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SeqScape Software Version 2.1 User Guide

1. Table 7 1 Project Views continued View Procedure Display Inverse Click Video L 5 vaga AA AciveLayer HL amp C CDS 7 Tab jumps to ned TEE ProjectNavigator E HLA C 31 00 iKnown Variants IHLA C_CDS ROIs All Variants QVs with From the Snippet or Dots view click Bul for sample QVs and or click fiat for Snippets or consensus QVs Dots IHLA C_CDS ROls ll Variants Summary GAAGTACAAGCGCCAGGCACAGWCTGMCCGAGTGAGCCTRYGKA INT Mariani Ble ica acs cias eC emn jai argon etapa at eo aes RR alge RC IR CR RR a c RR Index 200 210 220 230 Reference Reference AA Q K Y K R Q A T D R yv 3 L R Consensus w 3602 a ree TTEN rrr ee ree ret QVs GkncrTacascecccacccacacercWeccacrcaccetBBcpa 360 2 CX2F_0 Sample QVs 360 2 CX2R_02 3 gt dl Note A gray QV bar indicates that the base has been edited and that the QV no longer holds true until you restore the previous base Note Holding the pointer over a base displays the numerical QV assignment for that base 7 8 ABI PRISM SeqScape Software v2 1 User Guide Project Views Table 7 1 Project Views continued View Procedure Display Identification 1 Drag the split bar at the bottom of the window up until you reach the desired height 2 Select a base in a specimen sequence Project Navigator 2 HLA C 3100 Known Variants IHLA C CDS ROIs Il Variants S
2. View Procedure Display n lect th piam reece tab vajaa EE MIL Active LayerfHLA C_CDS Tab jumps to next v 8 U 3602 308 29 o amp aF25t 46 227 X182 482 792 S GACCGGGAGACACAGAAGTACAAGCGCCAGGCACAGVCTI DGAGTATTSGGACCGGGAGACACAGAAGTACAAQgCGCCAQGCACAQVCT B 179 189 199 3 209 219 BGAGTATIsGGaCCGGGAGaCACAGAAGTACAAGCgQCCAGGCACAGvVCTI 139 129 119 109 99 Dot 1 Select the ej wea ETNA Active Lyerje CDSE Tab jumps to next Assembly Assembly tab 2 Click 360 2 CX 3 i a 177 187 197 207 27 eA DTWTSUEN EC EE END ACECACRR ST AOA E CEC CAS Soo WO va Tq du ie i Pn zi Dm dod Ar js i Electro 1 Select the pherogram Assembly Assembly tab EE HLA C 3100 2 Selecta B xx d 309 28 sample in a 182 268 311 348 the sample 7 table 3 Click x CACAGVCT for multiple CACAQ vC TE EPS or click liz for one EP 7 12 ABI Prism SeqScape Software v2 1 User Guide Segment Views Table 7 2 Segment Views continued View Procedure Display QV Assembly 2 Click 1 Select the Assembly tab for sample QVs and or click n for consensus QVs 360 2 cx s 27 235 309 29 337 Joo 360 2 CX 3 227 182 1 209 88 187 SGAGTATTSGGACCGGGAG 197 D GAAGTACAAGCGCCAGGCACAGVCTq 335 309 29 227 182 268 311 ABI PRISM SeqSca
3. Chapter 3 1 Create Analysis Defaults and Display Settings Chapter 4 2 Ref Data G Add Reference Sequence ROls Create Reference Data Group gt Create ROIs gt taste Add nucleotide variants oH x eis Add amino acid variants Linka Library Setup variant styles Chapter 5 3 Create Project Template Select rF RDG Analysis Defaults Display Settings Chapter 6 4 Create and Analyze Project Select Project Template Software does automatically Add Specimens Import Sample Files Basecalling Filter Assembly Consensus Alignment Comparison Search Library Chapter 7 Data Project View Specimen View Segment View Sample View Report Manager Analysis QC Report Mutations Report AA Variants Report Specimen Statistics Report Sequence Confirmation Report Base Frequency report Library Search Report RDG Report Audit Trail Report Genotyping Report 5 View Data and Reports Chapter 8 6 Reanalyze and Edit Data Chapter 9 7 Export and Print Reports gt Aligned projects Consensus sequence Aligned sample sequence Sample files Figure 2 6 Typical Workflow for a Project 2 28 ABI PRISM SeqScape Software v2 1 User Guide Creating Analysis Defaults and Display Settings 3 This chapter contains Workflow for This Chapter 0 0 00 ee ee eee ee ee 3
4. Collapsed NT 31 Vs I Italicize Reverse Strand V oe Clear Range IV Index Show All amp v Reference r Expanded A Iv Summary v AA Variants Iv Index v Reference AA a In the General View Settings section click the buttons for the displays you want turned on in the project Select the Electropherogram view Note If it is not selected when the Assembly view is printed it appears that the peaks are not aligned Most of the buttons on this tab are the same as the viewing buttons on the lower row of the toolbar in the main SeqScape window Refer to Viewing Toolbar on page 2 25 b In the drop down lists select how you want to tab through the data c In the Sample View Settings section select the icon if you want to see the original sequence displayed 3 22 ABI Prism SeqScape Software v2 1 User Guide Specifying Display Settings d To differentiate forward and reverse sequences in the Specimen View Settings section select Italicize Reverse Strand e In the Project View Settings section enter the number of bases to be displayed for the Project view electropherogram snippets in the EP Window field the minimum is 3 7 Click Save to save the changes and close the dialog box The new display settings are added to the SeqScape Manager ABI PRISM SeqScape Software v2 1 User Guide 3 23 Chapter 3 Creating Analysis Defaults and Display Settings
5. Comment T Printto file r Print range Copies All Number of copies 1 C Pages from 1 to y ELS Selection yl Pl of d 6 Select a printer then click OK Both print dialog boxes close and printing begins Printing a Report To print a report 1 2 3 5 9 16 Open the project of interest then click E In the navigation pane select a report type Customize the report if desired see Customizing the Reports on page 7 42 Select File gt Print O 0x Print Properties Print Settings for Report 9 C Landscape Paper Letter 8 5 by 11 inches z HeaderiFooter Print preview Print Cancel Complete the Print Report dialog box a In the Print Properties section select the paper orientation and size b In the Print Settings for Report section select Print Preview if desired c Click Print ABI PRISM SeqScape Software v2 1 User Guide Printing Data and Reports 6 Use the following table to determine your next step If the Print preview option was Proceed to step Not selected 7 Selected 8 7 The Print dialog box opens m Printer Name HP C LaserJet 4500 PS Properties Status Ready Type HP C LaserJet 4500 PS Where IP_167 116 254 248 Comment T Printto file Print range Copies 6 All Number of copies 1 Ex C Pages from
6. 360 2 CX3R 02 360 2 CX3F 01 CGGCCCGTGAGGCGGAGCAG GAGAGCCTACCTGGAGGGCA AGCAG CGGCCCGTGAGGCGGAGCAGHPGAGAGCCTACCTGGAGGGCA AGCAGE GAGA rEcasA ABI Prism SeqScape Software v2 1 User Guide Viewing Variants Method 2 Open the project of interest Select Analysis gt Report Manager or click EJ In the navigation pane select the report you want to view Select Window gt Tile Uv de x get cR Review the positions by selecting a base change in the Mutation table This action brings the alignment view to the correct position in the alignment Saving Your Data When you finish select File gt Save Project or click fg IMPORTANT Any changes you make are saved and overwrite the existing project ABI PRISM SeqScape Software v2 1 User Guide 7 21 Chapter 7 Viewing the Results About the Reports Types of Reports Exporting and Printing Reports 7 22 After the data is analyzed you can view export and print reports Reports can help you troubleshoot your results because reports contain hyperlinks to the primary sequence data You can use reports with project results to evaluate your samples modify the analysis settings and edit the basecalling Ten reports are generated with every project analysis Each project has its own Report Manager window containing all of the following reports Analysis QC Mutations AA Variants Specimen Statistics Sequence Confi
7. Tab Displayed Information Annotation Information about the data and its analysis Sequence Sequence of the sample in NT codes For readability the display clusters the sequences into substrings of 10 characters each separated by blanks Features Calculated clear range and multiple base positions Electropherogram Electropherogram and basecall data for the sample The data excluded from the clear range is shown in gray Raw Raw data collected by the genetic analyzer Displaying the To display sample views Sample Views 1 Open the project of interest 2 In the navigation pane open a specimen then open a segment 3 Select a sample then select a tab see Table 7 3 4 Select a new tab to change the view 5 To view a different sample select a new sample from an open segment then select a tab Examples of the five tab views are displayed in Table 7 4 Sample Views on page 7 16 ABI PRISM SeqScape Software v2 1 User Guide 7 15 Chapter 7 Viewing the Results Table 7 4 Sample Views Tab Display Annotation Project Navigator E HLA 31 00_threeSpeci a Bg Unassembled 6 AF250557 BaF Bai 3R_02 Bai 2R_02 Blair 01 gi 7414348 em m A2 Was HLA 3100 threeSpecimens v2 Al AF250557 A1 3F_01 Annotation Sequence Features Electropherogram Raw Data Collection Sample name Model Number of Scans Length Start Run St
8. MV Use reference trimming Reference iy Multiple clear range methods are applied in order Smallest clear range is the result a If desired select one or more stop points for data analysis b If desired select to mask M13 primers from the clear range c Select Use reference trimming 8 10 ABI Prism SeqScape Software v2 1 User Guide Changing the Analysis Parameters in an Analysis Protocol 6 Select the Filter tab Analysis Protocol for A 1 2F O1 Specify the values for the filter Settings 7 Click OK to save the protocol and close the Analysis Protocol dialog box ABI PRISM SeqScape Software v2 1 User Guide 8 11 Chapter 8 Reanalyzing and Editing Data Applying the To apply an analysis protocol Analysis Protocol 1 Select the samples in the Sample Manager to apply the new settings to Click Apply Analysis Protocol Apply Analysis Protocol x Choose an Analysis Protocol that will be applied to the set of selected samples Analysis Protocol Bi D SR mixed z 3100SR mixed Select a protocol from the Analysis Protocol drop down list Click OK The spacing peak 1 and start and stop points change to zero Click Apply The Assembled indicator changes from green assembled to red unassembled and the Analysis button becomes active Click E ABI PRISM SeqScape Software v2 1 User Guide Editing the Data Editing the Data Ab
9. ich in Printout Print Properties Portrait Annotation C Landscape v Electropherogram Paper Letter8 5by1 WB Sequence Raw Data HeaderiFooter Print Cancel 4 Complete the dialog box a In the Print section select All data b In the Print Properties section select the paper orientation and size c In the Sample Settings section select a value in the Panels Per Page drop down list The range is 1 to 4 and the default is 4 d Select a value in the Points Per Panel value box The range is 100 to 12000 and the default is 1500 about 120 bases e In the Include a Printout section select the views you want to print f If you are printing a Segment assembly type a new value in the Bases per panel field in the Print Settings for Project section ABI Prism SeqScape Software v2 1 User Guide 9 13 Chapter 9 Exporting and Printing Data and Reports g Click Print six p Printer Name HP C LaserJet 4500 PS Properties Status Ready Type HP C LaserJet 4500 PS Where IP_167 116 254 248 Comment T Printto file r Print range Copies All Number of copies 1 C Pages from f1 to 5 DM yl el js T Collate Selection Uo K E 5 Select a printer then click OK Both print dialog boxes close and printing begins Printing Various To print different views of a project Views of a Project
10. ABI Prism SeqScape Software v2 1 User Guide Creating an RDG from Aligned Consensus Sequences The amino acid variants are imported and appear in the list in the AA Variants tab A sample of AA variants is shown below x General ROI NT Variants AA Variants Variant Se Type Layer Position Reference Variant Style Description Residue Change Layer 1 lao P Poor Yellow Residue Change Layer 180 D C160 Yellow Residue Change Layer 272 P P272C Yellow Residue Change Layer1 356 G G356T Yellow Residue Change HLA C_CDS 273 W lw273T Yellow Residue Change IHLA C CDS 92 H H927 Yellow Assigning Styles The Variant Styles tab allows you to define text coloring styles that to Variants identify different types of variants and change the display characteristics of variants in the Project view Use the Variant Style tab to assign styles to the variants as desired The table at the top of the dialog box displays the generic styles The table at the bottom of the dialog box lists the different types of variant conditions and their associated styles The styles you set appear in the Project view to identify the different types of variants ABI PRISM SeqScape Software v2 1 User Guide 4 39 Chapter 4 Creating a Reference Data Group To assign styles to the variants IMPORTANT When assigning color to text select light background colors so the text is easy to read 1 In the RDG Proper
11. Show Hide Consensus QV Inverse View of the electropherogram View Aligned EP Characters Dots Characters show basecalls that are different Dots show basecalls that are the same Collapsed Nucleotide View Full View of Electropherogram Ctrl Show Hide All Electropherograms Show Hide Sample QV Ctrl K Figure 2 4 Viewing Toolbar ABI PRISM SeqScape Software v2 1 User Guide View Original Sequence In Sample Electropherogram view View Column Selector 2 25 Chapter 2 Getting Started Menus on the Main SeqScape Window Figure 2 5 shows the menu structure of the main SeqScape window Submenus Main Menus Submenus SeqScape New Project Software C Open Project C New Project Wizard Close Project Save Project Save Project As Import Samples To Project C Import Text Segment Export Sample Sequence File in Sample view Print Consensus Sequence Last 4 Projects Opened in Specimen and Segment view Exit Undo Base Change Project Alignment Nucleotides in Project view Aligned Sample Sequence in Specimen and Segment view C V V SS OSS YY V VY aS Y Bind Project Alignment Amino Acid Report Find Again in Project view Delete C Rename C Toolbars General Show
12. Table Description Summary Displays project information and the specimens and samples in the report Audit Trail Displays a record of the edits and changes made to data in a project if the audit trail feature is on Ej Analysis OC Report E Mutations Report D AA Vanants Report LY Specimen Stativtcs Report li Sequence Confirmation Report E Base Frequency Report 18 Library Search Report D ROG Report LJ Genotyping Report Summary Acte Layer MAC COS Projest maca Project Coation Date 03 Dee 2002 4 220 04 PST Project Modification Date 13 May 2003 at 6 02 46 POT Povjest Template PT HUAC_S100POPO_mined_Slib PT Creation Date 03 Des 2002 1232008 PST PT Modification Date 13 May 2003 at 1602 46 POT Reference Data Group ROD MAC Erea noNT ROG Creation Date OIOI 2249 40 PLT ADO usainean Date 19 May 2000 at 10 02 45 POT TutonaituplaySetngr DS Creation Date 26 Now 2002 at 23 37 49 PST 13 May 2003 at 1602 46 POT Analysis Dataults AD 3300 amp POPR BOTvi mixed sus 03 Dee 2002 4232730 F 1 AD Modification Date 19 May 2003 at 10 0249 POT Specimens in Raport At A2 A3 Samples in Report jan Camper i selected Specimen Auda Trail Reason Desorption User Figure 7 11 Audit Trail Report ABI PRISM SeqScape Software v2 1 User Guide Genotyping Report Genotyping Report The Genotyping report contains five separate tables Table 7 15 Parts of the Genotypin
13. Creating a New RDG Using the Wizard 00 eee eee Using the Wizard to Learn the Software 2000 Setting Up the Reference Segment llsuuss Creating a New RDG Using SeqScape Manager Before You Begin 02 c eee eee Creating an RDG from SeqScape Manager About the Reference Sequence lllleesss Importing a Reference Segment lllslllssessnu Defining Regions of Interest ROI 0 00 cee eee eee Defining an ROlL 4 ssi et e Rn es yet eee Pace meis Pasting a Reference Segment llsllesseslss Deleting an ROI or Layer 0 cece eee Deleting a Reference Segment 00 e eee eee eee ROI Tab Descriptions 00 000 ee Layer Pane Functions 000 c eee eee eee The ROMP ane cae ae ee ERG e ox RERO ae Red Columns in the ROI Pane 2 20000 0c eee eee Creating aLibrary asispa tasir io EEE ee About the Library llsllleee eee Using Aligned FASTA Files 00 2 0 eee eee Using a Tool to Align the Files 2 0 0 0 c eee eee Setting Up Your Library 00 eee eee Creating New Layers 0 0 eee eee Adding a Reference Break in a Sequence ABI PRISM SeqScape Software v2 1 User Guide Chapter 5 Chapter 6 vi Declaring Variants into an RDG 20 eee ee 4 29 About NT Variants 2 0 00 cee eee 4 29 Creat
14. Incorporating Variants into the Project RDG Creating a New You can add a variant to a project by Variant in a Project Entering the type and position of the variant in the Variants tab of the RDG Properties dialog box Selecting the location on the reference sequence in the Sequence tab in the RDG Properties dialog box The appropriate information regarding the variant is automatically entered in the variant dialog box To create a new variant in the project From the Project window open the RDG Properties dialog box by selecting Analysis RDG Properties Click the ROI tab then select Add Variant RDG Properties ES xj General ROI NT Variants AA Variants Variant Style 1 Layer 1 settings _NewLayer_ Layer Name Layer1 Index Codon Number 1 Orientation Library z aol Translation Frame fz Right ROI Name Segment Seg Start Seg End ROIStart ROI Length Translation Color amp AF250557 AF250557 1 792 792 amp gi 7414348 emb AJ277e gi 7414348 emb AJ 1 276 276 HLA C exon2 AF250557 1 270 270 HLA C_gene AF250557 792 792 HLA C_intron2 AF250557 516 246 O40455 gctcccactc catgaggtat ttctacaccg ccgtgtcccg gcccddccgc ggagageccc gcttcatc c agtgd ctac gtggacgaca cgcagttcgt gcagttcgac agcgacgccg cgagtccaag aggggagccg cgggcgccgt qugtggagca dgaggggccg gagtattggg accgggagac acagaagtac aagcgccagg cacagactga ccgagtgagc ctgcugaacc tgcgcggcta ctacaaccag agcgaggccg gtgagtga
15. 1 Open a text file then click drag the region of interest you want to use as a reference segment Select Edit gt Copy In the RDG Properties ROI tab click Paste Ref Segment to use a reference segment that you copied to the clipboard The copied reference appears in the Reference Sequence pane If you want to delete the copied reference segment select it press Delete then click OK in the Confirmation dialog box ABI PRISM SeqScape Software v2 1 User Guide 4 15 Chapter 4 Creating a Reference Data Group Deleting an ROI To delete an ROI or layer reference segment orLayer Select the ROI layer or segment 2 Press Delete Only unlocked rows can be deleted 3 Check this on software IMPORTANT After you delete an object it cannot be undone Deleting a When RDG Properties window is open without being associated with Reference a project a reference segment can be deleted Right clicking a Segment selected reference segment in the Reference Sequence pane opens a pop up menu with selections to rename or delete the selected segment Note The reference segments and Layer 1 cannot be deleted by selecting them in the Layer pane then pressing Delete because they are in locked layers They cannot be deleted when they are part of an existing open project To delete a reference segment 1 In SeqScape Manager select the Reference Data Group tab and highlight the RDG in which you want to delete a reference segment
16. 3 24 ABI Prism SeqScape Software v2 1 User Guide Creating a Reference Data Group This chapter contains Workflow for This Chapter 0 00 0 c eee eee eee 4 2 Reference Data Group RDG 0 0 00 eee eee eee 4 3 Creating a New RDG Using the Wizard 4 6 Creating a New RDG Using SeqScape Manager 4 12 Defining Regions of Interest ROI 005 4 15 ROI Tab Descriptions 2 0 0 0 eee ee 4 18 Creating a Library 0 2 eee ee 4 20 Creating New Layers oner Con nee io eee eee 4 24 Declaring Variants into an RDG 000 5 4 29 Creating an RDG from Aligned Consensus Sequences 4 34 ABI PRISM SeqScape Software v2 1 User Guide 4 1 Chapter 4 Creating a Reference Data Group Workflow for This Chapter 4 2 Chapter 3 1 Create Analysis Defaults and Display Settings Chapter 4 Chapter 5 2 Create Reference Data Group Add Reference Sequence Create ROIs Create Layers Link a Library 3 Create Project Template Select RDG Chapter 6 Analysis Defaults Display Settings 4 Create and Analyze Project Select Project Template Add Specimens Import Sample Files Chapter 7 gt Software does automatically ROls Add nucleotide variants Add amino acid variants Setup variant styles Basecalling Filter Assemb
17. ABI PRISM SeqScape Software Version 2 1 User Guide Applied Biosystems Copyright 2003 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 SeqScape software has not undergone specific validation for human identification applications Human identification laboratories must perform their own validation studies 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 Applied Biosystems BigDye MicroSeq SeqScape and ViroSeq are registered trademarks of Applera Corporation or its subsidiaries in the U S and or certain other countries AB Design Applera GeneMapper POP 4 POP 5 POP 6 and POP 7 are trademarks of Applera Corporation or its subsidiaries in the U S and or certain other countries Wind
18. AT 2F 01 A1 2R 02 BaseCaller Basecaller 3100POP Basecaller 3100POP DT3100POP6 BDjv2 DyeSet Primer Spacing Peak 1 DT3100POP6 BD v2 4 84 946 946 B 41 3R_02 A1 3R 02 Basecaller 31 00POP DT31 00POP6 BDjv2 B 41 3F_01 AAT 3F 01 Basecaller 3100POP prat OOPOP6 BD v2 41 4R_02 Basecaller 3100POP DT3100POPB BD v2 lt 4F 01 Basecaller 3100POP DT3100POPB BD v2 A2 2R_04 Basecaller 3100POP DT3100POPB BD v2 jA2 2F 03 Basecaller 31 00POP DT3100POP5 BDjv2 42 3R_04 Basecaller 3100POP DT3100POPB BD v2 JA2 3F 03 Basecaller 3100POP DT3100POP6 BDjv2 A2 4R_04 Basecaller 3100POP DT3100POPB BD 2 JA2 AF 03 Basecaller 3100POP DT3100POP6 BDjv2 A3 4F_05 Basecaller 3100POP IDT3100POPB5 BD v2 2 D NG Dannnn Hav 24nnpoo Edit Analysis Protocol Apply Analysis Protocol Ir21nnpbopa mn Figure 8 2 Sample Files in the Sample Manager The Sample Manager window displays the following information Table 8 1 Information in the Sample Manager Column Heading Description Sample File Name Information from the plate record and project It cannot be changed in the Sample Manager Specimen Information from the plate record and project It cannot be changed in the Sample Manager Sam
19. Samples Basecalling mar mn Specimen Total amp Score Variants 6 E L1 L1 25 38 B Hi rj 25 48 il an Oo 24 31 Comments Complete E Partial Output No output Sample Analysis Sample Step Description Possible Heterozygous Indel Mutations Sample Position Size A1 2R 02 33 A1 4F 01 185 A2 4F 03 Report Settings Figure 10 5 Analysis QC Report ABI PRISM SeqScape Software v2 1 User Guide 10 11 Chapter 10 Sample and Consensus Quality Values Mutations Report QVs for each mutation and the average QV for the bases to the left and right of the deletion are provided in Mutations report Summary Active Layer HLA amp C CDS Project HLA C v2 Project Creation Date 03 Dec 2002 at Project Modification Date 13 May 2003 at 23 40 04 PST 16 02 46 PDT E Sequence Confirmation Report Project Template PT HLA C_3100POP6_mixe PT Creation Date 03 Dec 2002 at d 5lib 23 28 08 PST i Base Frequency Report Bn Library Search Report PT Modification Date 13 May 2003 at Reference Data Group RDG HLA C_Exon2 4_noNT 16 02 45 PDT gt RDG Report i i Audit Trail Report RDG Creation Date 03 Dec 2002 at RDG Modification Date 13 May 2003 at a 22 59 40 PST 16 02 46 PDT Genotyping Report gt AS Variants Report U Specimen Statistics Report Display Settings DS TutorialDisplaySettings DS Creation Date 26 Nov 2002 at 23 37 59 PST DS Modification Date 13 May 2
20. Select a results group or create a new one Select an instrument protocol or create one Select an analysis protocol or create one ow ND To add additional runs to the plate record a Select Edit gt Add Sample Instance b Select a results group instrument protocol and analysis protocol 10 Click OK ABI PRISM SeqScape Software v2 1 User Guide 11 21 Chapter 11 Automating Analysis Scheduling and Starting a Run To schedule and then start a run 1 Click the Run Scheduler icon in the navigation pane i roundation Data Collection ersion 2 0 Administrator user is logged in E 2 Anl xi File View Instrument Service Tools Wizards Help gt Be B AS B Boca Instruments B l Resuts Group K Database Manager Find Stacker Plat fo Add Plate S Type Plate ID amp Kga3730 in r Plate e Scan or Type Plate ID Plate Manager Input Stack Output Stack Protocol Manag s js Module Manage Plate ID Plate Name Plate Type Plate ID Plate Name Description Run History E G DakarPT3 E3 nstrument GA Instruments ga3730 DakarPT3 Run Scheduler p el i Search Up Do Remove B aray View Spectral vi AM Manual Cor Auto Sampler service Lo Plate ID Plate Name Plate Type Status Remove All i Clear Auta Fh Current Runs RuniD Application Run Protocol Status System Status
21. Showing the Printing and Exporting Steps Exporting Data Files Exporting Data Files File Names The default file name uses the project name and the report type Do not use the following characters in any file name 2 amp and space Format Options You can export a project specimen segment or sample file Table 9 1 summarizes the available format options Header and footer information is not incorporated in any data file Note Only one data file can be exported at a time Table 9 1 Export and File Format Options Export Option File Format Options Project Project Alignment Nucleotides FASTA Project Alignment Amino Acids Specimen and Segment Consensus Sequence FASTA SEQ or QUAL Aligned Sample Sequence FASTA Sample Sample Sequence File FASTA SEQ AB1 or PHD ABI PRISM SeqScape Software v2 1 User Guide 9 3 Chapter 9 Exporting and Printing Data and Reports 9 4 Exporting a To export a project alignment Project Alignment Open the project of interest In the navigation pane select the project icon Select File Export Project Alignment Nucleotides or Project Alignment Amino Acid FA Export Project AA Alignment x Look in fs AppliedBiosysterns X fl e al E Seq45 0 SeqScape File name IHLA C 3100_AaAlignmentfsta Export Files oftype FASTA format fsta S Cancel 4 Complete the Export Project dialog box a Se
22. What are the access control differences as you go from Admin to Scientist to Analyst Administrators can do everything that the application possibly lets you do This includes the admin specific tasks creating users viewing and changing user details importing and exporting users and changing the 21CFR Authentication and Audit features Scientists can do everything except the admin specific tasks Analysts can open projects and import samples but cannot affect other master objects For example the analyst cannot view modify import or export project templates RDGs analysis defaults etc This includes changing the RDG or analysis settings once inside a project although you can change the basecaller and dye set primer files from within the sample manager The Analyst is allowed to edit the project The Analyst also cannot perform any admin specific tasks For more information see Appendix D User Privileges Does the audit trail component add User ID and a Time Date stamp to each entry The Audit Trail report does include a user ID user first and last name and time date stamp for each audit event The Audit report also includes the reason why the user modified the data and it includes any comments that the user may have included ABI PRISM SeqScape Software v2 1 User Guide B 21 Appendix B Frequently Asked Questions B 22 ABI Prism SeqScape Software v2 1 User Guide Translation Tables Thi
23. 1 With the project open select the Analysis menu then select RDG Properties Analysis Defaults or Display Settings 2 Select any tab in any of these three dialog boxes then click Save to Manager As 3 In the Name field enter a new name or accept the default nameCopy and click OK The saved copy is available to import into another project ABI PRISM SeqScape Software v2 1 User Guide Creating a Project Template 5 This chapter contains Workflow for This Chapter eese 5 2 Creating a Project Template 0 0 0 00 00008 5 3 Saving Project Components 0 0 0 0 e eee eee 5 5 ABI PRISM SeqScape Software v2 1 User Guide 5 1 Chapter 5 Creating a Project Template Workflow for This Chapter Chapter 3 1 Create Analysis Defaults and Display Settings Chapter 4 2 Create Reference Data Group Add Reference Sequence ROIs Create ROIs Crente L Add nucleotide variants d e ayers Add amino acid variants Link a Library Setup variant styles Chapter 5 3 Create Project Template Select RDG Analysis Defaults Display Settings Chapter 6 4 Create and Analyze Project Select Project Template ___ Software does automatically Add Specimens Import Sample Files Basecalling Filter Assembly Consensus Alignment Comparison Search Library Chapter 7 5 View Data and Reports D
24. 24 25 29 20 32 a5 53 81 HLA C_exon2 HLA C_exon2 HLA C_exon2 HLA C_exon2 HLA C_exon2 HLA C_exon2 HLA C exon2 HLA C_exon2 HLA C exon2 HLA C exon2 Figure 7 8 Base Frequency Report 7 34 333 00 100 0 333 00 o0 00 333 66 6 ABI Prism SeqScape Software v2 1 User Guide Library Search Report Library Search Report The report contains five separate tables see Figure 7 9 on page 7 36 Table 7 12 Parts of the Library Search Report Table Description Summary Displays project information and the specimens in the report Library Displays the library and information used in the search Specimen Results Displays the match status crucial position and constant position errors for each specimen Hit List Displays the library matches found their scores closest match and mismatch information for each specimen Constant Positions Errors Displays the position specimen base and library base for each specimen and ROI The values in the Position column are hyperlinked to the project navigator ABI PRISM SeqScape Software v2 1 User Guide 7 35 Chapter 7 Viewing the Results Summary Active Layer HLA C_CDS Project HLACC 2 Project Creation Date 03 Dec 2002 at Project Modification Date 13 May 2003 at 23 40 04 PST 16 02 45 PDT Project Template PT HLA C_3100POP6_mixe PT Creation Date 03 Dec 2002 at d 5lib 23 20 08 PST PT Modification
25. 3 Open the project of interest In the navigation pane select a Segment icon Select File Export Consensus Sequence or Aligned Sample Sequence EE is bap fsta be HI C 310n dadlinnment feta FASTA format fsta ll Export Consensus Segment x Look in AppliedBiosystems x amp ek a 360 2_AF250557_ConsensusSequence fsta 360 2 gi 7414348 emb A 1277102 1 H84277102 Homo sapiens partial HLA File name Pw 0602 allele exon 4_ConsensusSequence fsta Export Files oftype FASTA format fsta x Cancel SEQ format seq QUAL format qual 4 Complete the Export dialog box a Select a folder location to store the file b Change the file name if desired The default file name uses the segment name and the FASTA extension c For the Consensus Sequence option select a file format in the Files of type drop down list d Click Export ABI Prism SeqScape Software v2 1 User Guide Exporting Data Files Exporting a To export a sample Sample 1 Open the project of interest 2 In the navigation pane select a Sample icon 3 Select File Export Sample Sequence File es lli Export Sample xj Look in E 4ppliedBiosystems bd EJ amp e ree 360 2 AF250557 ConsensusSequence fsta 360 2 gi 7414348 emb AJ277102 1 HS4277102 Homo sapiens partial HLA 360 2 gi 7414348 emb AJ277102 1 HS4277102 Homo sapiens partial HLA fe HI C
26. ABI Prism SeqScape Software v2 1 User Guide 4 17 Chapter 4 Creating a Reference Data Group ROI Tab Descriptions Layer Pane The Layer pane in the ROI tab refer to Figure 4 2 on page 4 14 has Functions the following functions Layers Shows the locked Reference Sequence in Layer 1 and ROIs associated with each layer New Layer button Adds a new layer to the end of the layer table Layer Number Settings The settings of the selected layer Each layer has its own unique settings Layer Name The name of the layer which can be edited Library Contains libraries to select if you are performing allele or haplotype identification Before you select a library to associate the Library field is blank A library can be copied into the RDG but is not associated until you select it from the Library drop down list Once it is selected the Library field shows the name of the library Index Codon Number Indicates the first amino acid number This number is always in relation to the number of the first reference segment base positive numbers only Translation Frame Sets the translation frame for the layer The values are 1 2 3 Orientation Sets the orientation of the layer right forward or left reverse The ROI Pane The ROI pane has the following features Clicking a row selects the ROI When you select an ROI in the RDG it selects and scrolls the reference segment and the associated sequence Pri
27. Analysis Reports FAQs B 19 Quality Values FAQs eee B 20 Printing and Exporting Results FAQs 0000 B 21 Audit Trail Security and Access Control FAQs B 22 ABI PRISM SeqScape Software v2 1 User Guide B 1 Appendix B Frequently Asked Questions Upgrading FAQs There are four versions of ABI PRISM SeqScape Software v2 1 available See SeqScape Software Versions on page 2 5 Table B 1 Upgrading Questions and Answers Question Answer How does SeqScape software v2 1 differ from v2 0 Refer to Chapter 1 Introduction to ABI PRISM SeqScape Software What happens to my data when upgrade my SeqScape software What happens to my data if do an install of the SeqScape software v2 1 If you install the upgrade version of SeqScape software version 2 1 data from SeqScape software v1 0 v1 1 or 2 0 will be stored in the Data Store backup folder that is named according to the version of SeqScape software previously installed i e Data v1 0 or Data v1 1 In addition all previous contents of the Data Store will be copied to the new Data Store If you install the full version of SeqScape software v2 1 you must first export the data objects from the SeqScape Manager in the previous version of SeqScape software then import them into SeqScape software v2 1 What happens to the data that created with the demo version when upgrade to the full version of SeqScape softwar
28. Figure 8 1 8 2 ABI PRISM SeqScape Software v2 1 User Guide Reanalyzing and Editing Data Step About Analysis Parameters About Analysis Parameters Introduction The analysis parameters basecaller and DyeSet Primer file associated with every sample file are used when the sample files are analyzed Sometimes poor project results can be corrected or improved by changing certain analysis settings and applying the new settings to the affected samples Common examples of errors that affect basecalling are Incorrect stop point selected Bad base spacing Poor quality data Incorrect basecaller and or dyeset primer used for basecalling Wrong peak 1 location and start point calculated by the software Note Refer to the ABI PrisM DNA Sequencing Analysis Software User Guide for instructions to define a new peak 1 and the start and stop point locations ABI PRISM SeqScape Software v2 1 User Guide 8 3 Chapter 8 Reanalyzing and Editing Data Viewing Analysis Parameters in the Sample Manager You can use the Sample Manager to display sample files and their current analysis information including the basecaller and DyeSet Primer files see Figure 8 2 on page 8 4 The analysis parameters can be modified and applied to samples You can apply these changes to one sample some samples or all samples in the Sample Manager Analysis parameters Assembly status B 41 2R 02 Specimen Sample Name
29. Genotyping Report 0 cece ee 10 14 Automating Analysis Integrating SeqScape and Data Collection Software 11 2 OVOrViOW sebiit gan get EE age eet ge eae ee pees 11 2 Software Relationships 20000 cee eee eee 11 3 Before You Start clues were tee eld Xa REI ek Geeta 11 4 Required Files Created in SeqScape Software 11 6 Launching Autoanalysis Manger 000 eee eee eee eee 11 7 Creating Required Files in the Data Collection Software 11 8 For More Information s oo ireen oe a cece ee eee 11 8 If the Files Already Exist lees 11 8 Creating an Instrument Protocol lllllslselesss 11 9 Creating an Analysis Protocol 00 eee eee 11 11 Creating a Results Group 1 eee ee 11 14 Creating a Plate Record 0 cee eee 11 18 Completing a Plate Record lllsllslsslessess 11 20 Scheduling and Starting a Run 00 ee ee 11 22 ABI PRISM SeqScape Software v2 1 User Guide Appendix A Appendix B Understanding the Autoanalysis Manager 11 24 OVGIVIEW 2x we held depo ad teats ew Pepe bee eed eet 11 24 Files Created iiu ee Sew ee Bad Be eee ee 11 24 Components eee pa a ee Regu See eee 11 24 General Tab iss donee ds ER Ree 11 25 oeqScape2 1 Tab ceeds iuge aW Re ug RUN 11 26 File Sharing Between Data Collection and SeqScape Software 11 28 Basecallers and DyeSet Primer Files Def
30. It is made up of one or more reference segments ABI Prism SeqScape Software v2 1 User Guide 4 7 Chapter 4 Creating a Reference Data Group 4 8 7 8 Click Add Ref Segment in the lower left to add a segment to the Reference Sequence A reference segment is a single sequence imported from a text file or GenBank file Mi x Add Reference Segments Add the Reference Segments that you want to include in the Reference Data Group Reference Sequence n l s O Add Ref Segment Paste Ref Segment Split Ref Segment lt lt Back Next gt gt Finish Cancel T Navigate to the file containing the reference sequence that you have stored such as a GenBank file the file may have a gb extension x Look in E Advanced Project Data gt amp amp c HLA C specimens File name JHLA C_ex2 3 gb Import Files of type GenBank format gb Span suede ne IMPORTANT The window opens to the directory that was set up during installation of the software If no default directory has been specified the window opens to the C drive If you need to set up the default directory select Tools gt Options gt General then click Browse to locate the directory ABI PRISM SeqScape Software v2 1 User Guide Creating a New RDG Using the Wizard 9 Click Import The imported sequence appears in the right pane of the dialog box as shown in the figure below x Add Reference Segments A
31. KB_3100_POP6_BDTv3 mob Saji Jez Jpuy 99u95 JUCAY OOLE WslHd IDV gt Table A 8 3100 Avant Basecaller and DyeSet Primer Files Used for Dye Terminator Chemistry continued A PIND I SN L ZA ejeMjjos edeosbes NSl d IJY DNA Sequencing Chemistry Capillary Array Length cm Basecaller DyeSet Primer ABI Basecalling ABI PRISM BigDye Terminator v1 0 and v1 1 36 ultra rapid Basecaller 3100APOP4UR bcp 80 long read Basecaller 3100APOP4_80cmv3 bcp DT3100POP4LR BD v1 mob 36 rapid read Basecaller 3100APOP6RRv2 bcp 50 std run Basecaller 3100APOP6SR bcp DT3100POP6 BD v2 mob ABI PRISM BigDye Terminator v3 0 and 3 1 36 ultra rapid Basecaller 3100APOP4UR bcp 80 long read Basecaller 3100APOP4_80cmv3 bcp DT3100POP4 BDv3 v1 mob 36 rapid read Basecaller 3100APOP6RRv2 bcp DT3100POP6 BDv3 v1 mob ABI PRISM dRhodamine Terminator 36 ultra rapid Basecaller 3100APOP4UR bcp 80 long read Basecaller 3100APOP4_80cmv3 bcp DT3100POP4 dRhod v2 mob 36 rapid read Basecaller 3100APOP6RRv2 bcp 50 std run Basecaller 3100APOP6SR bcp DT3100POP6 dRhod v2 mob seJrj JAW JaSekg pue siajjeoeseg v xipueddy QPIND Jesf L zA elemYyoS edeogbeg NSl d IJY GL V ABI PRISM 3700 DNA Analyzer Files Table A 9 3700 Basecaller and DyeSet Primer Files Used for Dye Terminator Chemistry DNA Sequencing Chemistry
32. Leu Ser AMB Trp G Leu Pro His Arg T E Leu Pro His Arg C Leu Pro Gin Arg A Leu Pro Gln Arg G lle Thr Asn Ser T 5 lle Thr Asn Ser C lle Thr Lys Arg A Met Thr Lys Arg G Val Ala Asp Gly F s Val Ala Asp Gly C Val Ala Glu Gly A Val Ala Glu Gly G Stop Codes AMBer OCHer OPA Note This chart is accessible from the Help menu C 4 ABI PRISM SeqScape Software v2 1 User Guide Amino Acid Abbreviations Table C 4 Amino Acid Abbreviations Amino Acid Abbreviations Amino Acid Three Letters One Letter Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic Acid Asp D Cysteine Cys C Glutamic Acid Glu E Glutamine Gin Q Glycine Gly G Histidine His H Isoleucine lle Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V Any Amino Acid X Note This chart is accessible from the Help menu ABI PRISM SeqScape Software v2 1 User Guide C 5 Appendix C Translation Tables C 6 ABI PRISM SeqScape Software v2 1 User Guide User Privileges Tables of User Privileges This appendix contains a list of privileges for users of the three cate gories Administrator Scientist and Analyst when they use the ABI PRISM SeqScape Software Table D 1 Access for Admin Level Description of access for users of Admin level only Admin Scientist
33. Open the project of interest 2 Inthe navigation pane select a view Project Specimen or Segment to print 3 If you are using WYSIWYG scroll to the area of the view you want to print 9 14 ABI Prism SeqScape Software v2 1 User Guide Printing Data and Reports 4 Select File gt Print or click amp j All data Print Properties Portrait C Landscape Paper Leter 8 5 by 11 inches z HeaderiFooter Print Cancel Print Specimen All data Print Properties Portrait C Landscape Paper Leter 8 5 by 11 inches z Print Cancel HeaderiFooter Bases per panel po All data Print Properties Portrait C Landscape Paper Leter 8 5 by 11 inches x HeaderiFooter Print Cancel 5 Complete the dialog box a In the Print section select Only the visible data WYSIWYG or All data if available b In the Print Properties section select the paper orientation and size c If you are printing a Segment assembly type a new value in the Bases per panel field in the Print Settings for Project section ABI Prism SeqScape Software v2 1 User Guide 9 15 Chapter 9 Exporting and Printing Data and Reports d Click Print zix j Printer Name HP C LaserJet 4500 PS Properties Status Ready Type HP C LaserJet 4500 PS Where IP 167 116 254 248
34. Translation codon table Layers which are units of analysis in any project and regions of interest ROIs grouped together into layers for display and translation Associated allele libraries e User defined styles for identification of variants in the project A reference segment is a contiguous segment of the reference sequence corresponding to a single contiguous DNA sequence It is also a region of interest The reference segment consists of Ananalyzed sample file Atext only format FASTA or seq file A GenBank format file ABI PRISM SeqScape Software v2 1 User Guide 4 3 Chapter 4 Creating a Reference Data Group GenBank Features 4 4 Every GenBank entry has a single contiguous sequence associated with it This is also referred to as the source feature This sequence is always numbered starting at 1 Because of this the sequence from a single GenBank entry translates into a single reference segment in the extended RDG Numbering of the base ROI on this segment is set by default to start at 1 Every GenBank entry has a feature table These features translate into regions of interest and layers in the extended RDG In the following table items in are qualifiers read for that feature key for example gene is the value of the gene qualifier If that qualifier doesn t exist then is substituted Table 4 1 GenBank feature table GenBank Feature Extended RDG equivalent source Skipped The
35. You insert a base The inserted base appears in lowercase and it has no SQV You delete a base The quality value for the base disappears You reinsert a deleted base The reinserted base appears in lowercase and it has no SQV ABI PRISM SeqScape Software v2 1 User Guide Cumulative Quality Value Scoring in Reports Cumulative Quality Value Scoring in Reports Quality values and scores are also displayed in several reports To view the reports select Analysis gt Report Manager or click BI Analysis QC Report Specimen Statistics Report Sequence Confirmation Report Base Frequency Report Library Search Report RDG Report Audit Trail Report ET Genotyping Report Active Layer Project Creation Date Project Template PT PT Modification Date RDG Creation Date Specimens in Report Consensus scores in an Analysis QC report are shown as an average quality value across the consensus sequence for each specimen Average consensus QV for all bases within the clear range Summary HLA C CDS 03 Dec 2002 at 23 40 04 PST HLA C_3100POP6 PT Creation Date mixed 5lib 13 May 2003 at 16 02 45 PDT 03 Dec 2002 at 22 58 40 PST Project Reference Data Gro RDG Modification D Project Modification Date hp RDG late HLA C v2 13 May 2003 at 16 02 46 PDT 03 Dec 2002 at 23 20 08 PST HLA C Exon2 4 n oNT 13 May 2003 at 16 02 46 PDT f A1 A2 A3 Specimen Analysis y
36. deleted by the uninstallation process Does not delete any files or folders created by users Any files that have been added to the application folders such as those created when the applications are run are not deleted by the uninstallation process Removing the To uninstall SeqScape software Software 1 Select Start gt Programs gt Applied Biosystems gt SeqScape gt Uninstall SeqScape vx x 2 Continue to follow the instructions to uninstall the software When the uninstallation is complete all the software program files are removed Your data files remain on the computer The uninstaller does not delete any folders or files created after installation If you want to delete any folders and files created after installation you must remove them manually ABI PRISM SeqScape Software v2 1 User Guide 2 9 Chapter 2 Getting Started Starting SeqScape Software for the First Time Before You Begin 2 10 File Naming Convention When you start the software for the first time you are prompted with a registration dialog box that creates an administrator account Log in to SeqScape software as Admin and enter the password you created To create new users you must log in as Admin Logging in with a user name allows SeqScape software to track each user s interactions with each project For information on the privileges for each category of user using the software refer to Appendix D User Privileges Some alpha
37. described 3 4 filtered sample sequence defined Glossary 3 first time user 2 10 format FASTA example E 3 options reports 9 8 frequently asked questions B 1 G Gap and Extension Penalties described 3 12 GenBank downloading file 4 5 features 4 4 general questions B 5 General tab described 3 4 genetic analyzer applications 1 5 Genotyping Report 7 39 H haplotype library defined Glossary 3 hard drive partitions 2 4 hardware and software requirements 2 3 Index 4 importing AA variant 4 38 and exporting about 6 34 from SeqScape Manager 6 34 NT variant 4 32 reference segment 4 13 samples automatically 6 11 variants 6 28 installation preparation 2 6 installing first time 2 7 preparation for 2 6 SeqScape software 2 5 upgrade 2 8 instrument protocol creating for autoanalysis 11 9 integrating SeqScape and data collection software 11 2 integration automation 1 3 invalid characters in names 2 10 IUB IUPAC defined Glossary 3 IUPAC diagrams C 3 IUPAC IUB codes C2 K KB basecaller 1 4 10 3 defined A 2 Glossary 3 key codes amino acid abbreviations C 5 complements C 3 IUPAC diagrams C 3 IUPAC IUB codes C 2 translation tables C 1 Universal Genetic Code C 4 L launching the software 2 10 layer creating 4 24 deleting 4 16 pane descriptions 4 18 layout view defined Glossary 3 ABI PRISM SeqScape Software v2 1 User Guide learning software wizard 4 6 library about 4 20 creating 4
38. is not connected to a genetic D default or analyzer C if D drive is not available has data collection software that is E connected to a genetic analyzer 2 4 ABI PRISM SeqScape Software v2 1 User Guide SeqScape Software Versions SeqScape Software Versions There are four versions of SeqScape Software v2 1 available Software Version Intended for a system that Existing Data SeqScape Software v2 1 full release version Has no SeqScape software installed or has the 30 day demo version installed Data created in v1 0 1 1 or 2 0 will be moved to version 2 1 SeqScape Software v2 1 upgrade Has SeqScape software v1 0 1 1 or 2 0 installed Data created in v1 0 1 1 or 2 0 will be moved to version 2 1 SeqScape Software v2 1 update Has SeqScape v2 0 installed Data created or saved in v2 0 will be moved to v2 1 IMPORTANT This update does not work on systems with SeqScape software v1 0 or 1 1 SeqScape Software v2 1 30 day demo Meets the minimum requirements You must export any files created in the 30 day demo version before the software expires You can then import any exported files into the full release version of SeqScape v2 1 ABI PRISM SeqScape Software v2 1 User Guide 2 5 Chapter 2 Getting Started Preparing to Install SeqScape Software Note An administrator should install the software and use it for the fir
39. llle 2 2 Hardware and Software Requirements 2 2 200005 2 3 Minimum System Requirements 000 eee eee eee 2 3 Hard Drive Partitions osse sd be aa a eee 2 4 SeqScape Software Versions 00 ccc eee 2 5 ABI PRISM SeqScape Software v2 1 User Guide iii Chapter 3 Preparing to Install SeqScape Software 000 0005 2 6 Installing the Full Version for the First Time 2 7 Upgrading from SeqScape Software Versions 1 0 1 1 or 2 0 2 8 About the Upgrade 0 cee ees 2 8 Upgrading to vaT wie pe eae ae RE EE 2 8 Existing USers s dai peaa Re eR ROO qi 2 8 Removing SeqScape Software 0 000 eee eee 2 9 What the Uninstallation Process Does 2 55 2 9 Removing the Software 0 00 e eee es 2 9 Starting SeqScape Software for the First Time 2 10 Before You Begin 00 eee E E ees 2 10 File Naming Convention 0 00 eect eee eee 2 10 Starting SeqScape Software 00 00 eee eee 2 11 Creating New Users 0 0 00 cece tees 2 13 Setting Up Authentication amp Audit 0 00000 2 15 Changing User Information 00000 eee eee 2 19 Setting Up the Default Directory 00000 2 20 New Users Logging In for the First Time 0 004 2 21 When New Users LogIn l lllllsllseleelsene 2 21 SeqScape Software Structure 0 0 ees 2 22 SeqScape Ma
40. specifying 3 12 viewing reports 7 23 what it entails 6 3 analysis and report questions B 18 analysis defaults creating new 3 3 defined Glossary 1 individual samples 3 17 setting 3 14 analysis parameters about 8 3 changing 8 7 analysis protocol applying 8 12 creating 3 3 editing 8 7 Analysis Protocol Editor tabs 3 4 Analysis QC Report 7 26 10 11 analysis changing base call settings 3 4 clear range method 3 8 filter settings 3 10 mixed base settings 3 8 Index 1 Analyst privileges D 5 existing users 2 8 analyzing data 6 21 project 6 1 Annotation tab sample 6 12 applying analysis protocol 8 12 template to existing project 6 23 Assembly view defined Glossary 2 assembly defined Glossary 2 assigning styles to variants 4 39 At PCR Stop check box 3 5 audit trail 1 3 Audit Trail Report 7 38 Authentication amp Audit setting 2 15 autoanalysis 11 2 Autoanalysis Manager components 11 24 explained 11 2 automated analysis 11 2 automatic analysis before you start 11 4 B Base Frequency Report 7 34 basecaller ABI 1 4 ABI defined A 2 and DyeSet Primer compatibility A 4 defined Glossary 2 KB 1 4 KB defined A 2 Basecalling tab described 3 4 before creating new RDG requirements 4 12 C changing basecaller files 8 6 DyeSet Primer files 8 6 settings within project examples 5 6 user information 2 19 Index 2 Clear Range tab described 3 4 widget using 8 17 clear range adjusting 8 16 changing 8
41. 1 User Guide Starting SeqScape Software for the First Time Changing User You can change the default settings for a user you are setting up Information IMPORTANT The Administrator is the only person who can set up and change the information in the Users tab The selections in this tab are inactive for all other users To change any of the information for a user 1 In the Options dialog box select the Users tab 2 Double click the name in the list to open the User Management dialog box lll User Management User Creation Update E x User Name Beenist First Name Scientist Last Name se Password pueen e Password must be 6 to 15 characters lon User Group Scientist x I Inactive Unlock Created 13 Sep 2002 at 13 52 10 PDT Last Modified 19 Nov 2002 at 17 41 33 PST Cancel 3 Change or correct the user information and click OK 4 If desired click the Export button in the Options dialog box to export the application configuration settings and or settings for a single user or multiple users in a zipped ctf format 5 Enter the path for exporting files in the Export User dialog box then click Export 6 Click OK to close the Options dialog box Note This process can be used by the first administrator to set up additional users or another administrator It is possible to import or export user settings from one computer to another For example an administrator can set
42. 17 defined Glossary 2 ClustalX software using F 1 comparison defined Glossary 2 compatibility basecaller and DyeSet Primer A 4 complements for reference C 3 completing plate record 11 20 components of project 6 4 computer configuration requirement G 1 technical support for altered configuration G 1 consensus caller defined Glossary 2 quality values defined Glossary 2 score 10 5 sequence defined Glossary 2 consensus quality values consensus score 10 5 explained 10 5 consensus sequence editing in project view 8 15 editing in specimen view 8 14 importing assembled sequences 6 20 importing text 6 20 constant position error defined Glossary 2 position defined Glossary 2 contig defined Glossary 2 ABI PRISM SeqScape Software v2 1 User Guide creating alibrary 4 20 an RDG about 4 34 analysis protocol for autoanalysis 11 11 analysis protocols 3 3 instrument protocol for autoanalysis 11 9 new layers 4 24 new NT variants 4 30 new project using project template 6 10 new users 2 13 plate record for autoanalysis 11 18 project 6 1 project template 5 1 project before you begin 6 3 project using New Project Wizard 6 5 RDG administrator privileges 4 12 RDG scientist privileges 4 12 results group for autoanalysis 11 14 crucial position defined Glossary 2 customizing data 7 44 header footer reports 9 11 reports 7 42 D data analyzing 6 21 display conventions 7 3 editing 8 1 reanalyzing 8 1 s
43. 2 Click Properties then click the ROI tab 3 In the Reference Sequence pane highlight the reference segment you want to delete 4 16 ABI PRISM SeqScape Software v2 1 User Guide Defining Regions of Interest ROI 4 5 Right click and select Delete in the pop up menu A confirmation dialog box opens nfirmation A Remove reference segment AF250557 partl This will remove the reference segment from the Layers and ROI tables and dis associate any ROIs You cannot undo this action Cancel Click OK in the confirmation dialog box Once you click OK the delete cannot be undone RDG Properties x General ROI NT Variants AA Variante Variant Style Layer 2 3 Al n Layer 1 settings _NewLayer_ LayerName Layer1 Index Codon Number fi Orientation LUbey OY S Translation Frame i Right ROI Name Segment Seg Start Seg End ROlStet _ ROI Length Translation Color on Layer AF250557 partt AF250557 pat 1 105 1 105 2 mum v E 2 fi AF250557 part2 part AF250557_part2_p 106 297 106 192 m EH 3 AF250557 part2 pari2 AF250557 part2 p 298 792 298 495 mM EN 4 HLAC exon2 1 AF250557 pari 1 105 1 105 mM mm 5 HLAC gene 1 AF250557 parl 1 105 1 105 2 mm zl 4 gt E Rete ET F Es 7 i 7 Rename a 80 par 81 105 pai AF250557 par M M Add Ref Segment Paste Ref Segment Split Ref Segment Add Variant Add ROI OK Cancel
44. 2400 Basecaller 377 bcp DT377 BDv3 v2 mob Terminator v3 0 and 3 1 36 amp 48 1200 Basecaller 377LR bcp DT377LR BDv3 v1 mob S OWL JaSekg pue siejjeeseg v xipueddy QPIND JASN L zA asemyoS edeogbeg NSl d IJY 6 V Table A 5 377 Basecaller and DyeSet Primer Files Used for Dye Primer Chemistry it eei WTR cm Basecaller DyeSet Primer ABI Basecalling ABI PRISM BigDye Primer 36 2400 Basecaller 377 bcp DP377 5 LR BD 21M13 mob dau 36 amp 48 1200 Basecaller 377 LR bcp DPS a SRE Tee ABI PRISM BigDye Primer 36 2400 Basecaller 377 bcp DP377 BDv3 21M13 v1 mob ee 36 amp 48 1200 Basecaller 377LR bcp Sii Dye Michevvinop sajl4 4eouenbegs YNA ZZE Wsidd IJV ABI PRISM 3100 Genetic Analyzer Files PIND Jasf L ZA ejeMjjos edeosbes NSl d IJY Table A 6 3100 Basecaller and DyeSet Primer Files Used for Dye Terminator Chemistry DNA Sequencing Chemistry Capillary Array Length cm Basecaller DyeSet Primer KB Basecalling ABI PRISM BigDye Terminator v1 0 and v1 1 36 ultra rapid 50 std read 80 long read KB bcp KB_3100_POP4_BDTv1 mob 36 rapid read 50 std read KB bcp KB_3100_POP6_BDTv1 mob ABI PRISM BigDye Terminator v3 0 and v3 1 36 ultra rapid 50 std read 80 long read KB bcp KB_3100_POP4_BDTv3_ mob 36 rapid read 50 std read KB bcp KB_3100_POP6_BDTv3 mob SO I 4 Jeuiug
45. 29 creating specimens 6 12 displaying views 7 4 expanded display viewing 7 11 exporting 9 4 importing variants 6 32 to 6 33 printing data views 9 14 reanalyzing with different template 6 22 saving your data 7 21 viewing results 7 25 Q quality values QV 10 1 to 10 13 consensus quality values 10 5 Index 5 cumulative QV scoring in reports 10 11 to 10 13 customizing display bars 10 7 defined Glossary 4 displaying QVs_ 7 3 sample quality values 10 3 table of values 10 3 questions analysis B 18 frequently asked B 1 general B 5 reports B 18 SeqScape Manager B 9 R RDG Reference Data Group about 4 3 creating 4 1 4 5 creating new 4 5 new using SeqScape Manager 4 12 new using the wizard 4 6 saving copy 4 41 Wizard 4 5 RDG Properties dialog box ROI tab graphic 4 14 RDG Report 7 37 reanalyzing data 8 1 data workflow 8 2 renaming and saving project 6 22 reference defined Glossary 4 sequence described 4 12 reference associated data defined Glossary 4 reference break adding in sequence 4 27 Reference Data Group RDG adding variants 6 29 defined Glossary 4 incorporating variants into projects 6 25 reference segment deleting 4 16 importing 4 13 pasting 4 15 setting up 4 10 registering software recording number 2 2 Index 6 removing software 2 9 Report Manager defined Glossary 4 reports AA Variants 7 31 Analysis QC 7 26 10 11 Audit Trail 7 38 Base Frequency 7 34
46. A1 3R_02 ab1 a Auto Add gt gt a A1 4R_02 ab1 i i A2 Automatically create la A2 2F 03 ab1 Specimen name based on the string between these fel A2 2P_04 ab1 delimiters in the sample s e A2 3F_03 ab1 sd A2 3R_O4 ab1 a A2 4F_03 ab1 E E A2 4R_04 ab1 Start a3 CI HLA C_Exon2 4_FASTA Libra End zl RN Use sample file name V Show ab Samples File Only grob OK Cancel 3 In the Samples To Add section on the right select the specimen into which to import the data ABI Prism SeqScape Software v2 1 User Guide 6 15 Chapter 6 Creating and Analyzing a Project 4 In the Files pane navigate to the samples you want to add 5 Select the first specimen in the Samples to Add pane Samples To Add Files Add Sample gt gt U Specimen2 Audio CD D Remove Sample lj Speciment Local Disk C Ua C3 AppliedBiosystems Daz eo SeqScape AppSeqScape 5 0 Tutorial Data H LA C Delete Specimen HLA C_3100POP6_Tutorial D a Bg a A1 2F 01 ab1 3 s SE Auto Add fa A1 3F_O1 abt ja Al 3R_02 ab1 Automatically create ie A1 4F_01 ab1 Specimen name based on je A1 4R_02 ab1 the string between these E C142 delimiters in the sample s a A2 2F 03 ab1 name a 42 2R_04 ab1 NES abl sten a A2 3R_04 ab1 fa A2 4F 03 ab1 End ha A2 4R_ 04 ab1 Use sample file name C Use sample name OK Cancel v Show ab1 Samples File On
47. AA Variants Report Specimen Statistics Report Sequence Confirmation Report Base Frequency report Library Search Report RDG Report Audit Trail Report Genotyping Report Create Analysis Defaults and Display Settings Step ABI PRISM SeqScape Software v2 1 User Guide Analysis Defaults Settings Analysis Defaults Settings Analysis Defaults are a component of the project template They are used to set the analysis settings for all the samples as they are imported into a project Creating Analysis Protocols Before you create analysis defaults you need to create the analysis protocol An analysis protocol in the ABI PRISM SeqScape Software Version 2 1 specifies the analysis conditions to be applied to your samples You can specify the analysis protocol settings for one or more samples You must select an analysis protocol before selecting analysis defaults The analysis protocol settings include Basecalling Mixed bases Clearrange Filtering Analysis Protocol Editor x General Basecalling Mixed Bases Clear Range Filter r Analysis Protocol Description wAnalysisProtoco Created 16 Aug 2002 at 14 28 10 PDT Created By NewUser New User Modified 23 Sep 2002 at 11 27 33 PDT Modified By N A Source N A Comments Figure 3 2 Analysis Protocol Editor Showing General Tab ABI PRISM SeqScape Software v2 1 User Guide 3 3 Chapter 3 Creating Analysis Defaults and D
48. Active Layer HLA C CDS Project HLACC 2 Pr Analysis QC Report Mutations Report a Project Creation Date 03 Dec 2002 at Project Modification Date 13 May 2003 at E AA Variants Report 23 40 04 PST 16 02 45 PDT Project Template PT HLA C_3100POP8_mixe PT Creation Date 03 Dec 2002 at d 5lib 2329 08 PST PT Modification Date 13 May 2003 at Reference Data Group RDG HLA C Exon2 4 noNT 16 02 45 PDT RDG Creation Date 03 Dec 2002 at RDG Modification Date 13 May 2003 at 22 59 40 PST 16 02 46 PDT Display Settings DS TutorialDisplaySettings DS Creation Date 26 Nov 2002 at 23 37 59 PST x Specimens in Report A1 A2 AS U Specimen Statistics Report ES Sequence Confirmation Report i Base Frequency Report Name Library Translation Index Orientation Number Frame Codon of ROIs Number Layer 1 1 1 Foward 2 HLA C CDS 3 1 Foward 3 ROIs ROI Parent ROI Stat Stop ROI ROI Translation On Layers Position Position Index Length 1 i AF250557 AF250557 782 1 HLA C_exon2 AF250557 270 HLA C_gene AF250557 782 HLA C intron2 AF250557 516 HLA C exon AF250557 782 HLA C_exond HSA277102 276 HLA C gene 1 HSA277102 276 HSA277402 HSA277102 276 Report Settings Figure 7 10 RDG Report ABI Prism SeqScape Software v2 1 User Guide 7 37 Chapter 7 Viewing the Results Audit Trail Report The report contains two separate tables 7 38 Table 7 14 Parts of the Audit Trail Report
49. Analysis Defaults AD 3100SR mixed v2 AD Creation Date 04 Sep 2002 at 19 04 47 PDT AD Modification Date 04 Sep 2002 at 19 41 57 PDT Specimens in Report A1 Specimen Analysis Specimen Assembly epscimen Total Samples ariants f m mm 1 Complete E Partial Output A No output 2 For Research Use Only Not For Use In Diagnostic Procedures Owner Paget v 4 1 of 2 ABI Prism SeqScape Software v2 1 User Guide Printing Data and Reports Table 9 3 Report Preview Button Functions Button s Function First Previous Next and Last Displays the various pages in a report only one page is visible at a time Print Opens the Print dialog box Select a printer then click OK to print the report Print Pages Opens the Page s dialog box Set the page range then click OK In the Print dialog box click OK to print the report Note Page s dialog box settings override the settings in the standard Print dialog box Close Closes the preview window without printing the report ABI PRISM SeqScape Software v2 1 User Guide 9 19 Chapter 9 Exporting and Printing Data and Reports 9 20 ABI Prism SeqScape Software v2 1 User Guide Sample and Consensus Quality 1 Values This chapter contains Types of Quality Values QVS 0 0 cc eee eee eee 10 2 Sample Quality Values 0 0 0 0 cece eee eee eee 10 3 Consensus Quality Values 10 5 Displaying Quality V
50. Analyst Admin only access Create User Accounts Exporting Importing User Accounts Export a Project PT RDG Library from the SeqScape Manager Import objects from outside the DataStore into the SeqScape Manger Install SeqScape for an automated analysis system Allowed Not Allowed Not Allowed ABI PRISM SeqScape Software v2 1 User Guide D 1 Appendix D User Privileges Table D 2 Access for Admin and Scientist Levels Description of access for users of Admin and Scientist levels Admin Scientist Analyst SeqScape 1 Delete an object from the SeqScape Allowed Allowed Not Manager manager Allowed 2 Delete a Project from the SeqScape manager 3 Save As an object in the SeqScape manager 4 Create a new object in the SeqScape Manager 5 Create a new Project Template in SeqScape Manager 6 Configure analysis defaults in SeqScape manager 7 Deleting entries from a library in the SeqScape manager 8 Re Configure an existing Project Template in the SeqScape Manager Analysis 9 Creating an analysis protocol Allowed Allowed Not Protocol amp Allowed Settings 10 Editing an existing analysis protocol 11 Apply an analysis protocol to a set of samples project sample specimen 12 Create new Primary Seq Analysis Protocols 13 Set Clear range determination in Analysis settings or analysis defaults 14 Set Mi
51. Clear Range dialog box Using the Clear To use the Clear Range widget to adjust the clear range Range Widget 1 Open asample from within a project 2 Select the Electropherogram tab 3 Locate and select the 5 CR start or 3 CR end widget The widget turns from gray to black when selected TEn 5 CR start widget 3 CR end widget 4 Drag the widget along the bases to the right or left as desired then release the cursor 5 If the audit feature is enabled an Audit Reason Editor opens lii Audit Reason Editor xj Event Setnew CR 12 293 Description 6 Complete the Audit Reason Editor dialog box then click OK The new clear range is displayed 7 Repeat the process to define a new clear range for the opposite end ABI PRISM SeqScape Software v2 1 User Guide 8 17 Chapter 8 Reanalyzing and Editing Data Using the Mouse To use the mouse to adjust the clear range 1 Open a sample from within a project 2 Select the Electropherogram tab 3 Right click between two bases where you want to move the 5 CR start or 3 CR end widget New widget position Original widget position Nakties unes CTM CA CICC TC M G UGG ATG tgTGGC R Set CR start at selection Set CR end at selection 4 Doone of the following If you are moving the Then select CR start widget Set CR start at selection CR end widget Set CR end at selection
52. Click OK to import the specimens and samples into the project Tonma Al E 4 f j 2 bs S Claw Range Known Variants Unknown Variants You can import the following types of sample data into specimens within a project e Sample data files from ABI PRISM instruments Database files Specimen text only files Table 6 2 Types of Sample Data To import See Sample data files Adding Specimens and Importing Data Files on page 6 15 Specimen text only files Importing Text Only Files on page 6 20 ABI Prism SeqScape Software v2 1 User Guide Adding Specimens and Importing Data into a Project Adding To import unanalyzed or analyzed sample data the files must be in Specimens and ABI format Sample data is imported into specimens in the project Importing Data New specimens are created in the Import Samples dialog box Files To add specimens and import sample data files 1 With the Project window open select File gt Import Samples To Project or click gx to open the Import Samples dialog box 2 Create a new specimen a Click New Specimen b Add two more specimens Import Samples xj Samples To Add EISE Add Sample Spec 3 lj Specimen J C AppliedBiosystems U Specimen B SeqScape Dat H O AppSeqScape U 42 B C Tutorial Data U A3 a HLa c E C HLA C_3100POP6_Tutorial D BC3A1 fa A1 2F_O1 ab1 a A1 2R 02 ab1 5 A1 3F_01 ab1 la
53. Creating a New RDG Using SeqScape Manager Importing a To form the reference sequence you need to import one or more segments Reference To import a reference segment 1 If itis not already open select the ROI tab The dialog box that opens shows Reference Sequence as a place holder in the lower left pane RDG Properties E E x General ROI NT Variants AA Variants Variant Syle y Layer 1 settings Js E Layer Name Layer 1 Index Codon Number 1 Orientation aye bay 7 d Translation Frame fz Right 2 Click Add Ref Segment in the lower left to add a segment to the reference sequence Navigate to the file containing the reference sequence It can be a GenBank file or a file that you stored on your computer the file may have a gb extension Click Import The reference sequence is on Layer 1 which is locked so it cannot be modified ABI PRISM SeqScape Software v2 1 User Guide 4 13 Chapter 4 Creating a Reference Data Group Multiple ROIs in layer Reference Sequence locked in Layer 1 Lock icon Layer pane Reference _ index base Reference _ segments Reference Sequence pane 4 14 RDG Properties neral ROI Layer i Reference break Library folder Layer 1 settings New Layer Layer Name ser Orientation Library Translation Frame T m Right ROI Name Segment Seg Start Seg End
54. Mutations Report 8 Print a report from the reports manager Project 9 Move sample data from one Specimen to Allowed Allowed Allowed View Display another 10 Display SQVs and CQVs 11 Re order aligned Specimen consensi 12 Change active Layer view 13 Show hide variants that result in silent mutations 14 Sort Summary Table in Specimen view 15 Display Sample and Consensus Scores 16 View Amino Acid tooltips for degenerate codons 17 View Amino Acid Alignment in Main Window 18 View Library Search Results in Alignment View Identification Pane ABI PRISM SeqScape Software v2 1 User Guide D 5 Appendix D User Privileges Table D 3 Access for Admin Scientist and Analyst Levels continued Description of access for users of Admin Admin Scientist and Analyst levels Scientist Analyst Project 19 View electropherogram data as aligned Allowed Allowed Allowed View Display peaks 20 View all objects in Project Navigator and Main Windows 21 View Specimen Layout 22 View Specimen Segment Assembly tab 23 View Unassembled data in the Project Navigator and Specimen Views 24 View Navigate through electropherogram snippets 25 View Navigate Specimen Segment electropherogram data 26 View a Project Navigate using the Overview pane 27 View Samples in the Sample Manager tab 28 View Navigate alignments using the display toolbar buttons Project Other 29 Appl
55. Open the project of interest Select a layer in the Active Layer drop down list Select Analysis Report Manager or click El In the navigation pane select the report you want to view Select Window gt Tile Click a hyperlink blue text in the report then view the data in the Project view File Edit View Tools Analysis Window Help cia IEI amp elUSSI s x IEIF miej fs e 9 RU ved ad E mf f US Active LayerfLayer 1 Tab jumps to next Muttiple Samples Basecalling Filter Assembly Specimen Total Comments Score Variants WAnalysis QC Repo Ng E AA Variants Report a a Li 2 a Specimen Statistics Report n oO E E Sequence Confirmation Report zi i 24 31 l Base Frequency Report amp Library Search Report Complete E Partial Output No output RDG Report Audit Trail Report Genotyping Report Sample Analysis Sample Step Description Possible Heterozygous Indel Mutations Sample Project view Project view AT2R 02 E A1 4F_01 Report Settings A2 4F 03 ILA C v2 Known Variants A P T P G s T P ScTecCcACTCCATGABe TA TTTCTBco cBEcco ro cceccccls GCcTCCCACTCCATGAGGTATTTCMacaccecloTeTccceccccl GCTCCCACTCCATGAGGTATTTcTACACCGCMGTGTCCCGGCCCE jl he active layer does not contain a library Figure 7 2 Viewing Results and Reports Together ABI Prism SeqScape Software v2 1 User Guide 7 25 Cha
56. PRISM SeqScape Software v2 1 User Guide Xv Preface Conventions Used in This Guide Text Conventions This guide uses the following text conventions xvi File Naming Convention User Attention Words e Bold indicates user action For example Type 0 then press Enter for each of the remaining fields e Titles of documents and CDs are shown in italics For example ABI PRISM SeqScape Software Version 2 1 User Guide e Italic text indicates new or important words and is also used for emphasis e A right arrow bracket gt separates successive commands you select from a drop down or shortcut menu For example Select File gt Open Project Right click the sample row then select View Filter gt View All Runs Some alphanumeric characters are not valid for user names or file names The characters that are illegal are listed below spaces M8 2 Two user attention words appear in Applied Biosystems user documentation Each word implies a particular level of observation or action as described below Note Provides information that may be of interest or help but is not critical to the use of the product IMPORTANT Provides information that is necessary for proper software operation Examples of the user attention words appear below Note Names for Reference Segments are not editable IMPORTANT Do not click OK until you have completed the RDG ABI PRISM SeqScape Software v2 1 User Guide Conventions U
57. Project Project Template View gt Analysis QC Report I Sample Parameters Reference Data Specimen gt Mutations Report L gt Analysis Protocol ou da AA Variants Report ariants Repo Analysis Protocols gt em dad Specimen Statistics Analysis Defaults IS Sample Report Display Settings ew gt Sequence Confirmation Report Libraries gt Base Frequency Report gt Library Search Report gt RDG Report gt Audit Trail Report L gt Genotyping Report View and Edit View Export or z Compare Data i Print Results Edit and Apply Figure 2 2 SeqScape Software Structure SeqScape In the SeqScape Manager you configure projects by creating project Manager Window templates The project templates can be reused in multiple projects and can be exported to be shared with other researchers The project templates contain Reference sequence information Analysis settings including analysis protocols Display settings 2 22 ABI Prism SeqScape Software v2 1 User Guide SeqScape Software Structure Project Window _ In the Project window you can view your data in the following ways Table 2 3 Views in the Project Window View Description Project View Shows the reference sequence each specimen consensus sequence and electropherogram snippets for each sample file in each specimen The Expanded Nucleotide View shows all the nucleotides The Collapsed Nucle
58. Report Mutations Report AA Variants Report Specimen Statistics Report Sequence Confirmation Report Base Frequency Report Library Search Report e RDG Report Audit Trail Report e Genotyping Report Note For more information on the reports see Viewing the Reports on page 7 23 What is the Analysis QC Report The Analysis QC report provides a summary of the project s history This report details the status of each specimen at each step of analysis In addition the Analysis QC report lists possible HIMs heterozygous insertion deletion mutations What does Segment Score mean in the Specimen Statistics report Segment Score gives an average of all the quality values within the clear range in that particular reference segment region What does Coverage mean in the Specimen Statistics and Sequence Confirmation reports Coverage gives a value for the number of samples in the consensus sequence Can edit sequences within a project while reports are open and see the updated information in the reports instantly or will have to close and re open the reports to see any changes The reports will stay open and the results are updated as edits are made ABI Prism SeqScape Software v2 1 User Guide Quality Values FAQs Table B 9 Analysis Reports Questions and Answers continued Question Answer Why are my sample files unassembled If y
59. Software Quick Reference Card Portable document format PDF versions of the documents listed above are available on the SeqScape software installation CD or by selecting Programs gt Applied Biosystems gt SeqScape If you do not have Acrobat Reader installed on your computer install it from the SeqScape software CD so you can open the pdf files Note For additional documentation see How to Obtain Services and Support on page xix Applied Biosystems welcomes your comments and suggestions for improving its user documents You can e mail your comments to techpubs appliedbiosystems com ABI PRISM SeqScape Software v2 1 User Guide How to Obtain Services and Support How to Obtain Services and Support For the latest services and support information for all locations go to http www appliedbiosystems com then click the Support tab At the Support page you can Search through frequently asked questions FAQs Submit a question directly to Technical Support Order Applied Biosystems user documents MSDSs certificates of analysis and other related documents Download PDF documents Obtain information about customer training Download software updates and patches In addition the Support page provides access to worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities ABI PRISM SeqScape Software v2 1 User Guide xix Preface XX ABI PRISM SeqScape Software v
60. Stacker DakarPT3 Changing stacker run mode to STACKER_MODE_MANUAL No Current Run 11 22 ABI Prism SeqScape Software v2 1 User Guide Scheduling and Starting a Run 2 Click Search in the Input Stack section The Add Plates to Input Stack dialog box opens Add Plates to Input Stack Type of Search Advanced x Condition Value 1 Value 2 eo SA Run Name Plate ID Not Equal Plate Name Type I Size Status ER Search Stop Clear Row Clear All Search Results I Append Results Name Type Description spec Spectral Calibration 01 Regular ddddd Regular Clear All Done 3 Complete the Add Plates to Input Stack dialog box a Select Advanced in the Type of Search drop down list b Enter your search criteria for your plate then click Search c Select the plate in the Search Results section then click Add d Click Done The plate is displayed in the current Runs section of the Run Scheduler window 4 Click gt Run to start the run ABI Prism SeqScape Software v2 1 User Guide 11 23 Chapter 11 Automating Analysis Understanding the Autoanalysis Manager Overview Autoanalysis occurs in the following sequence 1 When data collection software finishes a run the Message Service sends the message Run Completed 2 The Autoanalysis Manager receives the message and the job is submitted The job appe
61. The entries you define in this tab are used to identify known and unknown variants in your projects You can enter NT variants by Clicking Add Variant in the ROI tab then entering the variant attributes in the New NT Variant dialog box Creating a table of variants in a tab delimited format then saving the file and importing it into the NT variant file One way of creating a table of variants is by using Microsoft Excel The columns in the Excel table must map to the columns in the NT Variants tab as shown below RDG Properties x General ROI NT Variants aA variants Variant Syle Type Ro Position Reference Variant Style Description Used by all Change Base C exon3 75 Known lyes Change Base HLA C exon3 68 en yes Change Base AF250557 76 lyes Figure 4 3 NT Variants Tab Showing Table Column Names Importing an aligned FASTA file Selecting a sequence within a reference segment then clicking Add Variant This procedure is described below ABI PRISM SeqScape Software v2 1 User Guide 4 29 Chapter 4 Creating a Reference Data Group Creating New NT To create new NT variants Variants 1 In the SeqScape Manager select the Reference Data Group tab then click Properties 2 Select the ROI tab 3 Drag to select a sequence in the nucleotide sequence area of the tab then click Add Variant RDG Properties ES x General ROI NT Variants AA Variants Va
62. The variants now appear in the NT Variants tab of the RDG Properties dialog box ABI Prism SeqScape Software v2 1 User Guide 6 33 Chapter 6 Creating and Analyzing a Project Importing and Exporting Project Information About Importing and Exporting Importing from SeqScape Manager Exporting from SeqScape Manager 6 34 The purpose of importing and exporting project information is to transfer the project information to another computer You can export or import projects project templates reference data groups nucleotide and amino acid variant tables libraries and analysis defaults from the SeqScape Manager This allows you to examine and compare results from different Data Stores Note The export and import functions of SeqScape Manager use the file extension CTF To import from SeqScape Manager 1 Sc opes coL 9 Select Tools SeqScape Manager Select any tab into which you want to import Click Import Navigate to the file that you want to import Click Import The imported file appears in the list under the appropriate tab To export from SeqScape Manager 1 2 3 Select Tools SeqScape Manager Select any one of the tabs from which you want to export Select the file that you want to export from the list then click Export Navigate to a location to export Rename the file if necessary using the ctf extension Click Export The exported file is available to import into anoth
63. Use reference trimming Reference MENENEEEEEEEEENI L2 Y Multiple clear range methods are applied in order EN P3 p Reference QY gt X Smallest clear range is the result Because SeqScape software generates quality values for each base you can choose to use a region of sequence where a certain number of bases reach an appropriate quality value ABI Prism SeqScape Software v2 1 User Guide Creating Analysis Protocols Select Use clear range minimum and maximum then set the minimum first base and maximum end base or the number of bases to trim from the 3 end of the clear range Select Use quality values to remove bases until there are lt X number of bases per Z number of bases with QV lt Y This sets a window with a specified number of allowed low quality bases Select Use identification of N calls to remove bases until there are X number of Ns per Y number of bases This sets a window with a specified number of allowed ambiguous base calls Ns Select Mask M13 universal sequencing primers to exclude the M13 primer sequence from the clear range Select Use reference trimming to have the samples automatically trimmed to contain only sequences that align to the reference ABI PRISM SeqScape Software v2 1 User Guide 3 9 Chapter 3 Creating Analysis Defaults and Display Settings Specifying the Filter Settings 3 10 The Filter tab sets the criteria for rejecting sequenc
64. Values section click the colored bars to open a color chart then select the color if necessary To select the threshold values drag the divider bars between the colors Note The styles you specify here do not apply to variants For more information on quality values see Chapter 10 Sample and Consensus Quality Values ABI PRISM SeqScape Software v2 1 User Guide Specifying Display Settings 5 Select the Electropherogram tab then enter your preferences for viewing the electropherogram and axes a Enter your Scaling and Axes preferences b Select a Vertical Display setting Real Values or Relative Display Settings Real Values ABI Prism SeqScape Software v2 1 User Guide 3 21 Chapter 3 Creating Analysis Defaults and Display Settings 6 Select the Views tab then enter your preferences for the Project and Specimen views Select Electropherogram view General Bases Electropherograr Vi r General View Settings r Project View Settings Characters Dots E Display Mode 5 Iz gt Electropherograms Vj m Erao topno Shown GT Pos on Confidence Bars il Bu Hidden GT Pos r Expanded NT NT Tab Jump Multiple z F v Si A Tab Jump Multiple z ed M Show Overview Iv Variants View Colurnn Selector Z Iv index Reference Sample View Settings Iv IESISIETICS SUA Original Sequence i Specimen View Settings
65. Your To create your library in SeqScape software Library in 1 SeqScape Software Launch SeqScape software Select Tools gt SeqScape Manager Select the Library tab and click New 2 3 4 Enter a library name 5 Select the Entries tab and click Import 6 Select Display All File Types then navigate to and open the multi aligned FASTA file The software creates the library and displays the number of sequences added ABI PRISM SeqScape Software v2 1 User Guide F 7 Appendix F Creating a Multi Aligned FASTA File F 8 ABI PRISM SeqScape Software v2 1 User Guide Software Warranty Computer Configuration Applied Biosystems supplies or recommends certain configurations of computer hardware software and peripherals for use with its instrumentation Applied Biosystems reserves the right to decline support for or impose extra charges for supporting nonstandard computer configurations or components that have not been supplied or recommended by Applied Biosystems Applied Biosystems also reserves the right to require that computer hardware and software be restored to the standard configuration prior to providing service or technical support For systems that have built in computers or processing units installing unauthorized hardware or software may void the Warranty or Service Plan Limited Product Warranty Limited Warranty Applied Biosystems warrants that for a period of ninety 90 days from the date the warranty p
66. a 201 aagcgccagg cacagactga ccgagtgagc ctgcggaacc 240 241 tgcgcggcta ctacaaccag agcgaggccg gtdagtgacc 280 Find 281 ccggcccggg gcgcaggtca cgacccctcc ccatccccca 320 321 CGgacggccc gggtcgcccc gagtctcccg gtctgagatc 360 Add ROI 361 caccccgagg ctgcggaacc cgcccagacc ctcgaccgga 400 401 Gagagcccca gtcaccttta cccggtttca ttttcagttt 440 I lt lt Back Next gt gt Finish Cancel 2 Click Add ROI to add the segment to the ROI table in the ROI pane above the sequence Add as many ROIs as desired Click Next Follow the instructions to add layers and ROIs to layers Layer 1 is always the reference sequence which is generated by the software and is locked Click New Layer then name each layer that you add IMPORTANT To avoid confusion give each layer that you add a unique name ABI PRISM SeqScape Software v2 1 User Guide Creating a New RDG Using the Wizard 5 Click the new layer under the Layer label in the layer pane then select the ROI on Layer check box in the ROI pane to associate it with the selected layer Do this for each layer you create IMPORTANT In a layer you cannot define ROIs that overlap one another x Add ROIs to Layers Click New Layerto add a new layer Click ROls to associate with selected Layer a lt lt Back Finish Cancel 6 Click Finish or if you want to change any of the selections click Back The newly created RDG appears in the Reference Data Group l
67. a A reference break can be added in the Reference Sequence by using Reference Break the Split Ref Segment button Reference segments can be split if you in a Sequence Want to delete intervening reference sequences When reference segments are split the ROIs associated with the reference segment are also split To add a reference break 1 In the ROI tab Sequence pane select the base position where you want a split to occur then click Split Ref Segment RDG Properties General NT Variants AA Variants Variant Style Layer 4 2 Hacen 3 PAC akona LAC intron amp l 792 al 2 r Layer 1 settings _NewLayer_ Layer Name ver Index Codon Number 1 Orientation bey mj Tenslation Frame fz Right ROI Name Segment AF250557 AF250557 HLA C exon2 AF250557 HLA C_gene AF250557 ROI 1 AF250557 afe AF250557 4 Seg Start Seg End ROI Start ROI Length Translation Color on Layer 1 E Reference Sequence Beitr Add Ref Segment Paste Ref Segment Split Ref Segment Then click Split Ref uM a Segment button A break 2 A confirmation dialog box opens showing the position for the reference break Click OK ll Confirmation x 3 Split Ref Segment at position 271 ABI Prism SeqScape Software v2 1 User Guide 4 27 Chapter 4 Creating a Reference Data Group There is a new reference break in the Reference Sequence in the Layer pane as shown in the next
68. and corrects the color code changes due to the type of chemistry used to label the DNA DyeSet Primer files are sometimes referred to as mobility files DyeSet Primer DyeSet Primer files use the following name convention File Naming Conventions K B_3 73 e OP B DTv 3 m e b E File extension Chemistry Polymer Instrument KB basecaller DT31 00POP4 BDTv3 mob MEN File extension Chemistry Polymer Instrument Dye terminator and ABI basecaller Figure A 1 Examples of DyeSet Primer File Naming Convention A 2 ABI PRISM SeqScape Software v2 1 User Guide Definitions and Naming The DyeSet Primer file names use a combination of characters to indicate the basecaller instrument chemistry and polymer type as described in Table A 1 Table A 1 DyeSet Primer File Names Abbreviation For Runs Using Basecaller KB KB basecaller DP Dye primer chemistry and the ABI basecaller DT Dye terminator chemistry and the ABI basecaller Type of Polymer or Gel 596LR 96 Long Ranger in the gel 377 instrument only POP4 ABI PRISM POP 4 polymer POP5 ABI PRISM POP 5 polymer POP6 ABI PRISM POP 6 polymer POP7 ABI PRISM POP 7 polymer Chemistry BDTv3 BDv3 ABI PRIsM BigDye v3 0 and 3 1 Terminator chemistry BDv1 BD ABI PRIsM BigDye v1 0 and 1 1 Terminator
69. assemble analyze my samples without re basecalling my samples so that can conserve the existing basecalls Yes In order to assemble and analyze your sequences without basecalling open the Analysis Defaults for the project and select the Specimen tab Deselect the Basecall Samples check box How do analyze samples in one project with different basecallers dye set primer files To analyze samples in one project with different basecallers and dye set primer files you must open the Sample Manager from the Analysis menu Select the appropriate basecaller and dye set primer files and click Apply The project must be re analyzed for these changes to take effect Samples and specimens with a red slash indicate an unanalyzed status In the Sample Manager you can also choose to Edit the Analysis Protocol for the individual samples or Apply an Analysis Protocol What does a red line through the specimen icon indicate A red line indicates that analysis has not occurred A red line may also appear if the analysis settings have been changed and the project requires re analysis to apply these settings Click the green arrow run button at the top of the window to start analysis How can I edit my specimen name Select the specimen and select Edit gt Rename or right click and select Edit How can delete samples or specimens Select the item to be deleted then select Edit gt Delete click the Delete button on th
70. ator Insert ABI PRISM SeqScape Software v2 1 User Guide 9 11 Chapter 9 Exporting and Printing Data and Reports 3 To change the graphic if desired a In the Graphic section click Set b In the dialog box locate then select a graphic file c Click OK Note The graphic is displayed in the Headers amp Footers dialog box and in the upper left corner of printed or exported reports 4 To change the header and or footer information do one of the following Type text into any of the header and or footer text boxes e Use the autotext variables from the Autotext drop down list Insert the cursor in a text box select an autotext option in the drop down list then click Insert Usea combination of typing text and using the autotext variables 5 Save the changes a Click OK to close the Header amp Footer dialog box b Click Print in Print Report dialog box c In the Print dialog box click Cancel to save the changes without printing OK to save the changes and print Printing Views of To print different views of a sample file a Sample File 1 Open the project of interest 2 In the navigation pane select a sample to print 9 12 ABI PRISM SeqScape Software v2 1 User Guide Printing Data and Reports 3 Select File gt Print or click amp Print Sample Ai 2F_01 x Print r Sample Settings _ C Only the visible data Panels PerPage 4 E Points Per Panel 1500
71. called by the software e Use Find and Find Again to search for text 7 18 ABI Prism SeqScape Software v2 1 User Guide Sample Views Table 7 4 Sample Views continued Tab Display Raw E Es Bj Ej Bee pups FE m H a Active Layer HLA C CDS E Tab jumps to nex cimens_v2 LA 3100_threeSpecimens_v2 f Al AF250557 A Annotation Sequence Features Electropherogram i HLA 3100_threeg a ga Unassem E AF250557 Ba1 3F Ba1 3R Bai 2r Bir gi 741434 a g az Yas Jr ddl Jal ANI li I t J eiusd iM i MAN ABI PRISM SeqScape Software v2 1 User Guide 7 19 Chapter 7 Viewing the Results Viewing Variants Two methods to view variant data are presented here Note To edit variant data see Editing Variants on page 8 20 To view variant data Method 1 1 2 7 20 Open the project of interest Click a consensus base View the electropherogram snippets by clicking the triangle that appears next to the specimen name In the Tab Jump to Next drop down list select Multiple then select Known Variant and Unknown Variant Press Tab to move to the next variant or press Shift Tab to move to the previous variant Note Pressing Ctrl Z moves any electropherogram snippets of the selected variants to the middle of the view FIT Project Navigator ILA C 3100 2 HLA C 31 00 Known Variants
72. chemistry 21M13 Dye primer chemistry the 21M13 primer is labeled M13Rev Dye primer chemistry the M13Rev primer is labeled ABI PRISM SeqScape Software v2 1 User Guide A 3 Appendix A Basecallers and DyeSet Primer Files Basecaller and DyeSet Primer Compatibility The DyeSet Primer file must match the chemistry and basecaller type that you are using Note DyeSet Primer files are filtered based on the selected basecaller IMPORTANT However if you select the DyeSet Primer file then select a basecaller file no filtering of the basecaller list occurs If you select a KB DyeSet Primer file and an ABI basecaller for analysis or a DT DyeSet Primer file and an KB basecaller for analysis the following error dialog box opens see Figures A 2 and A 3 W BaseCaller DyeSet Primer Incompatibility e Please choose a compatible DyeSet Primer file for the BaseCaller which you have chosen Figure A 2 Error Message in the Sample Manager lif Incompatible mobility file chosen x Q Please choose a mobility file that matches the chosen basecaller Figure A 3 Error Message in the Analysis Protocol ABI PRISM SeqScape Software v2 1 User Guide opinc Jes zA asemyosg edeogbeg Wsikd IJV g v ABI PRISM 310 Genetic Analyzer Files Note 47 cm capillary array length 36 cm read length 61 cm capillary array length 50 cm read length Table A 2 310 Basecaller and DyeSet Primer Files Used
73. day demo version of the software installed IMPORTANT If you are using a SeqScape software 30 day demo export your data before the end of the 30 days and before you load the full version of SeqScape software To ensure data is not lost a Export your data Select Tools gt SeqScape Manager gt Target Files and click Export b Load the software as described in the procedure below c Re import your data To install the full version of SeqScape software for the first time 1 Insert the ABI PnisM SeqScape Software v2 1 CD into the computer CD ROM drive 2 If the installer does not start automatically double click setup exe on the CD 3 When the InstallShield Wizard Complete window opens click Finish After the software is installed the administrator must log into the software for the first time After the initial login the software can be set up for additional users ABI PRISM SeqScape Software v2 1 User Guide 2 7 Chapter 2 Getting Started Upgrading from SeqScape Software Versions 1 0 1 1 or 2 0 About the The SeqScape Software v2 1 installer automatically Upgrade Detects the previous version and backs up your data folder Removes the previous version then installs the new version of SeqScape software Follow the instructions on the installer Upgrading to v2 1 To upgrade your software 1 Insert the appropriate SeqScape software installation CD into the CD ROM drive If you are upgradin
74. fi to 5 5 Collate Selection Be eo ien Select the printer and define the page range then click OK ABI PRISM SeqScape Software v2 1 User Guide 9 17 Chapter 9 Exporting and Printing Data and Reports Print dialog box 8 The Report Preview dialog box opens Use the command buttons as described in Table 9 3 on page 9 19 forin dX Pintor Print Pages dialog box None LT 000 Stotys Reody Type HPCLasendet 4500S Whore _167 116 254248 cas nn x x Print range Copies CA Number ot copies os current 1 of 2 C from fi tok Use to view different pages of the report Print Preview dialog box P Report Preview x a SeqScape Analysis QC Report Appli ied Generated at04 Sep 2002 at 19 AS Biosystems 41 57 POT Summary Active Layer HLA C CDS Project HLA 3100 v2 Project Creation Date 04 Sep 2002 at 19 41 57 PDT Project Modification Date 04 Sep 2002 at 19 41 57 PDT Project Template PT HLA 3100 v2 PT Creation Date 04 Sep 2002 at 19 08 54 PDT PT Modification Date 04 Sep 2002 at 19 41 57 PDT Reference Data Group RDG HLA C exons2 4 v2 RDG Creation Date 04 Sep 2002 at 19 21 47 PDT RDG Modification Date 04 Sep 2002 at 19 41 57 PDT Display Settings DS DefaultDisplaySettings v2 DS Creation Date 20 Jul 2001 at 09 23 19 PDT DS Modification Date 04 Sep 2002 at 19 41 57 PDT
75. for Dye Terminator Chemistry DNA Sequencing Chemistry Capillary Array Length cm Basecaller DyeSet Primer KB Basecalling ABI PRISM BigDye Terminator 47 KB bcp KB 310 POP4 BDTv1 36Rapid mob dd KB 310 POP4 BDTv1 36Std mob 47 KB bcp KB 310 POP6 BDTv1 36Rapid mob 61 KB 310 POP6 BDTv1 50Std mob ABI PRISM BigDye Terminator 47 KB bcp KB 310 POPA4 BDTv3 36Rapid mob ASQ arid V3 1 KB_310_POP4_BDTv3_36Std mob 47 KB bcp KB_310_POP6_BDTv3_36Rapid mob 61 KB_310_POP6_BDTv3_50Std mob ABI Basecalling ABI PRISM BigDye Terminator 47 Basecaller 310POP4 bcp DT310POP4 BD v2 mob v1 0 and v1 1 47 Basecaller 310POP6 bcp DT310POP6 BD mob 61 DT310POP6 BD LR v3 mob Se9 j JOZA CUY oieuec OLE WSI4d gv 9 V epint Jes L ZA ejeMjjos edeosbes NSl d gv Table A 2 310 Basecaller and DyeSet Primer Files Used for Dye Terminator Chemistry continued DNA Sequencing Chemistry Capillary Array Length cm Basecaller DyeSet Primer ABI PRISM dRhodamine 47 Basecaller 310POP4 bcp DT310POP4 dRhod v1 mob Terminator 47 Basecaller 310POP6 bcp DT310POP6 dRhod v2 mob 61 ABI PRISM BigDye Terminator 47 Basecaller 310POP4 bcp DT310POP4 BDv3 v2 mob v3 0 and v3 1 47 Basecaller 310POP6 bcp DT310POP6 BDv3 v2 mob 61 Table A 3 310 Basecaller and DyeSet Primer Files Used for Dye Primer Chemistry DNA Sequencing Chem
76. have been created Autoanalysis Manager software is running e The 3730 3730xl or 3100 3100 Avant instrument is set up to run and samples are prepared For more information on setting up and using the 3730 Data Collection software refer to the Applied Biosystems 3730 3730xl Getting Started Guide PN 4347118 For more information on setting up and using the 3100 Data Collection software refer to the ABI PRISM 3100 3100 Avant User Guide PN 4347102 Files for a data collection software plate record are available For data collection and autoanalysis to be successful each run of samples must have an instrument protocol an analysis protocol and a results group assigned within a plate record The table below describes what each file specifies in the logical order of its use Table 11 1 File Specifications File Description Created in Instrument Contains everything needed Data collection Protocol to run the instrument software Analysis Protocol Contains everything needed Data collection to analyze sequencing data software or SeqScape software Results Group Defines the file type the file Data collection name file save locations software default analysis protocols linked to sample injections and user name and password Project Template Contains RDG analysis SeqScape software defaults and display settings 11 4 ABI PRISM SeqScape Software v2 1 User Guide Is SeqSc
77. more processing power in the system the better its performance Table 2 1 Minimum System Requirements System Component Minimum Requirements CPU 733 MHz or faster with an Intel Pentium III or IV processor The software does not run on computers with a dual processor or with a Xeon chip set CD ROM drive Any Operating system Microsoft Windows 2000 OS with Service Pack 3 Microsoft Windows XP with Service Pack 1 RAM 256 MB 512 MB is recommended Printer An HP 4500 8100 990cxi or an Epson 980 printer is recommended Monitor A 17 inch monitor or larger is recommended A monitor with 1024 x 768 resolution is recommended Disk space 1GB Storage requirements depend primarily on the quantity of data to be generated and stored It is common to store many SeqScape software project files on the analysis computer Install SeqScape software on a partition with enough space for the projects and their files because SeqScape software stores data files in the area where the program is installed ABI PRISM SeqScape Software v2 1 User Guide 2 3 Chapter 2 Getting Started Hard Drive The installer uses the following location for SeqScape software files Partitions drive letter Applied Biosystems SeqScape The drive letter is determined by the following conditions Table 2 2 Drive Letter Determination If the computer The installer selects drive
78. preparing your sequences before creating a library Method 1 Using SeqScape software to create a multi aligned FASTA file Method 2 Using ClustalX software to create a multi aligned FASTA file About ClustalX Software ClustalX software is a powerful multiple sequence alignment program available free of charge via the Internet See Jeanmougin et al 1998 Trends Biochem Sci 23 403 5 ABI PRISM SeqScape Software v2 1 User Guide F 1 Appendix F Creating a Multi Aligned FASTA File Method 1 Create a Multi Aligned FASTA File Using SeqScape Software 1 Create a Multi To create a multi aligned FASTA file using SeqScape software Aligned FASTA Fil Note You can try this procedure by obtaining HLA sequences from lle GenBank database The file names are listed below as bullet point items 1 Obtain the reference sequence gi 512471 emb X76776 1 HSHLADMBG 2 Obtain files that contain the sequences that will be used in your sequence library txt or fsta files gi 1045472 gb U32663 1 HSU32663 Human MHC class II antigen HLA DM beta chain HLA DMB gene gi 1373022 gb U31743 1 HSU31743 Human HLA DMB variant gene gi 2315188 emb Y 14395 1 HSHLADMB3 Homo sapiens HLA DMB gene gi 512471 emb X76776 1 HSHLADMBG H sapiens HLA DMB gene gi 881918 gb U16762 1 HSU16762 Human DMB gene 3 Launch SeqScape software and create an RDG using the file obtained in step 1 4 Create a project template 5 Start
79. r Report Display Settings Enables disables horizontal scrolling Horizontal Scrolling Selects font and Font arial font size size oz Wraps text in cells _ wrap Text Figure 7 13 Report Display Settings tab of the Report Settings dialog box ABI Prism SeqScape Software v2 1 User Guide Customizing the Reports 2 Select or deselect Horizontal Scrolling check box The default is off 3 Select a font type and font size in the appropriate drop down lists The default is font is Arial size 10 4 Select or deselect Wrap Text Examples of wrapped and unwrapped text are shown in Figure 7 14 Specimen Sample Step Description A3 A3 4F 05 Filter Exceeded maximum mixed base Unwrapped A3 AG 4AF 05 Assembly Incomplete results presented from x text Specimen Sample Step Description A3 A3 4aF 05 Filter Exceeded maximum mixed base Wrapped percentage 45 901638 gt 35 0 text A3 A3 aF 05 Assembly Incomplete results presented from previous stage Figure 7 14 Examples of unwrapped and wrapped text To set the display of AA variants 1 Click the AA Variants Settings tab Reports Settings o x 44 Variants Display Mode IUB C Three Letter C Full Name 2 Select display mode then click OK ABI Prism SeqScape Software v2 1 User Guide 7 43 Chapter 7 Viewing the Results Customizing the To customize the inform
80. reapplying a new project template see Reanalyzing a Project Using a Different Project Template on page 6 22 Gap and Project Tab Settings Extension Penalties The settings for Gap Penalty apply for alignment of different specimen consensus sequences to each other and the reference Analysis Defaults x General Settings Gap Penalty 20 0 Recommended value 30 0 Specimen Sample Extension Penalty fi 0 Recommended value 1 0 IV Couple the amino acid alignment to the nucleotide alignment Save To Manager As OK Cancel Figure 3 3 Analysis Defaults Project tab 3 12 ABI PRISM SeqScape Software v2 1 User Guide Specifying the Analysis Settings If you want to add gap and extension penalties these settings introduce gaps into sequence alignments allowing the alignment to be extended into regions where one sequence may have lost or gained characters not in the other gap penalty score G Ln G is gap penalty L is the length of gap and n is the number of bases A penalty is subtracted for each gap introduced into an alignment because gap increases uncertainty into an alignment Note Applied Biosystems recommends that you use the default values The default settings are already optimized for the current algorithm Specimen Tab Settings The settings of gap and extension penalty apply to setting alignment of samples to the reference Analysis Defaults x General Project ample S
81. segments regions of interest ROI layers and the name of the associated allele libraries What are the new features of the extended RDG When using a Genbank file to create the reference sequence the feature table of the Genbank file is pulled into the RDG and each feature is listed out in the ROI region of interest table These ROIs can be used to create new layers for sequence comparison For example if a Genbank file for a gene containing two exons and one intron is imported into the RDG the user can choose to create a layer that includes only the two exons When analysis occurs the specimens will be compared to this layer containing the two exons as well as the reference backbone layer that includes the two exons and the intron In addition the user can choose to turn translation on or off for specific ROIs A library containing aligned sequences can also be attached to a specific layer for comparison during analysis What is a reference sequence A reference sequence is the backbone sequence against which the software compares the consensus segments A reference sequence contains continuous or discontinuous sequences made up of one or more reference segments What is a reference segment A reference segment is a contiguous section of the reference backbone within the reference sequence that corresponds to a single contiguous DNA sequence The reference backbone is the entire reference sequence and can be comp
82. setup Click Back to change the setup if necessary 11 Do one of the following To analyze Then Now Click Finish Later 1 Deselect Analyze Project at the bottom left corner of the page 2 Click Finish 12 When you close the new project click Yes to save it This project is now available in the list of available projects in the SeqScape Manager ABI Prism SeqScape Software v2 1 User Guide 6 9 Chapter 6 Creating and Analyzing a Project Creating and Analyzing a New Project Using a Project Template You can use an existing project template to create a new project A project template contains A Reference Data Group RDG Analysis defaults Display settings About the Project For convenience one example project template is included in the Template software To create your own project template see Creating a Project Template on page 5 3 Table 6 1 Components of the Project Template Included in SeqScape Software Template Component File Name Project Template Name e HLA 3100 POP6mixed v2 e p53 exon7 v2 Reference Data Group HLA C exons2 4 noNT v2 e p53 Exon v2 Analysis Defaults e 3100 SR POP6 BDTv1 mixed v2 e 3700LR POP5 BDTv1 v2 Display Settings e DefaultDisplaySettings v2 Creating aNew Tocreate a new project using a project template Project Using a 1 Select File New Project Template 2 When the New Project window opens selec
83. the project of interest 2 Selecta layer in the Active Layer drop down list 3 Atthe top of the navigation pane select the project icon 4 Use the instructions in Table 7 1 to display the project views of interest Table 7 1 Project Views View Procedure Display Expanded Click HH m Nucleotide s PEE zj Daaa ue Active Layer HLA C_CDS s Tab jumps to nd Project Navigator ALA UU HL amp C 3100 Known Variants IHLA C CDS ROIs All Variants Summary ACMKCCGTGTCCCGGCCYGGCCGCGGAGAGCC HT Yai oe soos Ve rx s 9s as e idees Buel ay Ser wR BNR RR Index 20 30 40 50 Reference Reference AA R Y F T T A v 5 R P G R G E gt 3602 IREESEEEEEEEE ACMKCCGTGTCCCGGCCYGGCCGCGGAGAGCC 7 4 ABI Prism SeqScape Software v2 1 User Guide Project Views Table 7 1 Project Views continued View Procedure Display Collapsed Click Nucleotide Iz vajaa H MMB Active LayerJHLa C_COS 1 Project Navigator E HLA C 31 00 Known Variants IHLA C_CDS ROIs All Variants This view shows only those columns that differ from the aligned reference Bases that match the reference sequence are displayed as dots regardless of the state of the Dots setting Note Click a Expanded Nucleotide View to return to the expanded view pes MES 2 j s apa mp mp mmm pa NEU acieleve HLA C CDS T
84. to a project the software also automatically searches the library to find the closest match to each consensus sequence When the analysis is complete the software generates a project file that contains sample files a consensus sequence for each specimen and nine reports You can print and export your results ABI PRISM SeqScape Software v2 1 User Guide 1 7 Chapter 1 Introduction to ABI PRISM SeqScape Software 1 8 ABI Prism SeqScape Software v2 1 User Guide Getting Started This chapter contains Administrator Registering the Software 2 2 Hardware and Software Requirements 2 3 SeqScape Software Versions llle 2 5 Preparing to Install SeqScape Software 2 6 Installing the Full Version for the First Time 2 7 Upgrading from SeqScape Software Versions 1 0 1 1 or 2 0 2 8 Removing SeqScape Software 0 0 0 0c eee eee 2 10 Starting SeqScape Software for the First Time 2 11 New Users Logging In for the First Time 2 22 SeqScape Software Structure 0 0 cee eee eee ee 2 23 SeqScape Software Toolbar 0 0 0 0 0c eee eee eee 2 25 Menus on the Main SeqScape Window 2 27 Worktlowi cued ne reete e Ee cer ae Ge 2 29 ABI PRISM SeqScape Software v2 1 User Guide 2 1 Chapter 2 Getting Started Administrator Registering the Software This chapter provides informatio
85. to lift or move the computer or the monitor without the assistance of others Depending on the weight of the computer and or the monitor moving them may require two or more people ABI PRISM SeqScape Software v2 1 User Guide xvii Preface Workstation Safety Correct ergonomic configuration of your workstation can reduce or prevent effects such as fatigue pain and strain Minimize or eliminate these effects by configuring your workstation to promote neutral or relaxed working positions Nim MUSCULOSKELETAL AND REPETITIVE MOTION HAZARD These hazards are caused by potential risk factors that include but are not limited to repetitive motion awkward posture forceful exertion holding static unhealthy positions contact pressure and other workstation environmental factors To minimize musculoskeletal and repetitive motion risks Use equipment that comfortably supports you in neutral working positions and allows adequate accessibility to the keyboard monitor and mouse Position the keyboard mouse and monitor to promote relaxed body and head postures How to Obtain More Information Related Documentation Send Us Your Comments xviii The following related documents are shipped with the software SeqScape Online Help Provides procedures for common tasks Help is available from the Help menu in the main SeqScape window or by pressing F1 e ABI PRISM SeqScape Software Tutorial ABI PRISM SeqScape
86. to select the library before continuing with the procedure to create new layers To set up a library 1 In the main SeqScape window select Tools gt SeqScape Manager then select the Libraries tab Libraries tab in SeqScape Manager WR SeaScape Manager Projects Project Templates Reference Data Grou Library amp Length Created Create d Modified By Type HLA C ex244 822 18 May 200 N A 18 May 200 guest Appli diploid IHLA C ex2 822 16 May 200 NIA 18 Sep 200 guest Appli haploid I 10 Some HLA 2 Select New ABI Prism SeqScape Software v2 1 User Guide 4 21 Chapter 4 Creating a Reference Data Group 3 In the Library Editor General tab enter a name for the new library and select Haploid or Diploid Library Editor x Entries r Library Description Name HLA C ex24 v2 Created 16 May 2002 at 17 23 53 PDT Created By N A Modified N A Modified By N A Source N Haploid C Diploid Comments Some HLA C alleles OK Cancel 4 Select the Entries tab then click Import 5 Import the aligned multiple sequence FASTA file and click OK 6 In SeqScape Manager select the Reference Data Group tab and select the RDG that you want to link to the library 7 Click Properties then select the ROI tab Note At this point if you do not have layers in the RDG or you do not know how to create a la
87. up user information for many users then select all the user files and export them to other systems using SeqScape software ABI PRISM SeqScape Software v2 1 User Guide 2 19 Chapter 2 Getting Started Setting Up the The default directory should be set up for users for importing and Default Directory exporting data files If the directory path is not set up the default directory opens to C To set up the default directory path 1 In the SeqScape software main window select Tools gt Options 2 In the General tab select the appropriate check boxes for your setup if desired a Select the Display Reports after Analysis check box b Select the Export Reports after Analysis check box if desired then select the format in which to export them from the Format drop down list General Database Users Authentication amp Audit v Display Reports after Analysis v Export Reports after Analysis Format HTML z HTML PDF Default Path for rt IC A SeqSc2 0 4d XML ject_Datal Browse 3 Click Browse and navigate to the directory to use as the default for files to be stored 4 Click Open The exported reports are stored in the directory you select as the default 5 Click OK to save the directory path and close the dialog box 2 20 ABI PRISM SeqScape Software v2 1 User Guide New Users Logging In for the First Time New Users Logging In for the First Time When New Users After the installation
88. v2 1 User Guide 7 33 Chapter 7 Viewing the Results Base Frequency Report The report contains two separate tables Table 7 11 Parts of the Base Frequency Report Table Description Summary Displays project information and the specimens in the report Base Frequency Displays the reference ROI and the of each base and space for each variant position rt Manager Pa Analysis QC Report 2 Mutations Report E AA Variants Report Specimen Statistics Report Sequence Confirmation Report Bbase Freq port Library Search Report RDG Report 3 Audit Trail Report Genotyping Report Report Settings Active Layer Project Creation Date Project Template PT PT Modification Date RDG Creation Date Display Settings DS DS Modification Date Specimens in Report At A2 A3 Variant Reference Position HLA C CDS 03 Dec 2002 at 23 40 04 PST Summary Project Project Modification Date HLA C_3100POP6_mixe PT Creation Date d 5lib 13 May 2003 at 16 02 45 PDT 03 Dec 2002 at 22 59 40 PST TutorialDisplaySettings 43 May 2003 at 16 02 45 PDT ROI Reference Data Group RDG RDG Modification Date DS Creation Date Analysis Defaults AD Base Frequency A HLA C_v2 13 May 2003 at 16 02 45 PDT 03 Dec 2002 at 23 29 08 PST HLA C Exon2 4 noNT 13 May 2003 at 16 02 45 PDT 26 Nov 2002 at 23 37 59 PST 3100 SR POP BDTv1 mixed 5Lib 16
89. with a single line description Description followed by lines of sequence data The description line is distinguished from the sequence data by a greater than gt symbol in the first column FASTA Format An example sequence in FASTA format is as follows Example sary HXB2 Prt RTI 1 320 cctcaggtcactctttggcaacgacccctcgtcacaataaagataggggggcaactaaaggaag ctctattagatacaggagcagatgatacagtattagaagaaatgagtttgccaggaagatggaaa ccaaaaatgatagggggaattggaggttttatcaaagtaagacagtatgatcagatactcatagaa atctgtggacataaagctataggtacagtattagtaggacctacacctgtcaacataattggaaga aatctgttgactcagattggttgcactttaaattttcccattagccctattgagactgtaccagtaaaat taaagccaggaatggatggcccaaaagttaaacaatggccattgacagaagaaaaaataaaag cattagtagaaatttgtacagagatggaaaaggaagggaaaatttcaaaaattgggcctgaaaat ccatacaatactccagtatttgccataaagaaaaaagacagtactaaatggagaaaattagtagatt tcagagaacttaataagagaactcaagacttctgggaagttcaattaggaataccacatcccgca gggttaaaaaagaaaaaatcagtaacagtactggatgtggetgatgcatatttttcagttcccttag atgaagacttcaggaagtatactgcatttaccatacctagtataaacaatgagacaccagggatta gatatcagtacaatgtgcttccacagggatggaaaggatcaccagcaatattccaaagtagcatg acaaaaatcttagagccttttagaaaacaaaatccagacatagttatctatcaatacatggatgattt gtatgtaggatctgacttagaaatagggcagcatagaacaaaaatagaggagctgagacaacat ctgttgaggtggggacttaccacaccagacaaaaaacatcagaaagaacctccattcctttggat gggttatgaactccatcctgataaatggacagtacagcctatagtgctgccagaaaaagacagct ggactgtcaatgacatacagaagttagtggggaaattgaattgggcaagtcagatttacccaggg attaaagtaaggcaattatgtaaactccttagaggaaccaaagcacta
90. x Q Applying a new template will cause all analyzed data and associated results to be lost Continue The project opens containing all the specimens and samples but the data is unanalyzed 7 To analyze the data with the new template select Analysis gt Analyze Fite Ed View Tools Analysis Windew Help dew O e 08 2x m mm B mp oio o Em m m om M cem tec cos Tabuo neun 2 6 24 ABI Prism SeqScape Software v2 1 User Guide Incorporating Variants into the Project RDG Incorporating Variants into the Project RDG About You can incorporate variants into an active project RDG by doing one Incorporating of the following Variant Changing an unknown variant in a specimen to a known variant Sequences Adding a variant Importing a file containing variant sequences Importing a set of variants from a TXT file Note If you have a master RDG and want to include additional variants in the RDG you must incorporate them using the SeqScape Manager You can incorporate variants automatically by importing a file of a tab delimited text file of variant positions and descriptions By this method variants are created and styles are applied to all the variants in the file Alternatively you can change an unknown variant in a specimen to a known variant or you can create variants by adding them to the Reference Data Group Changing a To change an unknown variant to a known variant Single Unknown Vari
91. 003 at Analysis Defaults AD 3100 SR POP6 BDTw1 m ARNAR ONT mind RI ih Specimens in Report A1 A2 A3 Mutations Specime Base ROI Position Length Type av Known Effect Aa Descriptio n Change Change n AT 16g r HLA C ex 16 4 Sub 6 missens RS K R m on2 e 25a W HLA C ex 25 Sub 17 missens YS Y F on2 e 290 gt m HLA C_ex 29 Sub 25 silent on2 30g k HLA C ex 30 Sub missens AT1O A S on2 e 44o gt Y HLA C_ex 44 Sub silent on2 68g K HLA C ex 69 Sub missens A23 A S on2 e 103326 HLA C_ex 103 Sub missens Q34R on2 e 128a r HLA C ex 128 Sub silent an Report Settings Mutation quality values Figure 10 6 Mutations Report 10 12 ABI PRISM SeqScape Software v2 1 User Guide Cumulative Quality Value Scoring in Reports Specimen The Specimen Statistics table of this report displays the average Statistics Report consensus QV score for a segment in the Segment Score column The Sample Results table displays the average sample QV for the bases in the clear range in the Sample Score column Summary Active Layer HL amp C CDS Project HLA C v2 Project Creation Date 03 Dec 2002 at Project Modification Date 13 May 2003 at 23 40 04 PST 16 02 45 PDT Project Template PT HLA C 3100POP6 mixe PT Creation Date 03 Dec 2002 at d 5lib 23 29 08 PST Library Search Report PT Modification Date 13 May 2003 at Reference Data Group RDG HLA C Exon2 4 noNT 16 02 46 PDT RDG Report A
92. 0c eee ee eee eee Editing Variants 2 0 0 eee eee eee Adding a Genotype 2 0 0 eee cee eee ABI PRISM SeqScape Software v2 1 User Guide 8 1 Chapter 8 Reanalyzing and Editing Data Workflow for This Chapter Chapter 3 1 Create Analysis Defaults and Display Settings Chapter 4 2 Create Reference Data Group Add Reference Sequence Create ROIs Create Layers Linka Library ROls Chapter 5 3 Create Project Template Select Add nucleotide variants Add amino acid variants Setup variant styles RDG Analysis Defaults Display Settings Chapter 6 4 Create and Analyze Project Chapter 7 5 View Data and Reports Chapter 8 6 Reanalyze and Edit Data Chapter 9 Select Project Template Add Specimens Import Sample Files Data Project View Specimen View Segment View Sample View Software does automatically Basecalling Filter Assembly Consensus Alignment Comparison Search Library 7 Export and Print Reports Aligned projects Consensus sequence Aligned sample sequence Sample files Report Manager Analysis QC Report Mutations Report AA Variants Report Specimen Statistics Report Sequence Confirmation Report Base Frequency report Library Search Report RDG Report Audit Trail Report Genotyping Report
93. 0x instruments select Heat Sealing or Septa in the Plate Sealing drop down list h Type a name for the owner and operator i Click OK The SeqScape Plate Editor opens see Figure 11 4 on page 11 20 ABI Prism SeqScape Software v2 1 User Guide 11 19 Chapter 11 Automating Analysis 11 20 Completing a Plate Record To use the same specimen name for the entire plate enter the name then click Ctrl D to fill down the column i seqscape Plate Editor J x File Edit Well A0 B01 Cot D01 E01 Fo G01 H01 A02 B02 c02 D02 E02 an 1 Plate Name bar Operator bap Plate ID bar Owner bap Plate Sealing Septa E Sample Name Comment Project Project Template Specimen Results Group 1 Instrument Protocol 4 Analysis Protocol 1 a E Description Loo E Ok Cancel Figure 11 4 Blank Plate Editor To complete a plate record 1 Optional Add comments Select a project or create a new one If you select a project previously created in SeqScape software the project template field fills in automatically If you choose to create a new project in the plate editor you can select only a project template that is already registered in SeqScape software You cannot create a project template in the plate editor Select a project template Select a specimen or create one ABI PRISM SeqScape Software v2 1 User Guide Creating Required Files in the Data Collection Software
94. 1 181 GCCGTGGGTGGAGCAGGAG GCCGGAGTATTGGGACCG GA GC GG GGAGA jiBEgg cual amumuuusdHE HH Hu HR HH HERE RR RR GAGCCECGEGECGECCETGEGETEGAGCAGGAGEGECCEGAGTATTEGEACCEGEAGA 1 1 198 188 178 168 158 E x gi 741 a Tas Figu p 1 pee pz Nie bicis pe p bal px An 2p na o nn IEREHRHREE HHEE HRHERERR n a d E E LUI GAGCCGCGGGCGCCGTGGGTGGA AGGGGCCGGAGTATTGGGACCGGGAGA GAGCCECGGECECCGTGGGTGGAAN 3a GGEECCEGAGTATIGGGACCGGGAGA i i 7 198 188 178 168 158 GAGCCGCGGGCGCCGTGGGTGGAGCAGGAGGGGCCGGAGTATTGGGACCGGGAGA i 1 1 D 186 196 206 216 226 236 re 10 4 Displaying the Value of a Consensus QV Bar ABI PRISM SeqScape Software v2 1 User Guide 10 9 Chapter 10 Sample and Consensus Quality Values Editing Bases with Quality Values 10 10 Changing deleting and inserting a base affect the consensus or sample QVs displayed Table 10 3 Results of Editing Bases with Quality Values If Then The consensus caller calls a base not present in all the samples The new base is in uppercase in the consensus sequence and in lowercase in the samples that did not contain that basecall with a red dot You change a base The new base is in lowercase and the SQV has the same value but is displayed as a gray bar You change a base back to the original call The base appears in uppercase and the quality value bar color is restored
95. 1ege q pue siejjeeseg v xipueddy Table A 6 3100 Basecaller and DyeSet Primer Files Used for Dye Terminator Chemistry continued DNA Sequencing Chemistry Capillary Array Length cm Basecaller DyeSet Primer ABI Basecalling ABI PRISM BigDye Terminator v1 0 and v1 1 e ABI PRISM dGTP BigDye Terminator 36 ultra rapid Basecaller 3100POP4UR bcp 80 long read Basecaller 3100POP4_80cmv3 bcp DT3100POP4LR BD v1 mob 36 rapid read Basecaller 3100POP6RRv2 bcp 50 std read Basecaller 3100POP6SR bcp DT3100POP6 BD v2 mob e ABI PRISM BigDye Terminator v3 0 and 3 1 opinc Jes zA asemyosg edeosgbeg wWsikd IJV e ABI PRISM dGTP BigDye v3 0Terminator 36 ultra rapid Basecaller 3100POP4UR bcp 80 long read Basecaller 3100POP4_80cmv3 bcp DT3100POP4 BDv3 v1 mob 36 rapid read Basecaller 3100POP6RRv2 bcp 50 std read Basecaller 3100POP6SR bcp DT3100POP6 BDv3 v1 mob ABI PRISM dRhodamine Terminator 36 ultra rapid Basecaller 3100POP4UR bcp 80 long read Basecaller 3100POP4_80cmv3 bcp DT3100POP4 dRhod v2 mob 36 rapid read Basecaller 3100POP6RRv2 bcp 50 std read Basecaller 3100POP6SR bcp DT3100POP6 dRhod v2 mob LL V SJ Jez Jeuy DEUAD 001 NSl d IJV D N Table A 7 3100 Basecaller and DyeSet Primer Files Used for Dye Primer Chemistry PIND Jes L ZA ejeMjjos edeosbes NSl d IJY DNA
96. 2 Analysis Defaults Settings 0 0 0 0 eee eee eee 3 3 Creating Analysis Protocols 0 0 0 0 ce eee eee eee 3 3 Specifying the Analysis Settings 0 00000 3 12 Specifying Display Settings 0 0 0 eee eee eee 3 18 ABI PRISM SeqScape Software v2 1 User Guide 3 1 Chapter 3 Creating Analysis Defaults and Display Settings Workflow for This Chapter 3 2 Chapter 3 1 Create Analysis Defaults and Display Settings Chapter 4 2 Create Reference Data Group gt Create ROIs Create Layers Chapter 5 3 Create Project Template Link a Library Add Reference Sequence ROls Select Add nucleotide variants Add amino acid variants Set up variant styles RDG Chapter 6 Analysis Defaults Display Settings 4 Create and Analyze Project Chapter 7 5 View Data and Reports Select Project Template F Add Specimens Import Sample Files Software does automatically Basecalling Filter Assembly Consensus Alignment Comparison Search Library Data Project View Chapter 8 6 Reanalyze and Edit Data Specimen View Segment View Sample View Chapter 9 7 Export and Print Reports Aligned projects Figure 3 1 Consensus sequence Aligned sample sequence Sample files Report Manager Analysis QC Report Mutations Report
97. 2 1 User Guide Introduction to ABI PRISM SeqScape Software 1 This chapter contains New Features in SeqScape Software v2 1 0 1 2 For Users of Earlier Versions of SeqScape Software 1 3 About SeqScape Software llle 1 5 ABI PRISM SeqScape Software v2 1 User Guide 1 1 Chapter 1 Introduction to ABI PRISM SeqScape Software New Features in SeqScape Software v2 1 New Features ABI PRISM SeqScape Software Version 2 1 includes the following new features and enhancements for v2 1 Optimization Faster project load import and save times Analysis pipeline time is 33 faster Data analysis Automatic analysis Integration for automatic analysis with Applied Biosystems 3730 3730xl Data Collection Software version 2 0 and ABI PRISM 3100 3100 Avant Data Collection Software version 2 0 M13 primer trimming Trims the M13 universal primer sequence from the PCR product 3 clear range trimming Trims PCR primer sequences New Basecallers KB basecallers identify pure and mixed bases then assign quality values for data generated on ABI PRISM 310 3100 and 3100 Avant systems and Applied Biosystems 3730 and 3730xl systems Microsoft Windows XP SP1 OS compatibility Reports NT Mutations report Shows the amino acid effect of each mutation and hyperlinks link the nucleotide variant with the amino acid variant and vice versa New Genotyping report Id
98. 20 linking 1 7 match defined Glossary 3 searching 1 3 setting up 4 21 Library Search Report 7 35 license and warranty rights and responsibilities 2 2 login process user 2 10 M main toolbar 2 24 menu structure 2 26 Mixed Bases settings specifying 3 7 tab described 3 4 mixed bases identification 8 9 mobility files selecting A 13 to A 17 Mutations Report 7 28 10 12 N new AA variants entering 4 36 features 1 2 layers creating 4 24 NT variants creating 4 30 project template about 5 3 New Project Wizard creating project 6 5 new users creating 2 13 logging in 2 21 nibbler defined Glossary 4 NT variants about 4 29 creating 4 30 P password protection 1 3 performing analysis 1 7 ABI PRISM SeqScape Software v2 1 User Guide plate record completing 11 20 creating for autoanalysis 11 18 polymorphic position defined Glossary 4 prepare for installation 2 6 print preview 9 16 printing data views froma project 9 14 reports 9 16 privileges administrator D 1 analyst D 5 scientist D 2 using SeqScape D 1 project components 6 4 defined Glossary 4 overview workflow 6 2 summary sequence defined Glossary 4 project template about 5 3 6 10 creating 5 1 creating new analysis defaults 3 3 creating new project 6 10 creating procedure 5 4 defined Glossary 4 importing and exporting 6 34 saving 5 5 Project window overview 2 23 projects adding specimens 6 11 to 6 12 adding variants 6
99. 310n Sadlianment feta oft gt File name 380 2 CX3R 02 fsta Export Files of type FASTA format fsta z Cancel STA format fsta FA ABT NE peo format NE seq z format phd 1 MIU l 4 Complete the Export dialog box Select a folder location to store the file Change the file name if desired The default file name uses the sample name and the FASTA extension Select a file format in the Files of type drop down list d Click Export ABI PRISM SeqScape Software v2 1 User Guide 9 7 Chapter 9 Exporting and Printing Data and Reports Exporting Reports File Names The default file name uses the project name and the report type Do not use the following characters in a file name M gt and space Format Options You can export generated reports as portable document format pdf text HTML or XML Table 9 2 Note When selecting between HTML and XML use HTML for standard display and XML for scripting applications Table 9 2 File Formats and Corresponding Application Options File Format Open with PDF default Adobe Acrobat Reader HTML A web browser or any software that is able to display HTML files XML A web browser TXT Notepad Wordpad Microsoft Word or any text compatible software When exporting the report as HTML a folder is automatically created that may contain more than one HTML fil
100. 5 If the audit feature is enabled select a reason in the Audit Reason Editor then click OK The new clear range is displayed 6 Repeat the process to define a new CR widget position for the opposite end 8 18 ABI Prism SeqScape Software v2 1 User Guide Adjusting the Clear Range Using the Set To use the dialog box to adjust the clear range Clear Range Dialog Box 1 Open a sample from within a project 2 Inthe Electropherogram view or Specimen view determine your new beginning and ending base numbers 3 Select Tools gt Set Clear Range Set Clear Range for QA3 F QA3 F x Begin bp 2s End bp 380 Cael em 4 Enter the values determined in step 2 then click OK 5 Ifthe audit feature is enabled select a reason in the Audit Reason Editor then click OK The new clear range is displayed ABI PRISM SeqScape Software v2 1 User Guide 8 19 Chapter 8 Reanalyzing and Editing Data Editing Variants 8 20 Method 1 After you clean up errors in the sequences you can view and edit the variants Two methods to review variants follow To view and edit variant data 1 Open a project of interest 2 Click a summary base 3 Click the triangle that appears next to the specimen name to view the electropherogram snippets 4 Edit bases or spaces in the specimen consensus sequences or the shown sample data 5 Press Tab to move to the next variant or press Shift Tab to move t
101. 5 The New Project Wizard lesser 6 5 Creating and Analyzing a New Project Using a Project Template 6 10 About the Project Template 0000 0 eee eae 6 10 Creating a New Project Using a Template 6 10 ABI PRISM SeqScape Software v2 1 User Guide Adding Specimens and Importing Data into a Project 6 11 OVSE W i desea pitur Dae a ebb ee et aes aei 6 11 Adding Specimens and Importing Samples Automatically 6 11 Adding Specimens and Importing Samples Manually 6 14 Adding Specimens and Importing Data Files 6 15 Importing Text Only Files llle 6 20 Removing Samples or Specimens lsus 6 21 Analyzing the Data leeeleeseeeeeeee eese 6 21 Running an Analysis nnsa siau 00 00 eee eee eee 6 21 Reanalyzing a Project Using a Different Project Template 6 22 When You Would Want to Do This slllleessss 6 22 Saving a Project Before Reanalyzing lesse 6 22 Applying a Template to an Existing Project 6 23 Incorporating Variants into the Project RDG 6 25 About Incorporating Variant Sequences 6 25 Changing a Single Unknown Variant to a Known Variant 6 25 Changing Multiple Unknown Variants lle 6 27 Importing Variants 0 0 0 eee BR 6 28 Creating a New Variant in a Project 00055 6 29 Adding a Variant in t
102. 662 00 00 00 00 131 Genotype Settings NADO 100 0 40 4 1000 100000 00 OO 00 1000 40 4 1000 100000 00 00 00 Ej 333 m Figure 7 12 Genotyping Report ABI Prism SeqScape Software v2 1 User Guide 7 41 Chapter 7 Viewing the Results Customizing the Reports Customizing Text 7 42 You can customize the text and select the amino acid variant To customize the report settings 1 Click the Report Settings button on the Report Manager window X SeqScape DM DM is logged in 2 File Edit View Tools Analysis Window Help dsm Els Tela eso x gt S mI E 06 f sof 5f 9 a 09089 ved d E SE Active LayerfLayeri Z Tab jumps to nextfviutipie z Specimen Samples Basecaling riter Assembly Specimen Total Comments Score Variants Bhanalysis QC Repo Mutations Report 1 E r3 m m 25 38 joe o AA Variants Report E B m T m Specimen Statistics Report Sequence Confirmation Report Base Frequency Report Library Search Report Complete B Partial Output A No output y RDG Report Sample Analysis Audit Trail Report i Genotyping Report Sample Step Description 24 31 Possible Heterozygous Indel Mutations Sample Position 33 A1 2R_02 A1 4F_01 185 Report Settings A2 4F_03 172 The Report Settings dialog box opens displaying the Report Display Settings tab Reports Settings x Report Display Settings AA Variants Settings
103. A2 A3 Specimen Analysis Samples Basecalling Filter Assembly Specimen Total Comments Score Variants E E E 7 gt E m E bd B E E 2 Complete S Partial Output A No output Sample Analysis Sample Step Description Possible Heterozygous Indel Mutations Sample Position Size AVR 02 33 1 A1 4F_01 185 15 A2 4F_03 172 15 Figure 7 3 Analysis QC Report ABI PRISM SeqScape Software v2 1 User Guide 7 27 Chapter 7 Viewing the Results Mutations Report The report contains two separate tables All blue text is hyperlinked to the project navigator Table 7 6 Parts of the Mutation Report Table Description Summary Displays project information and the specimens in the report Mutation Displays the bases changed ROI position length type QV and effect information for each mutation detected in a specimen Summary Active Layer HLA C CDS Project HLA C v2 E Project Creation Date 03 Dec 2002 at Project Modification Date 13 May 2003 at Ss Variants Report 23 40 04 PST 16 02 45 PDT Specimen Statistics Report E Sequence Confirmation Report Project Template PT HLA C 3100POP8 mixe PT Creation Date 03 Dec 2002 at 4 5lib 23 20 08 PST Base Frequency Report p Library Search Report PT Modification Date 13 May 2003 at Reference Data Group RDG HLA C_Exon2 4_noNT 16 02 45 PDT m Lbs Repot RDG Creation Dat 03 Dec 2002 at RDG Modification Dat 43 May 2003 a
104. AND APPLIED BIOSYSTEMS MAKES NO OTHER WARRANTY OF ANY KIND WHATSOEVER EXPRESSED OR IMPLIED INCLUDING WITHOUT LIMITATION WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE WHETHER ARISING FROM A STATUTE OR OTHERWISE IN LAW OR FROM A COURSE OF DEALING OR USAGE OF TRADE ALL OF WHICH ARE EXPRESSLY DISCLAIMED ABI PRISM SeqScape Software v2 1 User Guide Limited Product Warranty Warranty THE REMEDIES PROVIDED HEREIN ARE THE BUYER S Limitations SOLE AND EXCLUSIVE REMEDIES WITHOUT LIMITING THE GENERALITY OF THE FOREGOING IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE INCLUDING WITHOUT LIMITATION ANY TRADE PRACTICE UNFAIR COMPETITION OR OTHER STATUTE OF SIMILAR IMPORT OR ON ANY OTHER BASIS FOR DIRECT INDIRECT PUNITIVE INCIDENTAL MULTIPLE CONSEQUENTIAL OR SPECIAL DAMAGES SUSTAINED BY THE BUYER OR ANY OTHER PERSON OR ENTITY WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT APPLIED BIOSYSTEMS IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES INCLUDING WITHOUT LIMITATION DAMAGES ARISING FROM OR RELATED TO LOSS OF USE LOSS OF DATA FAILURE OR INTERRUPTION IN THE OPERATION OF ANY EQUIPMENT OR SOFTWARE DELAY IN REPAIR OR REPLACEMENT OR FOR LOSS OF REVENUE OR PROFITS LOSS OF GOOD WILL LOSS OF BUSINESS OR OTHER FINANCIAL LOSS OR PERSONAL INJURY OR PROPERTY DAMAGE NO AGENT EMPLOYEE OR REPRESENTATIVE OF APPLIED BIOSYSTEMS HAS ANY AUTHORITY TO MODIFY THE TE
105. Analyze data using the BGB 68 Indicate that specific Samples are not to be basecalled D 8 ABI PRISM SeqScape Software v2 1 User Guide Tables of User Privileges Table D 3 Access for Admin Scientist and Analyst Levels continued Description of access for users of Admin Scientist and Analyst levels Admin Scientist Analyst Other 69 Browse Locate data in the file system 70 Exit SeqScape 71 Sort items in columns in any table in SeqScape 72 Install SeqScape on a clean system 73 Upgrade SeqScape Software v1 0 or v1 1 to v2 0 74 Uninstall SegScape 75 Launch SeqScape 76 Configure a sample in Data Collection for automated import into SeqScape ABI PRISM SeqScape Software v2 1 User Guide D 9 Appendix D User Privileges D 10 ABI PRISM SeqScape Software v2 1 User Guide Aligned Variant and FASTA File E Format This appendix contains Tab Delimited Files 2 2 00 00 000 erenneren ee eee E 2 FASTA File Format esee RR RII E 3 ABI PRISM SeqScape Software v2 1 User Guide E 1 Appendix E Aligned Variant and FASTA File Format Tab Delimited Files You can import variants into the ABI PRISM SeqScape Software if they are in the format of a tab delimited text file Creating a Variant SeqScape software tab delimited text files must conform to the Text File following rules One variant per line The f
106. CAGCAGGAGATGGAACCTTCCAGAAGTG T R A G D TTGTGGAGACCAGGCCAGCA AGATGGAACC P Minnn amnis oa HG ER ELE A T QV 23 Consensus QVs Note Holding the pointer over a base displays the numerical QV assignment for that base 7 6 ABI Prism SeqScape Software v2 1 User Guide Project Views Table 7 1 Project Views continued ME HLA C 3100 E G4 UN IT View Procedure Display Electro 1 In the Expanded Nucleotide or Dot view select a base in the summary or specimen pherogram sequence Snippet 2 Click the triangle next to the specimen name Project Navigator HLA C 31 06 Known Variants va gu A Active Layer HLA C_CDS Tab jumps tone Triangle 360 2 CX4R 02 360 2 CX4F 01 Electropherogram snippets IHLA C CDS ROls r T caccTGwGReGGeTGCC CagcTGWGREggtGCC GAAGTGGGCAGCTGUGRIGGTGCCTICTGGAGAAGAG view Note Pressing Ctrl Z centers any electropherogram snippets in the middle of the 100 Project Navigator 5 HLA C 31 00 Piknown Variants View Aligned EP Aires click LA EEEE EUM eet Oesl Tobin re IHLA C CDS ROls ACMKCCGTGTCCCGGCCYGGCCGCGGAGAGCC T A v 8 R P G R G E GCTTCUTCKCA ACMKCCGTGTCCCGGCCYGGCCGCGGAGAGCC GGAGAGCCEDE ABI PRISM SeqScape Software v2 1 User Guide Chapter 7 Viewing the Results
107. C_Exon2 4_GenBai 7 p53 gt config a i data Iv Show ab1 Samples File Only Add Sample gt gt Remove Sample New Specimen Delete Specimen Auto Add Automatically create Specimen name based on the string between these delimiters in the sample s name Start End F Use sample file name C Use sample name x r Samples To Add lt lt Back Next gt gt Cancel a In the Files section select a sample multiple samples or a folder then click Auto Add b Click Next Note For information on adding specimens see Adding Specimens and Importing Data into a Project on page 6 11 6 8 ABI Prism SeqScape Software v2 1 User Guide Using the New Project Wizard to Create and Analyze a Project New Project Wizard a X Finish Review the project setup summary then click Finish button The software will create the following elements Project NewProject2 Project Template NewProject2Template Reference Data Group HLA C ex2 4 with Variants Analysis Protocols NewProject2 Analysis Defaults NewProject2 This Project will contain the following specimens and samples HLA C 01 2222 Corr 1 KY80 ab1 M Analyze Project lt lt Back Finish Cancel When you use the Project Wizard for the first time master display settings are created These same settings are used if the wizard is used again 10 Review the
108. Capillary Array Length cm Basecaller DyeSet Primer ABI Basecalling Terminator ABI PRISM BigDye Terminator 50 Basecaller 3700POP6 bcp DT3700POP6 BD v5 mob v1 0 and 1 1 Basecaller 3700POP5LR bcp DT3700POP5 BD v3 mob ABI PRISM BigDye Terminator 50 Basecaller 3700POP6 bcp DT3700POP6 BDv3 v1 mob v3 0 and 3 1 Basecaller 3700POP5LR bcp DT3700POP5 BDv3 v1 mob ABI Prism dRhodamine 50 Basecaller 3700POP6 bcp DT3700POP6 dRhod v3 mob Basecaller 3700POP5LR bcp DT3700POP5 dRhod v1 mob SoJ4 4ezAJeuy YNA 004 Wsidd EV D o Table A 10 3700 Basecaller and DyeSet Primer Files Used for Dye Primer Chemistry PIND Jes L ZA ejeMjjos edeosbes Wslud IJY DNA Sequencing Chemistry Capillary Array Length cm Basecaller DyeSet Primer ABI Basecalling ABI PRISM BigDye Primer 50 Basecaller 3700POP6 bcp DP3700POP6 BD 21M13 v3 mob andy i DP3700POP6 BD M13Rev v2 mob Basecaller 3700POP5LR bcp DP3700POP5 BD 21M13 v1 mob DP3700POP5 BD M13Rev v1 mob ABI PRISM BigDye Primer 50 Basecaller 3700POP6 bcp DP3700POP6 BDv3 21M13 v1 mob v3 0 and v3 1 DP3700POP6 BDv3 M13Rev v1 mob Basecaller 3700POP5LR bcp DP3700POP5 BDv3 21M13 v1 mob DP3700POP5 BDv3 M13Rev v1 mob SO I4 Jeuiug 1eseKq pue siej eoeseg v xipueddy opinc Jes zA asemyosg edeosgbeg wWsikd IJV ZL V Applied Biosystems 3730 3730x DNA Analyzers Files Table A 11 3730 3730x Basecaller and D
109. Date 13 May 2003 at Reference Data Group RDG HLA C_Exon2 4_noNT 16 02 46 PDT 5 Analysis QC Report Mutations Report RDG Report W Audit Trail Report RDG Creation Date 03 Dec 2002 at RDG Modification Date 13 May 2003 at xl R Genotyping Report Specimens in Report Library is Length Haplotype Polymorphic Creation Date Modification Comments Date 822 yes 10 0 16 May 2002 at 13 May 2003 at Some HLA C 47 23 53 PDT 16 02 45 PDT alleles Specimen Results Perfect Crucial Constant Comments Match Positions Position Errors yes yes yes Hit List Library Sequence Score Mismatches Mismatches Total in Constant in Mismatches Pos Polymorphic Pos Cw12022 Cw 0102 980 o Cur 12021 0wr0102 Cw 0402 Cw1301 960 Cw O102 Cw 1203 840 Cw 0402 Cw 12042 920 Cw 02022 CW 07012 88 0 Constant Position Errors Position Specimen base Library base Figure 7 9 Library Search Report 7 36 ABI Prism SeqScape Software v2 1 User Guide RDG Report RDG Report The report contains three separate tables Table 7 13 Parts of the RDG Report Table Description Summary Displays project information and the specimens in the report Layers Displays a summary of the information for each layer in the project as defined in the RDG ROls Displays a summary of the information for each ROI as defined in the RDG ie HLA C v2 Report Manager Summary
110. FASTA File 9 Select Alignment gt Alignment Parameters gt Reset All Gaps Before Alignment 10 Select Alignment gt Do Complete Alignment 11 A dialog box appears asking you to select the location where the results should be saved Select the location 12 Click the ALIGN button and wait for the alignment to be completed Notice that dashes have been introduced where bases were missing E Clustalx 1 81 File Edit Alignment Trees Colors Quality Help Multiple Alignment Mode Font Size 10 FEE IE DE DE DE E E DEDE DE IE E EIE IEEE IE DEDE DE IE IE IE IE IE E MEIE IE JEDE E ME IE IE DE IE IE ME IE IE IE IE E ME AE AE IE IE MEMEA AEE HLA_HLA00490 HLA_HLA00493 CLUSTAL Alignment file created 13 Navigate to the file that was created The extension on this file is gde 3 Edit Characters To edit characters in the gde file from ClustalX using a text editor in the gde File 1 Launch a text editor such as Microsoft Word 2 Open the gde file that you just created 3 Search for all and replace with gt 4 Save this file as a text document with the file extension txt or fsta Note The format of the sequences in this document is commonly referred to as a multi aligned fasta format You can now use this file to create a library in SeqScape software F 6 ABI PRISM SeqScape Software v2 1 User Guide Method 2 Create a Multi Aligned FASTA File Using ClustalX Software 4 Create
111. Hide Electropherogram C Analysis in Assembly view Display in Assembly view ey All Electropherograms Full View Zoom In J C Zoom Out Show Hide Consensus QV in Project view C 2 Show Hide Sample QV Continued on next page Figure 2 5 Main SeqScape Window Menus 2 26 ABI PRISM SeqScape Software v2 1 User Guide Menus on the Main SeqScape Window Continued from previous page Main Menus Submenus Tools New Specimen Set Clear Range Add Genotype Genotype List Add Variant SeqScape Manager Options Change Password Analysis Analyze Sample Manager Report Manager Apply Project Template RDG Properties Analysis Defaults Analysis Settings Display Settings C Window Tile Cascade All open files C Help SeqScape Help Amino Aid Codes IUPAC Codes IUPAC Diagram IUPAC Hetero Diagram Genetic Codes Complement Codes Quality Values Chart My Science About SeqScape DPPDPDPD PYF Po FY NANI NA NUN NA NN NA ND NA YOY NU NA GUY VY ABI PRISM SeqScape Software v2 1 User Guide 2 27 Chapter 2 Getting Started Workflow A typical workflow using SeqScape software is shown below
112. ISM SeqScape Software v2 1 User Guide Appendix B Frequently Asked Questions Table B 4 General Questions and Answers continued Question Answer Can BLAST against a database To search a database using a sequence generated with SeqScape software in the Project view export the NT alignment as an aligned FASTA file by selecting File gt Export Open this file in a text viewer then cut and paste the sequence you would like to search for in your BLAST query Refer to Chapter 9 Exporting and Printing Data and Reports for detailed information on exporting Alignment What alignment algorithms are used in SeqScape software The sample assembly and specimen alignments are generated using a Smith Waterman local sequence alignment algorithm using parameters appropriate for DNA sequencing Alignment Can SeqScape software perform just the alignment for samples Yes In order to assemble and analyze sequences without basecalling open the Analysis Defaults for the project and select the Specimen tab Deselect the Basecall Samples check box B 8 ABI PRISM SeqScape Software v2 1 User Guide SeqScape Manager FAQs SeqScape Manager FAQs Table B 5 SeqScape Manager Questions and Answers Question Answer What is the SeqScape Manager SeqScape Manager allows you to import export create and delete projects project templates reference data groups analysis defaults libraries a
113. In the Specimen name delimiter field enter the delimiter text Note The delimiter text is derived from the sample ID name in the data collection software sample sheet or plate record In the figure in step 3 the delimiter is a dash The sample ID name from the data collection software appears in the Sample name section of the Annotation view of the sample 3 Select the folder containing the samples to be imported then click Auto Add Import Samples E x Samples To Add Files Add Sample gt gt Uu AppliedBi t U a2 pRO aS OO Remove Sample U a3 H E SeqScape B C SegScape v2 1 0ac3 m C data v2 0b7 EC data v2 0fc3 amp C DataStore New Specimen Bl Logging Ez Reports Delete Specimen B C data_v2 1 0ac3 EC DataStore BO Auto Add gt gt D H I analysisprotocols C applicationConfiguration C displaysettings the string between these libraries delimiters in the sample s E CJ projects LEN Automatically create Specimen name based on Start End Use sample file name NE a A1 3F_01 ab1 I Show ab1 Samples File Only C Use sample name Based on the text delimiter the samples are automatically imported into the appropriate specimens in this example the specimens are shown under HLA C specimens ABI Prism SeqScape Software v2 1 User Guide 6 13 Chapter 6 Creating and Analyzing a Project Adding Specimens and Importing Samples Manually 6 14 4
114. More information can be found at http www chem qmw ac uk iubmb misc naseq html 300 KB basecaller A new algorithm that calculates mixed or pure bases and determines sample quality values layout view Shows the layout of the sample assembly with arrows indicating the placement and orientation of samples library match The name of one allele or the combination of two alleles depending on the library type that agree closely with the specimen consensus sequence ABI PRISM SeqScape Software v2 1 User Guide Glossary 3 nibbler The algorithm that sets the clear range for each sample using the clear range settings specified in the analysis settings polymorphic position A position in the library alignment that differs for some alleles in the library See constant position project A group of related sequences that share the same reference or for which there is no explicit reference project summary sequence A summary of the alignment of the specimen consensi project template Contains an RDG analysis defaults display settings and output settings quality values Measure of certainty of the basecalling and consensus calling algorithms Higher values correspond to lower chance of algorithm error Sample quality values refer to the per base quality values for a sample and consensus quality values are per consensus quality values reference A nucleotide string that has
115. Printing Data and Reports 0 0 0 ce eee eee eee 9 11 ABI PRISM SeqScape Software v2 1 User Guide 9 1 Chapter 9 Exporting and Printing Data and Reports 9 2 Chapter 3 1 Create Analysis Defaults and Display Settings Chapter 4 2 Create Reference Data Group Chapter 5 Add Reference Sequence Create ROIs Create Layers Linka Library ROls 3 Create Project Template Select Chapter 6 RDG Analysis Defaults Display Settings Add nucleotide variants Add amino acid variants Setup variant styles 4 Create and Analyze Project Chapter 7 Select Project Template Add Specimens Import Sample Files Software does automatically Basecalling Filter Assembly Consensus Alignment Comparison Search Library 5 View Data and Reports Chapter 8 Data Project View Specimen View Segment View Sample View 6 Reanalyze and Edit Data Chapter 9 7 Export and Print Figure 9 1 Reports Aligned projects Consensus sequence Aligned sample sequence Sample files ABI PRISM SeqScape Software v2 1 User Guide Report Manager Analysis QC Report Mutations Report AA Variants Report Specimen Statistics Report Sequence Confirmation Report Base Frequency report Library Search Report RDG Report Audit Trail Report Genotyping Report
116. RMS OF THIS LIMITED WARRANTY STATEMENT OR TO BIND APPLIED BIOSYSTEMS TO ANY AFFIRMATION REPRESENTATION OR WARRANTY CONCERNING THE PRODUCT THAT IS NOT CONTAINED IN THIS LIMITED WARRANTY STATEMENT AND ANY SUCH MODIFICATION AFFIRMATION REPRESENTATION OR WARRANTY MADE BY ANY AGENT EMPLOYEE OR REPRESENTATIVE OF APPLIED BIOSYSTEMS WILL NOT BE BINDING ON APPLIED BIOSYSTEMS UNLESS IN A WRITING SIGNED BY AN EXECUTIVE OFFICER OF APPLIED BIOSYSTEMS THIS WARRANTY IS LIMITED TO THE BUYER OF THE PRODUCT FROM APPLIED BIOSYSTEMS AND IS NOT TRANSFERABLE ABI PRISM SeqScape Software v2 1 User Guide G 3 Appendix G Software Warranty Some countries or jurisdictions limit the scope of or preclude limitations or exclusion of warranties of liability such as liability for gross negligence or willful misconduct or of remedies or damages as or to the extent set forth above In such countries and jurisdictions the limitation or exclusion of warranties liability remedies or damages set forth above shall apply to the fullest extent permitted by law and shall not apply to the extent prohibited by law G 4 ABI PRISM SeqScape Software v2 1 User Guide Glossary ABI basecaller An algorithm used in earlier versions of ABI PRISM DNA Sequencing Analysis and ABI PRISM SeqScape Software administration The functions of SeqScape software relating to installing removing or updating the application aligned allele library A co
117. ROIStar ROI Length Translation Color ROI pane 4r250557 AF250557 1 792 792 v 7414348 emb AJ277 gil7414348 embiAs 1 276 276 2 AF250557 1 270 270 2 AF250557 792 792 2 AF250557 r Reference Sequence S 57 equence X gi 7414348 emb AJ277102 1 HS pane 281 321 Add Ref Segment Paste Ref Segment Figure 4 2 ROI Tab in the RDG Properties Dialog Box ABI Prism SeqScape Software v2 1 User Guide Defining Regions of Interest ROI Defining Regions of Interest ROI Defining an ROI Each reference segment has its own locked ROI Identify the ROIs you want to define on a piece of paper then use the information to define ROIs in the software However if you are using a GenBank file the ROIs or features will already be defined You can add additional ROIs where appropriate to your analysis To define an ROI 1 In the ROI tab select an empty layer or a layer where you want the ROI to appear Select a segment in the nucleotide sequence pane by dragging through the region of interest then click Add ROI Enter a name for the ROI under the ROI Name column in the ROI pane Define as many ROIs as appropriate by dragging through the regions of interest or by entering a number in the text box where the ROI should begin Pasting a You can create or enter a sequence in a text editor or word processing Reference program and copy the segment into the RDG at a later time Segment To define a reference segment by pasting
118. Reference Break in a Sequence on page 4 27 The wizard continues the instructions to add a new layer and regions of interest ROI to that layer An ROI is a region on a reference segment that defines exons introns splice junctions and other features ABI Prism SeqScape Software v2 1 User Guide 4 9 Chapter 4 Creating a Reference Data Group Setting Up the To set up the reference segment Reference Segment 4 10 1 Select the bases in the region of interest that you want to compare with the reference sequence or backbone In the Reference Segment pane drag through the bases you want to select or type the starting and ending bases under the Find ROI label New RDG Wizard 3 x Create ROIs Create Regions of Interest ROIs for Referense Segment AF250557 1 of 1 ROls for Reference Segment AF250857 ROI Name Seg Start Seg End ROlStat ROI Length Translation Color AF250557 1 792 1 792 2 NENNEN 2 HLAC exon2 1 270 1 270 2 Eas 3 HLAC_gene 1 792 1 792 2 4 HLAC intron2 271 516 1 246 r ue 5 HLAC exon3 517 792 1 276 v unn 1 rt Reference Segment AF250557 1 gctcccactc catgaggtat ttctacaccg ccgtgtceceg 40 Find ROI 41 GCccggccgc ggagagcccc gcttcatcgc agtgggctac g0 Starting with 81 Gtggacgaca cgcagttcgt gcagttcgac agegacgecy 120 121 cgagtccaag aggggagccg cdggcgccgt gugtggagca 160 Find 161 Ggaggggccg gagtattggg accgggagac acagaagtac 200 Enana
119. Sequencing Chemistry Capillary Array Length cm Basecaller DyeSet Primer ABI Basecalling ABI PRISM BigDye Primer v1 0 and v1 1 36 rapid read Basecaller 3100POP6RRv2 bcp 50 std read Basecaller 3100POP6SR bcp DP3100POP6 BD 21M13 v1 mob DP3100POP6 BD M13Rev v1 mob ABI PRISM BigDye Primer v3 0 and 3 1 36 rapid read Basecaller 3100POP6RRv2 bcp 50 std read Basecaller 3100POP6SR bcp DP3100POP6 BDv3 21M13 v1 mob DP3100POP6 BDv3 M13Rev v1 mob ABI PRISM BigDye v3 Primer All primers 36 ultra rapid Basecaller 3100POP4UR bcp 80 long read Basecaller 3100POP4_80cmv3 bcp DP3100POP4 BDv3 v1 mob SO I4 Jeuiug 1esge Kq pue siej eoeseg v xipueddy QPIND Jas L zA e4e jog edeogbeg NSl d IJY EL V ABI PRISM 3100 Avant Genetic Analyzer Files Table A 8 3100 Avant Basecaller and DyeSet Primer Files Used for Dye Terminator Chemistry DNA Sequencing Chemistry Capillary Array Length cm Basecaller DyeSet Primer KB Basecalling ABI PRISM BigDye Terminator v1 0 and v1 1 36 ultra rapid 50 std read 80 long read KB bcp KB_3100_POP4_BDTv1 mob 36 rapid read 50 std read KB bcp KB_3100_POP6_BDTv1 mob ABI PRISM BigDye Terminator v3 0 and v3 1 36 ultra rapid 50 std read 80 long read KB bcp KB_3100_POP4_BDTv3_ mob 36 rapid read 50 std read KB bcp
120. Tab jumps tone WE HLa c 3100 Acids Project Navigator LA C 3100 m EjHL amp C 3100 Known Variants translation LAC COS Rol of the EATLEFSWALCF PAEITLIWQFDGEDQTQDIELVETRPAGDGT nucleotide ae sequence EATLRSUALGFYPAEITLTUQFDGEDQTQD ELVETRPAGDGT WGC Serine Cysteine Note Holding the pointer over an amino acid displays the possible translations and the codon at that position Note Bold red characters default indicate the location of a degenerate codon ABI Prism SeqScape Software v2 1 User Guide 7 5 Chapter 7 Viewing the Results Table 7 1 Project Views continued View Procedure Display Dots Click Ei Has Ey BBL Aciveleyer HLA C CDS Tab jumps to ne RHL4 c 3100 Characters Dots Project Navigator HLA C 3100 Ei S HLA C 3100 Known Variants II Variants Summary TTCWTCKCAGTGGGCTACGTGGACGACACGCAGTTCGTSCGGT INTVariants J CI TITEL CD E IET nin Index 70 80 90 100 Reference ptcategcegtyggctacgtygacgacacucagttcgtgcagt Reference AA Fk 3 G6 o_o b 360 2 W 3 6 Note The characters that are identical to the reference are displayed as dots Consensus 1 Click m QV ef ess Em mm mp mm m fuma FY MYL Active LayerfHLA C_CDS p Tab jumps tone W HLa c 31 00 Project Navigator HLA C 3100 2 Click Wil s jHL amp c 3100 PMknown Variants HLA C CDS ROls All Variants TTGTGGAGACCAGGC
121. There are two segment views Layout view Displays a schematic of the location and orientation of the samples with respect to the consensus segment and the reference Assembly view Displays the nucleotide sequence of the consensus and samples sample electropherogram data and view position Note The view position in the Assembly view blue box is represented by red lines in the Layout view Click the Layout view to navigate to a desired position in the Assembly view Table 7 2 describes the multiple Assembly view types To display segment views 1 Open the project of interest 2 Selecta layer in the Active Layer drop down list 3 In the navigation pane open a specimen then select a segment 4 Use the procedures in Table 7 2 to display the segment views of interest Table 7 2 Segment Views View Procedure Display Layout pem e meme uu Ej eg wp ep veeu Active LeyerfHLAC_CDS Tab jumps to next LA C 3100 360 2 AF250557 E E HLA C 3100 P Layout Assembly UJ 360 2 CR Lengj CR Start CI 309 29 337 482 72 311 gie 705 1577 227 327 24cX2F 01 2 CX2R 02 360 2 CX3R 02 360 2 CX3F 0l ABI Prism SeqScape Software v2 1 User Guide 7 11 Chapter 7 Viewing the Results Table 7 2 Segment Views continued
122. a new project using the project template created 6 Add the files obtained in step 2 and analyze 7 Select File gt Export 8 Select Project Alignment Nucleotides You can now use this exported file to create a library in SeqScape software F 2 ABI PRISM SeqScape Software v2 1 User Guide Method 1 Create a Multi Aligned FASTA File Using SeqScape Software 2 Create Your To create your library in SeqScape software Library in 1 SeqScape Software Launch SeqScape software Select Tools gt SeqScape Manager Select the Library tab and click New 2 3 4 Enter a library name 5 Select the Entries tab and click Import 6 Select Display All File Types then navigate to and open the multi aligned FASTA file The software creates the library and displays the number of sequences added ABI PRISM SeqScape Software v2 1 User Guide F 3 Appendix F Creating a Multi Aligned FASTA File Method 2 Create a Multi Aligned FASTA File Using ClustalX Software 1 Obtain ClustalX To obtain ClustalX software Software 1 Launch an internet browser and search for ClustalX or go to http inn prot weizmann ac il software 2 Select ClustalX software from the list of software products available 3 Select the ClustalX software for your operating system 4 Download the software to your computer 2 Create a Multi To create a multi aligned FASTA file using ClustalX software Aligned FASTA File Note You can tr
123. a project template that is already registered in SeqScape software You cannot create a project template in the plate editor Before continuing we recommend that you Create the required data objects in SeqScape software Create an analysis protocol An analysis protocol can be created in SeqScape or data collection software Create the analysis defaults and link them to an analysis protocol Create a Reference Data Group RDG based on your samples Customize the display settings or use the default settings Create a project template and associate the objects you just created to it Create a new project associated with the project template you just created ABI PRISM SeqScape Software v2 1 User Guide Launching Autoanalysis Manger Launching Autoanalysis Manger Launch the Autoanalysis manager before continuing To Launch the Autoanalysis Manager 1 Select Start gt Applied Biosystems gt Autoanalysis Manager gt Autoanalysis Manager 2 0 14 Autoanalysis Manager 2 0 E inl xl File Edit Help i seascape 2 1 r Job Queue Analysis Order Application 3 of Samples Arrival Date Completed Date Status Message Delete Completed Jobs Move Job Up Move Job Down IMPORTANT Do not run other applications when the Autoanalysis manager and the data collection software are running Never open a project while it is in use by the Autoanalysis manager ABI PRISM SeqScap
124. ab are inactive for all other users ABI PRISM SeqScape Software v2 1 User Guide 2 13 Chapter 2 Getting Started To set up new users 1 Select Tools gt Options to open the Options dialog box 2 In the Options dialog box select the Users tab then click New xi General Database Users Authentication amp Audit User Admin 05 Sep 2002 at 09 28 11 PDT UserAdmin User Admin Eom 11 Sep 2002 at 14 29 45 PDT I pues sien Deaut Ural x Scientist Scientist User E AdminUser Admin User User Name D First Name o o n Last Name 1 Password Fl Password must be 6 to 15 characters lon User Group Scientist z I Inactive 3 Fill in the appropriate user name password first and last names then select the level of user from the Group drop down list Note Enter a User Name that contains only alphanumeric characters This field must not contain any spaces or characters that do not conform with the Microsoft Windows OS file system Refer to File Naming Convention on page 2 10 The new user appears in the list in the Users tab 4 To set up users on multiple computers we recommend that the administrator do the following a Create the list of users b Export the file c Install SeqScape software on the other computers d Import the user file New users can log in after exiting and then restarting SeqScape software 2 14 ABI PRISM SeqScape So
125. acagaagtaataccacta acagaagaagcagagctagaactggcagaaaacagagagattctaaaagaaccagtacatgg agtgtattatga ABI PRISM SeqScape Software v2 1 User Guide E 3 Appendix E Aligned Variant and FASTA File Format E 4 IUB IUPAC Codes Supported Nucleic Acid Codes Sequences are expected to be represented in the standard IUB IUPAC amino acid and nucleic acid codes with the following exceptions Lower case letters are accepted and are mapped into uppercase n amino acid sequences U and asterisk are acceptable letters see below Note Although FASTA codes allow a hyphen or dash to represent a gap in nucleotide sequences this practice is not acceptable for using FASTA format in SeqScape software Before importing a sequence any numerical digits or spaces in the sequence need to be either removed or replaced by appropriate letter codes for example N for unknown nucleic acid residue or X for unknown amino acid residue Table E 1 Accepted Nucleic Acid Codes Character Codes for A Adenosine C Cytidine G Guanine T Thymidine U Uridine R GA purine Y TC pyrimidine K GT keto M AC amino S GC strong W AT weak B GTC D GAT ABI PRISM SeqScape Software v2 1 User Guide Supported Amino Acid Codes FASTA File Format Table E 1 Accepted Nucleic Acid Codes continued Character Cod
126. ach sample Data Display The sequence data is displayed using the following conventions Conventions Every mixed base or choice of mixed bases is represented as a single IUB code For more information see Appendix C Translation Tables Spaces in aligned sequences are displayed as dashes and are not part of the original sequence In the Dots view and in the collapsed NT view characters that are identical to the reference are displayed as dots The aligned reference sequence appears at the top of the table and the aligned sequences appear in the rows below in the Project view Quality Value The QV quality value is displayed as a bar above each called base Display for the sample sequence and consensus sequence The height of a bar corresponds to a 1 99 value that is determined by the analysis Note For QVs from 50 to 99 all QV bars are identical in height and color Note For more information on quality values see Chapter 10 Sample and Consensus Quality Values Exporting and To export the project data see Exporting Data Files on page 9 3 Printing Project and to print data see Printing Data and Reports on page 9 11 Data ABI PRISM SeqScape Software v2 1 User Guide 7 3 Chapter 7 Viewing the Results Project Views There are three project views only one of which can be displayed at a time Expanded NT Collapsed NT Expanded AA Displaying the To display project views Project Views 1 Open
127. ag the CR bracket to reset the Clear Range You can also select Tools Set Clear Range to reset the sample clear range What are Filter Settings These allow you to set the maximum percentage of mixed bases allowed maximum Ns allowed minimum clear range length and the minimum sample score for each sample Samples failing the filter checks will not be included in the analysis What are Display Settings These control the font styles and colors for bases electropherogram display and axis scale display view for variants and display views for nucleotide translation Can export consensus sequences Yes Select the desired consensus sequence in the Project Navigator then select File Export ABI PRISM SeqScape Software v2 1 User Guide Appendix B Frequently Asked Questions Library FAQs Table B 6 Library Questions and Answers Question Answer What are the requirements of the library The library search feature is designed for use with libraries of alleles genotypes or haplotypes where all the sequences in the library are variations of the same sequence This feature is not designed for searching against a library of diverse sequences The library must have the following characteristics e All library sequences must be pre aligned and equal in length Alllibrary sequences must be variations of the same sequence variations must be less than 509 Alllibrary sequences must c
128. alues 0 0 cc eee eee eee ee 10 6 Editing Bases with Quality Values 0 10 10 Cumulative Quality Value Scoring in Reports 10 11 ABI PRISM SeqScape Software v2 1 User Guide 10 1 Chapter 10 Sample and Consensus Quality Values Types of Quality Values QVs Table 10 1 summarizes the types of QVs and where they are displayed Table 10 1 Quality Value Types Quality Value Type Definition Location Sample QV A per base estimate of basecaller accuracy e Sample view Specimen view Project view Sample Score The average quality value of the bases in the Specimen Statistics report clear range sequence for that sample Consensus QV A per base estimate of the accuracy of the e Specimen view consensus calling algorithm Project view Consensus Score The average quality value of the bases in the Analysis QC report consensus sequence for that specimen Specimen Statistics report Mutation QV A per base estimate of basecaller accuracy Mutations report QV for deletion Average of the quality values for the bases to Mutations report mutation the left and right of the deletion FGR Avg QV Average quality value of the consensus Genotyping report sequence across the target region 5 Reg Coverage Average quality value of the consensus Genotyping report Avg QV sequence at the 5 region Exon Avg QV Average quality value of the consensus Genotyping report sequence
129. ame Function Delete Job Deletes a pending job Delete Completed Jobs Deletes a completed job Move Job Up Moves a pending job higher in the queue Move Job Down Moves a pending job lower in the queue ABI Prism SeqScape Software v2 1 User Guide 11 25 Chapter 11 Automating Analysis SeqScape 2 1 The SeqScape 2 1 tab shows the jobs project and status information Tab lolx File Edit Help General is SeqScape Job Queue Status Message Details Resubmit Edit Properties Stop Processing Auto delete Jobs Delete Figure 11 6 Autoanalysis Manager SeqScape 2 1 tab Table 11 3 describes the functions of the command buttons in the SeqScape 2 1 tab Table 11 3 SeqScape 2 1 Tab Command Buttons Button Name Function Details Displays the project in the navigation pane Details for Job Run_SSIntegration_2002 08 23_16 59_1 SeqScape Results Gri HLA C 3100 amp D 360 2 B seq_005_F01_10301 Blseq 007 E01 10301 Bl seq 009 D01 10301 Bl sea 011 C01 10301 Bl seq 013 B01 10301 Bl seq 015 A01 10301 5 D 360 4 B seq_001_H01_10301 Bl seq_003_G01_10301 B seq_010_D02_10301 Resubmit If a job fails resubmit for analysis Edit Properties Edits the name and password active only if analysis failed Stop Processing Interrupts the processing of the current job this The job will be marked as failed and the auto analysis manager will proce
130. ample ID also appears in the Annotation view of the sample as indicated in Figure 6 3 Bat 3F_01 E amp gi 7414348 emb AJ23 Start Run Stop Run Collection Started Collection Stopped Dyeset Primer Lot number Expiration date Instrument name rate in Hz Channels Ave Basecaller Module file name Ave Signal Intensity 1 24 2001 12 15 59 42 1 24 2001 14 46 30 42 1 24 2001 12 15 59 42 1 24 2001 14 46 30 0 DT3LOOPOP6 BD 2 mob 0010008 2001 05 01 12 00 00 000a o0 40 SSTEST 1 7857142857142858 Collection version 1 0 Data Analysis Base Call Start 946 Base Call End 6140 Peak 1 Location 946 G 571 A 326 T 303 C 242 1 Basecaller 3100POP6SR bcp Basecaller Version BC 1 5 1b 3 Base spacing used 14 839999 Length to Detector 50 Tube Position Al StdSeq50_POP6DefaultModule Figure 6 3 Annotation Tab Specimen Sample name name Project Navigator HLA 3100 v2 A1 AF250557 A1 2F_01 Annotation Sequence Features lectropherogram Raw i U nassemble Data Collection Sample G AF250557 Sample name ID Model 3100 Bai 2F 01 Bai 2R_02 Number of Scans 11557 name Ba1 3R_02 Length 347 ABI Prism SeqScape Software v2 1 User Guide Adding Specimens and Importing Data into a Project To create a specimen and import samples automatically 1 With the Project window open select File gt Import Samples To Project or click 2
131. and setup are complete new users can log in to Log In the software To log in to SeqScape software 1 Start the software by double clicking the desktop shortcut E 2 The Log In dialog box opens showing the last user s name Enter your user name and password then click OK IMPORTANT If you have forgotten your user name or password contact your administrator x UserName Scientist Password pue o Note User Name and Password are case sensitive OK Exit SeqScape software is ready to use Note All existing users of an earlier version of SeqScape software will have Analyst privileges Only a user belonging to the Administrator group can change the user to Scientist or Analyst Users who existed in previous versions will be asked to set up their user profiles name and password when they try to use SeqScape Software v2 1 for the first time ABI PRISM SeqScape Software v2 1 User Guide 2 21 Chapter 2 Getting Started SeqScape Software Structure SeqScape software is organized around two main windows SeqScape Manager window from which you enter and manage the information necessary to perform analyses Project window from which you manage the results of analyses ESSISSNASSISORSESMS SINIT r Project Window Projects r Report Manager r Sample Manager
132. ank file containing Reference Data Group data New Project Wizard 1 Select Reference Data Group Select the Reference Data Group you want to use for the new project Created Created By Modified ModifiedBy 05 12 02 at4 14 2 0 at guest Ap HLA C ex2 4 with 12 01 87 at 2 59 4 N A 12 01 97 at 2 59 4 guest Application HXB2PrRT 09 22 00 at 11 59 N A 11 30 97 at 6 48 3 guest Application HXB2PrRT_v1 1 09 22 00 at 11 59 N A 11 30 97 at 6 48 3 guest Application lt lt Back Next gt gt Cancel Help Do one of the following Select Use a GenBank file containing Reference Data Group data then specify a GenBank file Or Select Use an existing Reference Data Group then select a Reference Data Group file in the list ABI Prism SeqScape Software v2 1 User Guide Click Next 6 7 Chapter 6 Creating and Analyzing a Project 9 Add specimens and import samples in the Create Specimens page New Project Wizard Create Specimens Create new specimens and import samples into specimens Files 3 ExportedReports gt HLAC_TutorialData_old G ProcessedData ai Run BoraBora 2002 07 10 14 39 C Run KONA 2002 07 10 14 14 1 B Seq data SegA5 0 Seqa5 1 Seqd_4331940B B SeqScape t AppSeqScape Ei Tutorial Data EM HLA C f HLA C 3100POPB Tutoi cma EM A2 a3 HLA C_Exon24_FASTA X HLA
133. ant to a Known Variant 1 Select a variant base in a specimen then right click it ABI PRISM SeqScape Software v2 1 User Guide 6 25 Chapter 6 Creating and Analyzing a Project 2 Select Add Variant from the shortcut menu to open the New NT Variant dialog box The information regarding the type and position of the variant appears in the New NT Variant dialog box New NT Variant E x Type ROI aF250557 Position bp NEEDS To i Reference base s 6 Variant base s NENNEN Style Yellow Description NENNEN M Used by all ROIs Create Another OK Cancel 3 Select a variant style from the Style drop down list 4 In the Description field enter text if desired then click OK 5 Repeat steps 1 through 4 for another variant 6 26 ABI Prism SeqScape Software v2 1 User Guide Changing Multiple Unknown Variants ABI PRISM SeqScape Software v2 1 User Guide Incorporating Variants into the Project RDG To change multiple unknown variants to known variants you need to export unknown variants in a project alignment file and then import them into the project To 1 3 change multiple unknown variants to known variants Open the project and select the specimen containing the unknown variants Select File gt Export gt Project Alignment Nucleotides Look in C HLA C specimens File name JHLA 3100_threeSpecimens_v2_NtAlignmentista Export Fi
134. ape properly installed Is SeqScape registered and have user IDs been created Have you created the required data objects a project template and a new project in SeqScape software Open Autoanalysis Manager Vv Set up instrument and prepare samples Y Do you have an Instrument Protocol No Analysis Protocol and Results Group Yes 4 wv Create and save plate record Reinstall see Chapter 2 Register software and create user IDs 1 Create data objects Analysis protocol Analysis defaults RDG Display settings optional 2 Create a project template 3 Create a new project You can create analysis protocols in either SeqScape or Data Collection software v Create Instrument Protocol Create Analysis Protocol Create Results Group Search for plate record in the Run Scheduler Vv Start run Figure 11 2 Autoanalysis Workflow ABI PRISM SeqScape Software v2 1 User Guide Before You Start 11 5 Chapter 11 Automating Analysis Required Files Created in SeqScape Software During autoanalysis setup in the data collection software you will be required to select data objects that have already been created For example if you choose to create a new project in the plate editor you can select only
135. ape software SeqScape software is a resequencing software tool designed to identify nucleotide variants amino acid variants and sequences in a library that match each consensus sequence SeqScape software can be SeqScape software can be used for SNP discovery and validation used for which sequencing mutation analysis and heterozygote identification sequence application confirmation for mutagenesis or clone construct confirmation studies and the identification of genotype allele and haplotype from a library of known sequences What is the overall workflow All analysis in SeqScape software occurs in a project Analysis and for analyzing and reviewing review of the data occur in six steps as follows parat 1 Create an analysis defaults and display settings or use one previously created 2 Create an RDG or use one previously created 3 Create a project template or use one previously created 4 Create and analyze a project by adding sample files to a project template 5 Review the results view edit the data and view the reports 6 Export print the results and reports What happens in the Each time you click the Analyze button the following pipeline is SeqScape software when applied to specimens that have not been analyzed click E Analyze 1 Basecall and determine Quality Values 2 Identify mixed bases This step may be bypassed if data was previously basecalled Trim low quality sequence ends F
136. ar AGGGCACGTG CGTGGAGTGG CTCCGCAGAT ACCTGGAGAA CGGGAAGGAG Bot ACGCTGCAGC GCGCGGGTAC CAGGGGCAGT GGGGAGCCTT CCCCATCTCC gil74143 YGTAGATCTC CCGGSATGGC CTCCCACGAG GAGGGGAGGA AAATGGGATC Ua AGCGCTAGAA TATCGCCCTC CCTTGAATGG AGAATGGGAT GAGTTTTCCT Uy a3 GAGTTTCCTC TGAGGGCCCC CTCTGCTCTC TAGGACAATT AAGGGATGAA GTCCTTGAGG AAATGGAGGG GAAGACAGTC CCTGGAATAC TGATCAGGGG TCCCCTITGA CCACTTTGAC CACTGCAGCA GCTGTGGTCA GGCTGCTGAC CTTTCTCTCA GGCCTTGTTC TCTGCCTCAC GCTCAATGTG TTTGAAGGTT TGATTCCAGC TTTTCTGAGT CCTTCGGCCT CCACTCAGGT CAGGACCAGA AGTCGCTGTT CCTCCCTCAG AGACTAGAAC TTTCCAATGA ATAGGAGATT ATCCCAGGTG CCTGTGTCCA GGCTGGCGTC TGGGTTCTGT BCCCCCTTCC CCACCCCRGG TGTCCTGTCC ATTCTCAGGA TGGTCMCATG GGCGCTGTTG GAGTGTCCCA AGAGAR ESETNENRENS IHLA 3100 threeSpecimens v2 A1 AF250557 A1 3F 01 Ei HLA 3100 threeSpeci Annotation 1S Features Electropherogram Raw gt Unassembled Iu a8 ETI i d I B AF250557 1 ACTOGGGOCG GGcCaGGGTC TCACACCCTC CAGWGGATGT avEECTECGA 50 A1 3F 01 LE TTE LETT BETTE LLTETT Ba 3R_02 51 CCTGGGGCCC GACGGGCGCC TCCTCCGCGG GTATGACCAG TMCGCCTACG 100 A1 2R 02 Blair 01 minm n u INIT e T 101 ACGGCAAGGA TTACATCGCC CTGAACGAGG ACCTGCGCTC CTGGACCGCY 150 gi 7414348 em Taz minim M IN m a3 151 GCGGACACSG CGGCTCAGAT CACCCAGCGC AaGTGGGAGG CGGCCCGTGA 200 miii u M t IN 1 ul al 201 GGCGGAGCAG YGGAGAGCCT ACCTGGAGGG CACGTGCGTG GAGTGGCTCC 250 UU pn Hin 251 GCAGATACCT GGAGAACGGG AAGGAGACGC TGCAGCGCGC GGGTACCAGG 300 ma on Features sa um B gEI B poet E
137. are installation CD ABI PRISM SeqScape Software v2 1 User Guide Integrating SeqScape and Data Collection Software IMPORTANT The data collection software must be running when installing SeqScape software on a computer that is connected to a 3730 3730xl DNA Analyzer or 3100 3100 Avant Genetic Analyzer If data collection software is not running SeqScape software does not register with the Data Service See Chapter 2 Getting Started for information on properly installing the software IMPORTANT After the initial installation of SeqScape software you must open Autoanalysis Manager software Autoanalysis Manager Open Open Nonuser Interface SeqScape Closes amp Reports Status Analysis Protocols Software Relationships Data Collection Run Complete Message Service Data Service Analysis Protocols Projects Specimens Projects Project Template Specimens Results Group Instrument Protocols v Project Template Litt Figure 11 1 Relationships Between Seqscape and Data Collection Software ABI PRISM SeqScape Software v2 1 User Guide Chapter 11 Automating Analysis Before You Start Successful automatic analysis requires that SeqScape software is installed properly SeqScape software is registered and the appropriate user IDs
138. ars in the General tab 3 The Autoanalysis Manager polls for jobs every 2 minutes and opens the automated processing SeqScape version to analyze the data in the projects 4 At the end of analysis the status in the Autoanalysis Manager is updated IMPORTANT Do not run other applications when the Autoanalysis manager and the data collection software are running Never open a project while it is in use by the Autoanalysis manager Files Created The data collection software stores the sample files in the location specified in the results group The Autoanalysis Manager copies the files into the DataStore for SeqScape software processing To maintain sufficient storage space on your hard drive delete the sample files created by data collection software that are no longer needed Components The Autoanalysis Manager has two or more tabs General SeqScape 2 1 GeneMapper 3 5 if loaded 11 24 ABI PRISM SeqScape Software v2 1 User Guide Understanding the Autoanalysis Manager General Tab The General tab shows the jobs that have been submitted and their status se Autoanalysis Manager 2 0 i m ni x File Edit Help Analysis Order Application of Samples Arrival Date Completed Date Status Message Delete Completed Jobs Figure 11 5 Autoanalysis Manager General Tab Table 11 2 describes the functions of the command buttons in the General tab Table 11 2 General Tab Command Buttons Button N
139. asecaller settings refer to the ABI PRISM DNA Sequencing Analysis Software User Guide ABI Prism SeqScape Software v2 1 User Guide 3 5 Chapter 3 Creating Analysis Defaults and Display Settings 9 10 For KB basecaller only select to display the data as either a true or flat profile in the Processed Data area True Profile Displays data as processed traces scaled uniformly so that the average height of peaks in the region of strongest signal is about equal to a fixed value The profile of the processed traces will be very similar to that of the raw traces Flat Profile Displays data as processed traces scaled semi locally so that the average height of peaks in any region is about equal to fixed value The profile of the processed traces will be flat on an intermediate scale gt about 40 bases For KB basecaller only set the quality threshold Call all bases and assign QV Use this setting to assign a base to every position as well as the QV Assign N for bases with QV Use this setting to assign Ns to bases with QVs less than the set point The QV will be grayed out IMPORTANT Applied Biosystems recommends that you use the KB basecaller to perform your analysis The KB basecaller was introduced with SeqScape software v2 0 and Sequencing Analysis software v5 0 We will continue to improve and develop this algorithm The ABI basecaller is an older algorithm that will be removed in future versions of
140. at the exon region Intron Avg QV Average quality value of the consensus Genotyping report sequence at the intron region 10 2 ABI PRISM SeqScape Software v2 1 User Guide Sample Quality Values Sample Quality Values Sample Quality A sample quality value SQV is a per base estimate of the basecaller Values accuracy There are two types of basecallers that generate SQVs KB A new algorithm that identifies mixed or pure bases and generates sample quality values e ABI Algorithm used in ABI PRISM Sequencing Analysis Software v3 7 that identifies pure bases Then the TraceTuner software identifies mixed bases and generates sample quality values KB and ABI algorithms can produce slightly different SQVs Interpreting the Per base SQVs are calibrated on a scale corresponding to Sample Quality V 101 P Values Bio Where Pe is the probability of error of the basecall The KB basecaller produces a QV range of 1 to 99 with 1 being low confidence and 99 being high confidence See Table 10 2 Quality Values and Probabilities of Error on page 10 4 for the probability of basecall errors for QVs ranging from 1 to 99 Mixed base calls yield lower SQVs than pure base calls The typical QV range for good data is 25 to 50 The size and color of QV bars for QV 50 to 99 are identical To view the actual QV number see Displaying the Quality Bars and Values on page 10 9 Sample Score A sample score i
141. ata Report Manager M Project View Analysis QC Report Specimen View Mutations Report Segment View AA Variants Report Sample View Specimen Statistics Chapter 8 Report Sequence Confirmation 6 Reanalyze and Edit Data Report Base Frequency report Library Search Report Chapter 9 RDG Report 7 Export and Print Reports ek Tra mopon Fo Aligned projects Se Gjananplng Raport Consensus sequence Aligned sample sequence Sample files Figure 5 1 Showing Create Project Template Step 5 2 ABI Prism SeqScape Software v2 1 User Guide Creating a Project Template Creating a Project Template Before you can effectively use the ABI PRISM SeqScape Software Version 2 1 you must create and configure a project template A project template contains all the reference data and settings needed to analyze your data automatically It defines how the software analyzes and displays your samples When project templates are created in the SeqScape Manager they can be imported exported and edited About Creating a When you create a new project template from the SeqScape Manager New Project you select Template A Reference Data Group Reference sequence and associated data to which all the specimens in a project are compared See Creating a New RDG Using SeqScape Manager on page 4 12 for more information Analysis defaults Settings that are used to analyze the data See Specifying
142. ation displayed in the report Data View 1 Right click any column heading of a table A list of the column headings in the table is displayed Specimens in Report A1 a2 a3 Specimen Analysis Specimen Basecalling Filter Assembly Specimen Total Comments Samples Score Variants v Specimen Brem EBEN A2 6 25 50 a v Filter A3 6 25 48 v Assembly v Specimen Score Comple V Total Variants A No output v Comments Sample Analysis Specimen Sample Step Description xf 2 To hide a column deselect the column heading 3 Repeat steps 1 and 2 to deselect additional headings 4 To redisplay a column right click any column heading then select the column heading 5 To sort the data A to Z or Z to A in a Sample Details or Errors table column double click the column heading Double click again to sort in the opposite direction 6 To customize the table header and footer information see Customizing Header and Footer Display on page 9 11 7 44 ABI PRISM SeqScape Software v2 1 User Guide Reanalyzing and Editing Data This chapter contains Workflow for This Chapter eese About Analysis Parameters 0 0 0 eee Changing the Analysis Parameters in the Sample Manager Changing the Analysis Parameters in an Analysis Protocol Editing the Data eae ee dto ainats etie Fe Editing a Sample or a Consensus Sequence Adjusting the Clear Range 0 0 0
143. ations Report f gt AA Variants Report Specimen Statistics Report 5 Sequence Confirmation Report Base Frequency Report I Library Search Report RDG Report i Audit Trail Report ping Report Report Settings Genotype Settings E EST Active LayerJHLA C_CDS z Tab jumps to next Active Layer Project Creation Date Project Template PT PT Modification Date RDG Creation Date Display Settings DS Specimens in Report Summary HLA C CDS 03 Dec 2002 at 23 40 04 PST HLA C_3100POP6_mixed_5li b 13 May 2003 at 16 02 45 PDT 03 Dec 2002 at 22 59 40 PST TutorialDisplaySettings Project Project Modification Date PT Creation Date Reference Data Group RDG RDG Modification Date DS Creation Date 13 May 2003 at 16 02 45 PDT 03 Dec 2002 at 23 29 08 PST HLA C Exon2 4 noNT 13 May 2003 at 16 02 45 PDT 26 Nov 2002 at 23 37 59 PST E A1 A2 A3 Gene Symbol Gene Part Number Gene Aliases Cytogenetic Band NCBI Gene Reference hCG Accession Number Layer oe ID Gene Summary Transc Assay Target Start Length Assay Target Gene Name Gene ID Chromosome Genomic Location Celera ID hCT Accession Numbers ript Table Regulatory Length lad Length ie Length No Data Speci FGR FGR men Cov Avg av ov Cov Resequencing Coverage 5 Reg 5 Reg 5 Reg 5 Reg Exon Cov Avg Fwd Rev Cov Avg Fwd Rev Cov Qv Cov Cov Exon Exon Exon Intron Intron I
144. aving 8 21 sources resequencing projects 1 6 when to edit 8 13 data collection software integration with SeqScape 11 2 more information 11 8 default directory setting up 2 20 defining an ROI 4 15 deleting alayer 4 16 anROI 4 16 reference segment 4 16 display settings defined Glossary 2 specifying 3 18 ABI PRISM SeqScape Software v2 1 User Guide displaying sample views 7 15 segment views 7 11 Dye Primer chemistry files A 12 A 16 Dye Terminator chemistry files A 5 A 13 DyeSet Primer files about parameter 8 7 defined Glossary 3 naming conventions A 2 DyeSet Primer files for specific instruments A 1 E editing analysis protocol 8 7 data 8 1 data workflow 8 2 existing users privileges 2 8 expanded display viewing 7 11 export defined Glossary 3 exporting about 6 34 all reports automatically 9 10 data file name and format options 9 3 from SeqScape Manager 6 34 project alignment 9 4 projects 9 4 report file name and format options 9 8 reports 9 9 samples 9 7 segments 9 6 specimens 9 5 extended reference data group 1 3 Extension Penalties described 3 12 Index 3 F FASTA codes E 4 creating a multi aligned FASTA file F 1 file formats E 2 to E 4 format description E 3 format defined Glossary 3 supported amino acid codes E 5 to E 6 supported nucleic acid codes E 4 text 4 34 features new 1 2 file sharing data collection and SeqScape 11 28 filter settings table of 3 11 Filter tab
145. cc ccggcccggg gcgcaggtca cgacccctcc ccatccccca ztgagatc Save To Manager As J OK Cancel 3 In the New NT Variant dialog box select the type of variant Base Change Insertion or Deletion ABI Prism SeqScape Software v2 1 User Guide 6 29 Chapter 6 Creating and Analyzing a Project 4 Select the Position and either To position or Variant base Note The Reference base is entered by the software based on the position x Type ROI AF250557 Position bp Bo To Reference base s bee y Variant base s OO Style Melow Description OO M Used by all ROIs Create Another OK Cancel 5 Select a style color with which you want the variant to be displayed then enter a description of the variant if desired 6 Click Create Another to add more variants or click OK to save the variant to the RDG 6 30 ABI Prism SeqScape Software v2 1 User Guide Incorporating Variants into the Project RDG Adding a Variant To add a variant in the project inthe Project 2 3 From the Project window select Analysis RDG Properties Select the ROI tab Indicate your variant by doing one of the following Select the base that corresponds to the substitution variant or range of bases for a deletion variant Click the position at which you want an insertion variant Select Add Variant RDG Properties x General ROI NT Variants AA Variants Variant Style 4
146. cing data generated from the following systems ABI PRISM 310 Genetic Analyzer ABI PRISM 377 DNA Sequencer ABI PRISM 3100 Avant Genetic Analyzer ABI PRISM 3100 Genetic Analyzer ABI PRISM 3700 DNA Analyzer Applied Biosystems 3730 DNA Analyzer Applied Biosystems 3730x DNA Analyzer Each project can contain Unanalyzed sample files ab1 Previously basecalled sample files ab1 Text sequences seq or fsta Aligned consensus sequences A single project can contain sample files from one or a mixture of instrument platforms The software analyzes the data displays several views of the analyzed project and reports on results for quality control and data review SeqScape software performs two levels of analysis It identifies variants positions that differ from the reference sequence and classifies those variants as known or unknown It searches a library of alleles or haplotypes to identify the alleles that most closely match the sample When you have added a reference sequence a library and sample files SeqScape software performs two levels of analysis Identification of nucleotide and amino acid variants The software identifies positions that differ from the reference sequence and classifies those variants as known or unknown variants Identification of genotypes alleles or haplotypes from a library In addition to identification of variants the software searches a library of genotypes alleles or haplot
147. cle means not assembled ABI PRISM SeqScape Software v2 1 User Guide 8 5 Chapter 8 Reanalyzing and Editing Data Changing the Analysis Parameters in the Sample Manager Adding Samples To add samples to the Sample Manager to the Sample 1 Open the project of interest Manager j dis 2 Selecta layer in the Active Layer drop down list 3 In the navigation pane To add all Select the Samples in a project Project icon Samples in a specimen Specimen Selected samples in a segment Segment Selected sample s Sample s Use the Shift key to select contiguous samples or use the Ctrl key to select noncontiguous samples 4 Select Analysis Sample Manager The selected files are displayed in the Sample Manager Changing Note Use the basecaller and DyeSet Primer tables in Appendix B to Basecaller and _ select the correct combination of files DyeSet Primer l Files TO change the basecaller and or DyeSet Primer file 1 In the Sample Manager select the sample you want to change 2 In the Basecaller drop down list select a new basecaller 3 In the DyeSet Primer drop down list select a new DyeSet Primer file To change multiple samples use the Fill Down function Click Apply Click OK Click 8 6 ABI PRISM SeqScape Software v2 1 User Guide aA mn R Changing the Analysis Parameters in an Analysis Protocol Changing the Analysis Parameters in an Analysis Pr
148. ct Alignment view Insert or delete a space in a Specimen consensus 49 Project Alignment view Insert delete Consensus bases 50 Project Navigator Rename Specimens 51 ROI tab Rename Segments in RDG 52 Specimen view Change a base ina sample 53 Specimen view Change basecalls in the consensus ABI PRISM SeqScape Software v2 1 User Guide D 7 Appendix D User Privileges Table D 3 Access for Admin Scientist and Analyst Levels continued Description of access for users of Admin Scientist and Analyst levels Admin Scientist Analyst Editing 54 Specimen view Change the Clear Range Allowed Allowed Allowed for sample data 55 Specimen view Insert or delete a base in a sample 56 Specimen view Insert or delete bases in consensus 57 Undo base edits SeqScape 58 Open the SeqScape Manager Allowed Allowed Allowed Manager 59 Save any SeqScape Manager Object Library 60 View the Libraries in the SeqScape Allowed Allowed Allowed Manager 61 View results of Library search in the Project Alignment View Analysis 62 View the Analysis Protocol Allowed Allowed Allowed Protocol and Settings 63 Change basecaller settings in an existing Sample within a Project 64 Reconfigure Analysis Defaults inside a Project 65 Configure Display Settings in Project or SeqScape Manager 66 Analyze data using the BGB without basecalling samples 67
149. ct the color in the Bar Code you want to change The Select a color dialog box opens color xj HSB RGB Ao ELLE rper VL es Preview a B Sample Text Sample Text E O a L1 Sample Text Sample Text Cancel Reset b Select a new color in the Swatches tab or use the HSB or RGB tabs to define a new color c Click OK The color dialog box closes 5 Do one of the following Click OK to save the changes to the samples you are working with Click Save to Manager As to save the changes to the SeqScape Manager 10 8 ABI PRISM SeqScape Software v2 1 User Guide Displaying Quality Values Displaying the If you do not see the QV bars when viewing samples or a consensus Quality Bars and in a project then follow the procedures below to display QV bars and Values values To view quality bars and values l 2 AF250 Open a project then open a specimen of interest Select the segment of interest then select the Assembly tab To view sample QVs select View gt Show Hide sample QV or click Bul To view consensus QVs select View gt Show Hide consensus QV or click uj To obtain a numerical value for a particular bar place the cursor over the bar for 2 sec The value is automatically displayed B amp gi 741 a as w qf eon E bb 558 jas 136 IAL UU 24 2 u8 sd n2 ICT eaz IFT 122 fon b 1a 151 161 17
150. cted positions in the reference 7 40 ABI PRISM SeqScape Software v2 1 User Guide Genotyping Report Summary Active Layer hCT1951704 Project pparg3700 Project Creation Date 04 Oct 2002 at 14 06 55 Project Modification Date 04 Oct 2002 at 14 41 14 PDT PDT Project Template PT PPARG 3700 PT Creation Date 04 Oct 2002 at 14 06 41 PDT PT Modification Date 04 Oct 2002 at 14 41 14 Reference Data Group RDG PPARG PDT Specimens in Report 4X0033 NA00131 NAD0333 NAO0546 NAD0607 NAD0893 NAD0946 NAD1805 NAD1814 NAN1953 NAO1954 NAD1990 NAO2254 NA05920 NAD8587 NA09947 NA10924 NA10959 NA12593 NA14439 NA14448 NA14454 NA149464 NA14474 NA14476 NA14480 NA14501 NA14503 NA14508 NA14511 NA14529 NA14532 NA14535 NA14548 NA14632 NA14649 NA14661 NA14663 NA14665 NA14672 Gene Summary Gene Symbol Gene Name Gene Part Number Gene ID Gene Aliases Chromosome Cytogenetic Band Genomic Location xl Transcript Table Amplicon ID Assay Target Assay Target 5 Regulatory Exon Length Intron Length Start Length Length Resequencing Coverage Spec FGR FGR FGR FGR 5 5 5 5 Exon Exon Exon Exon Intron Intron Intron Intron imen Cov Avg Fwd Rev Reg Reg Reg Reg Cov Avg Fwd Rev Cov Avg Fwd Rev Qv Cov Cov Cov Avg Fwd Rew Qv Cov Cov Qv Cov Cov Qv Cov Cov 4x00 100 0 402 10009896 00 OO OO OO 1000402 1000986 00 00 00 00 4 33 Report Settings NADO 743 39 0 743 662 00 00 00 00 743 390 743
151. customizing 7 42 customizing header footer 9 11 exporting 9 9 exporting all reports automatically 9 10 file name and format options 9 8 format options 9 8 Genotyping 7 39 Library Search 7 35 Mutations 7 28 10 12 new features 1 3 print preview 9 16 printing 9 16 RDG 7 37 Sequence Confirmation 7 33 Specimen Statistics 7 32 10 13 types 7 22 viewing 7 23 viewing the results 7 23 to 7 39 requirements hardware and software 2 3 resequencing applications common 1 5 data 1 5 Results Group Editor completing for autoanalysis 11 14 ROI defining 4 15 deleting 4 16 tab graphic 4 14 ROI pane columns described 4 19 descriptions 4 18 running an analysis 6 21 ABI PRISM SeqScape Software v2 1 User Guide S sample bases editing 8 14 data defined Glossary 4 editing 8 14 to 8 21 exporting 9 7 IDs 6 11 importing automatically 6 11 names 6 11 quality values defined Glossary 5 results viewing annotation results 7 15 score 10 3 score defined Glossary 5 view defined Glossary 5 Sample Manager defined Glossary 5 viewing analysis parameters 8 4 sample quality values explained 10 3 sample score 10 3 Save To Manager As button using 4 42 saving acopyofRDG 4 41 data 8 21 project template within project 5 5 RDG 4 41 template 5 5 scheduling a run 11 6 scientist privileges 4 12 D 2 segment defined Glossary 5 exporting 9 6 views displaying 7 11 SeqScape menus 2 26 typical workflow 2 28 SeqScape Manager creating new anal
152. d in sequencing analysis software Basecalling with ABI basecaller only is no longer available In SeqScape Software v1 1 you can choose to basecall data with ABI basecaller or ABI basecaller with TraceTuner Software In software v2 0 you do not have the option to basecall with ABI basecaller only The new options are Basecall with ABI and TraceTuner software automatic Basecall with KB basecaller Implicit Reference is no longer available In software v1 1 you can have an empty RDG and use the first specimen as your implicit reference sequence but this is no longer available in v2 0 However you can create an RDG and add an abl sample file as a reference sequence 1 4 ABI PRISM SeqScape Software v2 1 User Guide About SeqScape Software About SeqScape Software Genetic Analyzer Applications SeqScape Software Applications Resequencing Data with SeqScape Software SeqScape software is one of a suite of Applied Biosystems Genetic Analyzer software applications designed to control an instrument collect data and manage automated analysis This suite of data collection and analysis software systems includes TM GeneMapper Software Performs genotyping using fragment analysis methods ABI PRISM Sequencing Analysis Software Displays analyzes edits and prints sequencing files e ABI PRISM SeqScape Software Performs sequence comparisons for variants identification SNP discovery a
153. d layers containing all the ROIs and layers you previously defined on paper 5 You can edit the ROIs in the ROI pane by selecting the attributes then editing them directly in the table ABI Prism SeqScape Software v2 1 User Guide 4 25 Chapter 4 Creating a Reference Data Group 4 26 6 7 To include an existing ROI on an unlocked layer select or create the layer then select the On Layer check box for the ROI xi General ROT NT Variants AA Variants Variant Style Layer 1 4 bairz414348 embIAJ277102 11 2 HLA exon2 0 HAC exond 0 792 276 Al gt Layer 1 settings _NewLayer_ Layer Name Layer1 Index Codon Number 1 Orientation Libray zl iu Translation Frame fz Right ROI Name Segment Seg Start Seg End_ ROI Start_ ROI Length Translation Color AF250557 AF250557 1 792 792 2 amp gi T414348 emb AJ277e gi 7414348 emb AA 1 276 276 jHLA C exon2 AF250557 1 270 270 HLA C gene AF250557 1 792 792 HLA C_intron2 AF250557 271 4 5 Reference Sequence X gi 7414348 emb AJ277102 1 H8 Add Ref Segment Paste Ref Segment IMPORTANT If you want to add variants follow the procedure on page 4 29 Do not click OK If you do not want to add variants go to the next step When you finish adding ROIs and layers click OK The new RDG appears in the Reference Data Group list ABI Prism SeqScape Software v2 1 User Guide Creating New Layers Adding
154. d shortcuts for each button The top row of buttons Figure 2 3 are processing tools New Project Import Samples Sample Manager Ctrl N into Project Opens the Sample Creates a Ctrl M Manager window new project Analysis Settings Ctrl B Save Project Find Again Opens the Analysis Ctrl S Ctrl G Protocol window to Saves a new Finds a selected enter basecall mixed modified sequence again bases clear range project and filter settings for the selected sample Print Delete Ctrl P Deletes a selected MyScience object Links to www appliedbiosystems com ri E amp USB x SeqScape Manager Opens the SeqScape Manager window Open Project Ctrl O Opens an existing project New Specimen Opens a new specimen Find Ctrl F Finds a selected string IUB code in a sequence Undo Base Change Ctrl N Analyze Samples Ctrl R Figure 2 3 Main Toolbar 2 24 Report Manager Ctrl 1 Opens the Report Manager window RDG Properties Ctrl D Opens the RDG Properties window ABI PRISM SeqScape Software v2 1 User Guide Display Settings Ctrl Y Opens the Display Settings window for the project SeqScape Software Toolbar The second row of buttons Figure 2 4 are viewing options for the projects you create Expanded Nucleotide View Expanded Amino Acid View view Show Hide Electropherogram In Segment Assembly Zoom In Ctrl Zoom Out Ctrl
155. d text and FASTA file format can be imported into the software File import Can import multiple individual text files into one specimen rather than ab1 files Yes You can import seq format files or FASTA format files as sample files In order to see these files in the import dialog you need to deselect Show ab1 Samples File Only box These files can then be analyzed like normal files except no basecalling occurs File import Do my sample files need to be imported in the same reading frame as the reference sequence No the imported sample files do not need to be in the same reading frame as the reference sequence File format Can analyzed data be used in SeqScape software Analyzed data can be used However if it is in the ABI data format and not FASTA any prior analysis results and edits will be overwritten when the files are reanalyzed using SeqScape software ABI Prism SeqScape Software v2 1 User Guide SeqScape Manager FAQs Table B 5 SeqScape Manager Questions and Answers continued Question Answer File format What is FASTA format How can convert non FASTA files into the correct format A sequence in FASTA format begins with a single line description followed by lines of sequence data The description line is distinguished from the sequence data by a greater than gt symbol in the first column Note When creating a file in Microsoft Word be su
156. dd the Reference Segments that you wantto include in the Reference Data Group amp Reference Sequence al 1 gctcccactc catgaggtat ttctacaccg ccgtgtcccg 40 E x al gcccggccgc ggagagcccc gcttcatcgc agtgggctac 80 81 gtggacgaca cgcagttcgt gcagttcgac agcgacgccg 120 121 cgagtccaag aggggagccg cgdgcgecgt gugtggagca 160 161 ggaggggccg gagtattggg accgggagac acagaagtac 200 201 aagcgccagg cacagactga ccgagtgagc ctgcggaacc 240 24l tgcgcggcta ctacaaccag agcgaggccg gtgagtgacc 280 281 Cccddcccggg dcgcaggtca cgacccctec ccatccccca 320 321 cggacggece gggtcgcccc gagtctcccg gtctgagatc 360 361 Ccaccccgadg ctgcggaacc cgcccagacc ctcgaccgga 400 401 gagagcccca gtcaccttta cccggtttca ttttcagttt 440 44l aggccaaaat ccccgcgggt tgutcggggc tguggcgggg 480 481 ctcdggggac ggggctgacc acgdgggcgg guccagggtc 520 521 tcacaccctc cagaggatgt atggctgcga cctggggccc 560 561 gacgggegee tcctccgcgg gtataaccag ttcgcctacg 600 601 acdgcaagga ttacatcgcc ctgaatgagg acctgcgctc 640 641 ctggaccgcc gcggacaagg cggctcagat cacccagcgc 680 681 aagtgggagg cdgcccgtga ggcggagcag cggagagcct 720 721 acctggaggg cacgtgcgtg gagtggctcc gcagatacct 760 761 ggagaacggg aagaagacgc tgcagcgcgc gg 792 Paste Ref Segment Split Ref Segment lt lt Back Next gt gt Finish Cancel 10 Click Next Note For a procedure on using the Paste Ref Segment button refer to Pasting a Reference Segment on page 4 15 and for a procedure on using the Split Ref Segment button refer to Adding a
157. desired change the style and enter a description xj 7 Select the Used by all ROIs box if this NT variant is to be used by all ROIs ABI PRISM SeqScape Software v2 1 User Guide 4 31 Chapter 4 Creating a Reference Data Group Click Create Another or OK to save the changes After you click OK the variant additions appear in the list in the NT Variants tab RDG Properties General ROI NT Variants aA variants Variant Se Type ROI Position Reference Variant Style Description Used by all ROIs Change Base HLA C exon3 5 g M Known ves Change Base HLA C exon3 68 9 g Known Change Base AF250557 76 g c Red yes yes New pen Import Export elete Table Format General z Importing NT To import an NT variant from a tab delimited NT variant file Variants in Tab Delimited Format 4 32 In the SeqScape Manager click the Reference Data Group tab 2 Select the RDG in the list for which you want to import NT variants 3 Click Properties then select the NT Variants tab Click Import 5 Navigate to the tab delimited NT variants file and click OK ABI PRISM SeqScape Software v2 1 User Guide Declaring Variants into an RDG 6 An Import Results dialog box opens to show the number of variants imported as shown in the sample below Click OK to close the Import Results dialog box x G 331 Variants IMPORTED 185 were reconciled with the re
158. ding Specimens and Importing Data into a Project 10 If desired select each specimen and type a new name for the specimen then press the Enter key 11 The green arrow button gt on the toolbar indicates that the samples need to be analyzed Click this button After the samples are analyzed the red line through the specimen is gone and the samples are assembled as shown in the figure below Project Navigator Example Specimen3 5 E Example B U Speciment Unassembled Active Layer ROIs f gt AF250557 B 360 2 cx2R_02 Consensus B 360 2 cx2F_01 3 Coverage X gi 7414248 emblAJ21 5 Coverage B D Specimen2 Unassembled Samples B AF250557 Bl 360 2 0xaR_02 S 3 gi 414348 emb AJ21 B 360 2 cx4F_01 eng Reference ssembled AF250557 B amp gi 7414348 emblAJ21 B 360 2 cx4F_o1 B 360 2 cx4R 02 al gt j Legend ClearRange Known Variants Unknown Variants 12 Close the project and save it ABI Prism SeqScape Software v2 1 User Guide 6 19 Chapter 6 Creating and Analyzing a Project Importing Text You can import into a project a consensus sequence in text format as Only Files a text only specimen To import text or previously assembled sequences 1 In the Project Navigator select the project name then select File gt Import Text Segment Import Text Segment x Import Text Segment s automatically creates Specimens and Consensus Segments in your
159. diting the Data 0 2 a E E O ee 8 13 About Sequence Editing 00 anaana 8 13 When to Edit the Data 0 0 0 cece ee ee 8 13 Editing a Sample or a Consensus Sequence 8 14 Editing a Consensus Sequence in the Segment View 8 14 Editing Sample BaseS 2 00 naaar nananana 8 14 Editing a Consensus Sequence in the Project View 8 15 Adjusting the Clear Range 20 e eee eee eee 8 16 About the Clear Range 0c eee eee 8 16 Using the Clear Range Widget 0 ec eee eee 8 17 Using the Mouse 00 c ce eee eee eee 8 18 Using the Set Clear Range Dialog Box 0 2000 8 19 Editing Variants gs eae wanes ee oa ace ee ta ene 8 20 Method T cies tebe be cae eb eee ceed eee tales 8 20 Method 2 auis tup m sh NIRE A UE See ae UR QUERER 8 21 Saving Your Data aa sa na aian ee ene 8 21 Adding a Geriotype sen ESI vut den 8 22 Chapter 9 Exporting and Printing Data and Reports Exporting Data Files 1 EE RE Gee lee eee 9 3 File Names xz veceneng emer enbEMie E GR ERREUR SENE NN 9 3 Format Options 0 000 cee lees 9 3 Exporting a Project Alignment 00 ee eee eee eee 9 4 Exporting a Specimen lslsellllleeses 9 5 Exporting a Segment sr sessre reisa PEENES A ET DAROE ERE 9 6 Exporting a Sample 0 000 eee eee 9 7 Exporting Reports 0 a i a a Raia es 9 8 File Names idus metiera a p
160. e You need to export the data generated with the 30 day demo before it expires install SegScape software v2 1 then import this data into the full version of the SeqScape software v2 1 To export select the desired object from the SeqScape Manager window and click Export To import the object into the full copy of the SeqScape software v2 1 use the SeqScape Manager B 2 ABI PRISM SeqScape Software v2 1 User Guide Training and Documentation FAQs Training and Documentation FAQs Table B 2 Training and Documentation Questions and Answers Question Answer How do train myself on SeqScape software The best way to train yourself on SeqScape software is to use the training movie and the software tutorial included in the software package A printed tutorial is included with all SeqScape software versions except for the demo version The tutorial is also available online see below Where can get find resources documentation on SeqScape software To find documentation on SeqScape software go to www appliedbiosystems com gt Support gt Product and Service Literature In the Document Title Keyword dialog box at the bottom of this page enter SeqScape and click Search ABI PRISM SeqScape Software v2 1 User Guide B 3 Appendix B Frequently Asked Questions SeqScape Software Basics FAQs Table B 3 SeqScape Software Basics Questions and Answers Question Answer What is SeqSc
161. e Translation Orientation Index Codon Number 3 forward 4 AA Variants AA Change Position Length Type Known NT Change Description RS K R 5 4 Sub no 16grROL e HLA C_exon2 Ye v F 8 4 25a gt W ROI HLA C_exon2 AMOIA S 4 309 gt k ROI HLA C_exon2 AZ3IA S 1 69g K ROI HLA C_exon2 Q34R 103a gt 6 ROI HLA C_exon2 T72 T Al 216a rROI HLA C exon2 R98 R W 182 W ROI HLA C_exon3 Figure 7 5 AA Variant Report ABI PRISM SeqScape Software v2 1 User Guide 7 31 Chapter 7 Viewing the Results Specimen Statistics The report contains three separate tables Table 7 9 Parts of the Specimen Statistics Report Table Description Summary Displays project information and the specimens in the report Specimen Displays the bases changed ROI position length Statistics type QV and effect information for each mutation detected for each specimen Sample Results Displays the specimen segment assembly status calculated clear range sample score average QV and96 mixed bases for each sample port Manager Fa Analysis QC Ri Summam nalysis eport E Mutations Report Active Layer HLA C_CDS Project HLA C v2 r Project Creation Date 03 Dec 2002 at Project Modification Date 13 May 2003 at AA variants Report 23 40 04 PST 16 02 45 PDT Project Template PT HLA C 3100POP6 mixe PT Creation Date 03 Dec 2002 at d 5lib 23 29 08 PST nen Statistics Report Sequence Confir
162. e The file that uses only the report name contains all the data from the report 9 8 ABI PRISM SeqScape Software v2 1 User Guide Exporting Reports Exporting a To export a report Report 1 Open the project of interest then click EJ 2 Inthe navigation pane select a report type 3 Customize the report if desired See Customizing the Reports on page 7 42 4 Select File gt Export gt Report x Look in E Mv Exported Reports X amp amp ek Fes fa al Export Cancel 5 Complete the Export Report dialog box a Select a folder location to store the report b Change the file name of the report if desired The default file name uses the project name the report type and the pdf extension c Select a file format in the Files of type drop down list d Click Export ABI Prism SeqScape Software v2 1 User Guide 9 9 Chapter 9 Exporting and Printing Data and Reports Exporting All Reports can be automatically exported after analysis Reports Automatically To set up for automatic exporting of reports 1 Select Tool gt Options General Database Users Authentication amp Audit Display Reports after Analysis V Export Reports after Analysis Format HTML z HTML PDF Default Path for Text brt I XML Browse 2 Complete the General tab of the dialog box a Select the Display Reports after Analysis check box if desired b Select the Export Re
163. e in the Layer Name field 2 Select a layer by clicking it under the Layer label in the Layer pane If you need more information on libraries refer to Creating a Library on page 4 20 a Select a library from the Library drop down list if you are performing allele or haplotype identification b Select the library folder icon to open the Library Editor and view the entries 1 oil 7414348 e 792 Layer 1 settings New Layer Layer Name vert Index Codon Number 1 Library Translation Frame fz Click to open Library Editor Note The selected library in the Library drop down list is associated with the layer in the Layer Name field 4 24 ABI PRISM SeqScape Software v2 1 User Guide Creating New Layers The Library Editor opens as shown in the sample below Library Editor x ibrary Description Name HLA C_ex2 4 Created 16 May 2002 at 17 23 53 PDT Created By N A Modified N A Modified By N A Source N C Haploid Diploid Comments Some HLA C alleles OK Cancel c In the Layer pane enter the Index Codon Number d Select the Translation Frame e Select the Orientation 3 Select the appropriate Reference Segment in the Reference Sequence pane highlight the sequence representing the ROI then click Add ROI The ROI is added to the ROI pane and to the selected layer 4 Repeat the process to buil
164. e Software F 7 ABI PRISM SeqScape Software v2 1 User Guide Appendix G Software Warranty Computer Configuration 00 e eee ees G 1 Limited Product Warranty 0 0 0 0 c eee eee G 1 Limited Warranty ssseseeeeee eee G 1 Warranty Period Effective Date 2 2 000 2 ee eee G 2 Warranty Claims llle G 2 Warranty Exceptions llsleeeeeeee enne G 2 Warranty Limitations iese tea erana Ea V eee G 3 Glossary Index ABI PRISM SeqScape Software v2 1 User Guide Xiii xiv ABI PRISM SeqScape Software v2 1 User Guide Preface How to Use This Guide Purpose of This Guide Audience Assumptions What You Should Know Before Getting Started The Applied Biosystems ABI PRISM SeqScape Software Version 2 1 User Guide provides step by step instructions to use this software This guide is intended for novice and experienced analysts and scientists who are doing resequencing This manual uses conventions and terminology that assume a working knowledge of the Windows operating system the Internet and Web based browsers To make the best use of SeqScape Software Version 2 1 and the documentation be sure you are familiar with Microsoft XP with Service Pack 1 or Microsoft Windows 20009 with Service Pack 3 operating system The Internet and Web browser terminology DNA sequence detection and analysis methods DNA and amino acid coding conventions ABI
165. e Software v2 1 User Guide Chapter 11 Automating Analysis Creating Required Files in the Data Collection Software For More For more information on setting up and using the 3730 3730x DNA Information Analyzer and the 3730 Data Collection software refer to the Applied Biosystems 3730 3730xl Getting Started Guide PN 4347118 For more information on setting up and using the 3100 3100 Avant Genetic Analyzer and the 3100 Data Collection software refer to the ABI PRISM 3100 3100 Avant User Guide PN 4347102 If the Files If the appropriate instrument protocol analysis protocol and results Already Exist group have been created proceed to Creating a Plate Record on page 11 18 11 8 ABI PRISM SeqScape Software v2 1 User Guide Creating Required Files in the Data Collection Software Creating an Note The figures used in this section are from the 3730 data Instrument collection software v2 0 Protocol f To create an instrument protocol 1 In the navigation pane click the Protocol Manager icon to display the Protocol Manager Foundation Data Collection Version 2 0 Administrator user is logged in 5 x File View Help B Aca Instruments Results Group i Database Manager Instrument Protocols GA Instruments gt ga3730 Protocol Manager amp K ga3730 amp Plate Manager Find Protocol Taj Prot ad Uh Module Manage Name Run Module Dye Set Description Run History SpatialFi
166. e bars quality value Figure 10 1 Example of QV Bars in the Specimen View SQV bars in the sample Sequence view 109 301 Arnctapen Sesuence m m Electechergam Ree a pi Li cg LETTETTITIIIITI TRYGOETO ees ceccteerccecs SQV bars in the sample Electropherogram view Figure 10 2 Examples of SQVs in the Sample View 10 6 ABI Prism SeqScape Software v2 1 User Guide Displaying Quality Values Customizing the You can modify the low medium and high ranges and the color Quality Value associated with a QV Display To modify the QV display 1 Select Analysis gt Display Settings or click ds 2 Select the Bases tab 3 In the Quality Values section place the pointer between two colors it becomes a double headed arrow then click the slider on the color bar and drag it to left or right to the desired value Quality Values Bacooro MEN sc Use the criteria in the table below to define what values represent low medium and high ranges for your project Default Color Set the range to identify QV Bar and Range data that is Low Red Not acceptable Oto 14 Medium Yellow Needs manual review 15 to 19 High Blue Acceptable 20 or higher ABI PRISM SeqScape Software v2 1 User Guide 10 7 Chapter 10 Sample and Consensus Quality Values 4 Change the colors that represent low medium and or high QVs if desired a Sele
167. e detailed information on how to configure the software for the autoanalysis of these files see Chapter 11 Automating Analysis Sample files What instrument sample files can I use with the KB basecaller You can use the KB basecaller in SeqScape software to analyze sequencing sample files generated from the ABI PRISM 3100 and 3100 Avant genetic analyzers and Applied Biosystems 3730 3730xl DNA Analyzers How can share my work with someone at a different site What should send them All sample files analysis parameters reference sequence and analysis results are saved in every SeqScape project file You can share these files with anyone who has the software by exporting the project or data objects and importing them into their software on their computer There is no link between different SeqScape software installed on different computers You can also share project templates which contain the reference sequence and analysis parameters A colleague can then analyze sample files of their choice using the project templates to create a new project The analysis is identical to your own analysis with the same project template ABI PRISM SeqScape Software v2 1 User Guide B 5 Appendix B Frequently Asked Questions Table B 4 General Questions and Answers continued Question Answer Files What are the file types formats accepted by SeqScape software Reference Sequences SeqScape soft
168. e g 1000 The profile of the processed traces will be flat on an intermediate scale gt about 40 bases In the Quality Threshold group select either Call all bases and assign QV KB Basecaller calls all bases and assigns a QV Assign N for bases with QV lt x KB Basecaller calls Ns for all the bases that have QVs less than the value you enter ABI PRISM SeqScape Software v2 1 User Guide Changing the Analysis Parameters in an Analysis Protocol 4 Select the Mixed Bases tab Analysis Protocol for A1 2F O1 a If desired select Use Mixed Base Identification b Use the default detection level of 2596 or change the it by entering a new value or by dragging the line up or down ABI PRISM SeqScape Software v2 1 User Guide 8 9 Chapter 8 Reanalyzing and Editing Data 5 Select the ClearRange tab Analysis Protocol for d BE Basecalling Mixed Base E Clear Range Methods Use clear range minimum and maximum s First Base gt 20 End Base iso A z Bases ta trim from 3 end zo First bp Last bp I Use quality values Remove bases from the ends until Nbases Nbases fewer than 4 bases outof 20 have QVs less than 20 os x Qx J Use identification of N calls Rernove bases from the ends 5 until there are fewer than E Nscoutof 20 bases XN s per Z bases lt X N s per Z bases Mask M13 universal sequencing primers
169. e toolbar press the Delete key on the keyboard or right click and select Delete What is the TraceTuner basecaller module The ABI basecaller contains an algorithm that assigns bases and invokes a second algorithm the TraceTuner module TraceTuner module is responsible for generating per base sample quality values and identifying mixed bases What does a red dot mean in the analyzed project A red dot indicates a base that has been called by the consensus caller The consensus caller will edit this base in the relevant sample sequences of the specimen The edited base will appear in lower case and it will have a gray quality value bar Can change the settings of the tab jump key You can change the settings of the tab jump key in the Views tab of the project Display Settings You can also change the tab jump key settings when the project is open by selecting the Tab jumps to next option on the toolbar ABI PRISM SeqScape Software v2 1 User Guide B 17 Appendix B Frequently Asked Questions Analysis Reports FAQs Table B 9 Analysis Reports Questions and Answers Question Answer How can access my reports Access all reports by clicking the Report Manager button in the toolbar or by selecting Analysis gt Report Manager What are the different reports available in SeqScape software SeqScape software v2 1 has the ability to generate the following reports e Analysis QC
170. e v2 1 User Guide 2 11 Chapter 2 Getting Started oo UserName jdminUser Password e Note User Name and Password are case sensitive OK Exit 5 Enter your user name and password again then click OK The License dialog box opens if License Dialog Computer Configuration Applied Biosystems supplies or recommends certain configurations of computer hardware software and peripherals for use with its instrumentation Applied Biosystems reserves the right to decline support for or impose extra charges for supporting nonstandard computer configurations or components that have not been supplied or recommended by Applied Biosystems Applied Biosystems also reserves the right to require that computer hardware and software be restored to the standard configuration prior to providing service or technical support For systems that have built in computers or processing units installing unauthorized hardware or software may void the Warranty or Service Plan Limited Product Warranty Limited Warranty Applied Biosystems warrants that for a period of ninety 90 days from the date xl YOU ACKNOWLEDGE THAT YOU HAVE READ THIS END USER LICENSE AGREEMENT UNDERSTAND IT AND AGREE TO BE BOUND BY ITS TERMS AND CONDITIONS YOU FURTHER AGREE THAT THIS END USER LICENSE AGREEMENT I5 THE COMPLETE AND EXCLUSIVE AGREEMENT BETWEEN YOU AND APPLIED BIOSYSTEMS IF YOU ACCEPT AND AGREE TO THE FORGOING please select Accep
171. eak in the reference sequence between two reference segments where the reference is not contiguous ABI PRISM SeqScape Software v2 1 User Guide B 11 Appendix B Frequently Asked Questions Table B 5 SeqScape Manager Questions and Answers continued Question Answer What is a translation codon table A table that translates amino acid and genetic codes Refer to Appendix C Translation Tables What is a known variant An AA variant or NT variant that has been previously identified in the reference What is a region of interest ROI An ROI is a region on the reference segment with special numbering properties used for display The numbering for the ROI is continuous always increases from left to right and does not have to correspond to the numbering on the reference segment The ROI can also contain negative numbers ROIs can be grouped into layers for display or translational purposes How can configure a reference segment and ROIs within it After you import a reference sequence into the RDG use the ROI tab to reconfigure a reference segment and to add ROls What if do not have variant information Variants are not necessary to create a reference data group If you do import variants they must be in a tab delimited text file format or FASTA alignment of sequences File import What kinds of files can import into SeqScape software ABI sample files tab delimite
172. ect SeqScape then click OK The Analysis Protocol Editor dialog box opens Analysis Protocol Editor 3 In the General tab enter a unique name and description for the new protocol 11 12 ABI PRISM SeqScape Software v2 1 User Guide Creating Required Files in the Data Collection Software 4 Select the Basecalling tab then do the following a Select the appropriate basecaller and Dye Set Primer file For information on selecting the correct files see Appendix A Basecallers and DyeSet Primer Files IMPORTANT Make sure that the basecaller and the DyeSet Primer file types match b If desired select one or more stop points for data analysis Base your selection on the basecaller being used as indicated below Option Basecaller At PCR Stop check box KB or ABI After _ Ns in X bases check box ABI After __ Ns check box ABI After __ Bases check box KB or ABI c For the KB basecallers only select True Profile or Flat Profile for processed data and select a quality threshold 5 Select the Mixed Bases tab then Note This function is active with the KB basecaller only a For mixed bases only select Use Mixed Base Identification b Use the default setting of 25 or change the detection level by entering a new value or dragging the line up or down 6 Select the Clear Range tab then if desired select one or more stop points for data analysis Note The clear
173. ed in order from top to bottom with the clear range method never being lengthened based on the settings in subsequent algorithms The result is that the smallest clear range is used If you want to preserve the existing clear range in a sample when reapplying analysis protocol settings to a sample do not select any of the Clear Range methods IMPORTANT You can create a protocol without selecting a clear range method but it is recommended that at least one clear range method be selected for reference trimming The Clear Range tab enables you to set the part of the sequence that you consider to be good quality Good quality means that the sequence has the fewest errors and ambiguities and offers good base calling and spacing To set the way the clear range is determined 1 In the Analysis Protocol Editor select the Clear Range tab x General Basecalling Mixed Bases ilter Clear Range Methods Use clear range minimum and maximum a e First Base 20 End Base so a C Basesto trim from 3 end 120 First bp Last bp v Use quality values Remove bases from the ends until Nbases Nbases fewer than fa bases outof 20 have QVs less than 20 Q ovs x Use identification of N calls Rernove bases from the ends a Until there are fewer than j4 Ms aut of 20 bases X N s per Z bases lt X N s per Z bases w3 tud MEE Mask M13 universal sequencing primers v
174. ed to the next job 11 26 ABI Prism SeqScape Software v2 1 User Guide Understanding the Autoanalysis Manager Table 11 3 SeqScape 2 1 Tab Command Buttons Button Name Function Auto delete Jobs Automatically deletes the completed jobs from the auto analysis manager This option allows you to select when to delete in number of days the completed jobs This option is a convenient way to save disk space Delete Deletes a job from the Autoanalysis Manager ABI PRISM SeqScape Software v2 1 User Guide 11 27 Chapter 11 Automating Analysis File Sharing Between Data Collection and SeqScape Software In Table 11 4 the term files refers to projects project templates specimens and analysis protocols Table 11 4 File Sharing Table Conditions Result Corrective Action SeqScape software installed while data collection software was open proper installation e Files created in SeqScape software Data collection software open Files are registered in both applications and are available for use in the data collection software Files created in data collection software e SeqScape software open Files are registered in both applications and are available for use in SeqScape software SeqScape software installed while data collection software was closed improper installation Files created in SeqScape software or in data collection software O
175. eeaire d cy B cm t e hut 9 8 Format Options s s aaaea naana 9 8 Exporting a Beport cies x wit Eu RR RENE 9 9 Exporting All Reports Automatically 2 005 9 10 Printing Data and Reports 0 cee eee 9 11 Customizing Header and Footer Display 9 11 Printing Views of a Sample File 000 ee eee 9 12 Printing Various Views of a Project 00 ee eee 9 14 Printing a Report 9 16 ABI PRISM SeqScape Software v2 1 User Guide ix Chapter 10 Sample and Consensus Quality Values Chapter 11 Types of Quality Values QVs 000 cee ee 10 2 Sample Quality Values 0 0 cece ee 10 3 Sample Quality Values 0 0 2 0 cece eee 10 3 Interpreting the Sample Quality Values Less 10 3 Sample SCONE shs 2t e NR nie be dug 10 3 Consensus Quality Values lleeelleeee 10 5 Interpreting the Consensus Quality Values 10 5 Consensus Score 2 2 AA ERA ellen 10 5 Displaying Quality Values llle 10 6 Customizing the Quality Value Display 10 7 Displaying the Quality Bars and Values Less 10 9 Editing Bases with Quality Values leslseeesene 10 10 Cumulative Quality Value Scoring in Reports 10 11 Analysis QC Report 0 0 0 0 eee eens 10 11 Mutations Report 0 00 c eee ee 10 12 Specimen Statistics Report 00000 2 eee eee 10 13
176. eld indicates that the user will be locked out if the maximum number of attempts occur within the time entered in this field Enter a number or accept the default ABI PRISM SeqScape Software v2 1 User Guide 2 15 Chapter 2 Getting Started c The Maintain lockout for minutes field indicates the number of minutes that must elapse before the user can login again after being locked out of SeqScape software Enter the number of minutes or accept the defaults d The Change password every days field indicates the number of days before the users must enter a new password Enter a number of days or accept the default 2 In the Audit Trail pane select the Audit Trail On check box to have a dialog box open whenever an indicated reason occurs 2 16 ABI Prism SeqScape Software v2 1 User Guide Starting SeqScape Software for the First Time 3 In the Audit Reason pane enter reasons to provide an audit trail a Double click the Reason 1 field or highlight it and click New x Reason ase Change when a user makes a base change Description Inactive OK Cancel b In the Reason field type a reason for a change to the project to identify for example a base change or a variant that is imported Enter a description of the reason if desired d Click OK in the Audit Reason Editor The first reason appears in the list in the Options dialog box General Database Users Authenticatio
177. elect the second specimen select the samples and click Add Sample Repeat this for the third specimen r Samples To Add Ua GU Specimen3 Remove Sample Bl ai 2R_02 ab1 S U Specimen2 Seg 43319408 Bl At 2F_01 ab1 SeqScape B ai 2R_02 ab1 E AppSeqScape New Specimen Bl 41 3F_01 ab1 B Tutorial Data Bl a1 3R_02 ab1 E HLA C B HLA C_3100POP6_Tutorial Data ent Bl A2 3F_03 ab1 Delete Specimen B Bl 42 20 04 ab1 AT 2F 01 Bl A2 2F_03 ab1 Auto Add gt gt Bl A2 3R_04 ab1 Automatically create Specimen name based on the string between these delimiters in the sample s name Start A2 4F_03 ESN A2 4R_04 Use sample file name c Iv Show ab1 Samples File Only OC EI OK Cancel 9 Click OK to perform the imports and return to the Project window The project reflects the new specimens and samples with the specimens shown with a red line through them This indicates that the samples are unanalyzed and unassembled WR seascape Admin User is logged in E ADi xl Eh Ed View Tools Apabai Window Help dgs C 6 08 aa ex E ms oj o masy omansa se Maga Arey E Tabuo 21 S N Specimen 5 Unattembled i AF250557 5 gil7414348 emndiAN2 N Speamen 9 Unattembled 8 AF250557 8 gi7414348 embjAJ2 i Unassembled A AF250557 5 gi 414348 embjAJ21 ABI PRISM SeqScape Software v2 1 User Guide Ad
178. eletions and bases and the amount of coverage and whether it is continuous for each specimen eport Manager Summary Fa Analysis QC Report MI iduiatons Report Active Layer HLA C_CDS Project HLA C_v2 a Z Mutations Repo A oati E AA Variants Report Project Creation Date 03 Dec 2002 at Project Modification Date 13 May 2003 at 4 23 40 04 PST 16 02 45 PDT U Specimen Statistics Report Bequenks Confirmation Report Project Template PT HLA C_3100POP6_mixe PT Creation Date 03 Dec 2002 at p d 5lib 23 20 08 PST Base Frequency Report FS Library Search Report PT Modification Date 13 May 2003 at Reference Data Group RDG HLA C_Exon2 4_noNT 16 02 45 PDT RDG Report n E Audit Trail Report RDG Creation Date 03 Dec 2002 at RDG Modification Date 13 May 2003 at GennboinciRenbit 22 59 40 PST 16 02 45 PDT Display Settings DS TutorialDisplaySettings DS Creation Date 26 Nov 2002 at 23 37 59 PST DS Modification Date 13 May 2003 at Analysis Defaults AD 3100 SR POPB BDTwi 16 02 45 PDT mixed 5Lib AD Creation Date 03 Dec 2002 at AD Modification Date 13 May 2003 at xi Specimens in Report A1 A2 A3 Sequence Confirmation Specimen Matoh Insertions Deletions Base Coverage Continuous Comments Changes At ne o 4 37 0 6X no esu A2 no o 1 ar 0 4x no as m o 1 30 04x nme Report Settings Figure 7 7 Sequence Confirmation Report ABI Prism SeqScape Software
179. endix D Appendix E Appendix F Xii Translation Tables IUPAC IUB Codes i ahs vcre he E id awn ego RR ERR ARR s C 2 IUPAC Diagrams aidaa a o a RR III C 3 Complements cech koe ewe cae X OE ERE EE C 3 Universal Genetic Code 0 0 cc eee C 4 Amino Acid Abbreviations 0 0 0 0 cc eee C 5 User Privileges Tables of User Privileges 0 0 0c e ee ee D 1 Aligned Variant and FASTA File Format Tab Delimited Files 0 0 0 0 ccc E 2 Creating a Variant Text File 0 0 000 eee eee E 2 FASTA File Format 0 0c ccc E 3 FASTA Format Description 00020 cee eee E 3 FASTA Format Example 000 00 e eee eee eee E 3 IUB IUPAC Codes ra E E E teens E 4 Supported Nucleic Acid Codes 0 000 cece eee eee E 4 Supported Amino Acid Codes 0000 cece ee eee eee E 5 Creating a Multi Aligned FASTA File Introduction caves ee steed eed atest esie hrleieiugy5bIx F 1 Method 1 Create a Multi Aligned FASTA File Using SeqScape Software F 2 1 Create a Multi Aligned FASTA File 0 0050 F 2 2 Create Your Library in SeqScape Software F 3 Method 2 Create a Multi Aligned FASTA File Using ClustalX Software F 4 1 Obtain ClustalX Software 2 0 0 0 2c eee ee F 4 2 Create a Multi Aligned FASTA File 0 000 00 F 4 3 Edit Characters in the gde File 20 0 0 e eee F 6 4 Create Your Library in SeqScap
180. entifies the genotype at one or more positions of all the specimens in the project and provides coverage of the samples against the expected coverage based on the targeted sequence See page 7 39 Specimen Statistics report Provides the position of the clear range on the reference of the samples on the segment Amino Acid report Provides the option of displaying the amino acid full name or three letter symbol Specimen heterozygous indel mutation HIM identification Mutations report lists HIMs Amino acid alignment can be coupled to the nucleotide alignment Sequence Collector BioLIMS software is no longer supported ABI Prism SeqScape Software v2 1 User Guide For Users of Earlier Versions of SeqScape Software For Users of Earlier Versions of SeqScape Software Features of v2 0 The following features were introduced in SeqScape Software v2 0 Extended Reference Data Group SeqScape Software v2 0 contains an extended reference data group RDG The RDG contains a known reference sequence and any known nucleotide or amino acid variants The RDG available in this new version of the software enables analysis of simple or complex projects The Reference Sequence within the RDG can be a Contiguous reference sequence with a single reading frame Contiguous reference sequence with multiple reading frames Reference sequence constructed from several reference segments Each segment can come from different locat
181. enu select Nucleotide 3 Enter the nucleotide you want for the reference sequence in the for text box then click Go 4 After finding the desired sequence select the check box to the left of the accession number 5 Atthe top of the page next to Display select Genbank then select Send to File The file will be saved to the specified location This file can then be imported into the RDG The file can have a gb fcgi or cgi extension IMPORTANT Only a user from the Administrator or Scientist group can set up a new RDG Refer to Appendix D User Privileges for a list of the privileges that apply to each group You can create a new RDG in the following ways Usethe RDG wizard and follow the instructions Usethe SeqScape Manager window to open a blank RDG Follow the RDG wizard procedures below if desired to familiarize yourself with the windows of the RDG Then create subsequent RDGs by using the SeqScape Manager Refer to Creating a New RDG Using SeqScape Manager on page 4 12 ABI PRISM SeqScape Software v2 1 User Guide 4 5 Chapter 4 Creating a Reference Data Group Creating a New RDG Using the Wizard Using the Wizard The wizard in the SeqScape Manager will familiarize you with toLearn the setting up a new RDG Software To create an RDG using the RDG wizard 1 In the main SeqScape window select Tools gt SeqScape Manager 2 Select the Reference Data Group tab then click Wizard at the bottom of the pa
182. er is logged in 1 m ini xi File View Help AS B Aca Instruments GA Instruments ga3730 gt Plate Manager F Li Resuts Group b Database Manager Find Plates Matching These Criteria f ga3730 E Plate Manag jer Type of Search Barcode i E Protocol Manag F Hs Module Manage SENET END Run History y O E DakarPT3 Search Stop Find All Append Results Plate ID Plate Name Type Size Status Operator Last Modified Ei Clear Clear All i Edit Duplicate Delete Import Export 2 Click New 11 18 ABI Prism SeqScape Software v2 1 User Guide Creating Required Files in the Data Collection Software The New Plate Dialog dialog box opens new Plate Dialog X ID Barcode Jl Name Oe Description Application None h Plate Type fos wen bad Scheduling fi 234 Unavailable on the Plate Sealing one vi 3100 or 3100 Owner Name O Operator Name Avant instruments 3 Complete the information in the New Plate Dialog a Type an ID for the plate Type a name for the plate Type a description for the plate optional pns Select SeqScape YourlInstrumentName in the Application drop down list Select 96 well or 384 well in the Plate Type drop down list f For the 3730 3730x instruments define a scheduling pattern for a 384 well plate 9 g For the 3730 373
183. er project ABI PRISM SeqScape Software v2 1 User Guide Viewing the Results This chapter contains Workflow for This Chapter 7 2 About the Project Data 0 2 eee eee ee 7 3 Project VIEWS i258 ay bv RADO b E Melo 7 4 Specimen VIEWS cuui heed Weser ux AE ss 7 10 Segment VIEWS udo bidder b Pusat sehe dated Pase esas 7 11 Sample VIEWS zx esspEecesurektespkRDA ewe eer Es 7 15 Viewing Variant l l 7 20 About the Reports erir llle 7 22 Viewing the Reports 0 2c eee cece eh 7 23 Viewing the Reports and Project Results 7 25 Analysis QC Report 0 cece cece e 7 26 Mutations Report llle ee 7 28 AA Variants Report 0 0 00 cece ee eee eese 7 31 Specimen Statistics cisien oi eee eee ee 7 32 Sequence Confirmation Report 7 33 Base Frequency Report 0 0 cece cece eee eee eee 7 34 Library Search Report 0 0 0 eee eee 7 35 RDG Repott 3 2 554 86 ied e Ebb i EPI e SEL 7 37 Audit Trail Report 0 0 0 ee eee eee 7 38 Genotyping Report 2 0 2 eee eee eee 7 39 Customizing the Reports 2 0 0 0 cece eee eee eee 7 42 ABI PRISM SeqScape Software v2 1 User Guide 7 1 Chapter 7 Viewing the Results Workflow for This Chapter Chapter 3 1 Create Analysis Defaults and Display Settings Chapter 4 2 Create Reference Data Group Add Reference Sequence ROIs Create ROIs Cr ate E Add nucleot
184. eriod begins its ABI PRISM SeqScape Software Version 2 1 will perform substantially in accordance with the functions and features described in its accompanying documentation when properly installed on the instrument system for which it is designated and that for a period of ninety 90 days from the date the warranty period begins the tapes diskettes or other media bearing the software product will be free of defects in materials and workmanship under normal use If buyer believes that it has discovered a failure of the software to satisfy the foregoing warranty and if buyer notifies Applied Biosystems of such failure in writing during the ninety 90 day warranty period and if Applied Biosystems is able to reliably reproduce such failure then Applied Biosystems at its sole option will either 1 provide any software corrections or bug fixes of the identified failure if and when they become commercially available to buyer free of charge or ii notify buyer that Applied Biosystems will accept a return of the software from the buyer and upon such return and removal of the ABI PRISM SeqScape Software v2 1 User Guide G 1 Appendix G Software Warranty Warranty Period Effective Date Warranty Claims Warranty Exceptions software from buyer s systems terminate the license to use the software and refund the buyer s purchase price for the software If there is a defect in the media covered by the above warranty and the media
185. erogram Views Display Settings Description Display Settings Name Created N A Created By NiA Modified N A Modified By N A Source NIA r Comments Save Cancel 2 Click the Display Settings Name field then enter a name for the new display settings IMPORTANT The name cannot contain spaces or characters that do not conform with the Windows file system Refer to File Naming Convention on page 2 10 3 Enter any comments you want to record for the sample ABI Prism SeqScape Software v2 1 User Guide 3 19 Chapter 3 Creating Analysis Defaults and Display Settings 3 20 Select the Bases tab Display Settings 1 A f Electropherogram Views Quality Values Font Size 12 sarcooro EE so 15 20 Base Scale Show base number every 10 bases Base Colors Base Style Colored Text SE CEES Other N R Y EN Save Cancel a Enter the desired text style and color for each base In the Base Font section select your font preferences for the sequence bases or use the defaults b In the Base Scale section enter the frequency at which to display bases for the reference sequence in the Project view c Inthe Base Colors section select your color preferences for the sequence bases and electropherogram traces To select a color click the colored box next to A G C and T to open the color chart Select a new color then click OK In the Quality
186. es for H ACT V GCA N AGCT Table E 2 Accepted Amino Acid Codes Character Codes for A Alanine B Aspartate or asparagine C Cystine D Aspartate E Glutamate F Phenylalanine G Glycine H Histidine l Isoleucine K Lysine L Leucine M Methionine N Asparagine P Proline Q Glutamine R Arginine S Serine ABI PRISM SeqScape Software v2 1 User Guide E 5 Appendix E Aligned Variant and FASTA File Format Table E 2 Accepted Amino Acid Codes continued Character Codes for T Threonine U Selenocysteine V Valine WwW Tryptophan Y Tyrosine Z Glutamate or glutamine X Any 5 Translation stop Gap of indeterminate length E 6 ABI PRISM SeqScape Software v2 1 User Guide Creating a Multi Aligned FASTA File This appendix contains Introduction oe cen Wen RET RUPCERTIDA Es Ook F 1 Method 1 Create a Multi Aligned FASTA File Using SeqScape Software liess F 2 Method 2 Create a Multi Aligned FASTA File Using ClustalX Software 0 0 ees F 4 Introduction To use the Library Search feature in ABI PRISM SeqScape software you will need to create a sequence library All the sequences for the library must have equal lengths and must be aligned If you have sequences of equal length and they are already aligned go directly to SeqScape software to create your sequence library This appendix contains the instructions for two different methods of
187. es if they do not meet minimum standards Sequences not meeting the filter settings are not assembled Use the maximum percentage of mixed bases to look for frame shift Use the maximum percentage of ambiguities N and the minimum length settings to ensure that you are working with enough data for further analysis This filters data that may exceed a specified percentage of ambiguities Also use a minimum sample score to ensure that the quality of the sequences is high A setting of 20 indicates that the data is accepted if the mean quality value of all bases in the clear range is 20 or greater This corresponds to a 1 to 100 or 1 error rate To select the filter settings 1 In the Analysis Protocol Editor select the Filter tab Analysis Protocol Editor 3 x General Basecalling Mixed Bases Clear Range Filter Filter Settings Maximum Mixed Bases 5 0 Maximum Ns 96 f 0 0 Minimum Clear Length bp po Minimum Sample Score po ABI PRISM SeqScape Software v2 1 User Guide Creating Analysis Protocols 2 Enter your changes to the settings using the descriptions of the settings in the following table as a guide Parameter Description Maximum Mixed Bases Total maximum percentage of mixed bases that can occur in the clear range of a sample file Any more than this number causes the sample to fail analysis Maximum Ns Total maximum percentage of Ns that can occur in
188. ete or change a base in the Specimen view 1 Select the Segment icon in the navigation pane in the Project window The Specimen view opens in the project document window W SeqScape bapisloggedin lnixi File Edit View Tools Analysis Window Help them EB amp UB amp gnx EH SE E s BEEN a sj es e d vaga Fm By Active Layer HLA C CDS Tab jumps to next Muttiple WBHL4 c 3100 Project Navigator E E HLA C 3100 a D 360 2 Unassemblei a AF250557 ere re gi 7414348 e 276 212 72 91 101 iu 121 GAGATCACACTGACCTGGCAGYGGGATGGSGAGGACCAAACTCAGGACACCG GAGATCACACTGACCTGG CAMBY GGGATGGSGAGGACCAAACTCAGGACACCd 1 1 1 1 2 Select the Assembly tab then select a layer in the Active Layer drop down list 3 To change or delete a consensus base click the base you want to edit then delete or change the base 4 To insert a base in the consensus sequence click between two bases then insert the bases Note The changed bases appear in lowercase Note If the audit feature is enabled you must enter a reason for each base change base insertion and base deletion You can edit sample bases in the same manner as consensus bases However only the sample whose base is edited and the consensus sequence are affected by the changes ABI Prism SeqScape Software v2 1 User Guide Editing a Consensus Sequence in the Project View Editing a Sample or a Consensus Sequence Note Any chan
189. ettings Gap Penalty 225 Recommended value 22 5 Extension Penalty es Recommended value 8 5 Library Matches feo v Basecall Samples v Calculate Clear Range Save To Manager As OK Cancel Figure 3 4 Analysis Defaults Specimen tab ABI PRISM SeqScape Software v2 1 User Guide 3 13 Chapter 3 Creating Analysis Defaults and Display Settings Setting Analysis To create new Analysis Defaults Defaults 1 In the SeqScape Manager select the Analysis Defaults tab then click New 2 In the General tab of the New Analysis Settings dialog box enter an Analysis Defaults Name Note The name cannot contain spaces or characters that do not conform with the Windows file system Refer to File Naming Convention on page 2 10 x General Project Specimen Sample Name Analysis Defaults Name NewanalysisDefaults Created N Created By NIA Modified WA Modified By N A Source N A Comments 3 Enter any comments pertaining to the new analysis settings in the Comments box 3 14 ABI PRISM SeqScape Software v2 1 User Guide Specifying the Analysis Settings 4 Select the Project tab and if desired change the Penalty Settings The recommended gap penalty is 30 and the recommended extension penalty is 1 0 Note The gap and extension penalties refer only to the alignment algorithms that are used to align the consensus sequences to the re
190. ettings for this project 8 Click Close in the SeqScape Manager dialog box Selecting the Note Changing the analysis defaults does not affect the analysis Analysis Default settings of samples that are already in the project Settings for Individual TO select the analysis settings for each sample individually Samples 1 Select the sample in the Project view 2 Select Analysis gt Analysis Settings This opens the Analysis Protocol for that individual sample file 3 Make relevant changes to the settings then click Save ABI PRISM SeqScape Software v2 1 User Guide 3 17 Chapter 3 Creating Analysis Defaults and Display Settings Specifying Display Settings To accommodate personal preferences SeqScape software allows you to select the way results are displayed The display settings can be modified and then reapplied to a project The selected settings can also be saved in the SeqScape Manager to be used in a project template The display settings control Font colors and style for bases Electropherogram display and axis scale Display views for variants Display views for nucleotide translation Quality value display and thresholds ABI PRISM SeqScape Software v2 1 User Guide Specifying Display Settings To specify the display settings 1 In the SeqScape Manager select the Display Settings tab then click New The Display Settings dialog box opens displaying the General tab Display Settings xi ases Electroph
191. f you created an appropriate analysis protocol in SeqScape software you can use it in data collection software You can also create an analysis protocol in SeqScape software if desired Creating an Analysis Protocol To create an analysis protocol 1 In the Analysis Protocols section of the Protocol Manager click New lilroundation Data Collection Version 2 0 Administrator user is logged in ni x File View Help AS ACA Instruments GA Instruments gt ga3730 Protocol Manager H inesuts Group Database Manager instrument Protocols amp E Elgas730 E amp Plate Manager Find Protocol EE Tui Protocol Manad i s Module Manage Name Run Module Dye Set Description Clun History SpatialFill_1 SpatialFill_1 Created with populator E DakarPT3 n n SpatialNoFill 1 SpatialNoFill 1 Created with populator 8 Run StdSeq35 POP 1 Z BigDyev3 ud p New Edit Delete Import Export Analysis Protocols Find Protocol Application Sequenc S SequencingAnalysis SeqScape SeqScape 3100POP6 BDTwv3 KB DeNova v5 1 SeqScape AP1 3100 SR POPB BDTv1 mixed v2 isl e I Dese meet Ewo 11 11 ABI PRISM SeqScape Software v2 1 User Guide Chapter 11 Automating Analysis If more than one analysis application is installed on the data collection computer the Analysis Applications dialog box opens analysis Applications 2 Sel
192. ference sequence 3319 Variants were NOT IMPORTED 830 not valid 2489 duplicated existing variant s There were D parsing errors See the log file for more details 7 The new variants appear in the NT Variants list The Table Format options at the bottom of the window are General default and Hugo If desired select the format in the drop down list General ROI NT Variants AA variants Variant Style Type ROI Position Reference Variant Style Description Used by all ROls Insert After AF250557 16 lg lw Red lyes a Change Base AF250557_1 1 g c red 7 exonNe 354 yes Change Base AF250557_1 1 g A green 7 exonKG 1 ac yes Delete AF250557 1 1 3 get red 7 exonT1770s yes Change Base 3 t c yellow 7 exonYL98 3 yes Insert After 1 a GTT red 7 exonP5259s yes Change Base 2 c G red 7 exonMOTT8 yes Change Base 2 c A red T exonAMFS9 yes Change Base 1 a T red 7 exonRSBO0 yes Insert After 1 g GTT red 7 exonSEBsur yes Change Base B e IT red 7 exoncase2s yes Delete 4 ccc red 7 exon5Y00 B yes Change Base 5 c A red 7 exonLL 3sur yes Change Base 4 c A red 7 exonSKub85 yes Delete 7 act red 7 exonKs 1405 yes Insert After 7 a TCT red 7 exorVis sur yes Insert After 7 a ITCT fred 7 exonLuC89s yes Change Base 8 t G red T exonKC T12 yes C
193. ferences and to each other They do not affect the alignment of the samples to the reference for assembly New Analysis Settings ABI Prism SeqScape Software v2 1 User Guide 3 15 Chapter 3 Creating Analysis Defaults and Display Settings 5 Select the Specimen tab The Library Matches check box indicates the number of hits desired to match the library you select The recommended gap penalty is 22 5 and the recommended extension penalty is 8 5 New Analysis Settings xi General Project Sample Settings Gap Penalty 225 Recommended value 22 5 Extension Penalty a5 Recommended value 8 5 Library Matches feo v Basecall Samples Jv Calculate Clear Range a If desired change the settings b Select Basecall Samples to automatically calculate clear range and basecall samples If you do not select Basecall Samples the sample files are not basecalled and it is assumed that you have previously basecalled and edited the data When basecalling is skipped the software proceeds to filtering and assembly in the analysis pipeline 3 16 ABI Prism SeqScape Software v2 1 User Guide Specifying the Analysis Settings 6 Select the Sample tab then select the analysis protocol you just created from the Analysis Protocol drop down list New Analysis Settings q x General Project Specimen Sample Settings Analysis Protocol MEVANI See 7 Click Save to save the new s
194. ftware v2 1 User Guide Starting SeqScape Software for the First Time Setting Up Users belonging to the Administrator group can change the default Authentication amp settings in the Authentication amp Audit tab for security features of the Audit application Note The Administrator is the only person who can set up and change the information in the Authentication amp Audit tab The selections in this tab are inactive for all other users The Authentication amp Audit panes provide a way to track the changes in projects such as base change variants or processes you want to track You must turn Audit Trail On for tracking to occur To set up authentication and auditing 1 Select the Authentication amp Audit tab to change the defaults for the Authentication Settings ortions xl General Database Users Authentication amp Audit Authentication Settings Audit Trail Iv Audit Trail On Lockout user after B invalid login attempts Audit Reason within 1 minutes Reason Bi Reason 1 Maintain lockout for 1 minutes Reason 2 Reason 3 Reason 4 Automatic timeout after J60 minutes Change password every po days New Open Export OK Cancel a Lockout occurs when a user enters an incorrect password or user name the number of times you select for the Lockout user after invalid login attempts field Enter the number or accept the default b The within minutes fi
195. g Report Table Description Summary Displays project information and the specimens and samples in the report Specimens_in_ Lists the specimens included in the report Report Gene Summary These fields are not used at this time Transcript Table Displays the layout of the intended amplicons Lists all of the transcripts with their accompanying amplicons in the RDG e Layer Name of the transcript being reported on e Amplicon_ID Identity of an amplicon on this transcript there are many rows of amplicons for each transcript e Assay_Target_Start Start of the assay region of the amplicon in reference segment coordinates e Assay Target Length Designed length of the assay region e 5 Regulatory Length Amount of amplicon sequence covering 5 regulatory region Exon Length Amount of amplicon sequence covering exon region in this transcript ntron Length Amount of amplicon sequence covering intron region in this transcript ABI PRISM SeqScape Software v2 1 User Guide 7 39 Chapter 7 Viewing the Results Table 7 15 Parts of the Genotyping Report Table Description Resequencing Coverage Displays specimen coverage statistics for the active layer Specimen Name of specimen being reported on FGR Coverage Full gene region coverage Percent of specimen consensus sequence covering the entire targeted region FGR Avg QV Average quality value of the consensus sequence across the target re
196. g a Reference Data Group Entering New AA Variants 4 36 10 11 Click OK to close the Import Results dialog box The list of variants are displayed in the NT Variants tab RDG Properties E xl General Roi WT Variants Type RO Position Reference Variat Syle Description Used by all ROIs Change Base HLA C exon3 75 In M Known yes Change Base HLA C_exon3 68 lg g Known yes New p Import Export Table Format General z Save To Manager As OK Cancel Click OK to close the RDG Properties window The AA Variants tab lists the known amino acid variants associated with a reference sequence The entries you define in this tab are used to identify known and unknown amino acid variants in your projects You can enter AA variants in two ways 1 2 3 Click Add Variant in the ROI tab then enter the variant attributes in the New AA Variant dialog box Create a table of variants using Microsoft Excel then import the table The columns in the Excel table must map to the columns in the AA Variants tab Refer to Figure 4 3 on page 4 29 for the column names To enter a new AA variant In SeqScape Manager click the Reference Data Group tab Select a listed RDG and click Properties Select the AA Variants tab then click New ABI PRISM SeqScape Software v2 1 User Guide Creating an RDG from Aligned Consensus Sequences 4 Select the type of variant Insertion Dele
197. g from SeqScape software version Then insert the CD titled 2 0 ABI PRISVM SeqScape Software v2 1 Update CD 1 0 or 1 1 ABI PRISV SeqScape Software v2 1 Upgrade CD 2 Ifthe installer does not start automatically double click setup exe 3 In the SeqScape Registration Code dialog box enter your registration code for SeqScape software v2 0 4 Follow the instructions to upgrade the software After the software is installed you must register and log in as an Administrator user After you log in as Administrator you can set up additional users with Admin Scientist or Analyst permissions Existing Users All existing users of an earlier version of SeqScape software will have Analyst privileges Only a user belonging to the Administrator group can change the user to Scientist or Analyst A dialog box opens for users who existed in previous versions to set up their user profiles name and password when they try to use SeqScape Software v2 1 for the first time 2 8 ABI Prism SeqScape Software v2 1 User Guide Removing SeqScape Software Removing SeqScape Software What the To completely remove SeqScape software from your computer Uninstallation follow the procedure in this section The uninstallation process Process Does Deletes all folders and files installed by SeqScape software However if you moved SeqScape software folders or files from their original installed location they may not be found and
198. ge IE x Projects Project Templates Reference Data Group Libraries Analysis Defaults Analysis Protocols Display Settings Created Created By Modified Modified By Libraries Reference NtVariants Aa Variants Comments HLA C ex2 05 12 02 at NIA 06 28 02 at guest Appli 1068 HLA C ex2 12 01 97 at palmerrm 12 01 97 at palmerrm 1068 HXB2PrtRT 09 22 00 at NIA 11 30 97 at guest Appli 1256 HXB2PRRT 09 22 00 at NIA 11 30 97 at guest Appli 1256 4 6 ABI PRISM SeqScape Software v2 1 User Guide Creating a New RDG Using the Wizard 3 Enter a name for the new RDG that conforms with the Windows file system Refer to File Naming Convention on page 2 10 Codon Indicator Color Codon Table standard a z Name the Reference Data Group Enter the name and specify the general attributes of the Reference Data Group Reference Data Group Description Reference DataGroupName RDGExample SCS Created Created By Modified Modified By Source General Settings lt TUM Comments Next gt gt Finish Cancel 4 If desired click the Codon Indicator Color by clicking the yellow color box and select a new color 5 Then select the Codon Table to use 6 Click Next The next page shows the Reference Sequence pane The Reference Sequence forms the backbone for comparison
199. ge sequence for that sample A sample quality value is a per base estimate of basecaller accuracy How does the consensus quality value differ from the consensus score The consensus score is the average quality value of the bases in the consensus sequence for that specimen A consensus quality value is a per base estimate of the accuracy of the consensus calling algorithm ABI PRISM SeqScape Software v2 1 User Guide Appendix B Frequently Asked Questions Printing and Exporting Results FAQs Table B 11 Printing and Exporting Results Questions and Answers Question Answer Printing What can print in SeqScape software You can print the views only for sample specimen segment project and complete reports You can also print electropherograms complete reports and the visible data or all data for the project view Printing What printers are recommended for use with SeqScape software An HP 8100 4600 990cxi or an Epson 980 color printer is recommended File export What can export from SeqScape software User information projects project alignments project templates reports nucleotide and amino acid variants and libraries can be exported from the software Refer to Appendix D User Privileges File export Can export each consensus sequence individually Consensus sequences for a project can be exported as a group by using selecting File gt Exp
200. ges are reflected in the sample sequences within the specimen and in the summary To insert delete or change a base in the Project view 1 Select the project of interest in the navigation pane in the Project window The project view opens displaying the consensus sequences for each specimen WRseqscape bap is logged in E loj x File Edit View Tools Analysis Window Help dem Ej amp US Dx b E BE BG s OT He E HE ES vai gai peage Al Active Layer HLA C CDS Tab jumps to next Multiple W HLa c 3100 Project Navigator qHLA C 3100 E E HLA C 31 00 PiKnown Variants amp D 360 2 HLA C CDS ROIs Unassemblei All Variants AF250557 Summary E gil7414348le INT Variants GGGAGACAC R Q0 A4 Q T GGGAGACACRGAAGTACAAGCGCCAGGCACAGVCTGMCCGAGTGAGC To change or delete a consensus base click the base you want to edit then delete or change the base To insert a base in the consensus sequence click between two bases then insert the base Note The changed bases appear in lowercase To delete a space click the space to select then press the Delete key To insert a space click where you want to insert a space then press the dash key or space bar Note If the audit feature is enabled you must enter a reason for each base change base insertion and base deletion ABI Prism SeqScape Software v2 1 User Guide 8 15 Chapter 8 Reanalyz
201. gion FGR Fwd Coverage Percent of target region coverage by specimen consensus sequence generated from forward reads FGR Rev Coverage Percent of target region coverage by specimen consensus sequence generated from reverse reads 5 Reg Coverage 5 regulatory region coverage Percent of specimen consensus sequence covering the 5 regulatory region 5 Reg Coverage Avg QV Average quality value of the consensus sequence at the 5 region 5 Reg Fwd Coverage Percent of target region coverage by specimen consensus sequence generated at the 5 region for forward reads 5 Reg Rev Coverage Percent of target region coverage by specimen consensus sequence generated at the 5 region for reverse reads Exon Coverage Exon coverage for a specific gene Exon Avg QV Average quality value of the consensus sequence at the exon region Exon Fwd Coverage Quality value of the consensus sequence at the exon region for forward reads Exon Rev Coverage Quality value of the consensus sequence at the exon region for reverse reads Intron Coverage Intron coverage for a specific gene Intron Avg QV Average quality value of the consensus sequence at the intron region Intron Fwd Coverage Quality value of the consensus sequence at the intron region for forward reads Intron Rev Coverage Quality value of the consensus sequence at the intron region for reverse reads Genotyping Table Displays genotypes of all specimens at sele
202. gtgggctacgtggacgacg Reference AA P G R G E P R F I A v G Y wv D D b 3602 Smeg a gm nuno ee i ne CTTCUTCKCAGTGGGCTACGTGGACGACA 4 2 isl J Saving Your Data When you finish save your project Select File gt Save Project or click Save Project ABI PRISM SeqScape Software v2 1 User Guide 8 21 Chapter 8 Reanalyzing and Editing Data Adding a Genotype To add a genotype to the Genotype Report 1 In the Project View select a specimen base right click and then select Add Genotype COO O I I PUES LA C v2 Known Variants IHLA C_CDS ROls Il Variants Summary GeTcCCACTCCATGA TATTTcKWCACnkCyGTGTCCcGC Variants GcTcCCACTCCATGA lcETsTTTCc TMC ar GCTCCCACTCCATGA Add Variant GCTCCCACTCCATG A MESES Show Genotypes List nCGTGTCCcGC GTGTCCCGE GTGTCCCGE 2 To view the genotype list select the specimen base right click and then select Show Genotypes List Genotypes List x The following genotypes will appear in the Genotype Report when the Show checkbox is true Show Positian ROI v 175 R lName wm ROIName Wc AOIName vw 302 ROI Name 3 Select the Show checkbox to display that position in the Genotyping Report See Genotyping Report on page 7 39 8 22 ABI PRISM SeqScape Software v2 1 User Guide Exporting and Printing Data and Reports 9 This chapter contains Exporting Data Files Vue rm bre tea 9 3 Exporting Reports leeleeeeee eh 9 8
203. hange Base 8 c T red T exonBen1 2s yes Change Base E t A yellow 7 exonWH88 yes Delete cca blue T exoniWi 158 yes Change Base c T red 7 exonGB11su yes Change Base c A jred _ exonMG99 3 lyes Change Base c G red 7 exonMG99 4 yes Change Base a T red 7 exonYL A2su yes Change Base a G red 7 exonH8surg yes Insert After c GAC yellow 7 exonPX75xe yes Delete tga red 7 exonSh11su yes Change Base t A jred 7 exonNo7 35s yes Change Bace g A red T exonYS 58s yes Change Base t G blue 7 exonSC97 4 lyes Change Base t c red 7 exonMG98 1 yes Change Base a C red 7 exonGA020 yes Change Base c red 7 exon14Tsurg yes New Open Export Delete Table Format General z OK Cancel 8 Click OK to save the imported variants and close the RDG Properties window ABI Prism SeqScape Software v2 1 User Guide 4 33 Chapter 4 Creating a Reference Data Group Creating an RDG from Aligned Consensus Sequences About Creating an RDG Importing NT Variants from an Aligned FASTA File 4 34 SeqScape software will create a new reference sequence and variants from a set of aligned sequences imported into a blank RDG that contains no reference sequence The file format of the imported aligned sequences must be in FASTA text For more information on FASTA format see Appendix E Aligned Variant and FASTA File Format SeqScape software uses the first sequence in the set of aligned seq
204. he Delete key IMPORTANT This deletes the results and cannot be undone If you press Delete in error close the project without saving to restore the results 3 In the Confirm Delete dialog box click Yes Analyzing the Data After you import all your data you can run the analysis After new data is imported or analysis settings are changed for a sample the Analyze icon in the toolbar appears green indicating that there is unanalyzed data Running an To run an analysis in the project click E Analyze or select Analysis Analysis gt Analyze ABI PRISM SeqScape Software v2 1 User Guide 6 21 Chapter 6 Creating and Analyzing a Project Reanalyzing a Project Using a Different Project Template When You Would After you analyze an entire project that contains many samples you Want to Do This may want to reanalyze all the data using a project template that contains different settings or reference data Saving a Project IMPORTANT Applying a new project template discards all analyzed Before data including basecalls features alignments and manual edits To Reanalyzing avoid discarding the data rename the project to keep your original analysis if desired To save a project that you want to reanalyze 1 Select Tools SeqScape Manager 2 In the Project list select the project that you want to save before reanalyzing 3 Click Save As and rename the project 4 Click OK The project is saved under a new name and y
205. he Project 200 e eee eee 6 31 Importing Variants to the Project 220000 6 32 Importing and Exporting Project Information 6 34 About Importing and Exporting 2c eee eee eee 6 34 Importing from SeqScape Manager 22005 6 34 Exporting from SeqScape Manager 00ee eae 6 34 Chapter 7 Viewing the Results Workflow for This Chapter 00 000 eee eee eee 7 2 About the Project Data 0 naaa e ee ee 7 3 View Formats vic viscid dE ex Hee Ga ee Re e ese 7 3 Data Display Conventions 0 000 eee 7 3 Quality Value Display lessen 7 3 Exporting and Printing Project Data 2 005 7 3 Project VieWS cu eanta Bee Rude ea ote 7 4 Displaying the Project Views elles 7 4 Specimen Views 00000 een 7 10 Segment VIBWS web relidgu mE Legg eae ewe ween gee elles 7 11 Displaying the Segment Views llseselesseeens 7 11 ABI PRISM SeqScape Software v2 1 User Guide vii Chapter 8 viii Sample Views a n re ea a a E aaa a anaa iaa a ees 7 15 Displaying the Sample Views 0000 e eee eee eee 7 15 Viewing Variants s nniess ri f gemskkclemvGieL rkc rg ves 7 20 Saving Your Data nesa i eaea NEEE eee 7 21 About the Reports lesus ce eee eee eee 7 22 Types of Reports 0000 cee ee 7 22 Exporting and Printing Reports 00 e eee eee 7 22 V
206. he accession number At the top of the page next to Display select GenBank then select Send to File The file will be saved to the specified location This file can then be imported into the RDG The file can have a gb fcgi or cgi extension How can designate part of my sequence as untranslated intronic region You can designate part of the sequence as untranslated by deselecting the Translate check box for that ROI You can designate a region of the sequence as untranslated by first highlighting the desired section of the sequence in ROI tab and clicking on Add ROI This region will then appear in the ROI table Select the layer where you would like this ROI to appear and deselect the Translate check box How can change the number of the first base in the reference sequence How can reset the first codon You can designate the first base codon in the reference segment pane of the ROI tab This is the pane that shows selected reference sequence Click in the box on the top left of the pane and enter the desired number How can change the translation frame You can change the translation frame in the ROI tab of the RDG Can use an implicit reference sequence in SeqScape software No SeqScape software does not support the use of an implicit reference sequence However you can use ab1 files and genbank files as reference sequences What is a reference break A reference break is a br
207. i 7414348 emb AJ277102 1 H 792 276 2l Layer 1 settings _NewLayer Layer Name avert Index Codon Number ff Orientation Library x Translation Frame fz Risht ROI Name Segment Seg Start Seg End ROlBtet ROlLength Translation Color onLayeri AF250557 1 2 amp gi 7414348 emblAJ277e gi 7414348 emblAA 1 HLA C exon2 AF250557 HLA C gene AF250557 HLA C_intron2 AF250557 misc c c QCctcccactc catgaggtat ttctacaccg ccgtgtcccg 40 gcccggccgc ggagagcccc gcttcatc c agtgg ctac 80 gtggacgaca cgcagttcgt gcagttcgac agcgacgccg 120 cgagtccaag aggggagccg cgggcgccgt gugtggagca 160 ggaggggccg gagtattggg accgggagac acagaagtac 200 amp agcgccagg cacagactga ccgagtgagc ctgcggaacc 240 tgcdcggcta ctacaaccag agcgaggccg gtgagtgacc 280 ccggcccggg degcaggtca cgacccctcc ccatccccca 320 ztgagatc 360 zl Add ROI Save To Manager As OK Cancel In the New NT Variant dialog box note that the Position and the Reference base are already entered Select the type of variant by clicking Base Change Insertion or Deletion then enter the Variant base Select a style for the variant then enter a description of the variant if desired Click OK to save the variant to the project ABI Prism SeqScape Software v2 1 User Guide 6 31 Chapter 6 Creating and Analyzing a Project Importing When you import variants into a project they must be in one of the Variants to the following configurations P
208. ide variants nn S sel Add amino acid variants Link a Library Setup variant styles Chapter 5 3 Create Project Template Select gt RDG Analysis Defaults Display Settings Chapter 6 4 Create and Analyze Project Select Project Template p Software does automatically Add Specimens Import Sample Files Basecalling Filter Assembly Consensus Alignment Comparison Search Library Chapter 7 5 View Data and Reports Data Report Manager Project View Analysis QC Report Specimen View Mutations Report Segment View AA Variants Report Sample View Specimen Statistics Chapter 8 Report Sequence Confirmation 6 Reanalyze and Edit Data Report Base Frequency report Library Search Report Chapter 9 RDG Report Reports Audit Trail Report 7 Export and Print p Genotyping Report Aligned projects Consensus sequence Aligned sample sequence Sample files Figure 7 1 Showing the View Data and Reports Steps 7 2 ABI Prism SeqScape Software v2 1 User Guide About the Project Data About the Project Data View Formats You can view the results in multiple formats Project view A summary of all the specimen consensus sequences Specimen view A summary of all the segment sequences within each specimen Segment view A summary of all the sample sequences within each segment Sample view A summary of the data for e
209. iewing the Reports sisit d 00 cece eee 7 23 Viewing the Reports and Project Results 7 25 Analysis QC Report ei sarees nan aeaa a EE eh 7 26 Mutations Report 00000 eee 7 28 AA Variants Report 0000 eee 7 31 Specimen Statistics cre Robe RE ws ws dads EU 7 32 Sequence Confirmation Report s sssaaa naana 7 33 Base Frequency Report 0 00 cee eee ee 7 34 Library Search Report 0 cee eae 7 35 RDG Report sss dU RR EE pue Poe be ele A 7 37 Audit Trail Report 2 0 2 n erens e ER ato ap ia gee eee ele 7 38 Genotyping Report irsi arius nre eee ees 7 39 Customizing the Reports 0 0 0 cece eee eee 7 42 Customizing Text Settings 0 0 0 eee eee 7 42 Customizing the Data View 2 000 eee 7 44 Reanalyzing and Editing Data Workflow for This Chapter llle 8 2 About Analysis Parameters 00 0 eee 8 3 Introduction ERE Re eed Pee gw ee eee 8 3 Viewing Analysis Parameters in the Sample Manager 8 4 Changing the Analysis Parameters in the Sample Manager 8 6 Adding Samples to the Sample Manager 8 6 Changing Basecaller and DyeSet Primer Files 8 6 Changing the Analysis Parameters in an Analysis Protocol 8 7 Editing an Analysis Protocol 00 e eee eee eee 8 7 Applying the Analysis Protocol 000 eee eee 8 12 ABI PRISM SeqScape Software v2 1 User Guide E
210. ilter omit poor quality sequences Assemble sequences to the reference Generate a consensus sequence for each specimen Align consensus sequence to the reference sequence Compare each consensus to the reference O ON m mo Search the allele Library for matches to each consensus sequence This step may be bypassed if you do not have a need to identify allele matches 10 Generate Reports B 4 ABI PRISM SeqScape Software v2 1 User Guide General SeqScape Software FAQs General SeqScape Software FAQs Table B 4 General Questions and Answers Question Answer Instruments What Applied Biosystems instruments can use to generate data for SeqScape software SeqScape software analyzes sequence files generated from ABI PRISM 3700 3100 3100 Avant 377 310 and Applied Biosystems 3730 and 3730x genetic analyzers The software also accepts text sequences in FASTA format Instruments Can SeqScape software be used to analyze data that was generated on instruments other than ABI PRISM or Applied Biosystems instruments No Sequencing data generated on platforms other than Applied Biosystems platforms are not compatible with SeqScape software Sample files Can SeqScape software be used for autoanalysis of sample files SeqScape software v2 1 can be used to autoanalyze sample files from Applied Biosystems 3730 3730xl DNA Analyzers and ABI PRISM 3100 3100 Avant Genetic Analyzers For mor
211. imiter SeqScape software simultaneously and Specimens and automatically creates specimens and imports unanalyzed or analyzed Importing samples into a project Samples Automatically Sample IDs and Sample File Names To take advantage of this feature your sample ID which is created by the data collection software and stored within each sample file needs to contain the same prefix for all samples in each specimen IMPORTANT The sample ID sample name is the name that you assign to the sample in the data collection software You cannot modify the sample name The sample file name is longer than the sample ID and often is derived from the sample ID The sample file name is what you see when looking for the sample You can modify the sample file name ABI PRISM SeqScape Software v2 1 User Guide 6 11 Chapter 6 Creating and Analyzing a Project 6 12 The text delimiter is chosen from the sample ID Using this function a set of sample files that are grouped into the same folder and that share a similar delimiter can be imported into their corresponding specimens in a single step In the example shown in Figure 6 3 the delimiter is a dash Everything to the left of the delimiter determines the specimen name When you select Add Automatically the sample files are automatically transferred into specimens that are also created and named automatically In this example the first specimen includes all files that start with A1 The s
212. ing New NT Variants llle else 4 30 Importing NT Variants in Tab Delimited Format 4 32 Creating an RDG from Aligned Consensus Sequences 4 34 About Creating an RDG 2 2 ee ee 4 34 Importing NT Variants from an Aligned FASTA File 4 34 Entering New AA Variants llle 4 36 Importing AA Variants 2 2 0 cee ee 4 38 Assigning Styles to Variants lille 4 39 Saving a Copy of the RDG 2 eee 4 41 Saving the RDG for Other Projects 2000 4 41 Save To Manager As Button naaar eee eee 4 42 Creating a Project Template Workflow for This Chapter lees 5 2 Creating a Project Template 00 0c eee eee 5 3 About Creating a New Project Template 5 3 Creating a New Project Template 000 0005 5 4 Saving Project Components 220 0c eee eee eee 5 5 About Saving Template Components llus 5 5 Saving Template Components from Within a Project 5 5 Examples of Changing the Settings Within a Project 5 6 Creating and Analyzing a Project Workflow for This Chapter 00000 eee eee ee eee 6 2 Before You Begin Creating a Project 0 000 6 3 What an Analysis Entails 0 0 0 0 eee ee ee 6 3 Ways to Create and Analyze a New Project 22 6 4 Using the New Project Wizard to Create and Analyze a Project 6
213. ing and Editing Data Adjusting the Clear Range About the Clear 8 16 Range Sample data usually has unreadable or otherwise unusable sequence located at the beginning and end of the data Inclusion of this data causes errors in the alignments and erroneous variant detection The clear range is the area of continuous sequence that is the most error free In SeqScape software the clear range is set automatically for all samples during the analysis based on the Analysis Settings for that sample You can modify the clear range on a per sample basis IMPORTANT If you do not select Use Reference Trimming in the Analysis Settings you should manually set the clear range to remove any sample data that lies 5 of the 5 end or 3 of the 3 end of the reference if needed Any sample data that is outside the reference is not aligned IHLA C 3100 360 2 AF250557 360 2 CX3F 01 Annotation Sequence Features Electropherogram Raw i 2l 31 al S1 GGGCGGGG C GGG TCTCACAC CCT CCAG UGG ATGTRYGGCTGCGACCTGG Wi 3 ch 560 Data outside the Clear range data clear range Figure 8 3 Clear Range Data After changing the clear range the specimen is automatically reassembled then realigned and recompared to the reference ABI PRISM SeqScape Software v2 1 User Guide Adjusting the Clear Range The three methods for changing the clear range involve using the Clear range widgets Mouse Set
214. initions and Naming 0 0 cee eee BI A 2 Basecaller Algorithm llis A 2 DyeSet Primer File srs aaea e ee eee A 2 DyeSet Primer File Naming Conventions 4 A 2 Basecaller and DyeSet Primer Compatibility A 4 ABI PRISM 310 Genetic Analyzer Files 00 020 eee eaee A 5 ABI Prism 377 DNA Sequencer Files 200000 ee eee A 8 ABI PRISM 3100 Genetic Analyzer Files 200 000 A 10 ABI PRISM 3100 Avant Genetic Analyzer Files Ln A 13 ABI PRISM 3700 DNA Analyzer Files llle A 15 Applied Biosystems 3730 3730xl DNA Analyzers Files A 17 Frequently Asked Questions Upgrading FAQS sse DE Ear B 2 Training and Documentation FAQs llsleleleesesns B 3 SeqScape Software Basics FAQS 0 c cece eee ees B 4 General SeqScape Software FAQS 0 0c cece ee eee B 5 SeqScape Manager FAQs 0 000 e eee eee eee B 9 Library FAQS usto cute x eere eae epe CROP sen d GNU a e B 14 Mutation Variant HIM and HFM Detection FAQs B 15 Data Analysis FAQs 0 cece eee B 16 Analysis Reports FAQS 0 00 e eet eee B 18 Quality Values FAQS icit eere RELATA RODR USR IUE B 19 Printing and Exporting Results FAQs 00 00 0000 ee B 20 Audit Trail Security and Access Control FAQs B 21 xi ABI PRISM SeqScape Software v2 1 User Guide Appendix C App
215. ion and then data analysis without requiring user interaction Autoanalysis requires three software packages e 3730 3730xl or 3100 3100 Avant Data Collection software v2 0 Data collection software is used to run the instrument and collect fluorescent data from samples For autoanalysis to occur the data collection software must be set up properly to allow communication with the analysis software Data collection software uses a data service Data used for data collection as well as that created in SeqScape software can be accessed through the data service in data collection software Autoanalysis Manager Autoanalysis Manager software is part of the integration between data collection SeqScape and GeneMapper software It queues messages and tracks the status of their processing Each message is considered a batch job whether it contains a single sample samples from a result group or an entire run of samples Autoanalysis Manager is installed by SeqScape or GeneMapper software when loaded on a system with data collection software A version of SeqScape software with no user interface This version of SeqScape software is identical to the regular version of the software except that no user interface exists The Autoanalysis Manager opens and uses this version of software to analyze the data in the projects The automated processing version and the standard version of SeqScape software are installed from the SeqScape Softw
216. ions in the genome The reference sequence can contain features such as exons introns splice junctions primer binding sites and promoter regions Heterozygous insertion deletion mutation SeqScape identifies potential instances of this variant which often require manual review by trained personnel Library searching You can compare each consensus sequence to a sequence library to identify the closest match genotype allele or haplotype Enhanced reports You can customize reports Each variant in the report is hyperlinked to the sequence data providing rapid transition from results to data The results reports eliminate the need to manually record results You can automatically sort and reorganize any report Password protection and audit trail The software protects your data by providing password protection automatic lockout when the software is inactive and three levels of access control An audit trail records each manual insertion deletion or base modification with reasons for each change Integration automation The software uses an improved process for setting up samples for automated analysis on Applied Biosystems 3730 3730xl instruments ABI PRISM SeqScape Software v2 1 User Guide 1 3 Chapter 1 Introduction to ABI PRISM SeqScape Software New Basecallers The KB basecaller is a new algorithm that identifies mixed or pure bases and generates sample quality values The ABI basecaller is an algorithm use
217. is returned to Applied Biosystems within the ninety 90 day warranty period Applied Biosystems will replace the defective media Applied Biosystems does not warrant that the software will meet buyer s requirements or conform exactly to its documentation or that operation of the software will be uninterrupted or error free Any applicable warranty period under these sections begins on the earlier of the date of installation or ninety 90 days from the date of shipment for software installed by Applied Biosystems personnel For all software installed by the buyer or anyone other than Applied Biosystems the applicable warranty period begins the date the software is delivered to the buyer Warranty claims must be made within the applicable warranty period The above warranties do not apply to defects resulting from misuse neglect or accident including without limitation operation outside of the environmental or use specifications or not in conformance with the instructions for the instrument system software or accessories improper or inadequate maintenance by the user installation of software or interfacing or use in combination with software or products not supplied or authorized by Applied Biosystems and modification or repair of the product not authorized by Applied Biosystems THE FOREGOING PROVISIONS SET FORTH APPLIED BIOSYSTEMS SOLE AND EXCLUSIVE REPRESENTATIONS WARRANTIES AND OBLIGATIONS WITH RESPECT TO ITS PRODUCTS
218. isplay Settings Analysis Protocol The Analysis Protocol Editor tabs and descriptions are used to set up Editor Tabs the analysis protocol to select with the analysis defaults The General tab contains general information on the analysis protocol for example the name creation date and modification date Refer to Figure 3 2 The Basecalling tab has settings for how the software calls bases The basecaller you select is determined by the instrument and chemistry you are using For further details on basecalling files and dye primer set selections see Appendix A Basecallers and DyeSet Primer Files In the Mixed Bases tab the Use Mixed Bases Identification box generates calls following the international standard IUB code for heterozygous positions Mixed bases identification occurs only if secondary peak threshold is equal to or more than a specified percentage of the highest peak You set the level according to sample type reaction kit and purification reaction Clear Range is the region of the sequence that remains after excluding the low quality or error prone sequence at the 5 and 3 ends and the M13 primer sequence if applicable You can specify a range as a default It is recommended that you always select Use reference trimming The Filter tab sets the criteria for rejecting sequences if they do not meet minimum standards Sequences not meeting the filter settings are not assembled Specifying the To specify the basecall
219. ist ABI Prism SeqScape Software v2 1 User Guide 4 11 Chapter 4 Creating a Reference Data Group Creating a New RDG Using SeqScape Manager Before You Begin Creating an RDG from SeqScape Manager About the Reference Sequence You must have administrator or scientist privileges to create a new RDG using SeqScape Manager Before creating a new RDG make sure you Download a GenBank file a FASTA text file or have a reference sequence that is stored on your computer Define on paper the ROIs layers and segments to compare to the reference sequence Creating a Reference Data Group requires that you Import reference segments Create ROIs Create layers To create a new RDG from the SeqScape Manager 1 In the main SeqScape window select Tools gt SeqScape Manager then select the Reference Data Group tab 2 Click New 3 In the General tab enter a name in the Reference Data Group Name field The General tab contains general information about the RDG 4 Select a Codon table type and add comments if desired 5 Select the ROI tab IMPORTANT Do not click OK More steps are needed to set up the RDG The reference sequence is made up of one or more reference segments that become a backbone or reference to which all other sequences or regions of interest are compared After the reference sequence is imported into the RDG it cannot be changed or edited ABI PRISM SeqScape Software v2 1 User Guide
220. istry Capillary Array Length cm Basecaller DyeSet Primer ABI Basecaller ABI PRIsM BigDye Primer v1 0 and v1 1 47 Basecaller 310POP4 bcp DP310POP4 BD 21M13 v1 mob DP310POP4 M13Rev v1 mob 47 Basecaller 310POP6 bcp DP310POP6 BD 21M13 v1 mob 61 DP310POP6 M13Rev v1 mob SO I4 Jeuiug 1ege q pue siejeoeseg v xipueddy epint JasN zA auemyog edeogbeg slug Ig Z V Table A 3 310 Basecaller and DyeSet Primer Files Used for Dye Primer Chemistry continued ABI PRISM BigDye Primer v3 0 and v3 1 47 Basecaller 310POP4 bcp DP310POP4 BDv3 21M13 v1 mob DP310POP4 BDv3 M13Rev v1 mob 47 61 Basecaller 310POP6 bcp DP310POP6 BDv3 21M13 v1 mob DP310POP6 BDv3 M13Rev v1 mob SeJIj Jez Ajeuy 2neuec OLE slug gv gt ABI PRISM 377 DNA Sequencer Files PIND Jesf L ZA ejeMjjos edeosbes NSl d gv Table A 4 377 Basecaller and DyeSet Primer Files Used for Dye Terminator Chemistry DNA Sequencing Chemistry WTR cm Scan Rate scans hr Basecaller DyeSet Primer ABI Basecalling e ABI PRISM dGTP BigDye v3 0 Terminator e ABI PRISM BigDye 36 2400 Basecaller 377 bcp DT377 BD mob Terminator v1 0 and v1 1 ABI PRISM dGTP BigDye 36 amp 48 1200 Basecaller 377LR bcp Terminator ABI PRISM dRhodamine 36 2400 Basecaller 377 bcp DT377 dRhod mob Terminator 36 amp 48 1200 Basecaller 377LR bcp e ABI PRISM BigDye 36
221. ividual sample sequence strands The possible redundancy of calls from strands in the same orientation The possibility that the basecaller missed a mixed base The possible values for the QVs range from 1 to 50 Higher numbers indicate calls that the algorithm determined with a measure of confidence while lower numbers indicate calls that might require user inspection to verify the correct answer The consensus quality values are roughly calibrated to follow the same scale as the per base sample quality values Consensus Score Aconsensus score is generated from consensus QVs It represents the average quality value of the bases in the consensus sequence for a specimen ABI PRISM SeqScape Software v2 1 User Guide 10 5 Chapter 10 Sample and Consensus Quality Values Displaying Quality Values QVs are displayed as bars above each base in a sample Figures 10 1 and 10 2 The height and color of a bar indicates its value The taller the bar the higher the QV The color of a bar which is associated with its value is editable in the Display Settings Note QV bar height and color are identical for QVs 50 to 99 Project Navigator HLA C 3100 360 2 AF250557 amp HLA C 3100 amp UJ 360 2 Unassembled E amp AF250557 B 360 2 Cx2F_0 E 360 2 0x2R_0 Bl 360 2 CX3R B 3602 cxsF 0 gi 7414348 emblA Sample quality Numerical valu
222. lation Sets whether or not the ROI is translated Color Shows the color of the ROI Click to display the standard Color Picker dialog box if you want to select a different color for the ROI Note When an ROI is defined a default color is applied to the ROI based on the name of the ROI On Layer number Check box The label for this column changes based on the selected layer If the check box is selected the ROI appears on the selected layer ROIs can be associated with multiple layers However ROIs cannot overlap on a layer Therefore the check box is disabled if the Start Length range of the ROI overlaps with the range of an ROI already associated with the layer This prevents you from overlapping ROIs on the Layer table A dialog box appears if you try to select an overlapping ROI ABI PRISM SeqScape Software v2 1 User Guide 4 19 Chapter 4 Creating a Reference Data Group Creating a Library About the Library Using Aligned FASTA Files Using a Tool to 4 20 Align the Files You must classify your library as a haploid or diploid library and determine how many library matches you would like to see for each consensus sequence A library match is one allele or a pair of alleles that agree closely with each consensus sequence A haploid library contains sequences that have pure bases only AGCT When searching against a haploid library SeqScape software provides library matches and each library match contain
223. lay settings The parameters that govern the display of the data and results Glossary 2 ABI PRISM SeqScape Software v2 1 User Guide DyeSet Primer file The DyeSet Primer file compensates for the mobility differences between the dyes and primers and corrects the color code changes due to the chemistry used to label the DNA DyeSet Primer files are sometimes referred to as mobility files export Moving the data or settings from inside the SeqScape Software Data Store to outside the SeqScape Software Data Store either in ctf or txt format FASTA format A standard text based file format for storing one or more sequences filtered sample sequence A sample that has been processed by the basecaller factura filter algorithms of the pipeline genotype library A library where the allele sequences are either pure base or mixed base sequences When searching against a genotype library SeqScape attempts to find the best matches to the consensus sequence without trying different allele combinations Note This term is not used by SeqScape software haplotype library A library where the allele sequences are completely pure base sequences When searching against a haplotype library SeqScape attempts to combine haplotypes two at a time to find the best genotype match to the consensus sequence IUB IUPAC International Union of Biochemistry International Union of Pure and Applied Biochemistry
224. lect a folder location to store the project view b Change the file name if desired Note The default file name uses the project name with the element type suffix and the FASTA extension c Click Export ABI PRISM SeqScape Software v2 1 User Guide Exporting Data Files Exporting a To export a specimen Specimen 1 Open the project of interest 2 In the navigation pane select a Specimen icon 3 Select File Export Consensus Sequence or Aligned Sample Sequence FA Export Consensus Segments Folder x Look in E AppliedBiosystems amp e B E E SeqA5 0 SeqScape a HLA C 3100_Aadlignmentfsta File name fsta Export Files of type rasa format fsta ie Cancel FASTA format fsta 4 Complete the Export Consensus dialog box a Select a folder location to store the file Note The default file name uses the project name with the element type suffix and the FASTA extension If the number of segments in a project is Then c One Change the file name if desired Two or more Do not type a file name Note The individual segment names are used Any name you type is ignored b For the Consensus Sequence option select a file format in the Files of type drop down list c Click Export ABI Prism SeqScape Software v2 1 User Guide 9 5 Chapter 9 Exporting and Printing Data and Reports 9 6 Exporting a To export a segment Segment 2
225. les of type FASTA format fsta x Cancel In the Export Project NT Alignment dialog box select a destination for the exported data then click Export 6 27 Chapter 6 Creating and Analyzing a Project Importing To import variants Variants 10 6 28 Select Analysis RDG Properties 2 Select the NT Variants tab 3 4 In the Import NT Variants dialog box navigate to the project Select Import alignment file then select it Make sure the Files of type is set to All Files or Aligned Sequences x Look in 3 RPSeqScapeDataFiles J amp c zi wj HLA 3100_threeSpecimens_v2_NtAlignmentfsta s NewProject2 Ntlignment fsta File name JHLA 31 00_threeSpecimens_v2_NtAlignmentfsta Import Files of type aligned Sequences fsta Cancel Navigate to or select the file to import Click Import Select the reference segment in the drop down list in the Select Reference Segment dialog box then click OK The variants appear in the NT Variants table as Known variants The descriptions are the specimens in which the variants appear and the style is the default style for the variant type Select the Variant Style tab in the RDG Properties dialog box to change the default style in the RDG and enter a description of the imported variants Select the NT Variants tab to be sure the variants are Known Click OK to save the variants ABI PRISM SeqScape Software v2 1 User Guide
226. ll_1 Created with populator E DakarPT3 T SpatialNoFill 1 SpatialNoFill_1 Created with populator 8 Run StdSeq38 POP 1 ZBigDyeV3 El p New Edit Delete Import Export Analysis Protocols Find Protocol 3730BDTv3 KB DeNovo v5 3100POPB BDTv3 KB DeNovo v5 1 X SequencingAnalysis SeqScape_AP1 SeqScape 3100_SR_POP6_BDTv1_mixed_v2 SeqScape SequencingAnalysis ed New Edit Delete Import Export Figure 11 3 Protocol Manager Window ABI PRISM SeqScape Software v2 1 User Guide 11 9 Chapter 11 Automating Analysis 2 In the Instruments Protocols section click New or select an existing instrument protocol file then click Edit The Protocol Editor opens Name l Description Type REGULAR Run Module Fastseq50 POP7 1 b Dye Set 7 Bigdyev3 z a 3 Complete the Protocol Editor a Type a name for the protocol Type a description for the protocol optional Select Regular in the Type drop down list aoe p Select the correct run module for your run See the appropriate instrument user guide for information on selecting the correct run module e Select the correct Dye Set for your run See the appropriate instrument user guide for information on selecting the correct Dye Set f Click OK 11 10 ABI PRISM SeqScape Software v2 1 User Guide Creating Required Files in the Data Collection Software IMPORTANT I
227. llection of aligned sequences that are all variations of the same sequence This is the only type of library supported in SeqScape v2 0 An aligned allele library differs from a library of diverse sequences such as a library of different gene sequences and is also different from a library of unaligned sequences alignment The aligned reference sequence together with the aligned specimen consensus sequences alignment display A table of IUB codes space characters blanks and dots showing how the sequences within a project are aligned alignment score The number of mismatches between the aligned reference and the aligned consensus sequence for a given specimen allele An alternative form of a genetic locus analysis The complete procedure that SeqScape Software performs in a batch wise manner on sample data analysis defaults The default analysis settings that are stored in a project template analysis protocol The default settings basecalling mixed base identification clear range and trimming and filtering that govern sample analysis analysis settings The parameters that govern the basecalling trimming filtering and assembly of the analysis ABI PRISM SeqScape Software v2 1 User Guide Glossary 1 assembly The set of aligned and overlapping sample data that result from the sequencing of one PCR product or clone Assembly view Shows the specimen co
228. ly 6 16 ABI Prism SeqScape Software v2 1 User Guide Adding Specimens and Importing Data into a Project 6 In the Files section select the sample data files To import Then A single sample Select the single sample Multiple samples Ctrl Click to select contiguous or noncontiguous samples All samples in a folder Select the folder 3 Import Samples Ex Files l Audio CD D Local Disk C C AppliedBiosystems EC SeqScape H O AppSeqScape B Tutorial Data 5 0 HLA C B C HLA C_3100POP6_Tutorial D BC A1 fa A da AT 3F a A1 3R 02 1 fa A1 4F_01 ab1 fa A14R_02 ab1 Eam A2 a A2 2F_03 ab1 a A2 2R_04 ab1 a A2 3F 03 ab1 a A2 3R_04 ab1 a A2 4F_03 ab1 IV Show abl Samples File Only A2 4F 04 ab1 eae E Use sample file name C Use sample name Samples To Add Remove Sample lj Specimen2 lj Specimen New Specimen d a3 Delete Specimen ee Automatically create Specimen name based on the string between these delimiters in the sample s name Start End xi 7 Click Add Sample OK Cancel The sample data appears in the selected specimen showing where the data will be imported No data is actually imported into the project until you click OK ABI PRISM SeqScape Software v2 1 User Guide 6 17 Chapter 6 Creating and Analyzing a Project 6 18 8 S
229. ly Consensus Alignment Comparison Search Library 5 View Data and Reports Data m Project View Specimen View Chapter 8 6 Reanalyze and Edit Data Chapter 9 Segment View Sample View gt Analysis QC Report 7 Export and Print Reports FL Aligned projects Consensus sequence Figure 4 1 Aligned sample sequence Sample files Report Manager Mutations Report AA Variants Report Specimen Statistics Report Sequence Confirmation Report Base Frequency report Library Search Report RDG Report Audit Trail Report Genotyping Report Showing Create Reference Data Group Step ABI PRISM SeqScape Software v2 1 User Guide Reference Data Group RDG Reference Data Group RDG About the The Reference Data Group defines the sequence to which the Reference Data ABI PRISM SeqScape Software Version 2 1 compares the Group consensus segments to the reference sequence It contains the reference sequence and reference associated data The reference sequence is the entire backbone sequence for the project consisting of one or more reference segments separated by reference breaks The RDG contains all the gene analysis specific information consisting of Areference sequence containing continuous or discontinuous sequences made up of one or more reference segments Nucleotide variants Amino acid variants
230. m mi m Active Layer HLA C CDS z Tab jumps to nex Project Navigator LA 31 00 ethreeSpecimens 2 i Al AF250557 A1 3F 01 H Bea Annotation Sequence F 5 Electropherogram Raw B Al nasssm Feature key Panas Description G AF25055 lear Range 292 This is the clear range i A1 3F ABI_Multibase F w i BAR ABI_Multibase 42 y Bai 2R A amp BI Multibase 92 M Bar Bl Multibase 150 Y m ABI_Multibase 159 S 3 gij741434 BL Da ABl Mullibase 211 Y Tas ABI_Multibase 327 Y ABI_Multibase 341 S Bl Multibase 784 R ABI PRISM SeqScape Software v2 1 User Guide 7 17 Chapter 7 Viewing the Results Table 7 4 Sample Views continued Tab Display Electropherogram Active tools Fl HL4 3100_three Project Navigator 5 HLA 3100_threeS EESE T S E sj mieu E Active Layer HLA C CDS z Tab jumps to nex ELE Unassem B AF250557 Bai 3F BL C GC Y Bai 3r Blai 2n Blair E gi 741434 Ua a Da3 1628 1480 1332 1184 1036 888 740 592 444 296 148 o uu YGCGG ACACS mm 131 141 151 161 SGCGGCTCAGATCACCCAGCGCAAGTGGGAGG m TNNT 1568 1624 1680 1736 1792 1848 1904 Qu Use Se Show Hide Sample QV to display hide sample QVs e Use Inverse Video to switch between white and black background displays e Use View Original Sequence to display hide the original sequence
231. mary ROIs that are created when reference segments are imported are locked as indicated by the lock icon These primary ROIs cannot be deleted from the ROI table but can be deleted from the Reference Sequence navigation pane by right clicking and selecting Delete ABI Prism SeqScape Software v2 1 User Guide ROI Tab Descriptions Columns in the The ROI pane in the middle of the RDG Properties dialog box has the ROI Pane following columns ROI numbers The number of the ROI The Reference Sequence on Layer 1 is always locked Reference segments that make up the Reference Sequence are also locked Unlocked layers are below the reference segments and can be edited ROI Name Name of the ROI ROI names that are not locked can be edited The ROI Name must be unique Note Names for Reference Segments are not editable in the ROI pane They can be edited in the Reference Sequence navigation pane by right clicking and selecting Rename Segment Name of the segment to which the ROI is associated Seg Start The nucleotide number in the Reference Sequence where the ROI begins Seg End End of the ROI segment ROI Start The first nucleotide number you assign to this ROI The number can be positive or negative ROI Length Length of the ROI The value is automatically recalculated if you change the Segment Start or ROI Length values Entering a number into this cell automatically recalculates the ROI Length value Trans
232. mation Report 3 Base Frequency Report 4 Library Search Report PT Modification Date 13 May 2003 at Reference Data Group RDG HLA C_Exon2 4_noNT 16 02 45 PDT RDG Report RDG Creation Date 03 Dec 2002 at RDG Modification Date 13 May 2003 at pereant Sants sf ping Rep Specimens in Report A1 A2 AB Specimen Statistics Spec Seg User Insertio Deletio Base Range Lengt Segme Sampl Continuo Coverag Match imen ment Edite ns ns Chang on nt es jus es Referen Score ce 704 792 25 4 1276 276 27 2 1 794 1 276 794 792 1 276 276 Sample Results Specimen Segment Orientation Assembled Clear Range on Sample Mixed Range Reference Score Base At AF250557 foward B338 1336 21 565 M AF250557 reverse B1454 12861 21 3 08 A HSA277402 reverse 290 24 1276 28 144 M HSA277102 foward 1283 54276 21 179 Al AF250557 reverse 49 38 482702 33 257 M AF250557 foward 20 292 520702 33 256 AEORnERT v moomo moam on 7 Report Settings Figure 7 6 Specimen Statistics Report 7 32 ABI Prism SeqScape Software v2 1 User Guide Sequence Confirmation Report Sequence Confirmation Report The report contains two separate tables Table 7 10 Parts of the Sequence Confirmation Report Table Description Summary Displays project information and the specimens in the report Sequence Displays the match the number of insertions Confirmation d
233. n What is a layer A layer is a set of ROls that are grouped together for the purpose of display report or amino acid translation The ROls within a layer cannot overlap Example Your project may contain introns 1 2 3 4 5 6 and exons 1 2 3 4 5 You may choose to create a layer that contains only exons 1 4 5 or a layer containing intron1 exon1 intron2 exon2 intron3 exon3 and intron4 A layer may represent a transcript ABI PRISM SeqScape Software v2 1 User Guide B 9 Appendix B Frequently Asked Questions Table B 5 SeqScape Manager Questions and Answers continued Question Answer How do generate a new layer You can generate a new layer by clicking on New Layer in the ROI tab of the RDG Then choose to add the desired ROIs by checking the On Layer check box in the ROI table Also select whether you would like the ROI translation turned in the layer Can put samples from different individuals in the same specimen No Each individual sample should be in a different specimen Refer to Chapter 6 Can mix samples from different biological sources It is not possible to analyze data from different biological sources in the same specimen What is a reference data group RDG The RDG is an essential part of the project template that contains all of the analysis specific information including the reference sequence translation codon table known variants RDG name reference
234. n amp Audit rAuthentication Settings pAuditTrail V Audit Trail On Lockout user after B invalid login attempts Reason Reason amp Base Change Reason 2_ Reason 3 Reason4 within 1 minutes Maintain lockout for 30 minutes Automatic timeout after 60 minutes Change password every po days New Open Export ABI PRISM SeqScape Software v2 1 User Guide 2 17 Chapter 2 Getting Started e Whenever a change is made in any of the project views the Audit Reason Editor dialog box opens as shown below Select the reason for the change from the drop down list lli Audit Reason Editor 1 x Event Sample sequence substitution Reason EERO ETE Description mE Cancel 4 If desired click Export in the Options General tab and navigate to export the configuration settings to another computer The Import button allows configuration settings to be imported from another computer 5 Click OK in the Options dialog box to save the authentication and audit settings Note Itis possible to import or export Authentication amp Audit configurations from one computer to another For example an administrator may want to set up authentication and audit information for many users then select all the files and export them to other systems using SeqScape software 2 18 ABI PRISM SeqScape Software v2
235. n you need to know before installing and using the ABI PRISM SeqScape Software Version 2 1 The administrator must follow the procedures in this section Administrator Registering the Software through Starting SeqScape Software for the First Time on page 2 10 License and Before you begin read Appendix F Software Warranty Warranty Information Appendix F explains your rights and responsibilities regarding the SeqScape software During the installation process of the software you must accept the terms and conditions of the Software License Agreement before the software can be installed Registering Your To register your copy of SeqScape software complete the Software registration card included in this software package and return it to Applied Biosystems Registering the software enables Applied Biosystems to send you notification of software updates and any other future information that may be specific to SeqScape software owners IMPORTANT Your product registration number is located on the registration card Be sure to record the number here before you return the registration card Registration Number 2 2 ABI PRISM SeqScape Software v2 1 User Guide Hardware and Software Requirements Hardware and Software Requirements Minimum System SeqScape software can be installed on a computer that meets the Requirements minimum requirements summarized in Table 2 1 below In general the more memory and the
236. nager Window 000000 eee eaee 2 22 Project Window leeren 2 23 SeqScape Software Toolbar lille 2 24 Menus on the Main SeqScape Window 2 0000 05 2 26 Workflow mn b RR ee eg dh Cem ete 2 28 Creating Analysis Defaults and Display Settings Workflow for This Chapter 0 0000 eee eee 3 2 Analysis Defaults Settings llle 3 3 Creating Analysis Protocols 0 00 eee eee ees 3 3 Analysis Protocol Editor Tabs 0 00 cee e eee eee 3 4 Specifying the Basecall Settings 0 eee eee 3 4 Specifying the Mixed Bases Settings 0 3 7 Specifying Clear Range 0 ee 3 8 Specifying the Filter Settings llle 3 10 ABI PRISM SeqScape Software v2 1 User Guide Chapter 4 Specifying the Analysis Settings llle Gap and Extension Penalties 0 0 00 eee Setting Analysis Defaults 0 00 c eee eee Selecting the Analysis Default Settings for Individual Samples Specifying Display Settings 000 0c eee eee Creating a Reference Data Group Workflow for This Chapter 20000 cee eee eee Reference Data Group RDG 2 0 0000 eee ees About the Reference Data Group 200000 eee eee GenBank Features 000 00 eee ees Downloading a GenBank File 0020020 cece eee About Creating a New Reference Data Group RDG s
237. nalysis protocols and display settings Access SeqScape Manager by selecting it from the Tools menu What is an object An object is a named collection of data elements to perform certain functions for example analysis protocol How do create a new user You must be logged in as Admin user 1 Select Tools Options 2 Select the Users tab then click New 3 Enter the new user name be sure to omit any spaces in the user name then click OK 4 To log in with the new name exit the software then relaunch it 5 Log in with the new user name What is a project in SeqScape software A project is created using a project template Projects contain sample data files grouped into specimens What is a project template A project template is the mold from which projects are created Templates contain analysis defaults display settings and a reference data group What is a specimen A specimen contains all the sample data from a single biological source SeqScape software will assemble all sample data within a specimen and generate a consensus sequence For example a specimen contains forward strand PCR products for exons 3 4 and 5 of a gene and several reverse strand PCR products for the same exons The software will generate a single consensus sequence representing exons 3 4 and 5 and compare it to the reference sequence Do not mix products from different biological sources into a single specime
238. nalyzing data 8 2 typical 2 28 ABI PRISM SeqScape Software v2 1 User Guide 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 9 2003 Part Number 4346367 Rev B an Applera business
239. nd SNP validation Common resequencing applications include SNP discovery and validation Mutation analysis and heterozygote identification Sequence confirmation for mutagenesis or clone construct confirmation studies Identification of genotype allele and haplotype from a library of known sequences SeqScape software allows analysis of resequenced data comparing consensus sequences to a known reference sequence and optionally searching against a sequence library For example a simple project might contain one contiguous reference sequence in a single reading frame with no known nucleotide or amino acid variant information SeqScape software compares a consensus sequence to this reference sequence identifying any differences A more complex project might include a reference sequence constructed from several reference segments representing multiple exons and introns You can use SeqScape software to Build unique sequence layers composed of different groupings of reference sequence features Compare consensus sequences to each unique layer Identify differences Compare the sequence to a library of sequences to identify the closest match ABI PRISM SeqScape Software v2 1 User Guide 1 5 Chapter 1 Introduction to ABI PRISM SeqScape Software Data Sources for Resequencing Projects Levels of Automated Analysis What the Software Does 1 6 You can create projects in SeqScape software using sequen
240. nd identity in the Analysis QC Report Can the SeqScape software separate the HIM sequence traces No SeqScape software cannot separate HIM sequence traces However SeqScape software will report how many bases were deleted or inserted ABI PRISM SeqScape Software v2 1 User Guide Appendix B Frequently Asked Questions Data Analysis FAQs Table B 8 Data Analysis Questions and Answers Question Answer How do begin analysis Click E Analyze in the toolbar or select Analysis gt Analyze What does a red line across a specimen indicate Strike through symbols indicate that analysis needs to be performed Can the SeqScape software handle gaps in sequence SeqScape software automatically inserts gaps in the sample and consensus sequences if these gaps are necessary to produce clean sequence alignments Gaps should be removed before importing sequences from FASTA formatted files What does the Alignment Score mean in the Analysis Report The alignment score shows the number of characters that were inserted in each specimen consensus to create the project alignment A lower alignment score indicates more similarity between the specimen consensus and the reference How does editing affect my data What gets updated If you insert delete or change a base within a sample the change is reflected in the consensus sequence All samples change to reflect the consensus edits H
241. not real variants so you cannot predict a real effect Heterozygous Specimen level heterozygous indel mutation deletion insertion HIM identification 7 30 ABI Prism SeqScape Software v2 1 User Guide AA Variants Report AA Variants Report The report contains two separate tables All blue text is hyperlinked to the project navigator Table 7 8 Parts of the AA Variant Report Table Description Summary Displays project information and the specimens in the report AA Variant Displays the AA changed position length type and description for each variant detected in a specimen U Specimen Statistics Report Sequence Confirmation Report Base Frequency Report Library Search Report RDG Report Audit Trail Report Genotyping Report Report Settings Summary Active Layer HLA C CDS Project HLA C 2 Project Creation Date 03 Dec 2002 at Project Modification Date 43 May 2003 at 23 40 04 PST 16 02 45 PDT Project Template PT HLA C_3100POP6_mixe PT Creation Date 03 Dec 2002 at d 5lib 23 20 08 PST PT Modification Date 13 May 2003 at Reference Data Group RDG HLA C Exon2 4 noNT 16 02 45 PDT RDG Creation Date 03 Dec 2002 at RDG Modification Date 13 May 2003 at 22 59 40 PST 16 02 45 PDT Display Settings DS TutorialDisplaySettings DS Creation Date 26 Nov 2002 at 23 37 59 PST zl Specimens in Report A1 A2 A3 Layer Translation Layer Translated Translation Fram
242. nsensus sequence as well as the aligned sample sequences Electropherograms and quality values can also be viewed basecaller An algorithm that determines the bases within a sequence during analysis There are two types of basecallers KB basecallers and ABI basecallers clear range The region of sequence that remains after excluding the low quality or error prone sequence at both the 5 and 3 ends comparison The relationship between the aligned specimen consensi and the reference sequence and the associated reference data consensus quality values See quality values consensus caller The analysis algorithm that is responsible for generating an accurate consensus sequence with per base quality values consensus sequence The output of the assembly from a biologically related group of samples constant position A position in the library alignment that is identical for every allele in the library See polymorphic position constant position error A position in a specimen consensus sequence that corresponds to a constant position in the library and that disagrees with the library at that position contig The set of aligned and overlapping sample data that results from the sequencing of one PCR product or clone Also known as an assembly crucial position A position in a specimen consensus sequence that differs among the set of matches returned after a library search disp
243. nter a sample name or click Browse and navigate to the sample you want New Project Wizard E x Select Representative Sample Enter the path name of one sample from the samples you are adding to the new project This sample helps determine the analysis settings you could use to analyze all the samples Sample Name oiSeqSc2 0 Advanced_Project DatalHLa C specimens 360 2 CX2F_01 ab1 Browse lt lt Back Next gt gt Cancel 5 Select a sample with the ab1 extension then click Open ABI PRISM SeqScape Software v2 1 User Guide 6 5 Chapter 6 Creating and Analyzing a Project 6 6 6 Click Next The wizard uses analysis settings based on your sample choice x Verify Analysis Settings Based on the representative sample these are the recommended analysis settings to use for this project Basecalling Ending Base Basecaller At PCR Stop IBasecaller 31 004POPA4UR bcp Zl I7 after 5 Newer fio acer Dyset Primer IC After 20 Ns DT3100POP6 BD v2 mob z II After 800 Bases Note The Wizard uses default settings for Mixed Bases Clear Range and Filter lt lt Back Next gt gt Cancel 7 Verify the analysis settings Basecaller DyeSet Primer files and Ending Bases then click Next ABI Prism SeqScape Software v2 1 User Guide Using the New Project Wizard to Create and Analyze a Project 8 In the Select Reference Data Group page C Use a GenB
244. ntron Intron Cov Avg Fwd Rev av Cov Cov A1 1000 30 5 1000 325 A2 A3 1000 312 0 0 00 00 00 0 0 0 0 0 0 0 0 0 0 00 00 00 98 9 98 7 1000 305 93 1 1000 325 766 1000 312 575 Genotype Table No Data 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 100 0 0 0 0 0 0 0 0 0 Figure 10 8 Genotyping Report 10 14 ABI Prism SeqScape Software v2 1 User Guide Automating Analysis This chapter contains Integrating SeqScape and Data Collection Software 11 2 Before You Start 0 0 eee eee eee 11 4 Required Files Created in SeqScape Software 11 6 Launching Autoanalysis Manger 0 00005 11 7 Creating Required Files in the Data Collection Software 11 8 Scheduling and Starting a Run 000 5 11 22 Understanding the Autoanalysis Manager 11 24 File Sharing Between Data Collection and SeqScape Software 11 28 ABI PRISM SeqScape Software v2 1 User Guide 11 1 Chapter 11 Automating Analysis Integrating SeqScape and Data Collection Software Overview Sequencing data that is generated on Applied Biosystems 3730 3730xl DNA Analyzers and ABI PRISM 3100 3100 Avant Genetic Analyzers can be automatically analyzed for use in the ABI PRISM SeqScape Software Version 2 1 Autoanalysis can be performed only on the same instrument computer that collected the sample files You can configure the software packages to perform data collect
245. numeric characters are not valid for user names or file names The invalid characters are below spaces M82 lt gt l An error message is displayed if you use any of these characters You must remove the invalid character to continue IMPORTANT User names cannot be named seqscape admin in this version of the software If you have used this user name in a previous version of the software you must change the user name to follow the File Naming Convention shown above ABI PRISM SeqScape Software v2 1 User Guide Starting SeqScape Software for the First Time Starting To start the software for the first time SeqScape 1 Double click the SeqScape software desktop shortcut Software ae p The SeqScape Registration dialog box opens HR SeqScape Registration 1 X Product Registration UserName ldminUser First Name admin Last Name User Password Re enter Password p Group Admin Organization Yours Registration Code 2 In the SeqScape Registration dialog box enter all the information in the text fields The User Name and password must be 6 to 15 characters long The first user created is automatically assigned Administrator privileges 3 Enter the registration code on the registration card you received with your software 4 Click OK The splash screen appears while the program is loading then the Log In dialog box opens ABI PRISM SeqScape Softwar
246. o review the crucial positions identified among the set of library matches returned against the selected specimen consensus sequence In order to view this pane click on a base in a specimen consensus sequence in the Project Assembly view You can adjust the height of this pane by using the drag split bar The crucial position columns are hyperlinked to the specimen consensus sequence base positions highlighted by the column selector in the Project Assembly view ABI Prism SeqScape Software v2 1 User Guide Mutation Variant HIM and HFM Detection FAQs Table B 6 Library Questions and Answers continued Question Answer What is a crucial position error A crucial position error is a polymorphic position that occurs in all the allelic matches It is the position that makes each allele unique to one another What is a constant position error A constant position error is a position in a specimen consensus sequence that is different than the corresponding position in all the allelic matches All of the allelic matches have identical base for that particular position Mutation Variant HIM and HFM Detection FAQs Table B 7 Mutation Variant HIM and HFM Detection Questions and Answers Question Answer Does SeqScape software account for heterozygous indel mutations HIMs For example a sequence with an insertion of three bases Yes SeqScape software will show possible HIM location a
247. o the previous variant to view or edit more positions Note Pressing Ctrl Z centers the selected column in the display even if snippets are not showing Ll HLa c 3100 Project Navigator 2 HLA C 3100 GAGAGCCTACCTGGAGGGCA CGGCCCGTGAGGCGGAGCAGHBGAGAGCCTACCTGGAGGGCA AGCAGWEGAGA 360 2 CX3R_02 AGCAGWEGAGA 360 2 CX3F_01 la uls ae a ABI PRISM SeqScape Software v2 1 User Guide Editing Variants Method 2 To view and edit variant data 1 Open the project of interest m de i dq Select Analysis Report Manager In the navigation pane select the report you want to view Select Window Tile Review the positions by selecting a base change in the Mutations table This adjusts the alignment view to the correct position in the alignment 6 To add an unknown variant to the RDG right click the unknown variant position in a consensus sequence in the project alignment then click Add Variant File Edit View Tools Analysis Window Help a pied ial EB amp UR gxr E E lia Ej 5 lza B fo i S Riya Active Layer HLA C_CDS z Tab jumps to netu Project Navigator S HLA C 3100 Known Variants IHLA C CDS ROIs Il Variants Summary YGGCCGCGGAGAGCCCGGCTTCUTCKCAGTGGGCTACGTGGACGACA NrEVanante Mixsace aceccecae acie aree 1068 T1 RR Gri aie d Bee ee cae ewe Index 50 60 70 80 Reference Fcggccgcggagagccccgcttcatcgca
248. ollowing tab delimited column headings NT Variant Headings AA Variant Headings Type Type ROI Layer NT position AA position Reference Reference Variant Variant Style Style Description Description Used by all ROIs An example is provided in Figure E 1 Nucleotide E Type of position of Reference Variant Variant Description variant the variant base base style of the variant Lad DB11 98 type1FULL Notepad Bl x Ht Format He Reference variant E Descriptio A C blue 41 L Nuc RTI AZT M4lL T215v 60 70 fold base 418 A T blue M 41 L Nuc RTI AZT Md4lL T215v 60 70 fold base 418 c T blue A 62 v Multiple Nuc Res A62V alone has no effec base 491 A G blue K 65 R Nuc RTI ddI Infrequently observed in base 496 G A blue D 67 N Nuc RTI AZT D67N K7OR T215Y K2190 1 base 503 c G blue T6955G MultiNRTI with 506 insertion peAntoni97 after 506 T AGTGGT blue T6955G MultiNRTI DeAntoni97 base 502 A T blue T695ssS MultiNRTI with 506 insertion after 506 T AGTTCT blue T69S55G MultiNRTI base 503 c G blue T69SSA MultiNRTI with 506 insertion base 504 T blue T69SSA MultiNRTI with 506 insertion Figure E 1 Sample of Variant Tab Delimited Text Format E 2 ABI Prism SeqScape Software v2 1 User Guide FASTA File Format FASTA File Format Note The information on FASTA was obtained from http www ncbi nlm nih gov BLAST fasta html FASTA Format A sequence in FASTA format begins
249. op Run Collection Started Collection Stopped Dyeset Primer Lot number Expiration date Instrument name rate in Hz Collection version Data Analysis Base Call Start Base Call End Peak 1 Location Ave Signal Intensity Channels Ave Basecaller Basecaller Version Base spacing used Length to Detector Tube Position Module file name Al 3F 01 3100 11557 901 1 24 2001 14 47 13 0 1 24 2001 17 16 14 0 1 24 2001 14 47 13 0 1 24 2001 17 16 14 0 DT3100P0P6 ED v2 nob 0010008 2001 05 01 12 00 00 000 SSTEST 1 7857142857142858 1 0 566 11557 566 G 1442 A 885 T 633 C 556 1 Basecaller 3100POP65R bcp BC 1 5 1b 3 14 219999 50 A3 StdSeq50 POP6DefaultModule 7 16 ABI Prism SeqScape Software v2 1 User Guide Sample Views Table 7 4 Sample Views continued Tab Display Sequence q BESE SSS Mj WPMD Acivelayer HOC CDS Tebjumpsto Project Navigator HLA 3100 threeSpecimens v2 Al AF250557 A1 3F 01 Ei E HLA 3100_three Annotation Sequence Features Electropherogram Raw p U i 1 CTCCAGAGGA TGTRCYGGCT GCGACCTGGG GCCCGACGGG CGCCTCCTCC bon AGBS 51 GCGGGTATGA CCAGTMCGCC TACGACGGCA AGGATTACAT CGCCCTGAAC B TEE GAGGACCTGC GCTCCTGGAC CGCYGCGGAC ACSGCGGCTC AGATCACCCA S TE 151 GCGCAAGTGG GAGGCGGCCC GTGAGGCGGA GCAGYGGAGA GCCTACCTGG B
250. opy of To save a copy of the RDG the RDG 2 3 In SeqScape Manager select the RDG you want to save Click Save As When the confirmation window opens rename the RDG or click OK Saving the RDG Ifyou are working with an RDG that is embedded in a project or for Other Projects project template you can save a copy of the RDG into SeqScape Manager This is useful if you make edits to an RDG and want to reuse the RDG for other projects To save the RDG 1 With the project open in the RDG Properties window click Save To Manager As Enter a name for the RDG then click OK to save a copy of the RDG under a new name x Save HLA C_ex2 4_with ariants to SeqScape Manager AS Name RDG Name jH L C ex2 4 withVariantsCopy OK Cancel If you accept the default name a copy of the original RDG is saved with that name and is available to use with another project ABI PRISM SeqScape Software v2 1 User Guide 4 41 Chapter 4 Creating a Reference Data Group Save To Manager 4 42 As Button The selections on the Analysis menu RDG Properties Analysis Defaults and Display Settings have the Save To Manager As button available for all tabs in each dialog box The purpose of saving these elements of the project is to have them available to use for changes to the project template for that particular project or other projects that are created To use the Save To Manager As button
251. ort in the Project view What format can print export reports in You can export reports in pdf xml htm or txt file formats You can print the exported reports or you can choose to print an open report by selecting File gt Print Can export and print individual ab1 sample files from the project Yes You can export and print individual ab1 files from within the project To do this select the sample file in the Project Navigator view Then click on a sample file and go to File gt Export gt Sample Sequence File The sample file can be exported in four formats seq fsta FASTA phd 1 PHD and ab1 You can print individual sample files by selecting File gt Print B 20 ABI Prism SeqScape Software v2 1 User Guide Audit Trail Security and Access Control FAQs Audit Trail Security and Access Control FAQs Table B 12 Audit Trail Security and Access Control Questions and Answers Question Answer What security and audit trail features are included in SeqScape software SeqScape software v2 1 has the following security and audit trail features e Three levels of user access e User lockout after certain time frame has passed Password expiration e Audit trail that can be created for base change insertion or deletion e Audit trail also includes time date stamp and reason for change e User name that is displayed when logged into the software e Audit Trail report
252. osed of one or more reference segments What is the reference The reference backbone is the entire reference sequence that may be backbone comprised of many reference segments The backbone is the first layer of the RDG and this first layer may not be modified B 10 ABI Prism SeqScape Software v2 1 User Guide SeqScape Manager FAQs Table B 5 SeqScape Manager Questions and Answers continued Question Answer What does splitting the reference mean and how do split it Creating a split can represent that the sequences are not contiguous One side of the split may contain Exon3 and the other side may represent Exon8 On the ROI tab click the base position where you would like to split the reference segment and select Split Reference Segment Where can find information on the ROI tab You can find information by clicking Info on the bottom left of the ROI tab within the RDG Can the ROI contain negative numbering Yes the ROI can contain negative numbering You can assign a negative number to an ROI by entering the number into the ROI table of the RDG Can the reference sequence contain negative numbering The reference backbone sequence cannot contain negative numbering however individual ROIs within the reference backbone can contain negative numbers How do I save GenBank files After finding the desired sequence at the NCBI website select the check box to the left of t
253. otide View shows only variants of the nucleotides The Expanded Amino Acid View shows all the amino acids Characters NT or AA that are the same as the reference are shown as dots The Character Dots button switches to show or hide the view The Identification pane which shows the library search results appears at the bottom of the Project view Specimen View Shows the clear range and orientation of each sample and how they line up to the reference sequence and the overview pane with active ROls Segment View A table of sample information Clicking a row in the table shows the corresponding sample sequence below The Layout tab shows the direction of each sample within the segment The Assembly tab shows samples aligned to the consensus sequence An overview pane that represents forward and reverse sequences variants and ROls Electropherograms can be displayed for one or all sequences Sample View Shows pertinent information for the sample which includes annotation sequence electropherogram and raw data Refer to Chapter 10 Sample and Consensus Quality Values for detailed descriptions of these views ABI PRISM SeqScape Software v2 1 User Guide 2 23 Chapter 2 Getting Started SeqScape Software Toolbar The SeqScape software toolbar displays buttons for software functions that you are likely to use often Refer to the next two figures for the names descriptions and keyboar
254. otocol Editing an To edit an analysis protocol Analysis Protocol 1 Add the samples to the Sample Manager see Adding Samples to the Sample Manager on page 8 6 2 In the Sample Manager window click Edit Analysis Protocol Analysis Protocol for A1 2F_01 E i x Mixed Bases Clear Range Filter SS Ending Base At PCR Stop Basecaller Basecaller 3100POP6SR bcp zl I After 5 Ns in 9 bases DyeSet Primer I After 20 Ns DT31 00POP6 BD v2 mob be T After 800 Bases Processed Data r Quality Threshold Call all bases and assign QV True Profile C Assign N for bases with QV lt 15 C Flat Profile 3 In the Basecalling tab a In the Basecalling group select the correct Basecaller and DyeSet Primer files in the drop down lists b In the Editing Base group select one or more stop points for data analysis optional ABI Prism SeqScape Software v2 1 User Guide 8 7 Chapter 8 Reanalyzing and Editing Data c In the Processed Data group select either True Profile Scales the processed traces uniformly so that the average height of the peaks in the region of strongest signal is about equal to a fixed value e g 1000 The profile of the processed traces will be very similar to that of the raw traces Flat Profile Scales the processed traces semi locally so that the average height of peaks in any region is about equal to a fixed value
255. ou have samples in the unassembled node of a specimen check the Analysis QC report to determine the reason why the sample files were not assembled The analysis QC report will show whether or not the sample assembled as well as the reason for failure at a particular point in the analysis pipeline Quality Values FAQs Table B 10 QV Questions and Answers Question Answer What are quality values A quality value is an estimation of the certainty for a basecall in the sample Sample QV or consensus consensus QV Is there an option to basecall without generating quality values No all of the basecallers in SeqScape software generate quality values However you can choose to not display the quality values by deselecting the confidence bar icons in the Views tab of the project s Display Settings How is the basecaller quality value generated It is derived using an algorithm that is designed to examine the certainty of basecalls See Chapter 10 Sample and Consensus Quality Values for more information What is the quality value equation QV 0log PE where PE is the probability of error How are sample quality values generated They are generated using a statistical algorithm which is calibrated to estimate the certainty of basecalls How is a sample quality value different from the sample score The sample score is the average quality value of the bases in the clear ran
256. our original project remains in the list 6 22 ABI PRISM SeqScape Software v2 1 User Guide Reanalyzing a Project Using a Different Project Template Applying a To reanalyze the project with a different template Template to an Existing Project 1 Create a template containing the desired changed settings and or reference sequence see Creating a New Project Using a Template on page 6 10 Open the existing project that has the data analyzed using the old settings IMPORTANT Save the project under a new name if you want to keep the current project data to compare to the new project data If you do not save the project all the data is overwritten when you apply a new project template Select Analysis Apply Project Template to open the Apply New Project Template dialog box P Apply New Project Template x Name Project Template ProjectOne_Template Template Elements Selected Project Template AlE SAL Reference Data Group HLA C ex2 4 withVariants Analysis Defaults 3100SR mixed Display Settings JMSsettings OK Cancel 4 In the Selected Project Template drop down list select the project template that you want to apply to the project 5 Click OK ABI PRISM SeqScape Software v2 1 User Guide 6 23 Chapter 6 Creating and Analyzing a Project 6 A dialog box opens warning you that all analyzed data and results will be discarded To continue click Yes
257. out Sequence To edit a sequence you can Editing Adjust the clear range Add delete or change a base in a sample Add or delete a space in a sample Add delete or change a base in a specimen consensus Add or delete a space in a specimen consensus Add or delete a space in a reference You can edit sequences within a project The change is immediately reflected in the consensus sequence You can also edit the consensus sequence In this case all the samples change to reflect the consensus edits You can edit consensus sequences when viewing the data in the Specimen view or in the Project view Note An edited base change or insertion appears in lowercase to distinguish it from an unedited base This applies to both user edits and consensus caller edits See Editing Bases with Quality Values on page 10 10 for more information on editing bases with QVs When to Edit the After analysis is complete and you generate the analysis reports Data depending on the results in the reports you may want to Adjust the clear range for a sample see Adjusting the Clear Range on page 8 16 Edita base or space in a sample or specimen see Editing a Sample or a Consensus Sequence on page 8 14 ABI PRISM SeqScape Software v2 1 User Guide 8 13 Chapter 8 Reanalyzing and Editing Data Editing a Sample or a Consensus Sequence Editing a Consensus Sequence in the Segment View Editing Sample Bases 8 14 To insert del
258. over the same regions as the layer that is associated with that library for example for a layer that contains exons 2 3 7 and 8 a valid library should have sequences from exons 2 3 7 and 8 A library with sequences covering exons 2 3 4 5 7 and 8 would be invalid A library will be associated to its specific layer Is there a minimum maximum number of Library Search match returns that can define There is no maximum however it does not make sense to request more than the number of alleles in the library The minimum should be 1 You can set the number of library matches to return in the Analysis Defaults Specimen tab of the project What is the difference between a haploid and diploid library In a haploid library all the sequences are pure base sequences A diploid library contains both mixed base and pure base sequences A haploid library will return 2 possible matches while a diploid library will return 1 possible match Can add a library to an open project from within the RDG properties button and see the search results instantly or will have to re analyze the project for the library search to be initiated The library is automatically searched immediately after loading a new library for the active layer and closing the RDG dialog box What is the function of the Library Identification pane How can view the Library Identification pane You can use the Library Identification pane t
259. ow can distinguish between edited and non edited data Edited bases are displayed as lower case letters while unedited bases are displayed in upper case letters What will happen to my edited sequence when start analysis Once basecalling begins all current edits are overwritten Changes to the analysis settings that do not require re basecalling of the sample preserve edits and the reference sequence What happens if edit a consensus base The base changes to lowercase in the consensus and the quality bar turns gray All bases in the samples at that position that disagreed with the new basecall are changed to agree with the new consensus base and are shown in lowercase with a gray quality bar How do remove unwanted spaces in my samples To remove unwanted spaces in the sample double click the space and press the Delete key What can do if deleted too many bases Repeat the analysis Is there an option to basecall without generating quality values No All of the basecallers in SeqScape software will generate quality values However you do not have to display the quality values You can choose to not display the quality values by deselecting the confidence bar icons in the Views tab of the project s Display Settings ABI PRISM SeqScape Software v2 1 User Guide Data Analysis FAQs Table B 8 Data Analysis Questions and Answers continued Question Answer Can
260. ow can use sample files generated on the Macintosh computer with SeqScape software To use data files generated on a Macintosh computer with SeqScape software you must convert the files using the SCU Sample Conversion Utility This utility is available as a Macintosh application file on the SeqScape Analysis software CD The SCU must be loaded onto and launched from a Macintosh computer For more information see the Read Me file associated with the SCU B 6 ABI PRISM SeqScape Software v2 1 User Guide General SeqScape Software FAQs Table B 4 General Questions and Answers continued Question Answer Chemistry What Applied Biosystems chemistries are supported e ABI PnisM BigDye Terminator v3 1 v3 v2 v1 1 and v1 chemistries ABI PRISM BigDye Primers and dRhodamine dyes Computer What are the computer requirements for SeqScape software CPU 733 MHz or faster single processor Memory 512 MB RAM e OS Windows 2000 with Service Pack 3 or Windows XP with Service Pack 1 e 1 GB hard drive e Pentium III or IV chip not Xenon Computer What kind of performance can expect from my SeqScape software That depends on your computer specifications For example if you have a computer with an 850 MHz processor 256 MB RAM Pentium III chip running on Windows 2000 OS and are analyzing 100 samples files 10 specimens and a 1Kb reference the analysis time i
261. ows Windows NT Windows 2000 and Windows XP are registered trademarks of Microsoft Corporation All other trademarks are the sole property of their respective owners Part Number 4346367 Rev B 09 2003 Contents Preface How to Use This Guide 0 0 eee ee eens XV Conventions Used in This Guide 0 0000 c eee eee eee xvi Workstation Safety 20 0 cece ee eee xviii How to Obtain More Information 0 0000 eee eee xviii How to Obtain Services and Support 0020020e ee aee xix Chapter 1 Introduction to ABI PRISM SeqScape Software New Features in SeqScape Software v2 1 llllllsu 1 2 New Features for v2 1 2 2 2 0 eee 1 2 For Users of Earlier Versions of SeqScape Software 1 3 Features of V2 0 0 cee eee 1 3 About SeqScape Software 0 00 cee eee 1 5 Genetic Analyzer Applications 00000e eee eee 1 5 SeqScape Software Applications 00000 eee 1 5 Resequencing Data with SeqScape Software 1 5 Data Sources for Resequencing Projects 1 6 Levels of Automated Analysis 0 00 eee eee eee eee 1 6 What the Software Does 00000 cee eee 1 6 How the Software Performs Analyses lees 1 7 Chapter2 Getting Started Administrator Registering the Software 2 0 eae 2 2 License and Warranty isssseeeeeeeee 2 2 Registering Your Software
262. pe Software v2 1 User Guide 7 13 Chapter 7 Viewing the Results Table 7 2 Segment Views continued View Procedure Display Assembly 1 Select the OWS al Eg e p p le AWOL Active Layer HLAC_COS Tab jumps to next The EP Assembly TEENS and QV s reis Navigate l REUS AF250557 views can 2 Click fH E be used 46 with the T Sequence 3 Click B or Dot view 4 Click uu L N38 3 888 1 PEECTOTPSEDE Nytte SEES o dian si SUE EUREN Note A black dot above a base in the consensus sequence indicates a discrepancy between the consensus and the reference sequences and a red dot above a sample base indicates the base was edited by the consensus caller Options Use es E zoom tools to zoom in out the horizontal view e Use LA Aligned EP to scale horizontal EP peaks to be of even width and aligned to base calls e Use View Column Selector to turn on column selector indicating position where base call is aligned to EP peak of original not scaled EP Use F8 Inverse Video to switch between white and black background displays UseFind and Find Again to search for text 7 14 ABI Prism SeqScape Software v2 1 User Guide Sample Views Sample Views The sample result includes all the data characteristics of a sample Sample data characteristics are displayed in the following tabs Table 7 3 Sample View Tabs
263. ple Name Name of the sample taken from the plate record It can be changed Basecaller Algorithm used to call the bases It can be changed 8 4 ABI Prism SeqScape Software v2 1 User Guide About Analysis Parameters Table 8 1 Information in the Sample Manager continued Column Heading Description DyeSet Primer A DyeSet Primer file corrects for mobility shifts and color code changes depending on which chemistry was used DyeSet Primer files are sometimes known as mobility or mob files All mobility files have the extension mob It can be changed Spacing The number of scan points from the crest of one peak to the crest of the next peak During basecalling a spacing calibration curve is applied to the data to determine a base spacing value Peak 1 The first data point that is from the sample not including primer peaks in dye primer chemistries It is the reference point for the spacing and mobility corrections performed by the basecalling software Start The raw data point where the basecalling starts in the sample file The Start Point is normally the same as the beginning of the first base peak Stop Specifies the last raw data point to be included in the basecalling If the default Stop Point is used this endpoint is the last data point in the file Assembled Displays the assembly status of the sample A green box means assembled and a red cir
264. plit bar NTPos 15 23 24 28 29 Wo ss codo 5 8 8 9 10 To 23 T T T 2 2 2 CodonPos 2 1 2 3 1 E c Cw l 2022 Cw 0102 52 Cw 1203 Cw 0102 56 4 number of bases that differ Base R Cw 12021 Cw 0102 53 R R R between the consensus sequence E M K G K ees ues es pu m ett T Ww Wa Cw 0102 Cw 1301 54 The Diff column displays the and the allele sequence ABI Prism SeqScape Software v2 1 User Guide 7 9 Chapter 7 Viewing the Results Specimen Views The specimen result is displayed as a schematic of the location and orientation of all samples within a specimen with respect to the reference ROIs in the current layers and consensus sequence To display the specimen view 1 Open the project of interest 2 Select a layer in the Active Layer drop down list 3 In the navigation pane open the project if necessary then select a specimen icon File Edit View Tools Analysis Window Help ck uu Ej amp US 4Dx b EH E Daag SSS SS MA MaM Aciveleyer HUA C CDS Tab jumps tone I HLa c 3100 Project Navigator HLA C 3100 360 2 EV EIHLAC3100 PReterence 195260867 LoratasastemblAu27 zl 793 277 Active Layer ROIs Consensus 3 Coverage 5 Coverage Samples iud FE ClearRange Known Variants Unknown Variants 7 10 ABI Prism SeqScape Software v2 1 User Guide Segment Views Segment Views Displaying the Segment Views
265. port Web site iii xix technical support for computers with altered configuration G 1 template saving 5 5 toolbar main 2 24 viewing 2 25 translation tables amino acid abbreviations C 5 complements C 3 IUPAC diagrams C 3 IUPAC IUB codes C 2 Universal Genetic Code C 4 U uninstallation procedure 2 9 Universal Genetic Code C 4 upgrading from 1 0 1 1 or2 0 2 8 software 2 8 Use Mixed Base Identification check box 3 7 user creating new 2 13 information changing 2 19 login process 2 10 new login procedure 2 21 privileges D 1 using aligned FASTA files 4 20 Clear Range widget 8 17 Index 8 V Variant Styles tab described 4 39 variants adding to projects 6 29 aligned E 2 to E 4 assigning styles 4 39 changing unknown to known 6 25 6 27 creating text files E 2 editing data 8 20 importing 6 28 importing into a project 6 32 to 6 33 incorporating into project RDG 6 25 promoting unknown to known 6 25 viewing data 7 20 to 7 21 8 20 versions of SeqScape software available 2 5 viewing project results 7 25 toolbar 2 25 W warranty claims G 2 exceptions G 2 for computers with altered configuration G 1 limitations G 3 period G 1 rights and responsibilities 2 2 warranty period effective date G 2 wizard creating RDG 4 6 workflow analysis display 3 2 analyzing project 6 2 creating project 6 2 creating project template 5 2 creating RDG 4 2 editing data 8 2 for viewing results 7 2 rea
266. ports after Analysis check box then select an export format from the drop down list c Define a default location to save the exported files d Click OK 9 10 ABI PRISM SeqScape Software v2 1 User Guide Printing Data and Reports Printing Data and Reports You can print any viewable screen ina WYSIWYG what you see is what you get manner within the SeqScape software on one of the recommended printers HP 8100 4500 990cxi and Epson 980 You can print project specimen segment and sample views as well as the reports for a project Customizing Default header and footer information is included in all exported and Header and printed reports and in printed data views However headers and Footer Display footers are not included in exported data files To customize the header footer display in printed and exported reports 1 Select File gt Print li Print Report x Print Properties Print Settings for Report o if D Print preview C Landscape Paper Letter 8 5 by 11 inches Header Footer Print Cancel 2 Click Header Footer W Headers amp Footers X Graphic Set Reset r Header Left Center Right Applied Generated at DateTimeGenet Biosystems ated Footer Left Center Right Project Creator ProjectCreatot Page PageNumber Printed by CurrentUser Autotext CurrentUser CurrentUser DateTime Generated PageNumber ProjectName ProjectCre
267. project based on a list of sequence files amp single Specimen is created for each text sequence file FASTA files can be used to create multiple Specimens Each text sequence will be stored in a Specimen Consensus Segment associated with the selected Reference Segment 1 Enterthe sequence File s to import Delete Browse 2 Selectthe Reference Segment PHRSR E 2 Click Browse and navigate to the target segment 3 In the Import Text Only Segment dialog box select the text file fsta format then click Import The segment appears in the previously blank section in the Import Text Segment dialog box 4 Repeat steps 2 and 3 to add additional text segments 6 20 ABI PRISM SeqScape Software v2 1 User Guide Analyzing the Data 5 Click OK A new specimen is created with the name specified in the first line of the file New specimen in text format i L i 8eq_001_H01_1026343804062 ab1 no comment NewEroje JPisfgrence lh aitratasaelemblA 2771 Kl Seq 001 H01 1026343804067 i 792 278 TW lo 516 o T Active Layer RO s SHENRCSERSIIZENEIIEEIEEN 268 791 275 Consensus 3 Coverage 5 Coverage Samples Al Hi Legend ClearRange Known Variants Unknown Variants Removing To remove samples or a specimen from a project Samples or g 1 In the Project Navigator of the project select the samples or Specimens specimen you want to remove from the project 2 Press t
268. pter 7 Viewing the Results Analysis QC Report Applied Biosystems recommends that you click gt before viewing 7 26 the Analysis QC report This report contains four separate tables All blue text is hyperlinked to the project navigator Table 7 5 Parts of the Analysis QC Report Table Description Summary Displays project information and the specimens in the report Specimen Analysis Displays specimen analysis results specimen Score average consensus QV and total number of variants Sample Analysis Displays sample analysis errors and details Possible Heterozygous Indel Mutations Displays possible mutations and their positions and size for each specimen ABI PRISM SeqScape Software v2 1 User Guide Analysis QC Report Manager Summary E Active La HLA C CDS Project Mutations Report Ne YE E roject Project Creation Date 03 Dec 2002 at Project Modification Date 13 May 2003 at AA Variants Report 23 40 04 PST 16 02 46 PDT Project Template PT HLA C_3100POP6 PT Creation Date 03 Dec 2002 at mixed Slib 2329 08 PST PT Modification Date 13 May2003 at Reference Data Group RDG HLA C Exon2 4 n 16 02 46 PDT oNT RDG Modification Date 13 May 2003 at 16 02 45 PDT xl Specimen Statistics Report Sequence Confirmation Report Base Frequency Report Library Search Report RDG Report Audit Trail Report Weeds Oly EI Genotyping Report Specimens in Report A1
269. r analysis Assembles samples within each specimen to generate a consensus sequence Aligns each specimen consensus sequence to the reference sequence Compares the aligned consensus sequence to the reference sequence Displays analysis results ABI PRISM SeqScape Software v2 1 User Guide 6 3 Chapter 6 Creating and Analyzing a Project Ways to Create and Analyze a New Project After the analysis defaults are set up you can create a new project for data analysis by Using the New Project Wizard see page 6 5 Using an existing project template see page 6 10 SeqScape Project Project Template Sample Files RDG Analysis Protocol Analysis Defaults Display Settings ROI information Reference Sequence Reference Sequence Features Library Nucleotide Variant Amino Acid Variant z Variant Styles Figure 6 2 Components of a Project 6 4 ABI PRISM SeqScape Software v2 1 User Guide Using the New Project Wizard to Create and Analyze a Project Using the New Project Wizard to Create and Analyze a Project The New Project The New Project Wizard takes you through the process of setting up a Wizard new project To create a new project using the New Project wizard 1 Launch SeqScape software 2 Select File New Project Wizard 3 Enter a name for the new project in the Project Name field then click Next 4 E
270. range is the region of the sequence that remains after excluding the low quality or error prone sequence at both the 5 and 3 ends 7 Select the Filter tab then if desired change one or more of the settings 8 Select OK to save the protocol and close the Analysis Protocol Editor ABI PRISM SeqScape Software v2 1 User Guide 11 13 Chapter 11 Automating Analysis Creating a To create a results group Results Group 1 Click the Results Group icon in the navigation pane The Results Group Editor opens Results Group Editor E Untitled Results 2 Click New or select an existing group then click Edit 3 In the General tab a Type a Results Group Name The name can be used in naming and sorting sample files and it must be unique see File Naming Convention on page 2 10 for a list of accepted characters b Type a Results Group Owner The owner name can be used in naming and sorting sample files c Type a Results Group Comment optional 11 14 ABI PRISM SeqScape Software v2 1 User Guide Creating Required Files in the Data Collection Software 4 Select the Analysis tab results Group Editor 5 In the Analysis tab a In the Analysis Actions section select Do Autoanalysis b Select SeqScape_ YourInstrumentName in the Analysis Type drop down list c Type a valid SeqScape Login ID and Password in the text boxes IMPORTANT Failure to use the proper login and password ca
271. raw data and electropherogram data segment A contiguous segment of the reference sequence corresponding to a single contiguous DNA sequence SeqScape Manager The software component that manages the following settings SeqScape Software projects project template RDG analysis defaults and display settings space character A character in an aligned sequence is either an IUB code or space perhaps shown as a dash A space indicates a deleted base in this string or equivalently an inserted base in one of the other aligned strings specimen The container that holds all the sample data as assembled contigs from a biological source or PCR product specimen consensus quality value See quality values specimen consensus score The average overall of the consensus quality values in the consensus sequence specimen consensus sequence The output of the consensus calling algorithm from a biologically related group of samples specimen report A concatenated list of all the reported information on a per specimen basis ABI PRISM SeqScape Software v2 1 User Guide Glossary 5 specimen view A view in SeqScape software where you can see the consensus sequence and all sample files that were used to create that consensus sequence summary sequence The summary consensus sequence for the entire library alignment Pure positions in the summary sequence correspond to con
272. re to save it in text only format line breaks are OK but spaces are not OK gt HumMitoCamb from 15871 to 450 hard return aatactcaaatgggcctgtccttgtagtataaactaatacaccagtcttgtaaaccggagatg aaaaccttttccaaggacaaatcagagaaaaagtctttaactccaccattagcacccaaag ct hard return What are Analysis Settings The analysis settings determine the basecalling mixed base settings clear range and filter settings What is Clear Range Clear range defines the range of usable sample sequence to be included in the consensus Can the Clear Range be modified within a project Does changing the Clear Range require that the data be re analyzed You can change the Clear Range for the entire project by applying a new Project Template with a modified Analysis Protocol in which case all samples will need to be re analyzed and any sample basecall edits will be lost You can reset the Clear Range for an individual sample by modifying the sample s Analysis Protocol setting In this case the specimen containing affected sample s must be re analyzed If only the Clear Range tab was modified in the Analysis Protocol the analysis pipeline will be started from Clear Range determination onward So basecalls will not be overwritten You can also change the sample Clear Range within the sample file This will not require re analysis To change the clear range in this way right click a sample sequence and select Set CR at selection then click dr
273. rence Data Group RDG HLA C_Exon2 4_ 18 02 45 HDT noNT RDG Report I 1 Audit Trail Report RNG Creatibn Date NA Dea 2h at RDG Madifinatinn Date 48 Maw 20 at E3 Audit Trail Repo Specimens in Report Genotyping Report CES pecimen Analysis Specimen s Basecalling m Assembly Specimen Tolal Comments Samples Seore Variants Specimen3 0 NA NA NA 3 D Cc Complete S Partial Output No output Sample Analysis Specimen Sample Step Description No Data Possible Heterozygous Indel Mutations Specimen Sample Position Size No Data Report Settings lel x Select report type from this list 4 In the navigation pane select the report you want to view To view other reports select a different report in the navigation pane Note Only one report can be viewed at a time ABI Prism SeqScape Software v2 1 User Guide 7 23 Chapter 7 Viewing the Results 6 To update the reports with additional data a In the project select additional or different specimen samples b Click to update the data in the open report 7 24 ABI Prism SeqScape Software v2 1 User Guide Viewing the Reports and Project Results Viewing the Reports and Project Results Report Manager Hyperlinks to When you view reports you can tile the report with the Project window so that you can easily view the data when you use a hyperlink To view the project results and reports together 1 Ne Ue m
274. riant Style Layer 1 4 pAF250557 i 414348 embIAJ277102 1 H 4 PHLAC exon2 000000 792 E Al n Layer 1 settings New Layer J Layer Name layer SCS Index Codon Number 1 Orientation Ubay s d Translation Frame 1 Right gt Segment Seg Start Seg End ROIStart ROILength Translation Color lonLayeri AF250557 Iv e 2 amp gi 7414348embIAJ277e gi 414348 emblAd 1 2 3 HLAC exon2 AF250557 EE 4 B AF250557 r 5 5 HLA C_intron2 AF250557 x El gt a gctcccactc catgaggtat ttctacaccg ccgtgtcccg 40 gdcccggecgc ggagagcccc gcttcatc c agtgggctac 80 gtggacgaca cgcagttcgt gcagttcgac agcgacgccg 120 cgagtccaag aggggagccg cuggcgccgt gugtggagca 160 ggaggggccg gagtattggg accgggagac acagaagtac 200 aagcgccagg cacagactga ccgagtgagc ctgcggaacc 240 tgcgcggcta ctacaaccag agcgaggccg gtgagtgacc 280 ccggcccggg gcgcaggtca cgacccctcc ccatccccca 320 cugacggccc gggtcgccec gagtctcccg gtctgagatc 360 fa Bs aa E Add Ref Segment Paste Ref Segment Split Ref Segment Add Variant Add ROI 4 30 ABI Prism SeqScape Software v2 1 User Guide Declaring Variants into an RDG 4 Inthe New NT Variant dialog box select the type of variant Insertion Deletion or Base Change Type Base Change ROI AF250557 z Position bp re To Reference base s 5 Variant base s fe Style Red Description M Used by all ROIs Create Another OK Cancel 5 Enter the Variant base 6 If
275. riant codes for an amino acid substitution Nonsense The substitution variant codes for a terminator codon In a mixed codon if any codon is a terminator codon nonsense will be displayed Silent The substitution variant is in a coding region but does not code for an amino acid change Frameshift insertion The insertion variant is in a coding region and codes for a frameshift in translation the size of the insertion is not a multiple of three Frameshift deletion The deletion variant is in a coding region and codes for a frameshift in translation the size of the deletion is not a multiple of three In frame insertion The insertion variant is in a coding region and does not code for a frameshift in translation the size of the insertion is a multiple of three In frame deletion The deletion variant is in a coding region and does not code for a frameshift in translation the size of the deletion is a multiple of three Non coding The variant is not in a coding region Partial codon The variant is in a coding region but occurs at the beginning or end of the sequence where you do not know the full three base codon sequence ABI PRISM SeqScape Software v2 1 User Guide 7 29 Chapter 7 Viewing the Results Table 7 7 Predicted Effects of Nucleotide Variants No information The variant is a result of the consensus sequence not completely covering the reference sequence These are
276. rmation Base Frequency Library Search RDG Audit Trail Genotyping Note Only one report can be viewed at a time Common to all reports is a Summary table that includes project information and the specimens in the report To export a report see Exporting Reports on page 9 8 and to print a report see Printing Data and Reports on page 9 11 ABI PRISM SeqScape Software v2 1 User Guide Viewing the Reports Viewing the Reports The data in the reports is filtered based on the view project specimen segment or sample selected in the navigation pane of the project and the layer selected from the Active Layer drop down menu To view a report 1 Open the project of interest then select the active layer 2 In the navigation pane select the project specimen segment or sample view 3 Select Tools gt Report Manager or click El Ctrl drag to reorder Right click to show hide columns columns v2 Report Manager Summary ysis QC Report vibe Depo Active Layer HLA C_cD Project HLA C v2 a Z Mutations Report a Project Creption Date 03 Dec 20p2 at Project Modification Date 13 May 200 at AA Variante haport 23 40 04 RST 18 02 45 PDT Specimen Biatstics Report Project T en HLA C 31DOPOP PT Creation Dat 03 Dec 2002 roject Template m reation Date ec 2002 at Sequence Confirmation Report Besse Ecl Base Frequency Report Library Search Report PT Modification Date 43 May 2003 at Refe
277. roject Tab delimited text file format Text file format containing aligned sequences To import variants into a project 1 Open the RDG Properties dialog box and select the NT Variants tab 2 Click Import RDG Properti E xi General ROI Variants Variant Style Type ROI Position Reference Variant Style Description Used by all ROIs Change Base HLA C exon3 75 Known yes Change Base HLA C exon3 68 la Known yes New Import Export ete Table Format General z Save To Manager As OK Cancel 3 Browse to the appropriate file and select it Note The files must be tab delimited text files as indicated in the File Types field 6 32 ABI PRISM SeqScape Software v2 1 User Guide Incorporating Variants into the Project RDG 4 Click Import Look in L Tutorial Data amp c E QA1 edited QA2edited QA3 edited s HIV DB11 98 type1 bt File name vet Import Files of type rab Delimited Variants bxt 7 Cancel 5 Select the reference segment then click OK 6 After the data is imported the Imports Results dialog box opens with information regarding the import 7 Note the information then click OK x 15 Variants IMPORTED 6 were reconciled with the reference sequence 1 Variant was NOT IMPORTED 1 not valid 0 duplicated existing variant s There were D parsing errors See the log file for more details
278. s 2 min Note If SeqScape software is running slowly you can improve performance by archiving older projects To archive projects select Tools gt SeqScape Manager gt Project gt Export Software Does SeqScape software support BioLIMS Sequence Collector software No SeqScape software no longer supports BioLIMS Sequence Collector software Software How does SeqScape software compare to MicroSeq and ViroSeq software SeqScape software compares samples to a reference sequence MicroSeq software identifies bacteria ViroSeq software identifies genotype HIV 1 resistance mutations Software Do need Sequencing Analysis software if have SeqScape software Sequencing Analysis software is a multi purpose software used to analyze edit view display and print sequencing sample files SeqScape software is designed specifically for resequencing Sequencing Analysis software should be used in every laboratory for general troubleshooting and viewing of data Data objects Can transfer data objects like the RDG Display Settings Analysis Protocols etc from one computer to another Yes you can transfer data objects from one computer to another To export the data object to the SeqScape Manager select the object and click Export Send the exported file to the second computer and launch SeqScape software Open SeqScape Manager and click Import to import the file ABI PR
279. s a pair of sequences haplotypes that best match the genotype of each consensus sequence A diploid library contains sequences composed of pure bases only or pure bases and mixed bases When searching against a diploid library SeqScape software provides library matches and each match is a single sequence that best matches the genotype of each consensus sequence To use the library search feature you must import an aligned multiple sequence FASTA file into the SeqScape software If you have a series of FASTA sequences you must use a tool to align those sequences and create a single aligned multiple sequence FASTA file before importing the file into the software IMPORTANT All sequences in the library must be of equal length If some sequences are shorter than other sequences you can use dashes for missing bases If you have a series of text sequences or electropherograms you must create FASTA files then use a tool to align those sequences and create a single multi aligned FASTA file A common tool used to create aligned multiple sequence FASTA files is Clustal X For instructions on how to obtain and use Clustal X see Appendix F Creating a Multi Aligned FASTA File ABI Prism SeqScape Software v2 1 User Guide Creating a Library Setting Up Your Note Review the following procedure Setting Up Your Library Library then go to the next section Creating New Layers on page 4 24 Use the following procedure
280. s appendix contains IUPAC IUB Codes 0 0 0c ccc eet ne C 2 TUPAC Diagrams 23 see cate heals Lege Rew ERE RE oe eS C 3 Gomplements 22 ope ER eate UE ECKE C 3 Universal Genetic Code 0 0 eee C 4 Amino Acid Abbreviations nanasa 000 cece eee eee C 5 ABI PRISM SeqScape Software v2 1 User Guide C 1 Appendix C Translation Tables IUPAC IUB Codes Table C 1 IUPAC IUB Codes Code Translation A Adenosine C Cytidine G Guanosine T Thymidine B C G or T D A G or T H A C or T R A or G puRine Y C or T pYrimidine K G or T Keto M A or C aMino S G or C Strong 3 H bonds WwW A or T Weak 2 H bonds N aNy base V A C or G Note This chart is accessible from the Help menu C 2 ABI PRISM SeqScape Software v2 1 User Guide IUPAC Diagrams IUPAC Diagrams IUPAC IUPAC heterozygous Figure C 1 IUPAC Diagrams Complements Table C 2 Complements A T S S C G W W G C T A B V D H R Y H D Y R V B K M N N M K Note These charts are accessible from the Help menu ABI PRISM SeqScape Software v2 1 User Guide C 3 Appendix C Translation Tables Universal Genetic Code Table C 3 Universal Genetic Codes 5 End 2nd Position 3 End T C A G Phe Ser Tyr Cys T lL Phe Ser Tyr Cys C Leu Ser OCH OPA A
281. s generated from SQVs It is the average quality value of the bases in the clear range sequence for a sample ABI PRISM SeqScape Software v2 1 User Guide 10 3 Chapter 10 Sample and Consensus Quality Values Table 10 2 Quality Values and Probabilities of Error Quality Value 10log o Pe where Pe is probability of error KB basecaller generates QVs from 1 to 99 Typical high quality pure bases will have QV 20 50 Typical high quality mixed bases will have QV 10 50 Size and color of QVs bars are identical for QVs 50 99 0 79096 0 50096 0 31096 0 200 0 0012 0 000196 0 000001 96 0 00000012 10 4 ABI Prism SeqScape Software v2 1 User Guide Consensus Quality Values Consensus Quality Values A consensus quality value QV is a per base estimate of the accuracy of the consensus calling algorithm If the SQVs are generated from the KB basecaller then the KB consensus calling algorithm is used to generate the QVs If the SQVs are generated from an ABI basecaller and TraceTuner then the TraceTuner consensus calling algorithm is used to generate the QVs The KB and TraceTuner consensus calling algorithms can produce slightly different consensus QVs Interpreting the The degree of certainty of either consensus calling algorithm is Consensus reflected by the per base consensus QVs A consensus QV is derived Quality Values from a number of factors How large a quality value discrepancy exists between calls from the ind
282. screen shot The Reference Sequence shown in the ROI Layer is now in two locked layers one segment ending at position 270 and the second segment starting at position 271 The Sequence pane shows the first segment ending at position 270 New reference break at position 271 RDG Properties E xj General ROI NT variants AA Variants Vartgnt Ste Layer 4 274 gf 270 792 al 2 r Layer 1 settings _NewLayer_ Layer Name Layer1 Index Codon Number fi Orientation Libray zl i Translation Frame iz Risht ROI Name Segment Seg Start Seg End ROI Stat ROI Length Translation Color on Layer 1 AF250557 partl AF250557 pari 1 270 1 270 2 m v E AF250557 par2 AF250557 pat2 271 792 271 522 Iz L7 HLA C_exon2 AF250557 pat 1 270 1 270 Iz EN HLA C gene 1 AF250557 pati 1 270 1 270 Iv Em AF250557 pati 21 77 1 57 2 LLL Reference Sequence FEE art al X AF250557_part2 el 121 161 201 241 M v Add Ref Segment Paste Ref Segment m j Add Variant J Add ROI g Cancel 40 80 120 160 200 240 lr Two segments in the First segment Reference Sequence at position 4 28 ABI Prism SeqScape Software v2 1 User Guide Declaring Variants into an RDG Declaring Variants into an RDG About NT The NT Variants tab in the RDG Properties dialog box lists the Variants known nucleotide variants associated with a reference sequence
283. sed in This Guide Safety Safety Alert Words Conventions Four safety alert words appear in Applied Biosystems user documentation at points in the document where you need to be aware of relevant hazards Each alert word implies a particular level of observation or action as defined below Definitions IMPORTANT Indicates information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical Nexen Indicates a potentially hazardous situation that if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices Ne Indicates a potentially hazardous situation that if not avoided could result in death or serious injury N pela Indicates an imminently hazardous situation that if not avoided will result in death or serious injury This signal word is to be limited to the most extreme situations Examples The following examples show the use of IMPORTANT CAUTION and WARNING safety alert words IMPORTANT The sample name run folder name and path name combined can contain no more than 250 characters Nim MUSCULOSKELETAL AND REPETITIVE MOTION HAZARD These hazards are caused by potential risk factors that include but are not limited to repetitive motion awkward posture forceful exertion holding static unhealthy positions contact pressure and other workstation environmental factors Nee Do not attempt
284. settings Basecall Settings 2 Select Tools SeqScape Manager Select the Analysis Protocols tab then select the project in the list for which you want to change the settings Click Properties In the Analysis Protocol Editor select the Basecalling tab to view the basecalling settings ABI PRISM SeqScape Software v2 1 User Guide Creating Analysis Protocols Analysis Protocol Editor x General Basecalling Mixed Bases Clear Range Filter Basecalling Ending Base AtPCR Stop Basecaller rater Fe inf bases DyeSet Primer I After 120 Ns KB_3100_POP4_BDTv3 mob zl After eno Bases Processed Data Quality Threshold Call all bases and assign QV C Assign N for bases with QV lt 15 C Flat Profile 5 Select the appropriate basecaller dedicated to your instrument For more information refer to Appendix A Basecallers and DyeSet Primer Files 6 Selectthe DyeSet Primer settings mobility files having the mob extension for the instrument you are using For more information refer to Appendix A Basecallers and DyeSet Primer Files 7 If you have short PCR products you should end basecalling at the end of the PCR product In this case select the At PCR Stop check box 8 You can also stop basecalling after a specified number of ambiguities or Ns or after a certain number of bases Enter your changes to the settings For more information on b
285. source feature corresponds to the region of interest associated with the whole reference segment that is automatically created exon Region of interest is created called gene _exon number Translatable by default intron Region of interest is created called gene _intron number Not translatable by default gene Region of interest is created called gene _gene Translatable by default CDS Layer is created called gene product CDS If translatable regions of interest exist that correspond to this CDS then those are used for building the layer Otherwise new regions of interest are created as required New ROls are called layerName _region1 layerName _region2 etc Translation frame and orientation is taken from CDS qualifiers complement and codon_start misc_feature Region of interest is created called note Not translatable by default Unknown feature Region of interest is created called feature key Not translatable by default It is possible with this translation table to create many non uniquely named ROIs for example if the entry had lots of variation features ABI PRISM SeqScape Software v2 1 User Guide Reference Data Group RDG Downloading a To download a GenBank file from the Internet GenBank File About Creating a New Reference Data Group RDG s 1 Open your web browser and enter the following URL http www ncbi nlm nih gov 2 In the Search m
286. st time The administrator can set up the software for the analyst scientist or other administrator users To prepare for the installation 1 Ensure that your system meets the minimum requirements see Hardware and Software Requirements on page 2 3 Check that you have at least 1 GB of free disk space to accommodate SeqScape software and sufficient space for all projects and their sample files 2 Temporarily turn off any virus protection software 3 Exit all programs except the Applied Biosystems 3730 3730xl or ABI PRISM 3100 3100 Avant Data Collection software 1f applicable IMPORTANT To properly install SeqScape Software v2 1 on a computer that is connected to Applied Biosystems 3730 3730x1 DNA Analyzers or ABI PRISM 3100 3100 Avant Genetic Analyzers the data collection software must be running SeqScape software will not register with the Data Service if the data collection software is not running See Chapter 11 Automating Analysis for more information on file sharing and automation ABI PRISM SeqScape Software v2 1 User Guide Installing the Full Version for the First Time Installing the Full Version for the First Time The administrator of the software installs the software and sets up new users Follow this procedure to install the full version of SeqScape Software v2 1 only if you are installing on e A computer that does not have a previous version of the software or Acomputer that has a 30
287. stant positions and mixed base positions in the summary sequence correspond to polymorphic positions Glossary 6 ABI PRISM SeqScape Software v2 1 User Guide Index Numerics 310 Basecaller and DyeSet Primer files A 5 3100 Basecaller and DyeSet Primer files A 10 3100 3100 Avant software integration 11 2 3100 Avant Basecaller and DyeSet Primer files A 13 3700 Basecaller and DyeSet Primer files A 15 3730 automation required files analysis protocol 11 4 instrument protocol 11 4 results group 11 4 3730 3730xl Basecaller and DyeSet Primer files A 17 3730 3730xl software integration 11 2 377 Basecaller and DyeSet Primer files A 8 A AA variants entering 4 36 importing 4 38 AA Variants Report 7 31 ABI basecaller 1 4 10 3 defined A 2 Glossary 1 ABI data files importing sample data 6 15 adding samples 8 6 specimens 6 11 variant in project 6 31 adjusting clear range 8 16 with the mouse 8 18 administration defined Glossary 1 administrator privileges 4 12 D 1 ABI PRISM SeqScape Software v2 1 User Guide aligned allele library defined Glossary 1 FASTA files using in library 4 20 variants E 2 to E 4 alignment defined Glossary 1 display defined Glossary 1 score defined Glossary 1 allele defined Glossary 1 amino acid abbreviations C 5 analysis cumulative QV scoring inreports 10 11 defined Glossary 1 display workflow 3 2 protocol defined Glossary 1 settings defined Glossary 1 settings
288. t IF YOU DO NOT AGREE select Decline in which case the program vill exit and terminate or if installation has not been completed installation of the program will stop copy of this End User License Agreement is available as a text file Select the Print button to print out a copy If you have any questions regarding this agreement or if you wish to request any information from Applied Biosystems please use the contact information provided in the about box or enclosed with the disc or other media containing this program Decline Accept Service Print Figure 2 1 License Dialog 6 Read the license agreement then click Accept 2 12 ABI Prism SeqScape Software v2 1 User Guide Starting SeqScape Software for the First Time The SeqScape software main window opens DR Seascape Ruth Palmer is logged in fie Ede View Tools Analysis Window Heip ci u E 6 UBS xb E BB 6 Act Laver zZ tavwumef z ms ASS iseascape vzora Bl yenara me part stop ero mer sese un SEB num Creating New Because SeqScape software tracks the projects and settings for each Users user Applied Biosystems recommends that you create users for each individual who uses SeqScape software on the computer The Users tab allows exporting of user names and access privileges for these users IMPORTANT The administrator is the only person who can set up and change the information in the Users tab The selections in this t
289. t s reation Date ec at lodification le lay Audit Trail Report 22 59 40 PST 18 02 45 PDT Display Settings DS TutorialDisplaySettings DS Creation Date 26 Nov 2002 at 23 37 59 PST Genotyping Report DS Modification Date 43 May 2003 at Analysis Defaults AD 3100_5R_POP8_BDTV1 Specimens in Report A1 A2 AB m Mutations Specime Base ROI Position Length Type QV Known Effect Aa Descriptio Change Change n 16g HLAC ex 18 missens R5 K R a on2 e 25a gt W HLA C ex 25 missens YB Y F e 28c m HLA C ex 29 silent on2 30g gt k HLA C ex 30 missens A1D A S on2 e 440 gt HLA C ex 44 silent G g K HLAC ex 69 missens A23 A S on2 e 103a gt G HLA C ex 103 missens O34R on2 e 128a t HLA C ex 128 silent Report Settings Figure 7 4 Mutations Report 7 28 ABI Prism SeqScape Software v2 1 User Guide Mutations Report The Mutations report includes a column that provides a predicted effect for each nucleotide variant Table 7 7 describes the possible values in the Effect column In the Mutation report clicking an NT variant links to the corresponding base in the Project View The corresponding AA change in the Mutation Report links to the AA Variant in the AA Variants Report This AA variant is in turn linked to the AA in the Project AA view Table 7 7 Predicted Effects of Nucleotide Variants Effect Description Missense The substitution va
290. t a template from the list and enter a project name Note To see the whole name in the list click drag the Project Template heading to the right when the double headed arrow cursor appears on the column bar 3 Click New The new project using the selected template opens 6 10 ABI Prism SeqScape Software v2 1 User Guide Adding Specimens and Importing Data into a Project Adding Specimens and Importing Data into a Project Overview All sample data from a single biological source should be placed inside a specimen within a project All sample data inside a specimen is assembled and a consensus sequence is produced You can think of each specimen as holding the assembled samples from one PCR product for example The consensus that is generated is compared to the references and aligned to the other consensus sequences from other specimens If you have new unanalyzed data you need to create specimens in the project to hold the data You can add specimens to a project automatically or manually For more information on what types of data can be imported into a project see Adding Specimens and Importing Samples Manually on page 6 14 IMPORTANT Unanalyzed specimen and sample data show a red slash line through their icons indicating that analysis is needed IMPORTANT Specimen names can be edited only after they are imported Sample names cannot be edited from within SeqScape software at any time Adding Using a text del
291. the Analysis Settings on page 3 12 for more information Display settings Settings that are used to display the data See Specifying Display Settings on page 3 18 for more information ABI PRISM SeqScape Software v2 1 User Guide 5 3 Chapter 5 Creating a Project Template 5 4 Creating a New To create a new project template Project Template 1 In the SeqScape window select Tools SeqScape Manager 2 Select the Project Templates tab then click New New Project Template a x Project Template Description Project Template Name Created N A Created By N A Modified N A Modified By N A Source N A Template Elements Reference Data Group HLA C_ex2 4_withVariants z Analysis Defaults 5 O0SR mixed z Display Settings HLa Csettings z js 3 Enter a name for the project in the Project Template Name field Note The project template name must contain only characters that conform to the Windows file system Refer to File Naming Convention on page 2 10 for a list of all invalid characters Select the desired Template Elements from the drop down lists then click OK ABI PRISM SeqScape Software v2 1 User Guide Saving Project Components Saving Project Components About Saving _ If you modify RDG analysis or display settings within a project the Template changes are valid only in that one project However if you want to Components save those se
292. the clear range of a sample file Any more than this number causes the sample to fail analysis Minimum Clear Length bp Minimum length of bases required in the clear range of a sample file Any less than this number causes the sample to fail analysis Minimum Sample Score Minimum quality value score average of all sample QVs in the clear range that is acceptable The range is 1 50 see Sample Quality Values on page 10 3 3 When the analysis protocol is complete click OK to save the new settings If you do not want to save the new settings click Cancel to save the previous settings Note To implement the changes you must click OK to save them and then run the analysis If all filters pass then the assembly occurs ABI PRISM SeqScape Software v2 1 User Guide Chapter 3 Creating Analysis Defaults and Display Settings Specifying the Analysis Settings To accommodate sample variability and to ensure the quality of your results you can modify the settings used to analyze a sample and then reapply them to a sample You can save changes to the analysis defaults and display settings contained in a project and you can also save them in SeqScape Manager to be used in a project template The procedures in the following sections describe selecting the analysis settings for a set of samples These analysis settings can be saved as analysis defaults and saved in SeqScape Manager For information on
293. the following attributes it is contiguous it is not editable its orientation determines the project orientation and it is stored in the RDG reference associated data The things that are related or assigned to a particular base or ranges of bases on a reference There are two types of reference associated data structural and variant Reference Data Group RDG The data that contains the reference and the reference associated data Report Manager A window that contains nine separate reports detailing the success or failure of various portions of the analysis statistics mutations AA variants and library search information sample data The output of a single lane or capillary on a sequencing instrument that will be input into SeqScape Software Glossary 4 ABI PRISM SeqScape Software v2 1 User Guide Sample Manager A window that displays sample file name name and specimen last used basecaller and DyeSet Primer files calculated basecalling results spacing peak 1 start and stop and assembly status The sample name basecaller and or DyeSet Primer file can be changed here sample quality values See quality values sample score The average of the per base quality values for the bases in the clear range sequence for the sample sample view A view in the SeqScape software where you can see attributes of each ABI file including its annotation sequence features
294. the software ABI PRISM SeqScape Software v2 1 User Guide Creating Analysis Protocols Specifying the To specify the mixed bases settings Mixed Bases Inthe Analysis Protocol Editor select the Mixed Bases tab Settings Analysis Protocol Editor X General Basecalling Mixed Bases Clear Range Filter Mixed Bases Settings M Use Mixed Base Identification Call IUB if 2nd highest peak is gt fis of the highest peak 2 Select the Use Mixed Base Identification check box to generate calls according to the international standard IUB code for heterozygous positions Mixed bases identification occurs only if the second peak height is greater than or equal to a percentage of the main peak height 3 Set the level according to sample type reaction kit and purification reaction and expected or acceptable percentage Enter the threshold for calling a mixed base for the value of the primary peak IMPORTANT Applied Biosystems recommends that you do not enter a value less than the 15 default value If you decrease the default percentage to detect low percentage mixed bases the background signal may be higher and interfere with mixed base detection Be aware of this condition ABI Prism SeqScape Software v2 1 User Guide 3 7 Chapter 3 Creating Analysis Defaults and Display Settings Specifying Clear Range 3 8 You can apply all or a subset of the Clear Range Methods algorithms Each is appli
295. ther software open or SeqScape software was never registered in the Data Service no communication between the software 1 Uninstall SeqScape software Open the data collection software Reinstall SeqScape software closed 4 Register the software and define user IDs 11 28 ABI PRISM SeqScape Software v2 1 User Guide Basecallers and DyeSet Primer Files This appendix contains Definitions and Naming 0 0 0 eee eee eee eee A 2 ABI PRISM 310 Genetic Analyzer Files A 5 ABI PRISM 377 DNA Sequencer Files 04 A 8 ABI PRISM 3100 Genetic Analyzer Files A 10 ABI PRISM 3100 Avant Genetic Analyzer Files A 13 ABI PRISM 3700 DNA Analyzer Files A 15 Applied Biosystems 3730 3730xl DNA Analyzers Files A 17 ABI PRISM SeqScape Software v2 1 User Guide A 1 Appendix A Basecallers and DyeSet Primer Files Definitions and Naming Basecaller A basecaller is an algorithm that determines the bases within a Algorithm sequence during analysis There are two types of basecallers e KB basecaller An algorithm that calculates mixed or pure bases and sample quality values ABI basecaller An algorithm used in earlier versions of ABI PRISM Sequencing Analysis and ABI PRISM SeqScape Software DyeSet Primer The DyeSet Primer file compensates for the mobility differences File between the dyes and primers
296. ties dialog box select the Variant Style tab The Variant Styles pane shows the available default colors of the variants Variant Styles Foreground Background New Delete Variant Settings tariant Style NT Variants Known insertion Red Known deletion Black Unknown change base Cyan Unknown insertion Blue Unknown deletion Green Crucial position Red l M ren i 2 Selectthe colors in which you want the base changes insertions and deletions for known variants displayed a To add a new color and style click New b To name the variant style click the Foreground Color box select a new color in the color palette then click OK c Select a color from the color palette for the Background Color then click OK in both dialog boxes to set the new variant style The variant styles you set appear in the Project view to identify the different types of variants 4 40 ABI PRISM SeqScape Software v2 1 User Guide 3 Creating an RDG from Aligned Consensus Sequences To delete a color select the color then click Delete Note The first seven Foreground colors cannot be changed or deleted In the Variant Settings pane select the colors in which you want the base changes insertions and deletions for unknown variants to be displayed The Variant Styles area shows a list of the available default colors Saving a C
297. tion or Residue Change x Type Insertion ad Layer Layer 1 z Position codon ps To Reference fw Variant fe Style Red Description Create Another OK Cancel 5 Enter the Position codon in the reference sequence that you want changed The Reference appears after you enter the position in the sequence Enter the variant Select a color style and enter a description if desired Click OK The new variant appears in the AA Variants list O oND Click OK to save the new variant ABI PRISM SeqScape Software v2 1 User Guide 4 37 Chapter 4 Creating a Reference Data Group Importing AA To import an AA variant from a tab delimited file Variants 4 38 1 In SeqScape Manager select the Reference Data Group tab then select the RDG you created Click Properties then select the AA Variants tab Click Import then navigate to the variant data file It can bea tab delimited text file txt file Click Import ge Select RDG Layer E xi Select the Rdg Layer to associate with the imported 44 Variants OK Cancel 5 Select any layer from the drop down list and click OK 6 Click OK in the Import Results dialog box lli Import Results x G 6 Variants IMPORTED O were reconciled with the reference sequence 0 Variants were NOT IMPORTED 0 not valid 0 duplicated existing variant s There were 0 parsing errors See the log file for more details
298. ttings so they can be applied to other projects you can create new SeqScape Manager template components based on existing template components Saving Template To save project template components Components from Within a Project Within a project select the Analysis menu then select one of the template components that you want to modify RDG Properties Analysis Defaults Display Settings Make the desired modifications to the component Click Save To Manager As An appended name of the current template component appears in the Save xx to the SeqScape Manager As dialog box Leave the name unchanged or change it IMPORTANT You cannot save over an existing template component You must delete the existing master component from the SeqScape Manager before you can save a new template component Click OK To use the modified component for other projects make a new project template that uses the new components ABI PRISM SeqScape Software v2 1 User Guide 5 5 Chapter 5 Creating a Project Template Examples of Example 1 Changing the Settings Within a Project 2 Select Analysis gt Analysis Settings then change a sample analysis setting 1 Create a project template and apply it to a project The underlying Analysis Defaults are unchanged in the SeqScape Manager Example 2 1 Create a project template and apply it to a project 2 Modify each component of the template 3 Change a variant st
299. udit Trail Report RDG Creation Date 03 Dec 2002 at RDG Modification Date 13 May 2003 at 22 59 40 PST 16 02 45 PDT 44 Variants Report Specimen Statistics Report Es Sequence Confirmation Report Base Frequency Report EY Genotyping Report Specimens in Report A1 A2 A3 Specimen Statistics Spec Seg Deletio Base Range Lengt Segme Sampl Continuo Coverag Match imen ment ns Chang on h nt es us e es Referen Score ce 1 276 276 1 4X no 33 1 794 25 P 4 1 276 27 r 1 794 1 276 4 1794 792 12X El Sample Results y Specimen Segment Orientation Assembled Clear Range on Sample Mixed Range Reference Score Base Al AF250557 foward 5338 1336 21 565 A1 AF250557 reverse B14 54 1 261 21 3 08 A HSA277102 reverse 200 24 1276 28 144 A1 HSA277102 forward 81 283 54 276 179 A AF250557 reverse 48 38 482 792 2 57 A1 AF250557 fomward 20 202 520 792 256 1220 721 n4 26m 278 Report Settings Average sample QV for the bases is in the clear range Average consensus QV score for a segment Figure 10 7 Specimen Statistics Report ABI PRISM SeqScape Software v2 1 User Guide 10 13 Chapter 10 Sample and Consensus Quality Values Genotyping The Resequencing Coverage table lists several QVs Report BRSeqScape File Edit View Tools DM DM is logged in Analysis Window Help cie B S S 078 4e xmmm d 03 4 2 4 ar Analysis QC Report Mut
300. uences in the FASTA file as the reference The rest of the sequences will be evaluated relative to that first sequence to derive variants Any positions that are found to differ from the first sequence will be used to populate the variants table To import NT variants using an aligned FASTA file 1 Select Tools gt SeqScape Manager 2 Click the Reference Data Group tab then select the RDG for which the variant will be added Click Properties In the RDG Properties window select the NT Variants tab Click Import ONE EE 18 In the Import NT Variants dialog box navigate to and select an aligned sequence FASTA file fsta extension 7 Click Import ABI Prism SeqScape Software v2 1 User Guide Creating an RDG from Aligned Consensus Sequences 8 Inthe Select Reference Segment dialog box select the reference segment for which the variants are to be added W Select Reference Segment I xj Select the Reference Segment to associate with the imported NT Variant Exisiting Reference Segment AF250557 OK Cancel 9 Click OK After the data is imported the Import Results dialog box opens displaying information about the import The first sequence in the imported file will populate the reference The subsequent sequences will be used to derive variants by comparison to the first sequence These variants will appear in the Variants table ABI PRISM SeqScape Software v2 1 User Guide 4 35 Chapter 4 Creatin
301. uses your samples not to be analyzed automatically ABI PRISM SeqScape Software v2 1 User Guide 11 15 Chapter 11 Automating Analysis 6 Select the Destination tab MResults Group Editor 7 Inthe Destination tab use the default location or define a new location 8 To define a new location a Click Use Custom Location then click Browse to navigate to a different save location b Click Test to test the Location path name connection If it passes a message box displays Path Name test successful If it fails a message box displays Could not make the connection Please check that the Path Name is correct Click and retry to establish a connection c Click OK 11 16 ABI PRISM SeqScape Software v2 1 User Guide Creating Required Files in the Data Collection Software 9 Select the Naming tab then define custom names for sample file and run folder name if desired results Group Editor 10 Click OK to save the Results Group and close the Results Group Editor Note Specimens in the project can be created automatically based on the delimiter text present in the sample ID name See Annotation Tab on page 6 12 ABI Prism SeqScape Software v2 1 User Guide 11 17 Chapter 11 Automating Analysis Creating a Plate To create a new plate record Record 1 Click the Plate Manager icon in the navigation pane Foundation Data Collection Version 2 0 Administrator us
302. ware accepts the following file formats for reference sequences e Genbank File Format with a fcgi cgi or gb extension e txt text file format e ab1 file format e fsta FASTA file format e seq file format e Aligned sequences in fsta FASTA format The imported sequence will show a summary of all the sequences in the file by substituting the IUPAC codes for bases where there is a discrepancy in the sequences Data Sequences SeqScape software accepts the following file formats for data sequences used for analysis e ab1 files previously basecalled or not basecalled e txt TEXT files e fsta FASTA files e seq files Nucleotide Variants SeqScape software accepts the following file formats for nucleotide variants e fsta file containing a set of aligned sequences in FASTA format e tab delimited text txt file that lists one variant per line and eight column headings Type ROI NT position Reference Variant Style Description and Used by all ROls Amino Acid Variants SeqScape software accepts the following file formats for amino acid variants tab delimited text txt file that lists one variant per line and the following seven column headings Type Layer AA position Reference Variant Style and Description Files Can scf files be analyzed in SeqScape software No SeqScape software analyzes only sequencing data in ab1 files or text sequences Files H
303. x D User Privileges Table D 2 Access for Admin and Scientist Levels continued Description of access for users of Admin and Scientist levels Admin Scientist Analyst RDG AA variants Tab Import AA variants from a tab delimited text file into RDG RDG variant styles tab configure an RDG in Variant Styles tab Library overwriting appending sequences to an existing library Library editing sequence data in the library Library exporting data from the library as a Multi FASTA file Library viewing editing library types in the Library Type manager Library creating a new sequence library Sets General Preferences in Options Sets Sequence Collector Database Preferences in Options Add NT or AA variants from any data view Set specimen level analysis settings Set project level analysis settings D 4 ABI PRISM SeqScape Software v2 1 User Guide Table D 3 Access for Admin Scientist and Analyst Levels Tables of User Privileges Description of access for users of 3 e Admin Scientist and Analyst levels Admin Scientist Analyst Reports 1 View Reports Allowed Allowed Allowed 2 View Reports with enabled links back to primary data 3 View Reports while editing project 4 Export all reports 5 Export all customized reports 6 Print all reports 7 View heterozygous frame shifts links from
304. xed Base determination in Analysis settings or analysis defaults in a Project PT SS Manager D 2 ABI PRISM SeqScape Software v2 1 User Guide Table D 2 Access for Admin and Scientist Levels continued Tables of User Privileges Description of access for users of S Admin and Scientist levels Adnin Scientist Analyst RDG 15 RDG Import Variants and Reference into Allowed Allowed Not an RDG from a set of aligned FASTA files Allowed 16 RDG general tab configure an RDG in general tab 17 RDG ROI tab Edit a Reference Data Group RDG configure Layers 18 RDG ROI tab Edit a Reference Data Group RDG configure ROIs 19 RDG ROI tab Edit a Reference Data Group to use an implicit reference 20 RDG ROI tab adding modifying a Reference Segment 21 RDG ROI tab Change the Reference Segment index Base in an embedded RDG 22 RDG ROI tab deleting a Layer 23 RDG ROI tab deleting a Reference Segment 24 RDG ROI tab deleting an ROI 25 RDG ROI tab Import GenBank sequences into the RDG for automated Ref Segment and feature creation 26 RDG NT variants Tab Edit NT variants in an RDG 27 RDG NT variants Tab Import NT variants from a Tab Delimited Text into RDG 28 RDG AA variants Tab Add amino acid variants to an RDG 29 RDG AA variants Tab Edit AA variants in a RDG ABI PRISM SeqScape Software v2 1 User Guide D 3 Appendi
305. y anew Project Template to an Allowed Allowed Allowed Controls existing Project 30 Create a new Project from the SeqScape Toolbar 31 Delete Samples in Project Navigator 32 Delete Specimens in Project Navigator 33 Export Project Alignment in FASTA format 34 Export Sample data in SEQ FASTA or AB1 format 35 Export Specimen consensus or aligned sample sequences in FASTA format D 6 ABI PRISM SeqScape Software v2 1 User Guide Table D 3 Access for Admin Scientist and Analyst Levels continued Tables of User Privileges Description of access for users of Admin Scientist and Analyst levels Admin Scientist Analyst Project Other 36 Import a Text segment to a Text Specimen Allowed Allowed Allowed Controls 37 Import Samples to Project 38 Import Samples to Project from Database Sequence Collector 39 Import create a text only Specimen 40 Open an embedded Settings Object inside a Project 41 Open an existing Project 42 Print wrapped nucleotide or amino acid Project Alignments 43 Save Project from the Menu or Toolbar 44 Search for text strings in any sequence data Editing 45 Generate an Audit Trail event Allowed Allowed Allowed 46 Project Alignment view Change consensus basecalls 47 Project Alignment view Insert or delete a space in a Reference 48 Proje
306. y this procedure by obtaining HLA sequences from ftp ftp ebi ac uk pub databases imgt mhc hla and selecting DRB_nuc fasta 1 Open the folder that you downloaded 2 Double click the ClustalX icon File Edt Alignment Trees Colors Quality H Multiple Alignment Mode E FontSize 10 v 3 Select Multiple Alignment Mode 4 Select Alignment gt Output Format Options F 4 ABI PRISM SeqScape Software v2 1 User Guide Method 2 Create a Multi Aligned FASTA File Using ClustalX Software 5 Select GDE format and set the other parameters as indicated below Output Format Options 3 lel xi CLOSE Output Files I CLUSTAL format NBRF PIR format GCG MSF format PHYLIP format IV GDE format NEXUS format GDE output case Lower M CLUSTALW sequence numbers OFF bd AN Output order ALIGNED Parameter output OFF gt 6 Select File gt Load Sequences To obtain a file go to http www ebi ac uk imgt hla then select the Download tab 7 Browse to the location of your multiple sequence file and select the file E Clustalx 1 81 ini x File Edit Alignment Trees Colors Quality Help Multiple Alignment Mode Font Size 10 G o O OLG HLA HLA0049 HLA_HLA01083 File CADocuments and Settings doanqc De sktop HLA DMB_nuc fasta loaded 8 Select Edit gt Select All Sequences ABI Prism SeqScape Software v2 1 User Guide F 5 Appendix F Creating a Multi Aligned
307. yeSet Primer Files Used for Dye Terminator Chemistry DNA Sequencing Chemistry Capillary Array Length cm Basecaller DyeSet Primer KB Basecalling ABI PRISM BigDye v3 0 all lengths KB bcp KB 3730 POP7 BDTv3 mob Terminator ABI PRISM BigDye Terminator all lengths KB bcp KB 3730 POP7 BDTv1 mob ABI Basecalling ABI PRISM BigDye Terminator v1 0 and v1 1 36 rapid read Basecaller 3730POP7RR bcp 36 std read Basecaller 3730POP7SR bcp 50 long read Basecaller 3730POP7LR bcp DT3730POP7 BD mob ABI PRISM BigDye Terminator v3 0 and v3 1 36 rapid read Basecaller 3730POP7RR bcp 36 std read Basecaller 3730POP7SR bcp 50 long read Basecaller 3730POP7LR bcp DT3730POP7 BDv3 mob SeJ SIEZAEUY WNC IX064 064 suiejs soig paljday Appendix A Basecallers and DyeSet Primer Files A 18 ABI PRISM SeqScape Software v2 1 User Guide Frequently Asked Questions This appendix contains Upgrading FAQS rer iue eer REOR ht enm e B 2 Training and Documentation FAQs 0 020002 ee eee B 3 SeqScape Software Basics FAQs 0 0 0 0 cece eee eee B 4 General SeqScape Software FAQs 0000 e eee eee B 5 SeqScape Manager FAQs 2 0 eee eee eee eee B 9 Library EAQS ensi ie teesppePUMPPites ep r6i seseevntse B 15 Mutation Variant HIM and HFM Detection FAQs B 16 Data Analysis FAQs 000 cc cece eens B 17
308. yer go to Creating New Layers on page 4 24 Otherwise continue to the next step 4 22 ABI PRISM SeqScape Software v2 1 User Guide Creating a Library 8 Select a layer in the Layer pane 9 In the Library drop down list select the corresponding library that you created in steps 2 through 5 Library drop down list RDG Properties 792 276 4l Layer 1 settings New Layer Layer Name Layert Index Codon Number fi Orientation Library Translation Frame fz Right ROI Name Segment Seg Start Seg End RO Start ROI Length Translation Color on Layer 1 a ar250557 AF250557 1 792 1 792 Iv EN V 2 gi 7414348 emb AJ277e gi 414348 emblAJ 1 276 1 276 2 3 HLAC exon2 AF250557 1 270 0 270 2 2 r mm mr 4 HLAC gene AF250557 1 792 o 792 r r 5 HLA C intron2 AF250557 271 516 270 246 M ad p 1 al 81 121 161 201 241 281 321 Ep t Add Ref Segment Paste Ref Segment Split Ref Segment Add Variant Add ROI OK Cancel ABI Prism SeqScape Software v2 1 User Guide 4 23 Chapter 4 Creating a Reference Data Group Creating New Layers Layers organize groups of related nonoverlapping ROIs By organizing ROIs into layers results reviewing and library searching are faster and more focused The Layer table shows the organization of ROIs into layers To create new layers 1 In the ROI tab Layer pane click New Layer then enter a nam
309. yle in the RDG then select Save To Manager As A new RDG in the SeqScape Manager reflects this change but the old RDG in the SeqScape Manager remains unchanged Therefore the project template using the old RDG is also unchanged Note In both examples the open project displays the changes 5 6 ABI PRISM SeqScape Software v2 1 User Guide Creating and Analyzing a Project This chapter contains Workflow for This Chapter 6 2 Ways to Create and Analyze a New Project 6 4 Using the New Project Wizard to Create and Analyze a Project 6 5 Creating and Analyzing a New Project Using a Project Template6 10 Adding Specimens and Importing Data into a Project 6 11 Analyzing the Data llle 6 21 Reanalyzing a Project Using a Different Project Template 6 22 Incorporating Variants into the Project RDG 6 25 Importing and Exporting Project Information 6 34 ABI PRISM SeqScape Software v2 1 User Guide 6 1 Chapter 6 Creating and Analyzing a Project Chapter 3 1 Create Analysis Defaults and Display Settings Chapter 4 Workflow for This Chapter 2 Create Reference Data Group Chapter 5 3 Create Project Template Add Reference Sequence Create ROIs gt Create Layers Link a Library Select Chapter 6 4 Create and Analyze Project Chapter 7 5 View Data and Reports Chapter 8 RDG Anal
310. ypes and identifies the alleles that most closely match each consensus sequence ABI PRISM SeqScape Software v2 1 User Guide About SeqScape Software How the Software You provide the following information to the system before analysis Performs Analyses A reference sequence backbone made up of one or more reference segments and any known nucleotide variant information or amino acid variant information SeqScape software uses the backbone to classify all polymorphic positions as known variants or unknown variants An allele library a set of sequences for the alleles or haplotypes Using the reference sequence variants allele library and software settings you create a reusable project template With this template and the sequencing samples SeqScape software Performs basecalling quality value assignment and mixed base identification in that order Trims low quality bases from each sequence Identifies poor quality samples and removes them from further analysis Assembles the remaining samples against the reference sequence and generates a specimen consensus sequence Reviews the basecalling quality values and the sample assembly to confirm improve and assign quality values to the consensus sequence Identifies variants by aligning specimen sequences to the reference sequence and comparing the specimen consensus sequences to the reference sequence Generates nine detailed reports Note If you link a library
311. ysis Defaults Display Settings ROls Add nucleotide variants Add amino acid variants Setup variant styles Select Project Template gt Add Specimens Import Sample Files Data Project View Specimen View Segment View Sample View 6 Reanalyze and Edit Data Chapter 9 7 Export and Print Figure 6 1 Reports Aligned projects Consensus sequence Aligned sample sequence Sample files Software does automatically Basecalling Filter Assembly Consensus Alignment Comparison Search Library Report Manager Analysis QC Report Mutations Report AA Variants Report Specimen Statistics Report Sequence Confirmation Report Base Frequency report Library Search Report RDG Report Audit Trail Report Genotyping Report Showing Create and Analyze Project Steps ABI PRISM SeqScape Software v2 1 User Guide Workflow for This Chapter Before You Begin Before you can create a project in the ABI PRISM SeqScape Creating a Project Software Version 2 1 you must have created a project template that contains A Reference Data Group Analysis Defaults Display Settings What an Analysis The analysis in SeqScape software Entails Basecalls the raw data Assigns quality values and identifies mixed bases Filters out poor quality data and excludes that data from furthe
312. ysis defaults 3 3 creating new project templates 5 4 creating RDG 4 5 4 12 creating reference using aligned sequences 4 34 to 4 36 defined Glossary 5 exporting from 6 34 importing to 6 34 questions B 9 ABI PRISM SeqScape Software v2 1 User Guide RDG Reference Data Group Wizard 4 5 window described 2 22 Sequence Confirmation Report 7 33 sequence editing 8 13 sequencing data automating the analysis 11 2 mobility files A 13 to A 17 Set Clear Range using 8 19 setting analysis defaults 3 14 setting up default directory 2 20 library 4 21 new project using New Project Wizard 6 5 software integration 11 2 overview structure 2 23 registering 2 2 relationships for autoanalysis 11 3 upgrading 2 8 versions available 2 5 space character defined Glossary 5 specimen adding 6 11 adding sample data manually 6 14 to 6 21 adding to the project 6 11 to 6 12 consensus sequence defined Glossary 5 creating automatically 6 12 cumulative QV scoring in reports 10 13 defined Glossary 5 exporting 9 5 removing sample data 6 21 report defined Glossary 5 view defined Glossary 6 view displaying 7 10 specimen consensus quality value defined Glossary 5 score defined Glossary 5 Specimen Statistics Report 7 32 10 13 starting the software 2 10 summary sequence defined Glossary 6 system requirements minimum 2 3 Index 7 T tab delimited text file E 2 to E 4 importing NT variant 4 32 technical support Services and Sup

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