Create a Planned Run. Using the Ion AmpliSeq Pharmacogenomics Research Panel Plugin USER BULLETIN. Publication Number MAN Revision A.

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1 USER BULLETIN Create a Planned Run Using the Ion AmpliSeq Pharmacogenomics Research Panel Plugin Publication Number MAN Revision A.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. 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 Introduction... 4 Overview... 4 Import your PGx files from ampliseq.com and create template... 4 Create a Pharmacogenomics Research Panel Planned Run... 7 Review Run Results... 9 PGx Research Analysis results... 9 Appendix Upload the PGx Research Analysis Plugin to Torrent Suite Software Launch Plugins Manually Post-variant calling analysis options Translational TM Software Option 2: AlleleTyper Software (custom) Comparison of AlleleTyper and Translational TM Software Create a Planned Run Using the Ion AmpliSeq Pharmacogenomics Research Panel Plugin User Bulletin 3

4 Overview The Pharmacogenomics (PGx) Research Analysis plugin is designed to be used with the Ion AmpliSeq Pharmacogenomics research panel, which is a hotspot panel that allows the interrogation of Pharmacogenomics variants in samples using the Torrent Suite variant caller for genotyping and Coverage Analysis Plugin for CYP2D6 copy number detection. The figure below describes the pipeline of analyses. The following instructions are intended to assist Pharmacogenomics (PGx) research panel customers in setting up Torrent Suite planned runs incorporating the PGx research panel template and plugin. If you have Torrent Suite Software v5.0 installed, use the first set of instructions. If you have Torrent Suite Software v4.6 installed, use the second set of instructions. Import your PGx files from ampliseq.com and create template 1. Log into Torrent Suite Software. 2. Go to Plan4Templates. 4 Create a Planned Run Using the Ion AmpliSeq Pharmacogenomics Research Panel Plugin User Bulletin

5 Import your PGx files from ampliseq.com and create template 3. Click the AmpliSeq.com button and select AmpliSeq DNA. 4. Log in with your Ampliseq username and password. Select PGx Panel and click Import Selected. Note: Ion Proton P1 and Ion S5 540 chips are currently not supported. 5. Go to Plan4Templates. 6. Click Recently Created or Pharmacogenomics to find your template and select it. Create a Planned Run Using the Ion AmpliSeq Pharmacogenomics Research Panel Plugin User Bulletin 5

6 Import your PGx files from ampliseq.com and create template 7. On the Plan tab, verify Analysis Parameters is set to Default selection. 8. On the Application tab, verify Pharmacogenomics and AmpliSeq DNA are selected. 6 Create a Planned Run Using the Ion AmpliSeq Pharmacogenomics Research Panel Plugin User Bulletin

7 Create a Pharmacogenomics Research Panel Planned Run 9. On the Kits tab, select your Template Kit and Barcode Set. 10. On the Plugins tab, verify that variantcaller plugin is selected. 11. Also on the Plugins tab, select sampleid, coverageanalysis, and PGxAnalysis plugins. 12. Configure CoverageAnalysis as shown. 13. On the Save tab, verify the Target Regions and Hotspot BED files are correct. 14. Name and Save your template. Create a Pharmacogenomics Research Panel Planned Run 1. Click the name of the template you wish to create a planned run from. For this example, we chose PGx Research Panel. The Plan page of the wizard appears. Create a Planned Run Using the Ion AmpliSeq Pharmacogenomics Research Panel Plugin User Bulletin 7

8 Create a Pharmacogenomics Research Panel Planned Run 2. Enter a name for your run. 3. Select the appropriate Analysis Parameters for the chip you are using. To see the Default parameters, select Details button. Or, choose Custom to select other parameters. 4. Verify the settings on the Kits page. a. If you are using an Ion Chef instrument, verify this instrument is selected. 8 Create a Planned Run Using the Ion AmpliSeq Pharmacogenomics Research Panel Plugin User Bulletin

9 Review Run Results b. If you are using a Ion OneTouch 2 Instrument, verify this instrument is selected and go to the Kits chevron and select the Ion PGM Hi-Q OT2 Kit Enter Samples. 6. Once the table is complete, click on the Plan Run button. The planned run is ready to execute on the instrument specified in the plan (Ion Chef Instrument, Ion OneTouch 2 Instrument or the Ion PGM Sequencer). Review Run Results After your sequencing run completes, review results in the PGxAnalysis plugin output page. PGx Research Analysis results 1. Go to Data4Completed Runs & Results and search for your run. The Run Summary page appears. 2. Scroll down to the PGxAnalysis.html link and click on it. Here you see Overview, CYP2D6 CNV QC Analysis, and Variant Summary tables. The Overview table contains metadata associated with the run plan. You can download results as a VCF file, genotype Allele Typer format, or copy number variation Allele Typer format. VCF is the standard format used by downstream analysis tools. The two Allele Typer options are used by Allele Typer and Copy Caller tools. See the appendix for more information on downstream analysis tools. Create a Planned Run Using the Ion AmpliSeq Pharmacogenomics Research Panel Plugin User Bulletin 9

10 Review Run Results The VariantCaller results and ConverageAnalysis results links open the results in VariantCaller and CoverageAnalysis plugins, respectively. Click the VCF.ZIP, GENOTYPES_ALLELE_TYPER_FORMAT, or CNVS_ALLELE_TYPER_FORMAT buttons to export results. The CYP2D6 CNV QC Analysis table displays QC metrics for the CYP2D6 CNV calling algorithm. 10 Create a Planned Run Using the Ion AmpliSeq Pharmacogenomics Research Panel Plugin User Bulletin

11 Review Run Results The Variant Summary table lists all CNV variants, including: hotspots, novel variants, and CYP2D6 gene-level and exon 9-level CNVs and their confidence levels. Click barcode name links to see TVC results of individual samples. Column CYP2D6 Gene CNV CYP2D6 Exon 9 CNV CYP2D6 Gene CNV Confidence CYP2D6 Exon 9 CNV Confidence Description Copy number (gene copies, deletions, duplications, etc.) for CYP2D6 Gene (excluding exon 9) Copy number (gene copies, deletions, duplications, etc) for Exon 9 of CYP2D6 Phred quality score for CYP2D6 Gene level CNV call. A score of lower than 50 results in a no-call. Phred quality score for CYP2D6 Exon 9 CNV call. A score of lower than 15 results in a no-call Possible copy number states reported by the plugin are: CYP2D6 Gene CNV CYP2D6 Exon 9 CNV Explanation 0 0 homozygous deletion (CYP2D6*5/5) 1 1 single copy (carries one CYP2D6*5 deletion allele) 2 2 two copies 3 3 three copies (carries one CYP2D6 gene duplication allele) Create a Planned Run Using the Ion AmpliSeq Pharmacogenomics Research Panel Plugin User Bulletin 11

12 Review Run Results CYP2D6 Gene CNV CYP2D6 Exon 9 CNV Explanation 3 2 two normal and one 2D6/2D7 hybrid allele (e.g. CYP2D6*36 allele) 4 4 four copies (carries 2 CYP2D6 duplication or a multiplication) NOCALL NOCALL undetermined In most cases, CYP2D6 gene and exon 9 have the same copy number. However, some samples may contain hybrid alleles that formed by recombination between CYP2D6 and the highly homologous CYP2D7 pseudogene; e.g. CYP2D6*36 contains a gene conversion to CYP2D7 sequences in exon 9 adn is associated with negligible CYP2D6 enzyme activity. The CYP2D6*36 is most common in East Asian populations, and more rare in other populations. It is most typically found in tandem with a CYP2D6*10 allele. A sample carrying a nomral CYP2D6 gene on one chromosome and a CYP2D6*36+*10 arrangement on the other will have copy number 2 at the exon 9- level and copy number 3 at the gene-level. There are two circumstances where NOCALLs may happen, either at gene-level or exon 9-level. First, the samples did not pass one of the basic QC metrics Average coverage>=100 or Uniformity Rate >=80%. Second, as the plugin utilizes the coverage of CYP2D6 amplicons to infer the copy number status via a clustering approach, it can be sometimes difficult to make a reliable prediction due to unbalanced sample coverage or suboptimal sample preparation. Gene-level CNV tend to be more robust than the exon 9-level CNV, due to different numbers of amplicons used for the clustering algorithm (9 at gene-level versus 1 at exon 9-level). It is highly recommended to have a rerun including the samples with NOCALL, or verify those with orthogonal platforms (ex: qpcr TaqMan assay). This is especially important when a CYP2D6*10 containing sample is reported with copy number 3 at gene-level and NOCALL at exon 9-level, in order to distinguish between CYP2D6 duplication and CYP2D6*36 alleles. It is noted that Allele-specific CNV is not supported by the plugin. For more information on interpreting Torrent Variant Caller results, see user documentation on Ion Community: community/products/torrent-variant-caller. 12 Create a Planned Run Using the Ion AmpliSeq Pharmacogenomics Research Panel Plugin User Bulletin

13 Appendix This appendix describes how to upload the PGx Research Analysis plugin and launch it manually, as well as downstream analysis options. The plugin is included in Torrent Suite Software 5.0, but future versions may be released in between versions of the Torrent Suite Software. You can launch the plugin manually if you forgot to include it in your initial planned run. Upload the PGx Research Analysis Plugin to Torrent Suite Software 1. Click the Gear button and select Plugins. 2. Click the Install or Upgrade Plugin button. 3. Select Install via Zip Upload tab. 4. Select the PGxAnalysis.zip file and click Upload file. Launch Plugins Manually 1. Launch coverageanalysis and variantcaller plugin by clicking Select plugins to run. 2. After variantcaller and coverageanalysis results are ready, launch PGxAnalysis plugin. 3. Go to Data4Completed Runs & Results and locate your run report. 4. Scroll down to variantcaller and check that variantcaller completed. 5. Scroll to Plugin Summary and click Select plugins to run. Create a Planned Run Using the Ion AmpliSeq Pharmacogenomics Research Panel Plugin User Bulletin 13

14 Appendix Post-variant calling analysis options 6. Select PGxAnalysis. Post-variant calling analysis options The PGx Research Analysis plugin reports SNVs, Indels and CNVs in the following two formats 1. VCF format 1. AlleleTyper Format Any downstream analysis requiring star allele translation and/or Pharmacogenomics interpretation is out of the scope of this plugin. Below are two options for doing post variant-calling analysis, including star allele translation. Translational TM Software Translational TM Software provides a cloud-based service that incorporates guidelines from pioneering efforts in pharmacogenetics and makes them available to molecular research laboratories. Their service transforms raw test results into reports that can be used for analysis. The service provides both a leading-edge knowledgebase of PGx guidance and tools that integrate it into the workflow. Translational TM software full featured tool takes as input the VCF file generated by the Pharmacogenomics Research Panel Analysis Plugin and produces star allele translation along with a pharmacogenomics research report. Option 2: AlleleTyper Software (custom) The AlleleTyper cloud-based software interprets data that has been exported from the Ion AmpliSeq Pharmacogenomics Research Panel plugin. To enable our customer to use this AlleleTyper software, we are exporting our NGS outputs from VariantCaller plugin in the TaqMan Genotyper format via the PGx Research Analysis plugin. You can download the AlleleTyper Software, here: and search for "AlleleTyper". You will need to create a translation table to use AlleleTyper software. A translation table contains genetic patterns associated with a desired nomenclature, such as the star (*) allele nomenclature used to describe CYP P450 and other PGx gene haplotypes. AlleleTyper Software matches the genetic patterns in a biallelic translation table with sample genotype and copy number results and reports back the 14 Create a Planned Run Using the Ion AmpliSeq Pharmacogenomics Research Panel Plugin User Bulletin

15 Appendix Post-variant calling analysis options diplotype calls for each gene. For PGx community researchers, the PharmGKB website ( ) provides comprehensive haplotype translation tables for CYP gene variants found in the Human Cytochrome P450 (CYP) Allele Nomenclature Database ( ) as well as for other drug metabolism gene variants. These can be used as reference tables for creating AlleleTyper monoallelic translation tables containing haplotype genetic information for the targeted gene variants in your study. These are automatically converted by the AlleleTyper Software to biallelic translators containing diploid genetic patterns. Refer to the AlleleTyper Software User Guide for detailed information on creating translation tables. Note that this guide is written for the TaqMan assay workflow and TaqMan assay identifiers are used to identify targets. For the Ion AmpliSeq workflow, unique SNPID identifiers are used instead. The unique target identifier can be found in the SNPID field in the hotspot file "PGx_v4.12_IN_PGxTarget.hotspots_0713_2015.bed". Comparison of AlleleTyper and Translational TM Software The following table compares and summarizes the features of each of the two analysis options: AlleleTyper Software Cost Free For fee Translational TM Software Translational table built No Yes Provides Start Allele Translation Yes Yes Provides report No Yes Create a Planned Run Using the Ion AmpliSeq Pharmacogenomics Research Panel Plugin User Bulletin 15

16 For support visit thermofisher.com/support or thermofisher.com/lifescience 20 August 2015