Thermo Fisher Scientific Oncomine User Manual

Download

Oncomine™ Comprehensive Assay Plus

USER GUIDE

for use with: Oncomine™ Comprehensive Assay Plus, DNA panel Oncomine™ Comprehensive Assay Plus, RNA panel

Catalog Numbers A48577, A48578, A49667, and A49671

Publication Number MAN0018490

Revision C.0

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

Life Technologies Corporation | 7335 Executive Way | Frederick, Maryland 21704 USA For descriptions of symbols on product labels or product documents, go to thermoﬁsher.com/symbols-deﬁnition.

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. MAN0018490

Revision Date Description

C.0 9 February 2021 Full product launch, updates including:

•

Updated Ion Reporter™ Software analysis workﬂows:

–

Oncomine™ Comprehensive Plus - w2.1 - DNA and Fusions - Single Sample

–

Oncomine™ Comprehensive Plus - w2.1 - DNA and Fusions - (Low RMC Signal) - Single Sample

–

Oncomine™ Comprehensive Plus - w2.1 - DNA - Single Sample

–

Oncomine™ Comprehensive Plus - w2.1 - DNA - (Low RMC Signal) - Single Sample

–

Oncomine™ Comprehensive Plus - w2.1 - Fusions - Single Sample

–

Oncomine™ Comprehensive Plus - w2.1 - Annotate Variants - Single Sample

•

Updated analysis results in Ion Reporter™ Software w2.1 analysis workﬂows.

–

Automatically calculated tumor cellularity

–

Loss-of-heterozygosity determination

–

Gene fusion detection featuring FusionSync™ technology

•

Instructions for automated library preparation on Ion Chef™ Instrument

C.0 Early Access 18 May 2020 Updates for Oncomine™ Comprehensive Assay Plus, RNA panel early access

B.0 19 December 2019 Updates to user guide for product launch.

A.0 11 November 2019 This user guide provides instructions for library preparation, templating, sequencing,

customers.

•

Updated Ion Reporter™ Software analysis workﬂows:

–

Oncomine™ Comprehensive Plus - w2.0 - DNA and Fusions - (Manual Library Prep) - Single Sample

–

Oncomine™ Comprehensive Plus - w2.0 - DNA - (Manual Library Prep) Single Sample

–

Oncomine™ Comprehensive Plus - w2.0 - Fusions - (Manual Library Prep) Single Sample

–

Oncomine™ Comprehensive Plus - w2.0 - Annotate Variants - Single Sample

•

Updated analysis results in Ion Reporter™ Software w2.0 analysis workﬂows.

•

Inclusion of NTC in UDG treatment of FFPE DNA samples.

•

Expanded list of recommended controls.

•

Create planned runs from the provided template.

•

Update Ion Reporter™ Software analysis workﬂow name.

•

Include additional troubleshooting.

and results analysis of Oncomine™ Comprehensive Assay Plus libraries, speciﬁc for early access and restricted sales.

Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all applicable Limited Use Label Licenses.

Trademarks: All trademarks are the property of Thermo Fisher Scientiﬁc and its subsidiaries unless otherwise speciﬁed. TaqMan is a registered trademark of Roche Molecular Systems, Inc., used under permission and license. Eppendorf LoBind is a

trademark of Eppendorf AG. Agencourt and AMPure are trademarks of Beckman Coulter, Inc. Microsoft and Excel are trademarks of Microsoft Corporation.

■

CHAPTER 1 Product information .................................................. 7

Product description ............................................................. 7

Contents and storage ............................................................ 9

Oncomine™ Comprehensive Assay Plus – Manual Library Preparation ............. 9

Oncomine™ Comprehensive Assay Plus, RNA – Manual Library Preparation ....... 10

Oncomine™ Comprehensive Assay Plus – Automated Library Preparation ......... 11

Oncomine™ Comprehensive Assay Plus, RNA – Automated Library Preparation .... 12

Required materials not supplied ................................................. 13

Recommended materials ....................................................... 14

■

CHAPTER 2 Before you begin .................................................... 16

Procedural guidelines ........................................................... 16

Before each use of the kit ....................................................... 16

Guidelines for RNA isolation, quantiﬁcation, and input .............................. 17

Guidelines for DNA isolation, quantiﬁcation, and input .............................. 17

Library preparation from genomic DNA or RNA .................................... 18

■

CHAPTER 3 Automated library preparation on the Ion Chef™ System ....... 19

Create a sample set to prepare 2 pools of 4 libraries each ........................... 19

Automated RNA library preparation .............................................. 21

Reverse transcribe RNA for Chef Ready library preparation ..................... 21

Ion Chef™ Instrument setup information for automated RNA library preparation .... 23

Automated DNA library preparation ............................................... 24

RMC in DNA target ampliﬁcation reactions .................................... 24

Remove deaminated bases from FFPE DNA .................................. 24

Ion Chef™ Instrument setup information for automated DNA library preparation .... 27

Oncomine

™

Comprehensive Assay Plus User Guide

Contents

■

CHAPTER 4 Manual library preparation ......................................... 28

RNA preparation and cDNA ampliﬁcation ......................................... 28

Reverse transcribe RNA for manual library preparation ......................... 28

Prepare cDNA target ampliﬁcation reactions .................................. 29

Amplify the cDNA targets ................................................... 31

Combine cDNA target ampliﬁcation reactions ................................. 32

DNA preparation and ampliﬁcation ............................................... 33

RMC in DNA target ampliﬁcation reactions .................................... 33

Remove deaminated bases from FFPE DNA .................................. 33

Prepare DNA target ampliﬁcation reactions ................................... 34

Amplify the DNA targets .................................................... 35

Combine the DNA target ampliﬁcation reactions ............................... 36

Library preparation ............................................................. 36

Transfer the DNA amplicons ................................................. 36

Partially digest the DNA and cDNA amplicons ................................. 37

Ligate adapters to the amplicons and purify ................................... 38

Purify the unampliﬁed library ................................................ 39

Elute and dilute the library .................................................. 40

Quantify the library by qPCR and calculate the dilution factor ........................ 41

Combine libraries .............................................................. 43

Guidelines for templating and sequencing ......................................... 46

■

CHAPTER 5 Create a Planned Run .............................................. 47

About Planned Runs ........................................................... 47

Update Oncomine™ Comprehensive Assay Plus templates in Torrent Suite™ Software .. 48

Create a custom Planned Run template ........................................... 49

Create a Planned Run for manual library preparation ................................ 51

Create a Planned Run for automated library preparation using sample sets ............ 53

■

CHAPTER 6 Variant analysis ..................................................... 56

Analysis workﬂows in Ion Reporter™ Software ..................................... 56

Manually launch a DNA and Fusions analysis ...................................... 58

Manually launch a DNA analysis ................................................. 59

Manually launch a Fusions analysis ............................................... 59

View results ................................................................... 60

Fusion results ............................................................. 64

Visualize tumor mutational burden analysis results ................................. 70

QC metrics for tumor mutational burden ...................................... 71

Sample results ............................................................ 72

Oncomine™ Comprehensive Assay Plus User Guide

MSI analysis results ............................................................ 75

View MSI parameters ....................................................... 75

Visualize MSI analysis results ................................................ 76

Genomic segmentation analysis ................................................. 78

Visualization of genomic segmentation analysis, Allele Speciﬁc Copy

Number plots ........................................................... 79

Visualize exon level loss results .............................................. 81

Generate an Analysis Results Report ............................................. 82

Download Ion Reporter™ annotation VCF or TSV ﬁles ............................... 83

Requirements for variant annotation in Ion Reporter™ Software .................. 84

■

APPENDIX A Tips and troubleshooting ......................................... 87

Tips .......................................................................... 87

Troubleshooting ................................................................ 88

Library yield and quantiﬁcation .............................................. 88

Low amplicon uniformity (DNA only) .......................................... 89

Other ..................................................................... 89

Contents

■

APPENDIX B Supplemental information ........................................ 91

Update Oncomine™ Comprehensive Assay Plus templates in Torrent Suite™ Software .. 91

Install Oncomine™ Comprehensive Assay Plus workﬂows in Ion Reporter™ Software .... 91

Download and install BED ﬁles ................................................... 91

Conﬁgure the IonReporterUploader plugin in Torrent Suite™ Software ................. 92

Set TMB Classiﬁcation parameters ............................................... 93

■

APPENDIX C CNV baseline creation ............................................. 95

Use VCIB CNV baseline ......................................................... 95

Create a CNV baseline .......................................................... 96

Augment (add Samples to) an existing VCIB CNV baseline .......................... 97

Create an Ion Reporter analysis workﬂow ......................................... 99

Launch an analysis ............................................................ 100

■

APPENDIX D CNV somatic conﬁdence ﬁlter .................................. 102

CNV somatic conﬁdence range ................................................. 102

How to change the CI threshold default value .................................... 102

Oncomine™ Comprehensive Assay Plus User Guide

Contents

■

APPENDIX E Subset ﬁlter creation ............................................. 105

Create a gene-level ﬁlter ....................................................... 105

Create a variant-level ﬁlter ..................................................... 107

Create a new variantDB from the provided ﬁle ................................ 107

Create a new annotation set from the new variantDB and existing

Oncomine™ annotation sources .......................................... 108

Create a new ﬁlter chain using the new variantDB ............................. 109

Create a copied workﬂow with the new annotation set and ﬁlter chain ........... 110

Use the new workﬂow ..................................................... 111

APPENDIX F Safety .............................................................. 112

Chemical safety .............................................................. 113

Biological hazard safety ....................................................... 114

Documentation and Support ...................................................... 115

Related documentation ........................................................ 115

Customer and technical support ................................................ 115

Limited product warranty ...................................................... 116

Oncomine™ Comprehensive Assay Plus User Guide

Product description .................................................................... 7

■

Contents and storage .................................................................. 9

■

Required materials not supplied ........................................................ 13

■

Recommended materials .............................................................. 14

■

IMPORTANT! Before using this product, read and understand the information in the “Safety” appendix

in this document.

Product description

The Oncomine™ Comprehensive Assay Plus is a targeted, next-generation sequencing (NGS) assay that provides a comprehensive genomic proﬁling solution appropriate for formalin-ﬁxed paran-embedded (FFPE) tissues. The assay allows concurrent analysis of DNA and RNA to simultaneously detect multiple biomarkers associated with targeted and immune checkpoint therapies including comprehensive targets that are relevant in cancer, in a single workﬂow.

Product information

Features of the Oncomine™ Comprehensive Assay Plus include:

•

Enables analysis of variants across 500+ genes

•

Detection of SNVs, CNVs, InDels, TMB, MSI, and gene fusions

•

Robust performance from as little as 10 ng per pool of nucleic acid isolated from FFPE samples including ﬁne needle biopsies

•

Characterized with molecular standards and controls

This guide covers manual library preparation from DNA and RNA using the Oncomine™ Comprehensive Assay Plus, DNA and Oncomine™ Comprehensive Assay Plus, RNA panels, respectively. Each assay panel can be used with barcoded adapters, so that up to 4 paired DNA and RNA samples and DNA and RNA no-template controls (NTCs) can be combined and loaded onto a single Ion 550™ Chip in a single workﬂow to minimize the per-sample sequencing cost.

The Oncomine™ Comprehensive Assay Plus, DNA panel includes the Ion AmpliSeq™ Sample ID Panel primers to prevent research sample misidentiﬁcation and provide gender determination.

Note: This guide also covers automated library preparation on the Ion Chef™ System using the Oncomine™ Comprehensive Assay Plus – Automated Library Preparation kit (Cat. Nos. A49667 and

A49671). The kit provides the Oncomine™ Comprehensive Assay Plus, DNA (2-pools) and Oncomine

Comprehensive Assay Plus, RNA (2-pools) at 2X concentration pre-measured in barcoded primer pool tubes ready to load into an Ion AmpliSeq™ Chef Reagents DL8 cartridge.

™

Oncomine™ Comprehensive Assay Plus User Guide

Chapter 1 Product information

Product description

This guide includes protocols for using the following products:

•

Oncomine™ Comprehensive Assay Plus, DNA, Manual Library Preparation (Part No. A45615)

•

Oncomine™ Comprehensive Assay Plus, RNA, Manual Library Preparation (Part No. A45616)

•

Oncomine™ Comprehensive Assay Plus, DNA, Chef-Ready Library Preparation (Part No. A45617)

•

Oncomine™ Comprehensive Assay Plus, RNA, Chef-Ready Library Preparation (Part No. A45618)

•

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

•

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

•

Ion Torrent™ NGS Reverse Transcription Kit (Cat. No. A45003)

•

IonCode™ Barcode Adapters 1–384 Kit (Cat. No. A29751)

•

Ion Xpress™ Barcode Adapters (various Cat. Nos.)

•

Ion Library TaqMan™ Quantitation Kit (Cat. No. 4468802)

•

Uracil-DNA Glycoslyase, heat-labile (Cat. No. 78310100UN)

Oncomine™ Comprehensive Assay Plus User Guide

Chapter 1 Product information

Contents and storage

Oncomine™ Comprehensive Assay Plus – Manual Library Preparation

Oncomine™ Comprehensive Assay Plus (Cat. No. A48577) is designed to prepare barcoded sample libraries from DNA and RNA. The kits consist of the Oncomine™ Comprehensive Assay Plus, DNA, Manual Library Preparation panel (2‑pool) (Part No. A45615) and the Oncomine™ Comprehensive Assay Plus, RNA, Manual Library Preparation panel (2‑pool) (Part No. A45616), with two Ion AmpliSeq™ Library Kit Plus (Cat. No. 4488990). Sucient reagents are provided to prepare libraries from 24 samples.

Contents Amount Storage

Oncomine™ Comprehensive Assay Plus, DNA, Manual Library Preparation

2X DNA OCA Plus, Pool 1 of 2 (blue cap) 3 × 40 µL –30ºC to –10ºC

2X DNA OCA Plus, Pool 2 of 2 (blue cap) 3 × 40 µL

RMC 24 µL

Oncomine™ Comprehensive Assay Plus, RNA, Manual Library Preparation

5X RNA OCA Plus, Pool 1 of 2 (red cap) 3 × 16 µL –30ºC to –10ºC

5X RNA OCA Plus, Pool 2 of 2 (red cap) 3 × 16 µL

Ion AmpliSeq™ Library Kit Plus

5X Ion AmpliSeq™ HiFi Mix (red cap) 2 x 120 µL –30ºC to –10ºC

FuPa Reagent (brown cap) 2 x 48 µL

Switch Solution (yellow cap) 96 µL

DNA Ligase (blue cap) 2 x 48 µL

25X Library Amp Primers (pink cap) 2 x 48 µL

1X Library Amp Mix (black cap) 2 x 1.2 mL

Low TE 2 x 6 mL 15°C to 30°C

[1]

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

[1]

Oncomine™ Comprehensive Assay Plus User Guide

Chapter 1 Product information

Contents and storage

Oncomine™ Comprehensive Assay Plus, RNA – Manual Library Preparation

Oncomine™ Comprehensive Assay Plus, RNA – Manual Library Preparation (Cat. No. A48578) is designed to prepare barcoded sample libraries from RNA. The kits consist of the Oncomine

™

Comprehensive Assay Plus, RNA – Manual Library Preparation panel (2‑pool) (Part No. A45616), with one Ion AmpliSeq™ Library Kit Plus (Cat. No. 4488990). Sucient reagents are provided to prepare libraries from 24 samples.

Contents Amount Storage

Oncomine™ Comprehensive Assay Plus, RNA – Manual Library Preparation

5X RNA OCA Plus, Pool 1 of 2 (red cap) 3 × 16 µL –30ºC to –10ºC

5X RNA OCA Plus, Pool 2 of 2 (red cap) 3 × 16 µL

Ion AmpliSeq™ Library Kit Plus

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]

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

[1]

Oncomine™ Comprehensive Assay Plus User Guide

Chapter 1

Product information

Contents and storage

Oncomine™ Comprehensive Assay Plus – Automated Library Preparation

The Oncomine™ Comprehensive Assay Plus – Automated Library Preparation (Cat. No. A49667) provides the Oncomine™ Comprehensive Assay Plus, DNA, Chef-Ready panel (2‑pool) (Part No. A45617) and Oncomine™ Comprehensive Assay Plus, RNA, Chef-Ready panel (2‑pool) (Part No. A45618) at 2X concentration pre-measured in barcoded primer pool tubes ready to load into an Ion AmpliSeq™ Chef Reagents DL8 cartridge. In addition, the kit provides all the reagents and supplies in an Ion AmpliSeq™ Kit for Chef DL8 (Cat. No. A29024) sucient for preparing 32 samples.

Note: For detailed information on preparing Oncomine™ Comprehensive Assay Plus libraries on the Ion Chef™ System, see the Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide (Pub. No. MAN0013432).

Component Amount Storage

Oncomine™ Comprehensive Assay Plus, DNA, Chef Ready

2X DNA OCA Plus (pool 1 of 2) 4 × 150 µL –30°C to –10°C

2X DNA OCA Plus (pool 2 of 2) 4 × 150 µL

RMC 48 µL

Oncomine™ Comprehensive Assay Plus, RNA, Chef Ready

2X RNA OCA Plus (pool 1 of 2) 4 × 150 µL –30°C to –10°C

2X RNA OCA Plus (pool 2 of 2) 4 × 150 µL

Ion AmpliSeq™ Kit for Chef DL8

Ion AmpliSeq™ Kit for Chef DL8 (Part No. A29025) 2 × 4 cartridges –30°C to –10°C

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

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

•

Ion AmpliSeq™ Tip Cartridge L8

•

PCR Frame Seal

•

Enrichment Cartridge

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)

2 boxes with

4 inserts

2 sets of 4 plates 15°C to 30°C

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™ Comprehensive Assay Plus User Guide

Chapter 1 Product information

Contents and storage

Oncomine™ Comprehensive Assay Plus, RNA – Automated Library Preparation

The Oncomine™ Comprehensive Assay Plus, RNA – Automated Library Preparation (Cat. No. A49671) provides the Oncomine™ Comprehensive Assay Plus, RNA, Chef-Ready panel (2‑pool) (Part No. A45618) at 2X concentration pre-measured in barcoded primer pool tubes ready to load into an Ion AmpliSeq™ Chef Reagents DL8 cartridge. In addition, the kit provides all the reagents and supplies in an Ion AmpliSeq™ Kit for Chef DL8 (Cat. No. A29024) sucient for preparing 32 samples.

Component Amount Storage

Oncomine™ Comprehensive Assay Plus, RNA, Chef-Ready

2X RNA OCA Plus (pool 1 of 2) 4 × 150 µL –30°C to –10°C

2X RNA OCA Plus (pool 2 of 2) 4 × 150 µL

Ion AmpliSeq™ Kit for Chef DL8

Ion AmpliSeq™ Kit for Chef DL8 (Part No. A29025) 2 × 4 cartridges –30°C to –10°C

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

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

•

Ion AmpliSeq™ Tip Cartridge L8

•

PCR Frame Seal

•

Enrichment Cartridge

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]

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

2 boxes with

4 inserts

2 sets of 4 plates 15°C to 30°C

15°C to 30°C

[1]

Oncomine™ Comprehensive Assay Plus User Guide

Required materials not supplied

Unless otherwise indicated, all materials are available through thermoﬁsher.com. "MLS" indicates that the material is available from ﬁsherscientiﬁc.com or another major laboratory supplier.

Item Source

Chapter 1

Required materials not supplied

Product information

One of the following:

IonCode™ Barcode Adapters 1–384 Kit

Ion Xpress™ Barcode Adapters Kit

Ion Library TaqMan™ Quantitation Kit and real-time PCR system, see

4468802 (A26121)

A29751

4474517

[1]

“Recommended materials” on page 14.

Agencourt™ AMPure™ XP Kit NC9959336, NC9933872

ﬁsherscientiﬁc.com

(RNA only) Ion Torrent™ NGS Reverse Transcription Kit A45003

(DNA only) Uracil-DNA Glycoslyase, heat-labile 78310100UN

One of the following thermal cyclers, or equivalent:

•

ProFlex™ 96‑well PCR System

•

Veriti™ 96‑Well Thermal Cycler

•

2720 Thermal Cycler

•

GeneAmp™ PCR System 9700 96-Well GeneAmp™ PCR System 9700 Dual 96-Well

[2]

MicroAmp™ Optical 96-Well Reaction Plate or

MicroAmp™ Optical 96‑Well Reaction Plate with Barcode

Various

N8010560 or

4306737

MicroAmp™ Fast Optical 96-Well Reaction Plate 4346907

MicroAmp™ Clear Adhesive Film 4306311

MicroAmp™ Optical Film Compression Pad 4312639

DynaMag™–96 Side Magnet or other plate magnet 12331D

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

Nuclease-free Water AM9932

Ethanol, Absolute, Molecular Biology Grade BP2818500

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

[1]

Various kits are available. For more information, see thermofisher.com.

[2]

Supported but no longer available for purchase.

Oncomine™ Comprehensive Assay Plus User Guide

ﬁsherscientiﬁc.com

Chapter 1 Product information

Recommended materials

Unless otherwise indicated, all materials are available through thermoﬁsher.com. "MLS" indicates that the material is available from ﬁsherscientiﬁc.com or another major laboratory supplier.

Item Source

Recommended additional equipment

One of the following Applied Biosystems™ real‑time PCR instruments:

•

7500 Real-Time PCR System

•

7900HT Fast Real‑Time PCR System

•

StepOne™ Real-Time PCR System

•

StepOnePlus™ Real-Time PCR System

•

ViiA™ 7 Real-Time PCR System

•

QuantStudio™ 3 Real-Time PCR System

•

QuantStudio™ 5 Real‑Time PCR System

•

QuantStudio™ 7 Flex Real-Time PCR System

•

QuantStudio™ 12K Flex Real–Time PCR System

96-well plate centrifuge MLS

Qubit™ 4 Fluorometer

Recommended for nucleic acid isolation

MagMAX™ FFPE DNA/RNA Ultra Kit A31881

Recommended for nucleic acid quantiﬁcation

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

[2]

[1]

Various

Q33238

Qubit™ RNA HS Assay Kit (RNA) Q32852, Q32855

TaqMan™ RNase P Detection Reagents Kit 4316831

[1]

Supported but no longer available for purchase.

[2]

Qubit™ 2.0 Fluorometer or later are supported.

Recommended controls

AcroMetrix™ Oncology Hotspot Control Thermo Fisher Scientiﬁc 969056

Seraseq™ Tri‑Level Tumor Mutation DNA Mix v2 HC Seracare 0710‑0097

ATCC cell lines with CNV www.atcc.org ATCC® CRL-2327

Cell- Ref™ FFPE Cell Slide - HCC- 2998 (5 slides) AccuRef ASO-1010

Cell- Ref™ FFPE Cell Slide - T-47D (5 slides) AccuRef ASO-1032

Vendor Part number

ATCC® CRL-2336

ATCC® CRL-5868D

Oncomine™ Comprehensive Assay Plus User Guide

Chapter 1 Product information

Recommended materials

(continued)

Recommended controls Vendor Part number

Cell- Ref™ FFPE Cell Slide - A549 (5 slides) AccuRef ASO-1001

Cell- Ref™ FFPE Cell Slide - SK-MEL-2 (5 slides) AccuRef ASO-1028

Cell- Ref™ FFPE Cell Slide - H2228 (5 slides) AccuRef ASO-1006

Seraseq™ gDNA TMB Mix Score 7 Seracare 0710-1326

Seraseq™ gDNA TMB Mix Score 9 Seracare 0710-1325

Seraseq™ gDNA TMB Mix Score 20 Seracare 0710-1324

Seraseq™ gDNA TMB Mix Score 26 Seracare 0710-1323

Seraseq™ Fusion RNA Mix v4

18 RNA fusions: RET, ROS1, EGFRvIII, EGFR, ALK, NTRK3, FGFR3, NTRK1, METex14, PPARG1, BRAF, ERG

Seraseq™ FFPE NTRK Fusion RNA Reference Material

Horizon™ ALK RET ROS RNA fusion

RNA fusions: EML4-ALK, CCDC6-RET, and SLC34A2-ROS1

CancerSeq™ Plus Paran Tissue Curl (5 curls)

Copy number variation (CNV): CCNE1 ,EGFR, ERBB2, GNAS, KRAS, RB1 (-)

Seracare 0710‑0497

Seracare 0710‑1031

Horizon HD784

BioChain T2235152-SC

Lot No. B906046

Oncomine™ Comprehensive Assay Plus User Guide

Procedural guidelines ................................................................. 16

■

Before each use of the kit ............................................................. 16

■

Guidelines for RNA isolation, quantiﬁcation, and input ..................................... 17

■

Guidelines for DNA isolation, quantiﬁcation, and input .................................... 17

■

Library preparation from genomic DNA or RNA ........................................... 18

■

Procedural guidelines

•

Minimize freeze-thaw cycles of Oncomine™ Comprehensive Assay Plus panels and the RMC by aliquoting into low bind tubes as needed for your experiments. Panels can be stored at 4°C for 1 year. Store RMC at –30°C to –10°C.

•

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

•

Use a calibrated thermal cycler.

•

Pipet viscous solutions, such as 5X Ion AmpliSeq™ HiFi Mix, FuPa Reagent, Switch Solution, DNA Ligase, and panels, slowly and ensure complete mixing by vortexing or pipetting up and down several times.

•

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

Before you begin

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 ice during procedure. All other components, including primer pools, can be thawed at room temperature. Gently vortex and centrifuge before use.

•

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

•

Bring the Agencourt™ AMPure™ XP Reagent to room temperature.

IMPORTANT! Do NOT substitute a Dynabeads

AMPure™ XP Reagent.

™

-based puriﬁcation reagent for the Agencourt

Oncomine™ Comprehensive Assay Plus User Guide

™

Chapter 2 Before you begin

Guidelines for RNA isolation, quantiﬁcation, and input

•

We recommend the MagMAX™ FFPE DNA/RNA Ultra Kit (Cat. No. A31881) for isolating RNA.

•

We recommend the Qubit™ RNA HS Assay Kit (Cat. No. Q32855) for quantifying RNA.

•

Treat total RNA with DNase before use.

•

We recommend using 20 ng of total RNA for reverse transcription. Increasing the amount of total RNA will usually result in higher quality libraries, especially when RNA quality or quantity is unknown. With high-quality, well-quantiﬁed samples, as little as 1 ng total RNA can be used.

•

In general, library yield from high quality RNA is greater than from degraded samples. Library yield is not indicative of sequencing performance.

Guidelines for DNA isolation, quantiﬁcation, and input

•

We recommend the MagMAX™ FFPE DNA/RNA Ultra Kit (Cat. No. A31881) for isolating DNA.

•

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 used for quantiﬁcation, particularly for FFPE DNA, and highly degraded DNA samples.

•

Quantiﬁcation methods such as spectrophotometry (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 of sample DNA, under-seeding of the target ampliﬁcation reaction, low library yields, and poor chip loading.

•

We recommend using 20 ng of DNA for manual library preparation and automated library preparation. Increasing the amount of DNA results in higher-quality libraries, especially when DNA quality or quantity is unknown.

™

Oncomine™ Comprehensive Assay Plus User Guide

DNA or cDNA

Barcode Adapters

Barcoded library

Primer pairs

Amplicons

Amplify targets

Partially digest amplicons

Ligate adapters

Quantify libraries

Combine libraries (optional)

RMC reagent addition

Isolate and quantify DNA

UDG treat FFPE DNA

Reverse transcribe RNA

RNA

Isolate and quantify RNA

Chapter 2 Before you begin

Library preparation from genomic DNA or RNA

Oncomine™ Comprehensive Assay Plus User Guide

Automated library preparation on the

Ion Chef™ System

This chapter describes library preparation using the following components:

•

Oncomine™ Comprehensive Assay Plus, DNA, Chef-Ready Library Preparation (Part No. A45617)

•

Oncomine™ Comprehensive Assay Plus, RNA, Chef-Ready Library Preparation (Part No. A45618)

•

Ion Torrent™ NGS Reverse Transcription Kit (Cat No. A45003)

•

Uracil-DNA Glycoslyase, heat-labile (Cat No. 78310100UN)

•

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

Create a sample set to prepare 2 pools of 4 libraries each

IMPORTANT! The Oncomine

OCA Plus Library prep Protocol that limits the number of libraries to 4 per pool in order to generate sucient read depth when sequencing.

Note:

preparation. You can import new samples into Torrent Suite™ Software with the Import Samples from File feature, using a CSV template ﬁle that is available in Torrent Suite™ Software to simplify the

process. During this process, you can also create a new Sample Set for the new samples.

In Torrent Suite™ Software 5.16 or later Sample Sets are required for automated library

In the Plan tab, click Samples, then click Import Samples from File. For more information, see the Torrent Suite™ Software online help.

™

Comprehensive Assay Plus requires selection the 2 Library Pools -

Oncomine™ Comprehensive Assay Plus User Guide

Chapter 3

Create a sample set to prepare 2 pools of 4 libraries each

Automated library preparation on the Ion Chef™ System

In section 1 of the Import Samples section, click Sample File Format to download a sample CSV template.

The sample ﬁle format CSV contains the version of the CSV ﬁle in the top row, and sample attributes in separate columns.

Fill out the template CSV ﬁle as completely as possible, then save it to the location of your choice.

Note:

Required columns include: Sample name, PCR Plate Position (A–H), Barcode Kit (IonCode

Barcodes 1–32), and Barcode. Recommended columns include: Sample ID, Gender, Type (sample type, such as self), Group

(number that indicates the sample is a single sample, pair or trio), DNA/RNA, Cancer Type, and Cellularity %.

When the CSV ﬁle is ﬁlled out and saved, click Select File, navigate to the completed CSV ﬁle, then click Open.

Click Add Sample Set, then enter or select the required information in each ﬁeld.

Enter a Sample Set Name.

Select the Group Type.

Select the Library Prep Type—AmpliSeq on Chef.

Select the Library Prep Kit—Ion AmpliSeq Kit for Chef DL8.

Oncomine™ Comprehensive Assay Plus User Guide

Chapter 3

Select the Library Prep Protocol—2 Library Pools - OCA Plus.

Automated library preparation on the Ion Chef™ System

Automated RNA library preparation

Note: Completed libraries will be delivered to uncapped library Recovery Tubes in Position C (samples A–D) and Position D (samples E–H) in the Ion AmpliSeq™ Chef Reagents DL8 cartridge. Save the caps. To run 8 RNA samples simultaneously on the same chip do not select a Library Prep Protocol. All 8 sample libraries are combined in a single Recovery Tube v2 in Position D of the Ion AmpliSeq™ Chef Reagents DL8 cartridge.

Click Save & Finish. The software automatically imports the samples into the Sample Sets table.

Saved sample sets that enable 2 library pools for OCA Plus can then be selected on the Ion Chef Instrument user interface when setting up Ion AmpliSeq™ Kit for Chef DL8 library preparation runs.

Automated RNA library preparation

Reverse transcribe RNA for Chef Ready library preparation

If you are starting from RNA, you must ﬁrst reverse transcribe RNA to cDNA.

Remove and discard the plate seal from an IonCode™ 96‑well PCR Plate.

For each sample, add the following components into a single well in column 1 of the IonCode 96‑well plate (provided in the Ion AmpliSeq™ Kit for Chef DL8). Prepare a master mix without sample RNA for multiple reactions.

Component

Ion Torrent™ NGS 5X Reaction Buer 2 µL

Ion Torrent™ NGS 10X RT Enzyme Mix 1 µL

Total RNA (20 ng)

Nuclease-free Water to 10 µL

[1]

™

Volume

≤7 µL

Total volume per well 10 µL

[1]

If preparing an RNA positive control sample along with high quality RNA samples, use 20 ng positive control sample input. If preparing an RNA positive control sample along with FFPE RNA samples, reduce the positive control sample input to 2 ng. Substitute an equal volume of nuclease-free water or low TE to prepare a no-template control (NTC).

Oncomine™ Comprehensive Assay Plus User Guide

1 2

4 5 6

8 9

101112

Chapter 3

Automated RNA library preparation

Automated library preparation on the Ion Chef™ System

Column 1 wells contains a 10 μL reverse transcription reaction, or control reaction.

(Optional) Positive control or Non template control (NTC)

Each column 6 well contains a dried-down IonCode™ Barcode Adapter. The lowest barcode number is in A6, and the highest is in H6. All appear light blue in the actual plates.

Note:

If you are processing fewer than 8 samples, it is preferable to add replicates or positive control

samples to the run. Otherwise, pipet 15 µL of Nuclease-free Water as non-template control into column 1 wells that do not contain an RNA sample and balance the number of positive samples between rows A–D and E–H. We recommend processing at least 6 samples per run. If processing 5 or fewer samples, we

recommend that you quantify the output combined library by qPCR to ensure that an optimal concentration is used in templating reactions. If processing RNA samples that are to be combined with a paired DNA library ensure the

samples are processed in the correct rows A–D or E–H.

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

IMPORTANT! Oset the ﬁlm to the left so that the adhesive does not cover the barcode label. If

the barcode label becomes damaged, you can override the error during Deck Scan.

Place a MicroAmp™ Compression Pad on the plate, load the plate in the thermal cycler, then run the following program to synthesize cDNA.

Temperature

Time

25°C 10 minutes

50°C 10 minutes

85°C 5 minutes

10°C Hold

STOPPING POINT Samples can be stored at 10°C for up to 16 hours in the thermal cycler. For

longer term, store at −20°C.

Brieﬂy centrifuge the plate to collect any droplets at the bottom of the wells.

Oncomine™ Comprehensive Assay Plus User Guide

Chapter 3 Automated library preparation on the Ion Chef™ System

Automated RNA library preparation

Pipet 5 µL of nuclease-free water into each cDNA synthesis reaction in column 1 of the IonCode 96‑well plate.

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

IMPORTANT! Oset the ﬁlm to the left so that the adhesive does not cover the barcode label. If

the barcode label becomes damaged, you can override the error during Deck Scan.

Following completion of cDNA synthesis see "Thaw the reagents and prepare the instrument" in the Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide (Pub. No. MAN0013432) for instructions to prepare Oncomine™ Comprehensive Assay Plus libraries on the Ion Chef™ System.

For information on how to set up the Ion Chef™ Instrument, see “Ion Chef™ Instrument setup information for automated RNA library preparation” on page 23.

Ion Chef™ Instrument setup information for automated RNA library preparation

See the Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide (Pub. No. MAN0013432) for detailed information on preparing Oncomine™ Comprehensive Assay Plus libraries on the Ion Chef™ System.

™

IMPORTANT! When starting the library preparation run on the Ion Chef

correct Kit Type and Sample set are selected, and that 2 Library Pools - OCA Plus is displayed in order to properly prepare Oncomine™ Comprehensive Assay Plus–automated libraries. If 2 Library Pools

- OCA Plus is not displayed the default library preparation script is run which results in all 8 libraries combined into a single pool. The 2 Library Pools - OCA Plus library preparation script is only available in Torrent Suite™ Software 5.16 or later.

Figure 1 Example of a correct Oncomine™ Comprehensive Assay Plus setup

Ensure that 2 Library Pools - OCA Plus appears below the Sample set dropdown list.

™

Instrument ensure that the

Oncomine™ Comprehensive Assay Plus User Guide

Chapter 3

Automated DNA library preparation

Automated library preparation on the Ion Chef™ System

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

Stating material # of primer pools

High quality RNA

FFPE RNA

[1]

Due to the disparity in the required number of target amplification cycles for high quality and FFPE RNA we do NOT recommend running both high quality and FFPE samples on the same plate using the same input amount. If preparing a positive control (high quality) along with FFPE RNA samples, reduce the positive control sample input to 2 ng and use the FFPE cycling parameters.

[1]

2 23 4 minutes

2 29 4 minutes

Target ampliﬁcation

Automated DNA library preparation

RMC in DNA target ampliﬁcation reactions

Oncomine™ Comprehensive Assay Plus has been developed to support a wide range of biomarkers, including assessment of microsatellite instability (MSI). MSI arises from defects in the mismatch repair (MMR) system and is associated with hypermutability of short DNA sequence repeats (microsatellite locations) throughout the genome.

RMC is composed of in-sample standards that function as internal references in the analysis pipeline to ensure the robustness of MSI assessment in case of variations in sample preparation or run conditions. RMC is added to the DNA target ampliﬁcation reaction.

cycles

Anneal & extension time

Remove deaminated bases from FFPE DNA

Sample age, storage conditions, and FFPE preservation methods can lead to signiﬁcant cytosine deamination of the isolated DNA. This deamination can result in an artiﬁcially high deamination score when determining the tumor mutational burden result. We have demonstrated that deaminated cytosine (uracil) bases can be enzymatically removed by treatment with Uracil DNA Glycosylase (UDG).

Note: We recommend treating all samples including FFPE and high quality (for example, commercial controls or DNA isiolated from cell lines) DNA with UDG to remove deaminated bases before target

ampliﬁcation.

Remove and discard the plate seal from an IonCode™ Barcode Adapters 96‑well PCR plate.

For each FFPE DNA sample, add the following components to a single well in column 1 of the IonCode™ Barcode Adapters 96‑well PCR plate.

Component

20 ng FFPE DNA

Uracil-DNA Glycoslyase, heat-labile 1 µL

Low TE to 10 µL

[1]

Substitute an equal volume of nuclease-free water or low TE to prepare a no-template control (NTC).

[1]

Volume

≤9 µL

Oncomine™ Comprehensive Assay Plus User Guide

1 2

4 5 6

8 9

101112

Chapter 3 Automated library preparation on the Ion Chef™ System

Automated DNA library preparation

Column 1 wells contains 20 ng of FFPE DNA sample in 10 µL, or 10 µL Nuclease-free Water as non-template control. Samples A–D delivered to uncapped library Recovery Tube in Position C in the Ion AmpliSeq™ Chef Reagents DL8 cartridge.

Column 1 wells contains 20 ng of FFPE DNA sample in 10 µL, or 10 µL Nuclease-free Water as non-template control. Samples E–H delivered to uncapped library Recovery Tube in Position D in the Ion AmpliSeq™ Chef Reagents DL8 cartridge.

(Optional) Non template control (NTC)

Each column 6 well contains a dried-down IonCode™ Barcode Adapter. The lowest barcode number is in A6, and the highest is in H6. All appear light blue in the actual plates.

Note:

If you are processing fewer than 8 samples, it is preferable to add replicates or positive control

samples to the run. Otherwise, pipet 10 µL of Nuclease-free Water as non-template control into column 1 wells that do not contain a DNA sample and balance the number of positive samples between rows A–D and E–H. We recommend processing at least 6 samples per run. We do not recommend processing

1, 2 or 5 samples per run. If you do process 5 or fewer samples, we recommend that you quantify the output combined library by qPCR to ensure that an optimal concentration is used in templating reactions. If processing only 3 or 4 samples, group them together either in rows A–D or E–H.

Mix the reaction by pipetting at least half the total volume up and down at least 5 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 to collect the contents.

IMPORTANT! To prevent evaporation during UDG treatment, use an applicator tool to press the

ﬁlm securely around each reaction well and around the perimeter of the plate. Oset the ﬁlm to the left so that the adhesive does not cover the barcode label.

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

Temperature

Time

37°C 2 minutes

50°C 10 minutes

Oncomine™ Comprehensive Assay Plus User Guide

4°C Hold (≤1 hour)

Chapter 3 Automated library preparation on the Ion Chef™ System

Automated DNA library preparation

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

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

Carefully remove the plate seal, then add the following components to each well.

Note: If processing multiple samples, prepare a reaction master mix (+ 5–10% overage), then add 15 μL to each well.

Component Volume

RMC 1.5 µL

Nuclease-free Water 3.5 µL

Total volume per well 15 µL

Mix the reaction by pipetting at least half the total volume up and down at least 5 times, then carefully inspect each well for air bubbles. Remove any air bubbles by gentle pipetting. . Alternatively, seal the plate with MicroAmp™ Clear Adhesive Film, vortex for 5 seconds to mix the reactions, then centrifuge brieﬂy to collect the contents.

IMPORTANT! Oset the ﬁlm to the left so that the adhesive does not cover the barcode label.

If the barcode label becomes damaged, you can override the error during Deck Scan on the Ion Chef™ Instrument.

Proceed to "Thaw the reagents and prepare the instrument" in the Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide (Pub. No. MAN0013432) for instructions to prepare Oncomine

™

Comprehensive Assay Plus libraries on the Ion Chef™ System.

For information on how to set up the Ion Chef™ Instrument, see “Ion Chef™ Instrument setup information for automated DNA library preparation” on page 27.

Oncomine™ Comprehensive Assay Plus User Guide

Chapter 3 Automated library preparation on the Ion Chef™ System

Automated DNA library preparation

Ion Chef™ Instrument setup information for automated DNA library preparation

IMPORTANT! When starting the library preparation run on the Ion Chef

™

Instrument ensure that the correct Kit Type and Sample set are selected, and that 2 Library Pools - OCA Plus is displayed in order to properly prepare Oncomine™ Comprehensive Assay Plus–Chef Ready libraries. If 2 Library Pools OCA Plus is not displayed the default library preparation script is run which results in all 8 libraries combined into a single pool. This exceeds the capacity of the Ion 550™ Chip. The 2 Library Pools - OCA Plus library preparation script is only available in Torrent Suite™ Software 5.16 or later.

Figure 2 Example of a correct Oncomine™ Comprehensive Assay Plus setup

Ensure that 2 Library Pools - OCA Plus appears below the Sample set dropdown list.

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

Stating material

High quality DNA

FFPE DNA

[1]

Due to the disparity in the required number of target amplification cycles for high quality and FFPE DNA we do NOT recommend running both high quality and FFPE samples on the same plate using the same input amount.

Oncomine™ Comprehensive Assay Plus User Guide

[1]

# of primer pools

Target ampliﬁcation

cycles

2 13 16 minutes

2 16 16 minutes

Anneal & extension time

Manual library preparation

RNA preparation and cDNA ampliﬁcation ................................................ 28

■

DNA preparation and ampliﬁcation ...................................................... 33

■

Library preparation ................................................................... 36

■

Quantify the library by qPCR and calculate the dilution factor .............................. 41

■

Combine libraries ..................................................................... 43

■

Guidelines for templating and sequencing ............................................... 46

■

This chapter describes library preparation using the following kits:

•

Oncomine™ Comprehensive Assay Plus, DNA, Manual library preparation (Part No. A45615)

•

Oncomine™ Comprehensive Assay Plus, RNA, Manual library preparation (Part No. A45616)

•

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

•

Ion Torrent™ NGS Reverse Transcription Kit (Cat No. A45003)

•

Uracil-DNA Glycoslyase, heat-labile (Cat No. 78310100UN)

RNA preparation and cDNA ampliﬁcation

Use the components of the Oncomine™ Comprehensive Assay Plus, RNA (Cat. No. A45616) for the follow procedures.

Reverse transcribe RNA for manual library preparation

If the RNA was prepared from FFPE tissue and not previously heat-treated, heat at 80°C for 10 minutes, then cool to room temperature.

For each sample, add the following components into a single well of a 96-well PCR plate on ice or in a pre-chilled 4°C cold block. Prepare a master mix without sample RNA for multiple reactions.

Component

Ion Torrent™ NGS 5X Reaction Buer 2 µL

Ion Torrent™ NGS 10X RT Enzyme Mix 1 µL

Total RNA (20 ng)

Nuclease-free Water to 10 µL

Total volume per well 10 µL

[1]

Substitute an equal volume of nuclease-free water or low TE to prepare a no-template control (NTC).

[1]

Volume

≤7 µL

Oncomine™ Comprehensive Assay Plus User Guide

RNA plate

1 2

4 5 6 7 8 9 101112

Chapter 4 Manual library preparation

RNA preparation and cDNA ampliﬁcation

(Optional) Non template control (NTC)

RNA sample

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

Place a MicroAmp™ Optical Film Compression Pad on the plate, load the plate in the thermal cycler, then run the following program to synthesize cDNA.

Temperature

25°C 10 minutes

50°C 10 minutes

85°C 5 minutes

10°C Hold

STOPPING POINT Samples can be stored at 10°C for up to 16 hours in the thermal cycler. For

longer term, store at −20°C.

Brieﬂy centrifuge the plate to collect any droplets at the bottom of the wells, then proceed to the next step.

Prepare cDNA target ampliﬁcation reactions

IMPORTANT! The cDNA synthesis reaction, primer pools, and 5X Ion AmpliSeq

Pipet slowly and mix thoroughly.

Place the 96‑well plate in a pre-chilled cold block or on ice.

Thaw the 5X Ion AmpliSeq™ HiFi Mix on ice, gently vortex to mix, then brieﬂy centrifuge to collect.

Time

™

HiFi Mix are viscous.

Oncomine™ Comprehensive Assay Plus User Guide

RNA plate

1 2

4 5 6 7 8 9 101112

Chapter 4 Manual library preparation

RNA preparation and cDNA ampliﬁcation

To each cDNA synthesis reaction add:

cDNA synthesis reaction 10 µL

5X Ion AmpliSeq™ HiFi Mix (red cap) 4.5 µL

Nuclease-free Water 3.5 µL

Final volume 18 µL

Mix by pipetting at least half the total volume up and down at least 5 times, then transfer 8 µL to each of two adjacent wells (~2 µL overage remainder).

Component Volume

cDNA sample Non template control (NTC)

8 µL transferred cDNA target ampliﬁcation reaction.

Add 2 µL of 5X Oncomine™ Comprehensive Assay Plus, RNA primer pool‑1 into the ﬁrst well, then

~2 µL cDNA target ampliﬁcation reaction remaining.

add 2 µL of primer pool‑2 into the second well for a total of 10 µL in each well.

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

Proceed to “Amplify the cDNA targets” on page 31 .

Oncomine™ Comprehensive Assay Plus User Guide

Amplify the cDNA targets

IMPORTANT! When amplifying multiple samples in a single PCR plate, ensure that the input across all

samples is roughly equivalent so that the selected cycle number is optimal for all the samples in the run.

Place a MicroAmp™ Optical Film Compression Pad on the plate, then load the plate into the thermal cycler.

Run the following program to amplify the target regions.

Stage Step Temperature Time

Hold Activate the enzyme 99°C 2 min

Chapter 4 Manual library preparation

RNA preparation and cDNA ampliﬁcation

Cycle; set number according to Table 1

Denature 98°C 15 sec

Anneal and extend 60°C 4 min (RNA Panel)

Hold — 10°C Hold

Table 1 Recommended cycle number

Input nucleic acid

Recommended number

[1]

of cycles

[2]

Cycle number adjustment

[3]

10 ng RNA input 1 ng RNA input 100 ng RNA input

High quality RNA 22 +5 –3

FFPE RNA 28 +3 –3

[1]

[2]

Number of cycles can be increased when input material quality or quantity is questionable.

[3]

The recommended number of cycles is based on 10 ng RNA input per primer pool. Adjust the cycle number for lower or higher RNA input.

STOPPING POINT

Target ampliﬁcation reactions can be stored at 10°C overnight on the thermal

cycler. For longer periods, store at −20°C.

Oncomine™ Comprehensive Assay Plus User Guide

RNA plate

1 2

4 5 6 7 8 9 101112

Sample

NTC

Chapter 4 Manual library preparation

RNA preparation and cDNA ampliﬁcation

Combine cDNA target ampliﬁcation reactions

Note: If preparing both RNA and DNA sample libraries use the same FuPa digestion conditions,

samples may be combined onto a single plate for simultaneous processing.

Tap the plate gently on a hard ﬂat surface, or centrifuge brieﬂy to collect the contents at the bottom of the wells.

Carefully remove the plate seal.

For each sample, combine the 10-µL target ampliﬁcation reactions. The total volume for each sample should be 20 µL.

Oncomine™ Comprehensive Assay Plus User Guide

DNA preparation and ampliﬁcation

Use the components of the Oncomine™ Comprehensive Assay Plus, DNA, Manual Library Preparation (Cat. No. A47620) for the following procedures.

RMC in DNA target ampliﬁcation reactions

Remove deaminated bases from FFPE DNA

Chapter 4

Manual library preparation

DNA preparation and ampliﬁcation

Note: We recommend treating all samples including FFPE and high quality (for example, commercial controls or DNA isiolated from cell lines) DNA with UDG to remove deaminated bases before target

ampliﬁcation.

For each FFPE DNA sample, add the following components to a single well of a 96‑well PCR plate.

Component

20 ng FFPE DNA

Uracil-DNA Glycoslyase, heat-labile 1 µL

Low TE to 6.5 µL

[1]

Do not exceed 40 ng maximum FFPE DNA sample as input. Substitute an equal volume of nuclease-free water or low TE to prepare a no-template control (NTC).

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

[1]

Volume

≤5.5 µL

Oncomine™ Comprehensive Assay Plus User Guide

5 μL

2X primer

pool 1

Master

Mix

5 μL

2X primer

pool 2

Sample

Chapter 4

DNA preparation and ampliﬁcation

Manual library preparation

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

Temperature Time

37°C 2 minutes

50°C 10 minutes

4°C Hold (≤1 hour)

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

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

Carefully remove the plate seal, then proceed immediately to “Prepare DNA target ampliﬁcation reactions” on page 34, adding the target ampliﬁcation reaction components to the well containing

6.5 µL of UDG treated FFPE DNA.

Prepare DNA target ampliﬁcation reactions

IMPORTANT! Primer pools and 5X Ion AmpliSeq

thoroughly.

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

For each sample, prepare a target ampliﬁcation master mix without primers in a 1.5‑mL tube on ice.

Component

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

DNA (20 ng) (treated with UDG) 6.5 µL

RMC 1 µL

Nuclease-free Water to 12.5 µL

Mix thoroughly by pipetting up and down 5 times, then transfer 5 µL of each sample-speciﬁc master mix to 2 wells of a 96‑well PCR plate on ice or in a pre-chilled 4°C cold block.

Note: When using multi block thermal cyclers ensure each pair of samples is in the same temperature zone for ampliﬁcation. For example, use the same VeriFlex block on a Veriti™ Thermal Cycler.

Add 5 µL of 2X Oncomine™ Comprehensive Assay Plus, DNA primer pool 1 to the ﬁrst well, and 5 µL of primer pool 2 to the second well.

™

HiFi Mix are viscous. Pipet slowly and mix

Volume

Oncomine™ Comprehensive Assay Plus User Guide

Seal the plate with MicroAmp™ Clear Adhesive Film.

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 least 5 times before sealing the plate.

Proceed to “Amplify the DNA targets” .

Amplify the DNA targets

IMPORTANT! When amplifying multiple samples in a single PCR plate, make sure that the input

across all samples is roughly equivalent so that the selected cycle number is optimal for all the samples in the run.

Place a MicroAmp™ Optical Film Compression Pad on the plate, then load the plate into the thermal cycler.

Run the following program to amplify the target regions.

Chapter 4 Manual library preparation

DNA preparation and ampliﬁcation

Stage

Step Temperature Time

Hold Activate the enzyme 99°C 2 min

Cycle; set number according to Table 2

Denature 99°C 15 sec

Anneal and extend 60°C 16 min (DNA Panel)

Hold — 10°C Hold

Table 2 Recommended cycle number

Input nucleic acid

Recommended number

[1]

of cycles

[2]

Cycle number adjustment

[3]

10 ng DNA input 1 ng DNA input 20 ng DNA input

High quality DNA 12 +3 –1

FFPE DNA 15 +3 –1

[1]

If both high quality and FFPE nucleic acids are being used in the same reaction, use the FFPE parameters.

[2]

Number of cycles can be increased when input material quality or quantity is questionable.

[3]

The recommended number of cycles is based on 10 ng DNA input per primer pool. Adjust the cycle number for lower or higher DNA input.

STOPPING POINT

Target ampliﬁcation reactions may be stored at 10°C overnight on the thermal

cycler. For longer periods, store at −20°C.

Oncomine™ Comprehensive Assay Plus User Guide

1 2

4 5 6 7 8 9 101112

DNA plate

Sample

NTC

Chapter 4 Manual library preparation

Library preparation

Combine the DNA target ampliﬁcation reactions

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

Remove the DNA plate from the thermal cycler, then centrifuge brieﬂy to collect the contents.

Carefully remove the plate seal.

For each sample, combine both 10-µL DNA target ampliﬁcation reactions into a single well.

IMPORTANT! Accurate volume transfer in this step is critical. We recommend using a single-

channel pipettor. If you are using a multi-channel pipettor, visually check pipette tips to ensure that volumes are equivalent.

Note: In multi-zone thermal cyclers ensure samples are in the same zone for ampliﬁcation.

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

Library preparation

Transfer the DNA amplicons

If preparing both DNA and cDNA libraries with the same FuPa digestion conditions, library preparations can be transferred to a single plate for simultaneous processing. For more information, see “Partially digest the DNA and cDNA amplicons” on page 37.

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

Carefully remove the adhesive ﬁlm from the plate.

IMPORTANT! Be careful when removing the ﬁlm to minimize contamination.

Oncomine™ Comprehensive Assay Plus User Guide

Transfer DNA to RNA plate

RNA plate

1 2

4 5 6 7 8 9 101112

RNA plate

1 2

4 5 6 7 8 9 101112

1 2 3

4 5

6 7 8

9 10

DNA plate

Chapter 4 Manual library preparation

Library preparation

Transfer the amplicons from the DNA plate to the corresponding empty wells of the RNA/cDNA plate.

Sample DNA target ampliﬁcation reactions

Sample cDNA(RNA) target ampliﬁcation reactions

No template control (NTC) target ampliﬁcation reaction

Partially digest the DNA and cDNA amplicons

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

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

Add 2 µL of FuPa Reagent to each ampliﬁed DNA or cDNA sample. The total volume per well is ~22 µL.

Seal each DNA or cDNA plate with a clear adhesive ﬁlm, vortex thoroughly, then centrifuge brieﬂy to collect droplets. Alternatively, mix by pipetting at least half the total volume up and down at least 5 times before sealing the plate.

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

Temperature

50°C 20 min (cDNA/DNA)

55°C 20 min (cDNA/DNA)

60°C 20 min (cDNA/DNA)

10°C Hold (for up to 1 hour)

Time

Oncomine™ Comprehensive Assay Plus User Guide

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

Chapter 4 Manual library preparation

Library preparation

Ligate adapters to the amplicons and purify

When sequencing multiple libraries on a single run, you must ligate a dierent barcode to each library. DNA and RNA libraries from the same sample also require dierent barcodes.

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

Ion Xpress™ Barcode Adapters require handling and dilution as described in “Ion Xpress™ Barcode Adapters only: Combine and dilute adapters”.

IMPORTANT! When handling barcoded adapters, be careful to avoid cross contamination by changing

gloves frequently and opening one tube at a time.

Ion Xpress™ Barcode Adapters only: Combine and dilute adapters

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. Scale volumes as necessary. Use 2 μL of this barcode adapter mix in step 3 in “Perform the ligation reaction”.

For example, combine the volumes indicated in the following table.

Component

Ion P1 Adapter 2 µL

Ion Xpress™ Barcode X

Nuclease-free Water 4 µL

Total 8 µL

[1]

X = barcode chosen

Note: Store diluted adapters at –20°C.

[1]

Volume

2 µL

Perform the ligation reaction

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.

If you have not already done so, brieﬂy centrifuge the plate to collect the contents, then carefully remove the plate seal.

Oncomine™ Comprehensive Assay Plus User Guide

Chapter 4

Add the following components in the order listed to each well containing digested amplicons. If

Manual library preparation

Library preparation

preparing multiple non-barcoded libraries, a master mix of Switch Solution and adapters can be combined before addition.

IMPORTANT! Add the DNA Ligase last. Do not combine DNA Ligase and adapters before adding

to digested amplicons.

Order of addition

1 Switch Solution (yellow cap) 4 µL

2 Adapters (IonCode™ Barcode Adapters or diluted Ion Xpress

barcode adapter mix (for barcoded libraries))

3 DNA Ligase (blue cap) 2 µL

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

Seal the plate with a new MicroAmp™ Clear Adhesive Film, vortex thoroughly, then brieﬂy

Component Volume

™

centrifuge to collect droplets. Alternatively, mix by pipetting at least half the total volume up and down at least 5 times before sealing the plate.

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

Temperature

22°C 30 minutes

68°C 5 minutes

72°C 5 minutes

10°C Hold (for up to 24 hours)

Time

2 µL

STOPPING POINT Samples can be stored for up to 24 hours at 10°C on the thermal cycler. For

longer periods, store at –20°C.

Purify the unampliﬁed library

IMPORTANT! Bring the Agencourt

to disperse the beads before use. Pipet the solution slowly.

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

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

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

Incubate the mixture for 5 minutes at room temperature.

Oncomine™ Comprehensive Assay Plus User Guide

™

AMPure™ XP Reagent to room temperature and vortex thoroughly

Chapter 4 Manual library preparation

Library preparation

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

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

Note: If your magnet does not have two positions for shifting the beads, remove the plate from the magnet and gently pipet up and down 5 times (with the pipettor set at 100 μL), then return the plate to the magnet and incubate for 2 minutes or until the solution clears.

Repeat step 5 for a second wash.

Ensure that all ethanol droplets are removed from the wells. Keeping the plate in the magnet, air-dry the beads at room temperature for 5 minutes. Do not overdry.

IMPORTANT! Residual ethanol drops inhibit library ampliﬁcation. If needed, centrifuge the plate

and remove remaining ethanol before air-drying the beads.

Elute and dilute the library

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

Incubate at room temperature for at least 2 minutes.

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

STOPPING POINT

at –20°C. We recommend transferring the supernatant to a 1.5-mL Eppendorf LoBind™ tube for long-term storage.

Prepare a 100-fold dilution for quantiﬁcation. Remove 2 μL of supernatant, containing the library, then combine with 198 μL of Nuclease-free Water.

Proceed immediately to “Quantify the library by qPCR and calculate the dilution factor” on page 41.

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

Oncomine™ Comprehensive Assay Plus User Guide

Chapter 4

Quantify the library by qPCR and calculate the dilution factor

Manual library preparation

Quantify the library by qPCR and calculate the dilution factor

Determine the concentration of each library by qPCR with the Ion Library TaqMan™ Quantitation Kit (Cat. No. 4468802) using the following steps. Analyze each sample, standard, and negative control in duplicate 20‑μL reactions.

IMPORTANT! The following steps dier from those in the Ion AmpliSeq

™

Library Kit Plus User Guide.

Follow the steps below for quantifying your Oncomine™ Comprehensive Assay Plus libraries.

Prepare three 10-fold serial dilutions of the E. coli DH10B Ion Control Library (~68 pM, from the 2) at 6.8 pM, 0.68 pM, and 0.068 pM. Mark these tubes as standards, then use these concentrations in the qPCR instrument software.

Use the volumes per reaction in the following table to prepare a PCR Reaction Mix for all reactions. We recommend duplicate reactions for each sample library, standard, and NTC. Include a 5–10% overage to accommodate pipetting errors.

Note: Volumes are provided per reaction. Double the volume for duplicate reactions.

Component

2X Ion Library qPCR Master Mix 10 µL 5 µL

Ion Library TaqMan™ Quantitation Assay, 20X 1 µL 0.5 µL

Total 11 µL 5.5 µL

In a MicroAmp™ Optical Reaction Plate , set up duplicate PCR reactions for each sample,

96-well plate 384-well plate

Volume per reaction

standard, and NTC. To each well, add the following components:

Component

PCR Reaction Mix (from step 2) 11 µL 5.5 µL

1:100 dilution of the sample

[1]

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

[2]

High quality samples may require further dilution (5–10 fold) to give results within the range of the prepared standards. We recommend starting with 1000X dilution for RNA library quantification.

Seal the plate with a MicroAmp™ Optical Adhesive Film, vortex thoroughly, then brieﬂy centrifuge

[1,2]

to collect droplets.

Program your real-time instrument as follows:

Note: The fast cycling program was developed using the StepOnePlus™ Real-Time PCR System in Fast mode.

Enter the concentrations of the control library standards.

Oncomine™ Comprehensive Assay Plus User Guide

Volume per reaction

96-well plate 384-well plate

9 µL 4.5 µL

Chapter 4 Manual library preparation

Quantify the library by qPCR and calculate the dilution factor

Select ROX™ Reference Dye as the passive reference dye.

Select a reaction volume of 20 μL (96‑well plate) or 10 μL (384‑well plate).

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

Reaction plate format Run mode Stage Temperature Time

Hold (UDG incubation) 50°C 2 min

96-well Standard

384-well Standard

48- / 96-well Fast

384-well Standard

Following qPCR, calculate the average concentration of the undiluted library by multiplying the

Standard

Fast

Hold (polymerase

activation)

Cycle (40 cycles)

Hold (UDG incubation) 50°C 2 min

Hold (polymerase

activation)

Cycle (40 cycles)

95°C 2 min

95°C 15 sec

60°C 1 min

95°C 20 sec

95°C 1 sec

60°C 20 sec

determined concentration × 100.

Based on the calculated library concentration, determine the dilution that results in a concentration of ~50 pM for template preparation on the Ion Chef™ System.

For example:

•

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.

Note:

Good results have been observed with libraries ≤50 pM. Proceed to the next step without further

dilution. Libraries that yield signiﬁcantly less than 50 pM can be rescued with library ampliﬁcation. See

“Tips” on page 87.

Proceed to “Combine libraries” then template preparation, or store libraries as described below.

STOPPING POINT

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 an RNase‑free microcentrifuge tube for long-term storage. Alternatively, seal the plate with a new MicroAmp™ Clear Adhesive Film for long-term storage.

Oncomine™ Comprehensive Assay Plus User Guide

Combine libraries

% Reads, % Volume

of Combined Library

When comparing genomic DNA and RNA libraries that are prepared from the same sample, unequal volumes of libraries can be combined to produce dierent read depths for the paired DNA and RNA libraries.

•

Combine DNA and RNA library pools from the same samples prepared on an Ion Chef™ Instrument.

Note: Upon completion of the Ion Chef™ Instrument run libraries have already been adjusted to a ﬁnal concentration of ~100 pM each and pooled on the Ion Chef™ Instrument. Libraries from samples A–D are in the Recovery Tube in Position C and samples E–H in Position D of the Ion AmpliSeq™ Chef Reagents DL8 cartridge.

Combine the DNA library pool with the RNA library pool for a given set of 4 samples at a 90:10 ratio (DNA:RNA—18 µL of DNA library pool + 2 µL of RNA library pool).

Note: This ratio diers from previous Oncomine™ Comprehensive Assay protocols.

IMPORTANT!

The combined DNA and RNA library pools must be from matched Recovery Tubes. For

example, combine Position C libraries, do NOT combine sample DNA libraries from Position C (samples A–D) with sample RNA libraries from Position D (samples E–F) or vice versa. Each combined library must have a unique barcode.

Chapter 4 Manual library preparation

Combine libraries

Dilute the combined (90:10 DNA:RNA) pooled libraries 2‑fold (~50 pM ﬁnal concentration).

Note: We recommend sequencing up to 4 research samples (one library pool, either samples A–D or E–H) on a single Ion 550™ Chip.

DNA library pool

RNA library pool

Library pool Sample Barcode Fractional volume (90:10 DNA:RNA)

DNA–C DNA-1 BC_0101 0.225 0.9

DNA-2 BC_0102 0.225

DNA-3 BC_0103 0.225

DNA-4 BC_0104 0.225

RNA–C RNA-1 BC_0109 0.025 0.1

RNA-2 BC_0110 0.025

Oncomine™ Comprehensive Assay Plus User Guide

% Reads, % Volume

of Combined Library

Sample 1

4.0

6.0

8.0

10.0

2.0

% Volume of

Combined Library

NTC

4.0

6.0

8.0

10.0

2.0

DNA RNA

Chapter 4 Manual library preparation

Combine libraries

(continued)

Library pool Sample Barcode Fractional volume (90:10 DNA:RNA)

RNA–C RNA-3 0.1BC_0111 0.025

Sum — — 1.0 1.0

•

Combine manually prepared DNA and RNA libraries.

Combine each uniquely barcoded DNA and RNA library (~50 pM each) from the same sample at an 90:10 ratio (DNA:RNA—18 µL of DNA library + 2 µL of RNA library).

Note: This ratio diers from previous Oncomine™ Comprehensive Assay protocols.

Combine equal volumes of the paired libraries (90:10 DNA:RNA) to be sequenced on the same chip.

Note:

We recommend sequencing up to 5 samples (4 research samples plus 1 no-template

control) on a single Ion 550™ Chip. For runs that include a no-template control (NTC), add in the same fractional volumes of

undiluted DNA and RNA NTC libraries as is added for equivalent sample libraries.

RNA-4 BC_0112 0.025

Sample

DNA-1 BC_0101 0.18

RNA-1 BC_0102 0.02

DNA-2 BC_0103 0.18

RNA-2 BC_0104 0.02

DNA-3 BC_0105 0.18

RNA-3 BC_0106 0.02

DNA-4 BC_0107 0.18

RNA-4 BC_0108 0.02

Barcode Fractional volume (90:10 DNA:RNA)

Oncomine™ Comprehensive Assay Plus User Guide

Chapter 4 Manual library preparation

Combine libraries

(continued)

Sample Barcode Fractional volume (90:10 DNA:RNA)

DNA-5 (NTC) BC_0109 0.18

RNA-5 (NTC) BC_0110 0.02

Sum — 1.0

•

Combine only manually prepared RNA libraries to be sequenced on the same chip.

Note: We recommend sequencing up to 32 sample RNA libraries on a single Ion 550™ Chip.

Combine 2 μL of each uniquely barcoded RNA library (~50 pM each), then mix thoroughly.

•

Combine only RNA library pools prepared on an Ion Chef™ Instrument to be sequenced on the same chip.

Note:

When combining only RNA libraries, library pools in the Recovery Tube in Position C (samples

A–D) can be combined with library pools in the Recovery Tube in Position D (samples E–F). We recommend sequencing up to 32 sample RNA libraries on a single Ion 550™ Chip or Ion 540

Chip. We recommend sequencing up to 8 sample RNA libraries on a single Ion 530™ Chip.

™

Combine 5 μL from each of 4 pooled RNA libraries (~100 pM each).

Mix thoroughly, then dilute the combined library pools 2‑fold (~50 pM ﬁnal concentration).

•

Combine only manually prepared DNA libraries to be sequenced on the same chip.

Note: We recommend sequencing up to 4 sample DNA libraries on a single Ion 550™ Chip.

Combine 10 μL of each uniquely barcoded DNA library (~50 pM each).

•

Combine only DNA library pools prepared on an Ion Chef™ Instrument to be sequenced on the same chip.

Note: We recommend sequencing up to 4 sample DNA libraries on a single Ion 550™ Chip. Upon completion of the Ion Chef™ Instrument run each Recovery Tube contains 4 pooled libraries adjusted to a ﬁnal concentration of ~100 pM.

Dilute 15 μL of the pooled DNA libraries 2‑fold (~50 pM ﬁnal concentration), then mix thoroughly.

Oncomine™ Comprehensive Assay Plus User Guide

Chapter 4 Manual library preparation

Guidelines for templating and sequencing

Proceed to template preparation and sequencing using the following kits.

Chip

Ion 550™ Chip Ion Chef

Template

System

Sequencer Kit User Guide

™

Ion S5™ XL Sequencer, Ion GeneStudio™ S5 Plus Sequencer, or Ion GeneStudio™ S5 Prime Sequencer

Ion 550™ Kit − Chef (Cat. No. A34541)

Ion 550™ Single Chip Supplemental Kit (Cat. No. A36953)

Ion 550™ Kit − Chef User Guide

(Pub. No. MAN0017275)

To create a speciﬁc Run Plan for use in templating and sequencing, see Chapter 5, “Create a Planned Run”. Refer to the appropriate user guide listed in the table for more information.

Oncomine™ Comprehensive Assay Plus User Guide

Create a Planned Run

About Planned Runs .................................................................. 47

■

Update Oncomine™ Comprehensive Assay Plus templates in Torrent Suite™ Software ........ 48

■

Create a custom Planned Run template ................................................. 49

■

Create a Planned Run for manual library preparation ...................................... 51

■

Create a Planned Run for automated library preparation using sample sets .................. 53

■

IMPORTANT! This kit is compatible with Torrent Suite

Software 5.16 or later. Before proceeding, we recommend that you update to the latest available versions of Torrent Suite™, Ion Reporter™, and Ion Chef™ System software. Contact your service representative for assistance with upgrading the software.

Note: The IonReporterUploader 5.16 plugin must be installed in Torrent Suite™ Software. If your Ion Reporter™ account is not conﬁgured, conﬁgure it through Ion Reporter Conﬁgure settings (see “Conﬁgure the IonReporterUploader plugin in Torrent Suite™ Software” on page 92).

About Planned Runs

Planned Runs are digital instructions that are created in Torrent Suite™ Software for controlling the template preparation and sequencing instruments. Planned Runs contain settings such as number of ﬂows, kit types, barcodes, sample information, and reference ﬁles (if any). Planned Runs are also used to track samples, chips, and reagents throughout the workﬂow, from template preparation on the Ion Chef™ Instrument through sequencing on an Ion S5™ XL Sequencer, Ion GeneStudio™ S5 Plus Sequencer, or Ion GeneStudio™ S5 Prime Sequencer and subsequent data analysis. Each chip that is prepared in an Ion Chef™ run requires its own Planned Run.

IMPORTANT! For more information on creating a Planned Run in Torrent Suite

a complete description of each ﬁeld in the Create Plan workﬂow bar, see the Torrent Suite™ Software Help, available by clicking the Help button in the software.

™

Software 5.16 or later and Ion Reporter

™

Software, including

™

Oncomine™ Comprehensive Assay Plus User Guide

Chapter 5 Create a Planned Run

Update Oncomine™ Comprehensive Assay Plus templates in Torrent Suite™ Software

In Torrent Suite™ Software 5.16 or later, use the Oncomine™ Comprehensive Plus – DNA and Fusions template as the primary Planned Run template for the Oncomine™ Comprehensive Assay Plus.

Application category in

Torrent Suite™ Software

Oncology – Solid Tumor Oncomine™ Comprehensive Plus –

DNA and Fusions

Oncomine™ Comprehensive Plus – DNA

Oncomine™ Comprehensive Plus – Fusions

[1]

Available in Torrent Suite™ Software 5.16 or later.

Template name Description

DNA and RNA Planned Run template

DNA-only Planned Run template

RNA-only Planned Run template

[1]

Update Oncomine™ Comprehensive Assay Plus templates in Torrent Suite™ Software

In the upper right corner, click (Settings)4Updates, then scroll to the Update Products section.

In the Name column ﬁnd Oncomine™ Comprehensive Assay Plus, then in that row click Update.

The software update begins automatically and displays as Complete when ﬁnished.

Oncomine™ Comprehensive Assay Plus User Guide

Create a custom Planned Run template

Chapter 5

Create a custom Planned Run template

Create a Planned Run

IMPORTANT! Before creating a Planned Run, you may need to enable the Oncomine

™

Comprehensive Plus templates and upload the Reference Library, Target Regions, and Hotspots BED ﬁles on the Ion Torrent™ Server. For more information, see Appendix B, “Supplemental information”. Contact your local service representative to obtain the most current BED ﬁles.

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

Note: Copy number variation (CNV), microsatelite instability (MSI), and tumor mutational burden (TMB) functionalities may require copy/editing of the Ion Reporter™ Software analysis workﬂow with user deﬁned optimizations before creating a Planned Run. For more information on creating a CNV baseline (see Appendix C, “CNV baseline creation”), setting MSI thresholds (see “MSI parameters” on page 76), and setting the TMB classiﬁcation thresholds (see “Set TMB Classiﬁcation parameters” on page 93) or refer to the Ion Reporter™ Software Help.

In the Plan tab, in the Templates screen, click Oncology – Solid Tumor in the left navigation menu.

In the list of templates, ﬁnd Oncomine™ Comprehensive Plus – DNA and Fusions, then click

4Copy.

The Copy Template workﬂow opens to the Save step.

Enter or select the required information in each ﬁeld:

Entry or selection

Template Name Enter a name for the Planned Run template.

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

DNA Target Regions

[1]

Fusions Reference Library and Fusions Target Regions are not necessary for analysis in Torrent Suite™ Software.

[2]

Check with your service representative for updates to ensure that the most current files are used.

Click the Ion Reporter step, then select your Ion Reporter™ account (version 5.16 or later).

[1]

[2]

Select OCAPlus.20200727.designed.bed.

Action

Note: If the account is not conﬁgured, conﬁgure it through Ion Reporter Conﬁgure settings

(see “Conﬁgure the IonReporterUploader plugin in Torrent Suite™ Software” on page 92). For instructions on how to install the IonReporterUploader plugin, see the Torrent Suite™ Software Help.

In the Existing Workﬂow dropdown list, select the appropriate Ion Reporter™ analysis workﬂow (page 56) for your Planned Run (for example, Oncomine™ Comprehensive Plus - w2.1 - DNA and Fusions - Single Sample), then click Next.

Oncomine™ Comprehensive Assay Plus User Guide

Chapter 5 Create a Planned Run

Create a custom Planned Run template

In the Research Application step, verify that the appropriate Research Application and Target Technique are pre-selected, then click Next.

In the Kits step, verify that the Ion Chef radio button is selected for the Template Kit, then complete the following ﬁelds as described:

Field

Instrument Ion GeneStudio™ S5 System

Library Kit Type Ion AmpliSeq™ Library Kit Plus Ion AmpliSeq™ Kit for Chef DL8

Template Kit Ion 550™ Kit − Chef

Sequencing Kit Ion S5™ Sequencing Kit

Chip Type Ion 550™ Chip

Barcode Set IonXpress or IonCode IonCode

Flows 500

Select or edit the optional information ﬁelds appropriately for your run, then click Next.

10.

In the Plugins step, ensure that the sampleID and coverageAnalysis plugins are selected.

11.

Conﬁgure the coverageAnalysis plugin as follows:

Click conﬁgure next to coverageAnalysis.

Ensure that the Sample Tracking checkbox is selected, then click Save Changes.

Click Next.

Manual library preparation Automated library preparation

Selection

12.

In the Projects step, select the project appropriate to your run, then click Next.

13.

In the Save step, click Copy Template to save the new Planned Run template.

The customized template is now available in the Templates screen, under the Oncology – Solid Tumor application.

Oncomine™ Comprehensive Assay Plus User Guide

Chapter 5 Create a Planned Run

Create a Planned Run for manual library preparation

In the Plan tab, in the Templates screen, click Oncology – Solid Tumor in the left navigation menu.

In the list, click on your customized Planned Run template name, or click The Create Plan workﬂow opens to the Plan step.

Enter a name for the plan in the Run Plan Name ﬁeld.

Make or conﬁrm the following selections:

4Plan Run.

Field

Analysis Parameters Make sure that Default (Recommended) is selected.

Use same reference & BED ﬁles for all barcodes

Same sample for DNA and Fusions?

Number of barcodes The standard number of barcodes for an Oncomine™ Comprehensive

Sample Tube Label Enter or scan the barcode of the Ion Chef™ Library Sample Tube that will

Chip Barcode No entry required.

Oncology Make sure that this button is selected.

Pre-implantation Genetic Screening

Enter the sample and barcode information:

[1]

Field

Make sure that this checkbox is selected.

Assay Plus DNA and Fusions run is 8 without no-template controls (NTC) and 10 with NTCs. Enter the number of barcodes, then click button to the right of this ﬁeld.

be used in the run.

Make sure that this button is deselected.

Action

Barcode Use the default barcode selections, or select the barcode for each

sample from the dropdown list.

Sample Name Each sample must have a unique name. Type in the ﬁeld to overwrite the

default name. We recommend that you use unique names even between runs.

Control Type (expanded) Select No Template Control from the dropdown list to designate a

sample as a no template control.

Sample ID (Optional) Click in the ﬁeld, then enter a sample ID.

Sample Description (Optional) Click in the ﬁeld, then enter a sample description.

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

Oncomine™ Comprehensive Assay Plus User Guide

Chapter 5 Create a Planned Run

Create a Planned Run for manual library preparation

(continued)

[1]

Field

Cancer Type Select from the dropdown list. Click to copy the entry to all the

Cellularity % Each DNA sample must have a cellularity percentage. This ﬁeld is

Ion Reporter Workﬂow Ensure the correct workﬂow is selected.

Relation Ensure the correct value is auto-populated. Select from the dropdown

Gender Select from the dropdown list. Click to copy the entry to all the

Action

rows.

optional for RNA samples. Click to copy the same entry to all rows.

Note: If the workﬂows are not displayed in the dropdown list, select the Show All Workﬂows checkbox to view all workﬂows on the selected Ion

Reporter™ Server.

list to change.

rows.

Note: This is a required ﬁeld to perform CNV analysis. By default Ion Reporter™ Software treats samples as female.

IR Set ID

[2]

The IR Set ID links individual samples for analysis. Ensure the correct value is autopopulated. Select from the dropdown list to change.

IMPORTANT! Ensure that a unique value is entered for each sample.

[1]

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

[2]

Samples with the same IR Set ID are considered related samples and launched in the same analysis such as the DNA barcode and Fusions barcode of the same sample. Do not give unrelated samples the same IR Set ID value (even if that value is zero or blank).

Click Plan Run.

The run is listed in the Planned Runs screen under the name that you speciﬁed and is automatically used by the Ion Chef™ System when the associated Ion Chef™ Library Sample Tube is loaded on the instrument.

Oncomine™ Comprehensive Assay Plus User Guide

Chapter 5

Create a Planned Run for automated library preparation using sample sets

Create a Planned Run

Create a Planned Run for automated library preparation using sample sets

Automated Oncomine™ Comprehensive Assay Plus library preparation on the Ion Chef™ System requires use of sample sets for creating your planned run. For more information on creating a sample set, see “Create a sample set to prepare 2 pools of 4 libraries each” on page 19.

If combining more than one sample set, each sample set must correspond to Oncomine Comprehensive Assay Plus automated library preparations and use the same barcode kit to be included in a single Planned Run.

In the Plan tab, in the Samples screen, ﬁnd the Sample Sets that you want to add to the Planned Run.

Select one or more Sample Sets (for example a DNA Sample Set and an RNA Sample Set) to add to the Planned Run.

•

To plan a run using one Sample Set, click Set.

(Actions) 4Plan Run in the row of the Sample

™

•

To plan a run using multiple Sample Sets, select the checkboxes next to the Sample Sets you want to add to the Planned Run, then click Plan Run.

IMPORTANT! Ensure that all Sample Sets used in the Planned Run use the same barcode

kit. To verify the barcode kit used, expand the Sample Set entry to view its details.

The Select a Run Template to apply to this experiment dialog lists Planned Run templates that support your Sample Set.

Oncomine™ Comprehensive Assay Plus User Guide

Chapter 5 Create a Planned Run

Create a Planned Run for automated library preparation using sample sets

Select a Run Template to use for the experiment. Select the samples to Create Plan Run using: All (A–H: Pool 1, 2), Pool 1 (A–D), or Pool 2 (E–H) then click Plan Run.

Note: If you do not see the template that you are looking for, select Show All Templates, then look again for the template.

The Create Plan workﬂow bar opens to the Barcoding step with the Sample Sets that you selected:

In the Barcoding step in the workﬂow bar, make or conﬁrm the following selections.

Field

Analysis Parameters Make sure that Default (Recommended) is selected.

Use same reference & BED ﬁles for all barcodes

Same sample for DNA and Fusions?

Number of barcodes The standard number of barcodes for an Oncomine™ Comprehensive

Sample Tube Label Enter or scan the barcode of each Ion Chef™ Library Sample Tube that

Chip Barcode No entry required.

Oncology Make sure that this button is selected.

Pre-implantation Genetic Screening

Make sure your sample set information has been correctly imported. Edit entries in each ﬁeld as

Make sure that this checkbox is selected.

Assay Plus DNA and Fusions run is 8 without no-template controls (NTC) and 10 with NTCs. Enter the number of barcodes, then click button to the right of this ﬁeld.

will be used in the run.

Make sure that this button is deselected.

Action

needed in the Samples Table, then click Next.

[1]

Field

Barcode Use the imported barcode selections, or select the barcode for

each sample from the dropdown list.

Sample Name (required) Each sample must have a unique name. Type in the ﬁeld to

overwrite the default name. We recommend that you use unique names even between runs.

Control Type (expanded) Click on the Control Type column header to expand the Control

Type column. Select No Template Control from the dropdown list to designate a sample as a no template control.

Sample ID (Optional) Click in the ﬁeld, then enter a sample ID.

Description

Oncomine™ Comprehensive Assay Plus User Guide

Chapter 5

Create a Planned Run for automated library preparation using sample sets

Create a Planned Run

(continued)

[1]

Field

Description

Sample Description (Optional) Click in the ﬁeld, then enter a sample description.

DNA/Fusions For DNA and Fusions application, select DNA or Fusions from the

dropdown menu for each samples.

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

Cancer Type Select from the dropdown list. Click to copy the entry to all the

rows.

Cellularity % Each DNA sample must have a cellularity percentage. This ﬁeld is

optional for RNA samples. Click to copy the same entry to all rows.

Ion Reporter Workﬂow Ensure the correct workﬂow is selected.

Note: If the workﬂows are not displayed in the dropdown list, select the Show All Workﬂows checkbox to view all workﬂows on the selected Ion Reporter™ Server.

Relation Select sample relationship group.

Gender Select "Male", "Female", or "Unknown" from the dropdow list. Click

to copy the entry to all the rows.

Note: Gender must be deﬁned to perform CNV analysis. By default "Unknown" is treated as female.

IR Set ID

[2]

The IR Set ID links individual samples for analysis. Ensure the correct value is autopopulated. Set the IR Set ID to the same value for related samples.

IMPORTANT! Ensure that a unique value is entered for each

sample. Do not give unrelated samples the same Set ID value even if the value is zero or blank.

[1]

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

[2]

Note: You can save the samples table to a CSV ﬁle as a template for future use. Click Save Samples Table above the upper right corner of the Samples Table to save the CSV ﬁle to your

computer.

Review the Plugins and Projects tabs, make selections appropriate to your run, then click Next.

In the Save & Finish step in the workﬂow bar, enter a name for the Planned Run in the provided ﬁeld.

Click Save & Finish.

Oncomine™ Comprehensive Assay Plus User Guide

Variant analysis

Analysis workﬂows in Ion Reporter™ Software ........................................... 56

■

Manually launch a DNA and Fusions analysis ............................................ 58

■

Manually launch a DNA analysis ........................................................ 59

■

Manually launch a Fusions analysis ..................................................... 59

■

View results .......................................................................... 60

■

Visualize tumor mutational burden analysis results ........................................ 70

■

MSI analysis results ................................................................... 75

■

Genomic segmentation analysis ....................................................... 78

■

Generate an Analysis Results Report .................................................... 82

■

Download Ion Reporter™ annotation VCF or TSV ﬁles ..................................... 83

■

Analysis workﬂows in Ion Reporter™ Software

If you selected the appropriate Ion Reporter™ workﬂow when setting up your Planned Run in Torrent Suite™ Software, automated analysis has already been performed and you can view the Oncomine analysis results in Ion Reporter™ Software. For instructions to manually launch an analysis, see “Manually launch a DNA analysis” on page 59 and “Manually launch a Fusions analysis” on page 59.

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

Ion Reporter™ Software 5.16 or later includes the following analysis workﬂows:

Workﬂow

Oncomine™ Comprehensive Plus w2.1 - DNA and Fusions - Single Sample

name

[1]

Description

Detects and annotates low frequency somatic variants (SNPs, InDels, CNVs) from targeted Ion AmpliSeq™ DNA manual or Ion Chef™ automated libraries, computes automatic tumor cellularity, Loss-of-Heterozygosity, TMB and microsatellite instability (MSI), as well as gene fusions from targeted Ion AmpliSeq™ RNA manual or Ion Chef™ automated libraries, from Oncomine™ Comprehensive Assay Plus run on the Ion 550™ Chip. TMB uses the TMB (Non-Germline Mutations) ﬁlter chain with TMB algorithm v4.0. MSI status is computed using a baseline with MSI algorithm v1.2. Released with: Ion Reporter™ Software 5.16. Workﬂow Version: 2.1.

™

Oncomine™ Comprehensive Assay Plus User Guide

(continued)

Chapter 6 Variant analysis

Analysis workﬂows in Ion Reporter™ Software

Workﬂow name

[1]

Oncomine™ Comprehensive Plus w2.1 - DNA and Fusions - (Low RMC Signal) - Single Sample

Oncomine™ Comprehensive Plus w2.1 - DNA - Single Sample

Oncomine™ Comprehensive Plus w2.1 - DNA - (Low RMC Signal)- Single Sample

Description

™

Chip. TMB uses the TMB (Non-Germline Mutations) ﬁlter chain with TMB algorithm v4.0. MSI status is computed using a baseline (with MSI algorithm v1.2) that accommodates libraries with a lower range of RMC ﬂow signal. Released with: Ion Reporter™ Software 5.16. Workﬂow Version: 2.1.

Detects and annotates low frequency somatic variants (SNPs, InDels, CNVs), computes automatic tumor cellularity, Loss-of-Heterozygosity, TMB and microsatellite instability (MSI) from targeted Ion AmpliSeq™ DNA manual or Ion Chef™ automated libraries from Oncomine™ Comprehensive Assay Plus run on the Ion 550™ Chip. TMB uses the TMB (Non-Germline Mutations) ﬁlter chain with TMB algorithm v4.0. MSI status is computed using a baseline with MSI algorithm v1.2. Released with: Ion Reporter

™

Software 5.16. Workﬂow Version: 2.1.

Detects and annotates low frequency somatic variants (SNPs, InDels, CNVs), computes automatic tumor cellularity, Loss-of-Heterozygosity, TMB and microsatellite instability (MSI) from targeted Ion AmpliSeq™ DNA manual or Ion Chef™ automated libraries from Oncomine™ Comprehensive Assay Plus run on the Ion 550™ Chip. TMB uses the TMB (Non-Germline Mutations) ﬁlter chain with TMB algorithm v4.0. MSI status is computed using a baseline (with MSI algorithm v1.2) that accommodates libraries with a lower range of RMC ﬂow signal. Released with: Ion Reporter

™

Software 5.16. Workﬂow Version: 2.1.

Oncomine™ Comprehensive Plus w2.1 - Fusions - Single Sample

Detects and annotates gene fusions from targeted Ion AmpliSeq™ RNA manual or Ion Chef™ automated libraries from Oncomine™ Comprehensive Assay Plus run on the Ion 550™ Chip. Released with: Ion Reporter Software 5.16. Workﬂow Version: 2.1.

Oncomine™ Comprehensive Plus w2.1 - Annotate Variants - Single

Annotates VCF ﬁles from the Oncomine™ Comprehensive Assay Plus. Released with: Ion Reporter™ Software 5.16. Workﬂow Version: 2.1.

Sample

[1]

Workflow names can vary depending on the version of Ion Reporter™ Software.

Oncomine™ Comprehensive Assay Plus User Guide

™

Chapter 6 Variant analysis

Manually launch a DNA and Fusions analysis

In the Workﬂows tab, in the Overview screen, select DNA and Fusions from the Research Application dropdown list.

(Optional) In Search, enter a term, then click Go (or press Enter).

In the Workﬂow Name column, click the appropriate workﬂow (for example, Oncomine Comprehensive Plus - w2.1 - DNA and Fusions - Single Sample), then click Analysis in the Details pane.

Select a DNA sample.

In the Samples step, search by any unique identiﬁer you used to label your samples during setup, then ensure the sample Cellularity % , Sample Type and Gender are deﬁned.

Note: We recommend that you deﬁne the sample Gender to perform accurate CNV analysis. By default this is treated as female.

Click the checkbox to select a DNA sample, then click Add samples.

Select an RNA sample.

In the Samples step, search by any unique identiﬁer you used to label your samples during setup, then ensure the sample Sample Type is deﬁned.

Click the checkbox to select an RNA sample, then click Add samples.

Enter a Group Name, then click Add to Analysis.

In the Plugins step, ensure that the Oncomine™ Variant Annotator 3.1 plugin is selected, then click Next.

Actions4Launch

™

(Optional) Enter an Analysis Name and Description.

10.

Click Launch Analysis.

If you selected multiple samples, a separate analysis is launched for each sample for single-sample analysis

workﬂows.

Oncomine™ Comprehensive Assay Plus User Guide

Manually launch a DNA analysis

In the Workﬂows tab, in the Overview screen, select DNA from the Research Application dropdown list.

(Optional) In Search, enter a term, then click Go (or press Enter).

Chapter 6

Manually launch a DNA analysis

Variant analysis

In the Workﬂow Name column, click the appropriate workﬂow (for example, Oncomine Comprehensive Plus - w2.1 - DNA - Single Sample ), then click in the Details pane.

In the Samples step, search by any unique identiﬁer you used to label your samples during setup, then ensure the sample Cellularity % , Sample Type and Gender are deﬁned.

Note: We recommend that you deﬁne the sample Gender to perform accurate CNV analysis. By default this is treated as female.

Click the checkbox to select a DNA sample, then click Next.

In the Plugins step, ensure that the Oncomine™ Variant Annotator 3.1 plugin is selected, then click Next.

(Optional) Enter an Analysis Name and Description.

Click Launch Analysis.

Actions4Launch Analysis

™

Manually launch a Fusions analysis

In the Workﬂows tab, in the Overview screen, select Fusions from the Research Application dropdown list.

(Optional) In Search, enter a term, then click Go (or press Enter).

In the Workﬂow Name column, click the appropriate workﬂow (for example, Oncomine Comprehensive Plus - w2.1 - Fusions - Single Sample ), then click in the Details pane.

In the Samples step, search by any unique identiﬁer you used to label your samples during setup, then ensure that the Sample Type is deﬁned.

Click the checkbox to select a fusions sample, then click Next.

Oncomine™ Comprehensive Assay Plus User Guide

™

Actions4Launch Analysis

Chapter 6 Variant analysis

View results

In the Plugins step, ensure that the Oncomine™ Variant Annotator 3.1 plugin is selected, then click Next.

(Optional) Enter an Analysis Name and Description.

Click Launch Analysis.

View results

Ion Reporter™ Software analyses are performed automatically on uploading of the data ﬁles from the Torrent Suite™ Software.

Click the Analyses tab. The Overview screen displays a list of analyses in the Analyses table.

(Optional) Filter the Analyses table.

•

In the Overview screen, click More Filters4Research Application. In the Research

Application dropdown list, select the Oncomine™‑speciﬁc analyses (DNA, Fusions, DNA and Fusions, or Annotate Variants).

•

Enter a search term in the search ﬁeld, then click Go (or press Enter).

To further reﬁne the list of analyses, apply additional ﬁlters from the More Filters dropdown list or click the column headers. The Analyses table automatically updates based on the ﬁlter selections and search term.

Click on a row (but not on the sample data set hyperlink) to open the Details pane on the right side of the screen for that analysis.

In the Details pane, you can view Workﬂow Details and access the Actions dropdown list.

Oncomine™ Comprehensive Assay Plus User Guide

Chapter 6 Variant analysis

View results

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

The Analysis Results page opens to the Oncomine™ tab, and displays only variants relevant to cancer.

Item

MAPD (Median Absolute Pairwise Dierence)

Tumor Cellularity Percentage

Tumor Mutational Burden (Mutations/Mb)

Description

The MAPD metric is a measure of read coverage noise detected across all amplicons in a panel. Higher MAPD typically translates to lower coverage uniformity. Lower coverage uniformity can result in missed or erroneous CNV calls. MAPD score is viewable in downloadable VCF ﬁle or review of the Analysis Results of a single sample extended analysis. To make a CNV call the following criteria must be met:

•

MAPD <0.4

•

CNV Ratio for a copy number gain must be >2

The percent of tumor cells in the sample. Allowed values: Manually input Tumor Cellularity input per user or Auto-calculated tumor cellularity.

The value of the result for the tumor mutational burden calculation.

•

P-value <10

•

CNV Ratio for a copy number loss must be <0.85

-5

[1]

Oncomine™ Comprehensive Assay Plus User Guide

Chapter 6

View results

(continued)

Variant analysis

Item Description

TMB Classiﬁcation (based on speciﬁed parameters)

MSI Status

•

High—The Tumor Mutational Burden (Mutations/Mb) is greater than the value entered in the TMB-High Threshold parameter.

•

Low—The Tumor Mutational Burden (Mutations/Mb) is less than the value entered in the TMB-Low Threshold parameter.

•

Intermediate—The Tumor Mutational Burden (Mutations/Mb) is equal to or greater than the value entered in the TMB-Low Threshold parameter AND less than or equal to the value entered in the TMB-High Threshold parmeter.

•

Undeﬁned—TMB classiﬁcation thresholds settings were not included in the analysis workﬂow or were set incorrectly.

Note: Threshold values are not provided in the factory shipped analysis

workﬂow. The analysis workﬂow always reports the TMB classiﬁcation status as Undeﬁned unless a user sets the thresholds in the parameter settings. For more

information on setting TMB Classiﬁcation thresholds, see “Set TMB Classiﬁcation parameters” on page 93.

•

MSI-High—MSI score is greater than the threshold value set in the MSI-High Threshold parameter.

•

MSS—MSI score is less than the threshold value set in the MSS Threshold parameter.

•

No Call—MSI score is equal to or greater than the value entered in the MSS

Threshold parameter AND less than or equal to the value entered in the MSI- ThresholdHigh parmeter.

•

QCFail—Indicates determination of MSI status was not reliable due to the MSI baseline. For more information, see “MSI QC Failure” on page 90 or for more assistance contact support (see “Customer and technical support” on page 115).

MSI Score A sample-level MSI score that is calculated with individual MSI marker scores. The

overall score is used to determine the MSI status of the sample.

LOH Percentage Percentage of genomic segments with loss-of-heterozygosity (LOH) detected.

Calculated as the sum of the sizes of the genomic segments with LOH detected / total size of genomic segments assessed for LOH.

[1]

If tumor cellularity percentage is determined to be not reliable by auto-calculation, then "Manual" is displayed.

In the Analysis Results table, sort or ﬁlter the data using the Oncomine™‑speciﬁc annotations. See the Ion Reporter™ Software help menu for more options.

In the Filter Options pane, select the desired Filter Chain.

Note:

The default Filter Chain is Oncomine™ Extended, which limits the results that are

displayed to variants relevant to cancer research only. Each variant that is called must meet all the conditions of the ﬁlter chain to be ﬁltered‑in. For more information on ﬁlter chains, see the Ion Reporter™ Software Help. Select No Filter to view all the variant calls attempted by the variant caller.

Saving the analysis using a ﬁlter chain other than Oncomine™ Extended changes the

variant calls that are saved in the VCF ﬁle and can aect downstream workﬂows.

Oncomine™ Comprehensive Assay Plus User Guide

Chapter 6 Variant analysis

View results

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

In the Ontologies tab, click the column headers to sort the list by variant Type or Genes.

Click Pharmacogenomics to view the ClinVar column. Click the link in the ClinVar column for a selected variant to open an NCBI ClinVar website where information about the ClinVar variant annotation is available.

After you review, ﬁlter, and sort your Analysis Result, you can create a report (see “Generate an Analysis Results Report” on page 82), or download the analysis ﬁles (see “Download Ion Reporter™ annotation VCF or TSV ﬁles” on page 83). You can view the extracted ﬁles individually, or upload a VCF ﬁle to a software that requires VCF ﬁles, such as Ion Torrent™ Oncomine™ Reporter Software.

Oncomine™ Comprehensive Assay Plus User Guide

Chapter 6 Variant analysis

View results

Fusion results

The Fusions results table lists the calls and other information for the fusions, including intragenic MET exon skipping variants, analyzed in each sample in a run.

Click the Analyses tab. The Overview screen displays a list of analyses in the Analyses table.

Click a sample result hyperlink in the Analysis column to open the Analysis Results summary screen.

Click the Fusions tab to view the Fusions results table.

If using a Fusions analysis workﬂow, only the Fusions results table is displayed in the Analysis Results summary screen.

Column Description

Classiﬁcation A user-deﬁned classiﬁcation selected from the list.

Locus The chromosome positions in the reference genome that deﬁne the fusion junction.

Type Assay type (for example, Fusion, RNA exon variant (exon skipping), RNAExon Tile, Proc

Control).

Filter Indicates the presence or absence of a fusion or RNA exon variant. When the default ﬁlter

chain is applied, only the fusion/RNA exon variant calls that are designated with PASS are displayed in the results table. To view all calls, including calls that do not pass the required ﬁlter thresholds, apply the No Filter ﬁlter chain or download the Variants (.vcf) ﬁle.

•

PASS – indicates a high conﬁdence call that passes all ﬁlter thresholds at a given variant position.

•

FAIL – indicates the absence of a fusion due to the variant call failing internal quality control.

•

NO CALL – while some evidence for the presence of a fusion exists, the call does not pass one or more ﬁlters that are required for a high conﬁdence fusion call.

No Call Reason The reason for reporting a fusion as NOCALL in Filter column.

Genes (Exon) The name of fusion target and representative acceptor and donor exons.

Read Counts The frequency that the fusion was detected in the sample.

Oncomine™ Comprehensive Assay Plus User Guide

(continued)

Column Description

Chapter 6 Variant analysis

View results

Oncomine Variant Class

Oncomine Gene Class

Detection Returns: Present, Absent, Present-Non-Targeted or NoCall as suppported by the read

Ratio to Wild Type The ratio of a given variant within all wild type variants of that gene.

Norm Count Within Gene

Imbalance P-value The P-value calculated for a given variant Imbalance score.

Read Counts Per Million

Variants that are known Oncomine™ annotated hotspots.

Gain-of-function, or Loss-of-function

counts dependent upon the thresholds set for detection.

The ratio of a given variant within all variants of that gene.

The ratio of target fusion read counts within the total mapped reads X 10

View RNA Exon Tile Fusion Imbalance

The RNA Exon Tile Fusion Imbalance data view provides a visual representation of the RNA fusion imbalance analyses.

In the menu bar, click Analyses4Overview.

In the Analysis column, click the hyperlinked name of the analysis of interest.

Click the Variants tab, then click Fusions. The Fusions table opens to display fusions results.

In the top right corner of the screen, click Visualize, then select the RNA Exon Tile Fusion Imbalance tab to view the plots.

Oncomine™ Comprehensive Assay Plus User Guide

1 2

Chapter 6 Variant analysis

View results

Representative RNA Exon Tile Fusion Imbalance plots

The Fusion Imbalance Call: Imbalance Score & P-value plot shows the imbalance scores and p‑values for all the

genes in the selected sample. The dashed gray lines mark the threshold for an imbalance call, which is applied to all genes across all samples. Points that fall within the blue shaded area of the plot represent fusion‑positive genes ( ). All other points that are outside of the blue shaded area represent fusion‑negative genes ( ). Control genes are marked with

The Normalized Read Depth by Gene plot shows the mean read counts of each gene that is captured on the chip

for the selected sample. For each gene, the read counts are normalized to the number of amplicons.

Oncomine™ Comprehensive Assay Plus User Guide

Chapter 6

Variant analysis

View results

The Sample Coverage for Imbalance Analysis plots show the expression proﬁle for each exon‑exon tiling amplicon for each

gene. The Y‑axis represents the normalized coverage after baseline correction. The X‑axis represents individual exon‑exon junctions, which are listed from 5′ to 3′. The imbalance score and p‑value are listed in the panel of each gene that was called positive for fusion.

Baseline (a cluster of gray lines), generated from a fusion‑negative sample.

Test sample corrected read coverage (blue line), normalized to the baseline. Each point on the line represents a unique

exon‑exon junction that was covered by the assay and normalized to the baseline. Predicted range for the fusion break point for a fusion‑positive gene (dashed red line).

If the collected data are insucient to determine an imbalance score, the SAMPLE NOT VALID FOR <gene> IMBALANCE

ANALYSIS message appears in the panel for that gene. Sample coverage proﬁle for the control gene (orange line).

To return to the table view of fusions, click the browser Back arrow.

View RNA exon variants

The RNA Exon Variant data view displays a bar graph summary of intragenic exon rearrangements or fusions. The displayed RNA exon variants are deﬁned in the BED ﬁle that is associated with an assay. The RNA Exon Variant data view is available for all RNA and Fusion assays.

In the menu bar, click Analyses4Overview.

In the Analysis column, click the hyperlinked name of the analysis of interest.

Oncomine™ Comprehensive Assay Plus User Guide

Chapter 6 Variant analysis

View results

Click the Variants tab, then click Fusions. The Fusions table opens to display fusions results.

In the top right corner of the screen, click Visualization4RNA Exon Variant to view the RNA Exon Variant plot.

To return to the table view of fusions, click the browser Back arrow.

Oncomine™ Comprehensive Assay Plus User Guide

Representative RNA Exon Variant plots

1 2

Chapter 6 Variant analysis

View results

The X‑axis represents speciﬁc exon variants, where each variant is labeled with a gene ID followed by a sequence of adjacent exons. The Y‑axis measures the read counts for each variant, normalized to the wild type.

Example analysis where RNA exon 2–7 deletion occurred in the EFGR gene. The deletion of exons 2–7 resulted in an

increase of normalized read counts for the EFGR variant that contains the intragenic fusion of exon 1 and exon 8 (EGFREGFR.E1E8.DelPositive.2), and a decrease of normalized read counts for the wild type EFGR (EFGR.E6E7).

Example analysis where exon-skipping (i.e., exon deletion) of exon 14 in the MET gene was detected. Normalized read counts

for the variant representative of the exon-skip event (MET‑MET.M13M15.1) are higher relative to the MET wild-type assays (for example, MET.M13M14M15.ENST00000397752.WT and MET.M17M18M19M20.ENST00000397752.WT).

Oncomine™ Comprehensive Assay Plus User Guide

Chapter 6

Visualize tumor mutational burden analysis results

Variant analysis

Visualize tumor mutational burden analysis results

To visualize tumor mutational burden analysis results in Ion Reporter™ Software, the analysis workﬂow must be any DNA-single sample, or DNA and Fusions-single sample analysis workﬂow that has tumor mutational burden enabled.

Do one of the following to open tumor mutational burden analysis results:

Option Description

Visualize analysis results from an individual sample or from multiple samples simultaneously from the Analyses table.

Visualize analysis results individually from the Analysis Results screen.

The

Tumor Mutational Burden (Mutations/Mb) is displayed at the top of each section of sample

results.

Click Download Report to download the Tumor Mutational Burden Analysis Report in PDF format, which includes the results with graphs and metrics.

In the Analyses table, select an individual sample result row, or select the checkbox next to each sample result that you want to visualize simultaneously, then click Visualize. Alternatively, click Actions4Visualize.

In the Analyses table, click a sample result hyperlink in the

Analysis column to open the Analysis Results, then click Visualize.

Scroll down to view graphical representations of the analysis results and QC metrics, and download additional ﬁles.

Note: Multiple sample results are listed sequentially.

In the lower right corner of the screen:

• Click Download Variant Details TSV to download a tab-separated list of detected variants that contributed to the tumor mutational burden count. Open the ﬁle with compatible software, such as Microsoft™ Excel™.

• Click Download the TMB statistic.txt ﬁle to download the tumor mutational burden output ﬁle. The ﬁle is named <analysis name>_statistic.txt. For example, NCI-1395_c3495_2020-03-03-10-58-54-007_statistic.txt.

Oncomine™ Comprehensive Assay Plus User Guide

QC metrics for tumor mutational burden

Total base coverage across the genomic positions defined by the assay BED file

Number of bases in the DNA BED file

QC metrics for tumor mutational burden are listed at the top of Analysis Visualization in the TMB tab when you visualize analysis results with Ion Reporter™ Software.

QC metric Deﬁnition

Average Coverage The following formula is used to calculate average coverage:

If the average coverage is below 150, the analysis completes successfully but Tumor Mutational Burden is reported as -1.

Note: This setting cannot be changed in Ion Reporter™ Software.

Chapter 6 Variant analysis

Visualize tumor mutational burden analysis results

Number of bases used in calculating TMB

Number of variant calls

Deamination score Deamination score was previously reported as the estimated SNP proportion

The number of bases used as a denominator for the tumor mutational burden calculation. Only bases with sucient base coverage are used in the calculation, as deﬁned in the workﬂow parameters. In the parameters, you can also select only the exonic regions covered by the panel to be used instead of all genomic regions. See the Ion Reporter™ Software Help for more information.

The number of somatic variants that are identiﬁed in the sample. This value is reported in the statistic.txt ﬁle as Total Somatic Filtered Variants Count (numerator for TMB calculation) and Variant Count.

For more information, see Ion Reporter™ Software Help.

consistent with deamination (mainly FFPE). The deamination score can be used to determine the quality of the FFPE sample. For more information on how to minimize the impact of high deamination on a tumor mutational burden score, see Ion Reporter™ Software Help.

A deamination QC status of PASS/FAIL is also reported. The threshold for this QC can be adjusted in the analysis workﬂow parameter settings. For more information, see Ion Reporter™ Software Help.

Oncomine™ Comprehensive Assay Plus User Guide

Chapter 6 Variant analysis

Visualize tumor mutational burden analysis results

Sample results

Tumor mutation burden results are represented graphically in the TMB tab when you visualize analysis results with Ion Reporter™ Software.

Germline and Somatic Variants

This histogram shows the frequency distribution of allele ratio for total called germline and somatic variants. Listed below the ﬁgure is the combined total of called germline and somatic variants. The value is reported in the statistic.txt ﬁle as Variant count (Germline

+ somatic). For more information, see Ion Reporter Software Help.

Only Somatic Variants

This histogram shows the frequency distribution of allele ratio for only somatic mutations as determined by the selected TMB ﬁlter chain. Listed below the ﬁgure are:

•

The number of Total Somatic Variants , reported in the statistic.txt ﬁle as Total Somatic Filtered Variants Count (numerator for TMB calculation).

•

The number of variants determined to be nonsynonymous (detrimental) and synonymous (nondetrimental) as annotated by Ion Reporter™ Software. Values are reported by dierent Ion Reporter™ annotation types in the statistic.txt ﬁle.

•

The number of detected somatic variants found in the COSMIC database. The value is reported in the statistic.txt ﬁle as COSMIC Annotated Somatic Variants.

™

For more information, see step 4 of “Visualize tumor mutational burden analysis results” on page 70 .

Oncomine™ Comprehensive Assay Plus User Guide

Visualize tumor mutational burden analysis results

Substitution Type and Context of Somatic Mutations

Chapter 6 Variant analysis

C>A

C>G

C>T

Somatic 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 bases immediately 5′ and 3′ to each mutated base, 96 possible mutation types are in this classiﬁcation. These 96 mutation types are represented on the x‑axis, and variant frequency for mutation type on the y‑axis. Bars for each substitution class are grouped and displayed with dierent color. A summary TXT ﬁle of these results is also available, see “Download Ion Reporter™ annotation VCF or TSV ﬁles” on

page 83.

T>A

T>C

T>G

Substitution Type of Somatic Mutations

A 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.

C>T

C>G

C>A

Oncomine™ Comprehensive Assay Plus User Guide

T>G

T>C

T>A

Chapter 6 Variant analysis

Visualize tumor mutational burden analysis results

Signature Pattern of Somatic Mutations

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

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

In the pie chart, a small fraction of multiple signature types can be observed in the sample. However, signiﬁcant dominance of a single signature pattern often correlates to the respective 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. (Blue + Green + Yellow) For more information, see https://www.nature.com/articles/nature22071

•

High C>T at CpG is consistent with spontaneous deamination of 5‑methylcytosine. (Red + Blue). For more information, see https://www.nature.com/articles/nature12477

•

High C>A is consistent with smoking damage.(Orange) For more information, see https://science.sciencemag.org/content/354/6312/618.full

•

High C>T (site independent) is consistent with FFPE processing. (Green + Purple) For more information, see https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4032349/

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

Oncomine™ Comprehensive Assay Plus User Guide

MSI analysis results

You can determine whether microsatallite instability (MSI) is present using Oncomine™ Comprehensive Assay Plus - w2.1 - DNA (or DNA and Fusions) Ion Reporter™ Software analysis workﬂows. In these analysis workﬂows parameters are applied that detect MSI markers, which can identify a form of genomic instability in the replication of repetitive DNA.

Note: MSI parameters in the software are not applied when you use the Oncomine™ Comprehensive Plus - w2.1 - Fusions - Single Sample workﬂow. For more information, see Ion Reporter™ Software Help.

View MSI parameters

The parameters for MSI analysis are located in the Oncomine™ Comprehensive Plus - w2.1 - DNA Single Sample workﬂow in Ion Reporter™ Software.

Under the Workﬂows tab, in the Overview screen, search for the workﬂow, then select it in the list.

Chapter 6 Variant analysis

MSI analysis results

In the Details pane to the right of the list, scroll down, then click View next to Parameters.

In the Parameters dialog, select MSI from the left navigation bar.

Oncomine™ Comprehensive Assay Plus User Guide

Chapter 6

MSI analysis results

Variant analysis

MSI parameters

You can adjust MSI parameters to optimize your analysis results when you create or edit analysis

workﬂows in Ion Reporter™ Software. MSI parameters are available only for some Oncomine™ analysis workﬂows. For a complete description of all MSI parameters see the Ion Reporter™ Software Help.

Parameter name Description

Main tab

Enable MSI Detection When set to true, this parameter enables detection of MSI markers for the

workﬂow. These markers can identify a form of genomic instability in the replication of repetitive DNA. MSI often occurs in tumor cells. It leads to the appearance of multiple alleles at microsatellite loci, which can be easily

identiﬁed.

This parameter is set to True by default in the Oncomine™ Comprehensive Assay Plus analysis workﬂows. If you do not want to include MSI results in an analysis, you can set to the parameter to False. Do not change this setting in other Oncomine™ analysis workﬂows.

MSI-High Threshold The MSI score above which a sample is considered MSI-High. This score is

reported in analysis results.

MSS Threshold The MSI score below which a sample will report MSS in analysis results.

[1]

If the MSI Score falls between the MSI-High Threshold and the MSS Threshold, a No Call is reported in the analysis results.

[2]

The MSI-High and MSS thresholds are optimized based on the MSI baseline of the workflow. Do not change parameters from the default settings unless you understand how the change can affect your analysis.

Visualize MSI analysis results

There are two ways to visualize MSI analysis results in Ion Reporter™ Software:

Option

Visualize MSI results from one or more analyses simultaneously from the Analyses table.

Visualize analysis results individually from the Analysis Results screen.

The Analysis Visualization screen opens to the TMB tab.

[1,2]

Description

In the Analyses table, select a row for an analysis or select the checkbox next to one or more analyses that you want to visualize simultaneously, then click Visualize. Alternatively, click Actions4Visualize.

In the Analyses table, click an analysis hyperlink in the Analysis column to open the Analysis Results, then click Visualize.

Click the MSI tab. A table with MSI Status, MSI Score, and MSI Coverage is shown.

Oncomine™ Comprehensive Assay Plus User Guide

Chapter 6 Variant analysis

MSI analysis results

Item Description

MSI Status A sample is assigned an MSI status that is based on the MSI Score. The MSI

status can be one of the following:

•

MSI-High

•

MSS (MSI stable)

•

No Call (MSI status is intermediate, i.e., between the thresholds)

•

QC Fail (See step 3 to download the MSI results ﬁles. Review the MSIQC.json ﬁle to determine the failure mode.)

MSI parameter settings include thresholds that determine whether the status is MSI-High or MSS. A status of No Call or QC Fail is based on the MSI marker coverage.

MSI Score

MSI Coverage A combined sample-level coverage that is calculated with the individual MSI

Controls The in silico control deﬁned by the JSON ﬁle displayed.

Click Download Results to download a report of the MSI results. The report is downloaded to the

A sample-level MSI score that is calculated with individual MSI marker scores. The overall score is used to determine the MSI status of the sample.

marker-level coverage.

folder that is used for downloads, depending on the browser settings.

Oncomine™ Comprehensive Assay Plus User Guide

Chapter 6

Genomic segmentation analysis

Variant analysis

View downloaded MSI results ﬁles

The ZIP ﬁle of downloaded MSI results contains three ﬁles.

Filename File type Description

Details.tsv Tab separated values (TSV) File contains detailed information on each MSI marker in

the assay. Details include Ion Reporter™ Software sample and analysis name, marker level coverage, MSI score, and MSI algorithm version.

Summary.tsv Tab separated values (TSV) File contains sample level summary of MSI results. This

includes sample and analysis name in Ion Reporter Software, total coverage across all MSI markers, MSI score, MSI status, name of the control ﬁle used in the workﬂow, and MSI algorithm version.

MSIQC.json JavaScript Object Notation

(JSON)

File contains "ﬂags" and warning messages associated with MSI status used by support personnel. See “Customer and technical support” on page 115.

Extract the MSIResults_<date>.zip folder, then open the enclosed folder.

Genomic segmentation analysis

The Ion Reporter™ Software Oncomine™ Comprehensive Assay Plus, DNA analysis workﬂow utilizes heterozygous population SNPs covered by the assay to determine the ploidy levels of genomic segments. The genome is divided into contiguous segments of similar ploidy levels using a circular binary segmentation (CBS) algorithm. Log odd ratios for variant allele frequency of observed population SNPs (by TVC) and copy-number (CN) ratios (by CNV pipeline) for each segment are calculated. Log odd ratio and CN ratios are then used to infer:

•

Tumor cellularity percentage (percentage of tumor cells in the sample)

•

Loss-of-heterozygosity (LOH) for each genomic segment. Segment level LOH events are intersected with targeted gene boundaries to determine LOH events in selected genes. Segment level LOH events are also aggregated to determine sample level % LOH.

•

Exon-level copy number alterations in selected genes reported as:

™

Type

CNV BigDel

[1]

This CNV subtype detected by the Oncomine™ Comprehensive Assay Plus is not the same as one detected using assays without genomic segmentation (for example, the Oncomine™ BRCA Research Assay).

CNV Subtype Description

BigDup

[1]

Deletion is detected between exons and the genomic segment when it occurs at the beginning or end of a gene.

Duplication is detected between exons and the genomic segment when it occurs at the beginning or end of a gene.

Oncomine™ Comprehensive Assay Plus User Guide

Chapter 6 Variant analysis

Genomic segmentation analysis

Visualization of genomic segmentation analysis, Allele Speciﬁc Copy Number plots

There are two ways to visualize analysis results in Ion Reporter™ Software:

Option Description

Visualize analysis results from one or more analyses simultaneously from the Analyses table.

Visualize analysis results individually from the Analysis

Results screen.

Note: The Analysis Visualization screen opens to the TMB tab by default.

Click the Allele Speciﬁc Copy Number tab. A series of plots showing copy number variation across the entire genome is displayed. Click

Export Image to save a PNG image of all three plots to a location of your choice. If visualizing multiple analyses simultaneously, individual sample results are listed sequentially.

In the Analyses table, click an analysis hyperlink in the Analysis column to open the Analysis Results, then click Visualize.

Oncomine™ Comprehensive Assay Plus User Guide

Chapter 6 Variant analysis

Genomic segmentation analysis

This visualization shows identiﬁed genomic segments (shown as horizontal lines) using log2 ratios from the Copy Number Variation (CNV) analysis and log odds from the Torrent Variant Caller (TVC) analysis. Segments are identiﬁed using the heterozygous population SNPs (shown as dots in the upper two panels) targeted by assay amplicons.

Plot scale (x-axis)

logR = copy number log2 ratio

Log2 ratios (top panel) of the copy-number estimates relative to the baseline copynumber as calculated by the CNV algorithm for each amplicon in the assay. Ratio representative Genome segmentation overlay (horizontal brown bars) shows segments with similar log2 ratios clustered together.

Description

Oncomine™ Comprehensive Assay Plus User Guide

(continued)

Plot scale (x-axis) Description

Chapter 6

Genomic segmentation analysis

Variant analysis

log odds = ln(Tumor Reads / Reference Reads)

Tumor Copy Number Bottom panel shows the total (black horizontal line) and minor (red horizontal line)

Log odds (middle panel) for each heterozygous SNP in the assay. Log odds is calculated as the natural logarithm of the ratio of the sequencing reads with variant allele and reference allele that are obtained using Torrent Variant Caller (TVC). Genome segmentation overlay (horizontal brown bars) shows segments with similar log odds clustered together. Since the variant allele could be major or minor for any SNP, the corresponding log odds could be positive or negative and are therfore displayed as segments that are mirror images around 0.

copy-number estimates for each of the identiﬁed genomic segments (top two panels). Genomic segments where total CN ≥1 and minor CN = 0 are segments with LOH. There may exist genomic segments for which minor copy-number estimates can not be determined.

Visualize exon level loss results

Exon level loss is calculated for a subset of tumor suppressor genes. For a list of the genes covered contact support (see “Customer and technical support” on page 115).

Note: Current exon-deletion algorithm is genomic segmentation based where intersection between exons and genomic segment (with copy-number alteration) is assessed for reporting partial gene alterations from beginning or end of a gene. This approach does not provide detection of focal exon level changes.

There are two ways to visualize exon level loss analysis results in Ion Reporter™ Software:

Option

Visualize analysis results from one or more analyses simultaneously from the Analyses table.

Visualize analysis results individually from the Analysis Results screen.

In the Analyses table, click an analysis hyperlink in the Analysis column to open the Analysis Results, then click Visualize.

Description

The Analysis Visualization screen opens to the TMB tab.

Click the Exon CNV tab. A series of plots showing exon level copy number variation for each exon in queried genes is

displayed. Click Download All Plots to save a ZIP ﬁle contatining PNG images of each plot to a location of your choice. If visualizing multiple analyses simultaneously, individual sample results are listed sequentially.

Oncomine™ Comprehensive Assay Plus User Guide

Chapter 6 Variant analysis

Generate an Analysis Results Report

The visualization displays distribution of copy-number estimates (y-axis) of each amplicon from a gene across dierent exons (x-axis). Gene name is speciﬁed in the text on top of the plot. Each blue dot represents an amplicon. Dotted blue lines corresponds to the lower and upper range of observed CN estimates to the nearest integer value. Exon-level CNV plots are generated for a selected subset of tumor suppressor genes. Exon numbering (from left to right) represents strand orientation of the gene. For genes on the negative strand, exon numbering is in decreasing order.

Generate an Analysis Results Report

After you have reviewed, ﬁltered, and sorted your Analysis Result, you can download an Analysis Report. The procedure described here includes creating and formatting a report template.

In the Analysis Results screen for your sample, click Generate Report. The Generate Report workﬂow bar opens to the Conﬁguration step. The sections of the report

can be rearranged, deleted, or edited.

Hover over the various sections and icons to view instructional text to help you format your report output.

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

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

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

Click Lock & Publish to generate the ﬁnal Analysis Report.

Click Download.

Oncomine™ Comprehensive Assay Plus User Guide

Chapter 6 Variant analysis

Download Ion Reporter™ annotation VCF or TSV ﬁles

Variant call format (VCF), and tab separated values (TSV) ﬁles of the complete or ﬁltered results can be downloaded from the Analysis Results screen.

Click Download, then select All Variants, Filtered Variants, Current Results TSV, or Selected Variants.

•

All variants—A VCF ﬁle that contains all variants that are included in the analysis.

•

Filtered variants—A VCF ﬁle that contains the variants which were ﬁltered IN for the analysis.

•

Selected variants—A VCF ﬁle that contains your selected variants from the analysis. Each variant to be included in the selected variants VCF ﬁle must be selected in the Analysis Results before the ﬁles are downloaded.

•

Current Results TSV—A tab separated values (TSV) ﬁle of the analysis results of the current analysis.

Click Home4Notiﬁcations to open the Notiﬁcations screen, then click (Download) next to the ﬁle name to download your results.

Alternatively, select one or more rows, then click Download. The software generates a compressed directory named <analysis name>_All.zip that is

downloaded. Extract the contents of the ZIP ﬁle to access the following 6 folders: CNV_VCIB, QC, Variants, Results, MSI, and Workﬂow_Settings. Within the Variants folder, you’ll ﬁnd the Oncomine™ annotated VCF ﬁle.

Folder

CNV_VCIB Contains an image ﬁle (cn_results.png) of the copy number determination for all

amplicons.

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

Contents of folder

Oncomine™ Comprehensive Assay Plus User Guide

Chapter 6 Variant analysis

Download Ion Reporter™ annotation VCF or TSV ﬁles

(continued)

Folder Contents of folder

Variants Contains a folder with the following ﬁles:

•

Intermediate and Oncomine™-annotated .VCF ﬁles

•

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

Results If the Tumor Mutational Burden parameter is enabled a Results folder is generated.

The Results folder contains:

•

A PDF report of the tumor mutational burden results

•

TSV ﬁles that contain post-ﬁlter and somatic variants

•

Image ﬁles that graphically represent the results

•

TXT ﬁles that contain summaries of the TMB output

MSI Contains summary and detailed marker-level ﬁles with data about microsatellite

instability (MSI) markers. These markers can identify a form of genomic instability in the replication of repetitive DNA.

Workﬂow_Settings Contains folders with the following ﬁles:

•

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

•

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

You can view the extracted ﬁles individually, or upload a VCF ﬁles to an ancillary software application such as the Ion Torrent™ Oncomine™ Reporter for further analysis.

Requirements for variant annotation in Ion Reporter™ Software

The following table summarizes the requirements that must be met for the Oncomine™ Variant Annotator to annotate variants in Ion Reporter™ Software for the Oncomine™ Comprehensive Assay Plus.

For each variant type in this table, an annotation is generated only if all conditions in the corresponding Annotation Criteria column are satisﬁed.

Note: You can ﬁnd all relevant annotation criteria in VCF ﬁles.

Variant type

Copy number

ampliﬁcation

Copy number deletion

Oncomine Gene Class

Gain-of-Function Ampliﬁcation

Loss-of-Function Deletion

™

Oncomine

Variant Class

™

Annotation criteria

•

SVTYPE = "CNV"

•

FILTER = "GAIN"

•

Occurs in a designated copy-gain gene

•

SVTYPE = "CNV"

•

FILTER = "LOSS"

•

Occurs in a designated copy-loss gene

Oncomine™ Comprehensive Assay Plus User Guide

(continued)

™

Oncomine

Variant Class

Variant type

Copy number

Oncomine Gene Class

Loss-of-Function ExonDeletion

exon deletion

Loss of

Loss-of-Function LOH

Heterozygosity

Long deletion Loss-of-Function LongDel

Gene fusion Gain-of-Function Fusion

Chapter 6 Variant analysis

Download Ion Reporter™ annotation VCF or TSV ﬁles

™

Annotation criteria

•

SVTYPE = "CNV"

•

FILTER = "LOSS"

•

SUBTYPE = "BigDel"

•

SVTYPE = "LOH"

•

LOH = "1"

•

FILTER = "GAIN" or "LOSS"or "PASS"

•

Positive mutation call

•

SVTYPE = "LongDel"

•

SVTYPE = "Fusion"

•

FILTER = "PASS"

•

Is a targeted fusion isoform

RNA exon variant Gain-of-Function RNAExonVariant

Expression imbalance

Loss-of-function

Gain-of-Function ExpressionImbala

nce

Loss-of-Function Truncating truncating de novo mutation

Missense hotspot mutation

Gain-of-

Function / Loss-

Hotspot

of-Function

In-frame hotspot mutation

Gain-of-

Function / Loss-

Hotspot

of-Function

•

SVTYPE = "RNAExonVariant" or "Fusion"

•

FILTER = "PASS"

•

Is a targeted RNA exon variant

•

SVTYPE = "RNAExonTiles"

•

FILTER = "PASS"

•

Record meets Targeted Isoforms Detected Requirement

•

Positive mutation call

•

Functional impact is frameshift block substitution, frameshift deletion, frameshift insertion, or nonsense

•

Occurs in a loss-of-function gene

•

Positive mutation call

•

Functional impact is missense

•

Transcript and codon position occur in predeﬁned missense hotspot list

•

Positive mutation call

•

Function, transcript, and coding syntax occur in predeﬁned in-frame hotspot list

Splice site hotspot mutation

Gain-of-

Function / Loss-

Hotspot

of-Function

Oncomine™ Comprehensive Assay Plus User Guide

•

Positive mutation call

•

Transcript, location, and exon occur in predeﬁned splice site hotspot list

Chapter 6 Variant analysis

Download Ion Reporter™ annotation VCF or TSV ﬁles

(continued)

Variant type

Intronic hotspot mutation

Promoter hotspot mutation

Gain-of-function truncating hotspot mutation

Loss-of-function truncating hotspot mutation

Oncomine Gene Class

Gain-of-

™

Oncomine

Variant Class

Hotspot Function / Lossof-Function

Gain-of-

Hotspot Function / Lossof-Function

Gain-of-Function Hotspot

Loss-of-Function Truncating

™

Annotation criteria

•

Positive mutation call

•

Transcript, location, and coding syntax occur in predeﬁned intronic hotspot list

•

Positive mutation call

•

Transcript, location, and coding syntax occur in predeﬁned promoter hotspot list

•

Positive mutation call

•

Function, transcript, and coding syntax occur in predeﬁned truncating hotspot list

•

Occurs in a gain-of-function gene

•

Positive mutation call

•

Function, transcript, and coding syntax occur in predeﬁned truncating hotspot list

•

Occurs in a loss-of-function gene

MNV hotspot mutation

EGFR exon 19 deletion

EGFR exon 20 insertion

ERBB2 exon 20 insertion

MET exon 14 skipping

Gain-of-

Hotspot Function / Lossof-Function

Gain-of-Function EGFRExon19Dele

tion

Gain-of-Function EGFRExon20Inse

rtion

Gain-of-Function ERBB2Exon20Ins

ertion

Gain-of-Function METExon14Skipp

ing

•

Positive mutation call

•

Transcript and coding syntax occur in MNV hotspot list

•

Positive mutation call

•

Functional impact is nonframeshift deletion, or nonframeshift block substitution

•

Deletion impacts codons 744-761 of EGFR

•

Positive mutation call

•

Functional impact is nonframeshift insertion, or nonframeshift block substitution

•

Insertion impacts codons 762-775 of EGFR or variant is COSM26720

•

Positive mutation call

•

Functional impact is nonframeshift insertion, or nonframeshift block substitution

•

Insertion impacts codons 770-783 of ERBB2

•

Positive mutation call

•

Location is splice site in MET exon 14, is intronic >= 4bp deletion in 30 nucleotides preceding MET exon 14, or variant is in MET Exon 14 Skipping conﬁrmed list

Oncomine™ Comprehensive Assay Plus User Guide

Tips

Tips and troubleshooting

Tips ................................................................................ 87

■

Troubleshooting ...................................................................... 88

■

•

Arrange samples in alternating columns on the plate for easier pipetting with multichannel pipettes during puriﬁcation with the DynaMag™–96 Side Magnet.

•

Plate seals can be ﬁrmly applied using the applicator in the MicroAmp™ Adhesive Film Applicator. Plate seals can be removed with less eort when hot. Try removing seals right after taking the plate out of the thermal cycler.

•

Use IonCode™ Barcode Adapters to avoid handling and diluting adapters. Alternatively, combine and dilute Ion Xpress™ Barcode Adapters in large batches, then carefully aliquot into 96‑well plates.

•

If library yield is below 50 pM, libraries can still be sequenced by adding a proportionally larger volume to a combined library or template preparation.

•

If the unampliﬁed library yield is below 50 pM, libraries can be rescued with library ampliﬁcation. Combine 25 μL of unampliﬁed library with 71 μL of 1X Library Amp Mix and 4 μL of 25X Library Amp Primers. Seal the plate and perform 5–10 library ampliﬁcation cycles as follows:

Stage

Hold 98°C 2 minutes

5–10 cycles 98°C 15 seconds

Hold 10°C Hold (up to 1 hour)

•

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

Temperature Time

64°C 1 minute

Oncomine™ Comprehensive Assay Plus User Guide

Appendix A Tips and troubleshooting

Troubleshooting

Library yield and quantiﬁcation

Observation Possible cause Recommended action

Library concentration is low– general

Details: (Library concentration is NOT indicative of quality.)

Library concentration is too high

Sample DNA or RNA was mis-

quantiﬁed.

Sample DNA or RNA quality was low.

PCR, digestion, or ligation was

inecient.

Residual ethanol in the sample DNA or RNA inhibited target

ampliﬁcation.

Residual ethanol from AMPure

puriﬁcation inhibited library ampliﬁcation.

AMPure™ XP beads were overdried.

FFPE RNA was not heat treated before reverse transcription.

Sample DNA or RNA was mis-

quantiﬁed.

More than 100 ng of sample DNA/RNA was used.

Requantify sample DNA using the TaqMan RNase P Detection Reagents Kit.

Requantify sample RNA with a Qubit Fluorometer.

Add more DNA or RNA or increase target ampliﬁcation cycles.

Ensure proper dispensing and mixing of viscous components at each step.

Incubate uncapped tube in hood for 1 hour.

Speed-vac tube at room temperature for 5 minutes.

™

Carefully remove all drops of ethanol before library ampliﬁcation, then centrifuge plate, if needed.

Do not dry the AMPure™ XP beads more than 5 minutes.

Heat FFPE RNA at 80°C for 10 minutes, then cool to room temperature before reverse transcribing.

Requantify sample DNA using the TaqMan RNase P Detection Reagents Kit; quantify RNA with a Qubit™ Fluorometer.

Add less DNA/RNA, or decrease target ampliﬁcation cycles.

™

Oncomine™ Comprehensive Assay Plus User Guide

Low amplicon uniformity (DNA only)

Observation Possible cause Recommended action

Appendix A Tips and troubleshooting

Troubleshooting

Pool representation is not balanced

Details: Example of pool imbalance. Within the Coverage Analysis Plugin, mean read depth per primer pool is plotted for a 2‑pool Ion AmpliSeq Panel. In this example, Primer Pool 1 has approximately one quarter the reads of Primer Pool

Short amplicons are under-represented

™

Amount of DNA in target ampliﬁcation reactions varied.

Pipetting is inaccurate when pools are combined after target ampliﬁcation.

Puriﬁcation was poor. Vortex AMPure™ XP Reagent

Make a master mix for each sample DNA.

Centrifuge the plate after target ampliﬁcation. Ensure that the entire volume of each pool is removed and combined into a single pool.

thoroughly before use, and be sure to dispense the full volume.

100% ethanol is dicult to pipet accurately; it is essential to pre-wet pipette tips.

In post-ligation library puriﬁcation, increase AMPure XP Reagent volume from 45 μL (1.5X) to 50 μL (1.7X).

™

Other

Observation Possible cause Recommended action

The number of on-target reads is lower than expected

Percentage of polyclonal ISPs is high (>40%)

Low quality ISPs are present at high percentage (>15%)

Oncomine™ Comprehensive Assay Plus User Guide

Unknown. Increase the number of target ampliﬁcation

Sample ID Panel targets were counted as o-target reads.

Library input was too high. Decrease amount of library added to the

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

Other. Check the appropriate template preparation user

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

cycles by 2.

Add back the on-target reads from the Sample ID Panel.

template preparation reaction by 50%.

guide for more information.

preparation.

Use a fresh dilution of library prepared in a lowbind tube.

Appendix A Tips and troubleshooting

Troubleshooting

Observation Possible cause Recommended action

Low quality ISPs are present at high percentage (>15%)

(continued)

MSI QC Failure

Details: Error message: "Not enough markers with coverage X"

MSI QC Failure

Details: Error message: "RMC Out of Range Failure X"

MSI QC Failure

Details: Error messge: "RMC Calibration Failure"

Other. Check the appropriate template preparation user

guide for more information.

MSI marker(s) had insucient coverage to reliably calculate MSI. This is common in NTC samples.

RMC ﬂow signal not within range of QC threshold.

Insucient RMC read coverage for calibration. RMC reagent may have been omitted during library preparation.

If the sample is not an NTC, review the previous “Troubleshooting” topics to ensure sucient coverage. For further assistance, contact support (see “Customer and technical support” on page 115).

Contact support to determine whether reanalysis with "Low RMC Signal" analysis workﬂow may resolve the QC failure. See “Customer and technical support” on page 115.

Remake library including RMC in the target ampliﬁcation reaction, then sequence the new library.

Oncomine™ Comprehensive Assay Plus User Guide

Supplemental information

Update Oncomine™ Comprehensive Assay Plus templates in Torrent Suite™ Software ........ 91

■

Install Oncomine™ Comprehensive Assay Plus workﬂows in Ion Reporter™ Software ......... 91

■

Download and install BED ﬁles ......................................................... 91

■

Conﬁgure the IonReporterUploader plugin in Torrent Suite™ Software ....................... 92

■

Set TMB Classiﬁcation parameters ..................................................... 93

■

Update Oncomine™ Comprehensive Assay Plus templates in Torrent Suite™ Software

In the upper right corner, click (Settings)4Updates, then scroll to the Update Products section.

In the Name column ﬁnd Oncomine™ Comprehensive Assay Plus, then in that row click Update.

The software update begins automatically and displays as Complete when ﬁnished.

Install Oncomine™ Comprehensive Assay Plus workﬂows in Ion Reporter™ Software

To install or update the Oncomine™ Comprehensive Assay Plus workﬂows in Ion Reporter™ Software, contact your service representative to schedule an update.

Download and install BED ﬁles

Contact your service representative to obtain the latest versions of Oncomine™ Comprehensive Assay Plus BED ﬁles.

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

ﬁles.

Click the dropdown list.

Oncomine™ Comprehensive Assay Plus User Guide

(Settings) tab in the upper right of the screen, then select References from the

Appendix B

Conﬁgure the IonReporterUploader plugin in Torrent Suite™ Software

Supplemental information

Upload the target regions BED ﬁle:

In the left navigation menu, click Target Regions, then click the Add Target Regions button.

Select hg19 - Homo sapiens from the Reference dropdown list.

Click Select File, then navigate to and select the target regions BED ﬁle, which has the following extension: .

.designed.bed

Click Open, then click Upload Target Regions File.

Upload the hotspots BED ﬁle:

In the left navigation menu, click Hotspots, then click the Add Hotspots button.

Select hg19 - Homo sapiens from the Reference dropdown list.

Click Select File, then navigate to and select the hotspots ﬁle, which has the following extension:

.hotspots.bed

Click Open, then click Upload Hotspots File.

The installed BED ﬁles appear in the dropdown lists in the Ion Reporter™ Software.

Conﬁgure the IonReporterUploader plugin in Torrent Suite Software

Click (Settings)4Ion Reporter Conﬁgure.

™

In the Ion Reporter Uploader Account Conﬁguration screen, click Add Account4Ion Reporter.

Oncomine™ Comprehensive Assay Plus User Guide

Appendix B Supplemental information

Set TMB Classiﬁcation parameters

In the Add Ion Reporter account screen, enter the following information into the ﬁelds:

Field Directions

Server Type Select:

Display Name Enter a meaningful name of your choice. This name is used in the run plan

Server Enter:

Port Enter: 443

Username Enter your Ion Reporter™ Software username (your email address)

Password Enter your Ion Reporter™ Software password

[1]

Ask your Ion Reporter™ Server administrator for these values.

The "Default Account" is the account that is conﬁgured by default in run templates and run plans.

[1]

template wizard and is seen by other Torrent Suite™ Software users. Use only alphanumeric characters, spaces, and underscores.

[1]

If this account is the main account to be used for ﬁle transfers, enable the Default Account checkbox.

Note: You can always change this selection in the Planned Run template workﬂow bar and in the Upload to IR quick link.

Click Get Versions, select Ion Reporter 5.16 or later, then click ✓Add.

Set TMB Classiﬁcation parameters

Before the Ion Reporter™ Software analysis workﬂow can determine the TMB classiﬁcation the threshold parameters must be set in the analysis workﬂow. Default threshold values (0) result in a value of Undeﬁned.

Under the Workﬂows tab, in the Overview screen, search for the workﬂow, then select it in the list.

In the Details pane to the right of the list, click 4Copy (to create a new analysis workﬂow) or Edit to modify the existing analysis workﬂow.

Advance to the Parameters step in the workﬂow bar.

Click the Tumor Mutation Burden tab. The Tumor Muational Burden Filter Chain and the Tumor Mutational Burden Calculation

Version are displayed.

Oncomine™ Comprehensive Assay Plus User Guide

Appendix B Supplemental information

Set TMB Classiﬁcation parameters

Scroll to TMB-Low Threshold, then enter threshold values for the following parameters.

Item

TMB-Low Threshold Tumor Mutational Burden (mut/mb) threshold below which a sample is

deﬁned TMB-Low.

TMB-High Threshold Tumor Mutational Burden (mut/mb) threshold above which a sample is

deﬁned TMB-High.

Click Next to advance to the Conﬁrm step, then click Save Workﬂow.

Description

Oncomine™ Comprehensive Assay Plus User Guide

CNV baseline creation

Use VCIB CNV baseline ............................................................... 95

■

Create a CNV baseline ................................................................ 96

■

Augment (add Samples to) an existing VCIB CNV baseline ................................ 97

■

Create an Ion Reporter analysis workﬂow ................................................ 99

■

Launch an analysis .................................................................. 100

■

In Ion AmpliSeq™ assays, Copy Number estimates are made by counting reads for each amplicon, making adjustments to account for certain types of variability, comparing those read counts to expected counts for those amplicons in a "normal" sample, and then making further adjustment.

Known sources of variability include pool imbalance (when the assay has more than one pool of amplicons), total number of reads and per amplicon attributes of GC proportion, and length of the amplicon insert. In practice, we observe other variability that does not associate with known attributes yet is systematic. The method that we use trains on many diverse samples, captures systematic eects, and encodes these into a ﬁle (the "baseline").

When augmenting a baseline, new samples are run, the size of each systematic eect encoded in the baseline is estimated, and a correction is applied to remove the eect. These added samples need not be normal, and should be diverse so as to capture likely systematic variation.

The following instructions walk you through using the new Variability Correction Information Baseline (VCIB) CNV baseline, creating a new VCIB CNV baseline, or augmenting an existing VCIB CNV baseline for Oncomine™ Comprehensive Assay panels.

Use VCIB CNV baseline

If you want to use the VCIB CNV baseline included in Ion Reporter™ Software 5.16, simply select it when creating your workﬂow.

Note: The VCIB CNV baseline is currently noncompatible with the Ion GRCh38 human reference.

Oncomine™ Comprehensive Assay Plus User Guide

Appendix C

Create a CNV baseline

CNV baseline creation

Create a CNV baseline

Ion Reporter™ Software provides a wizard to guide you through Copy Number Variation (CNV) Baseline creation.

In the Workﬂows tab, in the Presets screen, click Create Preset, then select Copy Number Baseline

from the dropdown list.

Click AmpliSeq, select Oncomine™ Comprehensive Plus DNA Regions v1.2 as your Target Regions ﬁle, then click Next.

Select at least 48 samples, ﬂag at least 6 of the selected samples as "Normal" by selecting the checkbox in the Normal column, then click Next.

Note: Male or Female gender must be speciﬁed for Normal samples, but samples that are not ﬂagged as normal can be male, female, or unknown. You can use the Summary panel to see your

totals.

Enter a name for your baseline, then click Create Baseline.

Note: Log ﬁles for both successful and failed analyses include the BaselineCreation.log ﬁle, which has the BAM ﬁles named that were rejected due to similarity to other ﬁles in the baseline, as well as the map.TmapMergeActor-00.err ﬁle that has the BAM ﬁles named that were rejected due to QC failure.

To add this new baseline to your workﬂow, proceed to “Create an Ion Reporter analysis workﬂow” on

page 99.

Oncomine™ Comprehensive Assay Plus User Guide

Appendix C CNV baseline creation

Augment (add Samples to) an existing VCIB CNV baseline

Ion Reporter™ Software provides a wizard to guide you through Copy Number Variation (CNV) baseline creation. This example describes how to add additional samples to an existing CNV baseline.

Under the Workﬂows tab, in the Presets screen, click Create Preset, then select Copy Number Baseline

from the dropdown list.

Click AmpliSeq, select Oncomine™ Comprehensive Plus DNA Regions v1.2 as your Targets Region ﬁle, then click Next.

Select the Start with an existing CNV Baseline checkbox, then select a baseline from the dropdown list.

Note: By default, the software prompts you to add another 48 samples. However, you can set the number to 1 or more. Add non-Normal samples. Marking samples as "Normal" in the augmentation workﬂow has no eect, only the original Normals in the ﬁrst baseline creation are treated as Normals in the augmented baseline.

Click the Conﬁgure Parameters link.

Oncomine™ Comprehensive Assay Plus User Guide

Appendix C

Augment (add Samples to) an existing VCIB CNV baseline

CNV baseline creation

In the Conﬁgure Parameters dialog, click Cnv Baseline Creation, then Advanced. Set the Minimum number of samples required to add to an existing baseline to the number you are adding, click Done, then click Next.

Select additional samples, then click Next.

Enter a name for your baseline, then click Create Baseline to save.

Oncomine™ Comprehensive Assay Plus User Guide

Appendix C CNV baseline creation

Create an Ion Reporter analysis workﬂow

Ion Reporter™ Software provides a wizard to guide you through creating a workﬂow. However, it can be easier to copy an existing Oncomine™ workﬂow and edit it by adding your newly created baseline.

In the Workﬂows tab, in the Overview screen, select an appropriate Oncomine™ Comprehensive

workﬂow.

In the Details pane, click Actions4Copy. The workﬂow wizard opens to the Research Application step in the Create screen.

Click Next to advance to the Reference step.

Conﬁrm that hg19 is the selected Reference, then select a Target Regions, Hotspot Regions, and Fusions BED ﬁle from the respective dropdown lists. Click Next.

Select an Annotation Set from the dropdown list, then click Next.

Select a Filter Chain from the dropdown list, then click Next.

In the Copy Number step, select the baseline that you want to use from the Baseline dropdown list, then click Next.

In the Plugins step, ensure that all In-Analysis plugins are deselected, then click Next.

Select a Final Report Template from the dropdown list, then click Next.

10.

In the Parameters step, review the default settings, then click Next.

Note: Although Read Mapping parameters are exposed in workﬂow creation, it is not necessary to change any settings.

11.

In the Conﬁrm step, enter a Workﬂow Name and Description, then click Save Workﬂow.

Oncomine™ Comprehensive Assay Plus User Guide

Appendix C CNV baseline creation

Launch an analysis

In the Home tab, in the Dashboard screen, click Launch Analysis.

In the Launch Analysis wizard, in the Workﬂow step, select your custom workﬂow or one of the pre-installed Oncomine™ workﬂows, then click Next.

Select the samples to include in the analysis.

Use the Samples dropdown list to ﬁlter the available samples.

Click within a sample row to select each sample to include in a sample group, then click Add Samples in the Sample Groups pane.

Enter a Group Name, then click Add to Analysis.

100

Note: The Percentage Cellularity sample attribute is required for DNA samples.

Repeat substep 3b and substep 3c to add additional Sample Groups.

Click Next 2 times to move to the Conﬁrm & Launch step.

Oncomine™ Comprehensive Assay Plus User Guide

Thermo Fisher Scientific Oncomine User Manual

Specifications and Main Features

Frequently Asked Questions

User Manual

Contents

Product description

Contents and storage

Oncomine™ Comprehensive Assay Plus – Manual Library Preparation

Oncomine™ Comprehensive Assay Plus, RNA – Manual Library Preparation

Oncomine™ Comprehensive Assay Plus – Automated Library Preparation

Oncomine™ Comprehensive Assay Plus, RNA – Automated Library Preparation

Required materials not supplied

Recommended materials

Procedural guidelines

Before each use of the kit

Library preparation from genomic DNA or RNA

Create a sample set to prepare 2 pools of 4 libraries each

Automated RNA library preparation

Reverse transcribe RNA for Chef Ready library preparation

Ion Chef™ Instrument setup information for automated RNA library preparation

Automated DNA library preparation

Remove deaminated bases from FFPE DNA

Ion Chef™ Instrument setup information for automated DNA library preparation

Reverse transcribe RNA for manual library preparation

Amplify the cDNA targets

Remove deaminated bases from FFPE DNA

Amplify the DNA targets

Library preparation

Transfer the DNA amplicons

Partially digest the DNA and cDNA amplicons

Ligate adapters to the amplicons and purify

Ion Xpress™ Barcode Adapters only: Combine and dilute adapters

Perform the ligation reaction

Elute and dilute the library

Quantify the library by qPCR and calculate the dilution factor

Combine libraries

Guidelines for templating and sequencing

About Planned Runs

Update Oncomine™ Comprehensive Assay Plus templates in Torrent Suite™ Software

Create a custom Planned Run template

Create a Planned Run for manual library preparation

Create a Planned Run for automated library preparation using sample sets

Manually launch a DNA and Fusions analysis

Manually launch a DNA analysis

Manually launch a Fusions analysis

View results

Fusion results

View RNA Exon Tile Fusion Imbalance

View RNA exon variants

Visualize tumor mutational burden analysis results

QC metrics for tumor mutational burden

Sample results

MSI analysis results

View MSI parameters

MSI parameters

Visualize MSI analysis results

Genomic segmentation analysis

Visualize exon level loss results

Generate an Analysis Results Report

Requirements for variant annotation in Ion Reporter™ Software

Tips

Troubleshooting

Low amplicon uniformity (DNA only)

Other

Update Oncomine™ Comprehensive Assay Plus templates in Torrent Suite™ Software

Use VCIB CNV baseline

Create a CNV baseline

Augment (add Samples to) an existing VCIB CNV baseline

Launch an analysis