Guide to Reviewing and Approving Custom Designs

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1 Guide to Reviewing and Approving Custom Designs SeqCap EZ Designs, v4.1 Overview This document describes how to review and approve the proposed custom SeqCap EZ and SeqCap EZ Prime designs based on the customer-provided genomic regions. The purpose of reviewing designs is to verify that all desired regions are covered adequately and that the design meets your needs. This is part of our process, and must be completed by the customer prior to manufacturing of the design. If the regions vital to your experiment are not adequately covered, you may need to modify the parameters of your design. Before You Begin Roche provides design file(s) in three or four formats: BED (.bed) files: Viewable in the Roche SignalMap software or other genome browsers, such as in the Internet-based UCSC Genome browser, the Broad Institute's Integrative Genomics Viewer or ENSEMBL. For information about SignalMap and to download software, go to Coverage Summary Files (.txt) files: Viewable in any text editor. (Conditional) bedgraph (.bedgraph) file: Viewable in several genome browsers, such as the Internetbased UCSC Genome browser, the Broad Institute s Integrative Genomics Viewer, or ENSEMBL. BedGraph files are only included if targeted replication has been requested. This file type is not properly rendered in SignalMap. Design Summary (.pdf) file: This file contains relevant information on your design and can be opened in any PDF viewer. For Research Use Only. Not for use in diagnostic procedures.

2 Keep the following in mind while reviewing your design: For the purposes of visualizing coverage, use the three BED files provided with the design: primary_targets.bed, capture_targets.bed, and predicted_no_coverage_regions.bed. These files are described in detail in the appendix. If targeted replication was requested for the design, an additional file, a bedgraph, was included to visualize relative probe abundance. When reviewing the design files, focus on gaps in the capture_targets track that do not provide coverage for the primary_targets track. These gaps represent portions of your target region not covered by the design. Review the region-by-region coverage file for detailed coverage for each region. If two or more of your target regions overlap, Roche merges them into one region. In addition, for a SeqCap EZ or SeqCap EZ Prime design, regions that are less than 100 bp are padded to a minimum size of 100 bp. Regions not covered by the design are usually repetitive regions which, if included, cause capture of other homologous regions in the genome and decrease capture efficiency. Therefore, most Roche SeqCap experiments benefit from excluding these regions in the design. Check the region-by-region coverage file for information on regions not covered due to repetitive regions. The stringency filter Roche uses by default will not include low copy repeats in the design. If these regions are necessary to your research and you are working with the custom design group, indicate this requirement in the to Roche (refer to Step 3. Approve or Request Changes to the Design on page 9). Roche will use less stringent criteria in generating the design. Be aware that the less stringent design may provide more genomic coverage but at the cost of a decrease in capture efficiency and you may see more off-target reads when the captured DNA is sequenced. Please note that this parameter selection is not available in NimbleDesign. If you have questions, contact Roche Technical Support (refer to page 13 for contact details.) 2 Guide to Reviewing and Approving Custom Designs: SeqCap EZ Designs, v4.1

3 Review and Approve Step 1. Review the Design Summary File and Coverage Files The design summary files and coverage files describe the properties of your SeqCap custom design. 1. Open the Design Summary File (PDF), and check that all information is correct for the design specification requested. For example: Figure 1: Example Design Summary File. 2. Open the coverage_summary.txt file using a text editor, such as WordPad, Notepad, or Microsoft Excel. 3. Review each field to ensure the design meets the specifications for your Sequence Capture project. 4. Refer to the appendix to become familiar with the definitions of each field. Guide to Reviewing and Approving Custom Designs: SeqCap EZ Designs v4.1 3

Figure 2: Example Coverage Summary File Displayed in Excel. 5. Open the coverage.txt file. It is recommended that you view this file with a spreadsheet program, such as Microsoft Excel.

Review this file thoroughly to ensure the design meets the specifications for your Sequence Capture project.

4 Figure 2: Example Coverage Summary File Displayed in Excel. 5. Open the coverage.txt file. It is recommended that you view this file with a spreadsheet program, such as Microsoft Excel. Figure 3: Example Region-By-Region Coverage File Displayed in Excel. 6. This file displays region-by-region coverage information, with some details on why regions might not have full coverage. Review this file thoroughly to ensure the design meets the specifications for your Sequence Capture project. This file may be sorted by percent coverage to rapidly identify regions with little or no coverage in the design. See the Appendix for a detailed description of each column in the file. If two or more of your target regions overlap, Roche merges them into one region. In addition, for a SeqCap EZ or SeqCap EZ Prime design, regions that are less than 100 bp are padded to a minimum size of 100 bp. 4 Guide to Reviewing and Approving Custom Designs: SeqCap EZ Designs, v4.1

Step 2. Visual Review of the Design Refer to the appropriate instructions for the software you are using to review the design files. Using SignalMap Software to Review the Design 1.

5 Step 2. Visual Review of the Design Refer to the appropriate instructions for the software you are using to review the design files. Using SignalMap Software to Review the Design 1. Save the bed files provided via (if working with a designer) or the Review tab in NimbleDesign to your computer or a network drive. 2. Open SignalMap software. 3. Select File -> New to open the primary_targets.bed file. 4. Select File -> Import to import the capture_targets.bed, and, if desired, the predicted_uncovered_targets.bed files into the same panel. Figure 4 shows example primary and capture target files displayed in the SignalMap window. Figure 4: Example Design Files Displayed in the SignalMap Window. Review the design using the SignalMap software s functions, such as the following: Zoom: Click the magnifier button on the toolbar, and then click and drag to draw a bounding box around the region to magnify. Alternatively, hold down Ctrl and press + to zoom in or - to zoom out. Arrange chromosomes: View data tracks in many different ways by chromosome. Use the pane selector field below the toolbar to change the view to either all chromosomes (All Tracks) or a selected chromosome (e.g. chr2). Go to specific genomic location: Select View -> Go to position, select the chromosome, and enter the start and end positions. Refer to the SignalMap User s Guide available at for additional details about the software s capabilities. Guide to Reviewing and Approving Custom Designs: SeqCap EZ Designs v4.1 5

Using UCSC Genome Browser to Review the Design The images and results used in this document reflect the UCSC Genome Browser interface as of December 2015. 1. Save the primary_targets.

Open the UCSC Genome browser home page: http://genome.ucsc.edu. 3. In the left pane of the UCSC Genome browser home page, click Genomes. 4.

2009 (GRCh37/hg19) as the assembly. 5. Click the add custom tracks button.

6 Using UCSC Genome Browser to Review the Design The images and results used in this document reflect the UCSC Genome Browser interface as of December Save the primary_targets.bed, capture_targets.bed, and predicted_uncovered_targets.bed files provided via to your computer or a network drive. If a bedgraph was provided, save that as well. 2. Open the UCSC Genome browser home page: 3. In the left pane of the UCSC Genome browser home page, click Genomes. 4. In the Genome Browser Gateway page, choose the appropriate options in the genome and assembly list boxes for your design. This example uses Human for the build and Feb (GRCh37/hg19) as the assembly. 5. Click the add custom tracks button. If the Gateway page displays the manage custom tracks button, you may need to delete some of the existing tracks before viewing new data. 6. In the Add Custom Tracks page: a. Click the Choose file button to select and upload the BED file. b. Click the Submit button. 7. In the Manage Custom Tracks page: The Manage Custom Tracks page provides a link to name and describe your tracks. If you are loading multiple tracks, this will be necessary to help visualize the data. a. Change the configuration of existing tracks. Figure 5 shows an example of changing a track configuration. Figure 5: Example of Changing the Track Configuration in the UCSC Genome Browser. 6 Guide to Reviewing and Approving Custom Designs: SeqCap EZ Designs, v4.1

b. Add additional BED files as in Step 6, above. c. Click the go button to view the custom track(s). Figure 6 shows an example design displayed in the UCSC Genome browser.

7 b. Add additional BED files as in Step 6, above. c. Click the go button to view the custom track(s). Figure 6 shows an example design displayed in the UCSC Genome browser. Figure 6: Example Design File Displayed in the UCSC Genome Browser. 8. Review the design using the UCSC Genome browser s functions, such as the following: Zoom: Click the zoom in and zoom out buttons to zoom in or out on the center of the annotation tracks window by 1.5-, 3-, or 10-fold. Scroll: Click the move buttons to scroll to the left or right. Display a different position in the genome: Enter the coordinates in the position/search text box and click the jump button. View base composition: Click the base button to view the base composition of the sequence underlying the current annotation track display. Useful UCSC-provided tracks to load include mapability and RepeatMasker tracks. These can be used to diagnose regions uncovered due to repeats. 9. Click the Help link for additional details about the browser s capabilities. 10. If your design included per-region probe copy number adjustment (aka targeted replication ), you will have a bedgraph file in your deliverables. You may visualize this data in UCSC s genome browser, as follows: Please note, SignalMap treats bedgraph files as a BED file and does not currently plot the 4 th column. a. Load the bedgraph file as you would any other custom track (see above steps 4-6). Guide to Reviewing and Approving Custom Designs: SeqCap EZ Designs v4.1 7

8 b. Rename the track to something unique, or it may be replaced by any subsequent track additions (see above step 7). c. Click the go button to view custom track(s). Figure 7 shows an example bedgraph track displayed in the UCSC Genome browser. Note the bar height corresponds to the actual relative replicate value for the region. Figure 7: Example bedgraph File Displayed in the UCSC Genome Browser. 8 Guide to Reviewing and Approving Custom Designs: SeqCap EZ Designs, v4.1

9 Step 3. Approve or Request Changes to the Design For approving designs generated with NimbleDesign, please see the NimbleDesign User's Guide, available at For tracking purposes, reply to the that included the deliverables with the proposed design. In the reply, indicate you approve the design or specify any changes needed for approval. Roche Designers cannot finalize the design and send to production to manufacture the SeqCap probes until we receive your written approval. Guide to Reviewing and Approving Custom Designs: SeqCap EZ Designs v4.1 9

10 Appendix: Included files capture_targets.bed Overlapping probe coverage regions where each base is covered by at least one probe. This is a three column, tab-delimited coordinate file, with no header, in BED format ( suitable for loading into various genome browsers. primary_targets.bed Requested regions, padded to a minimal length and with overlapping ranges merged. This is a three column, tab-delimited coordinate file, with no header, in BED format ( suitable for loading into various genome browsers. Only regions with at least one selected capture probe appear in this file. predicted_no_coverage_regions.bed.bedgraph All positions from the primary_targets.bed that are not within 100 base pairs of a capture probe. This is a three column, tab-delimited coordinate file, with no header, in BED format ( suitable for loading into various genome browsers. All positions from the capture_targets.bed have associated values indicating the relative number of probes. This is a four column, tab-delimited coordinate file, with a header indicating track type, in BEDGRAPH format ( suitable for loading into various genome browsers where it will be rendered as a bar graph. coverage_summary.txt The global coverage properties of your SeqCap EZ Choice design. This file is tab-delimited text and can be opened in Excel or a text editor. A description of the fields included in this file can be found below. Genome build: Genome and build targeted by the design (e.g. GRCh37/hg19). Number of regions: Number of regions after padding and consolidation. Length of regions: Sum total of all region sizes (in basepairs) after padding and consolidation. Probe_Coverage column: Direct coverage; for the padded and consolidated regions, these values represent bases, covered or uncovered, directly by a capture probe. 10 Guide to Reviewing and Approving Custom Designs: SeqCap EZ Designs, v4.1

11 Estimated_Coverage column: Indirect or adjacent coverage; for the padded and consolidated regions, these values represent bases, covered or uncovered, that are within 100 bp of a capture probe s sequence position. This is an estimate of the actual amount of sequence that may be captured by a capture probe, determined in empirical tests, reflecting that capture probes may hybridize to the end of library insert and extend coverage away from the probe. The 100 bp capture padding was validated with Illumina dual-end sequencing, using a typical library size of ~200 bp. This number may not be accurate for libraries with much larger or smaller insert sizes, or single end reads. Target bases covered: Sum of all bases covered (in basepairs) by at least one capture probe or by predicted captured sequence. Calculations for Probe_Coverage and Estimated_Coverage are provided. Percent target bases covered: Percentage of all bases from the padded and consolidated regions which are covered by one or more capture probe or by predicted captured sequence. Calculations for Probe_Coverage and Estimated_Coverage are provided. Targets with no coverage: Number of padded and consolidated regions with no probes or no predicted captured sequence. Calculations for Probe_Coverage and Estimated_Coverage are provided. Target Bases Not Covered: The number of target bases from the padded and consolidated regions that are not covered by a capture probe or captured sequence. Calculations for Probe_Coverage and Estimated_Coverage are provided. Target Bases Not Covered (due to N's): The number of target bases from the padded and consolidated regions that are not covered by a capture probe or captured sequence due to the source genome having N s or ambiguous bases within the target range. Calculations for Probe_Coverage and Estimated_Coverage are provided. Target Bases Not Covered (due to repeats): The number of target bases from the padded and consolidated regions that are not covered by a capture probe or captured sequence due to the source genome having low complexity or highly repetitive DNA within the target range. Roche avoids selecting Sequence Capture probes in regions of low complexity or high repeat content to reduce the chance of capturing off-target sequence.calculations for Probe_Coverage and Estimated_Coverage are provided. Percent Target Bases Not Covered: The percentage of target bases from the padded and consolidated regions that are not covered by a capture probe or captured sequence. Calculations for Probe_Coverage and Estimated_Coverage are provided. Percent Target Bases Not Covered (due to N's): The percentage of target bases from the padded and consolidated regions that are not covered by a capture probe or captured sequence due to the source genome having N s or ambiguous bases within the target range. Calculations for Probe_Coverage and Estimated_Coverage are provided. Percent Target Bases Not Covered (due to repeats): The percentage of target bases from the padded and consolidated regions that are not covered by a capture probe or captured sequence due to the source genome having low complexity or highly repetitive DNA within the target range. Roche avoids selecting Sequence Capture probes in regions of low complexity or high repeat content to reduce the chance of capturing off-target sequence. Calculations for Probe_Coverage and Estimated_Coverage are provided. Total capture targets: The total number of regions in the capture target files. Total capture space (bp): The total number of bases covered by the capture targets. This can be very different from the primary target space, and provides an idea of the total amount of sequencing that will be needed for each sample. Guide to Reviewing and Approving Custom Designs: SeqCap EZ Designs v4.1 11

12 coverage.txt A tab-delimited text file show region-by-region coverage information of the padded an consolidated customer input regions. The headers are as follows: REGION_NAME: The customer-provided name from the 4th column of their input BED file(s). If no 4th column was provided, then a default name of the selection region will be used instead. This name takes the format of CHROMOSOME:START-STOP. CHROMOSOME: The target chromosome for the region. START: The region start coordinate. STOP: The region stop coordinate. LENGTH: The length of the region. BASES_PROBE_COVERAGE: The number of bases in the region which are directly covered by a capture probe. FRAC_PROBE_COVERAGE: The percent coverage of the region, as a fraction of 1, using direct coverage. For example, a value means that every base of the target is covered by one or more capture probes. A value of means that 46% of the region is covered by one or more capture probes. BASES_ESTIMATE_COVERAGE: The number of bases in the region directly covered by a probe or by indirect/adjacent coverage. This is an estimate of the actual amount of sequence that may be captured by a capture probe, determined in empirical tests, reflecting that capture probes may hybridize to the end of library insert and extend coverage away from the probe. The 100 bp capture padding was validated with Illumina dual-end sequencing, using a typical library size of ~200 bp. This number may not be accurate for libraries with much larger or smaller insert sizes, or single end reads. FRAC_ESTIMATED_COVERAGE: The percent coverage of the region, as a fraction of 1, using indirect/adjacent coverage. For example, a value means that 98.2% of the target is covered indirectly by one or more capture probes. PREDICTED_NO_COVERAGE_BASES: Number of bases in the region that are not covered indirectly and are likely to be missed during capture. BASES_W_NO_PROBE_COV: The number of bases in the region which are not directly covered by a capture probe. BASES_W_NO_PROBE_COV_DUE_TO_N: Number of bases in the region that are not covered directly by probes due to the region containing N s or ambiguous bases in the source. Roche cannot design probes against sequence containing Ns or non-acgt characters. BASES_W_NO_PROBE_COV_DUE_TO_REPEATS: Number of bases in the region that are not covered directly by probes due to the region containing low complexity or highly repetitive sequence. Roche avoids selecting Sequence Capture probes in regions of low complexity or high repeat content for the purposes of reducing off-target sequencing results. BASES_W_NO_EST_COV: The number of bases in the region not directly covered by a probe or by indirect/adjacent coverage. BASES_W_NO_EST_COV_DUE_TO_N: Number of bases in the region that are not covered indirectly due to the region containing N s or ambiguous bases in the source. BASES_W_NO_EST_COV_DUE_TO_REPEATS: Number of bases in the region that are not covered indirectly due to the region containing repetitive sequence. 12 Guide to Reviewing and Approving Custom Designs: SeqCap EZ Designs, v4.1

13 Contact Information Customer Service If you have questions, contact Roche Customer Service: Technical Support If you have technical questions, contact your local Roche Technical Support. Please go to for contact information. Patent License Limitations For patent license limitations for individual products, please refer to /17 For Research Use Only. Not for use in diagnostic procedures. NIMBLEDESIGN and SEQCAP are trademarks of Roche. All other product names and trademarks are the property of their respective owners. Roche Sequencing Solutions, Inc Hacienda Drive Pleasanton, CA USA sequencing.roche.com/support.html Roche Sequencing Solutions, Inc. All rights reserved. Guide to Reviewing and Approving Custom Designs: SeqCap Designs v4.1 13

Design and Annotation Files

Design and Annotation Files Release Notes SeqCap EZ Exome Target Enrichment System The design and annotation files provide information about genomic regions covered by the capture probes and the genes