Oncomine cfdna Assays Part III: Variant Analysis
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1 Oncomine cfdna Assays Part III: Variant Analysis USER GUIDE for use with: Oncomine Lung cfdna Assay Oncomine Colon cfdna Assay Oncomine Breast cfdna Assay Catalog Numbers A31149, A31182, A31183 Publication Number MAN Revision C.0 For Research Use Only. Not for use in diagnostic procedures.
2 The information in this guide is subject to change without notice. DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT. Revision history: Pub. No. MAN Revision Date Description C.0 14 November 2016 Update to include the following Ion Reporter workflows: Oncomine Breast Liquid Biopsy Oncomine Breast Tumor B.0 27 October 2016 Update to include the following Ion Reporter workflows: Oncomine Colon Liquid Biopsy Oncomine Colon Tumor A.0 29 June 2016 Initial release of variant analysis guide for Oncomine Lung Liquid Biopsy and Oncomine Lung Tumor workflows in Ion Reporter Software v5.2 and Torrent Variant Caller variant analysis for libraries made with the Oncomine cfdna Assay kits. Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all applicable Limited Use Label Licenses. Corporate entity: Life Technologies Corporation Carlsbad, CA USA Toll Free in USA TRADEMARKS: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified Thermo Fisher Scientific Inc. All rights reserved.
3 Contents About this guide... 4 CHAPTER 1 VariantCaller Plugin variant analysis... 5 Review Oncomine cfdna assay run results... 5 CHAPTER 2 Ion Reporter Variant Analysis... 9 Analysis workflows in Ion Reporter software... 9 View results and determine variants of interest... 9 Export results Oncomine annotations Manually launch an analysis APPENDIX A Documentation and support Related documentation Obtain information from the Help system Customer and technical support Limited product warranty Oncomine cfdna Assays, Part III: Variant Analysis User Guide 3
4 About this guide The Oncomine cfdna Assays can be analyzed with the variantcaller plugin in Torrent Suite Software v5.2.1 or in Ion Reporter Software v5.2. For robust detection of low frequency somatic variants from liquid biopsy samples, we recommend to multiplex up to: 8 Oncomine Lung cfdna Assay libraries on a single Ion 530 chip. 6 Oncomine Colon cfdna Assay libraries on a single Ion 530 chip. 12 Oncomine Breast cfdna Assay libraries on a single Ion 530 chip. In Torrent Suite Software, the Planned Run templates to be used with the Oncomine cfdna Assays are listed in the following table: Application Oncology - Liquid Biopsy Torrent Suite Software template Oncomine Lung Liquid Biopsy DNA Oncomine Colon Liquid Biopsy DNA Oncomine Breast Liquid Biopsy DNA Oncomine Lung Tumor DNA Oncomine Colon Tumor DNA Oncomine Breast Tumor DNA Description Planned run template for use with cell-free DNA (cfdna) research samples. Analysis parameters are optimized for the sensitive and specific detection of rare somatic variants (SNPs, InDels) present at 0.1% frequency in cfdna. Planned run template for use with solid tumor research samples from FFPE as well as fresh frozen tumor tissue. Analysis parameters are optimized for the sensitive and specific detection of rare somatic variants (SNPs, InDels) present at 0.5% frequency. Analysis parameters are optimized to eliminate false positives due to DNA damage resulting from formalin fixation. 4 Oncomine cfdna Assays, Part III: Variant Analysis User Guide
5 1 VariantCaller Plugin variant analysis Review Oncomine cfdna assay run results The Completed Runs Report from an Oncomine cfdna Assay run is similar to variantcaller plugin reports. The following outputs have been added. 1. After the run is complete, click Data4Completed Runs and Results, then click the Run Report for your results. 2. To view a summary of the variant analysis, scroll down to the variantcaller section, then click the appropriate button to download variant calls in.vcf or.xls formats. Oncomine cfdna Assays, Part III: Variant Analysis User Guide 5
6 1 Chapter 1 VariantCaller Plugin variant analysis Review Oncomine cfdna assay run results 3. Review the results in the Median Read Cov, Median Mol Cov, and Targets > 0.8MM columns. Column Median Read Coverage Targets >0.8 Median Molecular Coverage Median Read Coverage and Targets >0.8 Median Molecular Coverage Median Molecular Coverage Description Reports median coverage across targets. Median Molecular Coverage reports median number of individual interrogated DNA molecules across targets. Reports percent of targets with molecular coverage within 80% of the median coverage value. This is a new stricter definition of panel uniformity. Measures the quality of the sequencing run and library performance, while Median Molecular Coverage measures the amount and quality of the input DNA sample. Directly influences the limit of detection in a sample run. We always require two independent molecular families to identify a variant for it to be called. Lower median molecular coverage values result in less sensitive detection of variants at 0.1% frequency, although still sufficient for sensitive detection of variants with higher frequency. For example, Median Molecular Coverage of 700 is sufficient for accurate detection of variants at 0.5% frequency. For sensitive variant detection down to 0.1% frequency, we see optimal results when targeting a Median Read Coverage >25,000, Median Molecular Coverage > 2,500, and Targets >0.8 Median Molecular Coverage >60%. 4. Click a Barcode Name of interest to review Variant Calls by Allele. By default only hotspot alleles calls are shown in the variant table. We do not report hotspot alleles that did not meet our criteria for calling. However, we do provide at least one record for each hotspot position. This can include: novel allele call at hotspot position, hotspot allele call, or absent call when the first two are missing. 6 Oncomine cfdna Assays, Part III: Variant Analysis User Guide
7 Chapter 1 VariantCaller Plugin variant analysis Review Oncomine cfdna assay run results 1 Frequency LOD Column Description Reports the observed frequency of hotspot allele. Reports limit of detection at hotspot position, which is based on the number of interrogated DNA molecules (fragments) containing target. We use the term 0.1% LOD to mean we have data to support specific sensitivity and specificity claims (90% and 98%) at the 0.1% allelic frequency. By default, our analysis tool uses minimum alternative allele frequency threshold of 0.05% and we have a technical lower limit of detection of 0.03% for this method. Observed frequency can be lower than LOD due to sampling nature of the assay. If selected to display hotspot positions with absent variant call, then only one record per hotspot position is displayed and only one of the hotspot alleles at that position is displayed under "Allele Name". 5. Click View Coverage Metrics to view the total number of interrogated DNA molecules at hotspot positions (Molecular Coverage), and the number of molecules containing the variant (Allele Mol Cov). 6. You can modify the types of calls that are displayed in the Allele Calls dropdown list, by selecting or deselecting Absent, Heterozygous, Homozygous, or No Call. No calls are variant calls that are classified as systematic errors. Oncomine cfdna Assays, Part III: Variant Analysis User Guide 7
8 1 Chapter 1 VariantCaller Plugin variant analysis Review Oncomine cfdna assay run results 7. Select Absent in the Allele Call dropdown list to visualize hotspot positions without a valid variant call that meets our analysis criteria. We report one record per hotspot position with missing alternative call, and the alternative allele is an arbitrary value distinct from reference. LOD and molecular coverage metrics at those positions are measurements for variant absence among many interrogated molecules. 8. To view novel alleles, select Novel (sequenced allele that is different from the expected allele defined in the panel hotspot file) in the Allele Source dropdown list. 8 Oncomine cfdna Assays, Part III: Variant Analysis User Guide
9 2 Ion Reporter Variant Analysis Analysis workflows in Ion Reporter software Analysis workflows available in Ion Reporter Software include: Analysis Workflow Description Oncomine Lung/Colon/Breast Liquid Biopsy - w1.1 - DNA -Single Sample Detects and annotates low frequency (to 0.1% limit of detection) variants (SNPs, InDels) from targeted DNA libraries from the Oncomine cfdna Assay. This is compatible with DNA purified from cell-free DNA. Oncomine Lung/Colon/Breast Tumor - w1.1 - DNA -Single Sample Detects and annotates low frequency (to 0.5% limit of detection) variants (SNPs, InDels) from targeted DNA libraries from the Oncomine cfdna Assay. Due to deamination events caused by the FFPE process, the minimum alternative allele frequency is set to 0.3%. This makes it compatible with DNA purified from FFPE tumor tissue as well as fresh frozen tumor tissue. In Torrent Suite Software, you can plan your run to automatically transfer data to the appropriate Ion Reporter server and be analyzed through one of the Oncomine Lung workflows. View results and determine variants of interest Ion Reporter Software analyses are performed automatically on uploading of the data files from the Torrent Suite Software. To view the results: 1. Log in to the Ion Reporter Software. 2. Click the Analyses tab. Oncomine cfdna Assays, Part III: Variant Analysis User Guide 9
10 2 Chapter 2 Ion Reporter Variant Analysis View results and determine variants of interest 3. To view Oncomine cfdna Assay results, select Oncology-Liquid Biopsy in the Application dropdown list. The resulting Analyses screen displays the Oncology-Liquid Biopsy analyses. 4. In the Analyses screen you can: To Open an Analysis Results screen. View details Action Click the hyperlink (in the Analysis column). Click in the blank space of the analysis' row. Sort chronologically Click the Created On column header Open Analysis Results screen 2 View details 3 Sort chronologically 5. In the Analysis Results screen sort or filter the data using the Oncomine - specific annotations. See the software help menu for more options. In the Filter Chains panel, notice that the Oncomine Variants (5.2) filter chain has been applied by default. 10 Oncomine cfdna Assays, Part III: Variant Analysis User Guide
11 Chapter 2 Ion Reporter Variant Analysis View results and determine variants of interest 2 6. In the Liquid Biopsy column, view Mol Depth, Mol Counts, and other columns. Column Molecular Depth Molecular Counts Description Reports number of interrogated DNA molecules containing target. It defines limit of detection at hotspot position in a particular run and sample. For instance, if molecular depth is 1,500, you can have high confidence that no variant is present at 0.2%. If molecular depth is 2,500, you can have high confidence that no variant is present down to 0.1% LOD. For reference calls, Molecular Depth provides measurable metric that serves as confirmation for variant absence among a large number of interrogated molecules. Reports the number of detected DNA molecules containing variant allele. Oncomine cfdna Assays, Part III: Variant Analysis User Guide 11
12 2 Chapter 2 Ion Reporter Variant Analysis View results and determine variants of interest 7. In the Oncomine tab, click the column headers to sort the list of variants by Oncomine Variant Class and Oncomine Gene Class. Reference calls display chromosomal position with empty value in amino acid change field. 8. In the Ontologies tab, click the column headers to sort the list by variant Type or Genes to analyze your results. 12 Oncomine cfdna Assays, Part III: Variant Analysis User Guide
13 Chapter 2 Ion Reporter Variant Analysis Export results 2 Export results To export a report: 1. Click Download, then select All Variants, Filtered Variants or Current Results TSV. 2. Click Home4Notifications to open the Notifications screen, then click to download your results. The software generates a.zip file with three folders: QC, Variants, and Workflow_Settings. Within the Variants folder, you ll find the Oncomine annotated.vcf file, which is used by the Oncomine Knowledgebase Reporter. 3. Open the annotated.vcf file, then scroll to the Oncomine annotations. Oncomine annotations Variant Type Oncomine Gene Class Oncomine Variant Class Annotation Criteria Gain of Missense Hotspot Mutation Gain of In Frame Hotspot Mutation Loss of Missense Hotspot Mutation Gain-of- Gain-of- Loss-of- Hotspot Variant's functional impact is missense Variant occurs in Gain of gene Variant's transcript and codon position occur in predefined missense hotspot list Hotspot Variant occurs in Gain of gene Variant's function, transcript and coding syntax occur in pre-defined inframe hotspot list Hotspot Variant's functional impact is missense Variant occurs in Loss of gene Variant's transcript and codon position occur in predefined missense hotspot list Oncomine cfdna Assays, Part III: Variant Analysis User Guide 13
14 2 Chapter 2 Ion Reporter Variant Analysis Manually launch an analysis Variant Type Oncomine Gene Class Oncomine Variant Class Annotation Criteria Loss of In Frame Hotspot Mutation Gain of Splice Site Hotspot Mutation Loss of Synonymous Hotspot Mutation Loss-of- Gain-of- Loss-of- Hotspot Variant occurs in Loss of gene Variant's function, transcript and coding syntax occur in pre-defined inframe hotspot list Hotspot Variant occurs in Gain of gene Variant's transcript, location, and exon occur in pre-defined splice site hotspot list Hotspot Variant occurs in Loss of gene Variant's function, transcript, and coding syntax occur in pre-defined synonymous hotspot list Manually launch an analysis If your analysis did not automatically launch, you can launch it manually. 1. Click the Workflows tab (or Analyses tab). 2. In the Application dropdown menu, filter for Oncology-Liquid Biopsy. 3. In the Workflow Name column, click on the workflow appropriate to your assay. Oncomine Lung/Colon/Breast Tumor - w DNA - Single Sample or Oncomine Lung/Colon/Breast Liquid Biopsy - w DNA - Single Sample 4. Select Launch Analysis in the Actions dropdown. 5. Select one DNA sample. a. Search by any unique identifier you used to label the samples during setup. b. Click Add Samples. 14 Oncomine cfdna Assays, Part III: Variant Analysis User Guide
15 Chapter 2 Ion Reporter Variant Analysis Manually launch an analysis 2 The samples populate a field on the right side of the screen. 6. Click Next. 7. Confirm that the Oncomine Variant Annotator v2.1 plugin is selected, then click Next. 8. (Optional) Modify the name, then add a description. 9. Click Launch Analysis. 10. Follow the steps in the View results and determine variants of interest on page 9 and Export results on page 13 of this guide to sort, filter, and generate reports of your results. Oncomine cfdna Assays, Part III: Variant Analysis User Guide 15
16 A Documentation and support Related documentation Document Oncomine cfdna Assays, Part I: Library Preparation User Guide (Pub. No. MAN ) Oncomine cfdna Assays, Part II: Plan a Run, Template Preparation, and Sequencing User Guide (Pub. No. MAN ) Oncomine cfdna Assays, Part III: Variant Analysis User Guide (Pub. No. MAN ) Description Describes the preparation of Oncomine cfdna Assay libraries. Describes the automated template preparation of Oncomine cfdna Assay libraries using the Ion Chef System for sequencing on the Ion S5 System. Describes how to perform variant analysis on Oncomine cfdna Assay sequence data, and how to view, sort, and filter results. Note: For additional documentation, see Customer and technical support on page 16. Obtain information from the Help system The Torrent Suite Software has a Help system that describes how to use each feature of the user interface. In the toolbar of the Torrent Suite Software window, click Help4Software Help. You can use the Help system to find topics of interest by: Reviewing the table of contents Searching for a specific topic Customer and technical support Visit thermofisher.com/support for the latest in services and support, including: Worldwide contact telephone numbers Product support, including: Product FAQs Software, patches, and updates Training for many applications and instruments Order and web support 16 Oncomine cfdna Assays, Part III: Variant Analysis User Guide
17 Appendix A Documentation and support Limited product warranty A Product documentation, including: User guides, manuals, and protocols Certificates of Analysis Safety Data Sheets (SDSs; also known as MSDSs) Note: For SDSs for reagents and chemicals from other manufacturers, contact the manufacturer. Limited product warranty Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies' General Terms and Conditions of Sale found on Life Technologies' website at terms-and-conditions.html. If you have any questions, please contact Life Technologies at Oncomine cfdna Assays, Part III: Variant Analysis User Guide 17
18 For support visit thermofisher.com/support or thermofisher.com 14 November 2016
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