ABI PRISM SeqScape Software Version 2.x Tutorial Chapter 10
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. 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 and SeqScape are registered trademarks of Applera Corporation or its subsidiaries in the U.S. and/or certain other countries. AB (Design), Applera, and POP-6 are trademarks of Applera Corporation or its subsidiaries in the U.S. and/or certain other countries. Windows and Windows NT are registered trademarks of Microsoft Corporation. All other trademarks are the sole property of their respective owners. Part Number Part Number 4338877 Rev B 09/2003
........................................................ vii....................................................................... vii....................................................................... vii............................................................. vii.................................................................... vii...................................................................... viii............................................................ viii..................................................................... ix....................................................................... ix........................................................ ix 1......................................................................... 1-2................................................................ 1-2.......................................................... 1-3.............................................................. 1-4..................................................................... 1-5 2 Project Template Project Template............................................................... 2-2...................................................................... 2-2 Project Template................................................... 2-2 Project Template..................................... 2-2................................................................ 2-3...................................................................... 2-3............................................................ 2-3..................................................................... 2-6.................................................. 2-7 RDG............................................................. 2-7 RDG.............................................. 2-8 RDG................................................. 2-10 RDG............................................................... 2-10 Analysis Defaults......................................................... 2-11.................................................. 2-13................................................... 2-13 ABI PRISM SeqScape v2.x iii
Clear Range..................................................... 2-14.................................................... 2-15 Analysis Protocol...................................................... 2-15 Analysis Defaults...................................................... 2-17 Display Settings.......................................................... 2-17.................................................................... 2-17 Project Template........................................................ 2-18.................................................................... 2-18 3............................................................... 3-2..................................................... 3-2.......................................................... 3-2........................................... 3-3......................................................... 3-5..................................................................... 3-5 4.......................................................... 4-2........................................................... 4-2 Analysis QC.................................................... 4-2.................................................. 4-6........................................................ 4-8..................................... 4-9 Clear Range.......................................................... 4-11............................................................. 4-13......................................................... 4-14 Library Search................................................. 4-14 Library Identification............................................. 4-16.................................................... 4-18.................................................. 4-18....................................................... 4-18......................................................... 4-18 iv ABI PRISM SeqScape v2.x
A Software Warranty Information Computer Configuration.......................................................... A-2 Limited Product Warranty......................................................... A-2 Limited Warranty............................................................ A-2 Warranty Period Effective Date................................................. A-2 Warranty Claims............................................................ A-2 Warranty Exceptions......................................................... A-2 Warranty Limitations......................................................... A-3 ABI PRISM SeqScape v2.x v
vi ABI PRISM SeqScape v2.x
ABI PRISM SeqScape v2.x CD-ROM HLA- C SeqScape SeqScape SeqScape Administrator Quick Reference Card Microsoft Windows ABI PRISM SeqScape v2.x vii
0 [Enter] (>) File > Open > Spot Set View Filter > View All Runs Applied Biosystems 2 Control Console Oracle ID 1 96 1 Sample Entry viii ABI PRISM SeqScape v2.x
ABI PRISM SeqScape Quick Reference Card ABI PRISM SeqScape - SeqScape ABI PRISM SeqScape Quick Reference Card - SeqScape ABI PRISM SeqScape Training Movie - SeqScape SeqScape PDF SeqScape CD Applied Biosystems jptechsupport@appliedbiosystems.com http://www.appliedbiosystems.co.jp FAQ Applied Biosystems MSDS PDF Applied Biosystems ABI PRISM SeqScape v2.x ix
x ABI PRISM SeqScape v2.x
1.....................................................................1-2..........................................................1-2....................................................1-3.........................................................1-4................................................................1-5 ABI PRISM SeqScape v2.x 1-1
1 Administrator ABI PRISM SeqScape 2 3 2 3 4 HLA-C SeqScape ROI \ / : *? < > 1-2 ABI PRISM SeqScape v2.x
HLA-C AppliedBiosystems\SeqScape\Tutorial Data\HLA-C 1-1 1-1 3 1-1 HLA-C_Exon2-4_FASTA Library HLA-C-ex2-4_v2_LibraryEntries.fsta HLA-C_3100POP6_Tutorial Data ** A1 HLA-C_3100POP6_Tutorial Data A2 HLA-C_3100POP6_Tutorial Data A3 A1-2F_01.ab1 * A1-2R_02.ab1 A1-3F_01.ab1 A1-3R_02.ab1 A1-4F_01.ab1 A1-4R_02.ab1 A2-2F_03.ab1 A2-2R_04.ab1 A2-3F_03.ab1 A2-3R_04.ab1 A2-4F_03.ab1 A2-4R_04.ab1 A3-2F_05.ab1 A3-2R_06.ab1 A3-3F_05.ab1 A3-3R_06.ab1 A3-4F_05.ab1 A3-4R_06.ab1 HLA-C_Exon2-4_GenBank Refs HLA-C-ex-2-3_AF250557.gb HLA-C-ex-4_AJ277102.gb ** 3 A1 A2 A3 6 * A1-2F_01.ab1 2F 2 ABI PRISM SeqScape v2.x 1-3
1 SeqScape Manager 1-1 1-1 1-4 ABI PRISM SeqScape v2.x
HLA-C Project Template Project Template ABI PRISM SeqScape v2.x 1-5
1 1-6 ABI PRISM SeqScape v2.x
Project Template 2 Project Template...........................................................2-2..........................................................2-3...........................................2-7 Analysis Defaults....................................................2-11 Display Settings.....................................................2-17 Project Template...................................................2-18 ABI PRISM SeqScape v2.x 2-1
2 Project Template Project Template Project Template Project Template Project Template Project Template ABI PRISM SeqScape Project Template Project Template Project Template 2-1 RDG Analysis Protocol Analysis Defaults Display Settings ++ 2-1 Project Template Project Template 3 RDG Analysis Defaults Display Settings Project Template Project Template RDG - ROI Analysis Defaults - Analysis Protocol Analysis Defaults Analysis Protocol Display Settings - Display Settings Project Template - RDG Analysis Defaults Display Settings 2-2 ABI PRISM SeqScape v2.x
SeqScape Manager 2 3 4 48 HLA-C FASTA Project Template RDG SeqScape 1 1. Tools > SeqScape Manager Libraries 2-2 2-2 SeqScape Manager Libraries 2. New 2-2 3. General 2-3 a. TutorialLibrary b. Haploid ABI PRISM SeqScape v2.x 2-3
2 Project Template c. 2-3 Library Editor General 4. Entries Import 2-4 2-4 Library Editor Entries 2-4 ABI PRISM SeqScape v2.x
5. Import Library Sequence Entries a. AppliedBiosystems\SeqScape\Tutorial Data\HLA-C\ HLA-C_Exon2-4_FASTA Library HLA-C_ex2-4_v2_LibraryEntries.fsta 2-5 2-5 Import Library Sequence Entries b. Import 6. OK 2-6 2-6 Library Entries Import Statistics Library Editor 2-6 2-7 ABI PRISM SeqScape v2.x 2-5
2 Project Template 2-7 Library Editor 7. OK Library Editor SeqScape Manager 2-6 2-8 2-8 Library RDG 2-6 ABI PRISM SeqScape v2.x
RDG Project Template 1 RDG RDG 2-9 2-7 2-1 2-9 RDG 2-1 RDG ROI 1 FASTA GenBank ROI ROI RDG ABI PRISM SeqScape v2.x 2-7
2 Project Template 2-1 RDG RDG RDG - RDG - RDG - 2 GenBank.gb - RDG ROI ROI - RDG RDG RDG 1. SeqScape Tools > SeqScape Manager Reference Data Group 2. New RDG Properties 3. General Reference Data Group Name TutorialRDG 4. Codon Table Standard HLA-C tutorial, exons 2 to 4 OK RDG 2 GenBank SeqScape GenBank GenBank ABI PRISM SeqScape 4 1. RDG Properties ROI 2-8 ABI PRISM SeqScape v2.x
2. Add Ref. Segment Import Reference Sequence 3. HLA-C_Exon2-4_GenBank Refs GenBank HLA-C-ex-2-3_AF20557.gb 2-10 2-10 GenBank 4. Import RDG Properties 2-9 2-11 GenBank HLA-C_CDS GenBank ROI Layer 1 2 2 3 2 ROI HLA-C_CDS 2-9 2-11 2 ROI 3 ROI 1 2 Layer ROI 1 2-11 RDG GenBank ABI PRISM SeqScape v2.x 2-9
2 Project Template 5. 1 4 2 HLA-C-ex-4_AJ277102.gb 6. 2 4 ROI 2 a. Layer 2 b. ROI HLA-C_exon4 on Layer 2 2-10 2-12 c. HLA-C_exon4 Translation 2 4 ROI Layer ROI 2 2-12 RDG 2 GenBank RDG RDG RDG RDG RDG 1. Layer Layer 2 2. Layer Settings Library 2 ROI 2 2 3 4 ROI HLA-C RDG OK RDG SeqScape Manager Reference Data Group 2-10 ABI PRISM SeqScape v2.x
Analysis Defaults Analysis Defaults Project Template Analysis Defaults SeqScape Analysis Protocol Analysis Protocol 1 Analysis Defaults Analysis Protocol Analysis Defaults Analysis Protocol Analysis Defaults Analysis Protocol Analysis Defaults Analysis Protocol Analysis Protocol SeqScape Clear Range Analysis Protocol Basecalling Mixed Bases Clear Range Filter ABI PRISM SeqScape v2.x 2-11
2 Project Template Analysis Protocol 1. Tools > SeqScape Manager 2. Analysis Protocols 3. New Analysis Protocol NewAnalysisProtocol 2-12 2-13 Analysis Protocol Editor OK 2-13 Analysis Protocol 2-12 ABI PRISM SeqScape v2.x
Analysis Defaults Basecalling POP-6 50cm ABI PRISM 3100 SR BigDye Terminator v1.0 Basecaller 1. Analysis Protocol Editor Basecalling 2. Basecaller Basecaller-3100POP6SR.bcp 2-13 2-14 3. DyeSet/Primer DT3100POP6(BD)v2.mob 2-13 2-14 2-14 Basecalling 2 1. Analysis Protocol Editor Mixed Bases 2. IUB Use Mixed Bases Identification 2 25% ABI PRISM SeqScape v2.x 2-13
2 Project Template Clear Range Clear Range Spacing Clear Range 5 3 Clear Range 1. Analysis Protocol Editor Clear Range 2-15 2. Use quality values SeqScape Quality Value QV Quality Value Clear Range 20 20 QV 4 QV 20 = 1% QV ABI PRISM SeqScape 10 3. Use reference trimming Clear Range Clear Range 2-15 Clear Range 2-14 ABI PRISM SeqScape v2.x
Analysis Defaults Analysis Settings 1. Analysis Protocol Editor Filter 2-16 2. Maximum Mixed Bases (%) 20 35 2-16 Filter 3. 2-2 2-2 Maximum Mixed Bases (%) = 35 Maximum Ns (%) = 10 Minimum Clear Length (bp) = 50 Minimum Sample Score = 20 Clear Range Clear Range N Clear Range Quality Value Clear Range QV 1 50 20 Clear Range Quality Value 20 1 100 1% Analysis Protocol OK Analysis Protocol SeqScape Manager ABI PRISM SeqScape v2.x 2-15
2 Project Template Analysis Defaults Analysis Defaults 2 Analysis Defaults Analysis Defaults Analysis Settings Analysis Defaults Analysis Protocol Analysis Defaults 1. SeqScape Manager Analysis Defaults New New Analysis Settings 2-17 2. General NewAnalysisDefaults 3. Project 4. Specimen 2-17 a. Gap Penalty Extension Penalty b. # Library Matches 5 c. Basecall Samples 2-17 Specimen 2-16 ABI PRISM SeqScape v2.x
Display Settings 5. Sample 2-18 Analysis Protocol TutorialAnalysisProtocol 2-18 Sample Analysis Defaults Save Analysis Defaults SeqScape Manager Display Settings Project Template Display Settings Display Settings SeqScape Manager DefaultDisplaySettings_v2 Display Settings ABI PRISM SeqScape 3 Project Template ABI PRISM SeqScape v2.x 2-17
2 Project Template Project Template Project Template Project Template Project Template RDG Analysis Defaults Display Settings Project Template 3100 POP-6 BigDye Terminator v1.0 2 4 HLA-C Project Template Project Template 1. SeqScape Tools > SeqScape Manager 2. Project Templates New 2-19 Project Template Editor 3. Project Template MyHLA-CProjectTemplate 2-19 4. RDG Analysis Defaults Display Settings Reference Data Group TutorialRDG Analysis Defaults NewAnalysisDefaults Display Settings DefaultDisplaySettings_v2 5. OK Project Template SeqScape Manager Project Template 3 2-18 ABI PRISM SeqScape v2.x
3.........................................................3-2...................................................3-5 ABI PRISM SeqScape v2.x 3-1
3 2 Project Template HLA-C 1. SeqScape File > New Project 2. New Project 3-1 2 Project Template MyHLA-CProjectTemplate 3-1 3. Project Name HLA-C_Tutorial 3-1 4. New Project Template RDG Analysis Defaults Display Settings PCR 3 A1 A2 A3 DNA 3.ab1 3-2 ABI PRISM SeqScape v2.x
A1 A2 A3 - Import Samples 3-3 3-2 1. Import Samples to Project 2. HLA-C 3-3 3-2 1-3 3-2 HLA-C 3. Use sample file name 3-2 4. End - 3-2 5. Files A1 Auto Add 3-2 ABI PRISM SeqScape v2.x 3-3
3 6. A2 A3 Auto Add A1 A2 A3 Samples to Add 3-4 3-3 3-3 7. OK Import Samples 3-4 Project Navigator 3-4 3-4 ABI PRISM SeqScape v2.x
Analyze samples ABI PRISM SeqScape 1 Project Navigator Unassembled Consensus Segment 3-5 3-5 4 ABI PRISM SeqScape v2.x 3-5
3 3-6 ABI PRISM SeqScape v2.x
4...................................................4-2..........................................4-6.................................................4-8............................4-9.......................................................4-13..................................................4-14................................................4-18..................................................4-18 ABI PRISM SeqScape v2.x 4-1
4 Project Specimen SeqScape Active Layer HLA-C_CDS 4-1 Project ROI 2 3 4 ROI 4-1 Analysis QC Analysis QC Analysis QC 1. Project Navigator TutorialProject 4-2 4-2 TutorialProject 2. Tools > Report Manager 3. Report Manager Analysis QC Report 4-3 4-3 4-2 ABI PRISM SeqScape v2.x
4. Report Settings Wrap Text 4-3 5. Specimen Analysis Sample Analysis 4-3 4-4 Wrap Text 4-3 A1 A2 A3 Analysis QC Specimen Analysis Specimen Analysis 4-1 Specimen Score Total # Variants 4-1 - - - - - - Sample Analysis ABI PRISM SeqScape v2.x 4-3
4 Sample Analysis Partial Output No output 4-4 Sample Analysis A3 4-4 A3-4F_05 45.9% Analysis Protocol 35% 4-4 A3 A3-4F_05 4-5 Sample 4-5 4-4 ABI PRISM SeqScape v2.x
Electropherogram 4-6 Clear Range 4-6 A3-4F_05 Electropherogram Analysis Protocol ABI PRISM SeqScape v2.x 4-5
4 Analysis Protocol A3-4F_05 1. Project Navigator A3-4F_05 2. Analysis > Analysis Settings Analysis Protocol 3. Filter 4-7 4. Maximum Mixed Bases (%) 46.0 4-7 Analysis Protocol A3-4F_05 SeqScape ABI PRISM SeqScape 10 4-6 ABI PRISM SeqScape v2.x
5. OK Project Navigator 4-8 4-8 6. Analyze samples 4-9 HSA277102 4-9 7. Analysis QC Report ABI PRISM SeqScape v2.x 4-7
4 1. SeqScape HLA-C_CDS 2. Project Navigator A1 4-10 4-10 A1 3. ROI 4-10 Legend 4-8 ABI PRISM SeqScape v2.x
Specimen Consensus Segment Assembly 2 Layout - Assembly - 1. SeqScape HLA-C_CDS 2. Project Navigator AF250557 3. Layout 4-11 4-11 Layout ABI PRISM SeqScape v2.x 4-9
4 4. Assembly 4-12 Assembly Active Layer ROI Sample Assembly 4-12 Assembly 5. Sample Assembly 4-12 6. 4-13 Show/Hide All Electropherograms 4-13 4-10 ABI PRISM SeqScape v2.x
Clear Range Clear Range 5 3 Clear Range Clear Range Sample Assembly 4-14 A1-2F_01 A1-2R_02 Clear Range CR-Start Ref. Start A1-2F_01 CR 29 27 5 Clear Range 3 CR Sample Assembly 4-14 Assembly Clear Range 1. A1-2F_01 3 4-15 4-15 CR 2. Set CR start [ at selection CR CR ABI PRISM SeqScape v2.x 4-11
4 3. 1 2 A1-2R_02 CR Sample Assembly 1 4-16 Sample Assembly 4-16 Start Point 4-12 ABI PRISM SeqScape v2.x
Project Specimen ROI 1. SeqScape Active Layer HLA-C_CDS 4-17 4-17 2. A2 3. A2 4-18 4-18 A2 ABI PRISM SeqScape v2.x 4-13
4 RDG HLA-C Library Search Library Search 1. Project Navigator A2 2. Tools > Report Manager 3. Report Manager Library Search Report 4. Report Settings Wrap Text 5. Window > Tile Hit List 4-19 4-19 Constant Position Error 4-15 4-20 Position Project A2 2 4-14 ABI PRISM SeqScape v2.x
6. A2 4-20 A2-4R_04 4 45 K G T G T 1 G 4-20 A2-4R-04 45 7. QV 4-20 QV K QV 16 2.5% G G 8. G QV 4-21 4-21 ABI PRISM SeqScape v2.x 4-15
4 9. A2 Library Search 2 10. Library Search Library Identification Library Identification A2 Library Identification 1. A2 Identification 4-22 Identification 2. 4-22 Identification 4-22 Project Assembly Identification 3. View Column Selector 4-16 ABI PRISM SeqScape v2.x
4. Identification 4-23 4-23 Project Assembly Identification Project Assembly 2 197 268 4 210 A2 5. QV QV ABI PRISM SeqScape 10 ABI PRISM SeqScape v2.x 4-17
4 4-24 4-24 A2 A2 3 Cw*07011 07012 0706 Cw*02022 HLA-C 5 Cw*07012 Cw*07011 Cw*0706 5 DNA Library Search SeqScape Manager ABI PRISM SeqScape 9 4-18 ABI PRISM SeqScape v2.x
Software Warranty Information A Computer Configuration.................................................... A-2 Limited Product Warranty................................................... A-2 ABI PRISM SeqScape v2.x A-1
A Software Warranty Information 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 Warranty Period Effective Date Warranty Claims Warranty Exceptions Applied Biosystems warrants that for a period of ninety (90) days from the date the warranty period begins, its ABI PRISM SeqScape Software Version 2.x 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 (i) 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 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 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. A-2 ABI PRISM SeqScape v2.x
Limited Product Warranty The foregoing provisions set forth Applied Biosystems' sole and exclusive representations, warranties, and obligations with respect to its products, 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. Warranty Limitations The remedies provided herein are the buyer's 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 terms 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. 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. ABI PRISM SeqScape v2.x A-3
A Software Warranty Information A-4 ABI PRISM SeqScape v2.x