Applied Biosystems SOLiD™ 4 System SETS Software User Guide ...

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6 Chapter 6 Support Multiplexing Fragment and Paired-End Multiplexing Fragment and Paired-End Multiplexing Overview of Multiplexing and Barcodes Multiplexing in general, and barcodes in particular, enable the SOLiD 4 System to target many applications, from deep sequencing of a single source to extreme parallel sequencing of a large number of samples over limited regions - sometimes on the same slide. The additional stretches of DNA sequence, known as barcodes, can be used to segregate pooled library samples during sequencing back into their original (logical) single library groupings. Barcodes –in their simplest form– allow the pooling of samples on a slide without the loss of surface area creating geographic isolations. Multiplexing occurs at both the whole slide level as well as at the geographically isolated (mask) region level. A barcode, by definition, is a tag with a known sequence that is attached to a sequencing primer on a bead during library preparation. When sequenced, it is the barcode that identifies and classifies the bead to a named sample. This barcoding, or indexing, approach allows multiplexing at the sequencing level. Following sequencing of the target DNA, additional rounds of ligation-based sequencing are performed using primer sets complimentary to the barcode. The resulting reads can then be sorted by the barcode and aligned in groups to the reference sequence. Barcodes are assigned to a library both conceptually and physically (as specific tags) as part of the library preparation. If more than one barcode is assigned to a single library during the library preparation step, the conceptual grouping is referred to as a barcode group. All barcodes in the pool will assign the underlying beads to the same original library for analysis. The SOLiD 4 System supports multiplexing for both fragment sequencing and paired-end sequencing. See “Naming convention for sequencing reads” on page 11. Note: Multiplexing is not available for mate-paired libraries. 104 Applied Biosystems SOLiD 4 System SETS Software User Guide
View Multiplexing Series in SETS Access Multiplexing Series runs in SETS Applied Biosystems SOLiD 4 System SETS Software User Guide Chapter 6 Support Multiplexing View Multiplexing Series in SETS 6 The Multiplexing Series view in SETS shows all the barcode kits in the system to date. When a multiplexing series run is created in ICS, only the multiplexing series names are displayed. To view multiplexing series details, click View�Multiplexing Series. Select the multiplexing series run that you want to view. Every multiplexing series file includes: Name of the multiplexing kit Colorspace code Basespace sequence 105
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Applied Biosystems SOLiD 4 System
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How to use this guide Applied Biosy
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1 �� SETS So
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Naming convention for sequencing re
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SETS homepage Applied Biosystems SO
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2 �� SETS So
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SOLiD 4 System Software workflow A
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Log in to SETS Applied Biosystems S
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Define primary analysis settings Ap
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Primary analysis parameters 5. Clic
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Parameter Description Applied Biosy
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Parameter Description Applied Biosy
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Parameter Description Application D
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Define secondary analysis settings
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Chapter 2 Prepare Run Settings Set
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Load a Reference Sequence Specify a
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3 �� SETS So
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Note: For in-progress runs, you can
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Note: Refer to the Imaging Metrics
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Chapter 3 Monitor the Run in SETS V
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View sample data Applied Biosystems
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Troubleshoot failed jobs Sample ana
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Page 55 and 56: Filter Fasta fails Applied Biosyste
Page 57 and 58: View History View Run History in SE
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Page 61 and 62: System Logs Applied Biosystems SOLi
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Page 65 and 66: Imaging Metrics Report Applied Bios
Page 67 and 68: Cycle Heat Map Report Select a heat
Page 69 and 70: Chapter 4 View Reports View overall
Page 71 and 72: Exposure Time Report Applied Biosys
Page 73 and 74: Chapter 4 View Reports View overall
Page 75 and 76: Determine the optimal titration poi
Page 77 and 78: View sample reports Applied Biosyst
Page 79 and 80: Resizing images Applied Biosystems
Page 81 and 82: Scaled data Applied Biosystems SOLi
Page 83 and 84: Angle data Applied Biosystems SOLiD
Page 85 and 86: View Analysis Reports Applied Biosy
Page 87 and 88: View analysis Data Files View Analy
Page 89 and 90: Auto-Correlation Report Applied Bio
Page 91 and 92: Error Profiles Report Applied Biosy
Page 93 and 94: Master Report Tool Generate a Maste
Page 95 and 96: Chapter 4 View Reports Master Repor
Page 99 and 100: Chapter 5 Perform Reanalysis Primar
Page 101 and 102: Effect on the current run Applied B
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Page 107 and 108: Set up Multiplexing run in ICS Crea
Page 109 and 110: Multiplexing Assignment report Appl
Page 111 and 112: Reassign Run Applied Biosystems SOL
Page 115 and 116: Auto export in SETS Applied Biosyst
Page 117 and 118: Auto export with JMS broker and Exp
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Page 121 and 122: Manually configure the RSA keys App
Page 123 and 124: Change export for current run only
Page 125 and 126: Event notifications and email servi
Page 129 and 130: Delete runs Applied Biosystems SOLi
Page 131 and 132: Chapter 7 Manage Administrative Tas
Page 133 and 134: A Overview Applied Biosystems SOLiD
Page 135 and 136: Appendix A Web Services WSDL A Bind
Page 137 and 138: B Applied Biosystems SOLiD 4 Syste
Page 139 and 140: Appendix B Advanced Topic: Data Ana
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Page 147 and 148: Complementing color-space data Appl
Page 151 and 152: Data analysis considerations Unders
Page 153 and 154: SNPs and errors High accuracy for S
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Sampling Allele ratio Applied Biosy
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C Saving data Applied Biosystems SO
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D What is primary analysis? Applied
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Primary analysis workflow Applied B
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Primary analysis inputs and outputs
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Appendix D Advanced Topic: Primary
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Storage requirements for primary an
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Redo the read-filtering process App
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Image metrics Applied Biosystems SO
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Usage Validation and observed quali
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Key files generated by primary anal
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.spch files Applied Biosystems SOLi
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Troubleshooting analysis failure Ap
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E Applied Biosystems SOLiD 4 Syste
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LICENSE Appendix E Software License
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Appendix E Software License Agreeme
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Alignment Matrix The alignment matr
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Glossary WFA A Workflow Analysis (W
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Related documentation Document BioS
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Part Number 4448411 Rev. B 04/2010
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