Create a Planned Run - Thermo Fisher Scientifictools.thermofisher.com/content/sfs/manuals/MAN... · Create a Planned Run ... you have Torrent Suite™ Software v4.6 installed, use

USER BULLETIN

For Research Use Only. Not for use in diagnostic procedures.

Create a Planned RunUsing the Ion AmpliSeq™ PharmacogenomicsResearch Panel Plugin

Publication Number MAN0013730

Revision A.0

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.

©2015 Thermo Fisher Scientific Inc. All rights reserved.

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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9PGx Research Analysis results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

■ Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Upload the PGx Research Analysis Plugin to Torrent Suite™ Software . . . . . . . . . . . . . . . . . 13

Launch Plugins Manually . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Post-variant calling analysis options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14TranslationalTM Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Option 2: AlleleTyper™ Software (custom) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Comparison of AlleleTyper™ and TranslationalTM Software . . . . . . . . . . . . . . . . . . . . . 15

Create a Planned Run Using the Ion AmpliSeq™ Pharmacogenomics Research Panel Plugin User Bulletin 3

Introduction

Overview

The Pharmacogenomics (PGx) Research Analysis plugin is designed to be used withthe Ion AmpliSeq™ Pharmacogenomics research panel, which is a hotspot panel that allowsthe interrogation of Pharmacogenomics variants in samples using the Torrent Suite™

variant caller for genotyping and Coverage Analysis Plugin for CYP2D6 copy numberdetection. The figure below describes the pipeline of analyses.

The following instructions are intended to assist Pharmacogenomics (PGx) researchpanel customers in setting up Torrent Suite™ planned runs incorporating the PGxresearch panel template and plugin.

If you have Torrent Suite™ Software v5.0 installed, use the first set of instructions. Ifyou 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

3. Click the AmpliSeq.com button and select AmpliSeq DNA.

4. Log in with your Ampliseq username and password. Select PGx Panel and clickImport 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 selectit.

IntroductionImport 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 areselected.

IntroductionImport your PGx files from ampliseq.com and create template


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 PGxAnalysisplugins.

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 thisexample, we chose PGx Research Panel.The Plan page of the wizard appears.

IntroductionCreate 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 theDefault parameters, select Details button. Or, choose Custom to select otherparameters.

4. Verify the settings on the Kits page.a. If you are using an Ion Chef™ instrument, verify this instrument is selected.

IntroductionCreate a Pharmacogenomics Research Panel Planned Run


b. If you are using a Ion OneTouch™ 2 Instrument, verify this instrument isselected and go to the Kits chevron and select the Ion PGM Hi-Q OT2 Kit -200.

5. 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 (IonChef™ Instrument, Ion OneTouch™ 2 Instrument or the Ion PGM™ Sequencer).

Review Run Results

After your sequencing run completes, review results in the PGxAnalysis pluginoutput page.

1. Go to Data4Completed Runs & Results and search for your run. The RunSummary page appears.

2. Scroll down to the PGxAnalysis.html link and click on it. Here you seeOverview, CYP2D6 CNV QC Analysis, and Variant Summary tables.

The Overview table contains metadata associated with the run plan. You candownload results as a VCF file, genotype Allele Typer format, or copy numbervariation Allele Typer format.

VCF is the standard format used by downstream analysis tools. The two Allele Typeroptions are used by Allele Typer and Copy Caller tools. See the appendix for moreinformation on downstream analysis tools.

PGx ResearchAnalysis results

IntroductionReview Run Results


The VariantCaller results and ConverageAnalysis results links open the results inVariantCaller and CoverageAnalysis plugins, respectively.

Click the VCF.ZIP, GENOTYPES_ALLELE_TYPER_FORMAT, orCNVS_ALLELE_TYPER_FORMAT buttons to export results.

The CYP2D6 CNV QC Analysis table displays QC metrics for the CYP2D6 CNVcalling algorithm.



The Variant Summary table lists all CNV variants, including: hotspots, novelvariants, 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 Description

CYP2D6 Gene CNV Copy number (gene copies, deletions,duplications, etc.) for CYP2D6 Gene(excluding exon 9)

CYP2D6 Exon 9 CNV Copy number (gene copies, deletions,duplications, etc) for Exon 9 of CYP2D6

CYP2D6 Gene CNV Confidence Phred quality score for CYP2D6 Gene levelCNV call. A score of lower than 50 resultsin a no-call.

CYP2D6 Exon 9 CNV Confidence Phred quality score for CYP2D6 Exon 9 CNVcall. A score of lower than 15 results in ano-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 oneCYP2D6*5 deletion allele)

2 2 two copies

3 3 three copies (carries oneCYP2D6 gene duplicationallele)



CYP2D6 Gene CNV CYP2D6 Exon 9 CNV Explanation

3 2 two normal and one2D6/2D7 hybrid allele (e.g.CYP2D6*36 allele)

4 4 four copies (carries 2CYP2D6 duplication or amultiplication)

NOCALL NOCALL undetermined

In most cases, CYP2D6 gene and exon 9 have the same copy number. However, somesamples may contain hybrid alleles that formed by recombination between CYP2D6and the highly homologous CYP2D7 pseudogene; e.g. CYP2D6*36 contains a geneconversion to CYP2D7 sequences in exon 9 adn is associated with negligible CYP2D6enzyme activity. The CYP2D6*36 is most common in East Asian populations, andmore rare in other populations. It is most typically found in tandem with aCYP2D6*10 allele. A sample carrying a nomral CYP2D6 gene on one chromosome anda 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 orexon 9-level. First, the samples did not pass one of the basic QC metrics – ‘Averagecoverage>=100’ or ‘Uniformity Rate >=80%’. Second, as the plugin utilizes thecoverage of CYP2D6 amplicons to infer the copy number status via a clusteringapproach, it can be sometimes difficult to make a reliable prediction due tounbalanced sample coverage or suboptimal sample preparation. Gene-level CNV tendto be more robust than the exon 9-level CNV, due to different numbers of ampliconsused for the clustering algorithm (9 at gene-level versus 1 at exon 9-level). It is highlyrecommended to have a rerun including the samples with NOCALL, or verify thosewith orthogonal platforms (ex: qPCR TaqMan® assay). This is especially importantwhen a CYP2D6*10 containing sample is reported with copy number 3 at gene-leveland NOCALL at exon 9-level, in order to distinguish between CYP2D6 duplicationand 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 userdocumentation on Ion Community: http://ioncommunity.lifetechnologies.com/community/products/torrent-variant-caller.



Appendix

This appendix describes how to upload the PGx Research Analysis plugin and launchit manually, as well as downstream analysis options.

The plugin is included in Torrent Suite™ Software 5.0, but future versions may bereleased in between versions of the Torrent Suite™ Software. You can launch theplugin 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 torun.

2. After variantCaller and coverageAnalysis results are ready, launch PGxAnalysisplugin.

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.


6. Select PGxAnalysis.

Post-variant calling analysis options

The PGx Research Analysis plugin reports SNVs, Indels and CNVs in the followingtwo formats

1. VCF format

1. AlleleTyper Format

Any downstream analysis requiring star allele translation and/or Pharmacogenomicsinterpretation is out of the scope of this plugin.

Below are two options for doing post variant-calling analysis, including star alleletranslation.

TranslationalTM Software provides a cloud-based service that incorporates guidelinesfrom pioneering efforts in pharmacogenetics and makes them available to molecularresearch 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 guidanceand tools that integrate it into the workflow.http://translationalsoftware.com/

TranslationalTM software full featured tool takes as input the VCF file generated bythe Pharmacogenomics Research Panel Analysis Plugin and produces star alleletranslation along with a pharmacogenomics research report.

The AlleleTyper™ cloud-based software interprets data that has been exported fromthe Ion AmpliSeq™ Pharmacogenomics Research Panel plugin. To enable ourcustomer to use this AlleleTyper™ software, we are exporting our NGS outputs fromVariantCaller plugin in the TaqMan®Genotyper format via the PGx Research Analysisplugin. You can download the AlleleTyper™ Software, here: www.thermofisher.comand 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 genehaplotypes. AlleleTyper™ Software matches the genetic patterns in a biallelictranslation table with sample genotype and copy number results and reports back the

TranslationalTM

Software

Option 2:AlleleTyper™

Software (custom)

AppendixPost-variant calling analysis options


HTTP://TRANSLATIONALSOFTWARE.COM/

HTTP://WWW.THERMOFISHER.COM/US/EN/HOME/CLINICAL/CLINICAL-TRANSLATIONAL-RESEARCH/PHARMACOGENOMICS/ALLELETYPER-SOFTWARE.HTML

http://www.thermofisher.com/us/en/home/clinical/clinical-translational-research/pharmacogenomics/alleletyper-software.html

http://www.thermofisher.com/us/en/home/clinical/clinical-translational-research/pharmacogenomics/alleletyper-software.html

diplotype calls for each gene. For PGx community researchers, the PharmGKBwebsite (www.pharmgkb.org ) provides comprehensive haplotype translation tablesfor CYP gene variants found in the Human Cytochrome P450 (CYP) AlleleNomenclature Database (www.cypalleles.ki.se ) as well as for other drug metabolismgene variants. These can be used as reference tables for creating AlleleTyper™

monoallelic translation tables containing haplotype genetic information for thetargeted gene variants in your study. These are automatically converted by theAlleleTyper™ Software to biallelic translators containing diploid genetic patterns.Refer to the AlleleTyper™ Software User Guide for detailed information on creatingtranslation tables. Note that this guide is written for the TaqMan® assay workflow andTaqMan® 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".

The following table compares and summarizes the features of each of the two analysisoptions:

AlleleTyper™ Software TranslationalTM Software

Cost Free For fee

Translational table built No Yes

Provides Start AlleleTranslation

Yes Yes

Provides report No Yes

Comparison ofAlleleTyper™ andTranslationalTM

Software

AppendixPost-variant calling analysis options


HTTP://WWW.PHARMGKB.ORG

HTTP://WWW.CYPALLELES.KI.SE

For support visit thermofisher.com/support or email [email protected]

thermofisher.com/lifescience

20 August 2015

http://thermofisher.com/support

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http://thermofisher.com/lifescience

Documents

Create a Planned Run - Thermo Fisher Scientifictools.thermofisher.com/content/sfs/manuals/MAN... · Create a Planned Run ... you have Torrent Suite™ Software v4.6 installed, use