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

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D Appendix D Advanced Topic: Primary Analysis Understanding quality values in the SOLiD System together can estimate whether there is a localized problem over the whole sample (larger deviation with no outliers), a regional problem (outlier population that spans channels), or a transient problem (outliers in a single channel). A difference with the blur metric, however, is that whereas the blur metric is constant over the number of beads present in a panel, the exposure is more sensitive to the number of beads. A baseline exposure value per channel is determined based on a sampling strategy, but panels with either significantly more or fewer beads can be affected differently. As with the blur value, the reported value is the largest mean exposure value of the four channels over all panels in the sample. Understanding quality values in the SOLiD System The SOLiD primary analysis software analyzes the raw image data collected by the system and generates a color-space read for each tag. The software generates a quality value (QV) for each color call, which is an estimate of confidence for that color call. The SOLiD 4 System QVs are similar to those generated by Phred and the KB Basecaller software for capillary electrophoresis sequencing. The quality value q for a particular call is mathematically related to its probability of error p. The algorithm used to predict QVs for the SOLiD 4 System is similar to Phred and the KB Basecaller software. (Refer to the paper, Ewing B., Green P., 1998, Base-calling of automated sequencer traces using Phred. II. Error probabilities. Genome Research 8:186-194.) The algorithm relies on training or calibration to a large set of control data and color calls for which the correct call is known. In the SOLiD 4 System, the correct call is determined by mapping the read to a known reference sequence. 180 Applied Biosystems SOLiD 4 System SETS Software User Guide
Usage Validation and observed quality Applied Biosystems SOLiD 4 System SETS Software User Guide Appendix D Advanced Topic: Primary Analysis Understanding quality values in the SOLiD System D Given a read with QVs, the expected number of errors in the read can be estimated. If the QV of the i-th call is qi, then the expected number of errors in the read is: is the predicted probability of error for the i-th call. This probability includes the fact that QVs are conventionally rounded to the nearest integer. The average error rate for this read is m/n, which can be converted into an overall quality score for the whole read. The conventional method of validating QVs involves computing a so-called observed quality for all the calls with a particular predicted quality in a set of control data. The following figure shows such a validation. 181
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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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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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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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Colorcall Jobs Applied Biosystems S
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Filter Fasta fails Applied Biosyste
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View History View Run History in SE
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System Logs Applied Biosystems SOLi
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Imaging Metrics Report Applied Bios
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Cycle Heat Map Report Select a heat
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Chapter 4 View Reports View overall
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Exposure Time Report Applied Biosys
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Chapter 4 View Reports View overall
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Determine the optimal titration poi
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View sample reports Applied Biosyst
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Resizing images Applied Biosystems
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Scaled data Applied Biosystems SOLi
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Angle data Applied Biosystems SOLiD
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View Analysis Reports Applied Biosy
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View analysis Data Files View Analy
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Auto-Correlation Report Applied Bio
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Error Profiles Report Applied Biosy
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Master Report Tool Generate a Maste
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Chapter 4 View Reports Master Repor
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Chapter 5 Perform Reanalysis Primar
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Effect on the current run Applied B
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View Multiplexing Series in SETS Ac
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Set up Multiplexing run in ICS Crea
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Multiplexing Assignment report Appl
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Reassign Run Applied Biosystems SOL
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Auto export in SETS Applied Biosyst
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Auto export with JMS broker and Exp
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3. Run the following command: Appli
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Manually configure the RSA keys App
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Change export for current run only
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Event notifications and email servi
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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
Page 141 and 142: Applied Biosystems SOLiD 4 System
Page 145 and 146: Appendix B Advanced Topic: Data Ana
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
Page 155 and 156: Sampling Allele ratio Applied Biosy
Page 157 and 158: C Saving data Applied Biosystems SO
Page 161 and 162: D What is primary analysis? Applied
Page 163 and 164: Primary analysis workflow Applied B
Page 165 and 166: Primary analysis inputs and outputs
Page 167 and 168: Appendix D Advanced Topic: Primary
Page 169 and 170: Storage requirements for primary an
Page 175 and 176: Redo the read-filtering process App
Page 177 and 178: Image metrics Applied Biosystems SO
Page 179: Applied Biosystems SOLiD 4 System
Page 183 and 184: Key files generated by primary anal
Page 185 and 186: .spch files Applied Biosystems SOLi
Page 187 and 188: Troubleshooting analysis failure Ap
Page 191 and 192: E Applied Biosystems SOLiD 4 Syste
Page 193 and 194: LICENSE Appendix E Software License
Page 195 and 196: Appendix E Software License Agreeme
Page 201 and 202: Alignment Matrix The alignment matr
Page 205 and 206: Glossary WFA A Workflow Analysis (W
Page 207 and 208: Related documentation Document BioS
Page 209: Part Number 4448411 Rev. B 04/2010
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