oncomine brca research assay - thermo fisher scientific · 2019. 2. 26. · product information...

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

Oncomine™ BRCA Research AssayUSER GUIDE

For manual library preparation

Catalog Number A32840Publication Number MAN0014634

Revision B.0

Manufacturer: Multiple Life Technologies Corporation manufacturing sites are responsible for manufacturing the products associated withthe workflow covered in this guide.

Corporate entity: Life Technologies Corporation | Carlsbad, CA 92008 USA | Toll Free in USA 1 800 955 6288

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

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL,INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOURUSE OF IT.

Revision history: Pub. No.MAN0014634

Revision Date DescriptionB.0 8 June 2017 Support added for exon deletion functionality in updated Oncomine™ BRCA

Research Assay workflows in Ion Reporter™ Software 5.4.

A.0 12 October 2016 New user guide for the Oncomine™ BRCA Research Assay with detailedinstructions for manual library preparation.

Important Licensing Information: This product may be covered by one or more Limited Use Label Licenses. By use of this product, you accept theterms 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. Agencourt and AMPure aretrademarks of Beckman Coulter, Inc. Eppendorf and Eppendorf LoBind are trademarks of Eppendorf AG. TaqMan is a registered trademark of RocheMolecular Systems, Inc., used under permission and license. Agilent and Bioanalyzer are trademarks of Agilent Technologies, Inc.

©2017 Thermo Fisher Scientific Inc. All rights reserved.

Contents

■ CHAPTER 1 Product information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Kit contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Ion Library Equalizer™ Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Ion Xpress™ Barcode Adapters Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Recommended materials and equipment (optional) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Oncomine™ BRCA Research Assay, Chef-Ready Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Oncomine™ BRCA Research Assay manual workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

■ CHAPTER 2 Prepare Oncomine™ BRCA ResearchAssay libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Procedural guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Amplify the targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Combine the target amplification reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Partially digest primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Ligate adapters to the amplicons and purify . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16Combine and dilute the adapters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16Perform the ligation reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Purify the library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Option 1: Equalize the library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Amplify the library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Wash the Equalizer™ Beads (if not previously performed) . . . . . . . . . . . . . . . . . . . . . . 20Add Equalizer™ Capture to the amplified library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20Add Equalizer™ Beads and wash . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20Elute the equalized libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Option 2: Quantify the library by qPCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22Elute the library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22Quantify libraries by qPCR and calculate the dilution factor . . . . . . . . . . . . . . . . . . . . . 22

Oncomine™ BRCA Research Assay User Guide 3

Option 3: Quantify the amplified libraries by Qubit™ Fluorometer or Agilent™ 2100Bioanalyzer™ instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Amplify the library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24Purify the amplified library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24Qubit™ Fluorometer: Quantify the library and calculate the dilution factor . . . . . . . . . 26Agilent™ 2100 Bioanalyzer™ instrument: Quantify the library and calculatethe dilution factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Combine Oncomine™ BRCA Research Assay barcoded libraries for sequencing onthe same Ion chip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Guidelines for templating and sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

■ CHAPTER 3 Create a Planned Run and analyze results withan Ion Reporter™ workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

Configure the Torrent Browser to use your Ion Reporter™ account . . . . . . . . . . . . . . . . . . . 31

About Ion Reporter™ Oncomine™ BRCA Research workflows . . . . . . . . . . . . . . . . . . . . . . . . . 33

Upload Target Regions and Hotspots BED files into Torrent Suite™ Software . . . . . . . . . . . 34

Create a Planned Run with an Oncomine™ BRCA Research template . . . . . . . . . . . . . . . . . . 36

Using the Ion AmpliSeq™ Sample ID Panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

View Ion Reporter™ analysis results and generate a report . . . . . . . . . . . . . . . . . . . . . . . . . . 44

Download results or generate a report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

Edit an Ion Reporter™ Oncomine™ BRCA Research workflow with a newAnnotation Set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

Create a new Annotation Set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57Copy and edit a workflow to add a new Annotation Set . . . . . . . . . . . . . . . . . . . . . . . . . . 60

Oncomine™ Knowledgebase Reporter Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

■ APPENDIX A Tips and troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62Tips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63Library yield and quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63Other . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

■ APPENDIX B Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

■ Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

Contents

4 Oncomine™ BRCA Research Assay User Guide

Product information

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

Product description

The Oncomine™ BRCA Research Assay (Cat. No. A32840) provides reagents for themanual preparation from genomic DNA of up to 24 Oncomine™ BRCA ResearchAssay sample libraries targeting the BRCA1 and BRCA2 genes. Use of Ion Xpress™Barcode Adaptors in library preparation enables you to combine barcoded libraries intemplating reactions and sequencing runs. Up to 32 germline libraries, or up to8 somatic libraries, can be combined and sequenced on a single Ion 318™ chip, and upto 32 germline or somatic libraries can be combined and sequenced on an Ion 530™chip. The protocol requires 10 ng of DNA isolated from unfixed or formalin-fixedparaffin-embedded (FFPE) tissue per target amplification reaction (20 ng total).

This guide also provides optional protocols for normalizing library concentrationusing the Ion Library Equalizer™ Kit, quantifying library concentration using qPCR,and quantifying library concentration with the Qubit™ Fluorometer, or the Agilent™2100 Bioanalyzer™ instrument.

Note: The Oncomine™ BRCA Research Assay Chef-Ready Library Preparation Kit(Cat. No. A32841) is also available for automated library preparation (see “Oncomine™ BRCA Research Assay, Chef-Ready Kit“ on page 10). The kit providesOncomine™ BRCA Research Assay Pools 1 and 2 at 2X concentration pre-measured inbarcoded Primer Pool tubes ready to load into an Ion AmpliSeq™ Chef Reagents DL8cartridge.

1


Kit contents and storage

The Oncomine™ BRCA Research Assay (Cat. No. A32840) is a bundle of theOncomine™ BRCA Research Assay Manual Library Preparation panel and the IonAmpliSeq™ Library Kit Plus, sufficient for manually preparing 24 libraries.

Component Amount Storage

Oncomine™ BRCA Research Assay Manual Library Preparation, 5X concentration (PartNo. A32842)

Oncomine™ BRCA Pool 1 (blue cap) 48 µL –30ºC to –10ºC

Oncomine™ BRCA Pool 2 (red cap) 48 µL

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

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

FuPa Reagent (brown cap) 48 µL

Switch Solution (yellow cap) 96 µL

DNA Ligase (blue cap) 48 µL

25X Library Amp Primers[1] (pink cap) 48 µL

1X Library Amp Mix (black cap) 1.2 mL

Low TE (clear cap) 1 each 15°C to 30°C[2]

[1] Can be used with the Ion Library Equalizer™ Kit, and also for library amplification if quantifying amplified library with the Qubit™ 3.0 Fluorometer or Agilent™ 2100 Bioanalyzer™.

[2] Can be stored at −30ºC to −10ºC.

Chapter 1 Product informationKit contents and storage1


Ion Library Equalizer™ Kit

The Ion Library Equalizer™ Kit, ordered separately, provides a streamlined methodfor normalizing library concentration without quantification.

The Ion Library Equalizer™ Kit (Cat. No. 4482298) contains reagents for 96 reactions.

Component Amount Storage

Equalizer™ Primers (pink cap) 200 µL 2ºC to 8ºC

Equalizer™ Capture (purple cap) 1 mL

Equalizer™ Elution Buffer (clear cap) 10 mL

Equalizer™ Beads (orange cap) 300 µL

Equalizer™ Wash Buffer (clear cap) 35 mL

Ion Xpress™ Barcode Adapters Kits

Each kit, ordered separately, provides 16 different barcode adapters sufficient for 640total reactions.

Component Quantity Volume Storage

Ion Xpress™ P1 Adapter (violet cap) 1 tube 320 µL

–30ºC to –10ºCIon Xpress™ Barcode X (white cap) 16 tubes(1 per barcode)

20 µL each

The following Ion Xpress™ Barcode Adapters Kits are available:• Ion Xpress™ Barcode Adapters 1–16 (Cat. No. 4471250)• Ion Xpress™ Barcode Adapters 17–32 (Cat. No. 4474009)• Ion Xpress™ Barcode Adapters 33–48 (Cat. No. 4474518)• Ion Xpress™ Barcode Adapters 49–64 (Cat. No. 4474519)• Ion Xpress™ Barcode Adapters 65–80 (Cat. No. 4474520)• Ion Xpress™ Barcode Adapters 81–96 (Cat. No. 4474521)

Complete set of adapters:• Ion Xpress™ Barcode Adapters 1–96 (Cat. No. 4474517)

Chapter 1 Product informationIon Library Equalizer™ Kit 1


Required materials not supplied

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

Item Source

Veriti™ 96‑well Thermal Cycler, or equivalent See web product pages

MicroAmp™ Optical 96-well Reaction Plates N8010560

4306737 (with barcode)

MicroAmp™ Adhesive Film 4306311

MicroAmp™ Compression Pad 4312639

Agencourt™ AMPure™ XP Kit Fisher ScientificNC9959336, NC9933872

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

Nuclease-free Water AM9932

Absolute ethanol MLS

Pipettors, 2–1000 μL, and low-retention filtered pipette tips MLS

Eppendorf™ DNA LoBind™ Microcentrifuge Tubes Fisher Scientific13-698-791

96-well plate centrifuge MLS

Chapter 1 Product informationRequired materials not supplied1


http://www.thermofisher.comhttp://fisherscientific.com

Recommended materials and equipment (optional)

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

Item Source

Recommended additional equipment for qPCR

Real-time PCR instrument (for example, AppliedBiosystems™ 7900HT, 7500, StepOne™, StepOnePlus™,ViiA™ 7, QuantStudio™ 3/5 Systems, or QuantStudio™ 12KFlex Real-Time PCR System)

See web product pages

Recommended for gDNA isolation

Ion AmpliSeq™ Direct FFPE DNA Kit A31133, A31136

RecoverAll™ Total Nucleic Acid Isolation Kit AM1975

MagMAX™ FFPE Total Nucleic Acid Isolation Kit 4463365

PureLink™ Genomic DNA Mini Kit K182000

Recommended for gDNA quantification

TaqMan® RNase P Detection Reagents Kit 4316831

Qubit™ 3.0 Fluorometer or Qubit™ 2.0 Fluorometer[1]

Qubit™ dsDNA HS Assay Kit

Q33216

Q32851, Q32854

Recommended for library quantification (If you are NOT using the Ion LibraryEqualizer™ Kit for library normalization, select one of the following:)

Ion Library TaqMan® Quantitation Kit 4468802

Qubit™ 3.0 Fluorometer or Qubit™ 2.0 Fluorometer[1]

Qubit™ dsDNA HS Assay Kit

Q33216

Q32851, Q32854

Agilent™ 2100 Bioanalyzer™ Instrument

Agilent™ High Sensitivity DNA Kit

Agilent G2939AA

Agilent 5067-4626

Additional material for the Ion AmpliSeq™ Direct FFPE DNA Kit

(Optional) Uracil DNA Glycosylase (UDG) 18054015, or EN0361

[1] Supported but no longer available for purchase.

Chapter 1 Product informationRecommended materials and equipment (optional) 1


http://www.thermofisher.comhttp://fisherscientific.com

Oncomine™ BRCA Research Assay, Chef-Ready Kit

The Oncomine™ BRCA Research Assay, Chef-Ready Kit (Cat. No. A32841, orderedseparately) provides Oncomine™ BRCA Research Assay Pools 1 and 2 at 2Xconcentration pre-measured in barcoded Primer Pool tubes ready to load into an IonAmpliSeq™ Chef Reagents DL8 cartridge. In addition, the kit provides all the reagentsand supplies in an Ion AmpliSeq™ Kit for Chef DL8 sufficient for preparing32 libraries. See the Ion AmpliSeq™ Library Preparation on the Ion Chef™ System UserGuide (Pub. No. MAN0013432) for detailed information on preparing Oncomine™

BRCA Research Assay libraries on the Ion Chef™ System.

Component Part No. Quantity perkit Storage

Oncomine™ BRCA Research Assay Chef-Ready Library Preparation

Oncomine™ BRCA Tube 1 A32843 4 × 150 µL –30°C to–10°C

Oncomine™ BRCA Tube 2 4 × 150 µL

Ion AmpliSeq™ Kit for Chef DL8

Ion AmpliSeq™ Chef Reagents DL8 A29025 4 cartridges –30°C to–10°C

Ion AmpliSeq™ Chef Solutions DL8 A29026 4 cartridges 2°C to 8°C[1]

Ion AmpliSeq™ Chef Supplies DL8 (per insert)

• Ion AmpliSeq™ Tip Cartridge L8

• PCR Frame Seal

• Enrichment Cartridge

A29027 1 box with4 inserts

15°C to 30°C

IonCode™ 0101–0132 in 96 Well PCR Plates(dried)

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)

A29028 1 set of4 plates

15°C to 30°C

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

Chapter 1 Product informationOncomine™ BRCA Research Assay, Chef-Ready Kit1


Oncomine™ BRCA Research Assay manual workflow

Amplify DNA

Isolate and quantify gDNA

Oncomine BRCA Research Assay

Pool 2 Pool 1

Combine amplification reactions in a new column

Equalize, or quantify and dilute libraries

Digest primers

(18 cycles for non-FFPE gDNA, 21 cycles for FFPE gDNA)

Ligate barcode adapters

Purify amplicons

Combine libraries (optional)

TM

Primer pool 1 2

Sample A

B

C

D

E

F

G

H

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

Prepare master mix

Add DNA to individual wells

Processing of 8 gDNA samples and libraries at two wells per sample is shown, withSample 1 in wells A3 and A4, Sample 2 in wells B3 and B4, etc. After targetamplification in 10 µL reactions, Pool 1 and Pool 2 amplification reactions arecombined into new wells in the plate. After partial digestion of primers, ligation ofbarcode adapters, and amplicon purification, barcoded libraries are equalized to~100 pM concentration, or quantified and diluted to 100 pM concentration, and can becombined before template preparation.

Chapter 1 Product informationOncomine™ BRCA Research Assay manual workflow 1


Prepare Oncomine™ BRCA ResearchAssay libraries

■ Procedural guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12■ Amplify the targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14■ Combine the target amplification reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15■ Partially digest primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16■ Ligate adapters to the amplicons and purify . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16■ Option 1: Equalize the library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19■ Option 2: Quantify the library by qPCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22■ Option 3: Quantify the amplified libraries by Qubit™ Fluorometer or

Agilent™ 2100 Bioanalyzer™ instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24■ Combine Oncomine™ BRCA Research Assay barcoded libraries for

sequencing on the same Ion chip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28■ Guidelines for templating and sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Note: This chapter gives procedures for manually preparing Oncomine™ BRCAResearch Assay libraries. For procedures for using the Oncomine™ BRCA ResearchAssay, Chef-Ready Kit to prepare libraries on the Ion Chef™ System, see the IonAmpliSeq™ Library Preparation on the Ion Chef™ System User Guide (Pub. No.MAN0013432).

Procedural guidelines

Reagent preparation:• Thaw components that contain enzymes—such as 5X Ion AmpliSeq™ HiFi Mix,

FuPa Reagent, and DNA Ligase—on ice, and keep on ice during procedure.• Thaw kit components other than enzymes, including genomic DNA and primer

panels, at room temperature until no ice is present in the tubes. Vortex allreagents for 5 seconds (EXCEPT for enzymes, which should be flick-mixed) andpulse-centrifuge before use. A pulse-centrifugation is a 5-second centrifugation atmaximum speed.

• 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 dissolve.

• If using the Ion Library Equalizer™ Kit, minimize freeze-thawing of Equalizer™Primers by aliquoting as needed for your experiments. Primers can be stored at4°C for one year.

• Pipet viscous solutions slowly and ensure complete mixing by vigorous vortexingor pipetting up and down 5 times.

2


Laboratory and PCR setup:• Use good laboratory practices to minimize cross-contamination of products. If

possible, perform PCR setup in an area or room that is separate from templatepreparation. Always change pipette tips between samples.

• Before starting and after use, wash the working surface with 10% bleach followedby two water rinses.

• Use inner wells of PCR plate if possible, and skip wells or columns to preventcross-contamination between samples.

• Use a calibrated thermal cycler that is specified in “Required materials notsupplied“ on page 8.

• Ensure that the correct cycling protocol is being used before starting the thermalcycler.

• Store one 96-well cold block at –30°C to –10°C and one 96-well cold block at 2°Cto 8°C before use.

• Use the heated lid (105°C) for all thermal cycling conditions.

Pipetting recommendations:• Use aerosol-barrier pipette tips. Change pipette tips between samples.• Pipet viscous solutions (enzymes, beads, Switch Solution) slowly and ensure

complete mixing in the MicroAmp™ 96-well plate.• Avoid introducing air bubbles when pipetting by keeping the pipette tip at thebottom of the solution in the wells.

• Set pipette to the recommended volume for up and down mixing and insert tipinto solution with pipette plunger depressed to avoid introducing air bubbles.

• Visually check tips to ensure that volumes are equivalent if using a multi-channelpipette.

• Touch tip to side of well and slowly dispense reagent on the side of the well toform a droplet. This practice enables you both to pipet small volumes accuratelyand to see that you added reagent to the well.

• Ensure that reagent is adequately dispensed from the pipette tip.

Guidelines for DNA isolation and quantification:• For each target amplification reaction, use 3,000 copies (10 ng of genomic DNA in

≤6 µL) from peripheral blood, cell lines, or FFPE tissue. See “Required materialsnot supplied“ on page 8 for kits that are recommended for isolating DNA.

• We recommend the TaqMan® RNase P Detection Reagents Kit (Cat. No. 4316831)for quantifying amplifiable human genomic DNA. The Qubit™ dsDNA HS AssayKit (Cat. No. Q32851 or Q32854) can also be used with the Qubit™ 2.0 or Qubit™3.0 Fluorometer.

• We do not recommend methods such as densitometry (for example, NanoDrop™Spectrophotometers), because these methods do not discriminate between DNAand RNA and therefore are sensitive to small fragments of hydrolyzed RNA. Useof densitometry can lead to overestimation of the concentration of sample DNA,under-seeding of the target amplification reaction, low-quality libraries, and lowlibrary yields.

Chapter 2 Prepare Oncomine™ BRCA Research Assay librariesProcedural guidelines 2


Amplify the targets

IMPORTANT! Primer pools and 5X Ion AmpliSeq™ HiFi Mix are viscous. Pipet slowlyand mix thoroughly. We recommend PCR setup on ice or a cold block.

1. For each sample, add components to 2 adjacent wells of a 96-well plate using alow volume pipettor, as described in the following table. See the plate layoutexample following the table.

Note: If preparing multiple libraries, master mixes without DNA arerecommended. Add master mixes to the plate first.

IMPORTANT! Ion AmpliSeq™ Sample ID Panel primer pairs are included in theOncomine™ BRCA Research Assay. Do not add additional Ion AmpliSeq™Sample ID Panel primers to target amplification reactions.

Component Volume per well (Pool 1) Volume per well (Pool 2)

5X Ion AmpliSeq™ HiFi Mix(red cap)

2 µL 2 µL

Oncomine™ BRCA Pool 1(blue cap)

2 µL —

Oncomine™ BRCA Pool 2(red cap)

— 2 µL

DNA, 10 ng ≤6 µL ≤6 µL

Nuclease-free Water to 10 µL to 10 µL

Note: Add 10 ng of control DNA to 2 wells if running a control.

Primer Pool1 2

OncomineTM BRCA Research Assay

2 µL/well

Pool 1 Pool 2

A

B

C

D

E

F

G

H

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

Note: Avoid using columns on the periphery of the plate.

Chapter 2 Prepare Oncomine™ BRCA Research Assay librariesAmplify the targets2


2. Mix thoroughly by pipetting up and down, then seal the plate with MicroAmp™Adhesive Film. Alternatively, the plate can be vortexed after sealing, followed bycentrifugation at 100 × g for 30 seconds to collect volume at the bottom of thewells.

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

Stage Step Temperature Time

Hold Activate theenzyme

99°C 2 minutes

Cycle (18 cycles fornon-FFPE DNA,21 cycles for FFPEDNA)

Denature 99°C 15 seconds

Anneal and Extend 60°C 4 minutes

Hold — 10°C Hold (16 hoursmaximum)

Note: See the Ion AmpliSeq™ Library Kit 2.0 User Guide (Pub. No. MAN0006735)for guidance on cycle number if your gDNA is

Partially digest primers

Note: FuPa Reagent is viscous. Flick to mix, then pulse-centrifuge. To avoid carryingover excess enzyme, do not submerge the whole tip in the FuPa Reagent solution.Aspirate solution from the surface.

1. Add 2 µL of FuPa Reagent (brown cap) to each amplified sample. The totalvolume of each reaction is now ~22 µL.

2. Seal the plate with MicroAmp™ Adhesive Film, vortex thoroughly, thencentrifuge at 100 × g for 30 seconds to collect droplets.

3. Place a MicroAmp™ Compression Pad on the plate, load the plate in the thermalcycler, then run the following program:

Temperature Time

50°C 10 minutes

55°C 10 minutes

60°C 20 minutes

10°C Hold (for up to 1 hour)

4. Centrifuge the plate at 100 × g for 30 seconds before proceeding to the next step.

Ligate adapters to the amplicons and purify

For each Ion Xpress™ Barcode X chosen, prepare a mix of Ion P1 Adapter and IonXpress™ Barcode X at a final dilution of 1:4 for each adapter. Store diluted adapters at–20°C.

Combine the volumes indicated in the following table and use 2 µL of this barcodeadapter mix in step 1 below. Scale volumes as necessary.

Component Volume

Ion P1 Adapter 2 µL

Ion Xpress™ Barcode X[1] 2 µL

Nuclease-free Water 4 µL

Total 8 µL

[1] X = barcode chosen

Combine anddilute theadapters

Chapter 2 Prepare Oncomine™ BRCA Research Assay librariesPartially digest primers2


IMPORTANT! When handling barcode adapters, avoid cross-contamination bychanging gloves frequently and opening one tube at a time.

IMPORTANT! If there is visible precipitate in the Switch Solution, vortex or pipet upand down at room temperature to dissolve. Ensure that there is no precipitate in thetube cap.

1. Centrifuge the plate at 100 × g for 30 seconds in a plate centrifuge to collectcontents at the bottom of the wells.

2. Carefully remove the plate seal, then add the following components in the orderindicated to each well containing digested sample. After adding each component,mix by setting a pipette to 15 µL, then pipetting up and down slowly 5 times.

Note: Do not make a master mix of these components.

Order ofaddition Component Volume

1 Switch Solution (yellow cap) 4 µL

2 Ion Xpress™ Barcode Adaptor/P1 Adaptormix[1]

2 µL

3 DNA Ligase (blue cap) 2 µL

— Total volume (including ~22 µL of digestedamplicons)

~30 µL

[1] Prepared in "Combine and dilute the adaptors".

3. Seal the plate with a new MicroAmp™ Adhesive Film, vortex thoroughly, thencentrifuge at 100 × g for 30 seconds to collect droplets.

4. Place a MicroAmp™ Compression Pad on the plate, load the plate in the thermalcycler, then run the following program:

Temperature Time

22°C 30 minutes

68°C 5 minutes

72°C 5 minutes

10°C Hold (for up to 24 hours)

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

5. Centrifuge the plate at 100 × g for 30 seconds before proceeding to the next step.

Perform theligation reaction

Chapter 2 Prepare Oncomine™ BRCA Research Assay librariesLigate adapters to the amplicons and purify 2


IMPORTANT! Bring the AMPure™ XP Reagent to room temperature and vortexthoroughly to disperse the beads before use. Pipet solution slowly.

Do NOT substitute a Dynabeads™-based purification reagent for the AMPure™ XPReagent.

1. Prepare fresh 70% ethanol: Combine 230 µL of 100% ethanol with 100 µL ofNuclease-free Water per sample.

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

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

4. Place the plate in a DynaMag™-96 Side Magnet, then incubate for 2 minutes.Carefully remove and discard the supernatant without disturbing the pellet.

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

6. Repeat step 5 for a second wash.

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

IMPORTANT! Residual ethanol droplets inhibit library amplification. Ensurethat all ethanol droplets are removed from the wells. If residual ethanol ispresent, place the plate back on the magnet for 2 minutes and use a pipette toremove the ethanol.

Proceed immediately to one of the following:• Option 1: Equalize the library.• Option 2: Quantify the library by qPCR.• Option 3: Quantify the amplified library with the Qubit™ 2.0 or 3.0 Fluorometer,

or with the Agilent™ 2100 Bioanalyzer™ instrument.

Purify the library

Chapter 2 Prepare Oncomine™ BRCA Research Assay librariesLigate adapters to the amplicons and purify2


Option 1: Equalize the library

IMPORTANT! We recommend the Ion Library Equalizer™ Kit when unamplifiedlibrary concentration is consistently 100−500 pM after elution in 50 µL. If samplequality or quantity is variable or unknown, we recommend using the qPCR method(see “Option 2: Quantify the library by qPCR“ on page 22).

The Ion Library Equalizer™ Kit (Cat. No. 4482298) provides a method for normalizinglibrary concentration at ~100 pM without the need for special instrumentation forquantification. First amplify the Oncomine™ BRCA Research Assay library, thencapture the library on Equalizer™ Beads. After elution of the equalized library,proceed directly to combining libraries and/or template preparation.

Warm all the reagents in the Ion Library Equalizer™ Kit to room temperature. Vortexand centrifuge all reagents before use.

1. Prepare library amplification mix according to the following table. Prepare amaster mix for multiple reactions.

Order ofaddition Component

Volume perreaction

Volume for 8reactions[1]

1 1X Library Amp Mix (black cap) 50 µL 450 µL

2 Equalizer™ Primers (pink cap) 2 µL 18 µL

— Total 52 µL 468 µL

[1] One additional reaction added as overage to compensate for pipetting error.

2. Remove the plate from the magnet (at step 7 of "Purify the library"), then add52 µL of library amplification mix to each well containing air-dried beads.

3. Seal the plate with a new MicroAmp™ Adhesive Film, vortex thoroughly, thencentrifuge at 100 × g for 30 seconds to collect droplets.

4. Place the plate back on the magnet for 2 minutes, then carefully transfer ~50 µLof supernatant from each well to a new plate without disturbing the pellet.

5. Seal the plate with the adhesive film, place a MicroAmp™ Compression Pad onthe plate, load the plate in the thermal cycler, then run the following program:

Stage Temperature Time

Hold 98°C 2 minutes

Cycling (9 cycles)98°C 15 seconds

64°C 1 minute

Hold 10°C Hold(for up to 1 hour)

Note: During cycling, wash the Equalizer™ Beads for later use (see “Wash theEqualizer™ Beads (if not previously performed)“ on page 20).

Before you begin

Amplify the library

Chapter 2 Prepare Oncomine™ BRCA Research Assay librariesOption 1: Equalize the library 2


1. Bring the Equalizer™ Beads to room temperature, then mix thoroughly.

Note: Beads for multiple reactions can be prepared in bulk, and stored inEqualizer™ Wash Buffer at 4°C for up to 12 months until use. After 12 months, re-wash beads with an equal volume of Equalizer™ Wash Buffer.

2. For each reaction, pipet 3 µL of beads into a clean 1.5-mL tube, then add6 µL/reaction of Equalizer™ Wash Buffer.For example, if you have 4 reactions, add 12 µL of beads and 24 µL of washbuffer.

3. Place the tube in a magnetic rack for 3 minutes or until the solution is clear.

4. Carefully remove the supernatant without disturbing the pellet, then discard.

5. Remove the tube from the magnet, add 6 µL per reaction of Equalizer™ WashBuffer, then pipet up and down to resuspend.

1. Centrifuge the plate at 100 × g for 30 seconds in a plate centrifuge to collectcontents at the bottom of the wells.

2. Carefully remove the seal from the plate, then add 10 µL of Equalizer™ Captureto each library amplification reaction.

3. Seal the plate with a clear adhesive film, vortex thoroughly, then centrifuge tocollect droplets. Alternatively, mix by pipetting at least half the total volume upand down at least 5 times before sealing the plate.

4. Incubate at room temperature for 5 minutes.

1. Mix the washed Equalizer™ Beads by gentle vortexing or pipetting up and down.

2. Add 6 µL of washed beads to each plate well containing the captured library.

3. Set the pipette volume to 40 µL, then pipet the mixture up and down at least5 times to mix thoroughly.

4. Incubate at room temperature for 5 minutes. Briefly centrifuge the plate to collectall the liquid to the bottom of the plate wells.

5. Place the plate in the magnet, then incubate for 2 minutes or until the solution isclear.

6. Carefully remove the supernatant without disturbing the pellet.

Note: Check for droplets on the sides of the plate wells. If droplets are observed,seal the plate, then gently tap the plate on a hard, flat surface, or brieflycentrifuge to collect all the liquid to the bottom of the plate wells.

Note: Save the supernatant for repeat analysis if needed.

7. Add 150 µL of Equalizer™ Wash Buffer to each reaction.

Wash theEqualizer™ Beads(if not previouslyperformed)

Add Equalizer™

Capture to theamplified library

Add Equalizer™

Beads and wash

Chapter 2 Prepare Oncomine™ BRCA Research Assay librariesOption 1: Equalize the library2


8. Move the plate side-to-side in the two positions of the magnet to wash the beads.

Note: If your magnet does not have two positions for shifting the beads, removethe plate from the magnet and gently pipet up and down 5 times (with thepipettor set to at least half the total volume), then return the plate to the magnetand incubate for 2 minutes or until the solution clears.

9. With the plate still in the magnet, carefully remove, then discard the supernatantwithout disturbing the pellet.

10. Repeat the bead wash as described in steps 7–9.

Note: Ensure that as much wash buffer as possible is removed withoutdisturbing the pellet.

1. Remove the plate from the magnet, then add 100 µL of Equalizer™ Elution Bufferto each pellet.

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

Note: Centrifuge with sufficient force to collect droplets, but not pellet beads. Ifbeads are pelleted, vortex again and centrifuge more gently.

3. Elute the libraries by incubating in a thermal cycler at 32°C for 5 minutes.

4. Place the plate in the magnet, then incubate at room temperature for 5 minutes oruntil the solution is clear.The supernatant contains the equalized library at ~100 pM, which can be storedwith beads for up to 1 month at 4–8°C.

Proceed to “Combine Oncomine™ BRCA Research Assay barcoded libraries forsequencing on the same Ion chip“ on page 28 and template preparation, or storelibraries. Store libraries at 4−8°C for up to 1 month, or at −30°C to −10°C for longer-term storage.

Elute theequalized libraries

Chapter 2 Prepare Oncomine™ BRCA Research Assay librariesOption 1: Equalize the library 2


Option 2: Quantify the library by qPCR

Elute the library, then determine the concentration by qPCR with the Ion LibraryTaqMan® Quantitation Kit (Cat. No. 4468802). Libraries that have not undergone asecond round of amplification typically have yields of 100–500 pM. However, yield isnot indicative of library quality. After quantification, determine the dilution factor thatresults in a concentration of ~100 pM, which is suitable for template preparation usingan Ion template kit.

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

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

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

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

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

Determine the concentration of each Oncomine™ BRCA Research Assay library byqPCR with the Ion Library TaqMan® Quantitation Kit using the following steps.Analyze each sample, standard, and negative control in duplicate 20-µL reactions.

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

2. Prepare reaction mixtures. For each sample library, control, and standard,combine 20 µL of 2X TaqMan® MasterMix and 2 µL of 20X Ion TaqMan® Assay,then mix thoroughly. Dispense 11-µL aliquots into the wells of a PCR plate.

3. Add 9 µL of the diluted (1:100) Oncomine™ BRCA Research Assay library or 9 µLof each control dilution to each well (two wells per sample as noted before), for atotal reaction volume of 20 µL.

4. Program your real-time instrument as follows:a. Enter the concentrations of the control library standards.

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

c. Select a reaction volume of 20 µL.

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

Elute the library

Quantify librariesby qPCR andcalculate thedilution factor

Chapter 2 Prepare Oncomine™ BRCA Research Assay librariesOption 2: Quantify the library by qPCR2


e. The Ion Library TaqMan® qPCR Mix can be used on various instruments, aslisted in the following table. The fast cycling program was developed usingthe StepOnePlus™ System in Fast mode.

Real-time PCR System Reaction plate Run mode Stage Temperature Time

7500 Fast 96-well Fast

Fast

Hold (UDGincubation) 50°C 2 minutes

7900 HT7900 HT Fast 96-well Fast

384-well Standard

Hold (polymeraseactivation) 95°C 20 seconds

ViiA™ 7

Cycle (40 cycles)

95°C 1 secondQuantStudio™ 3 or 5

StepOne™

StepOnePlus™48-/96-well Fast 60°C 20 seconds

7300

96-well Standard Standard

Hold (UDGincubation) 50°C 2 minutes

7500 Hold (polymeraseactivation) 95°C 2 minutes

7900 HT7900 HT Fast

Cycle (40 cycles)

95°C 15 seconds

ViiA™ 760°C 1 minute

QuantStudio™ 3 or 5

5. Following qPCR, calculate the concentration of each undiluted Oncomine™BRCA Research Assay library by multiplying the concentration determined withqPCR by 100.

6. Based on the calculated library concentration, determine the dilution factor thatresult in a concentration for each library of ~100 pM.For example:

• The undiluted library concentration is 300 pM.• The dilution factor is 300 pM/100 pM = 3.• Therefore, 10 µL of library mixed with 20 µL of Low TE (1:3 dilution) yields

approximately 100 pM.

7. Dilute the libraries to ~100 pM with Low TE.

Note: A library that yields less than 100 pM can be rescued with libraryamplification. Combine 25 µL of unamplified library with 71 µL of 1X LibraryAmp Mix and 4 µL of 25X Library Amp Primers from the Ion AmpliSeq™ LibraryKit Plus. Perform 5–10 library amplification cycles (see step 4 of “Amplify thelibrary“ on page 19 or “Amplify the library“ on page 24 for cycling conditions).

Proceed to “Combine Oncomine™ BRCA Research Assay barcoded libraries forsequencing on the same Ion chip“ on page 28 and template preparation, or storelibraries at 4−8°C for up to 1 month, or at −20°C for longer-term storage.

Chapter 2 Prepare Oncomine™ BRCA Research Assay librariesOption 2: Quantify the library by qPCR 2


Option 3: Quantify the amplified libraries by Qubit™ Fluorometer orAgilent™ 2100 Bioanalyzer™ instrument

Oncomine™ BRCA Research Assay libraries must be amplified before quantification toenrich amplifiable material and obtain sufficient material for accurate quantification.Amplify the library using 1X Library Amp Mix, then purify. Quantify the libraryusing the Qubit™ 2.0 or 3.0 Fluorometer, or the Agilent™ 2100 Bioanalyzer™instrument. Amplified libraries typically have yields of 2,000–10,000 pM. Yield is notindicative of library quality. After quantification, determine the dilution factor thatresults in a concentration of ~100 pM, which is appropriate for template preparationusing an Ion template kit.

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

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

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

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

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

4. Seal the plate with MicroAmp™ Adhesive Film, place a MicroAmp™Compression Pad on the plate, load in the thermal cycler, then run the followingprogram:

Stage Temperature Time

Hold 98°C 2 minutes

5 cycles 98°C 15 seconds

64°C 1 minute

Hold 10°C Hold

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

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

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

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

Amplify the library

Purify theamplified library

Chapter 2 Prepare Oncomine™ BRCA Research Assay librariesOption 3: Quantify the amplified libraries by Qubit™ Fluorometer or Agilent™ 2100 Bioanalyzer™ instrument2


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

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

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

Agencourt™ AMPure™ XP Reagent.

First-round purification

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

2. Add 25 μL (0.5X sample volume) of Agencourt™ AMPure™ XP Reagent to eachplate well containing ~50 µL of sample. Pipet up and down 5 times to mix thebead suspension with the DNA thoroughly.


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

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

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

Second-round purification

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


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

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

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

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

5. Repeat step 4 for a second wash.

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

Chapter 2 Prepare Oncomine™ BRCA Research Assay librariesOption 3: Quantify the amplified libraries by Qubit™ Fluorometer or Agilent™ 2100 Bioanalyzer™ instrument 2


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

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

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

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

• "Qubit™ Fluorometer: Quantify the library and calculate the dilution factor" or• "Agilent™ 2100 Bioanalyzer™ instrument: Quantify the library and calculate

the dilution factor".

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

Analyze 10 µL of each amplified library using the Qubit™ 2.0 or 3.0 Fluorometer andthe Qubit™ dsDNA HS Assay Kit. Amplified libraries typically have concentrations of300–1500 ng/mL. Libraries below 300 ng/mL can still provide good quality sequences.For more information, see the Qubit™ dsDNA HS Assay Kits User Guide (Pub. No.MAN0002326).

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

Qubit™ dsDNA HS Buffer.

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

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

d. Measure the concentration on the Qubit™ 2.0 or Qubit™ 3.0 Fluorometer.

e. Calculate the concentration of the undiluted library by multiplying by 20.

2. Based on the calculated library concentration, determine the dilution that resultsin a concentration of ~100 pM.

Note: The Oncomine™ BRCA Research Assay panel has an average ampliconsize of approximately 140 bp, giving a library with average MW =140 bp × 650 Da/bp = 91,000 Da.A 100 pM concentration is equivalent to approximately 9 ng/mL:100 pmole/L × 91,000 pg/pmole = 9.1 × 106 pg/L = 9,100 pg/mL = 9.1 ng/mL

For example,• The library concentration is 450 ng/mL.• The dilution factor is 450 ng/mL divided by 9 ng/mL = 50.• Therefore, 10 µL of library mixed with 490 µL of Low TE (1:50 dilution)

yields 9 ng/mL (~100 pM).

3. Dilute library to ~100 pM with Low TE.

Qubit™

Fluorometer:Quantify thelibrary andcalculate thedilution factor

Chapter 2 Prepare Oncomine™ BRCA Research Assay librariesOption 3: Quantify the amplified libraries by Qubit™ Fluorometer or Agilent™ 2100 Bioanalyzer™ instrument2


Proceed to “Combine Oncomine™ BRCA Research Assay barcoded libraries forsequencing on the same Ion chip“ on page 28 and template preparation, or storelibraries at 4−8°C for up to 1 month, or at −20°C for longer-term storage.

Analyze 1 µL of amplified library on the Agilent™ 2100 Bioanalyzer™ instrument withthe Agilent™ High Sensitivity DNA Kit (Cat. No. 5067-4626). Amplicon librariesshould have multiple peaks in the 120–400 bp size range. Amplified libraries typicallyhave concentrations of 2000–10,000 pM. Yield is not indicative of library quality, andlibraries below 1,000 pM can still provide good quality sequences. If the libraryconcentration is over 20,000 pM, dilute the library 1:10 and repeat the quantification toobtain a more accurate measurement.

1. Determine the molar concentration of the amplified library using theBioanalyzer™ software. Ensure that the upper and lower marker peaks areidentified and assigned correctly. Follow the manufacturer’s instructions toperform a region analysis (smear analysis). Briefly:

a. Select the Data icon in the Contexts panel, then view the electropherogramof the sample to be quantified.

b. Select the Region Table tab below, then create a region spanning the desiredamplicon peaks. Correct the baseline if needed.

c. The molarity is automatically calculated and displayed in the table inpmol/L (pM).

2. Based on the calculated library concentration, determine the dilution that resultsin a concentration of ~100 pM.For example:

• The library concentration is 3,000 pM.• The dilution factor is 3,000 pM/100 pM = 30.• Therefore, 10 µL of library mixed with 290 µL of Low TE (1:30 dilution)

yields approximately 100 pM.

3. Dilute library to ~100 pM with Low TE.

Proceed to “Combine Oncomine™ BRCA Research Assay barcoded libraries forsequencing on the same Ion chip“ on page 28 and template preparation, or storelibraries at 4−8°C for up to 1 month, or at −20°C for longer term storage.

Agilent™ 2100Bioanalyzer™

instrument:Quantify thelibrary andcalculate thedilution factor

Chapter 2 Prepare Oncomine™ BRCA Research Assay librariesOption 3: Quantify the amplified libraries by Qubit™ Fluorometer or Agilent™ 2100 Bioanalyzer™ instrument 2


Combine Oncomine™ BRCA Research Assay barcoded libraries forsequencing on the same Ion chip

Multiple Oncomine™ BRCA Research Assay libraries can be sequenced on a single Ion318™ v2 BC or Ion 530™ Chip by combining equal volumes of each barcoded librarybefore template preparation. Recommendations for the maximum number of librariessequenced per chip are given in the following table:

Ion sequencing chip Germline librariessequenced per chipSomatic libraries

sequenced per chip

Ion 318™ v2 BC Chip 32 8

Ion 530™ Chip 32 32

Combined library

Prepare template

Sequence on one Ion 318™ v2 BC or Ion 530™ Chip

ion318

AX0005122

530 DAAG02858

ion torrent

Combine equal volumes ofbarcoded libraries afterequalization or quantification

Oncomine™ BRCA Research Assay libraries after amplication and barcoding

In this example, a barcoded library is generated from each of 8 samples using the twoOncomine™ BRCA Research Assay primer pools. After equalizing, or quantifying anddiluting to 100 pM, equal volumes of the 8 barcoded libraries are combined beforetemplate preparation for sequencing on one Ion 318™ v2 BC or Ion 530™ Chip.

Chapter 2 Prepare Oncomine™ BRCA Research Assay librariesCombine Oncomine™ BRCA Research Assay barcoded libraries for sequencing on the same Ion chip2


Guidelines for templating and sequencing

• Proceed to template preparation and sequencing using the following kitsappropriate to your instrument setup. See the appropriate user guide or guideslisted in the table for more information.

Kit Cat. No. User Guide

Ion PGM™ Hi‑Q™ View OT2 KitandIon PGM™ Hi‑Q™ ViewSequencing Kit

A29900A30044

Ion PGM™ Hi‑Q™ View OT2 KitUser Guide (Pub. No.MAN0014579)Ion PGM™ Hi‑Q™ ViewSequencing Kit User Guide (Pub.No. MAN0014583)

Ion PGM™ Hi‑Q™ View Chef Kit A29902 Ion PGM™ Hi‑Q™ View Chef KitsUser Guide (Pub. No.MAN0014571)

Ion 520™ & Ion 530™ Kit – Chef A27757A30010

Ion 520™ & Ion 530™ Kit – ChefUser Guide (Pub. No.MAN0010846)

• See Chapter 3, “Create a Planned Run and analyze results with an Ion Reporter™workflow“ for detailed instructions for using an Ion Reporter™ BRCA ResearchAssay workflow to analyze your results.

Chapter 2 Prepare Oncomine™ BRCA Research Assay librariesGuidelines for templating and sequencing 2


Create a Planned Run and analyzeresults with an Ion Reporter™

workflow

■ Configure the Torrent Browser to use your Ion Reporter™ account . . . . . . . . . 31■ About Ion Reporter™ Oncomine™ BRCA Research workflows . . . . . . . . . . . . . 33■ Upload Target Regions and Hotspots BED files into Torrent

Suite™ Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34■ Create a Planned Run with an Oncomine™ BRCA Research template . . . . . . . . 36■ Using the Ion AmpliSeq™ Sample ID Panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42■ View Ion Reporter™ analysis results and generate a report . . . . . . . . . . . . . . . . . 44■ Download results or generate a report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54■ Edit an Ion Reporter™ Oncomine™ BRCA Research workflow with a

new Annotation Set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57■ Oncomine™ Knowledgebase Reporter Software . . . . . . . . . . . . . . . . . . . . . . . . . . 61

Note: We recommend that you update to the latest available versions of TorrentSuite™ and Ion Reporter™ Software to enable the data analysis and reporting optionsdescribed in this chapter. Torrent Suite™ Software 5.2 is required to access the pre-made Planned Run templates. We recommend that you update to Ion Reporter™Software 5.4 to access new Ion Reporter™ workflows[1] and enable exon deletionanalysis.

3

[1] Torrent Suite™ Software and Ion Reporter™ Software are for Research Use Only. Not for use in diagnostic procedures.


Configure the Torrent Browser to use your Ion Reporter™ account

1. In the Torrent Browser, click Ion Reporter Configure in the Settings dropdown list.

2. Click Add Account, then select the appropriate Ion Reporter format from thedropdown list.

Chapter 3 Create a Planned Run and analyze results with an Ion Reporter™ workflowConfigure the Torrent Browser to use your Ion Reporter™ account 3


3. Enter Display Name, Username and Password for your IR account, then clickGet Versions.

4. Select Ion Reporter 5.4 for Version, then click 3Add.

Chapter 3 Create a Planned Run and analyze results with an Ion Reporter™ workflowConfigure the Torrent Browser to use your Ion Reporter™ account3


About Ion Reporter™ Oncomine™ BRCA Research workflows

Somatic and germline Ion Reporter™ Oncomine™ BRCA Research workflows areavailable for the Ion PGM™ and Ion S5™/Ion S5™ XL sequencing platforms. The w3.0versions of the workflows are enabled to detect BRCA1 and BRCA2 exon deletionsand duplications through inclusion of a Variability Correction Information Baseline(VCIB) CNV baseline (Oncomine BRCA DNA Baseline v1.0 (5.4)).

Ion Reporter™ Oncomine™ BRCA Research workflow Enabled for exondeletion detectionSequencing

platform

Oncomine™ BRCA Research Somatic - 318 - w2.1 - DNA - Single Sample No Ion PGM

Oncomine™ BRCA Research Germline - 318 - w2.1 - DNA - Single Sample

Oncomine™ BRCA Research Somatic - 318 - w3.0 - DNA - Single Sample Yes


Oncomine™ BRCA Research Somatic - 530 - w2.1 - DNA - Single Sample No Ion S5/S5 XL


Oncomine™ BRCA Research Somatic - 530 - w3.0 - DNA - Single Sample Yes


Note: The w2.1 workflows are visible to previous users of Ion Reporter™ 5.2 only.New Ion Reporter™ 5.4 users can only access the w3.0 workflows. Results with w2.1workflows in Ion Reporter™ 5.4 are the same as in 5.2, except that exon deletionanalysis is not functional.

IMPORTANT! To create a custom Ion Reporter™ Oncomine™ BRCA Researchworkflow, copy an existing Oncomine™ BRCA Research workflow and edit it. Youcannot create a workflow with full functionality starting from the Create workflowbutton on the Ion Reporter™ homepage. The Oncomine Variant Annotator (OVAT)v2.2 Plugin functionality is highly dependent on the panel used for the assay. Do notenable the OVAT plugin when copying or editing a non-Oncomine™ annotationworkflow.

Chapter 3 Create a Planned Run and analyze results with an Ion Reporter™ workflowAbout Ion Reporter™ Oncomine™ BRCA Research workflows 3


Upload Target Regions and Hotspots BED files into Torrent Suite™

Software

1. Upload the following BED files, required for your Oncomine™ BRCA ResearchIon Reporter™ workflow and provided in Ion Reporter™ Software 5.4, intoTorrent Suite™ Software:

• Oncomine_BRCA_Research_Assay.20170303.designed.bed• Oncomine_BRCA_Research_Assay.20170316.hotspots.blist.318.bed• Oncomine_BRCA_Research_Assay.20170316.hotspots.blist.530.bed

2. In the Torrent Browser, select References from the Settings dropdown list.

3. Select Target Regions, then click Add Target Regions.

4. Click Select File, then navigate to the Oncomine_BRCA_Research_Assay.20170303.designed.bed file on your Torrent Server. Click Upload Target RegionsFile.

Chapter 3 Create a Planned Run and analyze results with an Ion Reporter™ workflowUpload Target Regions and Hotspots BED files into Torrent Suite™ Software3


5. Select Hotspots from the list, then click Add Hotspots.

Note: There are no hotspots for the Oncomine™ BRCA Research Assay panel -the positions are better characterized as blacklist positions.

6. Click Select File, then navigate to the Oncomine_BRCA_Research_Assay.20170316.hotspots.blist.bed file on your drive appropriate to the 318 or 530workflow you are using. Click Upload Hotspots File.

Chapter 3 Create a Planned Run and analyze results with an Ion Reporter™ workflowUpload Target Regions and Hotspots BED files into Torrent Suite™ Software 3


Create a Planned Run with an Oncomine™ BRCA Research template

1. Sing in to the Torrent Server via the Torrent Browser, then navigate toPlan4Plan Template Run, Click Inherited Disease, or Oncology - Solid Tumorin the Applications list.

2. Select the applicable Oncomine™ BRCA Planned Run template:• Oncomine™ BRCA Research for S5• Oncomine™ BRCA Research for PGM

Alternatively, click Plan Run from the Settings dropdown list to the rightof the template

3. The Planned Run wizard opens to the Plan step. Enter or select the following:a. Run Plan name

b. Appropriate BED files from the Target and Hotspots Regions dropdownlists (if not selected)

c. Sample Tube Label and Chip ID (if using the Ion Chef™ Instrument)

d. The number of barcodes used in your sample

e. Sample information

Chapter 3 Create a Planned Run and analyze results with an Ion Reporter™ workflowCreate a Planned Run with an Oncomine™ BRCA Research template3


Note: Assign unique, descriptive names to your samples to ensure that resultsfor multiple samples are not combined in the run report.

Chapter 3 Create a Planned Run and analyze results with an Ion Reporter™ workflowCreate a Planned Run with an Oncomine™ BRCA Research template 3


4. Click the Ion Reporter step, select your Ion Reporter™ account, select anappropriate Oncomine™ BRCA Research workflow from the Existing Workflowlist, then click Next >.



5. In the Kits step, confirm settings and select Ion AmpliSeq Sample Id panel fromthe Control Sequence dropdown list.

Example of Kits page selections for the Ion S5™ System.

6. In the Plugins step,a. Select the sampleID plugin from the plugins list.



b. Select variantCaller from the plugins list, click Configure, then select ChipType and sample type. For Parameter Settings, select Custom, thennavigate to the matching json file. Click Save Changes.

Note: json file names:

json file name Sequencing platform

oncomine_brca_somatic_318_w2.1-somatic_lowstringency_pgm_parameters.json

Ion PGM

oncomine_brca_germline_318_w2.1-germline_lowstringency_pgm_parameters.json

oncomine_brca_somatic_318_w3.0-somatic_lowstringency_pgm_parameters.json

oncomine_brca_germline_318_w3.0-germline_lowstringency_pgm_parameters.json

oncomine_brca_somatic_530_w2.1-somatic_lowstringency_parameters.json

Ion S5/S5 XL

oncomine_brca_germline_530_w2.1-germline_lowstringency_parameters.json

oncomine_brca_somatic_530_w3.0-somatic_lowstringency_parameters.json

oncomine_brca_germline_530_w3.0-germline_lowstringency_parameters.json



c. Select coverageAnalysis from the plugins list, click Configure, click theSample Tracking checkbox, then click Save Changes.

Note: Checking Sample Tracking enables the analysis to produce a statisticfor reads mapped to Sample ID targets so that the level of off-target reads isaccurately represented in the Coverage Analysis Report.

d. Click Next on the lower right corner of the Plugins page to proceed to thenext page.

7. Enter optional information in the Projects step, then return to the Plan step. ClickPlan Run to complete the Planned Run.Your run is listed as pending in the Planned Runs List.

8. After completion of the sequencing run, navigate to your run in the TorrentBrowser Data4Completed Runs & Reports list to view your results. Go to theIon Reporter™ home page and click View Analyses, or the Analyses tab to viewyour Ion Reporter™ analysis.



Using the Ion AmpliSeq™ Sample ID Panel

The Oncomine™ BRCA Research Assay includes the nine primer pairs that make upthe Ion AmpliSeq™ Sample ID Panel, so do not add additional Sample ID primers totarget amplification reactions. If you have not done so already, use the followingprocedure to configure a Planned Run or Planned Run template and view the SampleID Report following a run.

Note: The Sample ID Panel may be used to match a tumor and normal sample.However, copy number variations in the tumor sample may distort the allele balancein the fingerprint.

IMPORTANT! Adding additional Ion AmpliSeq™ Sample ID Panel primer pairs caninterfere with target amplification reactions. Do not add additional primers.

1. When creating a new Planned Run in the Torrent Browser, select Ion AmpliSeqSample ID panel from the Control Sequence (optional): dropdown menu on theKits page.

2. When creating a new Planned Run in the Torrent Browser, select the sampleIDplugin on the Plugins page. Selection of the plugin automatically generates aSample ID Report after the run.

3. On the Plugins page, select the coverageAnalysis plugin checkbox, then clickConfigure. In the configuration dialog, select the Sample Tracking checkbox.This enables the analysis to produce a statistic for reads mapped to Sample IDtargets so that the level of off-target reads is accurately represented in theCoverage Analysis Report.

Note: If Sample Tracking is not selected, Sample ID reads are counted as off-target reads.

Chapter 3 Create a Planned Run and analyze results with an Ion Reporter™ workflowUsing the Ion AmpliSeq™ Sample ID Panel3


4. Following sequencing, select the Data tab in the Torrent Browser and selectCompleted Runs and Results. Open the report for your run and scroll down tothe Plugin Summary section, where you find the sampleID plugin results.

Chapter 3 Create a Planned Run and analyze results with an Ion Reporter™ workflowUsing the Ion AmpliSeq™ Sample ID Panel 3


View Ion Reporter™ analysis results and generate a report

This section describes how to use Ion Reporter™ Software to view analysis results, sortand classify variants, and generate a report. See the next section for information onhow to view exon deletion results. The Oncomine™ BRCA Research Assay used withIon Reporter™ Software 5.4 enables detection and visualization of whole exon andmultiple exon deletion in BRCA1 and BRCA2 genes in somatic and germline sampleswith high sensitivity. The following steps describe how to use the software tovisualize results and generate variant reports.

1. On the Ion Reporter™ home page, click View analyses, then navigate to youranalysis on the Analyses page and select it.

Chapter 3 Create a Planned Run and analyze results with an Ion Reporter™ workflowView Ion Reporter™ analysis results and generate a report3


2. On the Analysis Results page, view a summary of called variants and theirgenotypic and functional properties, and sort and select variants of interest byclicking the checkboxes at far left. Variant types and descriptions are listed in thefollowing table.

Description of variant types

Type CNV Subtype Description

CNV

BigDel Deletion of at least one exon

BigDup Duplication of at least one exon

GeneCNV Whole BRCA1/BRCA2 gene deletion orduplication

NOCALL Read count differs from baseline by non-integer amount; evidence for a BigDel orBigDup call is weak

REF Read count matches reference baseline

SNV — Single nucleotide substitution

MNP — Multiple nucleotide polymorphism at adjacentnucleotide positions

INDEL — Single or multiple nucleotide insertion ordeletion

Chapter 3 Create a Planned Run and analyze results with an Ion Reporter™ workflowView Ion Reporter™ analysis results and generate a report 3


Note: Scroll to the right using the scroll bar on the bottom to view additionalcolumns. You can customize the columns that appear in the Analyses andAnalysis Results tables by clicking Select Columns... in the Preferencesdropdown list in the upper right corner. Select or deselect columns in theAvailable Columns list, then click Apply.



3. Click the Summary link to view a summary of called variants. You can classifyvariants by selecting a classification from the dropdown list, if desired.



4. Click the Pharmacogenomics link to view the ClinVar column. Clicking theClinVar link (boxed) for a selected variant takes you to an NCBI ClinVar variant-specific home page where information regarding the ClinVar variant annotationis maintained.

Note: Other functional annotations in the Functional tab can also be used toclassify, sort, and filter variants. Sample ID results for a sample is displayed at thetop of the Analysis Results page, and on the Generate Report page in theSample Information section.



5. On the ClinVar variant home page, click the Variation Report link for the variant(boxed) to go to a Clinical assertion and Summary evidence page, whereadditional information about a selected variant can be viewed.



The following are examples of the type of information available in the ClinVarClinical assertions, Summary evidence, and Supporting observations tabs:

Clinical assertions tab



Summary evidence tab

Supporting observations tab



6. To view the exon deletion results, return to the Analysis Results page and clickVisualize at the upper right.

The BRCA Report on the Analysis Visualization page graphically shows the readcounts of BRCA1 and BRCA2 exons normalized to the Oncomine BRCA DNABaseline. Click Download Results to download a .png file of the graphic anda .tsv variant summary file.



7. Click the IRGV tab to display the IRGV view of exon deletions or duplicationson chromosomes 13 and 17.

2

1

3

4

5

8

7

6

1 Click to open an IRGV Details tab in your browser2 Zoom in and zoom out controls3 Click to view chromosome4 Scrollable alignment panel

5 Confidence filter6 MAPD filter7 Click to access more download options8 Oncomine BRCA Research Assay designed BED file

alignment

Note: The MAPD (Median of the Absolute values of all Pairwise Differences)metric is an estimate of coverage variability between adjacent amplicons, and isused in CNV scoring. The default threshold is 0.5, so sample results with aMAPD above this value should be viewed with lower confidence. For furtherinformation, see Ion Reporter™ Help.



Download results or generate a report

1. After selecting variants of interest on the Ion Reporter™ Analysis Results page,you can report your results in two ways:

• Download a .vcf (variant call format) file, or a .tsv (tab separated value) file ofcomplete or filtered results from the Download dropdown list, which you canthen customize to suit your reporting needs.

• Click Generate Report to produce a customizable IR report.

Note: These actions can also be performed in the Analysis Visualization pages.

Chapter 3 Create a Planned Run and analyze results with an Ion Reporter™ workflowDownload results or generate a report3


Clicking Generate Report takes you to the Configuration page where you cancustomize your report. Complete the required field and add any otherinformation.

2. Scroll down the Generate Report page to customize your report with theannotations that you want to include.

3. After making any additional comments and signing-off on the report, clickNext -> in the lower right corner.

Chapter 3 Create a Planned Run and analyze results with an Ion Reporter™ workflowDownload results or generate a report 3


4. On the Preview page, review the report, then click Lock & Publish to finalizeyour report. After generating the report, download it immediately.

Note: Only one report can be generated per analysis. Results require reanalysisto generate a second report. To capture all the information from the IonReporter™ annotation columns, generate a .tsv file using the Downloaddropdown list, then customize accordingly.

Example of an IR report

Chapter 3 Create a Planned Run and analyze results with an Ion Reporter™ workflowDownload results or generate a report3


Edit an Ion Reporter™ Oncomine™ BRCA Research workflow with anew Annotation Set

We recommend that you update the ClinVar annotation source in the Oncomine™BRCA Research Ion Reporter™ workflow Annotation Sets to the latest availableversion. This section describes how to create a new Annotation Set with an updatedClinVar annotation, then add it to a copy of an existing Oncomine™ BRCA Researchworkflow. See Ion Reporter™ Help for more information on how to update andcustomize Ion Reporter™ workflows.

1. Sign in to Ion Reporter™ Software, then click Workflows4 Presets.

2. On the Workflow Presets page, select Annotation Sets from the dropdown list,then click the Annotation Set that you want to update. Take note of the names ofthe annotations in the Details panel at right. You will add these annotationsources to the new set in the next steps.

Create a newAnnotation Set

Chapter 3 Create a Planned Run and analyze results with an Ion Reporter™ workflowEdit an Ion Reporter™ Oncomine™ BRCA Research workflow with a new Annotation Set 3


3. Select Annotation Set from the Create Preset dropdown list.

4. In the Create Annotation Set dialog, enter a new name for your Annotation Set,then select annotation sources from the dropdown list one at a time.

Chapter 3 Create a Planned Run and analyze results with an Ion Reporter™ workflowEdit an Ion Reporter™ Oncomine™ BRCA Research workflow with a new Annotation Set3


5. Select the Source Version of the annotation that you want to include in yourAnnotation set, then click Use. In this case, select the 20170404 Source Version toreplace the 20160802 Source Version in the Annotation Set. Repeat the selectionprocess to populate the remaining annotations in the Annotation Set, referring tothe list of annotations in the original Annotation Set.

6. When you have finished entering all the annotation sources, click Save.

Chapter 3 Create a Planned Run and analyze results with an Ion Reporter™ workflowEdit an Ion Reporter™ Oncomine™ BRCA Research workflow with a new Annotation Set 3


1. Click the Workflows tab, then select the Oncomine BRCA Research workflowthat you want to copy and edit. Select Copy from the Actions dropdown list.

2. Click the Annotation step in the Edit Workflow wizard, then select the newannotation set that you created from the Annotation Set dropdown list.

Copy and edit aworkflow to add anew AnnotationSet

Chapter 3 Create a Planned Run and analyze results with an Ion Reporter™ workflowEdit an Ion Reporter™ Oncomine™ BRCA Research workflow with a new Annotation Set3


3. Make any additional changes, or confirm settings in the other steps. In theConfirm step, enter a workflow name, then click Save Workflow. The newworkflow with an updated Annotation Set is listed on the Workflows page.

Oncomine™ Knowledgebase Reporter Software

You can also use Oncomine™ Knowledgebase Reporter Software to analyze yourOncomine™ BRCA Research Assay sequencing data, and manage and report results.To access the Thermo Fisher Cloud version of Oncomine™ Knowledgebase ReporterSoftware, go to: https://apps.thermofisher.com/apps/okr/index.html#/. Contact yourlocal Thermo Fisher Scientific representative if you have any questions regardingOncomine™ Knowledgebase Reporter Software.

Chapter 3 Create a Planned Run and analyze results with an Ion Reporter™ workflowOncomine™ Knowledgebase Reporter Software 3


https://apps.thermofisher.com/apps/okr/index.html#/

Tips and troubleshooting

Tips

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

• Plate seals can be firmly applied using the applicator in the MicroAmp™ OpticalAdhesive Film Kit. You can remove plate seals with much less effort when theplate is hot. Try removing seals right after taking the plate out of the thermalcycler.

• When transfer to a new plate is specified, you can transfer solutions to a cleanwell in the same plate instead, if desired.

A


Troubleshooting

Observation Possible cause Recommended action

Library yield is low Equalizer™ beads wereunwashed.

Be sure to wash Equalizer™ Beads before use.

Residual salt was present afterwash.

Carefully remove all of the Equalizer™ WashSoln before elution.

Input DNA was mis-quantified. Re-quantify input DNA using the TaqMan®

RNase P Detection Reagents Kit, or Qubit™ 2.0or Qubit™ 3.0 Fluorometer.

Input DNA was less than 10 ng. Add more DNA or increase target amplificationcycles.

PCR, digestion, or ligation wasinefficient.

Ensure proper dispensing and mixing ofviscous components at each step.

AMPure™ XP Beads wereoverdried.

Do not dry the AMPure™ XP Beads more than5 minutes. If the beads appear to be cracked,incubate the library amplification mix with thebeads for 5 minutes, mix well with a pipettoruntil completely resuspended before placingthe tube on the magnet to pellet the beads.

Residual ethanol inhibitedlibrary amplification.

Carefully remove all ethanol drops, using anadditional centrifugation and removal step, ifnecessary.

Library yield is high Input DNA was mis-quantified. Re-quantify input DNA using the TaqMan®

RNase P Detection Reagents Kit, or Qubit™ 2.0or Qubit™ 3.0 Fluorometer; quantify RNA withQubit™ 2.0 or Qubit™ 3.0 Fluorometer.

Input DNA was more than10 ng.

Add less DNA/RNA or decrease targetamplification cycles.

Library yield andquantification

Appendix A Tips and troubleshootingTroubleshooting A


Observation Possible cause Recommended action

Number of on-target reads islower than expected

Unknown causes. Increase the number of target amplificationcycles by two, or increase the anneal/extendtemperature of the target amplificationreaction from 60°C to 62°C for the first twocycles of the target amplification reaction.

Percentage of polyclonal ISPs ishigh

Library was over-seeded. Decrease amount of library added to thetemplate preparation reaction by 50%.

Other causes. Check an appropriate Ion PGM™ Hi‑Q™ Viewtemplate preparation guide for moreinformation.

Percentage of low quality ISPsis high

Library was under-seeded. Double the volume of library used in templatepreparation.

Use a fresh dilution of library prepared in anEppendorf LoBind™ Tube.

Other causes. Check an appropriate Ion PGM™ Hi‑Q™ Viewtemplate preparation guide for moreinformation.

Other

Appendix A Tips and troubleshootingTroubleshootingA


Safety

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

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

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

B


Chemical safety

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

· Read and understand the Safety Data Sheets (SDSs) provided by thechemical manufacturer before you store, handle, or work with any chemicalsor hazardous materials. To obtain SDSs, se

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