicating where the new Size was placed NOTE The height of the new Size in the Expected Size Standard trace is dependent on the height of the peak in the corresponding Sample ILS trace Delete Size Select Delete Size to remove the Size completely from the Size Standard Alternatively the Size can be disabled by deselecting Enabled in the Edit Size box or by placing a 0 in the Enabled column of the Expected Size Table NOTE Sizing is often more successful when there are many Sizes in the Size Standard Set Value to Column Makes all values in the column equal to the value in the highlighted cell Only available in the Expected Size Table 45 May 2012 Chapter 4 Fragment Sizing Standards Sample ILS The Sample ILS frame displays the selected sample s ILS trace C
112. S i 24 25 42 33 27 29 33 34 2 24 25 42 33 27 29 33 34 25 42 33 27 33 24 25 40 42 42 33 29 33 Ve SNPO06fsa 24 25 42 33 27 29 33 34 24 25 42 33 29 33 34 ENPANA fea 195 4 17 379 37 E sqri 2se2 2911 The report table is located beneath the electropherogram when the gel image is de selected Only peaks that fall within the bins of the panel are called in the SNP Analysis screen providing Abide by Panel functionality What to Expect SNaPshot and SNuPE implement single base extension technique by dye labeling primers with different colors for the different expected SNP alleles So in addition to the complimentary fragment appearing one basepair larger the different dye molecules will affect the mobility of the fragments differently This slight difference must be taken into 126 May 2012 Chapter 7 Special Applications account when creating Bins in the Panel Editor It is recommended to expand the Bin range slightly 0 7 on the Left and Right to accommodate variable mobility In the final SNaPshot Analysis window plot points in the Cluster Plot that approach the green group separation lines should be examined individually for accuracy After each Marker has been verified export the Report Table see the SNP Analysis Reporting section below SNPlex To fully utilize the diagnostic potential of SNPs a robust high throughput genotyping and data analysis system should be employed SNPlex Genotyping System Applied Biosy
113. Setting the minimum and maximum values changes the range of score at which the peak will be considered unstable Confident MSI calls are shown as red bars in the Histogram lesser confidence calls are displayed as green The default settings will display both gains and losses LOH in the final report Increase the negative value to 1 0 or more to filter out LOH reporting Show Loss in Histogram When deselected only peaks with a positive MSI score will be represented with a bar in the histogram Negative peaks or losses will not be shown Layout Settings Launches the MSI Display Settings box MSI Display Settings S Ctrl Sample Shift SE Change the amount of offset between the reference trace and the sample ese E trace in the Trace Overlay electropherogram Iw Peak Normalization Peak Normalization When selected Reference sample peaks will be normalized based on peak a height or area to the Experimental sample peaks in the Group Print Launches the MSI Print Settings box See Reports and Printing section below for more information What to Expect There are a few options for MSI display which will vary by personal preference In the MSI Analysis Settings box there are several options for filtering the allele call The peak detection thresholds Intensity and Local Region are designed to filter out noise near the baseline The Stutter Filter right and left thresholds are applied after the peak to peak comparis
114. Settings box and will be applied to the data without having to re analyze the data with Run Wizard or Auto Run Re analyze Individual Samples To re analyze an individual sample dye color or marker click the Call Allele icon in the main toolbar The arrow next to the icon opens the drop down menu with additional options Click an option from the drop down and the Recall Allele box will appear Adjust parameters as munnet pe necessary and click OK The new parameters will be applied Additional Promega_MSI l Iw Call Allele By ONE i All Samples Br Make BAT26 E L 4 Applies the new parameter settings to all samples in the r mmm gt dataset similar to Run Wizard and Auto Run Per Reject lt 100 Check ES lt Pass P Iw Auto Range bps fo aj for 3 fri 30 00 Peak Detection Threshold 25 Min Intensity 100 Max Intensity 30000 5 Global Max 2 1 2 Percentage gt obal Ma May 0 Local Region gt aj Local Max Iw Stutter Peak Filter 2 Iw Plus 4 Filter Left 90 Right 40 gt Cancel Chapter 2 General Procedure Open Samples Applies the new parameter settings only to samples that are checked in the Sample File Tree Current Sample Applies the new parameter settings only to the sample highlighted in the Sample File Tree Call the Dye Applies the new parameter settings to the dye selected in the Recall Allele Call Allele by Dye field Call the Marker Applies the new parameter sett
115. Size Standards To open the Size Template Editor select Tools Size Template Editor from the menu bar OR click the Size Template Editor icon in the Run Wizard Template Selection box Due to differential fragment mobility in capillary gel electrophoresis a sizing standard must be applied Each sample run through a CE instrument will contain an Internal Lane Standard ILS The ILS contains peaks of known size and is usually tagged with red or orange fluorescent dye Since the ILS dye labeled fragments migrate through the same capillary as the other dye labeled sample fragments they are subject to the same environmental conditions and can therefore be used as a guide to determine the size of the other fragments in the sample A Size Standard template is applied to each ILS and sizes between the known ILS peaks are interpolated NOTE GeneMarker is optimized to size fragments with linear mobility Larger fragments or those run through a high viscosity gel i e agarose do not migrate linearly and therefore cannot be analyzed with GeneMarker at this time Size Template Editor Size Standard List Expected Size Standard Sample List Expected Size Table Sample ILS d Size plate b ter D H Sus tacite 5120 ET400F i 50 100 Help ETSA ETSOD A GS 100 250 J 25 15 20 j 400 4 65200 6535 nl 25400 Gem frag Gen Hoi fea Match Scone 94 a T K H ane 20 000 To Hod e S o ee O 10 000 G Samples a
116. Standard selected and the Internal Lane Standard ILS of the individual samples Fragment mobility is from right to left with the smallest size fragments on the far left of the trace The Peak Table contains information about the called peaks currently displayed in the Electropherogram Electropherogram Trace Display Range The basepair size range x axis is as set in the Run Wizard Data Process Allele Call options box The RFU range y axis is variable and will re adjust according to the maximum peak height in the trace To manually set x and y axis ranges use the Set Axis icon in the main toolbar Cursor Locator The x and y axis position of the mouse pointer in the electropherogram is displayed in the upper right corner of the electropherogram Allele Call If a Panel is applied to the data then grey horizontal bar Markers will appear above the electropherogram indicating locus ranges Bin ranges appear as dye colored brackets above and below the sample trace Allele Labels appear below the electropherogram and are associated with the center of each called peak which is also marked by a light grey vertical line in the electropherogram If a Panel is not applied then Allele Labels for called peaks will only indicate the basepair size of the peak The red horizontal line seen here in the figure on the right is to alert analysts to trends in the data These areas Of the data have a more elevated baseline or noise to signal ratio often asso
117. T LENGTH POLYMORPHISM AED 76 PLEOCE UE A 76 Wat kN we 77 TERMINAL RESTRICTION FRAGMENT LENGTH POLYMORPHISM T RFLP ssseecccceesseccceeessecceseeeeecesseeeeecesseueeecessaeesses 79 NEVNT 79 O NN 79 WATT ENE ves See 79 MULTIPLEX LIGATION DEPENDENT PROBE AMPLIFICATION MLPA 0sccccccesseecccceeseccceeeeeecesseueeecesseueescesseeeeceessugnsses 80 VEVEN NNN 80 PEO COO A DETT EE EN ENE AN DA DE ED NE ie ons na 82 LOOMS ANA Ce Te EE 83 Wo 85 2 May 2012 Table of Contents Resa NG re 89 Microsphere MLPA Analysis Luminex 93 Methylation Specific MLPA Analysis MS MIDPAI 95 HUMAN IDENTITY HD va diia 101 Fe 101 WV TOU ee EXO COE E 102 EEN 102 PEDIGREE CHART HR 103 NN 103 Toe 106 JEONS ANA FUN CUINA aa 108 WAOTO EXD EOU a fis 109 NNN 110 VEV 110 EE 110 16005 GAG FUNCTIONS seede 111 SETENE 113 Importing Species Specific Allele Frequency and Mutation Rates oeseeoesenoesenoesensesereeseresserresereesrreeseresse 113 DATABASE SEARCH LOCATE DUPLICATE SAMPLES AND NEAREST RELATIWES sssssssssssessessreresessrerrereseoseerreresesseerrereseesee 114 NNN 114 Process 114 EON ANA FUNC UOS NN 115 SEMEN 116 Saving Genotypes from Two or More Multiplexes to the Dotobgee 117 PARENTAGE VERIFICATION FOR PURE BRED ANIMALS see 118 Fre NN 118 DOVE al EE EE EE RE EN EE REE 119 OVNEN See 120 Frede as 120 eelere 121 WV RGU 0 Exec aaa atisbo 121 SINGLE NUCLEOTIDE POLYMORPHISM SNP ANALYSIS ccccccessccccccsss
118. The Advanced option uses the trend in these data to adjust the slope This option works best if control probes have been assigned GeneMarker uses a cluster algorithm to differentiate well separated data points and then uses those clusters to calculate a corrected slope There are usually two populations clusters of peak ratios Initially the peak ratio plots are divided into approximately 4 equal parts The upper 25 peak ratios for each of the top two quadrants and the lower 25 peak ratios for each of the lower two quadrants are retained The middle values are not used in these statistics to avoid data points that are close to the quadrant boundary The retained values are used to calculate a slope for the upper two and the lower two quadrants These slopes are an estimate of the trend in the data and a corrected slope is plotted Verify Sample Lane Quality After normalization has been verified check the Sample List in the MLPA Analysis window to identify samples that received a lane score below the Minimum Lane Score Threshold Samples with low lane score will be disabled and grey out in the Sample List To view the disabled sample s probes in the Ratio Plot right click on the sample in the Sample List and select Show Control Sample Identification When MLPA Analysis is launched the software automatically selects a sample in the dataset to use as the control The sign next to the sample number indicates the automatically selected c
119. The normalized peak values are calculated using the exponential fit function and original peak height intensity Normalized Peak Value Original peak intensity Exponential fit peak intensity The square roots of the intensity ratios are calculated and the ratios are plotted to model a linear regression The median peak intensity is normalized to 3000 Population Normalization reduces errors Due to variations of PCR efficiencies from small to large DNA fragments or from sample to sample two selectable normalization methods are provided GeneMarker can normalize MLPA derived data by an internal control probe method or a population method The user is able to specify the normalization method in the Run Wizard window three Additional Settings prior to analysis Normalization using Control Probes removes the trend of dropping intensities as the DNA fragment size increases However the trend of peak intensities vary greatly from one sample to another with the internal control probes Also a small number of control probes or internal control probes that display atypical amplification kinetics often results in large errors in the intensity normalization Population Normalization addresses these problems yielding better results All of the probes control and test that are retained by the median filter are used to fit the exponential function which results in higher accuracy and lower false positive rates MLPA Analysis using normalized peak heigh
120. WAA Desege Native os mm ov Ch i 113 E SE MITA O JR e 2 113 14 Patenti _Oiyestod SCT Alan 1 b i NG 0 24 ATELIACA Sine bos i e 2322 95 May 2012 Chapter 7 Special Applications Sample Group List Samples in the Sample Group List appear within Group folders according to the information provided in the File Name Group Text file uploaded in the MS MLPA Analysis Settings box A sample is marked as the Reference because it contains a Control Identifier as set in the File Name Group tool See Chapter 8 Additional Tools Reference Control samples will appear in the first column of the File Name Group Text file Expand folders in the Sample Group List to view the Reference sample and Experimental sample in the group Double click the Reference sample to view just the Reference sample electropherogram Double click the Experimental sample to view the Experimental sample electropherogram overlaid on the Reference sample electropherogram and associated Dosage Histogram Electropherogram The Electropherogram displays a normalized electropherogram trace of the selected sample with the control sample reference trace displayed in light red behind it Change the degree of shift between the selected sample and the reference trace in the MS MLPA Display Settings box The allele or exon names are displayed below their corresponding bins The Dosage Histogram displays in bar chart form the ratio of normalized peak intensities b
121. ace Single left click samples in the Sample List to see additional samples OR use the Up Down Arrow keys The green triangle peak indicators appear atop peaks that correspond to the enabled Sizes in the Size Standard Trace The basepair size associated with the green triangle peak indicator is located above the electropherogram The peaks selected for size calling can be edited in the Sample ILS frame as described below Navigation in the Sample ILS frame is similar to navigation options in the Main Analysis window See Chapter 3 Main Analysis Overview Editing Size Call Single left click a green triangle peak indicator to select it The triangle that is currently selected will be yellow To move the green triangle hold down the CTRL key and left click and drag it to the desired position Right click the green triangle peak indicator or right click the top of an unmarked peak to see additional options 50 May 2012 Chapter 4 Fragment Sizing Standards Delete Peak Removes the green triangle peak indicator from the Sample ILS and the peak will not be considered in the Match Score calculation The Match Score calculation is updated when Update Calibration is selected Add Peak Right click at the peak position and select Add Peak A green triangle peak indicator will appear at the cursor position To move the green triangle hold down the CTRL key and left click and drag it to the desired position The newly added peak will be included in the Ma
122. ach of this Agreement User assumes all risk for use of the Software User further acknowledges that User is responsible for validating the Software for use in User s intended applications Due to the nature of computers software and installation procedures SoftGenetics cannot accept any liability or responsibility for validation of the Software in any of User s applications 3 Intellectual Property Rights The Software and any copies that you are authorized by SoftGenetics LLC to make are the intellectual property of and are owned by SoftGenetics LLC and its suppliers The structure organization and code of the Software are the valuable trade secrets and confidential information of SoftGenetics LLC and its suppliers The Software is protected by copyright including without limitation by United States Copyright Law international treaty provisions and applicable laws in the country in which it is being used You may not copy the Software except as set forth in Section 2 Software License Any copies that you are permitted to make pursuant to this Agreement must contain the same copyright and other proprietary notices that appear on or in the Software You also agree not to reverse engineer decompile disassemble or otherwise attempt to discover the source code of the Software except to the extent you may be expressly permitted to decompile under applicable law it is essential to do so in order to achieve operability of the Software with another so
123. al clustering is often represented as a dendrogram In GeneMarker the hierarchical algorithm is agglomerative and establishes clusters from the bottom up Overview The first step in hierarchical clustering is to select a distance measure GeneMarker distance options include Euclidean Distance Correlation Coefficient and Percentage of Same Genotypes Euclidean Distance is the straight line distance between two points in two or three dimensional space The equation is essentially the same as that for determining the length of the hypotenuse of a triangle computed by finding the square of the distance between each variable summing the squares and finding the square root of that sum We have simplified this equation below in GeneMarker The Correlation Coefficient is based on the Pearson Correlation equation and is a statistical concept that quantifies the level of relationship between two sets of measurements It is a measure of 136 May 2012 Chapter 7 Special Applications similarity where two values that are perfectly correlated have a distance of 1 00 Percentage of Same Genotypes is simply the number of similar genotypes divided by the total number of genotypes The following are GeneMarker s clustering algorithms Euclidean Distance In addition to a distance measure the type of linkage needs to be applied GeneMarker has three options Single Complete and Average linkage Single linkage measures the minimum distance between
124. al deviation is greater than 3 times the average deviation will be considered 92 May 2012 Chapter 7 Special Applications outlying to the data The outlying data points represent duplications and deletions Duplications will be shown above the plotted ratio line and deletions will be below the line A color coded triangle at the bottom right corner of each graph depicts the success of the standardization method for plotting of the peak ratios A green triangle represents successful standardization yellow represents standardization that needs to be verified for accuracy red means standardization failed for that sample or there are a significant number of probes displaying a copy number change Success or failure of the standardization is determined by calculating the relative deviation of the ratios The probe ratio relative deviation calculation uses the following formula 75th percentile sample peak ratio control peak ratio 25th percentile sample peak ratio control peak ratio 50th percentile sample peak ratio control peak ratio If the probe ratio relative deviation is gt 0 6 the triangle will be red If the probe ratio relative deviation is gt 0 4 lt 0 6 the triangle will be yellow If the probe ratio relative deviation is lt 0 4 the triangle will be green GeneMarker software MLPA Regression Analysis MLPA Regression analysis relies upon a T Distribution to form the regression line between the square roo
125. alization Adjusts peak intensities based on the intensities of the probes designated as controls in the Panel Editor 82 May 2012 Chapter 7 Special Applications Population Normalization Adjusts peak intensities based on an average of peak intensities from all probes of high quality samples NOTE Population Normalization is the recommended method for MLPA Analysis Advanced Available when Population Normalization method is selected Choose Advanced when samples contain deletion or duplications in more than half of the peaks probes V1 51 This option uses the normalization algorithm from GeneMarker version 1 51 MLPA Panel Selection and Modification After the data has been sized filtered and normalized with Run Wizard go to Tools Panel Editor to adjust the MRC Holland Panel selected To create a new Panel or for explanation of specific functions in Panel Editor see Chapter 5 Panel Editor 1 oe eh Se oe In Panel Editor select the appropriate MRC Holland Panel for the dataset from the Panel List a If the Panel does not appear in the Panel List go to File Import Pre Defined Panels b The C Program Files SoftGenetics GeneMarker 1 7 MLPA_Panel folder will appear c Select the correct Panel and click Open d The Panel will now appear in the Panel List NOTE Information regarding MRC Holland Panels can be located on their website at www inrc holland com d Panel Editor Adj
126. ame directory path and filename will appear in the Individual Sample Accordance File field if the SMP file is located in the same folder 8 If the SMP file is located in a different directory click the Open Files icon next to the Individual Sample Accordance File field select the correct directory and click Open 9 Click OK in the Load Pedigree File box 10 The information in the Pedigree and SMP files will recreate the Pedigree Tree and associate the nodes with the samples in the dataset Create an SMP File from a PED PRE File If an SMP file does not exist for a Pedigree File or has been lost a new SMP file can be generated with the Pedigree File Name Match Input PED PRE File C Users SoftGenetics Desktop TestPed pre Open SMP File _ tool Input Filenames 11 jenetics Desktop T estPed 279878 20_04 fsa Add 1 In the Main Analysis window menu bar select Tools Pedigree File Name Match The SMP File box appears Individual Identifier from character 7 to in file name Click the Open button re EET Select a previously created Pedigree File PED or PRE 7 Extract sting within seperators _ Click the Add button Select all the samples included in the Pedigree File Click the Process button The SMP Table box will appear with filenames aligned in family groups Click the Save As button Save the SMP file to the same directory that contains the Pedigree File Proceed through steps 1 10 above Family Identifie
127. ame height as the highest peak in the dye color Trisomy Print Report Below is a description of the Trisomy Print Report features For an explanation of functions within the Print Preview window see Chapter 6 Reports and Printing NOTE The analysis type chosen in the Trisomy Analysis Settings box Classic BPG or Aneuploidy determines the display of the resulting Print Report 147 May 2012 Chapter 7 Special Applications Classic Trisomy Print Report When Classic is selected as the analysis type in the Trisomy Analysis Settings box the Print Report example below is generated Trisomy Analysis Report SoftGenetics A Sample A11_1_gfpcr_07_06_07_b_01 fsa Conclusion Software GeneMarker V1 65 Analysis Type Trisomy me Date Panel QSTR_lite_2 i Classification Trisomy lt 0 70 or Trisomy gt 1 50 FE Report Time 16 10 2007 11 16 10 Report Type Peak Height Ratio Authorisation 1 Exp Time 07 06 2007 14 55 09 gt 07 06 2007 15 54 17 Plot Corrected Peak Height Ratio All Samples are Displayed Plot Legend Yellow Current Sample Red Trisomy Authorisation 2 D21511 D TIBIE i 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 430 Si 339 erch 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 Initial ight Ratic N Hi 1 000 0 Di a g E g S oo N 5 000 E 144 213 291 265 386 o E n S
128. ample drop down menu POG4_C12_06 fsa 6 The synthetic control sample can be saved as SCF 4 color data SG1 5 Serre color data or TXT file formats by clicking the Save icon ee P034_E11_09 fsa 7 To list the synthetic control samples in the MLPA Clinical Report click the EE Print Report icon EA ONDA 14 11 faa S The MLPA Print box will appear 9 Click the check box next to List Samples Used for Synthetic Control A maximum of 50 synthetic controls can be selected MLPA Analysis GeneMarker features two types of MLPA analyses MLPA Ratio and MLPA Regression Both MLPA analysis features identify data points that are outliers Outlying points are determined based on the deviation of each allele peak relative to the average deviation of all peaks Any individual peak whose residual deviation is ereater than 3 times the average deviation will be considered as outlying to the data These outlying points should be considered as possible duplications or deletions and need to be reviewed by the user MLPA Ratio Method MLPA Ratio plots the ratio of each sample compared to the panel In MLPA Ratio the ratio of the sample data to the control sample is standardized such that the median point within the data set is considered to be 1 All other data points are relative to the median value as depicted in the plots The outlying data points represent duplications and deletions Duplications will be shown above the plotted
129. amples of some of the pre defined Panels include MRC Holland s MLPA kits Promega s MSI kit and ABI s forensic human identity kits GeneMarker also offers the opportunity to create a new custom Panel if the pre defined Panels do not include a kit that the user is working with Below is a discussion of how to use the pre defined Panels or create a new Panel with GeneMarker s Panel Editor tool The Panels displayed by default include MLPA CMT1 P033 REX HID IDENTIFILER MLPA DMD P034 REX HID POWERPLEX 16 MLPA DMD P035 REX HID SGMPLUS MLPA MSH2 MLH1 P003 REX PROMEGA MSI MLPA VHL P016 REX SNPLEX 48PLEX 1 Pre Defined Panels In addition to the Panels displayed by default the user has the option to import standard Panels and Bins Text files and to access additional commercial kit panels including MRC Holland MLPA Panels Trisomy Panels for Aneufast Devyeser Finnzymes and GenProbe kits Cystic Fibrosis Panels for Abbott and GenProbe kits Import ABI Panels and Bins Files 1 In Panel Editor select File 0 Import ABI Panels from the menu bar A 2 The Import Panels from GeneMapper box appears EE 3 Click the access button next to the Panel File field A Windows Explorer kane EE window will appear 4 Navigate to the location of the Panels txt file and click Open 5 Next click the access button next to the Bins Load from File field and locate the Bins txt file 6 Click Open NOTE Select Bins Auto Build if a Bins txt fil
130. an analyze data from Multiplex Ligation dependent Probe Amplification MLPA MLPA was developed by the Microbiology Research Center MRC Holland in January 2002 and is a technique used to detect exon deletions in the genes for BRCA1 associated with breast cancer MSH2 associated with colon cancer and MLH1 associated with colon cancer MLPA can also be used to detect aneuploidy such as Trisomy 21 found in Down syndrome MLPA is a simple method for simultaneous quantification of up to 45 nucleic acid sequences in a single reaction With GeneMarker MLPA data analysis is an efficient quick and easy process with features such as customized reporting and printing In addition to the standard MLPA probe labeling technique Luminex Corporation has developed a method to uniquely label individual probes with different color beads GeneMarker software can quickly and accurately analyze data from the Luminex flow cytometry instruments Luminex 100 IS and Luminex 200 for microsphere detection Overview In the MLPA Analysis window duplications and deletions can be detected by four ways increased or decreased peak height in the Trace Overlay where the reference trace appears in light red behind the dye colored sample trace in the Dosage Histogram where the histogram bar appears larger for duplications and smaller or absent for deletions in the Ratio Plot where plot points represent the peak height or area ratio of the reference versus the sample
131. and independent assortment the Pedigree tool in GeneMarker is an excellent choice to determine genetic relationships between family members Overview The Pedigree module is designed to aid identification of inheritance patterns and abnormalities All individuals in the Pedigree Tree are directly linked to their corresponding Electropherograms To display the Electropherogram for a sample simply double click on the sample s node in the Pedigree Tree and the Electropherogram will appear in the Charts tab The link between the Pedigree Tree and Electropherogram Charts makes relationship analysis quick and efficient To activate the Pedigree function select Applications gt Pedigree from the Main Analysis window menu bar NOTE The dataset must be sized and a Panel applied prior to using the Pedigree function Pedigree Application Pedigree Tree Samples List Electropherogram Charts E Pedigree Edet New File A a lFf TE BIE fei Mier L t Makes Sech d a T Fre 1 Sarath hal i penedes Gage Eo Bi Sue 27987810 OG aa Sue BD im 279878 20_ Kira D 279878 10_O6 tra ak B J Arare iiia Gei hs 279878 30 D lea Be 279078 Die br 27987920 Sta hs Zem 201 Dt Da Fe CAT Bio 279879 30 Meisa Ar 27967910 12 fra Biz FETE 10 ON den Biz Ladder 1 fra i Ladder 2 Hrs Person ID 3 Peor Hare Lyn ard Ple BARA Dti Pedigree Tree The Pedigree Tree displays in a standard format the relationshi
132. and select Edit Allele The Edit Allele box appears Add or change the values in the Allele and or Size field The Allele field will be blank if no Panel has been applied to the dataset Check Confirm the Allele to automatically give the peak a Quality rank of Pass green Allele Comments Edit Allele Comments CS Right click an allele in the Electropherogram or Peak Table and select Edit Comments The Edit Allele Comments box appears Select a comment from the Comments Template list or enter a new comment in the Comments field Click OK and the comment will appear in the Comments column of the Peak Table Only one user edited comment can be added to a peak Comments automatically generated by the software cannot be removed Additional user comments will simply be added next to the software comment View History Opens the Show Edit History window Shows a record of all manual edits performed on the peak The Show Edit History window is only active when the Help User Management Settings Record Data Edit History option is selected See Chapter 9 User Management Report Table The Main Analysis window Report Table contains additional information about sample peaks Depending on the Analysis Type selected in the Run Wizard Template Selection box the Report Table will contain appropriate information See Chapter 6 Reports and Printing d GeneMarker Untitled Navigation o gt TET amp B QQBE DG BA Mate
133. anel Classification Loss lt 0 75 lt Equivalent lt 1 30 lt Gain ra fer 7 E Controk P034 C10 05f5a Report Value Type Peak Rabo gt a P034_H11_15 fsa CE EES ES 12 exos 2625 114880 13 Jee 3144 1514 18 exor 3538 144 15 Je 1383 Io r TI TE a T C PO 2 t om Sample Name 033855 LL P34 Machine 3100 Run Time 04 14 2005 13 27 40 gt 04 14 2005 14 17 43 PERF ERERSEE 3 g ee E noe EE ee 270 sl ee o mg SE o SES Authorization 1 m fa Jess jara Authorization 1 Klenges osr agoe ml 91 May 2012 Chapter 7 Special Applications MLPA Peak Height Normalization and Copy Number Determination MLPA Data Normalization using Peak Heights GeneMarker normalizes peak intensities based upon the statistically most probable median intensities calculating median intensities helps determine the trend of the data Median peak intensities are derived from the first five data points GeneMarker v1 70 and later GeneMarker v1 60 uses seven data points GeneMarker v1 51 and earlier use nine data points then sliding to data points 2 6 3 7 etc to ascertain the local median intensities Outliers are rejected after applying a median filter In order to correct for the peak intensity variation over size an exponential function a e bz is used to fit to the square root a statistical tool to reduce variation of the peak intensities where z is size a and b are fitting constants
134. anel List and saved in the SoftGenetics GeneMarker Panel directory Import Panels Opens a Windows Explorer window to the same folder the sample files were uploaded from Use the Import Panels option to find previously exported Panel Files xml on local or networked computers Import Pre Defined Panels Opens the SoftGenetics GeneMarker PreDefined Panels Folder This folder contains many panels from commercially available chemistries Aneuploidy Trisomy to import panels for Aneufast Devyser Elucigene and Finnzymes Cystic Fibrosis to import panels from Abbott and Elucigene and the MLPA Panel folder to import additional MRC Holland MLPA Panels Import ABI Panels Launches the Import Panels from GeneMapper box Opens Panels and Bins Text files and converts them to single Panel files in XML format for use in GeneMarker Export Panel Exports the currently selected Panel in the Panel List as an XML file to a specified directory on a local or network computer Exit Closes the Panel Editor tool Be sure to save changes to the Panel before exiting 63 May 2012 Chapter 5 Panel Editor Tools Menu Match Ladder Opens the Select Ladder box Choose an allelic ladder sample from the mams drop down menu Click OK and the Panel will adjust slightly to align zoomm with the peaks in the selected ladder sample a NOTE Large differences between peak and Bin position cannot be 1 resolved with the Match Ladder function Scroll Graphics Mo
135. as the Administrator Click the Add User button to add additional users AA Click the Access Rights button to set up user type access permissions a Be sure to select Run User Protection and click OK to exit mine Ea a e User Manager The User Manager tab displays user information and contains options for creating and deleting users User Window lol x Displays all users by name type and creation date User Manager History Settings Organization Admin Administrator 7 12 2011 10 56 49 AM E tech Analyst 7412 2011 11 04 37 AM nter your organiz ation name Jessica manager Lab Manager 7 12 2011 3 34 55 PM Ta Run User Protection When selected users will be prompted to log on with a user name and password When deselected any person can launch GeneMarker without a username and password Access Rights Change User Add User EE Organization General Hospital Run User Protection Launches the Add User box where a new username and password can be input This is also where the user type can be chosen A user can be deleted by right clicking the username and selecting Delete User NOTE Only the Administrator can add and delete users Eed Print Icon Launches the print preview to print the user management history or save as a pdf document My Password Launches the Change Password box where the user that is logged in can enter a new password The new password must Access Right of User Typer
136. as the analysis type in the Trisomy Analysis Settings box the Print Report example below is generated Grayed files indicate that the ratio is consistent with trisomy and indicates that the ratio is within the inconclusive range Report Header Contains information about the analysis project sample and parameters Signature Box Date and initial space for report reviewers Electropherogram Similar to the Trisomy analysis window displays all dye colors of the sample trace Report Table Displays selected peak and Marker values for the current sample Trisomy calls are highlighted grey An additional Check column is provided for indication of inconclusive range for trisomy and reviewer initials A ozs Dou ent CETE 13012 Io Jee ose lo fnar boloomlaeeae lo Corrected Ratio Plot Contains the entire dataset s plot points for all Markers in the dye color Symbol shapes represent different Markers and can be deciphered from the Symbol row in the Report Table Yellow filled symbols represent the current sample s data points Red outlined symbols represent trisomy calls NOTE The Corrected Ratio Plot appears on a second page for each sample only when Ratio Plot is selected in the Trisomy Print Report Settings box 149 May 2012 Chapter 7 Special Applications Loss of Heterozygosity LOH Lo
137. aseline Subtraction Selecting this option will remove the baseline completely so that the Y axis will be raised above the noise level This option is selected by default in the Run Wizard Auto Pull up Removal Automatically removes peaks caused by wavelength bleed through to other wavelengths This option is selected by default in the Run Wizard Manual Pull up Correction This allows the user to manually adjust larger pull up peaks in case the Auto Pull up Removal function has not corrected the problem It is recommend to de select Pull up Correction in the Run Wizard when using this function 2nd Derivative Trace This feature reduces high background noise and sharpens peaks Baseline fluctuation caused from dye blobs or the DNA template in PCR can also be reduced with this function It is recommended to de select Spike Removal in the Run Wizard when this function has been activated 17 May 2012 Chapter 2 General Procedure What to Expect The raw data correction icons can be selected individually in the Raw Data Analysis window The images below demonstrate how the data will look before left image and after right image the parameter is applied Range AutoRange Analyzes from 0 to end of trace for size call Manual Range user defined range Right click in gel image and select Get Start Point Gel Image Get Start Point Automatically Start Point 1000 Cancel Smooth Fourier frequency transformation FFT to dete
138. asure is changed Fig 1 amp 2 the basic overall structure is similar however on closer examination the fine structure of ordering within the main clusters differs The samples with 3 as the first character are grouped as are the samples with the number 4 The sole 7 sample is grouped in its own cluster in both examples These results are as expected Figure 1 and 2 also show an example of isomorphism in the dendrogram where the 3 group and 4 group positions are switched Fig 1 Euclidean Distance Single Linkage 2 Clustering Analysis SM e BR 0 4 0 3 0 2 0 1 0 0 100 0 0 451 442 537 4 D1 Y 528 B 4 G1 Y 535 4 E1 v 532 4 G2 Y 545 4 B2 Y 539 4 B3 Y 556 3 011 484 3 G11 Y 491 3 D11 v 485 3 F9 v 427 7 D7 Y 597 b Fig 2 Correlation Coefficient Single Linkage 138 May 2012 Chapter 7 Special Applications 2 Clustering Analysis SM Gray Soale 1 06 07 08 os 10 100 0 EE 0 170 3 011 7 484 3 G11 v 491 3 D11 Y 485 3 F9 1 427 442 537 4 E1 v 532 4 G2 Y 545 4 G1 7 535 4 D1 528 B 4 B2 Y 539 4 B3 Y 556 7 D7 Y 597 b When altering the analysis based just on linkage type and holding the distance measure constant Fig 2 4 we see that the overall structure remains the same however the finer structure is greatly affected Notice how in single linkage Fig 2 the three main groups are independent of one another where in complete Fig3 and average linkage Fig 4 the 3 gr
139. at a PIKI Same Individual 1 402 5000016910 2 852 xx 1212 6l6 5 29E 06 different individual in this animal granoa meme E e e e 4 405 5000016913 2 855 x 1212 6l6 5 29E 06 population has the same profile It is Deselect Node EE EE EE very likely that these are all replicate a Ree 7 ADS 5000016916 2 858 XX 1010 55 1 08E 06 Father Son G11 5000017096 2 933 ar co samples from the same individual Select Siblin 1 55E 00 g Mother D aughter Select Family Full Sibs 1 F02 5000017075 2 912 xx 712 1 36 00 Edit Node 2 E12 50000170722 910 x 712 1 36E 00 3 FO3 5000017076 2 913 XX 712 1 36E 00 Add Child Add Mate Half Sibs 1 H11_female_2008 0 945 x 712 9 55E 00 2 EO1_so00017054_2 899 xx 612 1 55E 00 Find Fami 3 E02 5000017055 2 900 xx B12 1 55E 00 ind Family 4 E 5000017056 2 901 ui 712 1 18E 00 5 xX 1 10E 00 Find Individual Delete Node Export Bitmap 114 May 2012 Chapter 7 Special Applications Sample in Pedigree Tree 1 Import or draw Pedigree fl Relationship Testing e Per Tree See Chapter 7 File DataBase Tools Pedigree Analysis and HT BIT IGG Family 1 8 1 Family 1 Famiy1 v Marker All Markers Search A Automated Pedigree Tree DS Samples lA Charts Report B Calculation Details _ FileName 1D_ Name XA Matched Alleles Matched Markers DI 2 Right click on the node and ene e select find family from the
140. ate to SoftGenetics GeneMarker the version that was just installed gt GeneMarker program The Configure Registration window appears Click Register Now to register the local license Click Register Local Hardware Key from the Choose Registration Method dialog box The Register Local Hardware Key window appears If the computer GeneMarker is being installed on has an internet connection select Online Registration If the computer does not have an internet connection or is connected to a proxy server select Offline Registration Proceed through the Registration steps as described in the Online Registration or Offline Registration section above Launch GeneMarker and begin analysis Network licensing Option The network licensing version of GeneMarker can be installed on any computer in a network configuration SoftGenetics uses the License Server Manager LSM to control the number of concurrent users accessing the network licensing option of GeneMarker v2 00 and above LSM uses text based registration no hardware is required Both software components are installed from the same EXE The computer where License Server Manager program is installed is considered the Server computer Computers on the network other than the Server are called Client computers x No security key has been detected Ifa local license was purchased click Register Now If a network license was purchased click Configure Network Client Configure
141. ated based on the area values of two peaks within a Marker Trisomy Ratio Apply Linear Correction When selected ratio values will be corrected for based on the slope of peak heights within a Marker The slope of all sample peaks within a single Marker is represented by the red Fit Line in the Ratio Plot Deselect this option to view raw uncorrected peak height or area ratios Trisomy Set lower and upper limits for Trisomy detection The values entered here are represented by the black lines in the Ratio Plot Inconclusive Range Samples that fall within an incolnclusive range will be identified by a in the report table if this option is selected as specified by the Best Practice Guidelines 2007 Statistics Plot Tab Provides display options for the Ratio Plot Coordinate Y Peak Ratio Evaluates trisomy based on differences in M intensity of peaks within a Marker Ae Statistics Pi Angle Evaluates trisomy based on the angle of a line that EE is formed between the center of the tops of the peaks in a TT Marker Ges R Show Ratio Raw Calculates the ratio of peak intensities based on raw data Corrected Calculates the ratio of peak intensities based on the normalized data Normalization is the process by which large fragment s relatively lower intensities are increased to equal small fragment intensity using a linear function Show Fit Line When selected a red center line is placed between the trisomy detection limits in t
142. ault and displays the angle of the Cluster Plot Layout Coordinates 12 5 C Cartesian Polar May 2012 Y Show Clustering Information Chapter 7 Special Applications vector connecting the peak intensities of the two alleles versus the square root of the sum of the square of the longer fragments and the square of the shorter fragment Cartesian displays the plot points on an Allele 1 versus Allele 2 graph Show Clustering Information When selected a statistical table will appear below the Cluster Plot with information for each group of plot points Statistical information reported includes number of samples in the group s population mean of the group and standard deviation SD of the group P ill SNP Analysis for SnapShot oo zz a B E FI Marker SNP1_G SNPI_A Sample D 4 IESSE F SNPODT fra SNPOD fa SMPOD2 fea SNPOOG fra SNPOO4 fsa SNPOO5 tsa SNPODE tsa SNPOO fea SNPODS fsa Ka SNPODS fea 6 000 10 SNPO10 fsa 11 SNPO11 fsa 5 000 13 SNPOIZ fsa 13 SNPOV3 sa 4 000 14 SNPON4 fra 15 SNPO15 tsa 3 000 16 SNPOIE fsa 17 SNPOI7 fsa 2 000 18 SNPOTS fa 19 SNPOIS fsa Kate 20 SNPOGO fsa 21 SNPOS fsa 22 SNPOZZ fsa 23 SNPO23 fsa 4 lm r Report Hide Synthetic Gel Image and display the report table under the electropherogram MI 1 H 3 4 5 B H H 3 EE f E d i Gap A SNP5 G SNP3_A SNP4 A SNP4C SNP2 TISNP2 C SNP3 T SNP3 C 1 SNPOOIfsa EE 33 33
143. available in the Open Data Files box to make data upload easier Add Used to locate and select raw data files for upload Click the arrow button next to the Add button to see the four most recently accessed directories Remove Used to remove samples from the Data File List Highlight the sample to remove by single left clicking it in the Data File List then click Remove Remove All Removes all sample files from the Data File List field Add Folder Click Add Folder to upload raw data files from a specific folder in the file directory tree Click the Default hyperlink to choose a folder to which GeneMarker will always open when the Add or Add Folder buttons are clicked d Set Channels for Data Importing 4 Colors 5 Colors Channels Color Channel 1 E Red vi Channels Charel Ml eue vi Opens the Set Channels dialog with 4 and 5 color tab options and tewes F vao l allows the user to choose from ABI MegaBACE and Beckman et Bn Coulter standard dye color orders The user can also manually Beset MegaBACE Channels Beckman Channels enter dye color and name The default channel color setup is ABI Set the dye color channels before clicking OK in the Open Data Files dialog box Canoe Raw Data Analysis Once the raw data files are uploaded the Raw Data Main Analysis window appears Double click the samples in the Sample Tree to open the individual Raw Data Traces The Synthetic
144. ays the peak area value for all Positive and Suspected peak positions Dollar signs separate values if more than one display option is selected Allele Count C Sample Name Ze File Name Orientation Horizontal Vertical m CG W Show Rejected Low Score Alleles Iv Show Intensity I Iw Show Peak Area Show Only Uncertain Alleles Hide Extra Sample Names Cancel Allele Report Settings x Report Style Options C Allele List Iw Abide By Panel C Marker Table Fragment Iw Show Type Symbol ME een eee np EI Bin Table AFLP MLPA Ge Negative 0 Peak Tabl sl ene Suspected Orientation A Horizontal Sample names appear on the left in rows and Markers appear at the top in columns Vertical Sample names appear in the far left column in rows Markers and Alleles in order of basepair size appear at the top in columns Show Only Uncertain Alleles When selected displays only the peaks with Quality ranks of Check yellow and Undetermined red Show Rejected Low Score Alleles When selected the peaks with peak scores below the Run Wizard Additional Settings Allele Evaluation Peak Score Reject setting will be displayed in the table Hide Extra Sample Names When data is 240 250 260 et TERS Raab DES A v Make N d 2 Report E Bin WR Chapter 6 Reports and Printing displayed in Vertical Orientation the samp
145. be selected from the drop down menu Save and Print Report Find Family Report right click on the nS S report to copy paste directly into an EHO e existing document or report al Or export as a txt file 293E 23 BE 0 MEL 207413 207413 7 24E 404 2 24E 402 JIE 02 E 01 Lan Lan set 01 286 401 R FRI Aa AR mn eee 4455644455 DURERERAARA RAR at m weet 116 May 2012 Chapter 7 Special Applications Saving Genotypes from Two or More Multiplexes to the Database B It is often necessary to run two or more multiplexes due to AMEL ASB23 marker overlap or incompatible PCR conditions for different ae primer sets Save the files with combined genotypes in a txt file ae and import into the Database using Save to Database dropdown 12776 XX menu Markers and allele calls can be tab delimited or a single column can contain both allele 1 and allele 2 AMEL AHT4 Select Load from TXT and navigate to the saved txt file to add individuals with these extended genotypes to the database x X H This process has been automated in GeneMarker 1 90 Please see Chapter 8 Merge Project Tool d Submit Genotypes Species Hu TXT Delimit Column pm Genotypes Clear All 117 May 2012 Chapter 7 Special Applications Parentage Verification for Pure Bred Animals Automated Pedigree Trio Diagrams and Analysis using Fa
146. ble x Show Dye Allows the user to select a single dye color to view in the Overlay View Cycle through the colors by left clicking on the icon DA A S MN Q Single left click to cycle through the options or use the drop down menu Trace Overlay displays all traces of the selected samples in the Samples List one dye color at a time Single click any trace in the Trace Overlay frame and the trace will become bold and the associated sample will be highlighted in the Sample List Max amp Average displays two traces in the electropherogram The darker color line corresponds to the maximum peak height at that position and the lighter color line corresponds to the average of all selected sample traces at that position Gel Image displays selected samples as a synthetic gel image Bin ranges in the Gel Image mode appear as white vertical lines and can be manipulated by holding down SHIFT and dragging the white lines left or right SIE Check Range in Edit When activated the software will warn the user if they set the left or right range of an allele to overlap with another allele This feature will prevent the user from setting allele boundaries too close to neighboring alleles This option is selected by default H 64 May 2012 Chapter 5 Panel Editor E it Major Adjustment of Panel Uses previously defined size and height information located in the Panel file to identify Marker and Bin positions To be use
147. can also be installed manually Select the Input Manually option and type in the Serial Number and Product name e Input Manually Serial Number 916354 Password vw Support Product Name NOTE Do not change the Password Product Name mutation 5 Click OK RE rn Cancel Registration 1 The Network Version Registration window appears 2 If the computer GeneMarker is being installed on has an internet connection select Online Registration If the computer does not have an internet connection select Offline Registration 3 Click Next Online Registration Ble Registration A Locate the Account and Password on the SoftGenetics CD o B Enter your E mail Address Account and Password information in the UserID QUORNENNV2x ETIVIRESSY1RNALVGTHNREO appropriate fields Account C Click Register Password D The USB key will automatically load the User ID information into the E registration field E Your software will be registered automatically A confirmation e mail sere Manan will be sent to you once registration is complete NOTE Some characters can commonly be misread If you get an error trying to register check for number 1 and lower case letter L or number 0 and upper case letter 0 confusion F Launch GeneMarker and begin analysis Release Version Registration Registration Offline Re gistration Click here for registration instructions A Copy and paste th
148. cation Enter the number of the beginning and ending character to identify how to group the samples The section of the filename specified will be highlighted red in the File Name List Control Identification Enter the number of the beginning and ending character to identify which part of the filename contains the control identifier The section of the filename specified will be highlighted green in the File Name List File Name Growp Editer Match by Group Order Allows the user to group samples that contain sequential identifiers Group Size Enter the number of groups for the analysis Standard MS MLPA analysis would use 2 groups an LOH analyses comparing one normal sample to three grades of tumor would use 4 groups Control Identifier Enter the character from the Control Identification section highlighted green that describes the control or reference sample Example N normal or R reference Select Case Sensitive if the Control Identifier needs to be identified by upper or lower case letters Control Match Mode Choose either Whole Words or Include Whole Words should be used if the characters entered into the Control Identifier field need to match exactly 173 May 2012 Chapter 8 Additional Tools Include should be selected if the characters in the Control Identifier field only need to be identified in the filename i e not an exact match Output Trace Data Tools
149. ccccceesesccsceeseccsseeuneeceseeeeesesseunecesseuneecessegeessess 122 NSM 122 MP 127 VIVIEN 129 re RED OF TING ass 130 MICROSATELLITE INSTABILITY MS EE 131 NNN 131 POE NNN 132 eet rte ee ee 132 MENN 133 Repons ana PN 134 PHYLOGENY CLUSTERING ANALYSIS Ree 136 Detecting EUCHOIGN PISTON CO EE 139 SULIS Er RED ONE NASA ai AD 140 Using Allele Bin Report from Merded Projects een EE 140 IRISOMY DETECTION saint is 142 DV 142 Preus 143 lons GING aere EE 143 HEEN 145 FP 146 Reie HETER ONO LON ae 150 3 May 2012 Table of Contents NVE 150 Proce dUe aid 151 lecons ana FUNCHUON eda 151 WOU TO EXPECT EE EEE EE EE EEE a a 152 Reports and PENNO EE 153 DENE NN 155 VEVEN NNN 155 FONN 156 ICONS GHG FUACTIONS ee 156 VINN 157 REDI Ne 158 HAPPENS 158 NNN 158 Poe 159 KON PNG 162 ARMS COMPARATIVE ANALYSIS FOR CYSTIC FIBROSIS ANALYSIS ssscccccseccccsececcsccccecceccusececaccecueececaucceecsseeeaaeeeees 162 NNN 162 Beier 163 KONST LNL 163 Moro edu 164 Reports Ona PINEDO NE 165 CHAPTER 8 ADDITIONAL TOOLS seede 167 BROWSE BALE CORORS iia 168 NERA 168 NERE NNN 169 PRO COGIC pr 169 IVIACROMOLECUTES Se 171 A OR ER POEL EEN 171 HEN ESN 172 POCERO S r 172 FILENAME GROUP EDITOR ae NE 172 PEO CC OUI NN 172 Icons and FINS ed 172 OUTPUT TRACE DI NN 174 Fe 174 PROJECT COMPARISON EE 174 Procedure varsel 174 CONS ana F uUnNCUONS E AAA ASS AAA AS EA TAS AAA AA A AS 175 CONVERT TAO DINA dios ie 176 ele e 176 EXPORT BERN 176 Fe 176
150. cess options For a Ref Trace Subtraction Ratio value of 1 5 the o vinPeak SAt Rao FI software will subtract 1 3 times the reference trace from the sample trace Data Range Signal to Noise ratio the software compares the called peak to other peaks in E E Whole Fragment Size bps 1049 the neighborhood Reference Data Range Enter the start and end size values for viewing the data Enter the C Selctad Same z expected size of the undigested fragment er Use Processed Data Cancel IS TILLING Analysis Setting Launches the TILLING Analysis Settings box from an open TILLING ANALYSIS WINDOW 156 May 2012 Chapter 7 Special Applications Tilling Analysis window Toolbar icons Show Hide Gel Image displays selected samples as a synthetic gel image Bin ranges in the Gel Image mode appear as white vertical lines and can be manipulated by holding down SHIFT and dragging the white lines left or right Show Color Allows the user to select all colors to view hide all colors or choose a single dye layer Choose a single dye by single left mouse clicking on the icon Zoom In Use the icon to zoom in on the image or hold down the left mouse button and draw a box from the top left corner to bottom right corner around the area you wish to zoom in on Zoom Out Use the icon to zoom out on the image or hold down the left mouse button and draw a box from the bottom right corner to top left corner Set Axi
151. ciated with poorly resolved peaks than the nearby regions of the trace which sometimes masks very minor peaks May 2012 Chapter 3 Main Analysis Overview Peak Table The Peak Table can be displayed below the Electropherogram by clicking the Show Chart Table icon in the main toolbar Right click in the Peak Table and select Show Columns The Show Columns fly out appears with column options B QQ6E Be Av Maker Hicre z a Dye Indicates the dye color of the peak VM 287 7 2512 x 0185535 Size Indicates the basepair size of the peak x axis Height Indicates the peak height in RFUs y axis Height Ratio E mg The value obtained when the peak s height is divided by Eu ms ES the height of the highest peak in the dye color or Marker E E S Area Indicates the area under the curve of the peak The area calculation begins and ends along the x axis as indicated by the Start and End columns of the Peak Table respectively Area Ratio The value obtained when the peak s area is divided by the area of the highest peak in the dye color or Marker Marker Panel Only Indicates which Marker Locus the peak is contained in Allele Panel Only Indicates which Bin the peak is contained in Difference Panel Only Indicates the absolute value in basepairs of how far the peak center is from the Bin center Quality Panel Only Assigns a Pass Check Undetermined qualit
152. ck the highlighted rows and select Merge Bins Hot Key CTRL M Click OK in the Report Bin Columns box when finished and the selected Bins will be averaged together Only Bins immediately adjacent to one another may be selected for merging Only the height and area for the first peak in the new merged Bin will be reported Peak Table The Peak Table Report Style displays user defined peak statistics Sample names _ alicierepor setting us are displayed in the far left column in rows and the Marker names are in the Report Style Options column adjacent to the sample names In columns at the top of the table are i a EE i the selected peak statistic information labels Peak Table is available for all C Bin Table AFLP MLPA TEE C Allele Count Columns C Sample Name Ze File Name Je Show when no allele call Show Only Uncertain Alleles Iw Show Rejected Low Score Alleles Orientation e Iw Hide Extra Sample Names 70 e mes May 2012 Chapter 6 Reports and Printing The column options available in the Peak Table Report Style are similar to the options available in the Peak Table that appears below the Electropherograms See Chapter 3 Main Analysis Overview for column option definitions Features Options Size Range bps When selected allows the user to define a specific basepair range Only the peaks within the range will be displayed within the Report Table Abide By Panel When selected the table
153. d the report on the right has only the FAM and VIC dyes selected 165 May 2012 Chapter 8 Additional Tools 166 May 2012 Chapter 8 Additional Tools Chapter 8 Additional Tools Chapter 8 Additional Tools Browse By All Colors Overlay View Merge Projects Macromolecules File Conversion Filename Group Editor Output Trace Data Project Comparison Convert TXT to Binary Export Electropherogram 167 May 2012 Chapter 8 Additional Tools Browse By All Colors Click the Browse by All Colors icon in the Main Analysis window toolbar Navigation and peak editing options in the All Color Browser is similar to the Main Analysis window See Chapter 3 Main Analysis Overview To scroll through samples in the All Color Browser click the drop down menu in the upper right corner and select a sample from the list Once a sample is selected in the drop down menu you can use the Up Down Arrow keys to scroll through samples Icons and Functions Zoom In Out CG a Use these icons to increase decrease the zoom aspect of the electropherograms Show Hide Mouse Cross Lines YE When selected x and y axis grid lines will appear at the tip of the mouse cursor along with the basepair size and RFU value of the mouse cursor position Show Hide Bin Ranges When selected the Bin brackets at the top and bottom of the electropherogram trace will appear de Auto Scale Markers When selected the RFU in
154. d when a Panel must be adjusted by 1 5 basepairs in order to align with the dataset peaks NOTE A Panel must be correctly aligned with peaks in the dataset before selecting Save Changes with Signal Info in order for the Major Panel Adjustment feature to work correctly Minor Adjustment of Panel Aligns the center of the Bin to the center of the nearest peak within one basepair of the Bin E What to Expect Once a Panel has been created it can be applied to the dataset Save the edited Panel in Panel Editor then exit the Panel Editor If the Panel Editor was accessed via the Run Wizard Template Selection box icon then the selected Panel will appear in the Panel field If the Panel Editor was accessed via the Tools menu then Click the Run Process icon in the Main Analysis toolbar The Run Wizard will appear Select the Panel from the Panel drop down menu in the Run Wizard Template Selection box Proceed through the other Run Wizard boxes and click OK when the Data Process window is complete The Panel will be applied After the Panel is applied to the dataset the Markers and Bins appear in the Electropherogram and Report Table In the Electropherogram the Markers are horizontal grey bars the Bins appear as dye colored brackets above and below the trace and the center of the peaks are marked with a vertical grey bar Peaks that fall outside of the Markers or Bins of the Panel are marked Off Ladder OL Off Ladder Allele A dil Ge
155. determine if the genes of interested have been deleted with the traditional MLPA technique Once the copy number has been determined digestion with methylation sensitive endonucleases can be performed to detect imprinting In the example below a normal patient s sample appears on the left and a sample from a patient suffering from Prader Willi Syndrome appears on the right In the normal patient s sample five green plot points appear between the green methylation threshold lines These plot points represent five genes of interest located in the PWS AS region on chromosome 15 As discussed previously unmethylated fragments are completely digested by the methylation sensitive endonuclease and methylated fragments remain undigested The five plot points that appear around Ratio 0 5 represent genes that contain one methylated allele and one unmethylated allele A normal patient without a deletion at the PWS AS site will express both the methylated and unmethylated genes from the mother and father respectively In the sample from the patient suffering from PWS none of the five genes of interest plot points appear between the methylation threshold lines Upon further inspection it is determined that the five peaks are in a 1 1 ratio with the undigested fragments Because it was determined previously with traditional MLPA analysis that this patients PWS AS region contained a deletion on one chromosome only the other parent s chromosome genes ar
156. dividuals can be compared to determine the likelihood that they have a specific relationship versus the likelihood that they are unrelated Kinship analysis compares STR profiles from individuals to determine likelihood of a family relationship versus the likelihood that two individuals with these STR profiles are unrelated The formulas used to calculate the level of kinship depend on 1 Probabilities that 2 1 or 0 alleles will be shared IBD identity by descent given a specific relationship 2 The probability of a specific genotype X given genotype Y at all loci under the conditions that X and Y have 2 1 or 0 alleles IBD GeneMarker uses established rigorous statistical analysis Kinship formulas from Brenner 2004 Eisenberg and Planz 2007 to calculate probabilities and likelihood ratios for different relationship levels including Parent child Siblings Half siblings Uncle Nephew Cousins and Grandparents P20y D2 Pry Pi Po amp y Do eq 1 Where P2 xy Probability of 2 alleles IBD I given the genotypes of sample x and sample y Piy Probability of 1 alleles IBD T given the genotypes of sample x and sample y Pony Probability of 0 alleles IBD O given the genotypes of sample x and sample Kinship Formula Transition Matrices Identity by Descent IBD d Pi Po for AA AAs AA each relationship category s e be AA 0 1 0 D gt Di Do Parent Child 0 1 0 S ch 8 d A Siblings 0 25 0 5 0 25 Half sibl
157. e Panel List The Panel List includes a list of all pre defined and custom Panels saved to the Panels folder in the SoftGenetics GeneMarker directory Single left click on the Panel name to display the Panel in the Overlay Trace frame Double click the Panel name to expand the folder and view the Markers associated with the Panel Single left click the Marker name to display that Marker in the Overlay Trace frame Additional Options To see additional options for each Panel right click the Panel name and the right click menu will appear with the following options Edit Panel Opens the Edit Panel box Editing the Panel Name field will change how the io Panel is labeled in Panel Editor Set the Ploidy from Monoploid 1 to Decaploid Panel Parameters 10 If the number of peaks within a Marker exceeds the Ploidy setting the api IA Meg additional peaks will be labeled Off Ladder OL and given the Undetermined ie ade 7 red Quality rank and PL Quality Reasoning See Chapter 7 Special g EC Applications SNaPshot regarding the SNaPshot feature in Edit Panel mem Delete Panel Select Delete Hot Key DEL to delete the Panel from the Panel List and from the SoftGenetics GeneMarker directory NOTE This action is irreversible Cancel 56 May 2012 Chapter 5 Panel Editor Export Panel Opens the Save As window Choose a directory folder and click Save The Panel will be copied to th
158. e Select samples Dyes and Image Size gt Output Trace Graph Es vw D2 fsa v 03 fsa Y D fea Y D5 fsa wv O6 fsa YD fsa wv D8 fsa wv D9 fsa Y 10 fsa wv 11 fsa Y 12 fsa wv 13 fsa Y 14 fsa wv 15 fsa wv 16 fsa Y 17 fsa Suffix 2008 Width 11024 pixels Height 768 pixels Export Format JPEG e Export Close 4 Use a dropdown menu to specify the export format JPEG and PNG are both available PNG is recommended 176 May 2012 Chapter 9 User Management Chapter 9 User Management Chapter 9 User Management Procedure User Manager History Settings Edit History Audit Trail 177 May 2012 Chapter 9 User Management Overview User management may be implemented after installation of GeneMarker The administrator activates User Managerment from the Help drop down menu User management provides control of user access rights automatically generates an audit trail of all edits to genotyping projects and is used to customize the Organization and Operator fields in final print reports of special applications such as MLPA and Trisomy d User Manager No user ZS User Manager History Settings User Nama Use Type Create Tine Procedure Select Help gt User Management The Login box appears Click Run User Protection to activate the setup Administrator here Boh Enter Organization Name an Administrator username and password O E Click OK tin on 5 You are now logged in
159. e Quantification method chosen in the MS MLPA Analysis Settings box Peak Height or Peak Area the digested sample trace is compared to the undigested reference trace The ratio value of the comparison is plotted by basepair size in the Ratio Plot below the electropherogram In the Ratio Plot the blue plot points represent the control probe peak ratios As expected the control probes of the sample trace are in a 1 1 ratio with the control probe peaks of the reference trace A green threshold line appears at Ratio 0 3 and represents the Methylated Peak Ratio value set in the MS MLPA Analysis Settings box Green plot points below this threshold do not contain a significant peak in the sample trace compared to the reference trace because the fragment was digested by the endonuclease Since the fragment was digested by the endonuclease it is assumed the gene is unmethylated Red plot points that appear above the green threshold line represent an amplified undigested fragment Undigested fragments which are not control probes are considered sites of methylation In 97 May 2012 Chapter 7 Special Applications Figure 1 one copy of gene ESR1 is methylated because its representative red plot point appears above the Methylated Peak Ratio threshold line at around Ratio 0 5 Promoter Methylation 5 gt Patient1 Digeste d SCF Genomic Imprinting Prior to detecting genomic imprinting with MS MLPA a researcher must
160. e user selected in the Main Analysis window Set Axis icon oe ial Iw Print Project Comments on Each Page I Word Wrap M Chart Height mm Specify the size of the printed electropherograms T Label Dyes amp Peak Numbers Print Markers Minimum 10mm maximum 100mm Iw Implement Y Axis Settings jw Print Alleles New Page for Each Sample Abide By Panel Print Markers The Marker label bars appear above the Grouped by Dye IV Auto Scale Markers electropherogram Chart Height mm 45 4 Print Alleles The Allele Labels appear below the electropherogram Bok Y Cane Abide by Panel Prints only alleles within a Panel Alleles that are outside the Panel are not included in the printed report New Page for Each Sample Prints a new page for each sample instead of continuing on the same page as the previous sample Auto Scale Markers When selected the RFU intensities of low peaks are adjusted to match the intensity of the highest peak in the dye color When low peaks are increased the intensity magnification factor is noted in the Marker 2X 8X Grouped by Dye Organizes the electropherograms in the Print Report such that samples are listed in order of dye color selected i e all samples in blue first then all samples in green etc 73 May 2012 Chapter 6 Reports and Printing Icons and Functions The following icons are available in the Print Preview window prior to printing the Print Report Print O
161. e 25 Recombination Values viarios aa aE 108 Record Data Edit History 179 Recover Old vValue 180 Reference Trace Compartson 76 Refresh Pedigree nren 112 116 162 KE EN e 7 Regi Tation ID E 8 10 12 regulatory SNPs rSNPS ccccccceecesssseeceeeeeeeeeeeeeeeeees 122 Relationship Testing 38 Relative Fluorescent Units RFUS coccc omooo 16 31 Reload Panel ass 57 Reload Size Standard 45 Remove files eneeennseenesrrsrerrsrrsrrresrrrsrerrsrers 16 EEGENEN EN ONE 74 Re Open Project Lusasuaqavavasss vudsadaksev kaken 35 Report Content OPTIONS pans ds cescivies cavcncaaseceesareevrereckevaeess 73 Report Save Options PNG ard PEG seas 74 Report Settings aiii 34 42 Report Tall vasse 33 68 Report Table Navigation o oooccccnccnoncnnnonanonnnonanonnnonos 33 Report Table Sort Options rrrrnnannrrrnnnnnrrnnnnnnrrnnnnneeene 34 restriction endonuclease ranuri iini ina ai 79 TT RENE 37 R n Method EE 35 RUN EEGENEN 20 40 RumUser Protection EE 178 RUN Wizard WEE 20 35 186 S Sample Comment ais A 29 sample File Tree Ljan 28 sample GrOUPIA md 29 Sample ILS ari 45 46 50 Sample Information 29 Sample LanecQuallity E 87 Saturated Peak Correction cccooooccncccnoconnnnnnoo 19 Saturation Correction cccccccccesecceeeceeeeeeseeeeeeees 17 Save as New Panel 63 Save as New Size Standard 47 Save Changes arr 48 Save Changes with Signal Info
162. e Chapter 3 Main Analysis Overview Click the Print icon to print the results See Chapter 6 Reports and Printing CODIS Report The Export CODIS tool was created for forensic labs that need to create reports for upload into the Federal Bureau of Investigation s Combined DNA Index System CODIS To access the CODIS report select Applications gt Export CODIS This tool allows the user to select profile descriptions from a drop down list for each individual sample and save the report as a CMF 3 2 CMF 3 0 xml file and CMF 1 0 dat file 102 May 2012 PR CODIS Export Header Version T CMF 32 mM C CMF 30 fami C CMF 1 0 fdat CODIS Header a pacmen Ty ource ORI ITE Submit By User ID Genemarker 4 Sample Nam Destinati on ORI Des ORI ample Inde PCR Kt Identities vi Y Export Batch Iderhlier Ei Urvecognized Locus Name zl gt Vabd Locus Name sl Ok ei d Source enti Cormctad Dilerdar orvicted Diterder false orwicted Ditender false Cormctad Diere fata onvicted Ditender false Cormctad Otlerdar fasa ormcted Ditender false Corwicted Dilendar fasa onvicted Dittender Chapter 7 Special Applications Pedigree Chart Relationship testing involves testing known genotypes in a familial relationship for simple Mendelian Inheritance laws Since the core loci selected for forensic genotyping demonstrate the Mendelian characteristics of segregation
163. e File menu allows the user to create save and export Size Standards The BestMatch menu contains options for selecting a Size Standard The Help menu shows navigation hints for Size Template Editor File Menu New Size Standard Opens the Input Dialog box with a field to enter a new Size Standard name Follow the steps above Custom Size Standard Creation Delete Current Size Standard Deletes the Size Standard that is currently highlighted in the Size Standard List NOTE This action is irreversible Save Changes Saves edits and changes to the Size Standard in the SoftGenetics GeneMarker Size Standard directory Save as New Size Standard Opens the Input Dialog box with a field to enter a new Size Standard name The Size Standard is added to the Size Standard List and saved in the SoftGenetics GeneMarker Size Standard directory Import Size Standard Opens a Windows Explorer window to the SoftGenetics GeneMarker Size Standard directory Use the Import Size Standard option to find previously exported Size Standard Files xml on local or networked computers Export Size Standard Exports the currently selected Size Standard in the Size Standard List as an XML file to a specified directory on a local or network computer Import ABI Size Standard Opens a Windows Explorer window to the same folder the sample files were uploaded from Export ABI Size Standard Exports the currently selected Size Standard in the Size Standard List as an XML
164. e ILS frame Cycle through the colors by left clicking the icon or use the drop down menu Size Match Automatically places the green size marker triangles atop the peaks of the sample trace and matches it with the selected Size Standard 2 m x m eE What to Expect Once the Size Standard is created it can be applied to the dataset Save the edited Size Standard in Size Template Editor then exit Size Template Editor If the Size Template Editor was accessed via the Run Wizard Template Selection box icon then the selected Size Standard will appear in the Size Standard field If the Size Template Editor was accessed via the Tools menu then click the Run Process icon in the Main Analysis toolbar The Run Wizard will appear Select the Size Standard from the Size Standard drop down menu in the Run Wizard Template Selection box Proceed through the other Run Wizard boxes and click OK when the Data Process window is complete The Size Standard will be applied The success of size calling for each sample is indicated by the green yellow and red sheet next to the sample filename in the Sample File Tree of the Main Analysis window The lane sizing quality is determined by the Match Score which in turn is a calculation of how closely the sample s ILS peaks match to the selected Size Standard If a sample receives a low Match Score the sample will be marked with a yellow sheet If the size calling failed the sample s ILS peaks could not be align
165. e Install window to restart the system 14 The Installation Wizard will close and the system will restart 15 Eject the SoftGenetics CD EE x This system must be restarted to complete the installation Click the OK button to restart this computer Press Cancel to return to Windows without restarting Cancel Register License Server Manager for GeneMarker Usage 1 Note A red star indicates the License server is not running The icon with a white star indicates the License Server is running properly 2 Click OK in the dialog box to proceed with registering License Server from the License Server Manager console 3 Select Register from the Help menu to activate the Register Product window EGG 4 Select GeneMarker from the Register Product Name drop down menu 5 Seleclf the computer License Server is being installed on ZS SoltGenetics License Server Open License Server from the System or Icon Tray by clicking the LSM icon SoftGenetics License Server has been successfully installed Click the Finish button to exit this installation IV Launch License Server Manager 60 a xl SoftGenetics License Server This program has not been registered To complete the registration process click on Help and select Register is Register Product loj xj GeneMarker Register Online Offine Registration Request ID MUIUTXhFRFEwR Ww pROmRETXURES4QURN in Register Product
166. e Mutant Green Wil Green Wild typd PEER Je FSS d 25 300 350 400 450 500 BEESSSESE TEF 3008 Seog BEES e 444gela4 1 Ce e o A ildtype 350 29 1717 1G A Bue Vuan Greeneviadtype 300 350 LI LA A A ALAM UE ul d Alt DM LL ML aen 9 wm A ee Oe ee IA SO E P OE A Comments TL BE i Trace Comparison with Linked Report Table Final Report with Trace Comparison Report Tables Header and Comments Section Procedure 1 Open GeneMarker and upload raw data files 2 Select a Panel Size Standard and Fragment Animal Analysis 3 Use the default settings in the second and third screen of the Run Wizard or customize as needed See Chapter 2 General Procedure 4 Verify size calls are accurate in the Main Analysis window and adjust parameter settings accordingly See Chapter 2 General Procedure 5 Tools gt Panel Editor to import panel and enter 0 in the Control Column for alleles that are Mutant Alleles and 1 for Wild Type Alleles See Chapter 5 Panel Editor Run Wizard with the panel and review allele calls File gt Save Project Project gt Apply Sample Grouping to pair both files for an individual See Chapter 9 Additional Tools Applications drop down menu select ARMS ComarativeAnalysis and select the appropriate option Select Mutant Wildtype to open the Trace Comparison Application of grouped files Select Mutant On
167. e does not exist 7 Click OK in the Import Panels from GeneMapper box C jie 8 All Panels in the Panels txt file will be uploaded into GeneMarker 9 Select a newly uploaded Panel from the Panel List 10 Edit the Markers and Bins so that they align with the peaks in the dataset 11 Save the edited Panel and close Panel Editor 12 Click the Run Project icon in the Main Analysis window 13 Select the Panel from the Panel field in the Run Wizard Template Selection box 14 Proceed through Run Wizard and data analysis See Chapter 2 General Procedure and Chapter 3 Main Analysis Overview 60 May 2012 Chapter 5 Panel Editor MLPA Panels 1 In Panel Editor select File Import Pre defined Panels from the menu bar 2 The SoftGenetics GeneMarker Pre defined Panels folder will appear 3 Open the MLPA Folder 4 Choose a MRC Holland MLPA Panel for upload NOTE Go to www mrc holland com products htm for additional information on MLPA Panels 5 Click Open 6 The MLPA Panel will appear in the Panel List 7 Follow steps 9 14 above Custom Panel Creation Follow the steps below to create a new Panel based on the dataset currently uploaded to GeneMarker Automatic Panel Creation 1 In Panel Editor select File Create New Panel from the menu bar or click the Create New Panel icon The Create New Panel box appears 2 3 Entera name for the Panel in the Name field This will be the Panel name that
168. e entire User ID string and type your Account and UserID QUOxNFNNV2xET3VJRESSY1RNdi VqTHANRED Password information into the body of an e mail Region B Send the email to tech_support softgenetics com C The Registration ID will be sent to you via email within one business day D Copy and paste the Registration ID from the e mail into the Registration dia ID field E Click Register F Launch GeneMarker and begin analysis Upgrade 12 May 2012 Chapter 2 General Procedure The upgrade version of GeneMarker Network should be installed on the server the computer running NetDog Server as well as all client computers First install upgrade network version on the server computer You will be prompted to configure the network enter server name or IP address Your software version is higher than NetDog key s do you want to update your Netdog 1 After installing and configuring the upgrade you should update NetDog on the server computer To do this launch the NetDog Upgrade tool NetDog Update can be accessed by going to Start menu All Programs _ SoftGenetics GeneMarker Network NetDogUpdate It is located in the same directory as the GeneMarker program The Confirm dialog box will appear if the versions of GeneMarker and NetDog are not consistent Click Yes and proceed to software registration Additional User Licenses If you are interested in purchasing additional user licenses please
169. e installed with the Software including all copies Updates and prior versions and all copies of font software converted into other formats to such person or entity b you retain no copies including backups and copies stored on a computer and c the receiving party accepts the terms and conditions of this Agreement and any other terms and conditions upon which you legally purchased a license to the Software Notwithstanding the foregoing you may not transfer education pre release or not for resale copies of the Software 5 Multiple Environment Software Multiple Language Software Dual Media Software Multiple Copies Bundles Updates If the Software supports multiple platforms or languages if you receive the Software on multiple media if you otherwise receive multiple copies of the Software or if you received the Software bundled with other software the total number of your computers on which all versions of the Software are installed may not exceed the Permitted Number You may not rent lease sublicense lend or transfer any versions or copies of such Software you do not Use If the Software is an Update to a previous version of the Software you must possess a valid license to such previous version in order to Use the Update You may continue to Use the previous version of the Software on your computer after you receive the Update to assist you in the transition to the Update provided that the Update and the previous version are ins
170. e is compared to a reference trace Samples with loss of heterozygosity are immediately apparent in two displays the electropherogram and the ratio plot In the electropherogram the reference trace is in light red behind the patient trace When a peak is absent in the patient trace only the reference trace peak remains In the ratio plot the absence of a patient trace peak is indicated by a red dot that falls outside the user defined bounds of the LOH ratio indicated by dashed lines Blue dots within the LOH ratio bounds indicate a patient trace with the expected number of peaks No Loss of Heterozygosity Loss of Heterozygosity Loss of Heterozygosity 152 May 2012 Chapter 7 Special Applications In addition to the ratio plot a clinician can use the LOH score parameter in the report table to qualify a given marker LOH score is calculated using the peak intensity or area ratio between normal and tumor samples For complementary peaks in both reference and sample trace the score will equal 1 0 In the case of complete loss of heterozygosity at a certain position the LOH score will be 0 0 Reports and Printing Loss of heterozygosity detection is a key component in separating cancerous from non cancerous tissue The ability to do this type of analysis quickly and accurately is an extreme advantage to clinicians determined to fight the disease In addition to GeneMarker s LOH analysis tool an easy to read report can be pr
171. e is known simply single left click the Size Standard name in the Size Standard List and click OK in Size Template Editor The selected Size Standard will then appear in the Size Standard field of Run Wizard Template Selection box and will be used to size the data Alternatively if the Size Standard name is not known follow the Best Match steps below 65600 The Size Standard with the best average Match Score across all samples s in the dataset will be highlighted in the Size Standard List and appear in tu the Expected Size Standard frame 519 600 SNFlex 48plex vi NOTE BestMatch will not always choose the correct Size Standard User hm one Analysis Time 14 85 1 In Size Template Editor select BestMatch Match All lt 2 The Data Processing box appears Events 3 GeneMarker cycles through all Size Standards Es 4 Click OK when Data Process is finished stn 5 inspection is required 6 Once the Size Standard is chosen click OK in the Size Template Editor 7 The selected Size Standard will then appear in the Size Standard field of Run Wizard Template Selection box and will be used to size the data Custom Size Standard Creation Follow the steps below to create a new Size Standard based on the dataset currently uploaded to GeneMarker 1 In Size Template Editor select File New Size Standard OR click the New pa Dialog amp Size Standard icon New Size Standard Name
172. e population Tetra and penta 4 and 5 nucleotide repeat units are also characteristic of the forensic core loci Several commercially available human typing kits are available from ABI and Promega and are included as pre defined Panels in GeneMarker For advanced human identification analysis please try GeneMarker HID software Post bone marrow transplant chimerism analysis and monitoring studies also use Human Identification PCR kits ChimerMarker software provides Short Tandem Repeat analysis automated chimerism analysis and monitoring Fully functional validation versions of the programs may be downloaded from http www softgenetics com downloads html Human Identity Analysis f T il GeneMarker Untitled lo e amp File View Project Applications Tools Help gt ER amp EH a BE LA Marker THO1 DI Y ISS Untitled al y D Report fd Y Help H Raw Data Ladder 1 fsa Allelic Ladder 181 4 487 x FG fro gar a B 273878 10_02 fsa 155 160 165 170 175 180 185 190 195 200 205 4988 1002 s3 gw gt SS 279878 20 03 fsa 2 279878 20_03 fs4 MY 8 3 3 B 273878 30_09 fsa 800 3 279878 30 09 f50 gt zz B 279878 10_06 fsa 4 E gt E gt B 279878 20_04 fsa 600 S g gt mo RB 279878 30_07 fsa me ala B 27987910 12 fsa e 279878 30 07 51 7 9 B 279879 20_05 fsa 400 7 279879 10 12 fs4gF 7 T e B 279879 30_11 fsa 8 279879 20_05 sd MF 3 3 279879 10_01 fsa 200 9 279879 30 11 550 gt 279879
173. e represented by the fragment peak In the PWS case since the remaining gene fragments were not digested it is deduced that the remaining chromosome is methylated and therefore comes from the mother The father s PWS AS genes have been deleted Fora patient suffering from Angelman Syndrome the mother s PWS AS genes would be deleted leaving the paternal chromosome genes The paternal chromosome is unmethylated in the PWS AS region therefore the fragments would be completely digested and no peak would appear in the electropherogram 98 May 2012 Chapter 7 Special Applications Genomic Imprinting Gee Patent Digested Ira HANN x E F S ao M mm 240 246 en e OM OG SMRPN Gel Beng H Da Dope Maiggrar LL LL 4 L Pate nt4thge 10 d fa 5 300 350 Sus Epa Reports and Printing GeneMarker automates the analysis process and creates professional clinical reports for accurate insertion deletion detection Report Table Displays peak ratio information for individual peaks of a sample compared to its designated reference A sample column will be highlighted blue when the sample is selected in the Sample Group List The Electropherogram trace will correspond to which ever peak cell is selected in the Report Table MS MLPA Report Option Genomic Imprinting Report Settings Report Contents Orientation Launches the MS MLPA Report Options dialog box I Status 0 1 2 Horizontal vw Peak Ratio C Ve
174. e score 0 to 100 that corresponds to the quality of the trace To view only the samples above a certain score threshold set the Minimum Lane Score Threshold to the desired value All samples below the selected value score will be deactivated and appear grey in the Sample List The default setting is 10 Save Parameters when Save Report Automatically generates an ini file of the MLPA Analysis Parameters when the MLPA report is saved Naming convention for the file is Report file name_MLPA_Settings ini Quantification Select whether to calculate peak ratios by area or height To view Advanced Settings click the double arrow button at the bottom of the dialog box Min T Distance Helps to reduce false positives in high quality samples by setting the minimum distance the alleles must be from one another to be considered true peaks The default setting is 0 05 Outlier Filter Confidence Limit Number of Probes Used This feature allows the user to define the confidence in the calling of outliers For example 99 confidence means that GeneMarker is 99 sure that the alleles considered as outliers are truly deletions and duplications The Number of Probes Used allows the user to set the percentage of probes used for each iteration of normalization in order to reach the desired confidence The default is 80 which means that 20 of the probes will be disregarded before each regression line fit Min Dosage Range The Minimum Dosage Range helps t
175. e selected directory and will also remain in the Panel List and SoftGenetics GeneMarker Panel directory The Panel will be exported as an XML file which can be opened with Internet Explorer Microsoft Excel or Notepad Reload Panel Click Reload to undo editing changes to the Panel The most recently saved Panel will be restored NOTE If the user selects Save Panel and then answers NO to the The Panel has been changed save changes to file the changes will remain in the Overlay View until the user chooses Reload Panel or GeneMarker program is closed Sample List The Sample List contains all the samples uploaded to GeneMarker in the current project Samples with a checkmark next to the filename will be displayed in the Overlay View Double click the sample filename OR right click the sample and choose Select De Select to enable disable it in the Overlay View Right click any sample in the list and choose Select All De Select All to display all or no sample traces in the Overlay View Sorting Options Sample Name Sorts the samples in alphanumeric descending order Sample Name sorting is the default option Size Score Sorts the samples by the lane size score as it appears in the Size Calibration Charts See Chapter 4 Fragment Sizing Standards Samples with higher scores will appear at the top of the list Overlay Trace The Overlay Trace displays all selected samples in the Sample List The Marker bars appear above the electrophero
176. eMarker will be considered the Reference project and the project uploaded to the Project Comparison tool will be considered the Sample project TA Project Comparison Settings San oes oe Launches the Project Comparison Settings box with several Pesk Matched By Peak Compare tems options for running the comparison green a M Dye Score M Allele Name Iw Deleted C Marker Name Peak Size i VW Size Iw Confirmed Iw Height Iw Comments Peak Matched By Allows the user to choose the principal parameters for comparison Dye Peak si itor Peak Comparison Threshold Peak Compare Items Options for which parameters should be MaxSize Dit 0 Max Height Dit TO x compared and marked as different 10 P Peak Comparison Threshold Allows the user to qualify the ranges for detecting differences in peak attributes File Grouping Tool Provides the ability to group duplicate files with different file names analyzed from different genetic analyzers If the projects have different file names they will appear in a list at the right side of the screen after selecting in the open folder process described above Use the file group tool gt match by section or fixed position gt Match gt OK T Project Compertion ml oes 8 896 GB 175 May 2012 Chapter 8 Additional To
177. ed with the Size Standard selected then the sample will be marked with a 48 May 2012 Chapter 4 Fragment Sizing Standards red strike through When low score or failed samples occur select the Size Calibration Charts icon in the main toolbar to correct the size calling Low Match Score and Failed Samples d GeneMarker Untitled le x File Vie Project Applications Tools Help aoe gt HEH amp Wo oO BE A y Marker None y E Y 5 Untitled 4 D E Raw Data 564 T Group1_0 07 fsa x E el amp Allele Call ee RSR S 50 100 150 200 250 300 350 400 450 500 B Group1_ _D5 fsa W 564 T Group1_0 07 fsa W 564 T Group1_102104_07 fsa 1 000 amp 745 8 Group1_0 09 fsa B 745 8 Group1_102104_09 fsa E 745 T Group1_0 11 fsa B 745 7 Group1_102104_11 fsa Page Up Page Down Size Calibration Charts The Size Calibration Charts tool is designed to aid the user in determining success or failure of size call after GeneMarker s automatic sizing is performed Click the Size Calibration Charts icon in the main toolbar of the Main Analysis window As mentioned previously once a Size Standard has been applied to the dataset Size Match Score indicators appear next to the filename in the Main Analysis window Sample File Tree Samples with a high Match Score are indicated by a green sheet those with a low Match Score have a yellow sheet Samples where size calling failed receive a red strike through To analyze how
178. efficient value to adjust the slope of the fit line Report Table The Report Table lists all samples in a column on the left Selected report values appear in columns along the top Click the Report Settings icon and select deselect reported values Double click values in the Report Table to link to the associated Electropherogram position Click the Save Report icon to save the Report Table as an Excel or tab delimited Text file See the Reports and Printing section below for more Report Table features Procedure 1 Open GeneMarker and upload raw data files 2 Select a Panel Size Standard and Fragment Animal Analysis 3 Verify allele calls are accurate in the Main Analysis window and adjust parameter settings accordingly See Chapter 2 General Procedure In the Applications menu select Trisomy Analysis The Trisomy Analysis Settings box appears Adjust settings and click OK The Trisomy Analysis window appears ES Icons and Functions Trisomy Analysis Settings e Opens the Trisomy Analysis Settings box Two tabs are available Analysis tab and Statistics Plot tab Analysis Tab Provides threshold setting options for Trisomy analysis To change default settings enter the desired ranges in the dialog box Analysis Classic When selected Quantification is by Peak Height and Trisomy Ratio default thresholds are lt 0 70 or gt 1 50 Additionally the Report Table in the Trisomy Analysis window will display Trisomy Score values and
179. eight e Peak Area Quantification LOH lt 0 70 or gt 1 30 1 51 Cancel May 2012 Chapter 7 Special Applications Peak Height When selected LOH peak ratios will be calculated based on the RFU intensity values of a peak Peak Area When selected LOH peak ratios will be calculated based on the area under the curve of the peak LOH The ratio between reference and sample based on height or area calculated for each individual peak to determine presence or absence of the peak Layout Settings Ctrl Sample Shift Adjust the amount of offset between the Reference trace and the Experimental Launches the LOH Display Settings box sample trace in the Trace Overlay electropherogram Peak Normalization When selected Reference sample peaks will be normalized based on peak height or area to the Experimental sample peaks in the Group Max Row Column Select the number of Ratio Plots to display at one time in the LOH Analysis window vd LOH Display Settings Lo fal SEE Chart Layout Ctrl Sample Shift 0 0 bps 0 Sbps Iw Peak Normalization Max Row 2 v Max Column 2 gt Cancel Print Launches the LOH Print Settings box See Reports and Printing section below for more information What to Expect The concept for determining LOH is the comparison of a suspect tissue sample to a normal tissue sample with all expected alleles In GeneMarker a patient trac
180. ent for combined reports that will be Horoma C Venea e Show Rejected Low Score Alleles Iw Hide Extra Sample Names m Carca used in Relationship Testing 8 Save the merged project report as a txt file Note The file name will automatically read from the current project Users may want to use a specific file name for the merged project report 169 May 2012 Chapter 8 Additional Tools fl Merge Projects a Project Edit Application Slel GO MB_K2774_DEC_Smarkers SGF Report RHH F7 K27774_D70904 3_G01 fsa gt ir B K27774_D70905 2_H01 fsa B K27774_D70905 2_A02 sa EE pr Load Files B K27774 D70907 2 BO2 fsa H 0 a B K27774 D70908 2 CO2 fsa 2 0 0 0 Le Activates a dialog box to open the project files to allow o K27774_D70908 0 0 merging of allele reports Icons and Functions 131 133 13 Renaming Tool This icon in the upper tool bar activates a renaming tool Use this function to transiently rename files that cannot be grouped by any of the methods in step 5 above Report Grouping Activates the report grouping to allow combining allele reports of the same samples that were produced from different multiplexes Allele Report Settings Allows selection of the appropriate allele report settings for each analysis type Save Save the combined allele report as a txt file for use in applications such as Cluster Analysis or Relationship Testing Func
181. ent background colors are used to make a distinctive look Left click to pop up a Save As dialog box and save this table to a txt file with specified name Using Allele Bin Report from Merged Projects A common challenge with cluster analysis in animal and plant populations is obtaining enough informative markers in one multiplex Overlapping marker ranges and or incompatible chemistry make it necessary to run the same samples multiple times with different sets of marker primers Cluster analysis using the combined results of two or more multiplexes or kits d Clustering Analysis 1 Select Load File gt Add Files and navigate to the TETSJES saved merged bin report Please see Merge Projects Chapter 8 2 Select Group Edit Enter values for Group Identification and Control Identification Select Match 3 OK 4 Select Finish Edit to display the Cluster Analysis of the combined project results Sa WW File Name Group Editor gt gt w File Name List ll Cluster Edit Form Report Q gt C Ueere SoltGer Report Style Bin FES0G4 She Fro Blue FEIN Blue FEI064 Blue FESOBA DB hue Fa Blue FE 13 1H 1 K27774_D7L0 0 127774 DNO 3 K27774_D7C0 4 K27774 DXO 5 K27774 DXO G 127774 DNO 7 127774 DNO 8 K27774_D7C0 3 K27774 DXO fio K27774_D7C0 m K27774 DNO 12 K274 DXO 13 127774 DXO K27774_D7C0 0000000000005 133 0 0000000000000 15 1 1 1 1 1 1 1 1 1
182. ental sample rows a 1 0 1 appear indicating the Gain Equivalent or Loss respectively of a peak in the Experimental trace as compared to the Reference trace Click the Report Settings icon and select deselect to display Gain Equivalent or Loss values Double click values in the Report Table to link to the associated Trace Overlay position Click the Save Report icon to save the Report Table as an Excel or tab delimited Text file Procedure 1 Import MSI raw data files and filter with Run Wizard Fragment Animal Analysis settings See Chapter 2 General Procedure Create a Panel for the data with the Panel Editor tool See Chapter 5 Panel Editor Apply the Panel Select Applications MSI Analysis The MSI Analysis Settings box appears Upload a tab delimited text file that identifies Reference samples first column and Experimental samples second column to the Group File field NOTE Use the File Name Group Tool to create a group file See Chapter 8 Additional Tools 7 Adjust MSI Analysis Settings as desired 8 Click OK 9 The MSI Analysis window appears 10 Click the Print icon to print the MSI Clinical Report Se eS Icons and Functions Load Group Information Choose a tab delimited Text txt file which contains information on how to group individual sample files For example pair Patient A s tumor sample to Patient A s normal sample by placing the normal sample filename in the first column and the
183. enter bar to select a peak Right click anywhere in the Electropherogram or Peak Table to see additional menu options Insert Allele a Right click at the place in the electropherogram where you would like to add an allele and select Insert Allele The basepair size or bin name will be Marker Penta D applied in the Allele Label and the peak specifications will be calculated and displayed in the Peak Table mie E Delete Undelete Allele Size 1817 Right click at the vertical grey bar indicating the center of the called peak or E Confirm the Allele the peak cell in the Peak Table and select Delete Hot Key DEL To call the allele again right click the peak and select Undelete Hot Key a pa SHIFT DEL EY ed Confirm Unconfirm Allele If a peak is given a low quality score it will receive a Check yellow or Undetermined red Quality ranking To give the peak a Pass green Quality ranking right click the peak center bar and select Confirm Hot Key CTRL M The peak will be marked Pass green and receive a Confirmed comment in the Peak Table To un confirm the allele select Unconfirm from the right click menu Hot Key CTRL ALT M Confirm Unconfirm All Confirm All and Unconfirm All options perform the same actions as the Confirm Unconfirm allele except that the Quality ranking for all peaks in that dye color for that sample will be affected Edit Allele Right click an allele in the Electropherogram or Peak Table
184. epair If selected the number will be rounded up to a whole number value Iw Auto Label Cancel Edit Group Allele LX To associate Bins with a different Marker hold down CTRL key and left click and drag across peaks at the edge of a Marker A light blue hashed box will appear Right click in the hashed box and select Change Marker The Edit Group Allele box will appear Select New Marker and a pre defined name will appear Use this Marker label or create a new name and click OK The highlighted Bins are now incorporated into the newly created Marker Cancel Delete Marker Right click the Marker bar and select Delete Marker OR right click the Marker name in the Panel List and select Delete Hot Key DEL Change Marker C Use Existing D135 42 H D135742 1 New Marker Bin Options Create Bin To create a Bin position right click in the electropherogram at the exact position to place the new Bin Select Insert Allele The Allele Editor box will appear Adjust parameters and click OK Allele Enter a name for the Bin All peaks that appear within the Bin will display this value in the Allele Label in the Main Analysis window Size Indicates the basepair position of the center of the Bin 58 May 2012 Chapter 5 Panel Editor Boundary Indicates the range of the Bin on either side Left and Right of the use tarer BER Bin center o E OK Marker Select w
185. eport Allows the user to Save Project or Print Report Selecting Print Report will launch the Print Report Settings box See Chapter 6 Reports and Printing Show Last Event Opens the last active Data Process action Help Help Menu User Management Help Launches a searchable version of this manual User Management Allows an administrator to assign access rights to different users Also used to set up the password protection feature Information from User Management is used to populate the header of some of the specialized application reports See Chapter 9 User Management About About About GeneMarker LS Displays information specific to the version of GeneMarker running on the computer Also contains links to email Technical Support and the SoftGenetics website SoftGenetics GeneMarker Version 1 60 Copyright SoftGenetics LLC All Rights Reserved Time 7 16 2007 ty tech _support softgenetics com www SoftGenetics com computer program is protected by copyright law and international treaties Unauthorized reproduction or distribution of this program or any portion of it may result in severe civil and criminal penalties and will be prosecuted to the maximum extent possible under the law Main Toolbar Icons DK Open Data Opens data input dialog box to begin analysis Run Project Opens Run Wizard for processing the data y e 80 TS IV Show Hide Toggles Displays or
186. ermine whether or not to analyze and report these results ARMS Comparative Analysis ARMS Comparative Print Settings Group File Groups jis CFEU2 fsa files June_matched_samples andB txt Gel All Groups C Selected Groups Iw PolyT Analysis Show Wildtype Size IT STR Check STR Check Cancel a i amp Ok x Cancel s gt z gt No Mutation_________ Wildtypel Mutant ARMS Comparative Analysis Report SoftGenetics R347H to Sample Trap 001 A07 1 B fsa frag 001 A01 1 A fsa 2 JR347P 40010 l 3 12789 5G gt A Mo Software GeneMarker V1 95 Analysis Type ARMS Comparative Analysis 4 3120 1G gt A WO z 711 1G gt T__ Project United Panet CF EU2 POP eaw a oo Exp Time 04 23 2010 18 01 49 gt 04 23 2010 18 32 46 Report Time 09 03 2010 14 35 43 9 13849 10kbC gt T Rn STRI Fei Blue Mutant Green Wildtypg mg T 150 200 250 300 350 400 450 500 Fs08del CFTRdele2 3 STRI Pakt Blue Mutant Green Wikitypq SR 1 20 50 d 250 300 350 400 450 500 POLA AL A AN OI Wl unt AU A4SSE a R1162X M 50 R1158X Ee ode Comments a ii aaa Pay 510 Authorization DER May 2012 Chapter 7 Special Applications The report table contains a 1 for any allele where a peak is detected and a 0 if no peak is detected This report is an example of on individual heterozygous for Cystic Fibrosis alleles F508del and CFTRdele2_3 with heterozygosity in the polyT re
187. error random cleavage and instrument spikes can easily be identified and removed Spikes often show multiple colors at the same location The peaks caused by these random processes seldom occur in a complementary size 157 May 2012 Chapter 7 Special Applications Reports Report ei v Show Low Quality Samples S N Rati Show No Peak 5 Save Report Choose to save the Report Table as ee no rek nampes SE en Hide Repeated Sample Names 101 4 2672 53992 an Excel file xls or a tab delimited Text file AT txt 73 platel_1111_AlOfs4 Green 2797 770 3 536 10183 74_platel_1111_B10 fsgHlo Pes 84 75_platel_1111_C10 fsghlo Pes 76 platet 1111 D10 fsdNo Pes 77 platet 1111 E10 fsaNo Pee 7 8 platet 1111 F10 fsafvo Pe 87 Report Options The report table can be Report E configured to report all results positive and Show Low Quality Samples S N Ratio Comm negative or to display samples containing only 2 Je Show No Peak Samples 478 5229 1220 3 Hide Repeated Sample Names 128 2814 71947 415 positive results 4 06 platel_1111_FO1 fsq Blue 948 9 2201 42685 579 5 16_platel_1111_HO2fsq Blue 6543 3957 614 5529 15 48 6 16 platet 1111 HG fi Green 653 2 332 2204 11 74 7 18 plate1_1111_B03 fsd Blue 1130 937 0 410 1975 14 50 8 18 plate1_1111_B03 fsd Blue 4662 5038 2077 13689 48 74 3 18 platet 1111 BO3 fsq Green 462 1 585 5433 1979 10 27 platel 1111_C04 fsd Blue 84 6
188. erview GeneMarker TILLING analysis software has been optimized for detecting SNPs generated by the TILLING technique By using the internal size standard to align each capillary and generating a reference trace from all of the samples in the run the reference can be subtracted from each individual sample trace yielding a plot highlighting the SNPs Additionally a table shows what the expected size of the complementary fragment should be to determine if each peak is a true variation The TILLING analysis window displays a synthetic gel image list of samples sample electropherogram and a report table The information contained in the report is interlinked to the sample traces Double clicking on the cell in the report highlights the cell corresponding sample ID in the sample list and locus in the electropherogram TILLING Analysis Reference Sample Subtracted sample mutation Traces Sample List Synthetic Gel Image Peak Table Mutation Report 2 Tilling Analysis S RM La Sampla 4 01 pl Gel Image 02 ple E 03 ple E 9 m m E 57 D p 937 3 1127 476 16730 493 e E 383 4 6 479 5157 11 45 07 pl 9483 10 50968 502 ae 6543 357 5510 14 89 10 pl 6532 8 323 2199 11 63 11 pl 130 9370 401 198 1418 ae eegen GA 466 2 5838 13489 5292 14 pl 462 1 9 584 5407 18 82 15 pl 845 9655 501 6351 449 een 968 8 l 671 43933 23 72 18 pl 1896 8604 869 4830 3072 19 pl i med 186 0 f 535 15218
189. es SoftGenetics GeneMarker version number EE 8 Click Next in the Select Program Manager Group window to accept the C Install License Server Manager Install GeneMarker and License Server Manager default Program Manager Group NOTE Changing the Program Manager Group default may affect program operability It is recommended to accept the default The License Server Manager must be installed on the Server It is not to be 9 Click Next in the Start Installation window to install GeneMarker E e 10 Click Finish in the Installation Complete window Important The license Server Manager is needed for users running this product in Network Configuration 11 The Installation Wizard will close came 12 Eject the SoftGenetics CD 13 Launch GeneMarker by double clicking the GeneMarker desktop xl icon OR open the Start menu and navigate to SoftGenetics If a local license was purchased click Register Now If a network license was purchased click Configure Network Client GeneMarker the version that was just installed gt GeneMarker program 14 The Configure window will appear Click Run Validation to launch Configure Network Client the software No previous network configuration has been detected 15 If the Run Validation button is grayed out this indicates the 35 day trial period has expired T l v Details Cancel 6 May 2012 Chapter 2 General Procedure Local licensing Option GeneMarker v2 00 and abo
190. es against a selected size standard to modify Convert Text to Binary Files and save the size standard for future use or create a customized size standard See Output Trace Data Chapter 4 Fragment Sizing Standards Export Electropherogram Pedigree File Name Match og Me ws Show Last Event Allows the user to automatically add additional files to a previously created pedigree tree A smp file is exported See Chapter 7 Special Applications Pedigree Chart Merge Project Combines the individual file results from different projects multiplexes into one complete genotype report that may be saved to print out separately or imported into Cluster Analysis or Relationship Testing Macromolecules Aligns the results for a project without a size standard Uses fragments that are consistent from one lane to another to correct for migration differences from one capillary to another File Conversion To combine data for X and Y axis from from unique instruments from a single file XY file or two files X file Y_file File Name Group Tool Used specifically with the MSI and LOH applications the Filename Group Tool allows users to define how reference samples and tumor samples should be grouped or paired A Text txt file is exported See Chapter 8 Additional Tools Output Trace Data Provides the option to output the raw or sized trace data as a TXT or SCF file Select the samples to include dye colors data type and the
191. es compared to a reference trace The reference and sample traces are overlaid for easy LOH identification MS MLPA Analysis Detects methylation sites within promoter regions and for genomic imprinting applications 38 May 2012 Chapter 3 Main Analysis Overview TILLING Detects SNPs generated by the TILLING technique By using the internal size standard to align each capillary and generating a reference trace from all of the samples in the run the reference can be subtracted from each individual sample trace yielding a plot highlighting the SNPs Additionally a table shows what the expected size of the complementary fragment should be to determine if each peak is a true variation Haplotype Analysis The program uses the allele calls of children and parents to assign phase of the alleles with a first order approximation ARMS Comparitive Analysis Allows comparison of pairs of files for a given individual includes electropherograms trace comparison and allele report in a patient report format Tools Menu IL Panel Editor The Tools menu contains the Panel and Size Editors in addition to other miscellaneous Size Template Editor modules Merge Project Macromolecules Panel Editor File Conversion Provides a variety of tools to adjust edit and create control Panels See Chapter 5 Project Comparison Panel Editor Pedigree File Name Match File Name Group Tool Size Template Editor Luminex MLPA Analysis Allows the comparison of sample fil
192. etween the reference and the sample trace Sample probes are represented by blue bars and control probes are black bars Allele editing options in the Trace Overlay frame are similar to those in the Main Analysis window Electropherogram See Chapter 3 Main Analysis Overview Ratio Plot The Ratio Plot displays in graphical form the ratio of normalized peak intensities between the reference and the sample trace Each point represents a sample peak or probe Enter the number of Ratio Plots to display simultaneously in the MS MLPA Display Settings box Green Peak is within the specified ratio limits Blue Peak is a control probe Red Peak is outside the specified ratio limits Yellow Current selected probe Double click a point in the Ratio Plot to view the peak in the Electropherogram and the ratio value in the Report Table Right click in the Ratio Plot and select Adjust by Control Probes to fit the data by only the control probes blue points in the sample Right click and select Reset to restore population fit calculation all control and sample probes in the sample are used to fit the data Report Table The Report Table lists in columns the probe or exon name according to the panel selected and the ratio for each sample probe as compared to the reference Double click a cell in the Report Table to display the corresponding peak in the Electropherogram and the plot point in the Ratio Plot See the Reports and Printing section below fo
193. file to a specified directory on a local or network computer 47 May 2012 Chapter 4 Fragment Sizing Standards Exit Closes the Size Standard Editor tool Be sure to save changes to the Size Standard before exiting BestMatch Match Selected When selected the Data Process box appears Each sample in the dataset is compared to the currently highlighted Size Standard in the Size Standard List The green triangle indicators are adjusted to give the best match possible Match All When selected the Data Process box appears All samples in the dataset are compared to each Size Standard The Match Scores for each sample are averaged together The Size Standard with the highest average Match Score for the dataset is chosen as the Best Match Help The Help menu contains a link to Hot Keys in Size Template Editor Click Hot Keys and the Size Editor Action Help box appears Toolbar Icons Size Template Editor Found in the Run Wizard Template Selection box or in the Tools menu Create New Size Standard Opens the Input Dialog box with a field to enter a new Size Standard name Allows for the creation of a new Size Standard Save Changes Saves modifications made to the Size Standard to the SoftGenetics GeneMarker Size Standard directory Delete Deletes the Size Standard that is currently highlighted in the Size Standard List NOTE This action is irreversible Show Dye Allows the user to select a single dye color to view in the Sampl
194. file uploaded in the LOH Analysis Settings box A sample is marked as the Reference because it contains a Control Identifier as set in the File Name Group tool See Chapter 8 Additional Tools Reference Control samples will appear in the first column of the File Name Group Text file Expand folders in the Group File Tree to view the Reference sample and Experimental sample in the group Double click the Reference sample to view just the Reference sample electropherogram Double click the Experimental sample to view the Experimental sample electropherogram overlaid on the Reference sample electropherogram and associated Gain Loss Histogram Use the Up Down Arrow keys to navigate through samples Hit the Enter key to open Group folders Right click an Experimental sample in the Group File Tree and select Set as Reference to mark the sample as the Group s Reference sample 150 May 2012 Chapter 7 Special Applications Trace Overlay The Trace Overlay displays the selected Experimental sample s trace superimposed over the selected Group s Reference trace The Experimental sample trace line color will appear in the Marker s associated dye color The Reference trace line color is displayed in light red The Reference trace is by default drawn directly behind the Experimental sample trace Click the LOH Display Settings icon to adjust the Reference trace offset as compared to the Experimental sample trace Additionally the RFU intensity or peak
195. for the file under analysis 111 May 2012 Relationship Testing Settings Chapter 7 Special Applications Samples e All Samples C Selected Samples s Default Allele Frequency Panel Name dentifiler Population US African American v vw Mutation Mutation Rate 44BB 2000 Max Mutation Markers Po Y STR Step Difference One Extra Step STR Mutation Ratio 0 100000 Paternal and Maternal Mutation Difference Prior Probability 0 0500 ma Genetic Analysis Settings allows setting the above options at the same time Tolerate mutations or genotyping error in wild life genotypes by setting the maximum mutation markers When this is selected the program will substitute the mutation rate mean PE for the kinship equation at that locus The resulting probability and likelihood ratio will be much lower but not zero as they would be if the mutation tolerance is de selected Search Report Display If gender information is present in the genotype select Gender determined to display Father Son and Mother Daughter potential relationships If gender information is not present as in the case of monoecious plants and many invertebrates de select Gender Determined All files with a potential parent child relationship will be displayed Limit the number of positive files retrieved by minimum likelihood ratio value or maximum file number Advanced settings should be used to select the relationship levels reported fo
196. frag DOT HO las D ET MT E 1 E IE Eu 1 pe 0 mee 150 200 250 300 350 400 456 mm e e me i m 5 Primei B ra 0H ED fa Elf r Pl hs ER DOT La frag DDE OM fr frag DOT BD tra ha O 507 fen B fag 009 HOR fra f frag DO GO las frag 011 Fida frag 012 EI fra B frag 013 DOG laa irag DA OUA Fea Mdddddddcdaded a BE i ee A AA Size Standard List The Size Standard List contains all pre defined Size Standards and any custom made Size Standards Single left click a Size Standard in the list to select it The Expected Size Standard trace and Size Table will appear on the right Additional Options To see additional options for each Size Standard right click the Size Standard name and the right click menu will appear with the following options 44 May 2012 Chapter 4 Fragment Sizing Standards Delete Size Standard Select Delete to delete the Size Standard from the Size Standard List and from the SoftGenetics GeneMarker Size Standard directory NOTE This action is irreversible Export Size Standard Opens the Save As window Choose a directory folder and click Save The Size Standard will be copied to the selected directory and will also remain in the Size Standard List and SoftGenetics GeneMarker Size Standard directory The Size Standard will be exported as an XML file which can be opened with Internet Explorer Microsoft Excel or Notepad Reload Size Standard Click Reload to undo editing changes to the Size Standard The
197. free form comments regarding the analysis These comments are saved with the project file and can be displayed in the Print Report Applications Menu The Applications menu contains individual modules for specific data and analysis types qa 8 These modules present advanced features and reporting options necessary for the Pte particular application See Chapter 7 Special Applications for more information on Export conis Overlay View each analysis application EA SNPlex SNaPshot Pedigree MSI Analysis Display and check genotype calls using a pedigree chart Data must be run with the SE correct size standard and Panel prior to using the Pedigree function Relationship Testing LOH Analysis MLPA Analysis supi Analyze data from Multiplex Ligation dependent Probe Amplification MLPA Offers eee SE two normalization analysis methods and includes a customizable patient report for snc e i clinical use Trace Comparison Used with AFLP data the Trace Comparison module was designed to identify length polymorphisms between closely related species See Chapter 7 Special Applications AFLP Export CODIS Developed for forensic scientists analyzing short tandem repeat fragment data Exports the CMF 3 0 xml and CMF 1 0 dat files for upload into the FBI s CODIS database See Chapter 7 Special Applications HID Overlay View Allows the user to graphically display any combination of samples and dye colors This feature includes a 2
198. ftware program and you have first requested SoftGenetics LLC to provide the information necessary to achieve such operability and SoftGenetics LLC has not made such information available SoftGenetics LLC has the right to impose reasonable conditions and to request a reasonable fee before providing such information Any information supplied by SoftGenetics LLC or obtained by you as permitted hereunder may only be used by you for the purpose described herein and may not be disclosed to any third party or used to create any software which is substantially similar to the expression of the Software Requests for information should be directed to SoftGenetics LLC Trademarks shall be used in accordance with accepted trademark practice including identification of trademarks owners names Trademarks can only be used to identify printed output produced by the Software and such use of any trademark does not give you any rights of ownership in that trademark Except as expressly stated above this Agreement does not grant you any intellectual property rights in the Software 4 Transfer You may not rent lease sublicense or authorize all or any portion of the Software to be copied onto another users computer except as may be expressly permitted herein You may however transfer all your rights to Use the Software to another person or legal entity provided that a you also transfer this Agreement the Software and all other software or hardware bundled or pr
199. gion in the 7 and 9 repeats ARMS Comparative Report Options Reports and Printing Output Status Peaks 1 r Report Contents Iw Loss 7 Status 1 0 1 Y Gain Launches the Report table options dialog box Iw Abide By Panel ces Save Icon Save the Report table as an xls or txt file ARMS Comparative Print Settings Groups All Groups C Selected Groups a d Printer Icon E Provides Print Settings if the polyT Ned Z PolyT Analysis 7 Show Wildtype Size information is not required it may be deselected 7 STR Check amp Ok X Cancel ARMS Comparative Analysis Report SoftGenetics EEE EE ARM S Comparative Analysis Report SoftGenetics Ke on ke pe cos Mutant R347H_ Cd Sam o o Sample frag_001_A07_1_B fsa frag_001_A01_1_A fsa 2 RaP M fo Sample trag 001 A07 1 Bfsaffrag 001 A01 1 Af5a EE E 001 A07 001 A01 1 Ee 2789564 ER ETE Hi Software GeneMarker V 1 95 Analysis Type ARMS Comparative Analysis At prica Mio 5 1711 16 T pm fo Panet CF EU POP7 5 1711 46 gt 1 Ro 6 Tr33aw hm Do SE 0 Others Chemistry Lot Report Time 0903 2010 15 09 44 10 stri Pot Blue Mutant Green Wildtypd e 300 150 200 250 350 400 Seet Green Widypd ER 250 300 350 400 46 3272 26A gt G ao 621 1G T O 48 A455 O 49 R1162X Oo 50 R1158X do The same patient report the left one has all fields selected an
200. gram and the Bins appear within the electropherogram as brackets at the top and bottom The center of the Bin is indicated by the vertical grey bar in the electropherogram only in Panel Editor The Overlay Trace view can be changed by clicking the Trace Mode icon in the toolbar Other options include Max amp Average and Gel Image Navigation in the Overlay Trace frame is similar to the navigation options in the Main Analysis window See Chapter 3 Main Analysis Overview Marker Options Create Marker Hold down CTRL key and left click and drag across peaks in the Overlay View A light blue hashed box will appear Right click in the hashed box and select Create Marker The Create Marker box appears Adjust parameters and click OK Marker Name Edit the Marker Name field to change how the Marker will be mg RW labeled in the Panel Boundary The basepair range of the Marker is defined by the range of the Er s light blue hashed box and is therefore inactive in the Create Marker box To GE Res ra edit the Boundary see Edit Marker below ee Nucleotide Repeat EE a Auto Detect Based on the peaks present in the Overlay View GeneMarker y will attempt to detect the number of nucleotides in each repeat unit of the M Fixed Bin Width 05 alleles and place Bins at the appropriate interval Y Auto Label Set by Manual Select this option if the number of nucleotides in the allele repeat unit is known and GeneMarker will place Bins at
201. h window where the user can select to open previously saved SoftGenetics GeneMarker project files sgf sfp Re Open Project Saves the last four projects that were opened by GeneMarker and allows the user to launch any one of those four projects directly Save Project Saves a SoftGenetics GeneMarker project sgf sfp to a specified directory Raw data files and analyzed data files with edits are saved within a project file Starting with GeneMarker v 1 96 the negative Y axis peaks of analyzed data are also saved in project files Pull down peaks may result from changes in optical alignment or polymer of the genetic analyzer Close All Closes a project without exiting the program NOTE It is recommended to select Close All before exiting the program Exit Closes the GeneMarker program View Menu The View menu contains options for how the data is displayed in the Main de Show Navigator Analysis window E Show Gel Image KE Show Report Show Navigator Gel Image Report Toggles the Sample File Tree Synthetic Gel Image or Report Table frames open and closed in the Main Analysis window Preference Preference Activates the three tab Preferences box Start up Settings The Start up Settings tab effective only at start up allows you to select the Run Method and General Settings Run Method ES Classic Appropriate for experienced users The user will move through the program data input settings and display optio
202. he Ratio Plot The Fit Line represents the slope of the peak heights for all the samples in the dataset at the selected Marker Iw Show Fit Line Print Report Launches the Trisomy Print Settings box See Reports and Printing section below for more information Show Bins Click the Show Bins icon to display Panel Bins in the Electropherogram The Show Bins option selected when the Print icon is clicked is the setting that will be applied to the Trisomy Print Report 144 May 2012 Chapter 7 Special Applications What to Expect GeneMarker has been designed to accurately detect aneuploidy using short tandem repeat markers derived from PCR DNA fragments Trisomy individuals will either show three fragments of equal intensity or two fragments at a 2 1 or 1 2 ratio Ratio 1 2 Ratio 2 1 0 155 D21511 225 230 235 240 245 250 255 260 265 Ratio 1 2 amp 2 1 Trisomy When DNA fragments are run through a PCR reaction the smaller fragments are preferentially amplified Electrophoresis injection also holds a bias toward smaller fragments Subsequently the smaller fragments peak intensity in an electropherogram will be higher than the larger fragments in the sample This is called preferential amplification and it is important in trisomy detection especially allele ratios that are 1 2 or 2 1 The 1 2 allele ratio occurs when the individual has 1 allele in the first position and 2 alleles in the
203. he peak to be accepted B Jor Jes AFLP Panel Creation After the data has been sized and filters have been applied go to Tools Panel Editor to create a Panel for the dataset For explanation of specific functions in Panel Editor see Chapter 5 Panel Editor 1 In Panel Editor select File Create New Panel OR click the Create New Panel icon Create New Panel z 2 The Create New Panel box appears Name AFLP Parel 3 Enter a name for the Panel and be sure AFLP is selected in the Type field EE 4 Select Automatically Create and Use All Samples e 5 Click OK htm Cea 6 Panel Editor will place one Marker in each dye color where peaks are detected GE 7 If only one dye color contains peaks delete Markers created in other dye colors 76 May 2012 Chapter 7 Special Applications 8 Click the Trace Mode icon until the Gel Image view eg appears in the Trace Overlay frame DB 9 Hold down SHIFT key and left click and drag Bin 2 edges to adjust the Bin range or drag the entire Bin to a E AFLP Paral da x E H 4 P Help ES al E new position E 10 Right click and select Add Delete Allele to add and gt ine remove Bins from the Panel 11 After editing select File Save Changes Hot Key CTRL S OR click Save Changes icon 12 Exit the Panel Editor 13 Click the Run Process icon in the Main Analysis Sez Left fange Fight Rang 1400 06 1411 14 1426 04 1443 as Dr
204. hich Marker to associate the Bin with The Markers to the see E Cancel right and left of the Bin position will be displayed as well as the option to Bounday Lett 05 mig 05 create a new Marker for the Bin All Bins must be associated with a Marker Marker ge Comments Free form text field to associate a comment with the Bin C New NONAME Control Gene Select Control Gene if the peaks that fall within the Bin are of WER Bes apes consistent height Peaks that appear within the Control Gene designated Bins will be used to normalize the data If a Bin is marked as a Control Gene a 1 will appear in the Control column of the Panel Table Additionally right click the center Bin vertical grey bar in the Overlay Trace and select Set as Control Non Control to enable disable Control Gene NOTE Control Gene is for use with MLPA analysis See Chapter 7 Special Applications MLPA Edit Bin Right click the vertical grey bar in the center of the Bin in the Overlay Trace Select Edit Allele and the Allele Editor box appears Adjust parameters and click OK See Create Bin section above for explanation of Allele Editor options To move a bin hold down SHIFT key and left click and drag the vertical grey bar in the center of the Bin to the right or left Let go of the SHIFT key and mouse button and the Bin will remain in place To edit the range of a Bin in the Overlay View click the Trace Mode icon to view the Gel Image Hold down
205. hides the Sample File Tree Synthetic Gel Image and Report Table frames respectively Print Report H Provides the user display options for the Print Report 40 May 2012 Chapter 3 Main Analysis Overview Show Color Allows the user to select all colors to view hide all colors or choose a single dye layer Choose a single dye by single left mouse clicking on the icon Zoom In Use the icon to zoom in on the image or hold down the left mouse button and draw a box from the top left corner to bottom right corner around the area you wish to zoom in D Zoom Out Use the icon to zoom out on the image or hold down the left mouse button and draw a box from the bottom right corner to top left corner 2 Set Axis The default setting automatically sets the Y axis according to the maximum peak intensity of the samples Two other options are available auto fit the Y axis using peak intensities of the alleles or the user can select the ranges for the X and Y axis E Browse by All Colors Displays a comparative view of sample electropherograms by dye color Individual samples can be selected from the drop down menu Gi Allele Call Icons These icons are only available after the raw data has been processed and the Sample File Tree Allele Call folder is selected Size Calibration Displays calibration charts for linearity of lane analysis E Show Chart Table Toggles display to show only the Peak Table the Peak Table and
206. hreshold 10 00 A e Quantification by Peak Height Minimum Lane Score Threshold 11 00 11 Click OK 12 The MLPA Analysis window appears Deal What to Expect Luminex Data Normalization Due to differences in laboratory requirements GeneMarker has the option to normalize MLPA derived data in two different ways by an internal control probe method or by a population method The user is able to specify the normalization method by selecting a control sample in the Luminex MLPA Data window or in the MLPA Analysis window s Control Sample drop down menu select Synthetic Control Sample to use population normalization Luminex Data Analysis After Control Genes and the Background Sample are selected in the Luminex MLPA Data window the MLPA Analysis window appears See the MLPA Analysis section above for more information about icons and functions A feature unique to the Luminex MLPA Analysis window is that samples that contain probes that do not meet the minimum bead threshold or Suspect Count are identified Suspect probes are labeled in red in the Overlay Trace and their data points appear fuchsia in the Ratio Plot MLPA data analysis proceeds as outlined in the MLPA Analysis section above 94 May 2012 Chapter 7 Special Applications Methylation Specific MLPA Analysis MS MLPA Detection of methylated sites in genomic DNA is important for two reasons First methylated DNA l
207. ht click menu and choose from the fly out menu to sort Ascending or Descending If Ascending is chosen then low quality peaks will be sorted to the top of the table If Descending is chosen then the lower quality peaks will be placed at the bottom of the table This option is only available with Marker Table and Allele Count Report Styles Sort by Column Select Sort by Column from the right click menu and choose from the fly out menu to sort Ascending or Descending If Ascending is chosen then lesser values will be sorted to the top of the table and greater values to the bottom the table and vice versa if Descending is chosen This option is available with all Report Styles Editing Peaks To edit peaks first left single or double click the cell in the Report Table then right click to see menu options or use Hot Keys Delete Peaks Right click the peak cell in the Report Table and select Delete Peaks Hot Key DEL The deleted peak will be removed from the Report Table Confirm Peaks If a peak is given a low quality score it will receive a Check yellow or Undetermined red Quality ranking To give the peak a Pass green Quality ranking right click the peak cell and select Confirm Peaks Hot Key CTRL M The peak will be marked Pass green Peak Information Hold down CTRL key and click the peak cell of interest The Allele Peak Info box will appear containing information such as Sample Dye Size Marker Allele Score and Comments
208. ic analyzer is used a separate panel for each genetic analyzer should be saved with signal information for example panelname_ABI3100 panelname_ABI3130 panelname_CEQ8000 Align all of the bins within a marker 1 Hold down the shift key 2 At the same time place the mouse over the gray marker name bar at the top of the electropherogram 3 The marker rectangle will be outlined Hud in red and the panel name will be in red font when the adjust feature is active 4 Drag the marker to align the bins with the peaks of the allelic ladder 5 Save the panel with signal information the turquoise save icon oa i ge to enable the major panel adjust a feature to work in future projects Sample TE r r r g r Ar g r r r di ols a x g rr bn PO02_B1_BRCA L I POGA_DMD_Parel 34 D EEETLECLCLLEEE CC fetes 53535 228 DINA D lo la aa je e pp pp R Or ebe f A yall fool Sel stb bp eee a eS Sg io 9 i i SRRRBARA CET Align an individual bin Select the gray vertical bar of the bin with the mouse the bar will turn blue Hold down the shift key and click on the gray vertical bar for the bin The vertical bar will be outlined in red and the panel name will be in red font Use the mouse to drag the gray vertical bar to the center of the peak Ep IO ES PO Sanges an 2 a a KL L a a a E PUD r
209. ies of the alleles or the user can select the ranges for the X and Y axis Browse by All Colors Displays a comparative view of sample electropherograms by dye color Individual samples can be selected from the drop down menu Parent nad Parece hid amp rile Ger Fase s JO 116 re Kinship analysis Save tables for export in txt files Or right click on the table to copy paste into an existing document or report Importing Species Specific Allele Frequency and Mutation Rates It is very easy to import region specific or species specific values for use in the kinship calculations Population specific allele frequency and mutation rate tables must follow the format of preloaded files and be saved as a txt tab delimited file Export one of the preloaded allele frequency tables and use it as a template to form an allele frequency table for the desired population Import the new tables by opening the Tools gt Allele Frequency gt Open folder icon gt Select file s gt Save 113 May 2012 Chapter 7 Special Applications Database Search Locate Duplicate Samples and Nearest Relatives Overview The database function is capable of closed system searches for same sample match identification of nearest relatives and calculating kinship statistics The Save to DataBase function allows easy updates of the relationship testing database The Data base searching tool is ideal for applications such as e Population diversity
210. in the far atid left column and peaks are numbered in columns at the top of the table Allele C Marker Table Fragment List is the default Report Style when SNPlex Analysis Type is selected OS Peak Table C Allele Count Features Naming Options C Sample Name e File Name Report each sample by sample name or by file name in the allele report pa Show Only Uncertain Alleles Iw Show Rejected Low Score Alleles Iw Hide Extra Sample Names Show Only Uncertain Alleles a SS When selected displays only the peaks with Quality ranks of Check yellow and Undetermined red Show Rejected Low Score Alleles When selected the peaks with peak scores below the Run Wizard po Er En Ee hm ee Additional Settings Allele Evaluation Peak Score Reject setting will be Oo E Jose noe scr FEE displayed in the table a besser We L Oo Kaze o BR e fosa Bos ser Oo mx Hide Extra Sample Names FAT ET When data is displayed in Vertical Orientation the sample names are Q ET repeated for each row of data that the sample is associated with If Hide fase Mi E ga E Extra Sample Names is selected then the sample name will only appear X 182 mf ise once in the first of the rows it is associated with Saree Qo w Qo wx he fers Bosser re gu wes Gase p eos Oo mz 18 Jaco DOG SCF T 06 Qo Quo Gase J Be Oo Marker Table Fragment Allele Report Settings The Marker Table Fragment Report Style displays the Quality rank
211. increase or decrease the zoom aspect of the Print Preview page I I E Save Project After a dataset is analyzed and edited the project can be saved as a SoftGenetics GeneMarker Project SGF Project files contain the raw unprocessed data files the sample files after processing the process parameters and all edits The project file does not contain any custom or modified Panels or Size Standards To export a custom Panel see Chapter 5 Panel Editor To export a custom Size Standard see Chapter 4 Fragment Sizing Standards NOTE Previous versions of GeneMarker saved projects in SFP format This format can be opened with current GeneMarker versions To save a project go to File Save Project in the Main Analysis window The Save Project box will open Select a directory and enter a project name Click Save To re open the project go to File Open Project in the Main Analysis window The last folder accessed by GeneMarker will appear Navigate to the directory containing the SGF or SFP file and click Open Additionally the last four projects that were opened by GeneMarker appear when the File Reopen Project fly out menu is selected Click a project from the fly out menu and it will be uploaded to GeneMarker 74 May 2012 Chapter 7 Special Applications Chapter 7 Special Applications Chapter 7 Special Applications AFLP T RFLP MLPA Human Identity Pedigree Chart Kinship Analysis Database Search Automa
212. information is displayed above the Signature box and contains sample information that includes number of control sample probes mean peak ratio for control sample probes and the standard deviation of the probes Signature Box Date and initial space for report reviewers MLPA Analysis Summary Page 3 Means 3 Duplication Deletion Report Table A list of all probes in the samples fragment basepair size and peak ratio information Peak ratio cells which meet the duplication deletion threshold criteria are highlighted grey Summary Report When Print Summary is selected in the MLPA Print Settings box additional print report sheets appear after all the sample sheets The Summary Report lists all the probes in the dataset and reports peak ratio information in sample columns Peak ratio cells which meet the insertion deletion threshold criteria are highlighted erey MLPA Clinical Report 90 May 2012 Chapter 7 Special Applications Final MLPA Report Probe Name Bn Size POS4_C12 06 m pas 903 por 2 ony me for ss er Panel P034 OMD Panel Classification Loss lt 075 lt Equvalent lt 130 lt Gan e as 4858 oe 7 xa28 3483 10971 P034_C12_06 fsa 8 fexor 1350 10954 9 fex02 1683 0919 P034 DMD 10 ex03 100 150 200 250 300 350 400
213. ing of non linear data To create the size standard for this method 1 2 oF 4 oo B B B B B D B B B D B B 2 HEI Pa NEB_ 0 Pr NEB B B BL NEDES2 PISNEB B B B D B B 5 NEOS4SE PINE NEDSA02 Pa NEB D NE 05540 PRNEB_D NEOMI 2 PISNEB D NE 09417 PAR D A NE 09446 PINE NE 09499 PINE NEOSEO1 PER NEOS621 PISNEB NE09536 PI NEB NE 0939 Pa NeE NE 09461 Pane NE09463 PtNE NEOMES E NE 09469 PIN NE 09473 EA NE 09478 PINE NE09480 PINE NE 09493 PaeNE e NS Ze Go D 0 D D o o o D o DO E F OD ooo d Ly i ei ed Su MEROS NE 09551 PINE B NE0 672 PRNEB NEQS657 PAR NE 09658 PaeNEB_ NID4427 PRES DU NEO3489 PISNEB NE09504 PieNEB NE 09409 PleNEB d Blank PRSNEB DUPI Import raw data files and Select None for Panel and Size Standard in the Run Wizard Select Tools gt Size Template Editor Select File gt New Size Standard Enter the Size information from the run without a size standard in the Size column of the Size standard Enter the Expected size bp in the comments column as seen in the figure below Save the size standard file Select Local Southern method to activate this size calling method in the Run Wizard 0 D a Es E D Coso 1 150 0 1 250 1 100 1 350 1 000 1 450 1 500 1550 1 600 A e NEO9526 PII NEB DUP09 2 Wel HOS eene GAAMVIC GER EE P09 017 0023152 eigenen EES A gt Enabled Comments
214. ing options are selected by default Unselect these options to change the Trace View settings Save Save the Trace View image as a Bitmap bmp file Merge Projects The Merge Projects Application is available directly from the opening GeneMarker Screen or from the Tools drop down menu After each project is analyzed with the appropriate panel the results may be combined using this tool A common challenge for Kinship calculations or cluster analysis in animal and plant populations is obtaining enough informative markers in one multiplex Overlapping marker ranges and or incompatible chemistry make it necessary to run the same samples multiple times with different sets of marker primers Human disease diagnostic research also my require more than one kit multiplex to obtain the full range of allele calls as with Duchenne s Muscular Dystrophy or Cystic Fibrosis By following a naming convention where the individual identifier and the panel identifiers are consistent researchers can combine the individual results from multiple panel analyses into one report providing a more complete genotype for each sample This merged report may be imported into other special applications such as Clustering Analysis or Relationship Testing to improve the robustness of the results by including information from more markers Procedure 1 Save each project for a set of individual files Please see Chapter 2 General Procedure Note Allele editing and reso
215. ings 0 0 5 0 5 A A A A AA Ge A A ALA Cousins 0 0 25 0 75 AA P Pz 0 AG Pr 2 Pipo P Uncle nephew 0 0 5 0 5 a KC O aa F 206 2 Grandparent 0 0 5 0 5 RE ERE KS e Grandchild AA o Di op Ah p 2 PiPz P2 From CC Li and Sachs 1554 All derivations of the Kinship equation 1 can be located at http www softgenetics com images formulasjpg Procedure 110 May 2012 Chapter 7 Special Applications Although allele calls can be edited in the Relationship Testing tool it is easier to begin a relationship test analysis with good clean traces In order to begin with the best sample traces complete size calling Panel alignment and allele editing in the Main Analysis see chapter 2 General Procedure window prior to launching the Relationship Testing tool File types accepted or generated by GeneMarker Relationship Testinge module Pre Ped files SGF TXT BMP JPG PNG CMF The Save to DataBase function allows easy updates of the relationship testing database directly from the genotyping results of GeneMarker Additionaly previously genotyped files can be submitted to the database when saved in a cmf file Select Applications Relationship Testing gt DataBase gt save to database gt load from CMF To launch the Kinship Analysis function select Applications Relationship Testing from the menu bar of the Main Analysis window 1 Open data file or previously saved project 2 Run Wizard to call alleles 3 Select
216. ings to the marker selected in the Recall Allele Call Allele by Marker field 26 May 2012 Chapter 3 Main Analysis Overview Chapter 3 Main Analysis Overview Chapter 3 Main Analysis Overview Main Analysis Window Menu Options Main Toolbar Icons 27 May 2012 Chapter 3 Main Analysis Overview Main Analysis Window The main window of GeneMarker has an easy to use layout The sample files are displayed on the left the Synthetic Gel Image is displayed at the top Electropherograms appear below the gel image and the Report Table is on the right side of the window To resize the frames in the Main Analysis window simply place the cursor over the partitions that separate the Synthetic Gel Image Electropherogram Sample File Tree Report Table The cursor will change to a two headed arrow bisected by two vertical lines Hold down the left mouse button and drag the gray vertical line in the direction you wish To open and close the frames use the Show Hide icons in the main toolbar Main Analysis Window Sample File Tree Report Table Synthetic Gel Image Electropherogram d CGeneharkeg Unitted e LISER A B QRGE De Mee D s UE aj E D Ree Data wi amp Alle Call r Lade d Es af E 62 Bla SCE B 052 HORSCF B 1 BOSSEF E i B 062 sa a d 063 DISSCF 121 Weit 144 D 065 FOSSCF oe i B GSE Drei HSC D 55 ADGSCF D Specs z B 55 HSSC 24 DE ie zelt E 919 BOG SKF B ao cso i 220 DES
217. inted for clinical review The LOH Clinical Report includes patient reference traces ratio plots and a table of peak statistics Report Table Displays peak loss information for individual samples compared to a reference Information for samples with suspected LOH will appear in blue font all others in gray The Overlay Trace window will correspond to which ever peak cell is selected in the Report Table vd LOH Report Settings Report Settings Report Content Allele Name Report Content C Peak Height Select to display Allele Name Peak Height Peak Area Peak Start and End and or LOH Ratio LOH Ratio is chosen by default oe eee Show Only LOH LOH Ratio When selected only the report content information for suspected LOH samples will be displayed Peak Area Show Only LOH Save Report Choose to save the Report Table as an Excel file xls or a tab delimited Text file txt LOH Clinical Report Settings Click the Print icon in the main tool bar to launch the LOH Print Settings box and preview print or save the LOH Analysis Report Groups LOH Print Settings zs Choose All Groups or select specific groups to display in the report SO Print Reference Sample All Groups C Selected Groups I When selected a separate page with just the reference sample will be printed for each group Print Reference Sample Markers Markers e All Markers Choose All Markers or select specific markers
218. ions ccccsseecceeeees 112 May 2012 Index Klinefelter Syndrome siura 142 L Label Dyes amp Peak Numbers rrrnnnnnnrrnnnnnnrrnnnnnnrrnnnnnnnene 73 BS NTT JJ 29 Large Size Algorithm uk 22 ater mobi 44 BMG A a 137 ut e BEE 109 EO EE 6 List Samples Used for Synthetic Control 89 Load Control Sample From a Database 85 87 Losa Deal en 24 Load Group Information aa 132 151 156 Local Region Percent nnooeennnsseeeesseeeessrrrrssrrresse 24 Local SOUTNEIN vr 22 Loci Description Ee 106 ess 178 Fu 150 LOH REN CN 38 151 LOH Analysis Settiggs 151 FOR Clinical Report 153 LOH Clinical Report Settinge 153 LOH Display Settings ooccccooooconconononoss 151 152 LOR PING eee 152 153 FORT PO ss 152 FORRAN 152 FOR RIO Plot 151 LOH Report El EE 151 153 LO Ee 153 Loss Of Heterozygosity ooccccccocccnnccnnccnnncnanonnnonanos 150 172 be NNN 80 93 Luminex Data Analysis occccccoocccncononcnnnonanonoss 94 Luminex Data Normalization oooococooooo 94 Luminex MLPA Analysis ccccooocccnccnnocnnnnnos 39 94 M Macintosh aars Seed 6 Macromolecule Analyse 171 Magic AV EE 40 41 Main Analysis Window 28 Man Toolbar e 40 Major Adjustment of Panel 65 Manual Calbration 52 Mantial Panel Creation usanne 61 Manual Pull p WE 19 Manual Pull up Correcton 17 Manual selection of Range 23 Mark
219. is module then click the SNP Analysis Settings icon Enter a minimum threshold value in the Min Intensity field to remove low false positive SNP calls Optimize peak calls by setting the Use Deviation when Intensity value higher than the Min Intensity value but below the highest peak height in the dataset Click OK in the SNP Analysis 128 May 2012 Chapter 7 Special Applications Settings box and the thresholds will be applied Use the Cartesian plot view to identify SNP calls in the gray region Evaluate the SNP call and edit as necessary Editing SNP Calls Marker PO x 1 sample_001 fsa Gel Image 2 sample 002 fsa 3 sample_O07 fsa 4 sample_008 fsa 5 sample_009 fsa 6 7 8 9 sample_010 fsa sample_015 fsa sample_016 fsa sample_017 fsa 10 sample_018 fsa 11 sample_023 fsa 12 sample_024 fsa 13 sample_025 fsa 14 sample_026 fsa 15 sample_031 fsa 16 sample_032 fsa 17 sample_033 fsa 18 sample 034 fsa 19 sample_039 fsa 20 sample_040 fsa 21 sample_041 fsa 1 15 27 28 29 30 31 32 33 34 35 36 37 38 39 40 4 l1g sample 34 3 sample 001 ba xn sample 34 3 200 pos Lea 3 000 Change SNP Type 36 ki 19 sample 34 34 36 28 29 30 31 32 33 34 35 36 37 38 39 40 20 sample 34 34 2 000 36 SNPWave One high throughput method to determine SNP genotypes is SNPWave Keygene N V SNPWave uses multiplex oligonucleotide ligation amplification of allele s
220. is closed the Size Match Score indicators next to the filenames in the Sample File Tree in the Main Analysis window will be updated Copy Current Calibration Data When selected the frame position and basepair position of the green triangle peak indicators for the selected sample will be copied to the Windows clipboard and can be pasted into a spreadsheet or word processing program such as Microsoft Excel or Word Calibration Plots The Calibration Plots chart the migration linearity of the ILS fragment peaks for each sample The charts plot the peak basepair positions on the y axis as a function of time raw data frame numbers on the x axis As the linearity of the line decreases so does the Match Score for the sample Incorrectly identified peaks will result in a low Match Score Double click a Calibration Plot to select the sample in the Sample List and display the sample in the Sample ILS The currently selected sample filename will appear red in the upper left corner of the Calibration Plot Procedure 993 FOS SCF 40 250 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500 rame After a Size Standard has been chosen and the data is processed by the Run Wizard the Size Calibration Charts can be used to correct improperly sized samples 1 Click the Size Calibration Charts icon in the main toolbar 51 May 2012 Chapter 4 Fragment Sizing Standards The Calibration Charts window appears Select a sample to edit in
221. is displayed in the Panel List 4 The Type will by default display the Analysis Type chosen initially in Run Wizard Template Selection Choose a different Type from the drop down list if required to match the Analysis Type selected a Fragment Animal Fragment Plant and HID Types several Markers per dye color will be created based on peak grouping in the dataset b AFLP MLPA and SNPlex will create just one Marker for each dye color c SNaPshot Type will associate nucleotides ATCG with each dye color 5 Select Automatically Create amp Create New Panel Type AFLP Y V Fixed Bin Width 0 5 a Use All Samples will create a Panel based on an overlay of all the sample peaks in the dataset Name Test Panel Parame ters Method C Manually Create Ze Automatically Create Ze Use All Samples Use Selected Samples b Use Selected Samples will create a Panel based only on the samples selected in the Panel Editor Sample List When finished click OK The new Panel will be created and added to the Panel List OOND Click the double arrow button to expand the dialog box and see additional parameters If required check the Fixed Bin Width option and enter a value for the left and right Bin ranges NOTE New Panels are created based on the Max amp Average View Mode More intense peaks are given higher priority for Bin placement when peaks do not overlap perfectly 10 Edit the Markers and
222. is selected then only 1 0 1 representing Loss Equivalent and Gain respectively will be displayed in the Report Table When Poisson Differentiation is selected then the Poisson Difference values will be displayed in the Report Table 78 May 2012 Classification Poisson Difference 1 1 Loss 0 20 lt Equivalent lt 0 20 lt Gain Dutput Status Peaks Iw Loss Equivalent Iw Gain Iw Abide By Panel Report Contents Status 1 0 1 Iw Poisson Differentiation Cancel Chapter 7 Special Applications Abide by Panel When selected and when a Panel has been applied to the data only peak positions that lie within the Marker range will be reported Terminal Restriction Fragment Length Polymorphism T RFLP Terminal Restriction Fragment Length Polymorphism T RFLP is a polymerase chain reaction PCR based genetic fingerprinting technique for the study of microbial community structure First DNA is extracted from a microbial community One of the primers of a primer pair is labeled with fluorescent dye and used to amplify a selected region of a gene of interest by PCR The resulting PCR fragment is digested with one sometimes two or more restriction endonuclease and the Terminal Restriction Fragments T RFs are separated with an automated DNA analyzer Microbial diversity in a community can be estimated by analyzing the number and peak heights of T RF patterns The aims and data analysis of T RFLP da
223. k 10 lt Pass NOTE If the Reject value is set too high a false negative call could result If the Reject value is set too low then a false positive call may occur soe at Back om C 124 May 2012 Chapter 7 Special Applications SNaPshot Panel Creation After the data has been sized and filters have been applied go to Tools Panel Editor to create a Panel for the dataset For explanation of specific functions in Panel Editor see Chapter 5 Panel Editor 1 In Panel Editor select File Create New Panel OR click the Create New Panel icon 2 The Create New Panel box appears 3 Enter a name for the Panel and be sure SNaPshot is selected in the Type field 4 Select Automatically Create and Use All Samples KE 5 Click OK e 6 Panel Editor will place Markers in each dye color where peaks are detected EN PRES 7 Right click the panel name in the Panel List mi mm 8 Select Edit Dea c e Dyes T 9 The Edit Panel box will appear 10 Set the Ploidy to 2 Diploid Cancel 11 Select SNaPshot and associate dye colors with nucleotides Sp 12 Click OK 13 In the Overlay Trace frame rename the Markers so that GeneMarker can associate SNPs in two different dye egen wem gg a colors For example if primer 1 was designed to detect 7 e ___ T would be to name one Marker peak of the pair as SNP1_A or BRCAla_A in the green dye color and the other Marker pea
224. k Saturation Iw Pull up Correction Size Call options I Enhanced Smooth Iw Baseline Subtraction Enhanced Baseline Subtraction Data Process SNPlex Analysis VW Spike Removal Local Souther Cubic Spline Large Size Load Default Save Default Allele Call 1 After the data has been sized select Tools Panel Editor 2 Select SNPlex_48plex_v1 Panel from the Panel List 3 Deselect the Check Range in Edit icon 4 Edit Markers and Bins to align with the dataset peaks See Chapter 5 Panel Editor NOTE Each Marker contains two Bins Allele 1 and Allele 2 5 Select File Save Changes Hot Key CTRL S OR click Save Changes icon Exit the Panel Editor Select Project gt AutoRun from the main menu bar Click OK in the Data Process box The adjusted Panel has been applied 10 Select Applications gt SNPlex SNaPshot 11 The SNPlex Analysis window appears OOND ds mo m n ml m s cl ds ml md mo md mo n al ms ml ds mo da ma a me SAA ge i STEELE EEE La zi RRS Icons and Functions See SNaPshot SNuPE Icons and Functions section above What to Expect FEIT TTT fe AP A AP An AP A AP At AP vn 05 05 05 05 0 Di 01 Since Peak Thresholds are not active in the Run Wizard when SNPlex Analysis Type is selected the SNP Analysis Settings must be adjusted to correctly identify SNP peaks Review the SNPlex results in the SNPlex Analys
225. k as SNP1_G or BRCAla_G in the blue dye color NOTE The naming schema is case and punctuation sensitive 14 Select File gt Save Changes Hot Key CTRL S OR click Save Changes icon 15 Exit the Panel Editor 16 Click the Run Process icon in the Main Analysis window 17 Select the newly created Panel from the Run Wizard i Template Selection Panel drop down menu 18 Proceed through Run Wizard and click OK in the Data Process box 19 The Panel has been applied 20 Select Applications gt SNPlex SNaPshot 21 The SNaPshot Analysis window appears SNPs A and G of BRCA1 an acceptable naming protocol En Icons and Functions Show Dye Left click to show individual colors in the Two Color Trace Overlay or use drop down menu to select Show All or Hide All dye colors SNP Analysis Settings 7 Launches the SNP Analysis Settings box vere EE Peak Options Peak Options Min Intensity Peaks with RFU heights below this value will not be called Use Deviation when Intensity Peaks with RFU heights below this value will be called with higher sensitivity using a deviation method to remove the influence of the baseline Min Intensity so Use Deviation when Intensity lt 200 OK Cancel Layout Settings Launches the Display Settings box Cluster Plot Layout Coordinates Cartesian Polar Select the format in which the Cluster Plot Display Settings GET will be displayed Polar is the def
226. k ratios to copy number values applying user defined parameters The Normal range in Settings MLPA Ratio is defined as 2 copies Deletion as 1 copy and Duplication as 3 copies The copy numbers will be applied to the Allele and Patient reports To view the MLPA Ratio to Copy Number option click the double arrow button at the bottom of the dialog box MLPA Regression T Distribution Uses the T distribution calculation to compare reference and sample peak heights or areas Using T distribution method requires confidence limits to be set Settings Regression Choose Use Control Probes to analyze each sample by the identified control probes MLPA Analysis Settings Advanced Settings Analysis Method Choose Use Population to analyze each sample by all EE Esseg Quantification by probes in the sample The Confidence Limit is aaa Een represented by the green lines in the Ratio Plot and can 6 Use Control Probes E Min T Distance O05 be used to determine which probe points are outliers as Use Population F Outier Fiter compared to all probes population or control probes Confidence Limit 98 00 Confidence Limit 3810 in the sample The default setting is 99 non Minimum Lane Score Threshold 10 00 Min Dosage Range 0 75 lt Ratio lt 1 30 Save Parameters when Save Report 84 May 2012 Chapter 7 Special Applications Minimum Lane Score Threshold Sample files are given a signal to noise typ
227. k1UTXhFRFEwRWwpROmR6TxURES4QURN Account 0 Register x Register Offline Password Email Back Cancel The SoftGenetics License Server Manager needs to be restarted for changes to take affect Please make sure that no clients are connected before restarting Restart Later Register Product z Register Online Offline Registration i x Register Product Name Register ID el Register Chapter 2 General Procedure Install GeneMarker software on the client computer 1 Proceed with installing GeneMarker software on the client computer as described in the Local licensing es Option Installation section above until the eee EEE PTE y Configure Registration window appears 8 is running property on the server computer 2 Click Configure Network Client to configure the client ee o 4 software to contact License Server Manager e SE 3 Click Configure Connection to License Server AS Manager from the Choose Network Configuration dialog box o Input Server Name or Server IP Address SeverPon PS 5 Click Configure and GeneMarker software will automatically open if connection is properly established and a license is available Oi comme Sap hoose Network Configuration e Upgrade of License Server Manager Activate the License Server Manager console Proceed with step 3 of Register License Server Manager for GeneMarker Usage section above Upgrade of Ge
228. ker V1 60 Analysis Type LOH Panel LOH_Panel Classification LOH lt 0 70 or LOH gt 1 30 Report Type Peak Area Ratio Group 1 Blue 138 140 142 144 146 Group 1 Green 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 Group 1 Yellow 134 138 140 142 144 146 148 150 152 154 May 2012 Chapter 7 Special Applications TILLING Analysis The techniques of Targeted Induced Local Lesions In Genomes TILLING and EcoTILLING have been widely used since 2000 to detect Single Nucleotide Polymorphisms SNPs During TILLING the test samples may be experimentally mutagenized ethylmethanesulfonate radiation etc or from natural populations or derived from tumors or diseased tissues Briefly the genes of interest are identified with gene specific primers and PCR amplified The amplicon s primers are labeled with two fluorescent dyes the forward is often labeled with FAM blue and the reverse primer is often labeled with HEX green The samples are mixed so heteroduplexes can be formed The hybridized fragments are cleaved at the heteroduplex site by CEL I or Surveyor Nuclease generating multiple pairs of fragments of complementary length and dye color The denatured samples can be run through gel electrophoresis or mixed with an internal size standard and run through capillary electrophoresis SNPs will yield two peaks of different color and the sum of the sizes will equal the amplicon length Ov
229. kers which contain two peaks in a 1 2 or 2 1 ratio are more difficult to detect Given user defined thresholds trisomy peaks in a 1 2 or 2 1 ratio appear as red triangles in the Ratio Plot and appear in dye colored font in the Report Table In this way the clinician can easily identify markers which contain trisomy peaks Trisomy Analysis Ratio Plot Electropherogram Sample List Report Table d Tegen Ange Corrected Kate es le En a amp BEGER maker Dasa Sample nl 1 tl report A A A cet 07 7 A ane Ngee 07 OT b SAR _ HAM 7 e es Market CN Blc 070507 b 25 m s 2M Ee 612 10 ape 07 0507 rere Js d EK 113 of 07 06 07 b 6 211 ape 07 06 07 foss DI 4 gp ONE 07 L Jomes O B D EN Til P D El2_13_ open BOT 1 D I d fps Or D6 D 2 HIE epes Or 16 Y b ER SSC f r EE les Eeer ees E amp c12 11 qoxnsennn a D z 4 E B 19 12 Disiance Sire 571811 Sample List The Sample List displays all current samples in the project Single left click a sample name or use the Up Down Arrow keys to scroll through the list Electropherogram The Electropherogram shows the trace for the sample by marker Select a marker to view from the Marker drop down menu in the main toolbar The allele editing options in the Electropherogram frame are similar to those in the Main Analysis window See Chapter 3 Main Analysis Overview 142 May 2012
230. l List The select Panel will appear in the Overlay Trace frame Click the Trace Mode icon until the Trace Overlay view appears in the Trace Overlay frame Right click in the Samples List and select Deselect All so that no samples are selected Double click only the Allelic Ladder samples in the Samples List Click the Major Adjustment of Panel icon Click the Minor Adjustment of Panel icon The Markers and Bins will shift to align with the average peak positions of the Allelic Ladder samples Verify Marker and Bin shift was accurate by clicking the Show Dye icon a Hold down SHIFT key and left click and drag Markers Bins to align correctly with the Allelic Ladders 10 After editing select File gt Save Changes with Signal Info OR click Save Changes with Signal Info icon 11 Exit the Panel Editor 12 Click the Run Process icon in the Main Analysis window 13 Select the newly adjusted Panel from the Run Wizard Template Selection Panel drop down menu 14 Proceed through Run Wizard and click OK in the Data Process box 15 The Panel has been applied What to Expect When HID Analysis Type is selected in Run Wizard the Main Analysis window automatically sets the selected Allelic Ladder sample as the top Electropherogram and the Report Table displays Marker Table Fragment Report Style Use the Marker drop down box to scroll through loci or click the All Color Browser icon to view all dye colors of a sample simultaneously Edit alleles as required Se
231. l alter the area value for the allele Procedure 1 Import raw data files and filter with Run Wizard See Chapter 2 General Procedure NOTE A Panel does not need to be applied to the dataset to use the Quantitative Analysis module Select Applications Quantitative Analysis The Quantitative Analysis window appears Click the Select Peak Number and Space icon The Quantitative Analysis Settings box will appear Choose either Curtain or Deconvolute analysis method ane 21 120 May 2012 Chapter 7 Special Applications Click OK The analysis method chosen is applied Edit the results and click Refresh to update the Report Table 10 Click Save Report to export the Report Table as a tab delimited Text txt file Se Icons and Functions Quantitative Analysis Settings Le Activates the Quantitative Analysis Settings dialog box Number of Peaks Allows the user to select the number of peaks that are expected in the Quentiatue Analysis settings ef 2 region Too 11 Space Between Neighboring Peaks Space Between Neighboring Peaks 30 bases Provides the option to specify the number of base pairs that separate the EE expected peaks amp Curtain C Deconvolute Analysis Method Sa eee ME The user can choose between the Curtain and Deconvolute methods See What to Expect section below ox Cancel Peak Boundary Peak Intensity Sets the start and end range points at the RFU position on the trace that equals
232. l in the Report Table to view the associated electropherogram Icons and Functions The SNP type call can be modified in the Report Table by right clicking a cell and selecting Change SNP Type Select the correct SNP call from the fly out menu and the Report Table will automatically update Additionally selecting Copy from the right click menu will place the selected cell s information into the Windows clipboard which can be pasted into a spreadsheet program Report Settings Launches the Report Settings box Peak Ratio Calculates the ratio for the intensity of the first d Report Settings nucleotide divided by the intensity of the second Report Content nucleotide in a SNP pair C Peak Ratio SNP Type Displays the nucleotide number of the peaks present in the SNP pair SNP Type is the default view for the Report Table Ok Cancel_ Probability Displays the SNP types present and calculates a number between 0 and 1 for the likelihood that the SNP call is correct The probability calculation takes into account the signal to noise ratio and peak intensities RH Save Report Saves the Report Table as an Excel xls or tab delimited Text txt file e SNP Type Probability 130 May 2012 Chapter 7 Special Applications Microsatellite Instability MSI Microsatellites are stretches of DNA where a 1 5 base pair sequence is repeated several times The most common microsatellite in humans is a dinucleo
233. lanation of specific functions in Run Wizard see Chapter 2 General Procedure Run Wizard Run Wizard Template Selection GEN Panel NONE OR chose custom Panel Size Standard User defined drm Analysis Type AFLP Bee pen p ll NOTE Depending on your experiment it may or may not be necessary to select a Panel for data analysis The Clustering Analysis module is only available when a Panel is applied Run Wizard Data Process Peak Detection Intensity Threshold 100 VE mn Global Percentage 1 Max Data Proces AFLP Analysis Local Percentage 1 Max Call Intensity 30 000 Stutter Peak Filter Left 5 Right 5 Plus A Filter Selected NOTE The correct settings for AFLP analysis should be a small global percentage plus a slightly larger or equal local percentage Itis suggested that the stutter peak filter be selected to allow the software to remove stutter peaks within 2 5 base pairs of each detected allele peak Addtional Settings AFLP Analysis Run Wizard Additional Settings iin oes tnt cs Allele Evaluation Peak Score Reject lt 1 Check 7 lt Pass NOTE If the Reject value is set too high a false negative call could result If the Reject value is set too low then a false positive call may occur carey AFLP Unconfidence at Rightside Score lt 30 AP Unner gal Sone MLPA Normalization Method NOTE This requires that two peaks to the right of the examined peak must have a score of at least 30 for t
234. lation Choose the distance or correlation percentage at which to start the grouping of individuals Distance Measure See Overview section above for definitions of Correlation Coefficient Percentage of Same Genotypes and Euclidian Distance Linkage See Overview section above for definitions of Single Complete and Average linkage Marker Selection Dye Marker choose which Markers to include in the clustering calculation and dendrogram Save Parameters when Save Report to automatically create an ini file of the AnalysisSettings 137 May 2012 Chapter 7 Special Applications Case Any Sens Analysis Layout Layout Build Dendrogram According to Similarity When selected will Fs show the distance correlation ruler above the dendrogram tree When deselected the distance correlation ruler will be removed o Cancel Show Gel Image provides flexibility of hiding or showing the gel image in the dendogram Save Dendrogram mj Allows the user to save the dendrogram image as a BMP image file or as a PNG file PNG uses lossless compression leading to greatly reduced image size Output Clustering Report Produces a matrix table that can be saved as a Text txt file Choose the analysis method from the drop down icon in the main toolbar What to Expect Notice how when just the distance me
235. ld expect individuals who are mosaic for a chromosome change tend to have a less severe form of the syndrome present than full trisomy individuals Critical examples of mosaicism are found in leukemia cases specifically chronic lymphocytic leukemia CLL which is a trisomy of chromosome 12 and acute myeloid leukemia AML prognosis which is a trisomy of chromosome 8 Detection of the smaller deviations from normal ratios caused by the presence of a population of trisomic cells in a single individual is possible with the GeneMarker Trisomy function Triallelic Homozygote It is possible that an individual with three chromosomes could potentially have the same allele on all three chromosomes In this instance the electropherogram trace for this allele would theoretically depict a peak three times the height of a peak with just one allele Since there is only one allele present in the marker and no other allele for intensity comparison the analyst must use their own knowledge and experience to determine if the individual is a triallelic homozygote Reports and Printing GeneMarker automates the analysis process and creates an easy to read report for aneuploidy determinations Report Table Displays peak ratio and Trisomy Score for individual Markers in a sample The Electropherogram frame will correspond to which ever peak cell is selected in the Report Table Report Settings Launches the Report Settings dialog box 146 May 2012 Chapter 7
236. le names are repeated for each row of data that the sample is associated with If Hide Extra Sample Names is selected then the sample name will only appear once in the first of the rows it is associated with Additional Functions Allele Editing Options The Bin Table Report Style offers additional options when a cell in the table is right clicked Insert a Peak at this Bin Site To indicate the presence of a peak at a position when it has been labeled with a Negative Type Symbol right click the cell and select Insert a Peak at this Bin Site The Negative Type Symbol will change to a Positive or Suspected Type Symbol depending on the Quality rank of the peak Hot Key INS Delete To indicate the absence of a peak at a position that has been labeled with a Positive or Suspect Type Symbol right click the peak cell and select Delete The Type Symbol will change to Negative Hot Key DEL Confirm To indicate the peak present at the position is truly a peak right click the peak cell and select Confirm Peaks Only peaks centered within a Panel Bin will change from Suspect Type Symbol to Positive Type Symbol when confirmed Once a peak is confirmed it cannot be unconfirmed only deleted Hot Key CTRL M Delete Bin Columns To completely eliminate an entire column in the Report Table left click any cell within the column then right click the cell and select Delete Bin Columns When Vertical Orientation is selected the Report Table rows which
237. lick and drag the Marker edge to increase or decrease the range OR right click the Marker bar and select Edit Marker The Edit Marker box appears Adjust the Boundary field values as necessary Additionally if a Marker needs only slight adjustment to the right or left right click the Marker bar and select Adjust Marker The Marker will move automatically to align with the closest peaks Edit Marker Bins Right click the Marker bar and select Update Alleles The Update Marker Alleles box will appear Adjust parameters and click OK Nucleotide Repeat Update Marker Alleles L ZS Auto Detect Based on the peaks present in the Overlay View GeneMarker will attempt to detect the number of nucleotides in each repeat unit of the alleles and place Bins at the appropriate interval Set by Manual Select this option if the number of nucleotides in the allele repeat unit is known and GeneMarker will place Bins at the specified interval Marker Name Ic 135634 Boundary bps 391 D To 4270 Nucleotide Repeat e Auto Detect C Set by Manual 2 a Auto Binning Vv Fixed Bin Width Check this option to enter the number of basepairs on Lee the right and left of the center of the Bins If 0 5 is selected as the Bin Width then the total Bin range will be 1 0 basepairs Auto Label When deselected the Bins will be automatically labeled with the basepair size of the Bin position to the nearest tenth of a bas
238. lick the Print Report icon in the Main Analysis window The Print Report options box will appear Select desired settings and click Preview to view the Print Report before printing or click OK to begin printing without previewing the report NOTE The View Preference gt Display Settings options will affect how the Print Report is displayed GeneMarker Print Report SoftGenetics Allele Report 8 28 2007 11 19 19 AM GeneMarker V1 60 Page 5 Trisomy Report Sample 5 Dye Blue 10 peaks 061_B05 SCF 250 150 200 300 350 Dye Green 9 peaks 061_B05 SCF p18s1002 Johss3g1 D135742 D185386 P135304 150 250 300 350 400 450 100 Dye Yellow 4 peaks 061_B05 SCF D2151411 300 Dye Red 0 peaks 061_B05 SCF 100 150 72 May 2012 Chapter 6 Reports and Printing Report Content Options The basic printing options allow the user to choose the Print Type Samples to print Dyes to include and Content options Each electropherogram will be automatically labeled with its respective sample file name in the printed report The Advanced button provides more options Print Type Normal All Print Report options are available when Normal Print Type is o selected EE C Chat Oveiap Chart Overlay Prints only the Electropherogram with the report ER pe Samples Selected Samples 7 Dye2 Contents M Dyes All Samples Prints all the samples in the project i Selected Samples Prints only those sample files that have
239. lick the Show Dye icon in the toolbar to cycle through the other dye colors Right click at a peak without a green triangle indicator and choose Insert Size The Edit Size box will appear Adjust as necessary and click OK The green triangle will now appear atop the peak and also in the Expected Size Standard Match Score Appears in the upper right corner of the Sample ILS and corresponds to the degree of pattern match between the sample s ILS and the Size Standard selected Perfect matches receive a score of 100 no correlation receives a score of 0 Navigation in the Sample ILS frame is similar to the navigation options in the Main Analysis window See Chapter 3 Main Analysis Overview Procedure As mentioned previously Size Standards are created to assign basepair size information to fragment peaks in a sample ILS The other dye color fragment peak positions are then interpolated based on a linear size scale from the basepair sizes assigned to the peaks in the ILS GeneMarker s Size Template Editor tool allows users to apply pre defined commercial Size Standards or create new custom Size Standards based on the dataset ILSs Pre defined Size Standards include 5C120 GS500 ET400 R GS500_1 ET550 R HD400 ET900 R ILS500 GS 100 250 ILS600 GS 75 300 12120 GS200 Rox1000 GS350 SEQ_600 GS400 SNPlex_48plex_v1 Pre Defined Size Standards There are two ways to choose a pre defined Size Standard for the dataset If the Size Standard nam
240. lution of any flagged allele calls must be done prior to saving each project and importing results into the Merge Project Tool 2 Open Merge Projects from the Tool Drop down menu or the Open Folder icon 3 Projects gt Merge Projects gt Add project files OK 4 Report Grouping icon activates the Grouping screen 5 Match by Section Fixed Position or Group Order gt Match OK Note To simplify this task separate the group identifier and the exact control identifier by an underscore or have the same number of spaces for the individual and group identifiers in a file name For example if the individual s identification number is 12345 and it will be analyzed with panel XYZ and panel ABC separate the sections by an underscore when applying the sample to the genetic analyzer D 0 ODIO Be PENE I 22d OUR ERRADA CA Cada caca ca ett ttt 4 Project with XYZ Project with ABC 12345 XYZ AUT 12345 ABC AO Allele Report Settings ls 12346 XYZ A02 12346 ABC A02 Repot Sy Open 12347 XYZ A03 12347 ABC A03 Gamm Agge C Marker Table Fragment Iw Show Type Symbol Bin Table AFLP MLPA Jak 6 Use the Report Icon to activate the Allele Report Settings aS ee 7 Select appropriate format Ks I Show Inensiy For example Select Bin Table and deselect Show Peak C Sample Name FieName T Area for combined reports that will be used in Cluster Analysis or Orientation e ei Select Marker Table Fragm
241. ly for analysis of a single file per individual to go directly to the print report preview 10 Select the Tools or parameter icon to select ARMS Comparative Analysis Parameters 11 Select the desired Parameter Options 12 Review analysis and select Print Preview or Print L A Po we EI o a e a a 2 a a e a a e a a a a a a ka e a a a a a LI a a a s LE O OND Icons and Functions En Comparative Analysis Parameters 163 May 2012 Chapter 7 Special Applications Launches the dialog box to select the analysis parameters Layout Settings Select the Comparative Analysis Display Settings Printer d Select the Printing Options for the final report Print from this screen or select Print Preview What to Expect The Header in the final report obtains Institution and Operator information from User Management All other information in the header is from the Panel and from the Project Parameters The Allele calls are entered into the Report Table from the Project The Control Column of the panel editor determines whether the allele call of the blue and green channels is placed in the Wildtype or Mutant column of the report table Some chemistries provide the option to amplify and report Poly T and unique STR information These settings are deselected in the default settings The analysis parameters and print settings in GeneMarker allow the analyst to det
242. lysis Settings dialog window will activate Choose the appropriate analysis parameters Click OK The MLPA Analysis window will appear Icons and Functions Display Settings Allows the user to select the preferred chart layout EET display A maximum of 5 rows and 5 columns is permitted Statistical on information will be included under each chart when the Show Statistics 0 83 May 2012 SC se Chapter 7 Special Applications Information option is selected MLPA Analysis Settings Launches the MLPA Analysis Settings box MLPA Ratio MLPA Ratio is a simple and easy method for detecting duplications and deletions The sample peak heights or areas are compared to a control reference sample and the ratio value is used to determine the addition or absence of the allele probe Settings MLPA Ratio Allows the user to input the deletion and duplication values for the analysis The mete Advanced Settings default setting is Deletion lt 0 75 lt Normal lt 1 30 lt MLPA Ratio C MLPA Regression n ra Duplication Sling MLPA Poi E Deletion lt 0 75 lt Nomal lt 1 30 lt Duplication z gid Ce EN Adjust by Control Probes When selected all samples FF Adjust by Control Probes 3Copies lt 1 75 lt 4 Copies with control probes designated in the panel will be plese ee adjusted by that sample s control probes The method Mirimum Lane Score Theshol 7000 of adjustment can be changed
243. mily Group Tool When a naming convention is followed the Family Group Tool enables matching of files into family groups If naming convention was not used for these files use File New Pedigree to draw the pedigree WW Fite Name Group Editor File Name List el PAT fea PAT IF ha 2 AT MP fsa 2 2 PAT 2 F fee Procedure 2 Ss Oo b P La fsa PAT_4_F fta 3 DP fee PAT_5_F fra PAT 6 F fee PAT_7_F fra foo PAT Ladder 2453 1 Select Applications Relationship Testing from the menu bar of the Main Analysis window 2 Match by Sections Positions or Group Order and then Match Whole Words 3 OK 3 The Pedigree for the families is drawn and displayed at the left of the Sample List SE Compass Character 56 18 DAT ne far Match by Sections Match by Feed Postion Group By Dad Whole words Indute Control identification Match to Ideas 73 CH Match BEI X Cancel d Relationship Testing TL 7 O File DataBase Tools lys M T 2 DIS FT a a Fam y 178 1 Fake 1 Esc EI Marker AR Markers vi Search d amp 4 Right click on a node for edit or analysis options gt Samples LO Chats Report Calculation Das 5 Select Family displays all m s a GE electropherograms for the pedigree iby em e e tree at the right EES mm 6 Select Node or parents siblings sees p Select Sibang displays electropherograms Select Family PAT_1_M fea x 7
244. most recently saved Size Standard will be restored NOTE If the user selects Save Size Standard and then answers NO to the Size Standard has been changed save changes the changes will remain in the Expected Size Standard and Size Table until the user chooses Reload or GeneMarker program is closed Sample List The Sample List contains a list of all the samples in the dataset Double click the filename and the sample s ILS trace will appear in the Sample ILS frame Use the Up Down Arrow keys to scroll through samples in the list Expected Size Standard and Size Table The Expected Size Standard frame displays as a trace all the known fragment peaks that are expected to appear in the Sample ILS Single left click a green triangle atop a peak to select the peak The green triangle will turn yellow when the peak is selected Additional Options Once a peak is selected right click anywhere in the Expected Size Standard frame The right click menu will appear with the following options Edit Size Edit Size EH The Edit Size box appears Adjust parameters and click OK Size Enter the expected basepair size of the ILS fragment Size Pe Comments Enter free form text regarding the Size Enabled When selected a 1 will appear in the Expected Size Table Deselect this option to disable the Size in the Size Standard Disabled sizes will be used Comments Y Enabled for pattern recognition in the sample IL
245. n the sample list and locus in the electropherogram Click the Report Settings icon to select display options Click the Save Report icon to save the Report Table as an Excel or tab delimited Text file Procedure E Launch GeneMarker software Import raw data files See Chapter 2 General Procedure N Select Tilling Analysis from Applications menu 3 Select Size Standard and Standard Color Data Process Parameters and Data Range Parameters from pull down menus 4 Click OK to process data 5 The report table can be saved in TXT file format Bil eremana United File View Project Applications Tools Help 1 gt gt D Pedigree Icons and Functions G a E CS EES Lis Y 01_A01 fsa 9 02 BOY fsa R Export CODIS Open files icon and navigate to the file of interest click Add navigate to 03 001 fo directory containing data of interest select data files up to 1000 lanes gt Fam click Open and OK Tilling Analysis IW Select Tilling Analysis from the Applications menu to activate the Tilling Analysis Options dialog box Select Size Standard TE CA ProgramFilesN SoftGenetics GeneMarkerl 1 90 Val SizeStd SGSize_Rox1 Size Standard fie Esa Sales Dance pg 000 xml if default installation location was accepted and dye color If a size ee sl standard used is other than one that is listed the user can quickly construct one a using the Size Template Editor See Chapter 4 a a LE Enter Data Pro
246. n window to install GeneMarker 10 Click Finish in the Installation Complete window 11 The Installation Wizard will close 12 Eject the SoftGenetics CD 13 Launch GeneMarker by double clicking the GeneMarker desktop icon OR open the Start menu and navigate to SoftGenetics GeneMarker the version that was just installed gt GeneMarker program 14 The Configure Registration window will appear Click Register Now to register the local license 15 Click Register Local Text based Key from the Choose Registration Method dialog box Registration 1 The Register Local Text based Key window appears 2 If the computer GeneMarker is being installed on has an internet connection select Online Registration If the computer does not have an internet connection or is connected to a proxy server select Offline Registration Online Registration A B C Locate the Account and Password on the SoftGenetics CD Enter your Account Password and e mail address information in the appropriate fields The Request Code information is automatically generated by GeneMarker Click Register 7 May 2012 2 SoftGenetics GeneMarker 2 2 0 x f Welcome to GeneMarker 2 2 0 Setup program This program f will install GeneMarker 2 2 0 on your computer It is strongly recommended that you exit all Windows programs before running this Setup Program Click Cancel to quit Setup and close any programs you have running Click Next to
247. nalyst See Chapter 5 Panel Editor ese eee ie 4 04 1 00 5 Save Allele report as txt file select report save icon marker table horizontal deselect extend heterozygous 6 Repeat steps 1 4 if a second kit or panel is required for a complete profile of the individual 7 Applications drop down menu select Haplotype Analysis 8 Tools or parameter icon to select Haplotype Analysis Settings 0 0 0 0 0 0 0 i 0 i D D 0 0 159 May 2012 Chapter 7 Special Applications Samples All or selected Samples Autosomal both haplobars are displayed X linked males have one haplobar displayed Display Genotypes lists the allele calls under each node Haplotype Analysis Settings Samples Selected Samples Pei Autosomal C linked Iw Display Genotypes Iw Display Locus Identifier Iw Color Pattern Ok Cancel Display Locus Identifier lists marker names in the pedigree diagram Color and pattern provides easily distinguishable haplobar for each individual Pattern alone provides distinguishable haplobar for analysts with color impaired vision or for black and white printers 9 File drop down menu New Pedigree File 10 Enter patient information 11 Enter Genotype results from one kit or two kits gt From one kit Select from Sample or From gg TT Database gt From two kits Select Manually Input and open file icon New Family Ne
248. nd allele editing in the Electropherogram Charts is similar to navigation options in the Main Analysis window See Chapter 3 Main Analysis Overview NOTE After alleles are edited in the Electropherogram Charts click the Update Sample Data icon in the toolbar Conflict and Suspect Marker identification will be adjusted accordingly Additionally allele changes made in the Pedigree tool will be applied in the Main Analysis window Edit Suspect Alleles i Pedigree Edit C Users SoftGenetics Desktop TestPed pre ld HT Lei D Family 2 2 2 Family 2 Jones v Marker 13 D18551 y Search d Fr da Samples WA Charts l BESCHE EHe a y 279878 30_07 fsa 298 2 757 x DB ty 260 280 300 320 340 Jon Tan 1 000 Edit Allele Edit Comments D Insert Allele 279878 10_06 fsa Dele Del SSS be 260 280 1 Undelete Shift Del 4 000 Confirm Ctrl M 2 000 Unconfirm Ctrl Alt M Confirm All 0 Unconfirm All W 279879 30_11 fsa View History 2 000 1 000 Person ID 4 Person Name Tam Sample File 279879 30_11 fsa D 105 May 2012 Chapter 7 Special Applications Procedure Although allele calls can be edited in the Pedigree tool it is easier to begin a relationship test analysis with good clean traces In order to begin with the best sample traces complete size calling Panel alignment and allele editing in the Main Analysis window prior to launching the Pedigree tool To launch the
249. ndard field See Chapter 4 Fragment Sizing Standards Size Template Editor This allows the user to check sample files against a selected size standard modify and save the size standard for future use or create a new size standard Standard Color Select the dye color which contains the internal lane standard Analysis Type Selecting an analysis type will set the options in the two subsequent boxes of Run Wizard Data Process and Additional Settings to the optimal settings for the analysis type selected See Chapter 7 Special Applications e Run Wizard lt mm Run Wizard Data Process Data Process Fragment Animal Analysis Set data process options Procedure 5 The Data Process window of Run Wizard will activate This Raw Data Anahi Allele Cal V Auto Range frame tal Iw Auto Range bps allows the user to select the peak filtering parameters The al ene oo Ser PR 3 em 33 settings will vary depending on the Analysis Type selected Y Smooth Enhanced Smooth Peak Detection Threshold 2 Iw Peak Saturation iv Intensity gt 100 Percentage gt 25 Max 6 Select the appropriate analysis settings in the Data Process LP nej E Loca window and click Next to continue Ss rr ER Local Southern Cubic Spline Large Size K Stutter Peak Filter 7 K Plus Filter Left 90 2 Right 40 lt Load Default Save Default Icons and Functions e lt Back Next gt Cancel Raw Data Analysis let er
250. ndrome ccccconccnncccnccnncnnnncnnnnnnnnonononaronononanos 98 Apply Linear Correction ooocccccoooncnnccnononnnonanonononanonoss 144 181 Apply Sample Grouping cccccccocccnnncnacnnnnnnnnnnnnnnanonnnnos 172 NNN 32 Area Ratio sssnsssscsscssscssecdsnsssonsevesswessvesssscsvessanseveseneesvonses 32 ARMS Comparative Analysis for CF analysis 162 FT 179 TR ll Un TE 57 58 Auto FIL Y EE 52 Auto Pull up Removal 17 MORE 21 23 Auto de 25 37 Auto Scale Markers vrnrunnnnnnnnnnnnnnnnnnnvnnnnnennnvnnnnnenn 73 168 Automated Pedigree Trio Diagrams 118 Automatic Panel Creation rrrrrnnnrrrnnnnnnrrnnnnnnrrnnnnnnnene 61 Automatically Re Sort Report 36 Automatically Scroll Charts to Alleles When Selected in FEI NNN 36 automatically selected Control 87 AUTOR INE E Luer 18 Average linkage cccooonccncccocnnnnononcnnnnnonocnnonononnnnnnnoos 137 B Background in White 31 36 Baseline Subtraction 17 18 21 basepair Size Pang cccoooccnnconoconnnononononononccnnononananonnnanonos 31 Best MN arr 46 BestMatch Match Al cccccoocccnncnnncnnnnnos 48 BestMatch Match Selected nossnnnoosnenessrenesssrresseeee 48 BIN 58 Bin Table AFLP MLPA cccccccccccseeeeeseeeeeeeeeeeeeeeeeees 69 Binning Options oaeneeennssensseessersseessserssersssrrsseene 70 gt EEE O RE 56 BNR EEEE 60 Browse by All Colors 41 113 116 Browse By All Colors 168 C Calibration Plot
251. neMarker Untitled File View Project Applications Tools Help ao gt GEN amp Wo oO Es Es LA Marker None y Y 4 D 064_E05 SCF x 240 250 260 270 280 290 300 3 500 3 000 2 500 2 000 1 500 1 000 500 o O 0 y MN ur Height 4464 3353 3160 5705 5430 3169 3039 2528 2296 6309 1036 Area 24033 20888 20559 41639 39585 23272 22700 24033 23875 69711 4069 Marker D1851002 D1851002 D185391 D185391 D135742 D135742 D185386 D185386 D135305 Allele OL 124 136 178 182 269 OL 377 392 450 OL Difference Quality Undeter Pass Pass Pass Pass Pass Undeter Pass Pass Pass Undeter Score 500 0 500 0 500 0 500 0 500 0 500 0 500 0 227 0 181 2 500 0 1 1 Quality Rea SP LS May 2012 65 Chapter 6 Reports and Printing 66 May 2012 Chapter 6 Reports and Printing Chapter 6 Reports and Printing Chapter 6 Reports and Printing Report Table Print Report Save Project 67 May 2012 Chapter 6 Reports and Printing Report Table The general features of the Report Table were outlined in Chapter 3 Main Analysis Overview Here we will discuss and give examples of each Report Style available in the Report Table Allele List The Allele List Report Style displays the basepair size or Allele Label if a Panel HEEE is applied of the called peaks The sample names are listed in rows
252. neMarker software on client computer Install GeneMarker software on the client computer by following the procedure in the Install GeneMarker software on the client computer section above If the network configuration has not changed the software should activate without configuring the IP address of License Server Install NetDog Server Management The following are instructions for installing the NetDog Server application See Local Version Installation section above for instructions on installing the GeneMarker program 1 Insert the NetDog Key USB parallel hardware key into the computer that will run the NetDog Server software The computer may be any client computer on your network that runs Microsoft Windows 2 Launch the setup file of NetDog Server NetDog Server Setup setup exe and install the server 3 After installation NetDog Server starts automatically e fa NetDog Server Management 4 Double click the icon on the task bar to open up the Es verinfematen instalfunnstal advanced Operaton Demo Period Heb NetDogServer Management application A The section in the figure that A is referring to shows Eat that the NetDog Server is able to detect the NetDog E Mode 3 If it is unable to detect the NetDog it will display berg Module 5 PAL a IF 8 odule please refer to the section about yet 10 E E E E d Module 6 E Module 9 11 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
253. ngle dye layer Choose a single dye by single left mouse clicking on the icon Zoom In Use the icon to zoom in on the image or hold down the left mouse button and draw a box from the top left corner to bottom right corner around the area you wish to zoom in Zoom Out Use the icon to zoom out on the image or hold down the left mouse button and draw a box from the bottom right corner to top left corner ARMS Comparative Analysis for Cystic Fibrosis Analysis Overview 162 May 2012 Chapter 7 Special Applications This analysis type allows comparison of pairs of files for a given individual within the same project includes electropherograms trace comparison and allele report in a patient report format For example if kits use one dye in a multiplex for most of the wild type alleles in one multiplex and a second dye in a second multiplex for the mutant alleles as is found in GeneProbe EU 2 for cystic fibrosis the ARMS Comparative Analysis application provides an accurate concise comparison of the two files for the individual ARMS Comparative Analysis Report SoftGenetics Mutation Wildtypel Mutant R347H zz Sample frag_001_A07_1_B fsa frag_001_A01_1_A fsa ao 2789 5G gt A Mo Jo Software GeneMarker V1 95 Analysis Type ARMS Comparative Analysis 3120 1G gt A Mo e 711 1G gt T eo il 6 rasew M bh istry sozda KI Others Chemistry Lot Zen Report Time 09 03 2010 14 35 48 S 849 Kb Blu
254. nnnn 36 Open Pedigree File 108 112 115 162 Open PrOlCE siii ida ici 35 74 ODORS iia A 37 OUTER INC ia 85 Output Status Peaks 89 135 Output Trace Data 39 174 185 Overlay VIEW Lee 38 168 Overtime Protection 179 P INTE d WEE 74 PON lit 20 Panel Creations ad 61 Panel Editor 21 39 56 115 Panel ito Na 56 het ET vene 59 Partitional clustering ooccccconoccnnnononononenanononananonoos 136 Passord vvs 178 Pata Lellgen E 142 PGR NG 79 Peak BOUNCSLY av 121 Peak Compare Items 0 175 Peak Comparison Histogram sssseccccsseeeeeeeeeeeees 133 Peak Comparison Threshold aoeeoe 175 peak detection threshold rrrrnnnrrrnnnnnnrrrnnnnnrvnnnnnnnene 79 Peak Detection Threshold oooocoooocccoooooo 24 Peak Differentiation rarrnrnrnrnnnnnnnrnrnvrnnnnnnnnnnnnnnnnnnr 89 Pealknftormaton Lae See 34 Peak EC TC WEE 36 Peak Matched Brisas DEE 175 Peak RAMO add 130 Peak Saturation ooccccncccnnccnnoccnnoccnnccnnnccnnancnnannnnn 21 PEAK SOE JG 24 Peak Taiana 32 70 179 Peak Table Features eee 31 AA 106 A Po oo A 38 103 Pedigree Aha 103 Pedigree FE JEG 106 Pedigree File Name March 39 Pedigree Parametere 108 112 116 162 EEN 103 PEeNeEraNteSuuam seen KGaA 108 IN tel e TEE 24 Personnr varar 104 Phylogeny Clustering Anahysis 76 136 Plus A Filer sara 24 Poisson Difference Histogram rrrrrnnnnrrnnnnnnrrnnnnnnrrnnnnner 78 Polar
255. nnnnnnennneee 178 Additional Settings RENE ENNER 24 additional user Iicenses 13 Adjust by Control Drobe s 81 96 PGIUSEIVIQIK asar 58 Adjusting Panels aaanneeenensseeneseenessreressrrrrssrrressn 62 ACMINISELATOF cceseccncsccncsecsssenseccnssccncsccassacsscenssceas 178 Advanced Population Normalization 25 83 86 87 Affected status 104 Affection Locus Description cccccoooccnnconoccnnnonanonnnnnos 108 A a 76 AFLP VY SIG isis 79 AFLP Panel Creation 76 AFLP Run Wizard Settings ooseenseseensssseenssrrrrsrrrressn 76 AFLP Unconfidence at Rightside Score 24 76 79 AFLP selective amplification coccoooccnnccnononnnnnos 129 Allele boundaries 64 Allele Call 23 31 Allele Call folder ooccooccccocccnncccncccnnacinnccns 28 Allele Comments reranrnrnnrrnnarrnnnnrnnnnrnnvnnnnnrnnnnrnnnee 32 33 Allele COUNT EE EE ieee 71 Allele Detection Bin Table ooooocccnnncnononocononoss 77 Allele Detection with Bin Table 76 Allele Editing Opntions 70 Allele Evaluation ooocccoccccncccnncccnoccnnncnnnarinninnns 24 Allele Label 35 108 E EEE EEE 68 Allelic Ladder aeeeeeenereesrnerererserrssrrsrrrers 24 Amplified Fragment Length Polymorphism AFLP 76 Analysis Display Settings ccccssscccccssseceeceeseeeeeeeeeees 35 NEI EC TEE 21 Aneuploidy E 142 Angelman Sv
256. ns without Dor prompting by simply following the program s sequential analysis flow C Ge Wead Wizard Activates the Run Wizard which will guide the user through the program s operation This setting is best for the inexperienced user EG SE General Settings F Show Gel mage Show Navigator When selected the Sample File Tree will automatically be displayed in the Main Analysis window after data processing Show Gel Image When selected the Synthetic Gel Image will an automatically be displayed in the Main Analysis window after data processing Show Report When selected the Report Table will automatically be displayed in the Main Analysis window after data processing The above settings take effect only at start up Preferences Display Settings p y 8 Start up Settings Display Settings Report Settings Others Allele Label Peak Label The Display Settings tab is used to set how the data is displayed in the Dem E ske Height electropherograms Se T Aea T Score T Use Size String for Label I Larger Font Allele Label Highlight abnormal allele Position Peak Top z Decimal Precision Select 0 to 2 decimal places for peak size labeling Dees wae Iw Max H of Open Charts 96 gt I Gray for Single Dye Max Chart in Page ls aj P 35 M Max llele LabelLayersi 6 May 201 2 Show Loci box with multi line Cancel Chapter 3 Main Analysis Overview Mark Off Allele
257. o reduce false positives by setting the thresholds that determine the calling of deletions and duplications to desired settings This is shown as red lines radiating from the origin 0 0 of the regression plot The default setting is Deletion lt 0 75 lt Normal lt 1 30 lt Duplication o E Print Report Launches the MLPA Print Settings box See Reports and Printing section below for more information Load Control Sample From a Database Individually load a control sample to be used in the analysis D Control Sample Selection ciri Launches the Control Sample Selection box which lists the dataset samples that can be selected and used as the synthetic control for MLPA analysis Save Synthetic Sample Saves the synthetic control sample as a Text txt or Trace scf or sg1 file What to Expect After initial analysis with Run Wizard and adjustment of analysis settings the sample data must be reviewed for normalization accuracy sample lane quality and control sample identification prior to final deletion duplication determination Verify Normalization Accuracy Normalization is the adjustment of peak heights in a sample to minimize or remove the effects of preferential amplification of smaller fragments during PCR Notice in the before and after example below how initially the sample s peaks are increasingly shorter as the fragment size increases After the normalization calculation is applied the peak heights are consistent f
258. ocations cannot be transcribed This is important when the methylation occurs in the promoter region of genes If the promoter region of a gene is methylated the gene will not be transcribed and its associated protein will not be produced Methylation of the promoter region for tumor suppressor genes is of great interest for cancer diagnostics When the tumor suppressor gene is switched off because of methylation in the promoter region the cell s growth will be unchecked and cancer may form Likewise if methylation occurs in mismatch repair gene promoter regions genetic mutations within a cell will proliferate thereby increasing the risk of tumorigenesis Second DNA methylation is important in discovering cases of genomic imprinting At conception two copies of each chromosome are inherited from an individual s parents one copy from the father and one copy from the mother Normally it cannot be determined from which parent each chromosome originated however in some cases differential expression of a gene occurs depending upon which parental chromosome the gene resides Additional research linked differential expression of genes with methylation patterns and or histone modifications around the gene of interest Methylation is useful in determining parental chromosome origin and is referred to as genomic imprinting Overview GeneMarker DNA analysis software has been successfully paired with the Multiplex Ligation dependent Probe Amplification
259. of analysis on similar data sets Run Wizard ES Run Wizard Additional Settings og Setings Fragment Animal Analysis Proc e dure Set additional options related to the different analysis type 7 The Run Wizard is almost complete Enter or change the Ze appropriate information for your analysis type Ge z 8 Once the settings have been adjusted to your analysis type click OK and a Data Processing window will appear while GeneMarker sizes the data based on the parameters set in the Run Wizard Click OK when the analysis has finished Peak Score Reject lt 1 Check lt Pass P 0 MLPA Normalization Method r Functions Allelic Ladder Permits the selection of a sample containing an allelic ladder This option is only available when Fragment Animal or HID analysis types are selected Allele Evaluation Peak Score User definable confidence level of the allele call Peak score is an algorithm that takes into account signal to noise ratio and peak morphology Rejected samples appear in red samples that need to be checked appear in yellow and samples that have passed appear in green AFLP Unconfidence at Rightside Score 24 May 2012 Chapter 2 General Procedure Available only when AFLP Analysis method is chosen When selected the user can enter the minimum score at which a peak to the right of the main peak will be called See Chapter 7 Special Applications AFLP MLPA Normalization Method Available
260. oftGenetics Gene Marker D atasets 4FLP frag_001_HO1 fsa 3 Th O H D Fil b al C SoftGenetics Gene Marker Datasets AFLP frag_002_G01 fsa Aa C SoftGenetics Gene Marker yD atasets 4FLP frag_003_F01 fsa i p en ata des DOX W1 appear CASoftGenetics Gene Marker Datasets 4FLP frag_004_E01 fsa rs nae 1 C SoftGenetics Gene Marker D atasets 4FLP frag_005_DO1 fsa 4 Click Add button C SoftGenetics Gene Marker Datasets 4FLP frag_006_CO1 fsa S C SoftGenetics Gene Marker D atasets 4FLP frag_007_B01 fsa 5 The Open dialog will appear C SoftGenetics Gene Marker Datasets AFLP rag_008_401 fsa Remove All oe CASoftGenetics Gene Marker Datasets AFLP frag_009_HO3 fsa 6 Navigate to directory containing raw data files C SoftGenetics Gene Marker Datasets AFLP frag_010_G03 fsa C SoftGenetics Gene MarkeryDatasets V FLPYfrag 011 FU fa 7 Select all files by CTRL A or use CTRL and or SHIFT C SoftGenetics Gene Marker Datasets AFLP frag_012_E03 fsa k 1 ai id 1 I yee ee R oa oe Add Folder ASoftGenetics Gene Marker Datasets rag 014 CO3 fsa eyo to select indtvidua samp ES Ch oftGenecch ene Marker D atasets 4FLP frag_015_B03 fsa Default 8 Click Open button in the Open dialog PASnftGenstics Gene MarkerDatasets AFl Piran MIA AN fra 9 The files selected will appear in the Data File List field i OK Cancel 10 Click OK button in the Open Data Files box and the samples will be uploaded to GeneMarker Features There are several features
261. ols Convert TXT to Binary Tools Convert Text to Binary Files The Convert Text to Binary tool allows the user to upload trace data information in Text txt file format for conversion into a four color SCF file or a five color SGI file The SCF and SG1 files can then be read by GeneMarker and translated into chromatograms This tool is useful for institutions developing their own fragment analysis instruments Procedure 1 Click the Load Text File button and select 1 sp Text txt files to convert 2 Once files are uploaded they will appear in the Text File field 3 The software will automatically calculate a Recommended Ratio for the user to condense the number of frames in a single trace 4 Enter a condense frames by XX number in the Condense Frames field id Convert Text to Binary File Iw Adjust Intensity Use Global Ratio 1 4 23399 65535 0 46 4 23399 65535 0 46 H Export to SG1 5 Click Export to SGI if exporting a five color trace click Export to SCF if exporting a four color trace Export Elecropherogram Tools Export Electropherogram The Export Electropherogram tool allows the user to export the trace images to a specified folder Procedure 1 Use a dropdown menu to specify the output folder 2 Specify the prefix and suffix for the exported file name The full file name will be Prefix Sample name _ Dye name Suffix Extension nam
262. on Surveyor NOTICE TO USER PLEASE READ THIS CONTRACT CAREFULLY BY USING ALL OR ANY PORTION OF THE SOFTWARE YOU ACCEPT ALL THE TERMS AND CONDITIONS OF THIS AGREEMENT INCLUDING IN PARTICULAR THE LIMITATIONS ON USE CONTAINED IN SECTION 2 TRANSFERABILITY IN SECTION 4 WARRANTY IN SECTION 6 AND 7 LIABILITY IN SECTION 8 YOU AGREE THAT THIS AGREEMENT IS ENFORCEABLE LIKE ANY WRITTEN NEGOTIATED AGREEMENT SIGNED BY YOU IF YOU DO NOT AGREE DO NOT USE THIS SOFTWARE IF YOU ACQUIRED THE SOFTWARE ON TANGIBLE MEDIA e g CD WITHOUT AN OPPORTUNITY TO REVIEW THIS LICENSE AND YOU DO NOT ACCEPT THIS AGREEMENT YOU MAY OBTAIN A REFUND OF THE AMOUNT YOU ORIGINALLY PAID IF YOU A DO NOT USE THE SOFTWARE AND B RETURN IT WITH PROOF OF PAYMENT TO THE LOCATION FROM WHICH IT WAS OBTAINED WITHIN THIRTY 30 DAYS OF THE PURCHASE DATE 1 Definitions Software means a all of the contents of the files disk s CD ROM s or other media with which this Agreement is provided including but not limited to i SoftGenetics LLC or third party computer information or software ii digital images stock photographs clip art sounds or other artistic works Stock Files iii related explanatory written materials or files Documentation and iv fonts and b upgrades modified versions updates additions and copies of the Software if any licensed to you by SoftGenetics LLC collectively Updates Use or Using means to access install download copy or
263. on contained in the report is interlinked Double clicking on the cell in the report highlights the cell corresponding sample ID in the sample list data point in the cluster plot and locus in the electropherogram SNaPshot Analysis Sample List Report Table 10 SNPOIOfsa 11 SNPOI1fsa 12 SNPOI2 Isa 13 SNPOIZIsa 5 14 SHPOV4 fsa 10 15 SNPOM fra 16 SNPOIG fra 17 SNPOI7 fsa Gel Image 18 SNP018 fsa 119 SNPOI fra 20 SHP020 ta 21 SNP fra 30 22 SNPO22 fra 3 SNPOZ Isa 24 SNPO24 fsa 25 SNPO2S fa b 26 SNHPO26 ba y 45 8 0 el Sample List The Sample List displays all current samples in the project Single left click a sample name or use the Up Down Arrow keys to scroll through the list Cluster Plot The Cluster Plot displays on a graph of peak angle vs Marker position points representing each sample Choose to show the Cluster Plot in Cartesian or Polar format in the Display Settings box Select a Marker from the Marker drop down menu in the toolbar to view its Cluster Plot Click on points in the graph to see individual sample traces in the Two Color Trace Overlay frame Plot points are separated into groups by green dashed lines The green group separation lines are derived from a RE PRO ARO statistical cluster analysis of the specific dataset and therefore a TT vary for each Marker Points between the green separation lines SES NERS ven 000 rep
264. on with the reference trace and are designed so the user can minimize the number of bars in the Peak Comparison Histogram The image below shows a low stutter filter setting Left 60 Right 40 compared to the same trace with a higher stutter filter setting applied Left 99 Right 99 Notice in the following example when the stutter filter is decreased the number of identified instability peaks increases and vice versa for a higher stutter filter setting 133 May 2012 Chapter 7 Special Applications MSI Stutter Filter Low Stutter Filter High Stutter Filter A second option to help a clinician display only the most relevant peaks in a microsatellite instable marker is through the MSI Score setting MSI Score is calculated using a log ratio plot of tumor sample versus reference In this way the normalized intensities of the reference sample can be accurately compared to the tumor sample thereby presenting a more meaningful analysis To normalize the data a single peak is divided by the average of all peak intensities across a dye color and the ratio is then used for peak to peak comparison In the example below a low MSI Score setting 2 2 is compared to a high MSI Score setting 2 4 for the same sample When the MSI Score threshold is lowered more peaks are identified as unstable red histogram bars As the MSI Score threshold is increased only the most relevant peaks are identified as instable MSI Score Low
265. only when MLPA Analysis method is chosen Two normalization methods are available to correct for variations of PCR efficiencies from small to large DNA fragments or from sample to sample See Chapter 7 Special Applications MLPA Internal Control Probe Normalization The traditional method of normalization using the control probes Population Normalization Unique to GeneMarker this method normalizes peak intensities based on the statistically most probable median intensities Advanced Available when Population Normalization is selected Advanced Population Normalization is meant to be used when more than half of the probes are deletions or duplications Adjust Analysis Parameters After the clicking OK in the Run Wizard Additional Settings box the Data Processing box appears The raw data is being processed and sized then the e filtering parameters are applied and finally a Panel is applied if selected Click 9 zt OK in the Data Processing box when analysis is complete DoD PT fs RR RR S de de ze rm Pi 2 oo anaaqaa a E 3 35 SPP BLAD DAS beer en ar nn e eae a eo amp Review the results in the Main Analysis window See Chapter 3 Main Analysis abre on Overview If you notice many false positive peak calls you may need to adjust o the analysis parameters There are three options for adjusting the analysis parameters as discussed below NOTE Manual
266. ons Deconvolute Method The Deconvolute method is best for very good data with only a few small secondary peaks This method allows users to view the Individual Peaks as well as the Complex Peaks The area calculation performed is only for the Individual peaks but the Complex peaks can be shown to give the user a general idea of the Complex Peak shape and distribution The Deconvolute method calculates the area under each peak including some of the area in between the peaks This method calculates a value by assigning the regions between the peaks to one peak or the other Deconvolute Method Single Nucleotide Polymorphism SNP Analysis Single nucleotide polymorphisms SNPs occur every 100 to 300 bases along the human genome and make up to 90 of human genetic variation Functional SNPs classified as non synonymous SNPs nsSNPs that occur in the coding region of a gene or as regulatory SNPs rSNPs that occur in the promoter region of a gene are often associated with altered protein function or gene expression Intronic or intergenic SNPs may not alter gene or protein function but can be used to address questions in evolutionary biology or in association studies with complex diseases drug response environmental insults quantitative trait loci QTL or genotyping plants and animals Various techniques have been developed to interrogate SNPs including SNPlex SNaPshotTM SNuPE SNPWave SNP chips and DNA sequence analysis
267. ons Tools Help e gt HER amp w e ok IB de Jo Maker f None v a Di PS Report H Bin Help frag_026_GO fsa x HE ET ME es eva fe fe axag 002 601 353 220 240 260 280 300 frag 002 GO1 fs e 1 e rag 019 FOS fs 03 04 05 07 1 17 18 Trace Comparison The Trace Comparison function for AFLP data is designed to identify length polymorphisms between closely related species Trace Comparison uses Poisson distribution to calculate significant allelic differences between samples or closely related species GeneMarker calculates the allelic differences between a user defined 77 May 2012 Chapter 7 Special Applications reference and sample traces the differences are displayed in a histogram located below each sample electropherogram To analyze the data with the Trace Comparison function it is not necessary to apply a Panel Overview After the data is uploaded and filtered with Run Wizard select Applications Trace Comparison from the main menu bar A new folder labeled Trace Compare will appear in the Main Analysis window Sample List When samples are selected in the Trace Compare folder the Main Analysis window will change to Trace Comparison mode Select a Reference sample from the Reference drop down menu in the main toolbar The Reference sample will appear as the top Electropherogram and all other samples selected in the Trace Compare folder will be compared to that
268. ons in the Selected Columns field to the All Columns field Click OK in the Set Peak Table Columns box and the Allele Report Settings box when finished The options in the Selected Column field will be displayed along the top of the table in columns Show when no allele call When selected this option allows the user to specify symbol or short word such as Null when there are no peaks in a marker If deselected the cell in the report would be empty Show Only Uncertain Alleles When selected displays only the peaks with Quality ranks of Check yellow and Undetermined red me ener finnest p ja ja Show Rejected Low Score Alleles When selected the EE I L R RS D135628 S 27 27 1 1051 9178 peaks with peak scores below the Run Wizard EE EE EET Additional Settings Allele Evaluation Peak Score Reject 1 fm 2852 setting will be displayed in the table l l pasaz Hide Extra Sample Names When data is displayed in Vertical 1 presse Orientation the sample names are repeated for each row of ooo data that the sample is associated with If Hide Extra Sample SS HE Names is selected then the sample name will only appear once p 3612 11004 6032 6315 6589 4304 3335 40647 47590 A Nm A ed in the first of the rows it is associated with EE sa LEE parae LP ors 3654 20542 Allele Count The Allele Count Report Style displays the number of alleles present in the Panel Marker SE
269. ontrol While the chosen sample may not actually be the experimental control this selection is based upon which sample presents the data with the fewest abnormal calls The user may change the control to reflect the experimental sample or another sample which they feel portrays the data in a meaningful fashion To select a control sample Click on the Control Sample drop down menu and select the desired control sample OR Select the Load Control Sample From a Database icon Selecting a control sample using this method requires the sample be in TEXT or SG1 file format Synthetic Control Sample GeneMarker s MLPA Analysis allows users to create a synthetic control sample that can be used as a control reference sample This sample is calculated as the median of all selected sample files 87 May 2012 Chapter 7 Special Applications To select sample files to be used in the calculation for the control sample 1 Click on the Control Sample Selection icon 2 The Synthetic Control Calculation box appears TA Synthetic Control Calculation Lelgl 2 3 Click the checkbox next to samples to include in the Synthetic Control PO34_AD9 02fsa 3 NOTE Sample files with a score below the Minimum Lane Score Threshold EI cannot be used in the control sample selection PO34 A1 02 fsa P034_B10_03 fsa 4 Click OK PO34_B11_03 fsa d i _B12_04 fsa 5 The Synthetic Control Sample is automatically selected in the Control SCC P034 C11 05 fsa S
270. ools gt Luminex MLPA Analysis The Open Luminex Data box appears dl Luminex MLPA Data Select a CSV file to analyze Click Open Dries The Luminex MLPA Data window appears van 133084 a Alleles Columns e Dept Users PAL MLPA NPCANPC MLPA 18 Project lxp b Samples Rows c Right click on a row to select the Background FE EET E O ee O ST 0 0 a on 10 I Le NR D An Set As Control Gene sample The software automatically chooses the Le ee overall least intense sample and labels it with a B E sete Background Sample d Right click on a column to select the Control Gene AE If you do not select control gene then the software e Jorge will automatically select the best control given your aE data set md oe e Suspect Count in the bottom left corner is set to Pte default 100 Suspect Count represents the e CN E threshold for bead number any sample with less than the Suspect Count threshold will be flagged for user inspection 8 Click OK in the Luminex MLPA Data window MLPA Analysis Settings 9 The MLPA Analysis Settings window opens Analysis Method Tee 10 Recommended Settings MLPA Ratio C MLPA Regression T Dist Fre TEGNE a Analysis Method MLPA Ratio ere tala MLPA Ratio to Copy Number b Settings Gm MLPA Ratio Deletion lt 0 75 lt Normal lt 1 25 lt Duplication Ock 0 00 ES c Deletion lt 0 75 lt Normal lt 1 30 lt Duplication mn d Minimum Lane Score T
271. or in the Report Table where peak ratio or simple loss gain information can be displayed All frames in the MLPA Analysis window are interlinked for example clicking on a cell in the Report Table will highlight the sample in the Sample List the peak in the Overlay Trace and the plot point in the Ratio Plot MLPA Analysis Trace Overlay amp Sample List Dosage Histogram Ratio Plot Report Table P E Fre box Iva 5 531 SET POH E12 1003 i PCS Fil 1 pa Han POL D 11 ies ap DEL 12 na BE sp TMS d WOH Gii Ota zF LPA Deiis TEEN A CDI he P lee 23 FM MI 15 ha Ee Fi HIT 18 628 FO HVA 16 ha PIM 012 04 fa a LJ gie D mer Dee poh ony der dl te ee AE A 164 200 Ipi 250 pin FO D42 OG ba 80 May 2012 Chapter 7 Special Applications Sample List The Sample List includes the filenames of all the samples in the dataset and their respective MLPA Score The MLPA Score is the signal to noise ratio that corresponds to the quality of the trace The larger the number is the higher the quality of the lane Additionally a number sign appears before the sample with the least variation that was automatically selected as the Control Sample This sample will also appear in the Control Sample drop down menu in the toolbar See the What to Expect section below for information regarding control samples and synthetic control creation Single left click a sample in the Sample List to display the sample s Overla
272. osition of the disabled peak is reported for each sample If the disabled peak is at the beginning or end of the Size Standard no basepair size position will reported Size Calibration Charts Sample List Calibration Plots Sample ILS Disabled Size Statistics Size Standard Trace core 5 ben 500 2500b 43000 00b o ye so lu Soo e a ww ERARIO Y DDD 00 00 00 40 40 40 40 40 40 DAD 5 S s S SS Q ZE RE BERE BRE E BE BEE EEE ESES Obos 500te 2500te Leggre 5 10 A 00 0 Disabled Size Statistics If Sizes were disabled in the Size Standard see previous section Size Template Editor then the Disabled Size Statistics table will appear in the bottom left corner of the Size Calibration Charts window The average basepair position the standard deviation and the difference between the maximum and minimum basepair positions across all samples are calculated for each ILS peak matched to the disabled peak s position No statistics will be calculated for disabled peaks at the beginning or end of the Size Standard Size Standard Trace The Size Standard Trace displays a synthetic trace of the selected Size Standard Enabled Sizes are red disabled Sizes are grey Each peak in the Size Standard Trace represents the expected basepair size of peaks in the sample ILS Sample ILS The Sample ILS displays the currently selected sample s ILS tr
273. otherwise benefit from using the functionality of the Software in accordance with the Documentation Permitted Number means one 1 unless otherwise indicated under a valid license e g volume license granted by SoftGenetics LLC Computer means an electronic device that accepts information in digital or similar form and manipulates it for a specific result based on a sequence of instructions SoftGenetics LLC means SoftGenetics LLC State College PA 16803 2 Software License As long as you comply with the terms of this End User License Agreement the Agreement and pay all license fees for the Software SoftGenetics LLC grants to you a non exclusive license to Use the Software for the purposes described in the Documentation Some third party materials included in the Software may be subject to other terms and conditions which are typically found in a Read Me file located near such materials 2 1 General Use You may install and Use a copy of the Software on your compatible computer use the Software on a computer file server provided concurrent use does not exceed the Permitted Number No other network use is permitted including but not limited to using the Software either directly or through commands data or instructions from or to a computer not part of your internal network for internet or web hosting services or by any user not licensed to use this copy of the Software through a valid license from SoftGenetics LLC and 2 2 Backu
274. oup and 4 group are derived from the same cluster It can also be seen from this example how average linkage is an amalgam of single and complete linkage Fig 3 Correlation Coefficient Complete Linkage 2 Clustering Analysis al Gray Scale 0 7 orrelation 3 C11 v 484 3 G11 v 491 3 D11 Y 485 3 F9 1 427 442 537 4 E1 v 532 4 G2 v 545 4 D1 Y 528 B 4 G1 Y 535 4 B2 Y 539 4 B3 Y 556 7 D7 v 597 b e 2 Clustering Analysis Eh al Gray Scale 0 6 0 7 0 989 3 C11 v 484 3 G11 Y 491 3 D11 Y 485 3 F9 1 427 442 537 4 E1 v 532 4 G2 Y 545 4 G1 Y 535 4 D1 Y 528 B 4 B2 Y 539 4 B3 Y 556 7 D7 v 597 b Detecting Euclidian Distance Press Ctrl and click anywhere in the linkage chart under the scale a dashed vertical line will show up indicating that distance Chapter 7 Special Applications Sub cluster Report and Saving Right click to obtain a pop up menu Left click to select SubCluster Report The table shows the grouping at that particular distance as well as the presence or absence of each allele for each individual sample with 1 indicating presence and 0 indicating absence The summary row of each group indicates the consistence or inconsistence at each allele for all individual samples in that group with 1 indicating consistence and 0 indicating inconsistence If all individual samples have value 1 at a certain allele then they are consistent Otherwise they are not Differ
275. ox determines the Print Settings initially chosen Samples Choose All Samples or select specific samples to display in the report Markers Choose All Markers or select specific Markers to display in the report Ratio Plot EEE When selected a separate page with the Ratio Plot for all Markers in a dye color a E E aaisa will be printed for each sample Choose to Show Population to include all ratio data points in the dataset Choose Show Sample Only to display only the sample s data points in the Ratio Plot amp Markers jw Ratio Plot Ze All Markers Show Population C Selected Markers C Show Sample Only Report T able Iw Show Allele Name Iw Show Peak Height Area Report Table Iw Show Peak Ratio Select the information to display in the Print Report s Report Table Electopherogem Size Range C Show Current Size Range Show Trisomy Scores Electropherogram Size Range C Show All Markers Size Range Show Custom Size Range Show Current Size Range Displays the Trisomy analysis window Electropherogram in Ste 50 pute Iw Scale data to highest peaks within size range its current zoom mode Show All Markers Size Range Electropherograms will be displayed so that all selected markers can be clearly viewed Show Custom Size Range Expands the Electropherogram view to the specified range Scale Data to Highest Peaks Within the Size Range Increases the peak heights of low peaks to approximately the s
276. p Copy You may make one backup copy of the Software provided your backup copy is not installed or used on any computer You may not transfer the rights to a backup copy unless you transfer all rights in the Software as provided under Section 4 2 3 Home Use You as the primary user of the computer on which the Software is installed may also install the Software on one of your home computers However the Software may not be used on your home computer at the same time the Software on the primary computer is being used 2 4 Stock Files Unless stated otherwise in the Read Me files associated with the Stock Files which may include specific rights and restrictions with respect to such materials you may display modify reproduce and distribute any of the Stock Files included with the Software However you may not distribute the Stock Files on a stand alone basis i e in circumstances in which the Stock Files constitute the primary value of the product being distributed Stock Files may not be used in the production of libelous defamatory fraudulent lewd obscene or pornographic material or any material that infringes upon any third party intellectual property rights or in any otherwise illegal manner You may not claim any trademark rights in the Stock Files or derivative works thereof 2 5 User acknowledges and agrees that the Software is licensed by SoftGenetics for research use only Any violation of this restriction on use shall constitute a bre
277. p between different nodes or individuals in the sample dataset Older generations appear at the top of the Pedigree Tree younger generations near the bottom Males are represented as square nodes females as circle nodes Horizontal lines indicate mates vertical lines indicate offspring Once a node is added to the Pedigree Tree right click the node to see additional options Select Deselect Individual Node To view the electropherogram for an individual right click their node and select Select Node OR double click the node and the electropherogram trace will appear in the Charts tab on the right To hide the sample s electropherogram right click the node and select Deselect Node When a node is selected in the Pedigree Tree the corresponding sample in the Sample List becomes highlighted 103 May 2012 Chapter 7 Special Applications Add Edit Delete Individual If an individual is the first to be added to the Pedigree Tree a family must be designated for the person If the person is already added to the Pedigree Tree right click and select Edit Node to change that person s characteristics To delete an individual from the Pedigree Tree right click the node and select Delete Node Family Name Enter a name for the family in the free form text box The Family Name field will not appear after the first individual is added to the Pedigree Tree All subsequent individuals added will be considered members of the family Person Info Name
278. pecific probes coupled with AFLP primer selective amplification SNPWave has the advantage of accurate high throughput genotyping of up to 100 SNPs Circularizing padlock ligation probes are constructed that are specific to the SNP and flanking sequences Locus specific probes will hybridize to complementary denatured genomic DNA Allele specificity is determined by the SNP at the 5 end of the padlock probe Probes that contain a 5 nucleotide complementary to the SNP will be ligated and amplified by PCR in subsequent reactions Probes that do not contain a 5 nucleotide complementary to the SNP will not be ligated and will not be amplified The padlock probes contain stuffer regions and primer binding sites for AFLP selective amplification The ligated padlock probe is amplified using fluorescently labeled 2 selective and unlabeled non selective AFLP primers The stuffer region provides length discrimination between alleles and among loci SNPs are separated by two basepairs and loci are separated by three basepairs The fragments are separated by size using capillary electrophoresis Fragment dye color and length indicate SNP locus and allele 129 May 2012 Chapter 7 Special Applications Analysis of SNPWave data follows the same procedure as SNPlex analysis See SNPlex section above SNP Analysis Reporting The Report Table in the SNP Analysis window by default displays the SNP type or alleles detected in the Marker Click a cel
279. pens the Print options box Select a printer the print range and the number of copies Export Report to Files Export Format JPEG Image DI f E JPEG Image File Naming M PNG Image Ze Named by sample name Export to File T Start by Page Number Opens the Export Report to Files box Save each page of the Print named by page number Report as an individual image file PNG or JPEG Export Directory C Program FilesySoftGeneticsyNGeneMarkeri1 710 EN Name Type Named by sample name saves each PNG or JEMES Pec Image 407_01_01 jpq JPEG Image JPEG under the sample name am 02 pg JPEG Image 408 02 Ol jpg JPEG Image E3 Pgi0 207 _05 jpg JPEG Image Start by Page Number combines the page number and the sample name for Pgli_c07_05 Ol jpg JPEG Image the saved file name Paz 08 Deg JPEG Image Named by page number saves each JPEG file by the page number within the Mame report Genemarker Pg1 jpg A GeneMarker_Pgq2 jpg Ss Genemarker Pgq3 jpg Page Setup Opens the Page Setup box Choose the paper size margins and orientation Portrait or Landscape O T F Content Options Opens the Print Report options box See the section above Report Content Options Zoom to Fit Zooms out to view the entire Print Preview page Zoom to Width Zooms in to view the Print Preview page at maximum width without losing information off the screen E Zoom Ratio Enter percentage numbers to
280. ppears and includes information like Sample Name Well ID Lane Number Instrument Name venue Machine MEGABACE 1000 and Chemistry The list of Properties varies depending on the file type Hot Weier 4 Chemistry ET ROX FAM NED HEX SES E e Edit Comments Sa a Opens the Edit Comments box Enter information in the Comments field The Ka aras last ten comments will be stored and can be subsequently selected for future fo EGIL samples The Sample Comments will appear on the Print Report See Chapter 6 Reports and Printing Hot Key F4 Disable Samples Disable Sample Opens the Input Disable Reason box and marks the sample with a red strike put Disable Reason e through A disabled sample cannot be selected for display in the Main E Analysis window and will not appear in the Report Table if View Preference Options Show Disabled Samples in Report is deselected Select a Comment Template or enter a new comment in the Comments field and click OK to disable the sample Hot Key CTRL DEL Comment Templates PCR Failed Bad Signal Water Sample Add Samples From the main toolbar select Project Add Samples to Project The Open Data Files box will appear Click the Add button to select additional samples to add to the project and click OK The added samples will be sized and the es allele calls will be filtered according to the parameters
281. print or save the MSI Analysis Report Groups MSI Print Settings ZS Choose All Groups or select specific groups to display in the report Groups Print Reference Sample All Groups S When selected a separate page with just the reference sample will be printed for each group Print Reference Sample Iw Show Difference Histogram Show Difference Histogram When selected the Gain Loss Histogram will be displayed below each Coen dye color s Overlay Trace electropherogram OP ob d il S P C Selected Markers ES Markers Choose All Markers or select specific markers to display in the report Shore Erect a netog ens K Cunent aie Hanga Show Electropherograms in Current Size Range E CS When selected will display Overlay Trace electropherograms in the same zoom mode as the main MSI Analysis window When deselected the electropherograms will be displayed so that all selected markers can be clearly viewed MSI Clinical Report Below is a description of the MSI Clinical Report features For an explanation of functions within the Print Preview window see Chapter 6 Reports and Printing Report Header Contains information about the analysis project sample and parameters Signature Box Date and initial space for report reviewers Overlay Trace Electropherogram Similar to the main analysis window displays the reference trace in light red behind the sample trace dye color Gain Loss Histogram Also
282. project originally loaded into GeneMarker will be marked as the Reference R gt and the second project uploaded to the Project Comparison tool is marked as the Sample S gt 7 Click the Project Comparison Settings icon to choose parameters to compare between the projects 8 Differences will be indicated in the report table on the right When a difference is selected each project s electropherogram and peak table will be displayed on the left Project Comparison Tool i Project Comparison Sm EECH al Ed R gt 052_404 5CF Sample Marker Allele1 lele2 Allele 3 D D2151437 115 DEI Ja D135528 327 1 400 0135634 m 1 200 D185535 473 1000 foresto Ja we pesma 178 P om Dese Ia gt 01353205 450 20 er Ia 0 D2151411 325 052_404 5CF 052102151437 1127 D21511 r de 247 D135634 380 390 400 410 420 430 411 S gt 052_A04 SCF Edited SGF D135634 380 390 400 410 420 430 x 1 400 1 200 1 000 D2151411 304 D2151437 127 in ae Je D21511 257 01355628 Loo 0135634 m D185535 473 HO D1851002 120 LL Jee 178 174 May 2012 Chapter 8 Additional Tools Icons and Functions Open Project to Compare el Opens a directory window for the user to identify a similar project to compare to the project already running in GeneMarker The first project in Gen
283. r additional options in the Report Table Procedure 1 Launch GeneMarker software MSMLPA Analysis Settings Group File 2 Import raw capillary electrophoresis data 5 3 Apply size standard and peak filtering options with Run Wizard A 22 el 4 Select a panel in Panel Editor and align Markers and Bins accordingly ee 5 Select Applications gt MS MLPA Promoter Methylation Genomic Imprinting 6 The MS MLPA Analysis Settings box will appear Quantification 7 Import grouping information created in the Filename Group Tool into the eee a Ge Group File field or import Common Reference SG1 5 color data SCF 4 Peak Ratios color data FSA file format into the Common Reference field or enter both a o Eee Group File and Common Reference A synthetic reference or control may be K Auto Slope Conection constructed and saved using this function in the MLPA analysis window ane eee see above A Common or Synthetic Reference must be imported into the Cancel project to be available for use click Project in the menu bar and select Add 96 May 2012 Chapter 7 Special Applications Samples to Project 8 Select an MS MLPA Analysis Type Promoter Methylation MSMLPA Analysis Settings 9 Select Promoter Methylation Analysis Type GE 10 Choose to analyze by Peak Height OR Peak Area Setzer 11 Enter the Peak Ratio value below which a peak will not be PA_ME028_SCF Synthetic Conto Sample 2 34 0f p MS MLPA Analysis T
284. r database searching Default setting will display most likely same individual father son mother daughter sibling and half siblings When multiple files are located for a specific kinship level they are ranked by likelihood ratio LR Search Report Display Gender Determined Minimum LA Value 11 000 Maximum File Number 10 jw Display Genotypes jw Display Locus Identifier Save Parameters when Save Report Search Scope Iw Sample Individual jw Father Son Mother Daughter jw Full Sibs Half Sibs Cancel e New Pedigree File Select New Pedigree File to create a new pedigree with multiple families OR select New Family to add a family to the pedigree file Enter the first family member s information into the New Family New Individual box and click OK to create a new Pedigree Tree E Open Pedigree File Launches the Load Pedigree File box automatically upload and click OK Select a PED or PRE file to upload the SMP file will Save Pedigree File Launches the Save Pedigree File box Enter filename and change directory to save the Pedigree Files PRE SMP DAT Show Individual Name When selected the individual ID will be displayed in the nodes of the Pedigree Tree Update Sample Data Select to refresh the Mendelian inheritance calculation after a node or allele is edited and after selection a different family when the show genotype display is used ICO Relationship Testing Parameters Launche
285. r from character 1 to 16 in file name Clear Close A e GA kA 106 May 2012 Chapter 7 Special Applications Create New Pedigree Edit Individual 1 In Pedigree tool click the New Pedigree File ier erson Into Affect Status or Mame Woe Unknown 2 The New Family New Individual box appears R 3 Enter a Family Name the Individual s Name Gender Male C Female C Unkown SE Gender and the Sample File to associate with Father 2 Ole the Pedigree Tree node 4 Click OK mik 8 5 The individual s node will appear in the Pedigree Tree and the individual ID information will appear next to the sample Cancel name in the Sample List 6 Continue to add family members by right clicking at the node and selecting Add Mate Child 7 Mouse over red highlighted nodes in the Pedigree Tree to view Conflicts and Suspect Markers see section above Pedigree Tree 8 Edit alleles in the Electropherogram Charts as required see section above Electropherogram Charts Sample File FPAT_3_F fea Save Pedigree File After the Pedigree has been created and modified there are two options for saving the information save the Pedigree File and or export the Pedigree Tree as an image Save Pedigree File 1 In Pedigree tool click the Save Pedigree File icon 2 The Save Pedigree File box appears hae ACE 3 Click the Save icon next to the Pedigree File field ee 4 The Windows Explorer directory
286. r installation of the software Installation 1 Insert the SoftGenetics CD into the CD ROM drive If your computer eeermmm x is not set to automatically open a CD navigate to the optical or CD Oe e vieeonetoGenelakerv220 Soup progam The progam ROM drive on the computer and open the directory Double click the GeneMarker Setup executable file EXE The Installation Wizard will launch Click the Next button in the Welcome window Read the SoftGenetics End User License Agreement and click the I Agree button in the Read Me File window 6 Select Install GeneMarker Recommended in the Select Program window and click Next 7 Click Next in the Destination Location window to install GeneMarker in the default folder Click the Browse button to choose a different It is strongly recommended that you exit all Windows programs before running this Setup Program TI Click Cancel to quit Setup and close any programs you have running Click Next to continue with the Setup program WARNING This program is protected by copyright law and international treaties SH es PIS Unauthorized reproduction or distribution of this program or any portion of it may result in severe civil and criminal penalties and will be prosecuted to the maximum extent possible under law x installation directory E i Select Program NOTE The default Destination Location for the GeneMarker program is Ke CA ProgramFil
287. reation section below Be default the detected alleles are reported in the table Other value display options include Peak Ratio and Probability See the SNP Analysis Reporting section below Procedure SNP analysis in GeneMarker requires the data to be sized and a Panel applied prior to launching the SNPlex SNaPshot module SNaPshot specifically requires the association of dye color to expected nucleotide which can be accomplished in the Panel Editor Below is the procedure for filtering with Run Wizard and creating a SNaPshot Panel SNaPshot Run Wizard Settings For explanation of specific functions in Run Wizard see Chapter 2 General Procedure Run Wizard Template Selection Set Run Wizard Template Selection ato Tri co Pa Panel NONE OR chose custom Panel Size Standard User defined tt a F5 Analysis Type SNaPshot eer EE NOTE A Panel is required to analyze SNaPshot data Choose NONE in A Se the Panel field only if a Panel has not yet been created ez KR Use leet template Run Wizard Data Process SNaPshot Analysis Run Wizard Data Process au Peak Detection Intensity Threshold 100 mee Global Percentage 1 Max 7 ha ana E Panama Local Percentage 30 smooth 3 Pes a Gees Max Call Intensity 30 000 Sr ences ak ae gee Stutter Peak Filter Left 25 Right 25 w Plus A Filter Selected Run Wizard Additional Settings peee Allele Evaluation Peak Score Reject lt 3 Chec
288. resent samples where both alleles are present in the Marker az N wars Points above and below the green separation lines contain only one peak or allele Statistical information about the Cluster Plot can be displayed by selecting Show Clustering Information in the Display Settings box 123 May 2012 Chapter 7 Special Applications Right click at a point in the Cluster Plot to modify the sample s allele call at the selected Marker A fly out menu will appear with options to change the SNP type When the SNP type is changed in the Cluster Plot the Report Table is automatically updated Two Color Trace Overlay The Two Color Trace Overlay displays the electropherogram traces for two associated dye colors based on the Marker naming convention used when the Panel was created See SNaPshot Panel Creation section below Since SNaPshot uses single base extension technique expect the associated alleles to be separated by approximately one base pair with some deviation with regard to the different dye sizes and weights The allele editing options in the Two Color Trace Overlay frame are similar to the Main Analysis window Electropherogram options See Chapter 3 Main Analysis Overview Report Table The Report Table lists the sample filenames in rows in the first column on the left The associated Markers appear as column headers Alleles in two dye colors are combined into one Marker by the Marker s naming convention See the SNaPshot Panel C
289. rmation rrrrnnnnrrnnnnnnrrnnnnnnrrnnnnnnnere 81 Show Toggles rrorrrrrnnnnrrrrnnnnnnrrnnnnnnrrnnnnnerrnnnnnssrnnnnnsssnnnnn 40 Show Type Symbol 69 Show Hide rrrvvnnnnnnnnnnvvnnnnnnnnnvvvnnnnnnnnnuvennnnnnnnnuvenene 28 Show Hide Toggles neeeeeeeeeeeeeseeesseeeeeeeesesees 40 MENES 137 Single nucleotide polymorphisSMmS sesssesssssenssssesssn 122 Size Calibraton 41 52 Size Calibration Charts 51 EN 22 Size Call Methode 22 TEN 48 ne decada 21 Size SEAN 21 44 Size Standard Custom 46 Size Standard LIST E 44 Size Standard TE EEN 50 Size Standarde 46 Size Standards bre Deftned 46 Size Table rrrrrrrnnnrrrnnnnnrrrnnnnnnrrnnnnnnrrnnnnnnrrnnnnnssernnnnsssnnnnn 45 Size Template Editor anen 21 39 44 48 Smooth vred 17 18 21 SNIP PIE Le 106 E e narrar ia 38 E e e O ENEN 122 SNaPshot Panel Creation oocccccccocnnnncnnnnnnnnnanonnnnnanos 124 SNaPshot Run Wizard Settings 124 lee 122 SNP Analysis Settings 000eneoeeeneeeeeenssreeene 125 128 T Hr 122 SNP Cluster Plot 123 124 125 126 127 SNP TYPO ee 130 PEN 38 122 ME 127 VE Pils 128 SNPlex Panel Adiustment 128 SNPlex Run Wizard Settings 000nnooseeeeseeeeeseen 128 MI 122 129 lee 122 Sort by Column E 34 NNN 34 Sort EE 29 Sorting el ele EE 29 Space Between Neighboring Peak 121 Species specific allele freguenchy 113 Spike Removal eeneeeenssrresrrresrrrsr
290. rmine frequency domain Use only top 40 of lowest frequencies Smoothing broadens peaks and therefore you can lose resolution Enhanced Smooth Same as Smooth but use only top 20 of lowest frequencies Baseline Subtraction Use 20 of lowest intensities to the right of the beginning of the range Looks at trace in 500 600 frame sections 18 May 2012 Chapter 2 General Procedure Pullup Correction Ax B A being the major coefficient Input matrix or use single dye adjustment up to 0 20 for small corrections When Manual Pullup correction is chosen a txt or mtx matrix file can be uploaded and used to deconvolute dye colors NOTE De select automatic Pullup Correction in the Run Wizard Data Process box if a manual matrix correction has been applied Saturated Peak Correction ABI instrument saturated peaks are typically gt 8000 RFU The top of a saturated peak looks split A small pullup peak may be present under the saturated peak GeneMarker takes the small pullup peak and adds it to the split in the saturated peak Spike Removal Caused by overheating of camera chip voltage spike etc Spikes usually only 1 2 frames wide peaks usually 5 10 frames wide Create a first derivative trace of the raw data Spikes are the 1st DT outliers 3 5 sigma 19 May 2012 Chapter 2 General Procedure Second Derivative Trace A1 A2 A2 A3 A1 A3 2 A2 Use when you have a fat base to your peaks ex Dye blob under peak e
291. rom the Edit History List to view changes in the Current Old Values table Changes will be highlighted in red To recover a change right click the row in the Edit History List and select Recover Old Value A star will appear in the Recover column Click OK and click Yes when the warning prompts you to confirm Click the printer icon for a print preview and print or save as pdf options Edits History Window BER Current Old Values Joe Fa betr se fases pe Maker falle ee oa Sone Pa Current Value 0 136 1 10097 1 00 60356 1 00 DarCP0026 136 OldValue Blue 136 1 10097 1 00 60356 1 00 QarCP0026 136 Edit History List _ Edit Time Deene NE Lesser 07 12 2011 15 42 42 General Hospital Admin Confirm Allele AlleleChart 180 May 2012 Index Index 2 EE 168 207 Derivative Trace 17 A PT 101 ABI SNPlex Panel 128 Abide by Panel cccssscsssessccssseccssssscasseccsssseaes 73 89 Abide By Panel rrrrnnrnnnnnnrnnnnrrnnnnnrnnnnnrnnnnvernnen 69 71 135 Tol 40 Access TE 178 Account and Password 7 8 10 12 acute myeloid leukemia LAML 146 LINE TTT 33 Add Family Members noonnneosennnsseenesssenesssrressseres 104 Add les 16 Add Folder sissccovcensccsndousscasduscessvdonsecessnsn cededbesieves penis nro 16 Add Samples to Project 29 37 Add Size Peak oooccccoccccccccnocinncccnaccnnaccnna nacos 51 Add User rarrnnnnrnnrnnnarnnnnnrnnnnrnnvnnnnnnnnnnnnnnnnn
292. rom the beginning to the end of the trace Normalization is important for MLPA analysis because the sample s peak heights areas are compared to a control reference trace If the peak heights are not consistent then false positive deletion duplication calls may result 85 May 2012 Chapter 7 Special Applications Before Normalization After Normalization S EN 3 il va UTE I Ad se E mal H Ve sa 1 BE Aa pee rr cer ser h her R Arn kote enhet oh men Bike Kr det me G rs ee m ne Ve te L j RT J Weu AA MOUK To verify normalization accuracy in the MLPA Analysis window locate the color coded triangle in the bottom right corner of the Ratio Plots A green triangle represents successful normalization yellow represents normalization that needs to be verified for accuracy and red indicates the normalization failed for that sample If several samples failed normalization go back to the Run Wizard and change the Internal Control Probe or Population Normalization Advanced Normalization option in the Additional Settings box NOTE A Panel must be applied to the dataset in order for normalization to occur Due to the variations of PCR efficiencies from small to large DNA fragments or from sample to sample two selectable normalization methods are provided GeneMarker can normalize MLPA derived data by an internal control probe method or by a population method In order to correct for the peak intensity variation over size
293. rrrsrrssn 17 19 22 Standard Color 21 Start up KN dTM 35 VE 40 stutter M EE EEEE E 79 Stutter Peak Filter 24 Sub cluster Report and Saving cccccsseseeeeeseeeeeeeees 140 SE E 105 EE 94 Synthetic Control Sample 87 Synthetic Gel Image oocccccccncccncc 16 28 30 31 35 40 system requirements rrnnanrnnanennenennernnnnvnnanennenennesnnnneenn 6 187 T T DISTIDU O Mera ia 84 Template Name occcooccncccnocccnnononocnnononononcnnanonnncnnannnos 20 Template Gelecton 20 Terminal Restriction Fragments cccccsssseceeeeeeeeeees 79 Terminal Restriction Fragment Length Polymorphism T PL 79 TILLING oooccccooccnnccnoconononononononononononnnonononnoncnnnnonanonos 39 TILLING Analysis cccccooccnncccnccnnncnoncnnnnnnnconnnonarononnnnoos 155 TILLING Mutation Report A 156 TV 40 Toole IY E 39 Trace Comparison neeenessensseeesseesseessrerssereseee 38 77 Trace Comparison Histogram ccseccceeccessccaeseeaeeeees 131 Trace Mode r rarnnnnnnnnnrnnnnnnnnnnnnnnnnnnrnnnnnnnnnnnnnvnnnvenne 57 64 festege 64 n E a N A E S eraceaaees 79 Triallelic Homozvgote 146 Trisomy Analysis ennneeeennesseennssreressreressreressreress 38 Trisomy Analysis Settings 143 Trisomy Best Practices Guidelne s 143 Trisomy Detection E 142 Trisomy detection limite 144 Trisomy inconclusive range rrrrnnnnnrnnnnnnnrnnnnnnnrnnnnnneene 144 Trisomy Print Report 147 Trisomy Print Report Set
294. rs and a list of Markers or loci with suspect peaks will appear Single left click 104 May 2012 Chapter 7 Special Applications the Marker name and the individual s electropherogram will appear in the Charts tab on the right The suspect Marker will appear red Show Inheritance Conflicts fl Pedigree Edit New File lyv a TI E Family 2 2 METTES Maker 3 D75820 y Search f e d Samples la Charts B QQ BE ral 4 y 279878 30_07 fsa x G Bil 245 250 255 260 265 270 275 280 285 290 295 300 WW E ft S Conflicts D75820 D195433 D18551 279878 10_06 fsa FGA 63 D75820 245 250 255 260 265 270 275 280 285 290 295 300 Suspect CSFIPO THO1 4 000 WA 2 000 279879 30_11 fsa 245 250 255 260 265 270 275 280 285 290 295 300 Person ID 5 Person Name Tam Sample File 279879 30 11 fsa Samples List The Samples List displays the filename and individual ID for each sample in the current dataset Drag and drop samples from the Sample List to nodes in the Pedigree Tree to associate individuals with the family Electropherogram Charts The electropherogram traces for the samples in the Sample List appear in the Charts tab Double click a node or single click the Marker name in the Conflict or Suspect list of the Pedigree Tree and the electropherogram trace for the Marker will appear If a Child node is selected the Mother and Father traces will also appear in the Charts tab Navigation a
295. rtical Report Contents Selecting Status will place a or sign in the cell for each detected allele to represent the presence or absence of alleles o shownamalPeaks respectively Selecting Peak Ratio will display the ratio value of the M ShowFatiomPercentage sample peak height or area compared to the reference peak Cancel Orientation Horizontal orientation displays the Report Table with samples listed in a column on the left and allele peak ratio values in rows Vertical orientation displays samples in a row along the top of the Report Table and allele peak ratio values in columns Show Normal Peaks Displays the peak height ratios of peaks within the methylation thresholds set in the MS MLPA Analysis Settings box Show Ratio in Percentage Displays peak height ratios as percentages Save Parameters when Save Report Automatically generates an ini file of the MS MLPA Analysis Parameters when the MS MLPA report is saved Naming convention for the file is Report file name MS MLPA Settings ini Save Report Choose to save the Report Table as an Excel file xls or a tab delimited Text file txt 99 May 2012 Chapter 7 Special Applications MS MLPA Print ReportSettings Click the Print Report icon to launch the MS MLPA Print dialog box and preview print or save the MS MLPA Print Report p MS MLPA Print ES Samples Select All Samples or just the samples you wish to report To Select EC
296. ry Audit Trail i User Manager Admin logged in User Manager History Settings V Overtime Protection Wat 30 minutes el ES Iw Record Data Edit History Iw Run User Protection When Record Data Edit History is selected in the User Manager Settings box see User Management Settings section above any change to allele calls in the analysis will be recorded Changes can also be recovered in the Edit History feature Procedure 1 Click the Show Chart Table icon in the Main Analysis window 2 The Peak Table will appear below the sample electropherogram 3 Make changes to allele calls by right clicking any cell in that allele s row in the Peak Table or right click the grey vertical bar at the center of the peak in the electropherogram 4 Choose to Edit Allele Edit Comments Add Delete Allele and Confirm See Chapter 3 Main Analysis Overview 5 Once a change has been made to the allele call notice the pink shading in the No column of the Peak Table This indicates a change has been made to that allele 6 Right click any changed allele and select View History 7 The Show Edit History window appears 179 May 2012 d GeneMarker HID Untitled B 2Q6E 72 lo anne d E a Marker Ss gt FER amp BS DR DI tea 0851179 15 we Comments Dusky Rea amp a e DDVUVDUDUODO Owaecworves Chapter 9 User Management 10 11 Select a change f
297. s enn 68 69 71 72 Hide LOSSES sara 40 Hierarchical clustering nnana 136 A A 179 Horizontal Movement 30 Horizontal Orientotion 69 72 elef EE 97 egent dl Ce 101 CONS aaa 40 Identity by Descente 110 mass DUNES ua 30 Implement Y Axis Settings cccccoocccnnccnoccnnnonaronnnonanonoos 73 Importa Panel ind 21 Import ABI Prev 60 63 Import ABI Size Standard 47 Import LE 63 Import Panels from GeneMapperf oeossseeesseeneseensn 60 Import Pre defined Pan elSs ooomccccccoonnnnnonanonnnonanos 61 Import Pre Defined Pan ls 0 63 83 Import Size Standard 47 independent assortment 103 Independent assortment 109 Mmdividual ID dass 104 Individual Node 103 Individual Peaks iodo dana 122 Individual Sample Accordance File 106 inheritance Contlicts aura 105 Insert a Peak at this Bin Site 00001000aeneoseennsseeeen 70 InsertAllele oia as 33 E SE ae 45 SAMA EE 6 Installation Wizard aiii 6 7 9 Instrument NaMe A 29 li ses coms o A T 24 Intensity Coefficients 0 nooeeeeeeseennsseeeesseeeesseerene 21 Internal Control Probe Normalization 25 86 Internal Lane Standard LS 31 44 Internal Probe Normalization rrrrnnnrrrnnnnnnrrnnnnnnrrvnnnnrr 82 International Date Format ooocccnnccnccnnnccnannnnnonanonononanos 37 K e pr 76 129 MS 1 E AEEA E AA P S A ET 110 Kinship Analysis Tolerate Mutat
298. s The default setting automatically sets the Y axis according to the maximum peak intensity of the samples Two other options are available auto fit the Y axis using peak intensities of the alleles or the user can select the ranges for the X and Y axis TE Editing peaks if a peak is called that you determine should be deleted S N ratio close to cut off value or no complementary fragment observed right click on peak in mutation chart trace and select delete from the pop up menu The deleted peak can be undeleted or an uncalled peak can be added using the same pop up menu What to Expect Increased sensitivity is a big advantage to using GeneMarker EcoTILLING samples which may contain one mutated portion out of 12 pooled samples may yield very low levels of the cleaved fragments We are able to detect variations in EcoTILLING samples where the variant may contribute to only 4 of sample Some researchers have found 30 more DNA variations by using this tool In some cases the intensity of the SNP may fall below the level of noise and making visual inspection impossible these variants can now be found when the reference noise is removed from the sample Because the sum of the sizes of each color should equal the whole amplicon size we are able to detect the cleavage sites in EcoTILLING sample using GeneMarker s Tilling Application module False Positives often caused by chemistry
299. s and Markers appear at the top in columns Vertical Sample names appear in the far left column in rows with Markers listed in the second column Alleles in order of basepair size appear at the top in columns Show when no allele call When selected this option allows the user to specify symbol or short word such as Null when there are no peaks in a marker If deselected the cell in the report would be empty Show Only Uncertain Alleles When selected displays only the peaks with Quality ranks of Check yellow and Undetermined red Show Rejected Low Score Alleles When selected the peaks with peak scores below the Run Wizard Additional Settings Allele Evaluation Peak Score Reject setting will be displayed in the table only appear once in the first of the rows it is associated with Bin Table AFLP MLPA E Sample 1 se CL ES CL Es es CL re ane ieee Jen sd 401 0 79 478 8 128 1 177 8 H CL Eer 326 7 H i ss LE i Loo CL Es CL ES Ecg CL Ee CL Ee 20 119 9 177 8 269 3 350 9 438 7 2 141 9 ET 1202 9 Hide Extra Sample Names When data is displayed in Vertical Orientation the sample names are repeated for each row of data that the sample is associated with If Hide Extra Sample Names is selected then the sample name will an y 555 127 2 611 48242 242 3 1434 1331 331 2 1282 3344 3401 401 0 1587 1491 490 9 906 S6gF132 132 2 2252 118
300. s electropherogram traces for the number of samples specified in the View Preference Display Settings Max Chart In Page field Hot Key Page Up Page Down Select Next Group In descending order selects the same number of samples previously selected by Select Page grouping options see Sample Grouping section below or double click option Select Max Opens electropherogram traces for the number of samples specified in the teten View Preference Display Settings gt Max Open Charts Fist Order File Name z Deselect All Second Order well 1D y Unselects all selected samples in the Sample File Tree list and closes the Third Order Size Score electropherogram traces Iw Ascend Sort jw Disabled to Bottom Sort Samples Case Sensitive Y Ordered by Group Opens the Sort Sample Options box Select First Second and Third Order Sar sorting from the drop down menu options Sample Type File Name Lane Cancel Number Well ID and Size Score Hot Key F3 Search Options Search File Opens the File Search box Enter any part of a filename to search for the sample in the list Click the Search button Left click and use CTRL or SHIFT key to highlight samples then click the Open Selected button The electropherograms of the selected samples will open in the Main Analysis window Hot Key CTRL F Sample Information Sample Info Se CS Opens the Sample Information box A list of Properties a
301. s the Relationship Testing Settings box Options for selected samples or all samples selecting the appropriate allele frequency for the population mutation rate and prior probabilitiy Show Conflict Toggle between Show Conflict with Parents and Show Conflict with Sibling Conflicting and suspected Markers based on Mendelian inheritance are highlighted Show Genotype e 112 May 2012 Chapter 7 Special Applications Toggle between Displaying and Not Displaying the Genotypes of the selected node Family 2 2 2 Family2 Jones Family Select a family from the currently uploaded pedigree file to view and edit Marker 12 TPOX Marker e E a Save and Print Report Select a Marker or Locus to view in the Electropherogram Charts Show Color Allows the user to select all colors to view hide all colors or choose a single dye layer Choose a single dye by single left mouse clicking on the icon Zoom In Use the icon to zoom in on the image or hold down the left mouse button and draw a box from the top left corner to bottom right corner around the area you wish to zoom in Zoom Out Use the icon to zoom out on the image or hold down the left mouse button and draw a box from the bottom right corner to top left corner Set Axis The default setting automatically sets the Y axis according to the maximum peak intensity of the samples Two other options are available auto fit the Y axis using peak intensit
302. sausesssaueessauaecesausesssuueessauasessaes 24 ADJUST ANALYSIS PARAMETERS ve 25 MN 25 RE GNOIVZE WITH AUTO RUN E 25 Rene Individual Samples reser 25 CHAPTER 3 MAIN ANALYSIS OVERVIEW sisas ias 27 MANANA IE WINDOW EE EE LENE EE 28 Sample EEE SE EEE EEE ENE ENE 28 Synthetic Gel Image and Electropherogram with Peak Table 30 SL e o e e ESO IA 33 WERTEN 34 FVN 34 VENN 35 TN 37 NNN 38 TN 39 Table of Contents Hep Mens 40 MAN TOOLBAR ICONS are ee 40 CHAPTER 4 FRAGMENT SIZING STANDARD sccccsccsccccsccccsccsceccsccccsscsceccsccccsscsceccsccccesesceccsccecssescecceceeces 43 ETNE 44 Ree e 46 ICONS GG FUNCTIONS Je 47 VG 10 l TO EXDCCL EE RER EN EE 48 SIZE CALIBRATION CHAR TS 49 Poet Jure 51 KONST Se 52 WV ROU TO EXO CCE irda via E cette NT 53 CHAPTER 5 PANEL EDITOR se 55 OVERVIEW NNN 56 POLL AA AAA AOS DA AA A dad NN 56 SMELTET 57 VET 57 FEN 59 REESEN 60 Pre Dene NENNE 60 CUSTOM Taner realiom es eeo TN 61 Adustno ana Calbroung e 62 IGCONSAND FUNCTIONS 1 NA 63 MENU OP TONS sarte erre 63 feiere 64 WHAT TO EXPECT ae 65 CHAPTER 6 REPORTS AND PRINTING eege negen gege eege Eege SEEERg EE g Eege 67 REPORT ET 68 MENN 68 Marker Table FINN ve en 68 BR TOR PEPE 69 Pee 70 AVE G1 vare sd 71 PRINT IRE ROR Lu 72 Report Content Oss 73 CONNOR 74 SAVE PROJECT EE 74 CHAPTER 7 SPECIAL APPLICATIONS ccscccoscsscsccssccccscsccccscccccscsccccnccccesceccccnscccesceccccuscccescsccscsscccescsccccscesces 75 AMPLIFIED FRAGMEN
303. set in the Run Wizard Sample Grouping From the main toolbar select Project Apply Sample Grouping The File Name Group Editor tool will appear See Chapter 8 Additional Tools Select Group and Control identifiers and click Match Click OK to apply the matched groups Group numbers will appear next to the filenames in the Sample File Tree Use the Select Next 29 May 2012 Chapter 3 Main Analysis Overview Group right click menu option OR CTRL PageUp Down to open samples in a group To disable the Sample Grouping feature go to View Preference gt Others and uncheck Enable Sample Grouping Synthetic Gel Image and Electropherogram with Peak Table The Synthetic Gel Image and Electropherogram displays are associated in the Main Analysis window Both display the fragment information in a visual form When GeneMarker is initially launched all dye colors are displayed in the Synthetic Gel Image and Electropherogram at once Single left click the Show Color icon in the main toolbar to cycle through the dye colors or use the Show Color drop down menu to disable individual colors or Show Hide All colors Navigation Zoom In Out In the Synthetic Gel Image or the Electropherogram hold down the left mouse button and drag a box from upper left to lower right around the area you would like to zoom in on To zoom back out hold down the left mouse button and drag a box in the opposite direction from lower right to upper left The
304. sis or mutation specific probes that have a value of 0 RFU for normal samples If the cause is due to mis alignment of the panel return to the main analysis screen select Tools Panel Editor and calibrate the panel Chapter 6 If the 81 May 2012 Chapter 7 Special Applications chemistry has mutation specific probes for example Tay Sachs and Parkinsons the empty square will be displayed at the origin indicating no peaks detected in the peak ratio plots of the analysis screen and the final reports in cases where both control and sample files are normal peak ratio will be reported as 1 0 0 is undefined Report Table The Report Table lists in columns the probe or exon name according to the panel selected the basepair size of that probe peak in the reference sample and the ratio for each sample probe outside the ratio limits Double click a cell in the Report Table to display the corresponding peak in the Trace Overlay and the plot point in the Ratio Plot See the Reports and Printing section below for additional options in the Report Table Procedure Analysis procedures for MLPA analysis include uploading the data sizing filtering and normalizing the data with Run Wizard creating or modifying a Panel and finally detecting probe insertions deletions in the MLPA Analysis module Below is a description of MLPA settings in Run Wizard and modifying pre defined MRC Holland Panels in the Panel Editor Subsequent analysis in the MLPA
305. so carries to the final reports in Special Applications such as MLPA Show Loci box with multi line Displays the loci boxes above the electropherogram markers staggered for clear view of the marker name when all multiple dyes are displayed at the same time in the electropherogram Peak Label Choose up to four labels size height area score to display as a flag next to individual peaks in the electropherogram Position Choose to place the peak label at either the top of the peak to the right side of the peak or in the allele label in the Electropherogram Gel Image Gray for Single Dye When selected will display and print the gel image with a black background and white bands When deselected the gel image will display a black background and colored bands depending on dye color chosen to view NOTE When all dye colors are selected the bands in the gel image will be displayed in color regardless if this option is selected Background in White Only available when Gray for Single Dye is selected Will invert the gel image so that the background will be white and the band fragments will be black Report Settings Automatically Re Sort Report Check this option if you would like eene GeneMarker to automatically re sort the report every time you modify alleles Un check this feature if you want the report to remain sorted until you choose to re sort Automatically Scroll Charts to Alleles When Selected in Report You may choose
306. ss of Heterozygosity LOH occurs when a somatic cell contains only one copy of an allele due to non disjunction during mitosis segregation during recombination or deletion of a chromosome segment LOH becomes critical when the remaining allele contains a point mutation that renders the gene inactive This is a common occurrence in cancers where a tumor suppressor gene is affected Tumor suppressor genes code for proteins that regulate the cell s life cycle Thus they are critical in preventing tumor formation Overview GeneMarker fragment analysis software has been developed to aid researchers and clinicians in the detection of LOH within cancer cells Using a patented allele calling algorithm GeneMarker uses the germ line reference trace to compare and detect LOH in patient samples LOH Analysis Trace Overlay Ratio Plot Group File Tree Report Table d LOH Anatys amp ala a a o ALE E Mee Samples 4 30 bog I 785 T Gent 102104 11 ba Gap Guo 3 Greup 1 Sue e 112 142 Grap 4 D Ret Control 7458 BH 745 TGroupl 1021 nes o CO 564 T Groupi 745 B Groupi 0 745 T Groupi Q0 00 745 B Groupi H HI N ii I 745 T Groupi_ a o ap o 1A o ip O o Ca CH o o O o o e GH o So So Group File Tree Samples in the Group File Tree appear within Group folders according to the information provided in the File Name Group Text
307. ss of Heterozygosity applications where tumor samples are compared to normal samples from the same patient Additionally the Filename Group Editor can be used in the Main Analysis window Sample File Tree Procedure SE 1 Select Tools File Name Group Tool OR Project Apply zm 77 Sample Grouping E 2 The File Name Group Editor window appears r pers til SCF meme aL SCF 3 Click the Load Files icon and select all files to pair ECKER Gemeen mee 4 Choose Match by Sections or Match by Fixed Position or Group by Order 5 Enter values for the Group Identification and Control Identification fields 6 Enter a Control Identifier value and click Match Tinchy Scare Mac y Fdo Ger Oo E O eten 7 The samples from the File Name List will be paired into cn EJ 0 le groups in the Matched Groups window Ee 8 When the samples are paired correctly click the Save Groups to File icon 9 Save the group information as a tab delimited Text txt file 10 Upload this filename group file into the Group File fields of the MSI or LOH analysis settings box OR the grouping will automatically be applied if Project Apply Sample Grouping was chosen Icons and Functions Load Files E Opens a directory window where raw data files can be located and uploaded to the Filename Group Editor Add Files Opens a directory window where additional raw data files can be uploaded into the Filename List field 172 May
308. stems can interrogate 48 SNPs simultaneously and has been used to investigate SNPs in 92 cancer related genes in breast cancer and to genotype plants GeneMarker genotyping software is designed for fast accurate and efficient analysis of SNPlex data Overview The SNPlex Analysis window displays a synthetic gel image list of samples cluster plot to analyze peak information and assign SNP genotypes sample electropherogram and SNP genotyping report The information contained in the report is interlinked Double clicking on the cell highlights the cell corresponding sample ID in the sample list data point in the cluster plot and locus in the electropherogram The Sample List Cluster Plot and Report Table in SNPlex analysis are all similar to SNaPshot analysis See SNaPshot SNuPe section above for explanation of functionality The Electropherogram frame in SNPlex analysis differs from SNaPshot analysis SNPlex technique uses primer length variation to identify SNP pairs SNP pairs in SNPlex analysis are typically separated by approximately two bases therefore an entire 48 plex SNP analysis can be accomplished with just two dye colors SNPlex Analysis Sample List Report Table Cluster Plot Electropherogram d SHP Analysk for Ste 1 5 H 007 ba 002 hes ape ampia angis DEZ ita apis a 3 4 4 3 DO fra 5 sample D lta b sample 7 E E UD za sample 075625 sample Tb t sample 1017 Lea sample Eiza 11
309. stitutions developing their own fragment analysis instruments that have variable time frames for collection of data points Procedure 1 Tools gt File Conversion to activate the File conversion screen 2 Select Load File as X Y if time and peak height information is contained md ml rn ae in the Same file en saa b 33514466580 Loading Data Num 18055 F 0 3351446657 S O51 446657 4 3351445657 end ree 0 3351446857 3351 3 Select Load X_File Y_File if the time jes mme muer IT Date Nun 96 sent and j 5081 446687 8 3051448687 0 3951446658 0 line Interval 0 2 0 3351446657 3381 and peak height information 1S 3951446658 3051446657 33514466580 Calculate Data Sum 3453 i 0 3351 445850 22514 33514465 8 0 o contained In two files 3051445650 3351445657 33514466580 0 18 4 Save the converted sgl file for swiss sss smuss 5 SE eene import into GeneMarker Se Se Se e Gees Ee e Seen es 3351446659 A 3351446669 0 3351446659 0 D 3351 446659 3351 446659 0 3351446659 0 0 3351 446659 33518 33514466596 3351445659 0 3351446653 0 D 59 0 3351446639 0 H 335 446539 33514 0 3051445559 8 3351445659 0 3351446653 0 0 3351 446659 3351 445559 3351446659 0 0 3351 446659 33614 Filename Group Editor Tools File Name Group Tool OR Project Apply Sample Grouping The Filename Group Editor was originally developed to be used in conjunction with the Microsatellite Instability and Lo
310. t as compared to the Experimental sample trace Additionally the RFU intensity or peak height of the Reference trace is normalized to the Experimental sample traces in the Group To disable Reference trace normalization click the MSI Display Settings icon and deselect Peak Normalization The uncorrected Reference trace peak heights will be displayed in the Trace Overlay The allele editing options in the Trace Overlay frame are similar to those in the Main Analysis window Electropherogram See Chapter 3 Main Analysis Overview Gain Loss Histogram The Gain Loss Histogram indicates the degree to which the Experimental sample peak at a given position differs from the Reference sample peak at the same position based on Peak Height or Peak Area as set in the MSI Analysis Settings box A Log2 function is applied to the Peak Height Area Ratio to determine MSI Score The MSI Score value is plotted in the Gain Loss Histogram Values above and below the minimum and maximum MSI Score thresholds set in the MSI Analysis Settings box will turn the Histogram bars red The minimum and maximum MSI Score thresholds can therefore be used as a visual confidence level gauge in the Gain Loss Histogram Report Table The Report Table lists all samples in a column on the left Reference samples are marked with a C which stands for Control In the Reference sample rows which appear in blue the basepair size positions of detected peaks are listed In the Experim
311. t of the sample and the square root of the control using either peak heights or areas GeneMarker utilizes an automatic iteration based regression method Using this method the software forms a best fit line retaining 80 of peaks with smaller deviations and 20 are rejected GeneMarker then iterates multiple times to reject and retain peaks with a confidence of 99 default setting confidence interval denoted by green lines on graphs This process is repeated until the regression line has reached the desired confidence Finally the removed data points are placed back into the plot and will show up either on the regression line normal copy number or as outliers possible copy number change Peak ratios normalized peak value for test sample normalized peak value for reference or control sample are plotted on a patient control graph These peak ratios are plotted as representations of the square root of the sample and the square root of the control used to determine the best fit line outliers and confidence interval The patient control graph combines peak ratio information to determine copy number changes with the confidence interval from regression analysis Microsphere MLPA Analysis Luminex An example of a DNA assay technique used to genotype patients with specific diseases is multiplex ligation dependent probe amplification MLPA MLPA relies on sequence specific probe hybridization to genomic DNA followed by amplification of
312. ta is similar with minor adjustments to the data analysis methods for AFLP Overview GeneMarker software has the ability to produce highly sensitive and reproducible results for T RFLP analysis by adjusting the AFLP Analysis default settings In the Run Wizard Template Selection box be sure to choose the correct Size Standard and set the Analysis Type as AFLP Procedure The Run Wizard Data Process box has many options for auto correction and peak detection thresholds Adjust the AFLP analysis parameters as follows h un Wiza mm Raw Data Analysis me Data Process AFLP Analysis Select Smooth Set data process options Allele Call Raw Data Analysis Allele Call Deselect Auto Range Iw Auto Range frame tal Auto Range bps Set Start to 60 i 1 2 an Stat 60 2 End 500 3 Set End to 500 S gen on z gielen deng D ee V Peak Saturation V Baseline Subtraction ntensity gt 2 ercentage gt Max Peak Detection Threshold E EE SC KT BE GE d jw Pull up Correction Y Spike Removal Intensity gt 40 Gg Max Call Intensity 30000 Stutter Pe ak Filter Ze Local Southern Cubic Spline Large Size K Stutter Peak Filter V PlusA Filter Left p a Right fo a Deselect or Load Default Save Default Set Left to 0 lt Back Cancel Set Right to 0 NOTE To detect low intensity peaks T RFLP analyses should include a low peak detection threshold with stutter filter turned off or set to zero The peak detection
313. talled on the same computer the previous version or copies thereof are not transferred to another party or computer unless all copies of the Update are also transferred to such party or computer and you acknowledge that any obligation SoftGenetics LLC may have to support the previous version of the Software may be ended upon availability of the Update 6 LIMITED WARRANTY SoftGenetics LLC warrants to the person or entity that purchases a license for the Software for use pursuant to the terms of this license that the Software will perform substantially in accordance with the Documentation for the ninety 90 day period following receipt of the Software when used on the recommended hardware configuration Non substantial variations of performance from the Documentation does not establish a warranty right THIS LIMITED WARRANTY DOES NOT APPLY TO UPDATES FONT SOFTWARE CONVERTED INTO OTHER FORMATS PRE RELEASE BETA TRYOUT PRODUCT SAMPLER OR NOT FOR RESALE NFR COPIES OF SOFTWARE To make a warranty claim you must return the Software to the location where you obtained it along with proof of purchase within such ninety 90 day period If the Software does not perform substantially in accordance with the Documentation the entire liability of SoftGenetics LLC and your exclusive remedy shall be limited to either at SoftGenetics LLC option the replacement of the Software or the refund of the license fee you paid for the Software THE LIMITED WARRANTY SET
314. tatus and deceased is saved and can be imported with the standard pedigree information for future edits in an extended pedigree Sample File Select the individual s sample file from the drop down list Only samples in the current dataset will be available If no sample file is chosen the node will be greyed out Additionally drag and drop a sample from the Sample List onto a node to associate the sample with the individual node Add Family Members To add family members to the Pedigree Tree right click a node and select Add Mate or Add Child The Add Mate or Add Child box will appear Enter the individual s information and click OK Display Conflicts Once a family is created in the Pedigree Tree GeneMarker will automatically identify any Mendelian inheritance conflicts between Parents and Children or between Siblings Click the Show Conflict with Parents Siblings icon in the toolbar to alternate between the two modes Nodes with conflicts will appear red Mouse over the red node and a list of Markers or loci with conflicts will appear Single left click the Marker name and the family members electropherograms will appear in the Charts tab on the right The conflicting Marker will appear red Additionally suspect Markers are indicated in the Pedigree Tree by a question mark symbol next to the individual s node Suspect Markers include those which contain additional peaks or low quality peaks Mouse over the node with suspect Marke
315. tc NOTE Do not use Znd DT with Spike Removal because real peaks look like spikes Process Data After the raw data files have been uploaded to GeneMarker they are ready to be processed The processing step includes application of a sizing standard filtering of noisy peaks and comparison to a known allelic Panel if desired GeneMarker combines all these steps in one simple tool called the Run Wizard To access the Run Wizard simply click the Run Project icon in the main toolbar The example below is for a basic fragment analysis For advanced applications see Chapter 7 Special Applications Run Wizard Template Selection TOF er gt Procedure je 1 Click the Run Project icon in the toolbar 2 The Run Wizard Template Selection dialog box will appear 6 Sel e ein eat or cts ne 3 Select a template a previously saved set of size standard rs PE Ger Swe standard color and analysis type named for future use OR Size Standard JS Jun select a new combination of size standard standard color standard Color Red z and analysis type EE Freonen Ars 4 Click Next when finished EN Bass does Icons and Functions Cae a Template Name Select from existing pre made templates or create your own by entering a Template Name and clicking the Save button To save the Run Parameters with the template use the Back arrow after setting the parameters in the second and third screens of the
316. tch Score calculation The Match Score calculation is updated when Update Calibration is selected NOTE Add Peak is only available when no other green triangle peak indicator is selected Fix Size When selected the Calibration Editor box appears Enter the correct basepair size of the peak and click OK NOTE Only Sizes that occur in the selected Size Standard can be entered in the Calibration Editor Size field The peak will be fixed at the specified basepair position and all green Calibration Editor LS triangle peak indicators to the left and right of the fixed peak will be adjusted to correctly align with the chosen Size Standard Peak 5085 UME The Fix Size feature is useful when the selected Size Standard has uniformly so ME spaced peaks and the sample ILS has additional peaks due to pull up or other experimental abnormalities thereby influencing the pattern recognition Cancel algorithm NOTE Fix Size is not active for manually added peaks or peaks outside the Size Standard range Reset Peaks Select Reset Peaks to eliminate manually added peaks and or extra green triangle peak indicators after Fix Size NOTE Deleted peaks will not be recalled when Reset Peaks is selected Update Calibration After editing peaks in the Sample ILS select Update Calibration The Match Score for the sample will be recalculated based on the edited peak indicator positions When Size Calibration Charts
317. te Undelete Allele cos 33 deletion duplicatiOn ci is 85 A A 88 Dendrogtai ii Menon 138 Difference nerona E 32 Disable une 29 disabled sample aa 36 Disabled Size Columns 49 Disabled Size Statistics Lua 50 Display BIOS EE 70 Display Con Hit sva 104 Display Se NES SEN 35 Distance Measure c occcocccncccoccnoconoconanonaconaconaconanoninos 137 Distanceikb GO Dosage HISLOGTAN used eege 80 ie e Bee gei EE 142 Ile ege TC 88 E EO TING va 155 Edit Aele vr 33 Edit BIN TS 59 Edit BS 58 EGON NPE 58 EI HS e V eee 179 EON EG 180 Edit Marker neeeeeeeseneressresressressrssrresrrerrrsrrseens 58 Edit Marker BIOS ers NS 58 Edit PAN Ol A A A 56 Ed SE 45 Ede Peksa 32 34 Editing SE una 50 Edwards Syndrome ANG 142 Electropherogram features cccccsssscceceeseeeeeeeeeeeees 31 Enable Sample Grouping 37 Enhanced Baseline Subtraction o e 21 Enhanced S MOON ida as 18 21 Euclidean Distance 137 Eutclidian Distante Rida aa occ 139 Event LOB Sia aio 41 May 2012 Index O RAA 35 Expected Size Standard 45 Export ABI Size Standard 47 EXport COD IS Lus 38 102 Export Hectropnbheroer am 40 176 Export Panel arica ara 57 63 Export Print Report 74 Export Size Standard iiiciniccionaicia in See aux 45 47 Export the Project Panel 64 Extend Diploid Homozvgous 68 Extended Pedigree Files ooomooooooomom o 108 F A ese Aion 113 116 162 Family Group TOO rs 118 Fam NING pe 104
318. ted Pedigree Drawing Quantitative Analysis SNP Analysis Microsatellite Instability Trisomy Detection Loss of Heterozygosity TILLING Analysis Haplotype Analysis ARMS Comparative Analysis Cystic Fibrosis 75 May 2012 Chapter 7 Special Applications Amplified Fragment Length Polymorphism AFLP Amplified Fragment Length Polymorphism AFLP is a polymerase chain reaction PCR based genetic fingerprinting technique developed in the early 1990s by Keygene AFLP uses restriction enzymes to cut genomic DNA followed by ligation of complementary double stranded adaptors to the ends of the restriction fragments A subset of the restriction fragments are amplified using two primers containing amplification selective nucleotides at their 3 ends complementary to the adaptor and restriction fragments These fragments are visualized on denaturing polyacrylamide gels using either autoradiographic or fluorescence methodologies Procedure Analysis procedures for AFLP analysis include uploading the data sizing and filtering the data with Run Wizard creating a Panel if desired and finally detecting subgroups within the population Below is an explanation of AFLP settings in Run Wizard and Panel creation for AFLP data Subsequent sections will describe in detail how the Allele Detection with Bin Table Reference Trace Comparison and or Phylogeny Clustering Analysis can facilitate subpopulation discovery AFLP Run Wizard Settings For exp
319. tensities of low peaks are adjusted to match the intensity of the highest peak in the dye color When low peaks are increased the intensity magnification factor is noted in the Marker 2X 8X Print Opens the Print Report settings box See Chapter 6 Reports and Printing Overlay View Applications Overlay View The Overlay View tool was developed as an easy way to compare several sample traces at once Procedure 1 Select several samples from the Sample List by placing a de el GT show 20 oma EF ES S check mark in the empty box to the left of the filename er am 2 Click OK ets S 3 The traces will appear in the window to the right SC ps wes 4 Slide the 2D Offset bar to the right to de convolute the traces ac Be 5 Select the Line List tab to open the list of traces present inthe 17 e 059 B05 5CF 3 539 HOS SCF 3 00 819 ADS SCF viewer Select any trace to bold the trace line OOODRDOOODDE 6 Select a marker from the drop down list to view one marker e008 SCF 200 i 620 DOG SCF 1 50 at a time Se 100 7 Select Show 3D to see the traces in a three dimensional view Gasen l LJ 848 DOG SCF Icons and Functions x Ge Dye Color Single click to scroll through the dye colors Zoom In Out Single click to zoom in or out on the center of the Trace View window 168 May 2012 Chapter 8 Additional Tools Tools All view
320. the Trisomy Print Report will be set to Classic view See the Reports and Printing section below BPG When selected Quantification is by Peak Area and Trisomy Ratio default thresholds are lt 0 80 or gt 1 40 Additionally the Report Table in the Trisomy Analysis window will display Peak Ratio values and the Trisomy Analysis Settings Analysis Statistics Plot Analysis by f Classic fe BPG f Aneuploidy Peak Height gt 50 Quantification by Height Ratio gt 30 00 Max f Peak Height Trisomy Ratio Iw Shorter Length Longer Length h Apply Linear Correction Trisomy Print Report will be set to BPG view See the Reports and Printing section below Aneuploidy When selected Quantification is by peak area and default thresholds are lt 0 80 or gt 1 40 Select 143 May 2012 Trisomy lt 0 80 or gt 1 40 W Inconclusive Range gt 0 65 to lt O 80 or gt 1 40 to lt 11 80 cave Chapter 7 Special Applications Shorter Length Longer Length The report table is similar to BPG with ability to report results on sex chromosomes as well as autosomes Thresholds Peak Height Minimum height a peak must reach to be called Height Ratio Maximum percentage of the main peak the second peak must reach in order for two alleles to be identified Quantification Peak Height Ratio calculations are generated based on the RFU values of two peaks within a Marker Peak Area Ratio calculations are gener
321. the percentage RFU value of the highest peak Save Report Saves the Quantitative Analysis Report Table information as a Text txt file Refresh Calculates the values of the added peaks and updates the Quantitative Analysis Report Table What to Expect Curtain Method The Curtain method is best for very complex traces as it is difficult to calculate the area for a primary peak when secondary peaks exist Using this method primary peaks will be calculated while secondary peaks will be removed from the calculation and will be left in an unassigned area between the peak boundaries This method integrates to calculate the area under each individual peak in the region The analysis shows gaps between the peaks in the region so the region between the peaks is not assigned to either peak for the area calculation To set the area between the peaks input the desired value in the Peak Boundary 15 peak intensity field A point along the curve must be at least x of the primary peak s intensity in order to be assigned as part of the primary peak and is used to calculate the area under that peak Editing Peak Boundaries 1 Hold down the Ctrl key and use the left mouse button to move the peak boundary 2 The peak boundary will be marked by a red diamond 3 After setting the new boundary release the key and mouse button 4 The new area will be automatically calculated Curtain Method 121 May 2012 Chapter 7 Special Applicati
322. tide repeat of CA which occurs tens of thousands of times across the genome Microsatellite instability MSI is a condition where repeat units are gained or lost within a locus resulting in length polymorphism Certain repeat regions are known to be highly polymorphic and hereditable MSI is most frequently detected in regions with long mononucleotide repeats where DNA slippage during the polymerase reaction commonly occurs Ultimately microsatellite instability within and around certain genes can have devastating effects due to the possibility of frameshift mutations Overview In GeneMarker s MSI Analysis module tumor samples are compared to normal samples based on peak to peak comparison Differences between the two traces are displayed in a Gain Loss Histogram below each trace overlay Within the electropherogram itself the tumor sample trace is overlain on a light red trace of the reference In this way the clinician can easily visualize where the areas of instability exist MSI Analysis Trace Overlay Gain Loss Histogram Group File Tree Report Table ai MSI Analysis cB ps aa AEB we or Samples D 6 Group I S gt 021 64 T fsa s Group 2 6 Group 3 90 H 6 Group 4 1 600 O Group 5 6 Group 6 Group 7 1 400 REF 021 63N fr e 021 64 T fsa 1 200 4 Group 8 12 gt Report H Help x a 1 1 015 46 t 130 1 000 Group File Tree Samples in the Group File Tree appear
323. timg s 147 Trisomy Ratio 144 Trisomy Ratio Plot 143 145 Trisomy Report Table ccoo 146 LEIS MN SCO gt EEE EE 145 TE 145 WMOr SUPPGSSO E 95 Tumor suppressor genes rrarrannnnnnnvrnunnnnnnvnnnenuennenere 150 Turner svndrome cooccccccnnccnncnonaconononaronononaronononanonononanos 142 Two Color Trace Overlay oooocccccoocccnconononoss 124 U Unconfidence at Rightside Score 24 NK A E E AA 6 Update Calibration ooooccccococcnnnonononononos 51 Update Sample Data 108 USB ROY Lea 6 7 8 10 USB POF GE 6 7 8 10 11 Use last template 20 Use Old Colbrotion 25 Use Size String for Label 36 USO io a 7 10 12 User Monogement 33 40 178 User Manager History rrrnnrnnnnnnnnnrnnnrnvnnrnnnnrnnnnnnnnsnne 179 User Manager Setting 179 User Window rrrrnnnnnrrnnnnnnrrnnnnnnrrnnnnnnsrnnnnnnsrnnnnnneene 178 V vertical gray bars casi 56 Vertical Orientation 69 72 May 2012 Index VIEW History svare 33 179 Z Vie IVICA EE 35 View Mode sne 52 Zoom Mii ieii 41 113 116 157 162 Zoom N O t ernie id W Zoom SE EE 41 113 116 157 162 MD ved 29 188 May 2012
324. tion Register Online Password Email Back Cancel Chapter 2 General Procedure E Your software will be registered automatically A confirmation e mail will be sent to you once registration is complete NOTE Some characters can commonly be misread If you get an error trying to register check for number 1 and lower case letter L or number 0 and upper case letter 0 confusion F Launch GeneMarker and begin analysis Offline Registration x A Copy and paste the entire Request Code string and type your Account and Rees Onine See Ong Password information from the SoftGenetics CD into the body of an e mail ee ee B Send the email to tech_support softgenetics com Reger dt C The Registration ID will be sent to you via email within one business day Gee D Copy and paste the Registration ID from the e mail into the Registration ID field E Click Register F Launch GeneMarker and begin analysis messe Upgrade There are two local licensing options available for GeneMarker v2 0 and above USB device required and text based The legacy local licensing option of GeneMarker available for previous versions v1 97 and earlier of the software is supported for v2 0 and above This option requires a USB device dongle key hardware based This option allows installation on many computers However the associated USB device must be inserted into the USB port of the computer to
325. tions Match by Sections Automatically separates the sample filenames into groups based on the specified Section Separators Group Identification Identifies how to match the filenames into groups based on the section entered into the Compare by Section field The section of the filename specified will be highlighted red in the File Name List Control Identification Identifies which section of the filename contains the reference vs sample information based on the section number entered in the Match to Identifier by Section field The section of the filename specified will be highlighted green in the File Name List Match by Fixed Position Allows the user to manually identify the characters of the filename for grouping the samples Section Separators like are counted as individual characters Group Identification Enter the number of the beginning and ending character to identify how to group the samples The section of the filename specified will be highlighted red in the File Name List Control Identification Enter the number of the beginning and ending character to identify which part of the filename contains the control identifier The section of the filename specified will be highlighted green in the File Name List File Name Group Editor Match by Group Order Allows the user to group samples that contain sequential identifiers 170 May 2012 Chapter 8 Additional Tools
326. to display in the report C Selected Markers Report Table Report Table Choose the contents of the report table K Show Allele Name T Show Allele Height MW Show Allele Area MW Show LOH Score Show Electropherograms in Current Size Range When selected will display Overlay Trace electropherograms in the same ee eee ee eee zoom mode as the main LOH Analysis window When deselected the electropherograms will be displayed so that all selected Markers can be E Ok Cancel clearly viewed LOH Clinical Report Below is a description of the LOH Clinical Report features For an explanation of functions within the Print Preview window see Chapter 6 Reports and Printing Report Header Contains information about the analysis project sample and parameters Signature Box 153 May 2012 Chapter 7 Special Applications Date and initial space for report reviewers Overlay Trace Electropherogram Similar to the LOH Analysis window displays the Reference trace in light red behind the Experimental trace Ratio Plot Also similar to the LOH Analysis window displays a ratio plot of reference versus sample based on peak height or area and can be adjusted in the LOH Analysis Settings box Report Table Identifies the marker and statistical information chosen in the LOH Print Settings box LOH Clinical Report LOH Analysis Report SoftGenetics Sample 745 T Group1_102104_11 fsa Control 745 B Group1_102104_09 fsa Software GeneMar
327. ts and Control Sample s When either MLPA Ratio or MLPA Regression Analyses are opened the software has automatically selected a sample to use as the control indicated by the sign next to the sample While the chosen sample may not actually be the experimental control this selection is based upon which sample presents the data with the fewest abnormal calls the sample with the least scatter of peak height ratios around the idealized ratio of 1 0 will be used as a control to calculate normalized height ratio of sample data to control data The user may change the control to reflect their experimental sample or another sample which they feel portrays the data in a meaningful fashion GeneMarker software MLPA Ratio Analysis In the MLPA analysis window the normalized samples are compared to a control sample to determine duplications deletions or no change A t test is used to determine which data points are outliers and which data points cluster together as the no change data points MLPA Ratio plots the ratio of each sample compared to the control The ratios of the sample data compared to the control sample control s defined by the user or software are standardized such that the median point within the data set is considered to be 1 All other data points are relative to the median value Outlying points are determined based upon the deviation of each allele peak relative to the average deviation of all peaks Any individual peak whose residu
328. tumor sample in the second column See Chapter 8 Additional Tools Filename Group Tool Analysis Settings E Opens the MSI Analysis Settings box 132 May 2012 Chapter 7 Special Applications Group File Click Open File icon to upload a txt file that groups samples by filename MSI Analysis Settings Allele Call EE Peak Detection Threshold EAUser SotGenetics Desktop DatacetsWMSNTral g Intensity Sets the minimum RFU intensity level at which peaks will be Allele Call called Peak Detection Threshold Local Region Sets the minimum percentage of the major peak in the dye gp ER color at which peaks will be called elite E Stutter Peak Filter Sets the minimum percentage of the major peak in a K Stutter Peak Fiter marker at which peaks to the right and left of the major peak will be Left 8000 Righe 4000 called Quantification amp Normalization Peak Height Peak Area Quantification and Normalization Peak Height When selected MSI Score and correcting for preferential MSI Score Loge lt 1250 ar FS amplification of smaller fragments normalization is calculated based on T Show Loss in Histogram the RFU intensity values of a peak Peak Area The same as Peak Height except calculations are based on the ES area under a peak MSI Score Log2 A logarithmic value based on height or area calculated for each individual peak The MSI Score value is represented as a bar in the Gain Loss Histogram
329. two clusters Clustering using single linkage tends to produce an effect called chaining where single genes are added to clusters one at a time Complete linkage is the opposite of single linkage It measures the distance between the farthest two points in the clusters Complete linkage performs well when the clusters are well defined with minimal noise Average linkage defines the distance between two clusters as the mean distance between all points in the clusters NOTE Choosing different linkage measures results in different cluster diagrams Procedure 1 Upload and filter the data with Run Wizard See Chapter 2 General Procedure Apply a Panel to the dataset See Chapter 5 Panel Editor Select Applications Clustering Analysis The Clustering Analysis module will appear Click the Clustering Analysis Settings icon The Clustering Analysis Settings box will appear Adjust settings Click OK Click the Save Dendrogram icon to save the image as a BMP file 0 Click the Output Clustering Report icon to save the matrix values as a tab delimited Text file Gomm m mt n Icons and Functions Clustering Analysis Settings a ee Select this option to choose between two tabs Analysis and Layout l Ciusteerag Rule Distance Measure Block Number 2 Sandanty Analysis The Analysis tab is used to set cluster calculation parameters Clustering Rule Block Number Choose the number of expected major groups in the dendrogram Max Distance Min Corre
330. um computer requirement cccoccccnccccncncnnnnnnnns 6 Minimum DOSAGE Range rrrnnnrnnnnrnnnnvnnnnnnnnrrnnnvnnnnsnnneee 85 Minimum Lane Score Threshold 0 000 84 85 87 Minimum TRDISTONCO 0 E E 85 Minor Adjustment of Panel 65 MIX DV OS ae 73 MLPA Adjust by Control Probes eee 84 MLPA Analhysis 38 80 88 MLPA Analysis Settings 84 156 MLPA Clinical REDON SE 89 MLPA Control Sample Identification oo 87 MLPA Display Settings ooocccccccoocnnnncnaconnnonanonononanonoos 83 MLPA Dosage Hietogronm 81 96 MLPA Normalization Method 25 82 MLPA Panel Modification 83 MLPA Panel Selection cccccsssssessssssvecsessssccneessssens 83 MLPA Print e e GE 89 MERA R ee See 88 MLPA ROO eet 81 84 MEPA Rotioto Copy Nunes paa 84 MLPA Regression 84 MEPARESFESS ION ua 88 MLPA Regression Analysis rrrnnrrrrnnnnnrrrrnnnnnrrnnnnnnrrnnnnner 81 MLPA Report Contents noeneesessessessessessessrsrrssee 89 MLPA Report Table 89 MLPA Run Wizard Settings neseeennsssenesseenessrrresseeee 82 MVEPA SOE oar AE ea 81 MLPA Statistics Information 90 MLPA Summary Report 90 MONS Tee 142 MOSS Mis ve 146 MRC Holland MLPA rroonnnrnnnonnrrnnnnnnrrnnnnnnrrnnnnnsssennnn 60 61 May 2012 Index RER RE RiSa 131 MSINEG Lunn ica 136 MSHPOS aged 136 KREE 38 131 MSI Analysis Settings iviioriiiionnin ii its 132 MSIECINICAL REDO aaa aa 135 MSI Display Settings
331. user may also use the Zoom icons in the main toolbar to zoom in and out The main analysis window also allows the user to manually set the x and y axis with the Set Axis icon Horizontal Movement The Synthetic Gel Image and the Electropherogram are synchronized to allow the user to view both images at once To move the images in the horizontal direction use the top slider bar below the toolbar to scroll the image in either direction or hold down the right mouse button and drag the trace right or left Marker Locus Specific Viewing To scroll through individual markers loci select a marker from the Marker drop down list in the main toolbar To view subsequent markers use the Up Down Arrow keys Synthetic Gel Image Features The Synthetic Gel Image displays all samples in the dataset vertically The direction of fragment mobility is horizontal with the small size fragments on the left and the larger fragments on the right so that the gel aligns with the electropherogram trace display Move the mouse pointer over the Synthetic Gel Image to reveal the sample lane filename Image Utilities Click the Image Utilities icon in the upper left corner of the Synthetic Gel Image A fly out menu appears with the following options Copy to Clipboard Copies the Synthetic Gel Image to the Windows clipboard for pasting into other applications such as Microsoft PowerPoint Save Image Allows the user to save the Synthetic Gel Image as a
332. ust the Markers and Bins to align with sample SEDE RA be enn peaks mene a Identify the Control Probe peaks BS sik L e POs ze a Right click a Bin in the Overlay Trace or Panel dE Table Select Edit Allele Click the Control des D Gene checkbox in the Allele Editor dialog box fre s H 4 amp Sample a 0 OR Click OK ER a th d lye Market ee EG j TAF EE a teal EN AIS at Ginz epp o 1 CECR b Enter a 1 in the Control column of the Panel ES SE 2 Joe Ee ia 8 pz I 28 5 Blue P034 DMD 1430 1 2 1 2 ent o 660 00 Table and enter a 1 for quality control among lfe me mm en 0 es fragments Ange JEE mmo ins 12 GI Sr d gege SIE EE UE ite ag E D E OR ROLEI Atlee 2 Blue POJ4DMD 2023 1 2 12 Xp22 1 0 00 val Ei di 13 Blue PO34DMD 2092 12 1 2 ex03 0 361 00 c Right click a Bin in the Overlay Trace or Panel Ze E A PO34_F11_11 tes Maker Title P034 DMD Table and select Set as Control Set as Non E Gemeng A P034_G11_13fsa control After editing select File Save Changes Hot Key CTRL S OR click the Save Changes icon Exit the Panel Editor Click the Run Process icon in the Main Analysis window Select the adjusted Panel from the Run Wizard Template Selection Panel drop down menu Proceed through Run Wizard and click OK in the Data Process box The Panel has been applied Launch MLPA Analysis Module 10 11 12 13 14 In the main toolbar select Applications gt MLPA Analysis The MLPA Ana
333. ve complete pedigree including edits and re opened to edit as more information becomes available Allele conflicts flagged with red font potential uniparental disomy is identified E E eal gt gt 158 May 2012 Chapter 7 Special Applications Haplotype Analysis X linked nomenclature Assigned probable phase of alleles Allele conflict possible mutation in red font Haplotype Analysis New File File DataBase Tools J e T Bie a ED Family 1 1 _ dadA mom JULYTEST JULYTEST SRS GT 338 338 334 STR62 453 475 453 STR49 293 293 288 STRI2 264 262 262 STR 268 271 Dysil 147 147 STRMP 365 STRA4 169 STR25 161 STRA4 243 STR45 223 DXS1214 266 STR 9_GT2 264 daughter son JULYTEST JULYTEST STR 9_GT3 338 334 334 STR62 452 453 453 STR49 293 288 288 sTR12 264 262 262 STR2 268 266 266 Dysll 147 187 197 STRMP 372 380 380 STR4 16 167 167 STR25 181 181 181 STR44 241 243 241 STR45 225 225 225 DXS1214 267 271 271 STR 9 GT2 296 264 264 Procedure 1 Open GeneMarker and upload raw data files 2 Select a Panel Size Standard and Fragment Animal Analysis 3 Verify allele calls are accurate in the Main Analysis window and adjust parameter settings accordingly See Chapter 2 General Procedure 4 Panel Editor to input Mb distance and cM Mb distance is used to order the markers in the pedigree diagram and cM is used to report information on genetic distance entered by the tbe Tea A A E RNE a
334. ve mouse left or right while holding the right mouse Export the Project Panel Exports the currently selected Panel in the Panel List as an XML file toa specified directory on a local or network computer Delete an Allele Right click at the allele to select Delete Allele Insert an Allele Delete Alleles Help Menu The Help menu contains a link to Hot Keys in Panel Editor Click Hot Keys and the Panel Editor Action Help box appears Hold Cul key bold left mouse and move the mouse to select a region drawn cyan shadow then right click at the mouse to delete alleles covered The markers that are covered will be erased Toolbar Icons Panel Editor Found in the Run Wizard Template Selection box OR the Tools menu Create New Panel Opens the Create New Panel box Follow the steps in the Create a Custom Panel section above Save Changes Permanently saves Panel edits to the currently opened Panel file which is located in the SoftGenetics GeneMarker Panel directory for Save Changes with Signal Info Permanently saves all Panel edits including height information which is used with the Major Panel Adjustment feature NOTE A Panel must be correctly aligned with peaks in the dataset before selecting Save Changes with Signal Info in order for the Major Panel Adjustment feature to work correctly Delete Current Panel Marker Deletes whichever Panel or Marker is currently highlighted in the Panel List This action is irreversi
335. ve supports text based registration for the local licensing option no USB device dongle key or hardware is required This text based registration ID is registered to one specific PC If the license needs to be transferred to a different PC registration for that one license PC must be inactivated first before the software will be registered to the new PC Installation 1 Sl ha Insert the SoftGenetics CD into the optical or CD ROM drive If your computer is not set to automatically open a CD navigate to the optical or CD ROM drive on the computer and open the directory Double click the GeneMarker Setup executable file EXE The Installation Wizard will launch Click the Next button in the Welcome window Read the SoftGenetics End User License Agreement and click the I Agree button in the Read Me File window Select Install GeneMarker Recommended in the Select Program window and click Next Click Next in the Destination Location window to install GeneMarker in the default folder Click the Browse button to choose a different installation directory NOTE The default Destination Location for the GeneMarker program is CA ProgramfFilesN SoftGenetics GeneMarker ver 8 Click Next in the Select Program Manager Group window to accept the default Program Manager Group NOTE Changing the Program Manager Group default may affect program operability It is recommended to accept the default 9 Click Next in the Start Installatio
336. w Individual Family Name New Individual Person Info Affect Status Individual ID f C Unknown Sumame smith C Unaffected Forenames eck e Affected Gender e Male Female Unknown Carrier Date of Birth 1 22 1987 Pie Age Gestation Pregnancy O Deceased c StillBirth c r Genotypes From Sample File None e 7 From Database None sl Ok Cancel o Navigate to the saved Allele Reports with the open folder icon o Select kit 1 and kit 2 results for the individual o Ok 160 May 2012 Genotype Editor ee TXT Delimit KEE Gel Column Mt Genotypes Marker Allele1 Allele 2 All pp 235 D55637 295 297 SMA Family1 A amp Kit E12 fsa SMA Family1 B Kit4 D12 fsa SMA Family1 C Kit4 F12 fsa SM Family1 4 KitB C02 fsa SM A Family1 B KitB B02 fsa SMA Family1 C KitB DO02 fsa 415 417 323 325 420 422 Ok Cancel Chapter 7 Special Applications 12 Right click on the node to activate a pop up menu edit or add mate and child ren d Haplotype Anal Select Sibhng File DataBase 1 Poe TO BIF 2 9 rem fm Je Ge NM eem Q E Family 1 1 1 Fa jack jane SMITH SMITH b 1 2 1960 b 1 12 1970 A B D552046 b 203 207 200 207 D58679 b 250 246 259 246 AEE D5S681 b 287 287 287 287 Espert image jack jane D55435 b 182 178 170 170 SMITH SMITH D55629 a 420 422 415 415 D55823 8 184 184
337. window appears EAUser SoftGenetis Desktop TestPedpre Aa 5 Enter a filename and select a directory v sieben File SMP IV Auto Load 6 Click Gave C Users SoftGenetics Desktop T estPed smp al P 7 The directory path and filename will appear in all three m LociDesciption Fie day fields of the Save Pedigree File box led dll a 8 Click OK 9 Three files will be saved to the specified directory PRE een SMP and DAT 10 To reopen the files in Pedigree tool follow steps 1 10 of the Upload Previously Created Pedigree section above Save Pedigree Tree 1 Right click anywhere in the Pedigree Tree 2 Select Export Bitmap 3 The Save As window appears 4 Enter a filename choose a directory and click Save 5 The Pedigree Tree will be saved as an image file BMP BRSRSRSSSSSSSSSRRSSRRSRRRIZ i 107 May 2012 Chapter 7 Special Applications Extended Pedigree Files ep TB az r All personal information is saved and retrieved with S the pedigree file including affected status and deceased Extended family trees can be built saved and B added to as additional information becomes available Pees Fie t Pa Pad le Usere ScltGenetcs Desktop Relatonshp TemngWNew AT pr gal Y indu Sampie Accordance Fie SMP W Liso Load E Waers SoltGenetcs Desktop Relatonshp TesngiNem RT o Gel Seer SBUBRARBRIS l gt El T ei oP Family 1 8 1 Family1 Familyl Marker All Markers D Search d ry
338. within Group folders according to the information provided in the File Name Group Text file uploaded in the MSI Analysis Settings box A sample is marked as the Reference because it contains a Control Identifier as set in the File Name Group tool See Chapter 8 Additional Tools Reference Control samples will appear in the first column of the File Name Group Text file Expand folders in the Group File Tree to view the Reference sample and Experimental sample in the group Double click the Reference sample to view just the Reference sample electropherogram Double click the Experimental sample to view the Experimental sample electropherogram overlaid on the Reference sample electropherogram and associated Gain Loss Histogram Use the Up Down Arrow keys to navigate through samples Hit the Enter key to open Group folders Right click an Experimental sample in the Group File Tree and select Set as Reference to mark the sample as the Group s Reference sample 131 May 2012 Chapter 7 Special Applications Trace Overlay The Trace Overlay displays the selected Experimental sample s trace superimposed over the selected Group s Reference trace The Experimental sample trace line color will appear in the Marker s associated dye color The Reference trace line color is displayed in light red The Reference trace is by default drawn directly behind the Experimental sample trace Click the MSI Display Settings icon to adjust the Reference trace offse
339. y Trace and Ratio Plot Use the Up Down Arrow keys to scroll through the sample list Right click and select Hide to disable a sample Disabled samples cannot be used in the Synthetic Control calculation and will not appear in the MLPA Clinical Report Right click and select Show to enable a sample Trace Overlay and Dosage Histogram The Trace Overlay displays a normalized electropherogram trace of the selected sample with the control sample reference trace displayed in light red behind it The allele or exon names are displayed below their corresponding bins See the What to Expect section below for information regarding trace normalization The Dosage Histogram displays in bar chart form the ratio of normalized peak intensities between the reference and the sample trace Sample probes are represented by blue bars and control probes are black bars Allele editing options in the Trace Overlay frame are similar to those in the Main Analysis window Electropherogram See Chapter 3 Main Analysis Overview Ratio Plots The Ratio Plot displays in graphical form the ratio of normalized peak intensities between the reference and the sample trace Each point represents a sample peak or probe Select Ratio or Regression Analysis in the MLPA Analysis Settings box to change graphical views Additionally statistical information is displayed below the Ratio Plot when Show Statistics Information is selected in the Display Settings box Green Peak is within
340. y ranking for each peak with regard to the peak Score as set in the Run Wizard Additional Settings box See Chapter 2 General Procedure and or software editing of the original raw data such as correction of saturated peaks SAT Repaired Score The peak quality score is calculated based on signal to noise ratio and peak shape or morphology Lower scores indicate poorer quality peaks Additionally the Score value is a based on an exponential curve Start End Indicate the beginning and end of the Area calculation for the peak Comments Software and user edited comments appear in the Comments column Quality Reasons Indicates the reason why a peak received a Quality rank of Check or Undetermined For explanation of the two and three letter codes click the Help icon above the Report Table Save Peak Table Click the Save Peak Table icon in the main toolbar to export the Peak Table information currently being displayed in Excel xls or tab delimited Text txt format All samples peak information for only the dye colors selected will be exported in the table Additionally the user can right click in the Peak Table and select Copy Table Hot Key CTRL C to place the current table information onto the Windows clipboard The information can then be pasted into most common spreadsheet or word processing programs including Microsoft Excel Editing Peaks 32 May 2012 Chapter 3 Main Analysis Overview Double click the vertical grey peak c
341. ype considered methylated Ponte Hidden Garon np 12 Click OK Quantification 13 Analyze data for non control peaks above the Methylated Peak 9 Ratio set in the Analysis Settings box 14 Click the Print icon to create an MS MLPA Clinical Report Peak Ratios OAlleles lt 0 30 lt Normal lt 0 70 lt Other Auto Slope Correction Cancel Genomic Imprinting 9 Select Genomic Imprinting Analysis Type 10 Choose to analyze by Peak Height OR Peak Area 11 Enter the Peak Ratio values between which a peak will be considered normal 12 Click OK 13 Analyze data for non control peaks above the Other Peak Ratio set in the Analysis Settings box 14 Click the Print icon to create an MS MLPA Clinical Report What to Expect The MRC Holland MS MLPA kits take advantage of methylation sensitive endonucleases Hhal and Hpall which will cleave unmethylated DNA fragments Methylated fragments are not digested by the endonucleases and will therefore produce a peak signal in the electropherogram In addition to novel methylation detection probes MRC Holland includes multiple control probe genes in the MS MLPA kit for monitoring the efficiency of the endonuclease activity Promoter Methylation In trace electropherogram the undigested patient DNA sample appears as the red background trace in the electropherogram and is used as the reference The same patient s digested DNA sample appears as the blue trace in the electropherogram Based on th

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