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

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2 Chapter 2 Prepare Run Settings Set analysis parameters Parameter Description Exposure Value Typically the ideal exposure level of an image is one that maximizes the dynamic range of the content with some signal occupying the lowest (darkest) range and some information extending just to the saturation (highest) point. An overexposed image is one that compresses too much of the content at the top end of the range, creating a non-linearity in response and making it impossible to differentiate between the highest signals. An underexposed image is one that does not extend the content of the image across the possible range, thereby creating a low-end compression and failing to use the available range of the system. The calculated exposure value is a measure of how close the system is to capturing an image that maximizes dynamic range. Looking at the tail behavior of the signal distribution makes the determination. If the tail does not extend to the saturation point, the image is underexposed. In the case of overexposure, the tail has a peak at the far right side that is a measurement of the saturation. To get an estimate of the overexposure, the slope of the curve prior to the peak is extrapolated to a distance equal to the area of the saturated peak. The computed exposure quality metric is two-sided, extending from an ideal of 0 in the negative direction for underexposure and in the positive direction for overexposure and uses a log (2) scale. To fit this value into the traffic light model, the distance from ideal performance (not necessarily a value of 0) is used. Applied Biosystems has found that for purposes of our color calling algorithms, slightly overexposing the images yields better results. As with the blur value, the reported value is the largest exposure value of the four channels over all panels in the sample. Exposure Population As with the blur population, rather than providing the average exposure quality value, the exposure population value reports the number of panels that have an exposure quality beyond a desired threshold. A larger than average size can be due to a small number of panels with large outlier values or a large number of panels with a larger size. The two values, taken together, can estimate whether there is an endemic 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 from the blur metric, though, is that blur metric is invariant to the number of beads present in a panel where 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. 30 Applied Biosystems SOLiD 4 System SETS Software User Guide
Parameter Description Application Descriptions Applied Biosystems SOLiD 4 System SETS Software User Guide Chapter 2 Prepare Run Settings Set analysis parameters 2 Exposure Time In the SOLiD 4 System, every panel could technically have a different exposure value within a channel. This metric tracks the mean exposure time over all panels and reports the highest of the four channels. Increasing exposure times can indicate problems in the sequence. If the exposure time jumps too high too quickly, the signal on the beads is decreasing too quickly cycle over cycle, and there will likely be problems achieving longer read lengths. Vibration, or other issues, can also up the exposure time as the beads become larger due to blur and the exposures increase in order to get sufficient signal spread over a larger area. An increase in exposure time by itself is not a problem, but it is a warning sign that is valuable when taken into consideration with the other available run metrics to diagnose potential problems. Exposure times are ‘effective’ exposure times; they are a combination of both the exposure time and the gain. There are two types of applications available for the secondary analysis settings SingleTag IQ 31
Page 1 and 2: Applied Biosystems SOLiD 4 System
Page 7 and 8: How to use this guide Applied Biosy
Page 9 and 10: 1 �� SETS So
Page 11 and 12: Naming convention for sequencing re
Page 13 and 14: SETS homepage Applied Biosystems SO
Page 19 and 20: SOLiD 4 System Software workflow A
Page 21 and 22: Log in to SETS Applied Biosystems S
Page 23 and 24: Define primary analysis settings Ap
Page 25 and 26: Primary analysis parameters 5. Clic
Page 27 and 28: Parameter Description Applied Biosy
Page 29: Parameter Description Applied Biosy
Page 33 and 34: Define secondary analysis settings
Page 35 and 36: Chapter 2 Prepare Run Settings Set
Page 37 and 38: Load a Reference Sequence Specify a
Page 43 and 44: Note: For in-progress runs, you can
Page 45 and 46: Note: Refer to the Imaging Metrics
Page 47 and 48: Chapter 3 Monitor the Run in SETS V
Page 49 and 50: View sample data Applied Biosystems
Page 51 and 52: Troubleshoot failed jobs Sample ana
Page 53 and 54: Colorcall Jobs Applied Biosystems S
Page 55 and 56: Filter Fasta fails Applied Biosyste
Page 57 and 58: View History View Run History in SE
Page 61 and 62: System Logs Applied Biosystems SOLi
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
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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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5 �� SETS So
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Chapter 5 Perform Reanalysis Primar
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Effect on the current run Applied B
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6 �� SETS So
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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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7 �� SETS So
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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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Applied Biosystems SOLiD 4 System
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Delete runs Applied Biosystems SOLi
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Chapter 7 Manage Administrative Tas
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A Overview Applied Biosystems SOLiD
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Appendix A Web Services WSDL A Bind
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B Applied Biosystems SOLiD 4 Syste
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Appendix B Advanced Topic: Data Ana
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Appendix B Advanced Topic: Data Ana
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Complementing color-space data Appl
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Data analysis considerations Unders
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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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