ABI PRISM® GeneMapper™ Software Version 3.0 Microsatellite
Contents
1. ABI Prism GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial 2 9 Chapter 2 Working With Panels and Bins Setting Up Bin Sets Overview Once you have sized reference data for the panel you wish to use you are ready to set up bin sets The steps required are e Creating a bin set e Performing autobinning Note If you create a bin set with tutorial data it is only applicable to the tutorial Creating a Bin To create a new bin set Set 1 In the Project window select Tools gt Panel Manager The Panel Manager window opens 2 In the navigation pane select the Microsatellite Tutorial folder The panels contained in this folder are displayed in the right pane of the Panel Manager window and the Create New binset icon ml on the toolbar is enabled 43 Panel Manager x File Edit Bins View B BB onset Ea emme mm m anger iN TE 1 futoriatPaneig none _o Cancel Apply Click the New binset icon 2 10 ABI PRISM GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial Setting Up Bin Sets To create a new bin set continued 3 Select Bins gt New Bin Set The New Bin Set dialog box opens 4 Type Microsatellite Tutorial BinSet and click OK The new bin set name is added to the Bin Set drop down list at the top of the Panel Manager window 5 In the navigation pane a Expand the Microsatellite Tutorial folder b Select the Tuto2. Examine the analyzed data e Adjust panels or bins e Edit allele calls See Chapter 2 and Chapter 4 See Creating an Analysis Method on page 3 5 See the User Manual See Importing Reference Data into a New Project on page 2 5 See Applying Project Settings on page 3 10 See Examining the Analyzed Data on page 3 13 ABI Prism GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial Working With Panels and Bins In This Chapter Assumptions This chapter includes the following topics Process Flowchart cece ee eee eee eee nee 2 2 Importing Panels 0 0 eee eee eee eee 2 3 Importing and Sizing Reference Data 0 4 2 5 Setting Up Bin Sets crane peard ess enea nii i i i 2 10 This chapter guides you through setting up a project with ABI PRISM GeneMapper Software Version 3 0 in order to analyze microsatellite data either yours or the sample files provided with this tutorial It is assumed that you have already installed and logged on to the GeneMapper software Note This chapter provides instructions for setting up a GeneMapper software project using imported panels If you want to create your own panels refer to Chapter 4 ABI PRISM GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial 2 1 Chapter 2 Working With Panels and Bins Process Flowchart Setting Upa The flowchart below p
3. 2 In the navigation pane select the Panel Manager root node ABI Prism GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial 2 3 Chapter 2 Working With Panels and Bins To import panels into the Panel Manager window continued 3 Select File gt Import Panels The Import Panels dialog box opens Li Look in E Panels x c a Install Standards Panels bt a LMS HD5 25 Panels bet a LMS MD10 2 5 Panels bt Microsatellite Tutorial Panel bt File name Microsatellite Tutorial Panel tet Import Files of type au Files Cancel Navigate to the disk directory containing the GeneMapper software application Import a panel from the Panels folder to this project as follows a Open the following folders in this order GeneMapper gt Panels b Select the Microsatellite Tutorial Panel txt file c Click Import The Import Panels dialog box closes and a new kit folder entitled Microsatellite Tutorial is created in the navigation pane of the Panel Manager window Click OK to close the Panel Manager window 2 4 ABI Prism GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial Importing and Sizing Reference Data Importing and Sizing Reference Data Overview The steps required to import and size reference data are e Converting the sample files if necessary e Importing reference data That is Linkage Mapping samp
4. To create an Analysis Method continued 4 Note Refer to Appendix A for further information on Peak Detection Algorithms Select the Peak Detector tab then a Select Basic from the Peak Detection Algorithm drop down list Be sure to use the Basic mode for this tutorial the Advanced and Classic algorithm modes are described in the ABI PrisM GeneMapper Genotyping Software User s Manual PN 4335526 b Select User specified rfu in the Minimum Peak Height pane c Leave the default values for each color dye 50 relative fluorescent units Note When you make changes to the Minimum Peak Height setting the automatic setting is equivalent to 10X the noise level for each sample individually Analysis Method Editor Microsatellite x General Allele Peak Detector Peak Quality Quality Flags Peak Detection Algorithm Basic x gt Minimum Peak Height C Automatic Factory Defaults OK Cancel ABI Prism GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial 3 7 Chapter 3 Data Analysis To create an Analysis Method continued 5 Select the Peak Quality tab No changes need to be made the defaults are satisfactory for this tutorial Analysis Method Editor Microsatellite q x General Allele Peak Detector Peak Quality Quality Flags Signal level Homozygous min peak height 200 0
5. ABI PRIsM GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial Applied Biosystems ABI PRismM GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial BSS BE stems Copyright 2002 Applied Biosystems All rights reserved For Research Use Only Not for use in diagnostic procedures Information in this document is subject to change without notice Applied Biosystems assumes no responsibility for any errors that may appear in this document This document is believed to be complete and accurate at the time of publication In no event shall Applied Biosystems be liable for incidental special multiple or consequential damages in connection with or arising from the use of this document Notice to Purchaser License Disclaimer Purchase of this software product alone does not imply any license under any process instrument or other apparatus system composition reagent or kit rights under patent claims owned or otherwise controlled by Applera Corporation either expressly or by estoppel TRADEMARKS ABI PRISM and its design Applied Biosystems GeneScan Genotyper SNaPshot LIZ and VIC are registered trademarks of Applera Corporation or its subsidiaries in the U S and certain other countries AB Design ABI Applera GeneMapper NED PET ROX and 6 FAM are trademarks of Applera Corporation or its subsidiaries in the U S and certain other countries Macintosh is a registered trademark of
6. Note If you are in the Samples Plot window the Separate Dyes mode must be on in order to view microsatellite labels Click the desired peak to select it Right click the selected peak to bring up the editing options The options are different for labeled and unlabeled peaks as described below If you selected You can a labeled peak edit or delete the allele call an unlabeled peak you can only add an allele call ABI Prism GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial 3 19 Chapter 3 Data Analysis To edit microsatellite alleles in the Plot window continued 5 When you finish editing note that e In the Plot window the edited allele s display the allele edit history comments This can be turned off by selecting View gt Allele Changes e In the Project window the edited allele s display gray quality flags These are displayed in the Genotypes tab only Editing Marker To edit marker and bin information Select Alleles gt Editing Mode gt and Bin Binning Information If necessary the Plot window will automatically switch to a view a appropriate for editing marker and bin information To Perform this action Edita a Click the red triangle marker indicators marker size b Click and drag the size range handles to range the desired locations Edit a bin a Click a bin to select it b Right click the selected b
7. Vv In the Peak Detector tab select a peak detection algorithm for your application Vv In the Peak Quality tab set the minimum requirements Vv In the Quality Flags tab adjust the PQV and threshold settings Vv Click OK to save the new Analysis Method Analysis Method Editor Window ABI Prism GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial 3 3 3 4 Chapter 3 Data Analysis Accessing the To access the Analysis Method Editor Analysis Method Editor 1 In the Project window select Tools gt GeneMapper Manager The GeneMapper Manager window opens GeneMapper Manager 5 E x Projects Analysis Methods Tabie Settings Plot Settings Matrices Size Standards Name Last Saved Owner instrument analysis Type Description 1 3730 DS 33 Install 2002 06 19 16 16 22 0 Jam 3730 Microsatellite 2 Microsatellite Default 2002 06 19 16 16 22 0 am Factory Provided Microsatellite Factory Provided 20 1916 16 22 0 gm New Open Save As Import Export Delete 2 Select the Analysis Methods tab then click New The New Analysis Method dialog box opens x Select analysis type C HID C SNaPshot Microsatellite OK Cancel 3 Select Microsatellite as the analysis type then click OK The Analysis Method Editor window opens Note The analysis type you select e Sets the analy
8. 0 25 Genotype Quality From jo 7s to 1 0 From 0 0 to fo 26 Factory Defaults Note When analyzing your own data the Quality Flags tab allows you to manipulate the importance level of the individual quality values The quality values are on a scale of 0 to 1 with 0 being of no importance and 1 being of high importance Changing these values will affect your final GQ value Click OK to close the Analysis Method Editor window and save the new Analysis Method Note Clicking Cancel will close the window without saving your changes Click Done to exit the GeneMapper Manager window ABI Prism GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial 3 9 Chapter 3 Data Analysis Performing Analysis on Microsatellite Data Overview The steps required to perform analysis on microsatellite data are e Converting the sample files if necessary e Adding samples files to the project e Applying project settings and starting the analysis Converting Note The information below is not necessary for this tutorial Sample Files however you may find it useful when you are working with your own sample files If necessary convert ABI PRISM GeneScan Analysis Software sample files generated by the Apple Macintosh software to the fsa format The conversion is described in the ABI PRISM GeneMapper Genotyping Software User s Manual PN 4335526 Adding Sample If you have followed along through this
9. converting 2 5 sample files converting 3 10 setting up panels and bin sets 2 10 size standard definition 1 6 selecting 2 7 sizing analysis performing 2 7 T tutorial default settings 1 4 panel information 1 4 sample files 1 6 terms usedin 1 2 ABI PRISM GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial Index 1 Index 2 ABI Prism GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free In North America 1 800 345 5224 Fax 1 650 638 5884 Worldwide Sales and Support Applied Biosystems vast distribution and service network composed of highly trained support and applications personnel reaches 150 countries on six continents For sales office locations and technical support please call our local office or refer to our Web site at www appliedbiosystems com www appliedbiosystems com Applied Biosystems Applera Corporation is committed to providing the world s leading technology and information for life scientists Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses Printed in the USA 09 2002 Part Number 4335525 Rev B an Applera business
10. is displayed as follows e The progress indicator at the bottom of the Project window shows progress in two ways Asa bar graph extending from the left Asa percentage indicator e The current sample undergoing analysis is indicated by the sample row in the table displayed in green or red if analysis failed for the sample When the program has finished analyzing the samples the message Analysis Completed appears on the Status bar of the Project window lower left corner Note Auto saving takes place after every 10 sample files are analyzed or before the Analysis Completed message displays 8 Click the Genotypes tab in the Project window to view analysis results The GQ column informs you of the quality of the allele call e Green square Pass e Yellow triangle Check e Red circle Fail Note As the samples are analyzed the results display in the Project window Genotypes tab This tab was previously unavailable because you were sizing the samples By selecting a bin set allele calls are made and saved 3 12 ABI PRISM GeneM apper Software Version 3 0 Microsatellite Analysis Tutorial Examining the Analyzed Data Examining the Analyzed Data Overview You can examine the analyzed data in both the Samples and Genotypes tabs of the Project window The steps included are e Customizing the Project window optional e Displaying samples in the Samples Plot window e Displaying samples
11. samples are displayed in Full View e The X axis is displayed in Basepairs Allele call labels bins and marker indicators cannot be viewed in the Combine Dyes mode To view these items switch to the Separate Dyes mode by selecting View gt Plots gt Separate Dyes The default options may be changed Take a moment now to test all of the available options in the Samples Plot window To do this select the menu items and toolbar icons If you would like to learn more about all of the available options in the window refer to e Performing Tasks in the Plot Windows on page 3 18 e The ABI PRISM GeneMapper Genotyping Software User s Manual PN 4335526 Use the Genotypes Plot window to display electropherograms on a per marker basis To display genotypes in the Genotypes Plot window 1 In the Project window select the Genotypes tab 2 For each genotype you wish to display select the genotype row number ABI Prism GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial 3 15 Chapter 3 Data Analysis To display genotypes in the Genotypes Plot window continued 3 Select Analysis gt Display Plots The Genotypes Plot window opens with an electropherogram displayed for each selected marker Note If you had left the Samples Plot view open and performed steps 1 and 2 above the view would automatically refresh and display the selected genotypes Yi
12. to List Your project is added to the list in the right pane Click Add A dialog box appears indicating that the reference data has been added to the new bin set Click OK to close the Add Microsatellite Reference Data dialog box and return to the Panel Manager window Performing To perform autobinning Autobinning 1 Select Bins gt Auto Bin The Auto Bin dialog box opens 2 For this tutorial select the following options a Under Allele Naming Scheme select Rounded basepair b Under Auto Bin Options select Auto Bin ABI PRISM GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial Setting Up Bin Sets To perform autobinning continued 3 To initiate Autobinning click OK 4 When the Autobinning completed message displays click OK 5 To view the bins created select a marker from the navigation pane The bins established by the reference data are displayed The red cross hatches indicate the positions of the reference alleles The Y axis scale marks the GQ values qq Panel Manager E File Edit Bins View Bx cai N Bin seticrosatenite tutorial gin H Sa a a mm Tutorial Panel Sal D65264 D6S1574 179 180 182 D65276 D55408 D6s308 D65287 068292 D68434 058426 D551981 D6S446 D58641 D58433 D58422 D58406 EI Reference Samples C006_E019_Run_c008 C006_E029_Run_c008 CO06_E039_Run_C006 C006_E109_Run_C
13. 008 0 0 162 164 166 168 170 172 174 176 178 180 182 184 186 188 190 192 194 196 198 200 DES257 ok Cancel Apply ABI Prism GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial 2 13 Chapter 2 Working With Panels and Bins To perform autobinning continued 6 If you select a reference sample the electropherogram is displayed to show placement of the bins Edit bin and allele size information a To edit create or delete bins Use the options under the Bins menu or Right click within the pane to make a new bin on an existing bin to edit or delete it b To edit allele size ranges select the marker name and drag the left or right handles of the marker range to the desired location Note To correct an undesired change click Edit gt Undo 7 To view the current project alleles select the Show Project Alleles icon The project alleles are displayed as blue asterisks Note You can only view the project alleles after you have analyzed your data with the new bin set At this point they are the same as the reference alleles gp Panel Manager File Edit Bins View SX RW ainsetpcresatemme woran H Lae Se i i 5 6 Panel Manager B g5 Microsatellite Tutor g5 Tutorial Panel 1 0 D6S264 Dests74 BE 065276 0 3 D55408 D68308 D65287 173 170 180 182 Reference Samples C006_E018_Ru
14. 2 5 SNaPshot Tutorial Primer Focus Tutorial DRE H A c Double click the panel you wish to use The Select a Panel window closes and the selected panel is entered into the first cell of the Panel column in the Samples tab Note This step is not necessary for this tutorial However you may find it necessary to select a size standard when you are working with your own sample files To select a size standard a Select the top cell in the Size Standard column b Select an appropriate size standard from the drop down list Apply the selections to the selected samples as follows a Click and drag the mouse across the three column headings Analysis Method Panel and Size Standard to select the entire columns b Select Edit gt Fill Down or press Ctrl D to apply the selections to the selected samples The Status column is now selected indicating that the analysis needs to be performed ABI PRISM GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial 3 11 Chapter 3 Data Analysis To apply project settings continued 6 Select Analysis gt Analyze Note Since this project is the one you created in Chapter 2 the Save As dialog box does not appear Your project will be saved automatically under the same name 7 The program initiates analysis then saves each analyzed sample to the project While analysis is proceeding progress
15. Apple Computer Inc All other trademarks are the sole property of their respective owners Part Number 4335525 Rev B 09 2002 Contents Chapter 1 Chapter 2 Chapter 3 Chapter 4 Introduction About the Microsatellite Tutorial cc eee DatairProvided s 2 pafiecan4 ewe dt aha uae vat aldo bath eed New Features in GeneMapper Software Version 3 0 Process FIOWE hart ni i eee ti ata ia at pu la Ale ceda d ta i Working With Panels and Bins Process Flowchart ns pese pir aaa tg a de a poa e e importing Panels is siraga ad at ee hohe uate e dt Aa Importing and Sizing Reference Data 0 0 cece eee Setting Up Bin Sets 2 c iia Sie eee He hea ea eas Data Analysis Process Flowchart ia eine ada belie win aia eas he ae 4 Creating an Analysis Method 0 0 0 cece eee eee Performing Analysis on Microsatellite Data Examining the Analyzed Data 0 0 cece eee eee Performing Tasks in the Plot Windows uc cc Creating Custom Kits Panels and Markers Process Flowchart rekta orn EU Op EA N E UA EAA Creating Kits Panels and Markers 0 0 cc eee eee teens ABI Prism GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial Appendix A Peak Detection Algorithms Index iv ABI PRISM GeneM apper Software Version 3 0 Microsatellite Analysis Tutorial Introduction In This Chapter This chapter includes the
16. Genotypes Plot I8l Ja a El EEEE Bich File Edit View Tools Alleles Help Microsatellite Default MH afe Maf Some splen Pan nt 0s Sv ona sea se am Dm LEN 370 Ame an lee L cQ 100 FO RU OD IEZI AC RC ee fe oe ele le oea aos mor 109 12325107 oa o e as O O O g n A A W Ti T 125 03 Ez 134 95 ES RRES 2B SNL BL LCS BB fer E E E E IE E A IE IE IE IN IE IE IE E Select Microsatellite Default from the Plot Setting pull down menu on the left side of the toolbar Note The Microsatellite Default Plot Setting has been configured for use with microsatellite data and is appropriate for this tutorial When analyzing your own data you can create and or edit Plot Settings using the GeneMapper Manager For more information refer to the ABI PRISM GeneMapper Genotyping Software User s Manual PN 4335526 ABI PRISM GeneM apper Software Version 3 0 Microsatellite Analysis Tutorial Examining the Analyzed Data To display genotypes in the Genotypes Plot window continued 5 Test all of the options in the Genotypes Plot window By default each electropherogram is displayed with the following options e Only the marker size range of the selected genotype is shown together with a marker margin of five basepairs A marker margin is how many basepairs beyond the marker size r
17. Heterozygous min peak height fi 00 0 Heterozygote balance Min peak height ratio jos Peak morphology Max peak width basepairs fi 5 mPull up peak Pull up percentage for Allele number Max expected alleles p Factory Defaults Note When analyzing your own data adjusting these values triggers specific PQV flags within a sample For example if a homozygous peak is detected with a signal level below 200 RFUs then the Low Peak Height value will be flagged For more information refer to the ABI PRISME GeneMapper Genotyping Software User s Manual PN 4335526 Note When analyzing your own data you can change the Allele Number value to allow for analysis of polyploid samples 3 8 ABI PRISM GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial Creating an Analysis Method To create an Analysis Method continued 6 Select the Quality Flags tab No changes need to be made the defaults are satisfactory for this tutorial Analysis Method Editor Microsatellite General Allele Peak Detector Peak Quality Quality Flags Quality weights are between 0 and 1 Quality Flag Settings Spectral Pull up ps Control Concordance ps Broad Peak ps Low Peak Height ps Single Peak Artifact 0 5 _ Off scale ps Sharp Poak bs Peak Height Ratio 05 One Basepair Allele ps Out of Bin Allele ps Split Peak jos MPAY Thresholds Sizing Quality From 75 to 1 0 From 0 0 to
18. ange is shown on the screen both right and left sides of the range e The X axis is displayed in Basepairs e The Bins mode is on e The Marker Range mode is on e The Indicators mode is on e The allele call labels are displayed horizontally 6 The default options may be changed Take a moment now to test all of the available options in the Genotypes Plot window To do this select the menu items and toolbar icons If you would like to learn more about all of the available options in the Genotypes Plot window refer to e Performing Tasks in the Plot Windows on page 3 18 e The ABI PRISM GeneMapper Genotyping Software User s Manual PN 4335526 ABI Prism GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial 3 17 Chapter 3 Data Analysis Performing Tasks in the Plot Windows Overview This section provides information about some common tasks performed in the Samples and Genotypes Plot windows These are Zooming Adjusting the X Axis Scale Adjusting the Y Axis Scale Editing Allele Calls Editing Marker and Bin Information If you would like to learn more about all of the available options in the Samples and Genotypes Plot windows refer to the ABI PRISM GeneMapper Genotyping Software User s Manual PN 4335526 Zooming Table 3 1 Zooming Choices If you want to Then zoom in on a specific click the X or Y axis and drag it to the desired region region zo
19. arker Name Dye Color etc x File Edit Bins View 2x ome IM ese o ee a fe i Panel Manager H E GeneScan Installation SNPdemo 9 New Microsatellite Kit 2 me New Panel New Marker 1 NewMarker blue 0 0 0 0 amp Reference Samples OK Cancel Apply 4 To create other new markers associated with this panel repeat steps 2 and 3 You can create as many markers as you d like 5 Click OK Completing To complete the GeneMapper software project setup continue with Project Setup the following procedures e Importing and Sizing Reference Data on page 2 5 e Setting Up Bin Sets on page 2 10 IMPORTANT While following the procedures be sure to make adjustments for your customized panels You cannot simply use the tutorial defaults with your custom settings ABI Prism GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial 4 5 Chapter 4 Creating Custom Kits Panels and Markers 4 6 ABI PRISM GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial Peak Detection Algorithms 3 Types of Peak Three types of Peak Detection algorithms are available Detection user control over data analysis for desired results Basic Contains limited parameters that may not provide enough e Classic Includes the same parameters and the same size caller and produces similar results to GeneScan software version 3 1 2 designed for use with th
20. displayed for each selected sample wi Samples Plot ioi x File Edit View Tools Alleles Help Microsateti 7 Panes e ME ee Maaf i Sample File Sample Name Panel os Isa 006_E019_Rum_ E019 Tutorial Pan 0 965 a EE Mi Bik Mx A 39 79 119 159 199 239 279 319 359 399 439 479 519 559 599 639 679 719 n C006_E029_Run_C E029 Tutorial Pan J 0 96 4 39 79 119 459 199 239 279 39 359 399 439 479 519 559 599 639 679 719 o eo De 4 Select Microsatellite Default from the Plot Setting pull down menu on the left side of the toolbar Note The Microsatellite Default Plot Setting has been configured for use with microsatellite data and is appropriate for this tutorial When analyzing your own data you can create and or edit Plot Settings using the GeneMapper Manager For more information refer to the ABI PRISM GeneMapper Genotyping Software User s Manual PN 4335526 3 14 ABI PRISM GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial Displaying Genotypes in the Genotypes Plot Window Examining the Analyzed Data To display samples in the Samples Plot window continued 5 Test all of the available options in the Samples Plot window By default each electropherogram is displayed with the following options e The Combine Dyes mode is on that is all dye colors for a sample are overlaid within a single pane e The
21. e Macintosh operating system e Advanced Includes the same parameters and the same size caller and produces similar results to GeneScan software designed for use with the Microsoft Windows NT operating system Also it includes a new size caller with the exception of the smoothing function Usage Guidelines The table below lists general guidelines for selecting an algorithm to use based on the instrument Instrument Peak Detection Algorithm 310 and 377 Classic Multi capillary Advanced ABI Prism GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial A 1 Appendix A Peak Detection Algorithms A 2 ABI Prism GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial Index A adding reference SNP samples to projects 2 5 adding sample files 3 10 Analysis Method definition 1 2 analysis method creating 3 2 editor 3 4 autobinning 2 12 bin set creating 2 10 definition 1 2 bin definition of 1 2 C converting sample files 2 5 3 10 creating an SNP bin set and panel 2 10 creating an SNP kit 4 3 D data analysis performing 3 2 data provided in tutorial 1 4 editor analysis method 3 4 M marker definition 1 3 microsatellite data examining data and results 3 13 performing analysis 3 10 New Features in GeneMapper Software Version 3 0 1 7 P panel definition of 1 3 Project 2 2 project settings applying 3 10 definition 1 3 Project window 2 6 S sample files
22. following topics About the Microsatellite Tutorial 0 0 0 0 0 00 00 c eee eee 1 2 Data Provided 24 0350 524s BE eA eae ed ae o Bea ed 1 4 New Features in GeneMapper Software Version 3 0 1 7 Process Flowchart eee 1 8 ABI Prism GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial 1 1 Chapter 1 Introduction About the Microsatellite Tutorial Overview Goals How to Use This Tutorial Terms Used in This Tutorial 1 2 This tutorial is intended to help you get quickly up to speed in performing analyses of microsatellite data with ABI PRISM GeneMapper Software Version 3 0 Note This tutorial contains basic information only For more advanced information please refer to the ABI PRISM GeneMapper Genotyping Software User s Manual PN 4335526 After completing this tutorial you should be able to e Set up a GeneMapper software project e Import sample files for analysis e Create bins using the autobinning feature When you are following the procedures in this tutorial it is important that you e Perform every step in the order listed e Do not introduce any extraneous samples or bins The procedures are written using menu selections for example Select Tools gt GeneMapper Manager You may also use the icons located on the toolbar The following terms are used in this tutorial Table 1 1 Terms and Definitions Term Definition Analysis Method A collection of
23. in the Genotypes Plot window Customizing the As you examine your data you may want to customize the Project Project Window window to better view your data Customize the Project window by e Resizing the Project window e Creating a new Table Setting or using the provided default table setting These tasks are optional For procedures refer to the ABI PRISM GeneMapper Genotyping Software User s Manual PN 4335526 About C reating For this tutorial you do not need to create your own Plot Setting Use Plot Settings the default Plot Setting provided named Microsatellite Default When examining your own data however you may wish to create your own Plot Settings to customize the information that is displayed For procedures refer to the ABI PrisM GeneMapper Genotyping Software User s Manual PN 4335526 Displaying Use the Samples Plot window to display electropherograms on a per Samples in the sample basis Samples Plot Window T display samples in the Samples Plot window 1 In the Project window select the Samples tab 2 For each sample you wish to display select e The sample row number or e The sample file name ABI Prism GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial 3 13 Chapter 3 Data Analysis To display samples in the Samples Plot window continued 3 Select Analysis gt Display Plots The Samples Plot window opens with an electropherogram
24. in to delete or edit the bin information Move a bin a Click a bin to select it b Click and drag the center of the bin to the desired location Resize a bin a Click a bin to select it b Drag the left and right bin range handles to the desired locations When you are finished making changes close the Plot window and save the changes 3 20 ABI PRISM GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial Creating Custom Kits Panels and Markers In This Chapter This chapter includes the following topics Process Flowchart nos cet heen aaa aa Prada cd ila edna 4 2 Creating Kits Panels and Markers 0 0000005 4 3 Assumptions In Chapter 2 you used predefined panels to set up a project if not using LMS v2 5 It is possible to set up a project using custom kits panels and markers It is assumed that you have already installed and logged on to the ABI PRISM GeneMapper Software Version 3 0 ABI Prism GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial 4 1 Chapter 4 Creating Custom Kits Panels and Markers Process Flowchart Setting Upa The flowchart below provides an overview of the tasks required to Project setup a GeneMapper software project when you are creating custom kits panels and markers Create a new kit Create new markers le J gt Create a new panel Panel Manager Im
25. l a Select the top cell in the Panel column The Select a Panel window opens b Expand the Microsatellite Tutorial folder c Double click Tutorial Panel 9 The Select a Panel window closes and Tutorial Panel 9 is entered into the first cell of the Panel column in the Samples tab 4 Select a size standard a Select the top cell in the Size Standard column b Select GS500 250 LIZ from the drop down list Note The GeneScan 500 250 LIZ size standard has been predefined for you To learn how to create your own size standard refer to the ABI PRISM GeneMapper Genotyping Software User s Manual PN 4335526 5 Apply the selections to the selected samples as follows a Click and drag the mouse across the three column headings Analysis Method Panel and Size Standard to select the entire columns b Select Edit gt Fill Down or press Ctrl D ABI Prism GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial 2 7 Chapter 2 Working With Panels and Bins To perform sizing analysis on the reference data continued 6 Select Analysis gt Analyze The Save Project dialog box opens Gi Project name e Cancel Type Microsatellite Tutorial and click OK The program initiates analysis then saves each analyzed sample to the project you have just named While analysis is proceeding progress is displayed as follows e The progress indicator at the bottom of
26. le files into a new project e Performing sizing analysis on the reference data which results in sample files that can be used for creating bins Converting Note The information below is not necessary for this tutorial Sample Files however you may find it useful when you are working with your own sample files If necessary convert ABI PRISM GeneScan Analysis Software sample files generated by the Apple Macintosh software to the fsa format The conversion is described in the ABI PRISM GeneMapper Genotyping Software User s Manual PN 4335526 Importing To import reference data into a new project Reference Data into a New 1 In the Project window select File gt Add Samples to Project Project The Add Samples to Project window opens 2 Navigate to the disk directory containing the GeneMapper software application ABI Prism GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial 2 5 Chapter 2 Working With Panels and Bins 2 6 To import reference data into a new project continued 3 Add sample files from the Microsatellite folder to this project as follows a Expand the following folders in this order GeneMapper gt Tutorial Data gt Microsatellite b Select the Microsatellite folder c Click Add To List The Microsatellite folder and all sample files within it moves to the Samples To Add list in the Add Samples to Project window If you selected an i
27. n Size Standard The following size standard definitions are provided for use with Definitions microsatellite data GeneScan 400HD size standard e GeneScan 500 size standard e GeneScan 500 250 size standard e GeneScan 500 250 LIZ size standard e GeneScan 500 LIZ size standard Note Custom size standard definitions can be created For procedures refer to the ABI PRISM GeneMapper Genotyping Software User s Manual PN 4335526 Sample Files Four microsatellite sample files are provided for use with this tutorial The files were generated on a 3100 instrument using LMS MD 10 panel 9 with 5 custom PET markers We used the GeneScan 500 LIZ size standard 1 6 ABI Prism GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial New Features in GeneM apper Software Version 3 0 New Features in GeneMapper Software Version 3 0 List of New Features How These Features Affect Data Analysis GeneMapper Software Version 3 0 is an enhancement of GeneMapper Software Version 2 0 The new features in GeneMapper Software Version 3 0 are listed below ABI PRISM GeneScan software functionality including EPT Data tab added to the Samples tab accessed via the Project window Sizing Table tab added to the Plot Settings accessed via the Plots window Standard Curve tab added to the Size Match Editor ABI PRISM Genotyper software functionality including Enhanced allele call label
28. n_Co06 C006_E029_Run_C006 0 3 C006_E039_Run_C008 C006_E109_Run_C006 0 0 162 164 166 168 170 172 174 176 178 180 182 184 186 188 190 192 194 196 198 200 D65257 Lal ll coca aso 2 14 ABI PRISM GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial Setting Up Bin Sets To perform autobinning continued 8 Click OK to accept the new bin set This also closes the Panel Manager window ABI Prism GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial 2 15 Chapter 2 Working With Panels and Bins 2 16 ABI PRISM GeneM apper Software Version 3 0 Microsatellite Analysis Tutorial Data Analysis In This Chapter Assumptions This chapter includes the following topics Process Flowchart esii 0 0 cc eee e eee eee eee ene 3 2 Creating an Analysis Method 0 0 0 00 00 eee 3 2 Performing Analysis on Microsatellite Data 3 10 Examining the Analyzed Data 0 0 0 0 000 005 3 13 Performing Tasks in the Plot Windows 3 18 This chapter guides you through analyzing microsatellite data with ABI PRISM GeneMapper Software Version 3 0 It is assumed that you have already e Installed and logged on to the GeneMapper software e Set up panels and bin sets as discussed in Chapter 2 ABI Prism GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial 3 1 Chapter 3 Data Analysi
29. ndividual sample only that sample is added Note If you move the wrong folder to the Samples To Add list select the folder and click Clear to remove it Click Add to import the sample files into this new project and close the Add Samples to Project window The Project window now shows the imported sample files displayed in the Samples tab lelzi File Edit Analysis View Tools Help sd BR OO AWB p 6 Tableseting Deru la BS S Project Samples Genotypes Microsatel Status Sample File Sample Name Comments Sample Type Specimen Ca Analysis Method Panel CO06_E019_RI C006_E029_Ri E029 Sample no export C006_E039_Ri E039 Sample C006_E109_Ri E109 Sample no export no export ll Progress Status ABI Prism GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial Importing and Sizing Reference Data Performing Sizing To perform sizing analysis on the reference data Analysis 1 Go to the Samples tab view in the Project window 2 Select an Analysis Method a Select the top cell in the Analysis Method column b Select Microsatellite Default from the drop down list Note When you are analyzing your own microsatellite reference data make sure the Analysis Method you choose has Microsatellite as the analysis type and a bin set of None For more information see page Creating an Analysis Method on page 3 2 3 Select a pane
30. nel information is provided for e LMS HD5 Version 2 5 and LMS MD10 Version 2 5 kits using allele size ranges for each marker determined by data generated with the ABI PRISME 3100 Genetic Analyzer e GeneScan Installation Standard DS 33 6 FAM VICE NED and PET dyes e GeneScan Installation Standard DS 30 6 FAM HEX NED and ROX dyes e This tutorial Note For more information refer to the specific panel guide for the kit you are using And if you are using custom kits refer to Chapter 4 for more information Bin information is provided for GeneScan Installation Standard DS 33 with GeneScan 500LIZ_3730 size standard for the Applied Biosystems 3730 3730x1 DNA Analyzer only The following microsatellite default settings are provided Table 1 3 Default Settings Type Name Parameters Analysis Microsatellite Analysis Type Microsatellite Method Default Bin Set none e Analysis Algorithm Basic DS 33 Install e Analysis Type Microsatellite Bin Set 3730 DS 33 Install Bins Analysis Algorithm Basic ABI PRISM GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial Table 1 3 Default Settings Data Provided Table Setting Microsatellite Default Type Name Parameters Plot Setting Microsatellite N A Default Sizing ABI Prism GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial 1 5 Chapter 1 Introductio
31. om out to the full view double click the X or Y axis access additional zoom right click the X or Y axis options Adjusting the X To adjust the X axis scale Axis Scale 1 Select View gt X Axis Scale 2 Select one of the following options Basepairs default e Data Points Note If you choose the Data Points option be aware that the point where sample extraction begins while always labeled 0 or zero in GeneMapper may correspond to a later scan number in the gel processing application 3 18 ABI PRISM GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial Performing Tasks in the Plot Windows Adjusting the Y To adjust the Y axis scale Axis Scale 1 Select View gt Y Axis Scale 2 Select one of the following options e Scale Individually default This option scales each electropherogram to its individual maximum height e Scale to maximum Y If you are viewing multiple electropherograms this option scales all panes to the global maximum height e Scale To This option allows you to set the Y scale for all of the electropherograms you are currently viewing Editing Allele To edit microsatellite alleles in the Plot window Calls Select Alleles gt Editing Mode gt Peak Selection If labels are not shown turn them on by selecting e View gt Labels gt Horizontal Labels or e View gt Labels gt Vertical Labels
32. parameters set by the user to determine the bin set and analysis algorithms See Creating an Analysis Method on page 3 2 Bin A fragment size or basepair range and dye color that define an allele Bin set A set of bins allele definitions for one source or set of experimental conditions usually an instrument Bin sets are available inside a kit ABI PRISM GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial About the Microsatellite Tutorial Table 1 1 Terms and Definitions continued Term Definition Marker A known microsatellite location Panel A set of markers The grouping of markers in panels is determined by the kit provider or user Project settings Parameters set by the user to prepare a project for analysis See Applying Project Settings on page 3 10 For More For more information about the GeneMapper software refer to the Information documents listed below Table 1 2 Documents Document Part Number ABI PRISM GeneMapper Genotyping Software 4335526 User s Manual SNP Genotyping with ABI Prism GeneMapper 4335524 Software Version 3 0 Tutorial ABI Prism GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial 1 3 Chapter 1 Introduction Data Provided Overview Panel Information Bin Information Default Settings 1 4 GeneMapper Software Version 3 0 comes with the data listed below Pa
33. port reference data into a new project y Size reference data e Select the Analysis Method with Bin Set of none Select the size standard Select the panel Project Window V Create a new bin set V Add reference data N Perform autobinning Panel Manager 4 2 ABI PRISM GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial Creating Kits Panels and Markers Creating Kits Panels and Markers Overview To begin set up of a GeneMapper software project for this tutorial you may create your own custom kits panels and markers The steps required are e Creating a microsatellite kit e Creating a panel e Creating markers Note A panel is a set of markers The grouping of markers in panels is determined by the kit provider or user C reating a To create a microsatellite kit Microsatellite Kit 1 In the Project window select Tools gt Panel Manager The Panel Manager window opens 2 Select the Panel Manager root node in the navigation pane 3 Select File gt New Kit The New Kit dialog box opens Kit name J Kit type Microsatellite gt OK Cancel 4 Complete the following information a In the Kit name text field type a name for the kit b From the Kit type drop down list select Microsatellite c Click OK The Panel Manager window now shows with the new Microsatellite kit displayed in the naviga
34. rial Panel 9 folder The markers contained in this folder are displayed in the right pane of the Panel Manager window and the Add Reference Data icon f on the toolbar is enabled x Panel Manager File Edit Bins View Ma S E Panel Manager Add Reference Data Microsatellite Tutorial LLALA LU e D6S1574 0 0 none Des276 oo none D55408 oo none D65308 0 0___ none D65287 0 0 _ none D68292 0 0 _ none D68434 none D55426 none D551981 none D65257 none D65446 none D55641 none D58433 none D55422 none 058406 2 oo none D55400 none D65309 none Reference Samples oK Cancel Apply ABI PRISM GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial 2 11 Chapter 2 Working With Panels and Bins 2 12 To create a new bin set continued 6 Select Bins gt Add Reference Data The Add Microsatellite Reference Data dialog box opens A folder representing your project is visible in the bottom pane Note The bottom pane will also show folders for all projects that have been sized using this panel if any exist Ef Add Microsatellite Reference Data x Edit Samples To Add j Cancel Search fa Microsatellite Tutorial Select your project or Microsatellite Tutorial and click Add
35. rovides an overview of the tasks required to Project setup a GeneMapper software project when you are importing the LMS kit panels provided with the GeneMapper software or your own customized panels D Import panels lt T E oO a Vv Import reference data into a new project Vv gt Size reference data 3 Selectan Analysis Method with a bin set of none e Select a size standard 9 D e Selecta panel 2 Q y Create a new bin set T AA 2 Add reference data 5 Y y amp Perform autobinning 2 2 ABI PRISM GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial Importing Panels Importing Panels Overview To begin setup of a GeneMapper software project for this tutorial you need to import the predefined tutorial panel into the Panel Manager window The imported panel is in the form of a text file txt extension provided with the GeneMapper software Note A panel is a set of markers The grouping of markers in panels is determined by the kit provider or user Importing Panels Use this procedure for importing LMS panels or your own panels To import panels into the Panel Manager window 1 In the Project window select Tools gt Panel Manager The Panel Manager window opens 4g Panel Manager x File Edit Bins View Ce Mm mm jaf Panel Manager
36. s Process Flowchart Performing Data The flowchart below provides an overview of the tasks required to Analysis perform data analysis Create an Analysis Method Add sample files to your project 4 Apply project settings e Select the Analysis Method Select the size standard Select the panel Vv Start analysis Vv Examine the analyzed data Vv Perform tasks in the Plot windows optional Creating an Analysis Method You can preconfigure project settings through the Add Samples tab in the Options window see the User Manual Overview Creating an Analysis Method allows you to reference the correct bin set and analysis algorithms for your data The steps required are 3 2 e Accessing the Analysis Method Editor via the GeneMapper Manager window e Creating the Analysis Method ABI Prism GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial Creating an Analysis Method Process The flowchart illustrates the tasks required to create an Flowchart analysis method y E Select Tools gt GeneMapper Manager os E vV gt Select the Analysis Methods tab 3 TA eat dS y o Click New G o U O 5 6 v l os Select the analysis type Microsatellite Vv In the General tab type a name for the new Analysis Method Vv In the Allele tab select the bin set to use
37. s in the Plots window Customized allele labeling options in the Plots window Marker and bin editing within the Plots window User interface consistency across products including new or revised menus and shortcut keys For detailed information about these new features refer to the ABI PRISM GeneMapper Genotyping Software User s Manual PN 4335526 When you perform microsatellite data analysis with GeneMapper Software Version 3 0 you will Be able to view electropherograms within the Panel Manager Have several algorithm options which can increase analysis flexibility Have increased allele bin and marker editing flexibility ABI Prism GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial 1 7 Chapter 1 Introduction Process Flowchart 1 8 Analyzing Microsatellite Data The flowchart below provides an overview of the tasks required to analyze microsatellite data with the GeneMapper software For detailed procedures see the references below Set up a new project e Import panels for ABI PRISME LMS kits or create custom panels Use the Autobinning feature to create bins i Create an Analysis Method Select the analysis type Microsatellite Select a bin set Select an analysis algorithm V Define a size standard if necessary V Import data e Convert Macintosh computer files if necessary Analyze data Apply project settings
38. sis algorithm e Displays the appropriate fields for that type of analysis in the Analysis Method Editor windows ABI Prism GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial Creating an Analysis Method C reating an To create an Analysis Method Analysis Method 1 In the Analysis Method Editor window select the General tab Analysis Method Editor Microsatellite Allele Peak Detector Peak Quality Quality Flags Analysis Method Description Name Description Instrument Analysis Type Microsatellite oc ca 2 Complete the following information a In the Name text field type Microsatellite Tutorial b In the Description text field type a description of the Analysis Method if desired c In the Instrument text field type your instrument name serial number if desired ABI Prism GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial 3 5 Chapter 3 Data Analysis To create an Analysis Method continued 3 Select the Allele tab then a Select Microsatellite Tutorial Binset from the Bin Set drop down list This is the bin set you created earlier see 2 10 b Leave the default values as they are for all other fields Analysis Method Editor Microsatellite 3 6 ABI PRISM GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial Creating an Analysis Method
39. the Project window shows progress in two ways As a bar graph extending from the left As a percentage indicator e The current sample undergoing analysis is indicated by the sample row in the table displayed in green or red if analysis failed for the sample When the program has finished analyzing the samples the message Analysis Completed appears on the Status bar of the Project window lower left corner Note Auto saving takes place after every 10 sample files are analyzed or before the Analysis Completed message appears When samples are sized successfully e A green square is displayed in the SQ column you may need to scroll to see this column e The Status column is cleared 2 8 ABI Prism GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial Importing and Sizing Reference Data To perform sizing analysis on the reference data continued 8 Verify the size calling a Select a sample b Select Analysis gt Size Match Editor The Size Match Editor window opens showing how the size standard peaks were labeled Note The sizing should be successful for this tutorial When analyzing your own data if the size standard failed or if labels were assigned to the wrong peaks you would be able to make changes in the Size Match Editor window For more information on using this window refer to the ABI PRISME GeneMapper Genotyping Software Users Manual PN 4335526
40. tion pane ABI Prism GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial 4 3 Chapter 4 Creating Custom Kits Panels and Markers Creating a Panel To create a panel 1 In the navigation pane of the Panel Manager window select the kit you just created see page 4 3 2 Select File gt New Panel This creates a new panel which will be associated with the new Microsatellite kit 3 On the right side of the Panel Manager window type a name for the new panel a Click in the text box b Type name c Press Enter File Edit Bins view WX mimi ie eset OO E ma el fi B S Panel Manager H E GeneScan Installatio H SNPdemo o ae New Microsatellite Kit New Panel SI Reference Samples OK Cancel Apply 4 To create other new panels associated with this kit repeat steps 2 and 3 You can create as many panels as you d like Creating Markers To create markers 1 In the navigation pane of the Panel Manager window select a panel you want to add markers to 2 Select File gt New Marker This creates a new marker which will be associated with the panel you selected 4 4 ABI PRISM GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial Creating Kits Panels and Markers To create markers continued 3 On the right side of the Panel Manager window enter the relevant information for the marker for example M
41. tutorial you should already Files have added your sample files to the Project window These are the same files that you used for sizing see Importing and Sizing Reference Data on page 2 5 You will notice that a blue checkmark is displayed in the REF column indicating that these samples were used as reference data for creating a bin set Applying Project The project settings include Settings The desired Analysis Method containing the appropriate bin sets e The appropriate panel e The appropriate size standard To apply project settings 1 In the Project window select the Samples tab 2 Select an Analysis Method a Select the top cell in the Analysis Method column b Select Microsatellite Tutorial from the drop down list This is the Analysis Method you created earlier see 3 5 3 10 ABI PRISM GeneMapper Software Version 3 0 Microsatellite Analysis Tutorial Performing Analysis on Microsatellite Data To apply project settings continued RA Note This step is not necessary for this tutorial Tutorial Panel 9 is already selected However you may find it necessary to select a panel when you are working with your own sample files To select a panel a Select the top cell in the Panel column The Select a Panel window opens b Click a folder to expand it xi e None 24 Microsatellite Tutorial o IMENE GeneScan Installation Standards LMS HD5 2 5 E LMS MD10 V
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