Thermo Fisher Scientific Oncomine User Manual

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).

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4 5 6

8 9

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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

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8 9

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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

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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

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