Oncomine Tumor Mutation Load Assay - assets.thermofisher.com · tumor samples—10 ng of DNA per primer pool—using the Oncomine™ Tumor Mutation Load Assay library preparation

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

Oncomine™ Tumor Mutation Load AssayUSER GUIDE

Catalog Numbers A37909, A37910Publication Number MAN0017042

Revision C.0

Manufacturer: Life Technologies Corporation | 6055 Sunol Blvd | Pleasanton, CA 94566

The information in this guide is subject to change without notice.

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. 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. MAN0017042

Revision Date DescriptionC.0 4 October 2018 • Include troubleshooting information for FFPE DNA samples with

high levels of deamination

• Include Uracil DNA Glycosylase (UDG) treatment supplementalprotocol

B.0 31 August 2018 • Updated Mutation Load calculation

• Updated to include information for use with the Ion 550™ Chip

• Updated for use with Ion Reporter™ Software 5.10

A.0 20 December 2017 Oncomine™ Tumor Mutation Load Assay User Guide, provides instructionfor library preparation, templating, sequencing, and results analysis ofthe Oncomine™ Tumor Mutation Load Assay.

Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you acceptthe terms and conditions of all applicable Limited Use Label Licenses.TRADEMARKS: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Microsoft™ and Excel™ aretrademarks of Microsoft Corporation.

©2018 Thermo Fisher Scientific Inc. All rights reserved.

Contents

■ CHAPTER 1 Product information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Definition of Mutation Load . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Oncomine™ Tumor Mutation Load Assay – Manual Library Preparation Kit . . . . . . . . . 9Oncomine™ Tumor Mutation Load Assay – Chef‑Ready Library Preparation . . . . . . . 10

Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Recommended materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Sequencer compatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

■ CHAPTER 2 Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Procedural guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Before each use of the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

■ CHAPTER 3 Library preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Ion Chef™ Instrument setup information for Chef Ready kit users . . . . . . . . . . . . . . . . . . . . 14

Set up DNA target amplification reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Guidelines for DNA isolation and quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Guidelines for the amount of DNA needed per target amplification reaction . . . . . . . 14

Prepare DNA target amplification reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Amplify the targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Combine target amplification reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Partially digest the amplicons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Ligate adapters to the amplicons and purify . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Ion Xpress™ adapters only: Combine and dilute adapters . . . . . . . . . . . . . . . . . . . . . . . 18Perform the ligation reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18Purify the library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Elute the library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Quantify library by qPCR and calculate the dilution factor . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Combine libraries for equal depth of coverage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Guidelines for templating and sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Oncomine™ Tumor Mutation Load Assay User Guide 3

■ CHAPTER 4 Create an assay-specific Planned Run . . . . . . . . . . . . . . . . . 25

About Planned Runs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Create a custom Planned Run template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Create a Planned Run from a template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

■ CHAPTER 5 Mutation load analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Review sequencing run results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Ion Reporter™ analysis workflows . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

View results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32Generate an Analysis Results Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35View the Mutation Load statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Visualize Mutation Load analysis results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37Sample Results tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38Sample QC tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

■ APPENDIX A Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

Tips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

■ APPENDIX B Quantify the amplified library with theQubit™ Fluorometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

Amplify the library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

Purify the amplified library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45First-round purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45Second-round purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

Qubit™ Fluorometer: Quantify the library and calculate the dilution factor . . . . . . . . . . . . . 47

Store libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

■ APPENDIX C Supplemental information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

Torrent Suite™ Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48Install the IonReporter Uploader plugin on your Torrent Server . . . . . . . . . . . . . . . . . . 48Configure the IonReporter Uploader plugin in the Torrent Browser . . . . . . . . . . . . . . . 49Download and install BED files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Ion Reporter™ Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50Oncomine™ Tumor Mutation Load Assay filter chain criteria . . . . . . . . . . . . . . . . . . . . . 50Enable Mutation Load calculation in existing workflows . . . . . . . . . . . . . . . . . . . . . . . . 51Manually launch an analysis in Ion Reporter™ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52Customize the Ion Reporter™ workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

Remove deaminated bases from FFPE DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

Contents

4 Oncomine™ Tumor Mutation Load Assay User Guide

■ APPENDIX D Example reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

Example Analysis Results Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

Oncomine™ Tumor Mutation Load Assay report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

■ APPENDIX E Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

■ Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

Contents


Product information

Definition of Mutation Load

Mutation Load, also known as Tumor Mutational Burden (TMB), as determined bythe Oncomine™ Tumor Mutation Load Assay is defined as the number ofnonsynonymous variants (missense and nonsense single nucleotide variants (SNVs)),plus insertion and deletion variants (INDELs) detected per megabase (Mb) of exonicsequence. The Ion Reporter™ Software TMB algorithm filters out germline mutationsusing the Mutation Load Calculation Filter Chain when calculating the Mutation Loadresult.

Table 1 Mutation Load algorithm versions and descriptions

Version[1] Description

TMB algorithm v2.5 Used in the Oncomine™ Tumor Mutation Load - w2.0 - DNA - Single Sample workflow. TheMutation Load Calculation Filter Chain parameter = Mutation Load (Somatic Mutations) bydefault.

precalibration Mutation Load = (Nonsynonymous Somatic Mutations x 106) Total Exonic Bases with Sufficient Coverage

IF precalibration Mutation Load ≥25, THEN Mutation Load = (precalibration Mutation Load −25) x Calibration Slope + 25

IF precalibration Mutation Load <25, THEN no calibration is requiredMutation Load = precalibration Mutation Load x 1.0

• Includes exonic INDELs in the Mutation Load calculation.

• Calibration factor applied to the Mutation Load calculation.

Note: To enable other workflows to apply the TMB algorithm v2.5 you must copy then editthe Oncomine™ Tumor Mutation Load - w2.0 - DNA - Single Sample workflow to match theworkflow of choice (for example, Oncomine™Comprehensive v3 - w3.2 - DNA and Fusions -Single Sample). For more information, see “Enable Mutation Load calculation in existingworkflows“ on page 51.

1


Version[1] Description

TMB algorithm v2.0 Used in workflows enabled by the user to include the mutation load calculation by selecting aMutation Load Calculation Filter Chain parameter[2].

Mutation Load = (Nonsynonymous Somatic Mutations x 106) Total Exonic Bases with Sufficient Coverage

• Includes only exonic nonsynonymous somatic SNVs in the Mutation Load calculation.INDELs are not included in the Mutation Load calculation even if the selected filter chainfiltered‑in INDELs.

• The Mutation Load Calculation Filter Chain parameter can be applied to any "DNA -Single Sample" or "DNA and Fusions - Single Sample" workflow.

TMB algorithm v1.0[3] Used in the Oncomine™ Tumor Mutation Load - w1.0 - DNA - Single Sample workflow.Includes all exonic and intronic synonymous and nonsynonymous somatic SNVs in theMutation Load calculation.

[1] To determine the TMB algorithm version used for analysis of a particular sample, look in the statistic.txt file. For more information, see page 36.

[2] We recommend setting the Mutation Load Calculation Filter Chain parameter to "Mutation Load (Somatic SNVs)" filter chain. For more information, see the Ion Reporter™ Software online help.

[3] Hidden from view in Ion Reporter™ Software 5.6.

Note: Reanalysis of samples previously run with TMB algorithm v1.0 (or v2.0) withthe new Ion Reporter™ Software 5.10 TMB algorithm v2.5 may result in a differentMutation Load result due to changes in the algorithm.

Chapter 1 Product informationDefinition of Mutation Load 1


Product description

The Oncomine™ Tumor Mutation Load Assay is a targeted next-generationsequencing (NGS) assay that is designed for tumor profiling by annotating cancerdriver variants and provides an accurate assessment of Mutation Load(mutations/Mb). The assay detects and annotates low frequency somatic variants(SNPs and INDELs) from 409 genes, spanning ~1.7 Mb of genomic space,encompassing 1.2 Mb of exonic sequence. This assay is designed to facilitatesuccessful selection and identification of samples most likely to derive responses fromtargeted therapies to immunotherapies in cancer research.

This guide covers library preparation from formalin-fixed paraffin-embedded (FFPE)tumor samples—10 ng of DNA per primer pool—using the Oncomine™ TumorMutation Load Assay library preparation kits (manual and Chef-Ready), throughresults analysis. The assay is used with barcoded adapters so that multiple librariescan be combined and loaded onto a single chip to minimize the per-samplesequencing cost.

Barcoded libraries per chip

Ion 540™ Chip Ion 550™ Chip[1]

2–8[2] 2–16[2]

[1] The Ion 550™ Chip is only compatible with the Ion GeneStudio™ S5 Plus Sequencer, Ion GeneStudio™ S5 Prime Sequencer, or Ion S5™ XL Sequencer. The Ion 550™ Chip cannot be sequenced on the Ion S5™ Sequencer,or base model Ion GeneStudio™ S5 Sequencer.

[2] Calculation of the Mutation Load alone does not require as many reads as variant calling. We recommend multiplex sequencing of no more than 4 libraries (Ion 540™ Chip)—6 libraries (Ion 550™ Chip)— to achieve sufficient read depth for variant calls at a ≥5% allele frequency.

The Ion Reporter™ Software 5.10 analysis workflow uses a custom variant calling andgermline variant filtering algorithm to accurately estimate somatic variants in cancerresearch samples, with no matched normal sample required. The software provides adetailed report, feature-rich visualization, annotation of low frequency cancer drivervariants (SNVs and INDELs), and the normalized Mutation Load (mutations/Mb).Included in the results are the percentage of mutations consistent with UV damage,tobacco smoke damage, deamination, and specific substitutions.

This guide covers the following products:• Oncomine™ Tumor Mutation Load Assay – Manual primer panel (2-pools)

(Cat. No. A37907)• Ion AmpliSeq™ Library Kit Plus (Cat. No. 4488990)• Ion Xpress™ Barcode Adapters (various Cat. Nos.)• IonCode™ Barcode Adapters (Cat. No. A29751)

Note: Oncomine™ Tumor Mutation Load Assay – Chef-Ready Library Preparation(Cat. No. A37910) is also available for automated library preparation (see page 10).The kit provides the Oncomine™ Tumor Mutation Load Assay – Chef-Ready primerpanel (2-pools) at 2X concentration pre-measured in barcoded Primer Pool tubesready to load into an Ion AmpliSeq™ Chef Reagents DL8 cartridge.

Chapter 1 Product informationProduct description1


Contents and storage

The Oncomine™ Tumor Mutation Load Assay – Manual Library Preparation Kit(Cat. No. A37909) consists of the Oncomine™ Tumor Mutation Load Assay – ManualLibrary Preparation primer panel (2-pools), and the Ion AmpliSeq™ Library Kit Plus.Sufficient reagents are provided for the rapid preparation of 24 barcoded samplelibraries from DNA.

Contents Amount Storage

Oncomine™ Tumor Mutation Load Assay – Manual Library Preparation primer panel(Part No. A37907, 24 reactions)

Oncomine™ Tumor Mutation Load Assay (2X)(blue cap) (pool 1 of 2)

120 µL –30ºC to –10ºC

Oncomine™ Tumor Mutation Load Assay (2X)(blue cap) (pool 2 of 2)

120 µL

Ion AmpliSeq™ Library Kit Plus (Part No. 4488990)

5X Ion AmpliSeq™ HiFi Mix (red cap) 120 µL –30ºC to –10ºC

FuPa Reagent (brown cap) 48 µL

Switch Solution (yellow cap) 96 µL

DNA Ligase (blue cap) 48 µL

25X Library Amp Primers (pink cap) 48 µL

1X Library Amp Mix (black cap) 1.2 mL

Low TE 6 mL 15°C to 30°C[1]

[1] Can be stored at –30ºC to –10ºC for convenience.

Oncomine™ TumorMutation LoadAssay – ManualLibraryPreparation Kit

Chapter 1 Product informationContents and storage 1


The Oncomine™ Tumor Mutation Load Assay – Chef-Ready Library Preparation Kit(Cat. No. A37910) provides the Oncomine™ Tumor Mutation Load Assay –Chef-Ready Library Preparation primer panel at 2X concentration pre-measured inbarcoded Primer Pool tubes ready to load into an Ion AmpliSeq™ Chef Reagents DL8cartridge. In addition, the kit provides all the reagents and supplies in an IonAmpliSeq™ Kit for Chef DL8 sufficient for preparing 32 libraries. See the IonAmpliSeq™ Library Preparation on the Ion Chef™ System User Guide (Pub. No.MAN0013432) for detailed information on preparing Oncomine™ Tumor MutationLoad Assay libraries on the Ion Chef™ System.

Component Amount Storage

Oncomine™ Tumor Mutation Load Assay – Chef‑Ready (Part No. A37908, 32 reactions)

Oncomine™ Tumor Mutation Load Assay – Manual (2X)(pool 1 of 2)

4 × 150 µL –30°C to –10°C

Oncomine™ Tumor Mutation Load Assay – Manual (2X)(pool 2 of 2)

4 × 150 µL

Ion AmpliSeq™ Kit for Chef DL8 (Cat. No. A29024)

Ion AmpliSeq™ Chef Reagents DL8 (Part No. A29025) 4 cartridges –30°C to –10°C

Ion AmpliSeq™ Chef Solutions DL8 (Part No. A29026) 4 cartridges 2°C to 8°C[1]

Ion AmpliSeq™ Chef Supplies DL8 (per insert)(Part No. A29027)

• Ion AmpliSeq™ Tip Cartridge L8

• PCR Frame Seal

• Enrichment Cartridge

1 box with4 inserts

15°C to 30°C

IonCode™ 0101–0132 in 96 Well PCR Plates (dried)(Part No. A29028)

Set includes 4 PCR plates:

• IonCode™ 0101–0108 in 96 Well PCR Plate (red)

• IonCode™ 0109–0116 in 96 Well PCR Plate (yellow)

• IonCode™ 0117–0124 in 96 Well PCR Plate (green)

• IonCode™ 0125–0132 in 96 Well PCR Plate (blue)

1 set of4 plates

15°C to 30°C

[1] Ion AmpliSeq™ Chef Solutions DL8 cartridges are shipped at ambient temperature, but need to be stored at 2°C to 8°C upon arrival.

Oncomine™ TumorMutation LoadAssay –Chef‑ReadyLibraryPreparation

Chapter 1 Product informationContents and storage1


Required materials not supplied

Unless otherwise indicated, all materials are available through thermofisher.com.MLS: Fisher Scientific (fisherscientific.com) or other major laboratory supplier.

Item Source

IonCode™ Barcode Adapters 1–384 Kit

OR

Ion Xpress™ Barcode Adapters

A29751

4471250, 4474009, 4474518,4474519, 4474520, 4474521,

or 4474517

Agencourt™ AMPure™ XP Kit Beckman Coulter, A63880 orA63881

One of the following:

• GeneAmp™ PCR System 9700 or Dual 96-well ThermalCycler

• AB™ 2720 Thermal Cycler

• Veriti™ 96-well Thermal Cycler

• ProFlex™ 96‑well PCR System

See web product pages

MicroAmp™ Optical 96-well Reaction Plate N8010560

4306737 (with barcode)

MicroAmp™ Fast Optical 96-Well Reaction Plate 4346907

MicroAmp™ Optical Adhesive Film 4311971

MicroAmp™ Adhesive Film 4306311

MicroAmp™ Compression Pad 4312639

DynaMag™-96 Side Magnet, or other plate magnet 12331D

Eppendorf™ DNA LoBind™ Microcentrifuge Tubes, 1.5 mL 13-698-791

fisherscientific.com

Nuclease-free Water AM9932

Absolute ethanol MLS

Pipettors, 2–200 μL, and low-retention filtered pipette tips MLS

Chapter 1 Product informationRequired materials not supplied 1


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http://fisherscientific.com


Recommended materials

Unless otherwise indicated, all materials are available through thermofisher.com.MLS: Fisher Scientific (fisherscientific.com) or other major laboratory supplier.

Item Source

Recommended additional equipment

Real-time PCR instrument (e.g.,Applied Biosystems™

7900HT, 7500, StepOne™, StepOnePlus™, ViiA™ 7 Systems,or QuantStudio™ 12K Flex Real-Time PCR System)

See web product pages

96-well plate centrifuge MLS

Qubit™ 4 Fluorometer[1] Q33226

Recommended for nucleic acid isolation

RecoverAll™ Multi-Sample RNA/DNA Workflow A26069

RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE AM1975

MagMAX™ FFPE Total Nucleic Acid Isolation Kit 4463365

Recommended for nucleic acid quantification

Qubit™ dsDNA HS Assay Kit (DNA) Q32851/Q32854

Recommended for library quantification

Ion Library TaqMan® Quantitation Kit 4468802

Recommended for Uracil-DNA Glycosylase treatment

Uracil-DNA Glycosylase (UDG), heat labile 78310100UN

[1] Qubit™ 2.0 & Qubit™ 3.0 Fluorometers are supported but no longer available for purchase.

Sequencer compatibility

Ion GeneStudio™ S5 Series System refers generically to the three Ion GeneStudio™ S5Systems, unless otherwise specified:

• Ion GeneStudio™ S5 System (Cat. No. A38194)• Ion GeneStudio™ S5 Plus System (Cat. No. A38195)• Ion GeneStudio™ S5 Prime System (Cat. No. A38196)

Chapter 1 Product informationRecommended materials1


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Before you begin

Procedural guidelines

• Minimize freeze-thaw cycles of Oncomine™ Tumor Mutation Load Assay panelsby aliquoting as needed for your experiments. Panels can be stored at 4°C for oneyear.

• Use good laboratory practices to minimize cross-contamination of products. Ifpossible, perform PCR setup in an area or room that is free of ampliconcontamination. Always change pipette tips between samples.

• Use a calibrated thermal cycler specified in “Required materials not supplied“.• Pipet viscous solutions, such as 5X Ion AmpliSeq™ HiFi Mix, FuPa Reagent,

Switch Solution, DNA Ligase, and panels, slowly and ensure complete mixing byvortexing or pipetting up and down several times.

• Arrange samples in alternating columns on the plate for easier pipetting withmultichannel pipettes during purification with the DynaMag™ Side Magnet.

Before each use of the kit

• Thaw components that contain enzymes—such as 5X Ion AmpliSeq™ HiFi Mix,FuPa Reagent, DNA Ligase, and 1X Library Amp Mix—on ice, and keep on iceduring procedure. All other components, including primer pools, can be thawedat room temperature. Gently vortex and centrifuge before use.

• If there is visible precipitate in the Switch Solution after thawing, vortex or pipetup and down at room temperature to resuspend.

2


Library preparation

IMPORTANT! Oncomine™ Tumor Mutation Load Assay – Chef-Ready LibraryPreparation Kit (Cat. No. A37910) users.

· For instructions to prepare Oncomine™ Tumor Mutation Load Assay libraries onthe Ion Chef™ System, see the Ion AmpliSeq™ Library Preparation on the Ion Chef™

System User Guide (Pub. No. MAN0013432).· When templating the combined libraries, use at 50 pM concentration.

Ion Chef™ Instrument setup information for Chef Ready kit users

During Ion Chef™ Instrument setup, enter the following parameters when prompted.

Stating material # of primer pools Target amplificationcycles

Anneal & extensiontime

FFPE DNA 2 16 16 minutes

Set up DNA target amplification reactions

• We recommend the RecoverAll™ Multi-Sample RNA/DNA Workflow(Cat. No. A26069) for isolating gDNA.

• We recommend the TaqMan® RNase P Detection Reagents Kit (Cat. No. 4316831)for quantifying amplifiable human genomic DNA (see Demonstrated Protocol:Sample Quantification for Ion AmpliSeq™ Library Preparation Using the TaqMan®

RNAse P Detection Reagents Kit (Pub. No. MAN0007732) available at thermofisher.com).

• The Qubit™ dsDNA HS Assay Kit (Cat. No. Q32851 or Q32854) can also be usedfor quantification, particularly for FFPE DNA, and highly degraded DNAsamples.

• Quantification methods such as densitometry (for example, using a NanoDrop™

spectrophotometer) are not recommended, because they are not specific for DNA.Use of these methods can lead to gross overestimation of the concentration ofsample DNA, under-seeding of the target amplification reaction, low libraryyields, and poor chip loading.

• For each target amplification reaction, use 300–30,000 copies of DNA (10 ng ofmammalian gDNA) from normal or FFPE tissue.

• Increasing the amount of DNA results in higher-quality libraries, especially whenDNA quality or quantity is unknown. We recommend using 1 ng gDNA(300 copies) only with high-quality, well-quantified samples.

3

Guidelines forDNA isolation andquantification

Guidelines for theamount of DNAneeded per targetamplificationreaction


https://www.thermofisher.com

Prepare DNA target amplification reactions

IMPORTANT! Primer pools and 5X Ion AmpliSeq™ HiFi Mix are viscous. Pipet slowlyand mix thoroughly.

1. Place a 1.5-mL tube and 96-well plate on ice or in a pre-chilled 4°C cold block.

2. For each sample, prepare a target amplification master mix without primers in a1.5-mL tube on ice.

Component Volume

5X Ion AmpliSeq™ HiFi Mix (red cap) 5 µL

DNA (20 ng)[1] ≤7.5 µL

Nuclease-free Water to 12.5 µL

[1] Substitute 5 µL AcroMetrix™ Oncology Hotspot Control to prepare a control library.

3. Mix thoroughly by pipetting up anddown 5 times, then transfer 5 µL ofeach sample-specific master mix to 2wells of a 96-well PCR plate on ice orin a pre-chilled 4°C cold block.

4. Add 5 µL of Oncomine™ TumorMutation Load Assay (2X) manuallibrary preparation primer pool 1into the first well, and 5 µL of primerpool 2 to the second well.

5. Seal the plate with a MicroAmp™

Adhesive Film.

6. Vortex for 5 seconds to mix, then briefly centrifuge to collect the contents.Alternatively, mix by pipetting at least half the total volume up and down at least5 times before sealing the plate.

5 μL 2X primer

pool 1

MasterMix

5 μL 5 μL

5 μL2X primer

pool 2

Sample

Chapter 3 Library preparationPrepare DNA target amplification reactions 3


Amplify the targets

IMPORTANT! When amplifying multiple samples in a single PCR plate, make surethat the input across all samples is roughly equivalent so that the selected cyclenumber is optimal for all the samples in the run.

1. Place a MicroAmp™ Compression Pad on the plate, then load the plate into thethermal cycler.

2. Run the following program to amplify the target regions.

Stage Step Temperature Time

Hold Activate theenzyme

99°C 2 min

15 Cycles Denature 99°C 15 sec

Anneal and extend 60°C 16 min

Hold — 10°C Hold

STOPPING POINT Target amplification reactions may be stored at 10°C overnighton the thermal cycler. For longer periods, store at −20°C.

Combine target amplification reactions

Note: Perform the following steps on ice or in a pre-chilled 4°C cold block.

1. Remove the plate from the thermal cycler.

2. Centrifuge briefly to collect the contents, then carefully remove the plate seal.

3. For each sample, combine both 10-µL target amplification reactions into a singlewell.

IMPORTANT! Accurate volume transfer in this step is critical. We recommendusing a single-channel pipettor. If you are using a multi-channel pipettor, visuallycheck pipette tips to ensure that volumes are equivalent.

1 2 3 4 5 6 7 8 9 10 11 12A

B

C

D

EF

G

H

1 2 3 4 5 6 7 8 9 10 11 12A

B

C

D

EF

G

H

1 2 3

1 Pool 12 Pool 2

3 Combined pools

The total volume for each sample should be ~20 µL.

Chapter 3 Library preparationAmplify the targets3


Partially digest the amplicons

IMPORTANT! Keep the plate on ice or in a pre-chilled 4°C cold block while preparingthe reactions.

1. Thaw the FuPa Reagent (brown cap) on ice, gently vortex to mix, then centrifugebriefly to collect.

2. Add 2 µL of FuPa Reagent to each amplified sample. The total volume is ~22 µL.

3. Seal the plate with a clear adhesive film, vortex thoroughly, then centrifugebriefly to collect droplets. Alternatively, mix by pipetting at least half the totalvolume up and down at least 5 times before sealing the plate.

4. Place a compression pad on the plate, load in the thermal cycler, then run thefollowing program:

Temperature Time

50°C 20 minutes

55°C 20 minutes

60°C 20 minutes

10°C Hold (for up to 1 hour)

STOPPING POINT Store plate at –20°C for longer periods.

Ligate adapters to the amplicons and purify

When sequencing multiple libraries on a single chip, you must ligate a differentbarcode adapter to each library. DNA and RNA libraries from the same sample alsorequire different barcodes.

IonCode™ Adapters are provided at the appropriate concentration and includeforward and reverse adapters in a single well. No further handling is necessary.

Ion Xpress™ adapters require handling and dilution as described below.

IMPORTANT! When handling barcoded adapters, be careful to avoid crosscontamination by changing gloves frequently and opening one tube at a time.

Chapter 3 Library preparationPartially digest the amplicons 3


For each barcode X selected, prepare a mix of Ion P1 Adapter and Ion Xpress™

Barcode X at a final dilution of 1:4 for each adapter. For example, combine thevolumes indicated in the following table. Scale volumes as necessary. Use 2 µL of thisbarcode adapter mix in step 3 below.

Component Volume

Ion P1 Adapter 2 µL

Ion Xpress™ Barcode X[1] 2 µL

Nuclease-free Water 4 µL

Total 8 µL

[1] X = barcode chosen

Note: Store diluted adapters at –20°C.

1. If there is visible precipitate in the Switch Solution or the tube cap after thawing,vortex or pipet up and down at room temperature to resuspend before pipetting.

2. Briefly centrifuge the plate to collect the contents.

3. Carefully remove the plate seal, then add the following components in the orderlisted to each well containing digested amplicons. If preparing multiple non-barcoded libraries, a master mix of Switch Solution and adapters can becombined before addition.

IMPORTANT! Add the DNA Ligase last. Do not combine DNA Ligase andadapters before adding to digested amplicons.

Order ofaddition Component Volume

1 Switch Solution (yellow cap) 4 µL

2 IonCode™ Adapters ordiluted Ion Xpress™ barcode adapter mix (for barcodedlibraries)

2 µL

3 DNA Ligase (blue cap) 2 µL

— Total volume (including ~22 µL of digested amplicon) ~30 µL

4. Seal the plate with a new MicroAmp™ Clear Adhesive Film, vortex thoroughly,then briefly centrifuge to collect droplets. Alternatively, mix by pipetting at leasthalf the total volume up and down at least 5 times before sealing the plate.

Ion Xpress™

adapters only:Combine anddilute adapters

Perform theligation reaction

Chapter 3 Library preparationLigate adapters to the amplicons and purify3


5. Place a MicroAmp™ Compression Pad on the plate, load in the thermal cycler,then run the following program:

Temperature Time

22°C 30 minutes

68°C 5 minutes

72°C 5 minutes

10°C Hold (for up to 24 hours)

STOPPING POINT Samples can be stored for up to 24 hours at 10°C on the thermalcycler. For longer periods, store at –20°C.

IMPORTANT! Bring Agencourt™ AMPure™ XP Reagent to room temperature andvortex thoroughly to disperse the beads before use. Pipet the solution slowly.

Do NOT substitute a Dynabeads™-based purification reagent for the Agencourt™

AMPure™ XP Reagent.

1. Prepare 70% ethanol (350 µL × number of samples) fresh daily.

2. Briefly centrifuge the plate to collect the contents in the bottom of the wells.

3. Carefully remove the plate seal, then add 45 µL (1.5X sample volume) ofAgencourt™ AMPure™ XP Reagent to each library. Pipet up and down 5 times tomix the bead suspension with the DNA thoroughly.

Note: Visually inspect each well to ensure that the mixture is homogeneous.

4. Incubate the mixture for 5 minutes at room temperature.

5. Place the plate in a magnetic rack such as the DynaMag™-96 Side Magnet, thenincubate for 2 minutes or until the solution clears. Carefully remove, then discardthe supernatant without disturbing the pellet.

6. Add 150 µL of freshly prepared 70% ethanol, then move the plate side-to-side inthe two positions of the magnet to wash the beads. Carefully remove, thendiscard the supernatant without disturbing the pellet.

Note: If your magnet does not have two positions for shifting the beads, removethe plate from the magnet and gently pipet up and down 5 times (with thepipettor set at 100 µL), then return the plate to the magnet and incubate for2 minutes or until the solution clears.

7. Repeat step 6 for a second wash.

Purify the library

Chapter 3 Library preparationLigate adapters to the amplicons and purify 3


8. Ensure that all ethanol droplets are removed from the wells. Keeping the plate inthe magnet, air-dry the beads at room temperature for 2−5 minutes. Do notoverdry.

IMPORTANT! Residual ethanol drops inhibit library amplification. If needed,centrifuge the plate and remove remaining ethanol before air-drying the beads.Under conditions of low relative humidity the beads air-dry rapidly. Do notoverdry.

Proceed immediately to “Elute the library“.

1. Remove the plate with purified libraries from the plate magnet, then add 50 μLof Low TE to the pellet to disperse the beads.

2. Seal the plate with MicroAmp™ Clear Adhesive Film, vortex thoroughly, thenbriefly centrifuge to collect droplets. Alternatively, mix by pipetting at least halfthe total volume up and down at least 5 times before sealing the plate.

3. Incubate at room temperature for at least 2 minutes.

4. Place the plate on the magnet for at least 2 minutes.

STOPPING POINT Libraries can be stored at 4–8°C for up to 1 month. For longerterm, store at –20°C. We recommend transferring the supernatant to a 1.5-mLEppendorf LoBind™ tube for long-term storage.

5. Prepare a 500-fold dilution for quantification. Remove 2 µL of supernatant,containing the library, then combine with 998 µL of Nuclease-free Water.

Elute the library

Chapter 3 Library preparationLigate adapters to the amplicons and purify3


Quantify library by qPCR and calculate the dilution factor

Determine the concentration of each Oncomine™ Tumor Mutation Load Assay libraryby qPCR with the Ion Library TaqMan® Quantitation Kit (Cat. No. 4468802). Analyzeeach sample, standard, and negative control in duplicate reactions. Libraries that havenot undergone a second round of amplification typically have yields of 100–500 pM.However, yield is not indicative of library quality. After quantification, determine thedilution factor that results in a concentration of ~50 pM.

Note: Alternatively, quantify the libraries using a Qubit™ 4 or 3.0 Fluorometer, see Appendix B, “Quantify the amplified library with the Qubit™ Fluorometer“ for moreinformation.

1. Prepare three 10-fold serial dilutions of the E. coli DH10B Ion Control Library(~68 pM; from the Ion Library TaqMan® Quantitation Kit) at 6.8 pM, 0.68 pM, and0.068 pM. Mark these tubes as standards, then use these concentrations in theqPCR instrument software.

Standard ControlLibrary

Nuclease-freeWater[1] Dilution factor Concentration

1 5 µL undilutedControl Library

45 µL 1:10 6.8 pM

2 5 µL Std 1 45 µL 1:100 0.68 pM

3 5 µL Std 2 45 µL 1:1000 0.068 pM

[1] Not DEPC-treated.

Note: When you program the qPCR instrument, enter the concentration of eachstandard in the "Amount" field.

2. Calculate, then prepare the required volume of PCR master mix for duplicatereactions of each library sample, standard, and NTC using the following table.Include a 5−10% overage to accommodate pipetting errors.

Component Volume per reaction (96-well plate)

2X TaqMan® Master Mix 10 µL

20X Ion TaqMan® Assay 1 µL

Total 11 µL

3. In an Optical PCR plate, set up duplicate PCR reactions for each sample,standard, and NTC. To each well add the following components:

Component Volume per reaction (96-well plate)

PCR Master Mix 11 µL

1:500 dilution of the sample[1] 9 µL

[1] Substitute E. coli DH10B standards prepared in step 1 for standards. Substitute nuclease-free water for NTC.

4. Program your real-time instrument as described in the following table.a. Enter the concentrations of the control library standards.

Chapter 3 Library preparationQuantify library by qPCR and calculate the dilution factor 3


b. Select ROX™ Reference Dye as the passive reference dye.

c. Select a reaction volume of 20 µL.

d. Select FAM™ dye/MGB as the TaqMan® probe reporter/quencher.

Note: The fast cycling program was developed using the StepOnePlus™ Systemin Fast mode.

Reaction plateformat Run mode Stage Temperature Time

48- / 96-well Fast Fast

Hold (UDGincubation) 50°C 2 min

Hold (polymeraseactivation) 95°C 20 sec

Cycle (40 cycles)95°C 1 sec

60°C 20 sec

96-well Standard Standard

Hold (UDGincubation) 50°C 2 min

Hold (polymeraseactivation) 95°C 2 min

Cycle (40 cycles)95°C 15 sec

60°C 1 min

5. Following qPCR, calculate the average concentration of the undilutedOncomine™ Tumor Mutation Load Assay library by multiplying the determinedconcentration × 500.

6. Based on the calculated library concentration, determine the dilution that resultsin a concentration of 50 pM for Ion Chef™ template preparation.

• The undiluted library concentration is 300 pM.• The dilution factor is 300 pM/50 pM = 6.• Therefore, 10 µL of library that is mixed with 50 µL of Low TE (1:6 dilution)

yields approximately 50 pM.

7. Store undiluted libraries, or proceed to “Combine libraries for equal depth ofcoverage“ and template preparation.

STOPPING POINT Undiluted libraries can be stored at 4–8°C for up to 1 month.For longer term, store at –20°C. We recommend transferring the supernatant to a1.5-mL Eppendorf LoBind™ tube for long-term storage.

Chapter 3 Library preparationQuantify library by qPCR and calculate the dilution factor3


Combine libraries for equal depth of coverage

Multiple barcoded Oncomine™ Tumor Mutation Load Assay libraries can besequenced on a single chip by combining equal volumes of each barcoded librarybefore template preparation.

IMPORTANT! You can prepare barcoded libraries from different samples usingIonCode™ or Ion Xpress™ barcode adapters. However, do NOT combine libraries thatare prepared with IonCode™ barcode adapters with libraries that are prepared withIon Xpress™ barcode adapters.

We recommend combining up to a maximum of 8 libraries (Ion 540™ Chip), or 16libraries (Ion 550™ Chip) when determining the Mutation Load only. For tumorprofiling as well as the Mutation Load calculation we recommend combining up to 4libraries (Ion 540™ Chip), or 6 libraries (Ion 550™ Chip), as described below.

Ion sequencing chip Number of libraries Volume to combine (X)[1]

Ion 540™ Chip 2 13.5 µL

4 6.8 µL

8 3.4 µL

Ion 550™ Chip 2 13.5 µL

6 4.5 µL

16 2.0 µL

[1] Provides sufficient volume of combined libraries for 1 templating reaction plus overage to accommodate pipetting error.

Note: Due to coverage variability in FFPE samples that may be due to sample quality,increased sequencing depth may be necessary to achieve sufficient coverage.Increased sequencing depth can be achieved by reducing the number of samples perchip.

To prepare a combined library:

1. Dilute all individual barcoded libraries to 50-pM concentration.

2. Combine X-µL of each library in a single 1.5-mL Eppendorf LoBind™ tube.

3. After adding the last library, pipet up and down 5 times to mix, then centrifugebriefly to collect in the bottom of the tube.

IMPORTANT! Do not dilute the combined library further before templating, useat 50-pM concentration.

STOPPING POINT Libraries can be stored at 4–8°C for up to 1 month. For longer term,store at –20°C.

Chapter 3 Library preparationCombine libraries for equal depth of coverage 3


Guidelines for templating and sequencing

Proceed to template preparation and sequencing using the following kits.

TemplateSystem Sequencer Kit User Guide

Ion Chef™ IonGeneStudio™

S5 System

Ion 540™ Kit – Chef(Cat. Nos. A27759, A30011)

Ion 540™ Kit – Chef UserGuide(Pub. No. MAN0010851)

Ion 550™ Kit − Chef(Cat. No. A34541)

Ion 550™ Kit − Chef UserGuide(Pub. No. MAN0017275)

IMPORTANT! When templating the combined libraries, use at 50 pM concentration.Do NOT dilute further before loading the Ion Chef™ Instrument.

To create a specific Run Plan for use in templating and sequencing see “Create acustom Planned Run template“ on page 26. See the appropriate user guide listed inthe table for more information.

Chapter 3 Library preparationGuidelines for templating and sequencing3


Create an assay-specific PlannedRun

IMPORTANT!· We recommend that you update to the latest available versions of Torrent Suite™

and Ion Reporter™ Software to enable the data analysis and reporting options thatare described in this chapter. Torrent Suite™ Software 5.6 is required to access thepreinstalled Planned Run templates.

· These kits are compatible with Torrent Suite™ Software 5.6 or later and IonReporter™ Software 5.10 or later. Before proceeding, check for updates to theTorrent Suite™, Ion Reporter™, and Ion Chef™ System software, and install theupdates if available.

· Sequencing on an Ion 550™ Chip requires Torrent Suite™ Software 5.8 or later.· If running Ion Reporter™ Software 5.10 you must install the IonReporterUploader

5.10 plugin. See “Install the IonReporter Uploader plugin on your TorrentServer“ on page 48 for more information.

About Planned Runs

Planned Runs are digital instructions that are created in Torrent Suite™ Software forcontrolling the template preparation and sequencing instruments. Planned Runscontain settings such as number of flows, kit types, barcodes, sample information, andreference files (if any). Planned Runs are also used to track samples, chips, andreagents throughout the sequencing workflow, from template preparation on theIon Chef™ Instrument through sequencing on an Ion GeneStudio™ S5 Series Sequenceror Ion S5™/Ion S5™ XL Sequencer and subsequent data analysis. Each chip prepared inan Ion Chef™ run requires its own Planned Run.

In Torrent Suite™ Software 5.6 or later, the primary Planned Run template to be usedwith the Oncomine™ Tumor Mutation Load Assay is the Oncomine™ TumorMutation Load template.

Application Torrent Suite™ Software template Description

Mutation Load Oncomine™ Tumor Mutation Load DNA-only planned run template

4


Create a custom Planned Run template

IMPORTANT! Before creating a Planned Run you must enable the Oncomine™ TumorMutation Load Assay template, and upload the Reference Library, and TargetRegions BED files to the Torrent Suite™ Software. For more information, see Appendix C, “Supplemental information“. Contact your local service representativeto obtain the most current BED files.

We recommend setting up a customized Planned Run template for reuse when thesame conditions will be used for multiple runs. For more information about creatingPlanned Runs manually or from the generic application template, see the online helpavailable in the Torrent Suite™ Software.

1. Sign in to the Torrent Suite™ Software.

2. In the Plan tab, click Templates, then click Mutation Load in the left navigationmenu.

3. In the Mutation Load list, find the Oncomine™ Tumor Mutation Load PlannedRun template from which you want to create your custom Planned Run, thenclick (Actions)4Copy.The Copy Template worklow bar opens to the Save step.

4. Enter or select the required information in each field in the Save step.

Field Action…

Template Name Enter a name for the Planned Run template.

DNA Reference Library Select hg19(Human (hg19)).

Set as Favorite Select the Set as Favorite checkbox to add your customtemplate to the Favorites list.

DNA Target Regions[1] Select Oncomine_TML.20180605.designed.bed

DNA Hotspot Regions[2] —

[1] Check with your service representative for updates to ensure the most current files are being used. For BED file installation instructions, see page 49.

[2] Not required for the Oncomine™ Tumor Mutation Load Assay. Leave this field blank if a DNA Hotspot Regions file is not used.

5. Click the Ion Reporter step, then select your Ion Reporter™ 5.10 account.

Note: If the Ion Reporter™ 5.10 account is not configured, configure it throughIon Reporter Configure settings (see “Configure the IonReporter Uploaderplugin in the Torrent Browser“ on page 49 for more information).

6. In the Existing Workflow dropdown list, select Oncomine™ Tumor MutationLoad - w2.0 - DNA - Single Sample , then click Next.

Note: You must update to Ion Reporter™ Software 5.10, and have an off-cyclesoftware package installed by your service representative for the Oncomine™

Tumor Mutation Load Assay Ion Reporter™ workflow to appear in the Existingworkflow dropdown list.

Chapter 4 Create an assay-specific Planned RunCreate a custom Planned Run template4


7. In the Research Application step, verify that Mutation Load and AmpliSeqDNA are selected for Application and Target Technique respectively, then clickNext.

8. In the Kits step, verify the Ion Chef Template Kit radio button is selected, andthe following fields are completed:

FieldSelection

Ion 540™ Chip Ion 550™ Chip[1]

Instrument Ion GeneStudio™ S5 System

Library Kit Type Ion AmpliSeq™ Library Kit Plus

Template Kit Ion 540™ Kit – Chef Ion 550™ Kit − Chef

Sequencing Kit Ion S5™ Sequencing Kit

Base Calibration Mode Default Calibration

Chip Type Ion 540™ Chip Ion 550™ Chip

Barcode Set IonCode™ Barcodes 1–32

Flows 400

[1] The Ion 550™ Chip is only compatible with the Ion GeneStudio™ S5 Plus Sequencer, Ion GeneStudio™ S5 Prime Sequencer, or Ion S5™ XL Sequencer. The Ion 550™ Chip cannot be sequenced on the Ion S5™ Sequencer,or base model Ion GeneStudio™ S5 Sequencer.

9. Select or edit the optional information fields appropriately for your run, thenclick Next.

10. Review the Plugins and Projects steps and make selections appropriate to yourrun, then click Next.

11. In the Save step, click Copy Template to save the new run template.

The customized template is now available in the Templates screen of the MutationLoad Research Application. If you set your customized template to favorites, it alsoappears in the Favorites list.

Create a Planned Run from a template


2. In the Plan tab, click Templates, then click Mutation Load in the left navigationmenu.

3. In the Mutation Load list, click on your customized Planned Run template nameor the preinstalled system template. Alternatively, in the row of the templateclick 4Plan Run.The Create Plan workflow bar opens to the Plan step.

Chapter 4 Create an assay-specific Planned RunCreate a Planned Run from a template 4


4. Enter or select the following information. Row numbers in the table correspondto the callouts in the following figure.

Callout Field Action

1 Run Plan Name Enter a Run Plan name.

2 Analysis Parameters Ensure the Default (Recommended) radiobutton is selected.

3 Use same reference &BED files for all barcodes

Ensure that the checkbox is selected.

4 Number of barcodes Enter the number of barcodes that will beused in this run, then click the button tothe right of this field.

5 Sample Tube Label Enter or scan the barcode of the Ion Chef™

sample tube that will be used in the run.

6 Chip ID No entry required.

7 Oncology Ensure that the radio button is selected.

8 Pre-implantation GeneticScreening

Ensure that the radio button is unselected.

4

1

2

3

5

6

7

8

Chapter 4 Create an assay-specific Planned RunCreate a Planned Run from a template4


5. Enter sample information. Row numbers in the table correspond to the calloutsin the following figure.

Callout Field[1] Action

1 Barcode For each sample select the Barcode that willidentify it from the dropdown list.

2 Sample Name Accept the auto-populated sample names orclick in a field, then enter a unique samplename. We recommend sample names (eitherauto-populated or user defined) be uniqueeven between runs.

3 Control Type (expanded) Select No Template Control from thedropdown list to designate a sample as a notemplate control.

4 Sample ID (Optional) Click in the field, then enter asample ID.

5 Sample Description (Optional) Click in the field, then enter asample description.

6 Annotations (expanded) Click to reveal Cancer Type and Cellularity %.

7 Cancer Type (Optional) Click in the field, then select fromthe dropdown list. Click to copy the entryto all the rows.

8 Cellularity % (Optional) Click in the field, then enter avalue. Click to copy the entry to all therows.

9 Ion Reporter Workflow Ensure the correct workflow is selected.

10 Relation (Optional) Click in the field, then ensure thecorrect value is auto-populated. Select fromthe dropdown list to change.

11 Gender (Optional) Click in the field, then select fromthe dropdown list. Click to copy the entryto all the rows.

12 IR Set ID The IR Set ID links individual samples foranalysis. Ensure the auto-populated value isunique to each sample. Select from thedropdown list to change.

[1] Click vertical column headers (Control Type, Reference, Annotations) to reveal additional columns.

Chapter 4 Create an assay-specific Planned RunCreate a Planned Run from a template 4


2 43 5

6 7 119 10

1

8 12

6. Click Plan Run.

The run is listed in the Planned Run List under the name that you specified and isautomatically used by the Ion Chef™ System when the associated Ion Chef™ LibrarySample Tube is loaded on the instrument.

Chapter 4 Create an assay-specific Planned RunCreate a Planned Run from a template4


Mutation load analysis

Review sequencing run results

If when creating your Planned Run you selected the data upload option to Reviewresults after run completion, then upload to Ion Reporter you can review yoursequencing results in the Torrent Suite™ Software by running the coverageAnalysisplugin. The coverageAnalysis plugin generates a report that describes the level ofsequence coverage produced for targeted genomic regions.

1. After the sequencing run is complete, in the Data tab, click Completed Runs &Reports.

2. In the list of runs, find the run of interest, then click the link in the Report Namecolumn.

3. In the left navigation menu, click coverageAnalysis to view the plugin summary.

4. Review the results in the Mean Depth, and Uniformity columns to ensure thatthe thresholds have been met for each barcoded library.

Column Description Threshold

Mean Depth Reports mean read depth across all targetedregions.

≥1100[1]

Uniformity The percentage of bases in all targeted regions(or whole‑genome) covered by at least 20% of theaverage base coverage depth reads. (Cumulativecoverage is linearly interpolated between nearestinteger base read depths.)

≥80%

[1] Provides sufficient read depth for variant calling at ≥5% allele frequency. If only determining the Mutation Load, ≥300 mean read depth is sufficient.

Note: Due to coverage variability in FFPE samples that may be due to samplequality, increased sequencing depth may be necessary to achieve sufficientcoverage. Increased sequencing depth can be achieved by reducing the numberof samples per chip.

5


Ion Reporter™ analysis workflows

If you selected the appropriate Ion Reporter™ workflow when setting up your PlannedRun in Torrent Suite™ Software, automated analysis has already been performed andyou can view the Mutation Load analysis results in Ion Reporter™ Software. Forinstructions on manually launching an analysis, see page 52.

Note: Microsoft™ Excel™, or other spreadsheet tool, is required for viewing VCF, CSV,and TSV files.

Analysis Workflow Description

Oncomine™ Tumor Mutation Load- w2.0 - DNA - Single Sample

Detects and annotates low frequency somaticvariants (SNPs and INDELs) from targeted DNAlibraries from the Oncomine™ Tumor Mutation LoadAssay run on the Ion 540™ or Ion 550™ Chip.Computes tumor mutational burden (TMB) from asingle sample using the Mutation Load (SomaticMutations) filter chain and TMB Algorithm Version2.5. Released with: Ion Reporter™ Software 5.10.Workflow version: 2.0.

Oncomine™ Tumor Mutation Load- w1.0 - DNA - Single Sample

Compute tumor mutation load from single sample.Released with: Ion Reporter Software 5.6. WorkflowVersion: 1.0.

View results

Ion Reporter™ Software analyses are performed automatically on uploading of thedata files from the Torrent Suite™ Software. To view the results and calculatedMutation Load (mutations/Mb):

1. Sign in to the Ion Reporter™ Software.

2. Click the Analyses tab.

3. In the Workflow dropdown list, select Oncomine™ Tumor Mutation Load - w2.0- DNA - Single Sample to limit the list of results to analyses using this workflow.

You can further refine the list of analyses by applying More Filters, or clickingcolumn headers. The Analyses table automatically filters the content appropriateto your selection.

Chapter 5 Mutation load analysisIon Reporter™ analysis workflows5


4. Click within a row (but not on thesample data set hyperlink) to view theDetails of the analysis.In the Details pane you can viewWorkflow Details, and access theActions dropdown list.

5. Click a sample result hyperlink in the Analysis column to open the AnalysisResults page.

The mutation load is displayed at the top of the results page in mutations perMb. The Analysis Results page opens to the Oncomine tab displaying onlyOncomine™ annotated variants that are known to be cancer drivers.

6. In the Analysis Results table, sort or filter the data using the Oncomine™-specificannotations. See the Ion Reporter™ Software help menu for more options.

a. In the Filter Options pane, select the desired Filter Chain.For example, selecting the Mutation Load (Somatic Mutations) filter chaindisplays all somatic mutations used in the Mutation Load analysis.

Note:· The default Filter Chain is Oncomine Variants (5.10) which limits the

results displayed to cancer driver variants only. Each variant called mustmeet all the conditions of the filter chain to have been filtered-in. Formore information, see “Oncomine™ Tumor Mutation Load Assay filterchain criteria“ on page 50 .

· Select No Filter to view all the variant calls attempted by the variantcaller. To view only the variant calls used in calculating the MutationLoad select Mutation Load (Somatic Mutations) (5.10).

· Saving the analysis using a filter chain other than Oncomine Variants(5.10) changes the variant calls that are saved in the VCF file and canaffect downstream workflows, such as with Oncomine™ KnowledgebaseReporter Software.

b. In the Oncomine tab, click the column headers to sort the list of variants byOncomine Variant Class or Oncomine Gene Class.

Chapter 5 Mutation load analysisView results 5


c. In the Ontologies tab, click the column headers to sort the list by variantType or Genes.

7. To download a results file.a. Click Download , then select All Variants, Filtered Variants, or Current

Results TSV.

b. In the Home tab, click Notifications, then click next to the file name.Alternatively, select one or more rows, then click Download.

The software generates a ZIP file with 4 folders: RESULTS, QC, Variants, andWorkflow_Settings.

Folder Contents of folder

RESULTS Contains:

• TSV files: filtered_variants.tsv and somatic_variants.tsv. Bothcontain somatic mutations, or the mutations that are left after applying MutationLoad filter chain, from both exonic and non-exonic regions of the panel. The sumof variants in both files equals the "Total Somatic Variants Count" in thestatistic.txt file. Only the missense and nonsense SNVs, and frameshift andnon‑frameshift INDELs of these files, are included in the TMB calculation.

• PDF of the Mutation Load analysis report.

• File named statistic.txt, which contains mutation load statistics.

Note: The Variant Details TSV file can be downloaded separately if you prefer. For moreinformation see “Visualize Mutation Load analysis results“ on page 37.

Also contains PNG image files:

• allele frequency distribution of germ-line and somatic variants

• allele frequency distribution of only somatic variants

• bar plot of signature type and context of somatic mutations

• pie chart of substitution type of somatic mutations

• pie chart of signature pattern of somatic mutations

QC Contains a PDF of the QC report, and a folder containing coverage statistics files.

Variants • Contains intermediate and Oncomine™ annotated .VCF files, which are used byOncomine™ Knowledgebase Reporter Software. For more information, see theOncomine™ Knowledgebase Reporter 3.0 User Guide (Pub. No. MAN0017300).

• TSV files that contain Oncomine™-filtered and all somatic variants.

Chapter 5 Mutation load analysisView results5


Folder Contents of folder

Workflow_Settings Contains folders with:

• A text file that describes settings used for the analysis. Open the file with a texteditor.

• Configuration files used by the Ion Reporter™ Software in the workflow settings.

After you have reviewed, filtered, and sorted your Analysis Result, you can downloadan Analysis Report. The procedure described here includes creating and formatting areport template.

1. In the Analysis Results screen for your sample, click Generate Report.The Generate Report workflow bar opens to the Configuration step. The sectionsof the report can be rearranged, deleted, or edited.

2. Hover over the various sections, and icons to view instructional text to help youformat your report output.

3. Enter information in editable fields (for example, edit the report name or enterbackground information).

4. (Optional) Click Save As New Template to save your reconfigured reporttemplate for future use with other sample results.

5. Click Next, a live preview of your report is displayed.

6. Click Lock & Publish to generate the final Analysis Report.

7. Click Download.

To view an example Analysis Report, see page 57. A separate Mutation LoadAnalysis Report including graphical representations of the data can be downloadedfrom the Analysis Visualization page, for more information see “Visualize MutationLoad analysis results“ on page 37.

Generate anAnalysis ResultsReport

Chapter 5 Mutation load analysisView results 5


The Mutation Load value is calculated automatically when the Oncomine™ TumorMutation Load - w2.0 - DNA - Single Sample workflow is run. To view more details ofthe analysis (for example, the TMB algorithm version used), and how the MutationLoad is calculated.

1. Download, then extract a results file to your hard drive.

2. In the RESULTS folder, open the statistic.txt file in a compatible texteditor. The Mutation Load and the data that are used to calculate its value arelisted.

Values used in calculating the Mutation Load (red box) are highlighted (yellow).

Mutation Load calculation

precalibration Mutation Load = (Nonsynonymous Somatic Mutations x 106) Total Exonic Bases with Sufficient Coverage

IF, precalibration Mutation Load ≥25, THEN Mutation Load = (precalibrationMutation Load − 25) x Calibration Slope + 25

IF, precalibration Mutation Load <25, THEN no calibration is required,Mutation Load = precalibration Mutation Load x 1.0

View the MutationLoad statistics

Chapter 5 Mutation load analysisView results5


Visualize Mutation Load analysis results

Visualizing the Ion Reporter™ Software analyses allows you to view a single sampleresult or multiple sample results simultaneously. Individual sample results can bevisualized from either the Analyses table or Analysis Results screen.

• To visualize sample results individually from the Analyses table.a. In the Analyses table select a sample result row, then click Visualize.

Alternatively, click Actions4Visualize.

The Analysis Visualization screen opens to the Mutation Load tab with thetumor mutation load (Mutations/Mb) displayed at the top.

b. Select either the Sample Results or Sample QC tab, then select the graphicalrepresentation of the data from the Views dropdown list.

c. Click Download Report.A 2-page PDF Mutation Load Analysis Report of the results including copiesof the graphs is automatically downloaded, see page 58.

d. In the lower right corner, click Download Variant Details TSV.A tab separated values list of each variant detected is automaticallydownloaded. Open the file with a TSV compatible viewer to see details ofeach individual variant that contributed to the Mutation Load count.

• To visualize sample results individually from the Analysis Results page.a. In the Analyses table, click a sample result hyperlink in the Analysis column

to open the Analysis Results.

b. Click Visualize.

c. Select either the Sample Results or Sample QC tab, then select the graphicalrepresentation of the data from the Views dropdown list.

d. Click Download Report.A 2-page PDF Mutation Load Analysis Report of the results including copiesof the graphs is automatically downloaded, see page 58.

e. In the lower right corner, click Download Variant Details TSV.A tab separated values list of each variant detected is automaticallydownloaded. Open the file with a TSV compatible viewer to see details ofeach individual variant that contributed to the Mutation Load count.

Chapter 5 Mutation load analysisVisualize Mutation Load analysis results 5


• To visualize multiple sample results simultaneously.a. In the Analyses table, select the checkbox next to each sample result you

want to visualize together, then click Visualize. Alternatively, in the SelectedAnalyses pane, click Actions4Visualize.

b. Select either the Sample Results or Sample QC tab, then select the graphicalrepresentation of the data from the Views dropdown list.

c. Click Download Report for each individual sample result.A 2-page PDF Mutation Load Analysis Report of the results including copiesof the graphs is automatically downloaded, see page 58.

d. In the lower right corner, click Download Variant Details TSV.A tab separated values list of each variant detected is automaticallydownloaded. Open the file with a TSV compatible viewer to see details ofeach individual variant that contributed to the Mutation Load count.

The Oncomine™ Tumor Mutation Load Assay results are represented graphically.Select the Sample Results to view from the Views dropdown list.

Figure 1 Allele ratio of Germline and Somatic VariantsA histogram showing frequency distribution of allele ratio for total called germline and somaticvariants. Listed below the figure is the combined total of called germline and somatic variants.Value is reported in the statistic.txt file as Total Variant Count, see“View the MutationLoad statistics“ on page 36.

Sample Resultstab

Chapter 5 Mutation load analysisVisualize Mutation Load analysis results5


Figure 2 Allele ratio of Only Somatic VariantsA histogram showing frequency distribution of allele ratio for only somatic mutations asdetermined by the Mutation Load (Somatic Mutations) filter chain. Listed below the figure are:

• The number of Total Somatic Variants (Value is reported in the statistic.txt file asTotal Somatic Variant Count, see“View the Mutation Load statistics“ on page 36).

• A break down of the number of mutations determined to be nonsynonymous (detrimental)and synonymous (non-detrimental) as annotated by Ion Reporter™ Software. Values arereported in the statistic.txt file, see“View the Mutation Load statistics“ onpage 36.

• The number of detected somatic variants found in the COSMIC database. Value is reportedin the statistic.txt file as COSMIC Annotated Somatic Variants, see“View theMutation Load statistics“ on page 36.

Figure 3 Substitution Type and Context of Somatic Mutations

C>A

C>G

C>T

T>A

T>C

T>GSomatic mutations can be divided into 6 base substitution classes (that is, C>A, C>G, C>T, T>A,T>C, T>G) based on their substitution type. After incorporating information on the basesimmediately 5′ and 3′ to each mutated base, there are 96 possible mutation types in thisclassification. These 96 mutation types are represented on the x‑axis, and variant frequency formutation type on the y‑axis. Bars for each substitution class are grouped and displayed withdifferent color.



Figure 4 Substitution type of Somatic Mutations

C>T

C>G

C>A

T>G

T>C

T>AA pie chart dividing somatic mutations into 6 base substitution classes(that is, C>A, C>G, C>T, T>A, T>C, T>G) based on their substitution type.

Figure 5 Signature Pattern of Somatic Mutations

C>T at [AG]CG

C>T at [CT]CG

C>T at NCC,CC[ACT],TC[ACT]

C>T at other sites

C>A

T>A and T>C

Rest

A pie chart dividing somatic mutations in groups consistent with specific mechanisms.

In Figure 5, a small fraction of multiple signature types can be observed in the sample.However, significant dominance of a single signature pattern often correlates to therespective tumor type. For example, 56.7% of the variants detected (sum of blue,green, and yellow) are an observed UV damage signature in this sample.

• High C>T at CpC, CpC, TpC, T>A, and T>C is consistent with UV damage(Hayward, 2017). (Blue + Green + Yellow)

• High C>T at CpG is consistent with spontaneous deamination of5-methylcytosine (Alexandrov, 2013). (Red + Blue)

• High C>A is consistent with smoking damage (Alexandrov, 2016). (Orange)• High C>T (site independent) is consistent with FFPE processing (Wong, 2014).

(Green + Purple)

Chapter 5 Mutation load analysisVisualize Mutation Load analysis results5


Note: Underlined bases represent the reference base being substituted (for example,CpG in the first bullet is same as to [ACGT]CG).

The sample QC tab lists the:

QC metric Definition

Average Coverage The average read depth across covered nucleotide bases. Valueis reported in the statistic.txt file as Average Coverage,see “View the Mutation Load statistics“ on page 36.

Number of Calls The number of variants identified in the sample. Value isreported in the statistic.txt file as Total Variant Count,see “View the Mutation Load statistics“ on page 36.

Estimated SNPproportion consistentwith Deamination(mainly FFPE)

The proportion of identified mutations that are likely the resultof FFPE sample deamination during processing. Value isreported in the statistic.txt file as Deamination, see “View the Mutation Load statistics“ on page 36.

Sample QC tab



Troubleshooting

Tips

• Arrange samples in alternating columns on the plate for easier pipetting withmultichannel pipettes during purification with the DynaMag™ Side Magnet.

• Plate seals can be firmly applied using the applicator in the MicroAmp™ OpticalAdhesive Film Kit. Plate seals can be removed with much less effort when hot.Try removing seals right after taking the plate out of the thermal cycler.

• If library yield is below 50 pM, libraries can still be sequenced by using aproportionally larger volume into a combined library or into templatepreparation.

• If the unamplified library yield is below 50 pM, libraries can be rescued withlibrary amplification. Combine 25 µL of unamplified library with 72 µL of 1XLibrary Amp Mix and 4 µL of 25X Library Amp Primers. Perform 5–10 libraryamplification cycles using the following cycling conditions.

Stage Temperature Time

Hold 98°C 2 minutes

5– 10 cycles 98°C 15 seconds

64°C 2 minutes

Hold 10°C Hold

• When amplifying multiple samples in a single PCR plate, ensure that the inputacross the samples is roughly equivalent so that the selected cycle number isoptimal for all the samples in the run.

Observation Possible cause Recommended action

Sample resulted in a highdeamination score

• Old FFPE sample.

• Improperly fixed FFPEsample.

• Improper sample storageconditions.

Edit the Ion Reporter™ workflow, thenreanalyze the data at a higher minimum allelefrequency. For more information, see “Customize the Ion Reporter™ workflow“ onpage 53.

Treat the FFPE sample DNA with Uracil DNAGlycosylase (UDG), then resequence. For moreinformation, see “Remove deaminated basesfrom FFPE DNA“ on page 55.

Library concentration is low–general

(Library concentration is NOTindicative of quality.)

Sample DNA was mis-quantified.

Requantify sample DNA using the TaqMan®

RNase P Detection Reagents Kit.

Sample DNA quality was low. Add more DNA or increase target amplificationcycles.

PCR, digestion, or ligation wasinefficient.

Ensure proper dispensing and mixing ofviscous components at each step.

A


Observation Possible cause Recommended action

Library concentration is low–general

(Library concentration is NOTindicative of quality.)

Residual ethanol in the sampleDNA inhibited targetamplification.

Incubate uncapped tube in hood for 1 hour.

Speed-vac tube at room temperature for 5minutes.

Residual ethanol fromAMPure™ purification inhibitedlibrary amplification.

Carefully remove all drops of ethanol beforelibrary amplification, then centrifuge plate, ifnecessary.

AMPure™ XP Beads were over-dried.

Do not dry the AMPure™ XP Beads more than5 minutes.

Percentage of polyclonal ISPs ishigh (>40%)

Library input was too high. Decrease amount of library added to thetemplate preparation reaction by 50%.

Library was mis-quantified. Ensure that library was quantified accurately.

Other. Check the appropriate template preparationuser guide for more information.

Low quality ISPs are present athigh percentage (>15%)

Library input was too low. Double the volume of library used in templatepreparation.

Use a fresh dilution of library prepared in alow-bind tube.

Other. Check the appropriate template preparationuser guide for more information.

Appendix A TroubleshootingTips A


Quantify the amplified library withthe Qubit™ Fluorometer

Oncomine™ Tumor Mutation Load Assay libraries must be amplified beforequantification to enrich amplifiable material and obtain sufficient material for accuratequantification. Amplify the library using 1X Library Amp Mix, then purify. Quantifythe library using the Qubit™ 4 Fluorometer. Amplified libraries typically have yieldsof 2,000–10,000 pM. Yield is not indicative of library quality. After quantification,determine the dilution factor that results in a concentration of ~50 pM, which isappropriate for template preparation using an Ion template kit.

Alternatively, the Ion Library TaqMan® Quantitation Kit can be used to quantifyamplified libraries.

Amplify the library

1. Remove the plate with purified libraries from the plate magnet, then add 50 μLof 1X Library Amp Mix and 2 μL of 25X Library Amp Primers to each beadpellet.

Note: The 1X Library Amp Mix and 25X Library Amp Primers and can becombined before addition.

2. Seal the plate with MicroAmp™ Adhesive Film, vortex thoroughly, thencentrifuge briefly to collect droplets. Alternatively, mix by pipetting at least halfthe total volume up and down at least 5 times before sealing the plate.

3. Place the plate back on the magnet for at least 2 minutes, then carefully transfer~50 µL of supernatant from each well to a new well or a new plate withoutdisturbing the pellet.

4. Seal the plate with MicroAmp™ Adhesive Film, place a MicroAmp™

Compression Pad on the plate, load in the thermal cycler, then run the followingprogram:

Stage Temperature Time

Hold 98°C 2 minutes

5 cycles 98°C 15 seconds

64°C 1 minute

Hold 10°C Hold

STOPPING POINT Samples can be stored at –20°C.

B


Purify the amplified library

Perform a two-round purification process with the Agencourt™ AMPure™ XP Reagent:• First round at 0.5X bead-to-sample-volume ratio: High molecular-weight DNA

is bound to beads, while amplicons and primers remain in solution. Save thesupernatant.

• Second round at 1.2X bead-to-original-sample-volume ratio: Amplicons arebound to beads, and primers remain in solution. Save the bead pellet, and elutethe amplicons from the beads.

IMPORTANT!· Bring Agencourt™ AMPure™ XP Reagent to room temperature and vortex

thoroughly to disperse the beads before use. Pipet the solution slowly.· Use freshly prepared 70% ethanol for the next steps. Combine 230 µL of ethanol

with 100 µL of Nuclease-free Water per sample.· Do NOT substitute a Dynabeads™-based purification reagent for the Agencourt™

Agencourt™ AMPure™ XP Reagent.

1. Tap the plate gently on a hard flat surface, or centrifuge briefly to collect thecontents at the bottom of the wells, then remove the plate seal.

2. Add 25 μL (0.5X sample volume) of Agencourt™ AMPure™ XP Reagent to eachplate well containing ~50 µL of sample. Mix the bead suspension with the DNAthoroughly by pipetting up and down 5 times.


4. Place the plate in a magnet such as the DynaMag™–96 Side Magnet for at least5 minutes, or until the solution is clear.

5. Carefully transfer the supernatant from each well to a new well of the 96-wellPCR plate without disturbing the pellet.

IMPORTANT! The supernatant contains the desired amplicons. Do not discard!

1. To the supernatant from step 4 above, add 60 μL (1.2X original sample volume)of Agencourt™ AMPure™ XP Reagent. Pipet up and down 5 times to mix thebead suspension with the DNA thoroughly.


3. Place the plate in the magnet for 3 minutes or until the solution is clear. Carefullyremove, then discard the supernatant without disturbing the pellet.

IMPORTANT! The amplicons are bound to the beads. Save the bead pellet.

First-roundpurification

Second-roundpurification

Appendix B Quantify the amplified library with the Qubit™ FluorometerPurify the amplified library B


4. Add 150 μL of freshly prepared 70% ethanol to each well, then move the plateside to side in the magnet to wash the beads. Remove and discard thesupernatant without disturbing the pellet.

Note: If your magnet does not have two positions for shifting the beads, removethe plate from the magnet and gently pipet up and down five times (with thepipettor set at 100 µL), then return the plate to the magnet and incubate for2 minutes or until the solution clears.

5. Repeat step 4 for a second wash.

6. Ensure that all ethanol droplets are removed from the wells. Keeping the plate inthe magnet, air-dry the beads at room temperature for 2−5 minutes. Do notoverdry.

7. Remove the plate from the magnet, then add 50 μL of Low TE to the pellet todisperse the beads.

8. Seal the plate with MicroAmp™ Adhesive Film, vortex thoroughly, thencentrifuge to collect droplets. Alternatively, mix by setting a pipettor to 40 µL,then pipet the mixture up and down at least 5 times before sealing the plate.

9. Incubate at room temperature for at least 2 minutes.

10. Place the plate in the magnet for at least 2 minutes, then analyze an aliquot of thesupernatant as described in:

• “Qubit™ Fluorometer: Quantify the library and calculate the dilutionfactor“ on page 47 or

• “Quantify library by qPCR and calculate the dilution factor“ on page 21

IMPORTANT! The supernatant contains the desired amplicons. Do not discard!

Appendix B Quantify the amplified library with the Qubit™ FluorometerPurify the amplified libraryB


Qubit™ Fluorometer: Quantify the library and calculate the dilutionfactor

Analyze 10 µL of each amplified library using a Qubit™ Fluorometer and the Qubit™

dsDNA HS Assay Kit. Amplified libraries typically have concentrations of 300–1500 ng/mL. Libraries below 300 ng/mL can still provide good quality sequences. Formore information, see the Qubit™ dsDNA HS Assay Kits User Guide (Pub. No.MAN0002326).

1. Determine the amplified library concentration:a. Make a 1:200 working dilution of Qubit™ dsDNA HS reagent using the

Qubit™ dsDNA HS Buffer.

b. Combine 10 µL of the amplified Ion AmpliSeq™ library with 190 µL of dyereagent, mix well, then incubate for at least 2 minutes.

c. Prepare each Qubit™ standard as directed in the user guide.

d. Measure the concentration on the Qubit™ Fluorometer.

e. (Qubit™ 2.0 Fluorometer only) Calculate the concentration of the undilutedlibrary by multiplying by 20. Alternatively, use the "Calculate Stock Conc."feature on your instrument.

2. Based on the calculated library concentration, determine the dilution that resultsin a concentration of 40–60 pM:

Average amplicon size Concentration in ng/mL (40–60 pM)

175 bp 5.5

225 bp 7.5

For example, with a FFPE-compatible 125–175 bp design (avg. 225 bp withadapters):

• The library concentration is 450 ng/mL.• The dilution factor is 450 ng/mL divided by 7.5 ng/mL = 60.• Therefore, 5 µL of library that is mixed with 295 µL of Low TE (1:60 dilution)

yields approximately 7.5 ng/mL (~50 pM).

3. Dilute library to ~50 pM as described, combine, then proceed to templatepreparation, or store libraries as described below.

Store libraries

You can store libraries at 4–8°C for up to 1 month. For longer lengths of time, store at–30°C to –10°C.

Appendix B Quantify the amplified library with the Qubit™ FluorometerQubit™ Fluorometer: Quantify the library and calculate the dilution factor B


Supplemental information

Torrent Suite™ Software

The IonReporterUploader 5.10 plugin is automatically installed on Torrent Serverwhen you update to a new release.

Note: If your instrument server does not have internet access, you must downloadand install the latest file named IonReporterUploader_<version>.deb from http://iru.ionreporter.thermofisher.com/ before proceeding.

To reinstall or update the IonReporterUploader 5.10 plugin for Torrent Suite™

Software 5.2 or later.

Note: An administrative ionadmin account is not required for this procedure.

1. Sign in to Ion Reporter™ Software, then click (Settings)4Download IonReporter Uploader.

2. Click the filename IonReporterUploader.zip, then download the file to your localmachine.

3. Sign in to Torrent Suite™ Software, then click (Settings)4Plugins.

4. Click Install or Upgrade Plugin.

5. Click Upload a Plugin file, then browse to the IonReporterUploader.zip file.Click Open, click Upload, then click Install.

C

Install theIonReporterUploader pluginon your TorrentServer


http://iru.ionreporter.thermofisher.com/


2. Click the (Settings) tab (upper right), then select Ion Reporter Configure.

3. In the Ion Reporter Uploader Account Configuration dialog, click + AddAccount, then select Ion Reporter.

4. In the Add Ion Reporter account screen, enter the following:

Field Directions

Server Type Select:[1]

Display Name Enter a meaningful name of your choice. This name is used inthe run plan template wizard and is seen by other TorrentBrowser users. Use only alphanumeric characters, spaces,and underscores.

Server Enter:[1]

Port Enter: 443

Username Enter your Ion Reporter™ Software username (your emailaddress)

Password Enter your Ion Reporter™ Software password

[1] Ask your local Ion Reporter™ Server System administrator for these values.

5. The "Default Account" is the account that is configured by default in runtemplates and run plans. If this account is the main account to be used for filetransfers, enable the Default Account checkbox. (You can always change thisselection in the run plan template wizard and in the Upload to IR quick link.)

6. Click Get Versions, select Ion Reporter 5.10 , then click ✓Add.

Contact your field service representative to obtain the latest versions of Oncomine™

Tumor Mutation Load Assay BED files.

1. Extract the BED file containing ZIP file to a location of your choice.

2. Sign in to the Torrent Suite™ Software where you want to install the TargetRegions BED file.

3. In the upper right of the screen, click (Settings)4References.

4. Upload the Target Regions panel BED file:a. In the left navigation menu, click Target Regions, then click Add Target

Regions.

b. Select hg19 - Homo sapiens from the Reference dropdown list.

Configure theIonReporterUploader plugin inthe TorrentBrowser

Download andinstall BED files

Appendix C Supplemental informationTorrent Suite™ Software C


c. Click Select File, then navigate to and select the Target Regions file:Oncomine_TML.20180605.designed.bed

d. Click Open, then click Upload Target Regions File.

The Target Regions BED file uploads to your Torrent Server and appears in the TargetRegions dropdown list.

Ion Reporter™ Software

Oncomine™ Tumor Mutation Load Assay filter chain criteria

Variant Type Oncomine™ GeneClass

Oncomine™

Variant Class Annotation Criteria

Loss of FunctionMutation

Loss-of-Function Deleterious • Positive mutation call

• Variant's functional impact is frameshift blocksubstitution, frameshift insertion, frameshiftdeletion, or nonsense

• Variant occurs in a loss-of-function gene

Gain of FunctionMissense HotspotMutation

Gain-of-Function Hotspot • Positive mutation call

• Variant's functional impact is missense

• Variant occurs in a gain-of-function gene

• Variant's transcript and codon position occur inpredefined missense hotspot list

Loss of FunctionMissense HotspotMutation

Loss-of-Function Hotspot • Positive mutation call

• Variant's functional impact is missense


• Variant's transcript and codon position occur inpredefined missense hotspot list

Gain of FunctionIn Frame HotspotMutation



• Variant's function, transcript, and coding syntaxoccur in predefined in-frame hotspot list

Appendix C Supplemental informationIon Reporter™ SoftwareC


Variant Type Oncomine™ GeneClass

Oncomine™

Variant Class Annotation Criteria

Loss of FunctionIn Frame HotspotMutation



• Variant's function, transcript, and coding syntaxoccur in predefined in-frame hotspot list

Gain of FunctionSplice SiteHotspot Mutation



• Variant's transcript, location, and exon occur inpredefined splice site hotspot list

Loss of FunctionSplice SiteHotspot Mutation



• Variant's transcript, location, and exon occur inpredefined splice site hotspot list

Gain of FunctionPromoter HotspotMutations



• Variant's transcript, location, and coding syntaxoccur in predefined promoter hotspot list

Note: This feature is only available in Ion Reporter™ Software 5.10 or later.

You can enable any DNA – Single Sample, or DNA and Fusions – Single Sampleworkflow to include the mutation load calculation in addition to the other analysisresults expected from that workflow.

A workflow that is enabled for Mutation Load calculates mutations per megabase(Mb) and adds graphics and other information about the mutations to Ion Reporter™

Software analysis results and visualizations.

To enable Mutation Load, you must edit a workflow, then set a parameter in theworkflow.

Note: You can copy and edit any Ion Reporter™ predefined workflow, and you canalso edit an existing custom workflow if is not locked.

1. In the Workflows tab, click Overview.

2. In the list of workflows, select the row for the DNA – Single Sample, or DNA andFusions – Single Sample workflow that you want to copy. Click

(Actions)4Copy.The Edit workflow bar opens to the Research Application step with theResearch Application and Sample Groups preselected. When you copyworkflow templates, you cannot change these settings.

3. Click Next multiple times to proceed to the Parameters step.

Enable MutationLoad calculationin existingworkflows

Appendix C Supplemental informationIon Reporter™ Software C


4. In the Parameters step, find the Mutation Load Calculation Filter Chainparameter, then change the value in the dropdown list to Mutation Load(Somatic SNVs).

5. Click Next, then enter a Workflow Name and Description (Optional) for theworkflow.

6. Click Save Workflow.The custom workflow is saved and is added to the Workflows table.

To ensure that the workflow was saved, click the Workflows tab, then click Overview,and search for the workflow name. To analyze samples with your new Mutation Loadenabled workflow, see “Manually launch an analysis in Ion Reporter™“ on page 52.

1. Sign in to the Ion Reporter™ Software.

2. Launch the analysis.

From the… Directions

Home tab 1. In the Dashboard screen, click Launch analysis.

2. In the Launch Analysis screen, select DNA from the ResearchApplication dropdown list.

3. In the Research Category dropdown list, select Oncology -ImmunoOncology, then click in the Oncomine™ Tumor MutationLoad - w2.0 - DNA - Single Sample row.

4. Click Next.

Analyses tab 1. Click Launch Analysis, then select Manual from the dropdownlist.

2. In the Launch Analysis screen, select Mutation Load from theResearch Application dropdown list.

3. Click in the Oncomine™ Tumor Mutation Load - w2.0 - DNA -Single Sample row, then click Next.

Workflowstab

1. Select Mutation Load from the Research Application dropdownlist.

2. Click in the Oncomine™ Tumor Mutation Load - w2.0 - DNA -Single Sample row, then select Launch Analysis from theActions dropdown list in the Details pane.

3. Search by any unique identifier you used to label your samples during setup,select one or more samples to include in the analysis, then click Next.

4. (Optional) Select plugins to run with your analysis.

Note: Oncomine Variant Annotator v2.4 is applied be default and cannot beedited or removed from the workflow.

a. Click adjacent to the selected plugin.

b. Configure the plugin parameters as necessary, click Submit, then clickClose.

Manually launchan analysis in IonReporter™



5. Click Next.

6. Enter an Analysis Name and Description (Optional), then click Launch Analysis.

The Ion Reporter™ Software performs the analysis and the results will appear on theAnalyses page.

The Ion Reporter™ workflow Oncomine™ Tumor Mutation Load - w2.0 - DNA -Single Sample calls variants at ≥5% allelic frequency at positions with read coverage≥60 for Mutation Load calculation. Poor sample fixation can artificially increase thedetermined Mutation Load due to deamination. Deamination is reported as theEstimated SNP proportion consistent with Deamination (mainly FFPE) under theSample QC tab. When considering the Estimated SNP proportion consistent withDeamination (mainly FFPE) number, note that samples with low tumor content mayhave true biological somatic C:G>T:A mutations that are counted towardsdeamination.

Samples of low quality that exhibit deamination can be analyzed using an analysisworkflow with higher minimum allele frequency (for example, 10%) for MutationLoad calculation to reduce the impact of deamination on the reported Mutation Load.It is important to consider tumor content of a given sample when increasing theminimum allele frequency of the Mutation Load calculation. Samples with low tumorcontent can have many true somatic mutations that are excluded from the MutationLoad calculation when analyzed using a workflow with a higher minimum allelefrequency filter. For samples with a high Estimated SNP proportion consistent withDeamination (mainly FFPE) number (for example, >100), Mutation Load values maynot be reliable and adjusting the minimum allele frequency in the analysis parameterswill not help. The mean depth and uniformity of the sample as determined by thecoverageAnalysis plugin (see page 31) can also indicate sample quality.

To modify the existing Oncomine™ workflow to achieve higher minimum allelefrequency for Mutation Load calculation, you must create a new filter chain, thencopy and edit the Oncomine™ Tumor Mutation Load - w2.0 - DNA - Single Sampleworkflow as described.

1. In the Workflows tab, click Presets, then click Create Presets4Filter Chain.

2. In the Create Filter Chain dialog, enter a Name, and Description (not required).

Note: The Name field accepts alphanumeric characters, underscore, hyphen,period, and space only.

3. Set the filters to apply to the filter chain.a. Click Choose Filter, type the filter name into the Find Filter field, then select

the filter.

b. Configure the filter with the provided selections.

c. Click Set.

Customize the IonReporter™

workflow

Appendix C Supplemental informationIon Reporter™ Software C


4. Repeat step 3 for each filter listed in the following table.

Filter name Value

Selections

Includeboundary

values

Includeunannotated

variants

Filtered Coverage GREATER_THAN = 60 Select Select

Variant Type SNV, INDEL n/a n/a

Minor Allele Frequency From = 0.0, To = 0.000001 Select Select

5000Exomes Global MAF(20161108)

From = 0.0, To = 0.000001 Select Select

ExAC GAF (1) From = 0.0, To = 0.000001 Select Select

UCSC Common SNPs Filter value = Not In n/a n/a

Homopolymer Length From = 0, To = 4 Select Select

Allele Frequency From = X[1], To = 1.0 Select Select

[1] Set "From =" value to 0.05 for ≥5%, to 0.1 for ≥10% allele frequency

The FilterChain Query and Selected Filters panes should look like this.

5. Click Save, the Filter Chain is now available to apply to a custom workflow.



6. Copy, then edit the Oncomine™ Tumor Mutation Load - w2.0 - DNA - SingleSample workflow.

a. In the Workflows tab, click Overview, then select the Oncomine™ TumorMutation Load - w2.0 - DNA - Single Sample workflow.

b. Click (Actions)4Copy.

c. Click Next multiple times to advance to the Parameters step.

d. In the Mutation Load Calculation Filter Chain parameter dropdown list,select your new Filter Chain.

e. Click Next, then in the Confirm tab, enter a Workflow Name anddescription, then click Save Workflow.

The modified workflow is now available for use in the Workflows tab, Overviewscreen.

Remove deaminated bases from FFPE DNA

IMPORTANT! We have demonstrated that deaminated cytosine (uracil) bases can beenzymatically removed by treatment with Uracil-DNA Glycosylase (UDG) before thetarget amplification reaction of the Ion AmpliSeq™ Library Kit Plus librarypreparation protocol (page 15). However, the following protocol has not been fullyverified for use with the Oncomine™ Tumor Mutation Load Assay. In a subset ofFormalin-fixed paraffin-embedded (FFPE) samples tested, UDG treatment wasdemonstrated to reduce the Oncomine™ Tumor Mutation Load Assay Estimated SNPproportion consistent with Deamination (mainly FFPE) number.

Sample age, storage conditions, and FFPE preservation methods can lead tosignificant cytosine deamination of the isolated DNA. This deamination can result inan artificially high Estimated SNP proportion consistent with Deamination number(see “Sample QC tab“ on page 41) when determining the Mutation Load result. FFPEsamples that yield a high Estimated SNP proportion consistent with Deaminationnumber may benefit from treatment with Uracil-DNA Glycosylase (UDG) to removedeaminated bases before target amplification.

1. For each FFPE DNA sample, add the following components to a single well of a96-well PCR plate.

Component Volume

20 ng FFPE DNA ≤6.5 µL

UDG, heat‑labile 1 µL

Low TE to 7.5 µL

Appendix C Supplemental informationRemove deaminated bases from FFPE DNA C


2. Mix the reaction by pipetting at least half the total volume up and down at least5 times, then seal the plate with MicroAmp™ Clear Adhesive Film. Alternatively,seal the plate, vortex for 5 seconds to mix the reactions, then centrifuge briefly tocollect the contents.

Note: To prevent evaporation during UDG treatment, use an applicator tool topress the film securely around each reaction well and around the perimeter ofthe plate.

3. Place a MicroAmp™ Optical Film Compression Pad on the plate, load the plateinto the thermal cycler, then run the following program.

Temperature Time

37°C 2 minutes

50°C 10 minutes

4°C Hold (≤1 hour)

4. Remove the plate from the thermal cycler, then centrifuge briefly to collect thecontents.

STOPPING POINT Reactions can be stored at −20°C long term.

5. Carefully remove the plate seal, then proceed immediately to “Prepare DNAtarget amplification reactions“ on page 15, adding the target amplificationreaction components to the well containing 7.5 µL of UDG treated FFPE DNA.

Appendix C Supplemental informationRemove deaminated bases from FFPE DNAC


Example reports

Example Analysis Results Report

D


Oncomine™ Tumor Mutation Load Assay report

Appendix D Example reportsOncomine™ Tumor Mutation Load Assay reportD


Appendix D Example reportsOncomine™ Tumor Mutation Load Assay report D


Safety

WARNING! GENERAL SAFETY. Using this product in a manner not specifiedin the user documentation may result in personal injury or damage to theinstrument or device. Ensure that anyone using this product has receivedinstructions in general safety practices for laboratories and the safetyinformation provided in this document.

· Before using an instrument or device, read and understand the safetyinformation provided in the user documentation provided by themanufacturer of the instrument or device.

· Before handling chemicals, read and understand all applicable Safety DataSheets (SDSs) and use appropriate personal protective equipment (gloves,gowns, eye protection, and so on). To obtain SDSs, see the “Documentationand Support” section in this document.

E


Chemical safety

WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards,ensure laboratory personnel read and practice the general safety guidelines forchemical usage, storage, and waste provided below. Consult the relevant SDSfor specific precautions and instructions:

· Read and understand the Safety Data Sheets (SDSs) provided by thechemical manufacturer before you store, handle, or work with any chemicalsor hazardous materials. To obtain SDSs, see the “Documentation andSupport” section in this document.

· Minimize contact with chemicals. Wear appropriate personal protectiveequipment when handling chemicals (for example, safety glasses, gloves, orprotective clothing).

· Minimize the inhalation of chemicals. Do not leave chemical containers open.Use only with adequate ventilation (for example, fume hood).

· Check regularly for chemical leaks or spills. If a leak or spill occurs, followthe manufacturer's cleanup procedures as recommended in the SDS.

· Handle chemical wastes in a fume hood.· Ensure use of primary and secondary waste containers. (A primary waste

container holds the immediate waste. A secondary container contains spillsor leaks from the primary container. Both containers must be compatiblewith the waste material and meet federal, state, and local requirements forcontainer storage.)

· After emptying a waste container, seal it with the cap provided.· Characterize (by analysis if necessary) the waste generated by the particular

applications, reagents, and substrates used in your laboratory.· Ensure that the waste is stored, transferred, transported, and disposed of

according to all local, state/provincial, and/or national regulations.· IMPORTANT! Radioactive or biohazardous materials may require special

handling, and disposal limitations may apply.

Appendix E SafetyChemical safety E


Biological hazard safety

WARNING! BIOHAZARD. Biological samples such as tissues, body fluids,infectious agents, and blood of humans and other animals have the potential totransmit infectious diseases. Conduct all work in properly equipped facilitieswith the appropriate safety equipment (for example, physical containmentdevices). Safety equipment can also include items for personal protection, suchas gloves, coats, gowns, shoe covers, boots, respirators, face shields, safetyglasses, or goggles. Individuals should be trained according to applicableregulatory and company/ institution requirements before working withpotentially biohazardous materials. Follow all applicable local, state/provincial,and/or national regulations. The following references provide generalguidelines when handling biological samples in laboratory environment.

· U.S. Department of Health and Human Services, Biosafety in Microbiologicaland Biomedical Laboratories (BMBL), 5th Edition, HHS Publication No. (CDC)21-1112, Revised December 2009; found at:www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf

· World Health Organization, Laboratory Biosafety Manual, 3rd Edition,WHO/CDS/CSR/LYO/2004.11; found at:www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf

Appendix E SafetyBiological hazard safetyE


http://www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf

http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf

Documentation and support

Related documentation

Document Description

Ion AmpliSeq™ Library Kit Plus User Guide(Pub. No. MAN0017003)

Comprehensive instruction and troubleshooting for thepreparation of Oncomine™ and Ion AmpliSeq™ libraries.

Ion AmpliSeq™ Library Preparation on the Ion Chef™

System User Guide (Pub. No. MAN0013432)Describes the automated preparation and templating ofOncomine™ Tumor Mutation Load Assay libraries usingthe Ion Chef™ System.

Ion 540™ Kit – Chef User Guide (Pub. No. MAN0010851) Describes the automated template preparation ofOncomine™ Tumor Mutation Load Assay libraries usingthe Ion Chef™ System for sequencing on an IonGeneStudio™ S5 Series System or Ion S5™/Ion S5™ XLSystem.

Ion 550™ Kit − Chef User Guide (Pub. No. MAN0017275) Describes the automated template preparation ofOncomine™ Tumor Mutation Load Assay libraries usingthe Ion Chef™ System for sequencing on an IonGeneStudio™ S5 Series System or Ion S5™ XL System. TheIon 550™ Chip is not compatible with the Ion S5™

Sequencer.

Ion Reporter™ Software 5.10 User Guide (Pub. No. MAN0017605)

Includes software installation and set up information inaddition to Ion Reporter™ Software Help content.

Torrent Suite™ Software 5.10 User Guide(Pub. No. MAN0017598)

Includes software installation and set up information inaddition to Torrent Suite™ Software Help content.

Ion Library TaqMan® Quantitation Kit User Guide(Pub. No. MAN0015802)

Provides detailed instruction and troubleshooting for useof the Ion Library TaqMan® Quantitation Kit

Demonstrated Protocol: Sample Quantification for IonAmpliSeq™ Library Preparation Using the TaqMan®

RNAse P Detection Reagents Kit (Pub. No. MAN0007732)

Provides detailed instruction for sample quantificationusing the TaqMan® RNase P Detection Reagents Kit.

Note: For additional documentation, see “Customer and technical support“ onpage 64.


Customer and technical support

Visit thermofisher.com/support for the latest service and support information.• Worldwide contact telephone numbers• Product support information

– Product FAQs– Software, patches, and updates– Training for many applications and instruments

• Order and web support• Product documentation

– 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 forthin the Life Technologies' General Terms and Conditions of Sale at www.thermofisher.com/us/en/home/global/terms-and-conditions.html. If you haveany questions, please contact Life Technologies at www.thermofisher.com/support.

Documentation and supportCustomer and technical support


http://thermofisher.com/support

http://www.thermofisher.com/us/en/home/global/terms-and-conditions.html

http://www.thermofisher.com/support

References

Alexandrov, LB et al. (2013) Signatures of mutational processes in human cancer.Nature 500:415–421.

Alexandrov, LB et al. (2016) Mutational signatures associated with tobacco smoking inhuman cancer. Science 354:618–622.

Hayward, NK et al. (2017) Whole-Genome Landscapes of Major Melanoma Subtypes.Nature 545:175–180.

Wong, SQ et al. (2014) Sequence artefacts in a prospective series of formalin-fixedtumours tested for mutations in hotspot regions by massively parallel sequencing.BMC Medical Genomics 7:23.


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4 October 2018

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Documents

Oncomine Tumor Mutation Load Assay - assets.thermofisher.com · tumor samples—10 ng of DNA per primer pool—using the Oncomine™ Tumor Mutation Load Assay library preparation