Thermo Fisher Scientific Oncomine BRCA Research Assay User guide

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

Oncomine™ BRCA Research Assay

USER GUIDE

For manual library preparation

Catalog Number A32840

Publication Number MAN0014634

Revision B.0

Manufacturer: Multiple Life Technologies Corporation manufacturing sites are responsible for manufacturing the products associated with

the 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 YOUR

USE OF IT.

Revision history: Pub. No.MAN0014634

Revision Date Description

B.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 detailed

instructions 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 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. Agencourt and AMPure are

trademarks of Beckman Coulter, Inc. Eppendorf and Eppendorf LoBind are trademarks of Eppendorf AG. TaqMan is a registered trademark of Roche

Molecular Systems, Inc., used under permission and license. Agilent and Bioanalyzer are trademarks of Agilent Technologies, Inc.

Contents

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

Product 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 Research

Assay 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 ....................................... 16

Combine and dilute the adapters ............................................ 16

Perform the ligation reaction ................................................ 17

Purify the library .......................................................... 18

Option 1: Equalize the library .................................................... 19

Before you begin ........................................................... 19

Amplify the library ......................................................... 19

Wash the Equalizer™ Beads

(if not previously performed)

...................... 20

Add Equalizer™ Capture to the ampliﬁed library ............................... 20

Add Equalizer™ Beads and wash ............................................. 20

Elute the equalized libraries ................................................ 21

Option 2: Quantify the library by qPCR ............................................ 22

Elute the library ........................................................... 22

Quantify 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™ 2100

Bioanalyzer™ instrument ........................................................ 24

Amplify the library ......................................................... 24

Purify the ampliﬁed library ................................................. 24

Qubit™ Fluorometer: Quantify the library and calculate the dilution factor ......... 26

Agilent™ 2100 Bioanalyzer™ instrument: Quantify the library and calculate

the dilution factor .......................................................... 27

Combine Oncomine™ BRCA Research Assay barcoded libraries for sequencing on

the same Ion chip .............................................................. 28

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

■CHAPTER 3 Create a Planned Run and analyze results with

an 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 new

Annotation Set ................................................................. 57

Create a new Annotation Set ................................................ 57

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

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

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

Tips .......................................................................... 62

Troubleshooting ............................................................... 63

Library yield and quantiﬁcation .............................................. 63

Other ..................................................................... 64

■APPENDIX B Safety ..................................................... 65

Chemical safety ................................................................ 66

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

■Documentation and support ............................................. 68

Customer and technical support ................................................. 68

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

Contents

4

Oncomine

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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 the

manual preparation from genomic DNA of up to 24 Oncomine™ BRCA Research

Assay sample libraries targeting the BRCA1 and BRCA2 genes. Use of Ion Xpress™

Barcode Adaptors in library preparation enables you to combine barcoded libraries in

templating reactions and sequencing runs. Up to 32 germline libraries, or up to

8 somatic libraries, can be combined and sequenced on a single Ion 318™ chip, and up

to 32 germline or somatic libraries can be combined and sequenced on an Ion 530™

chip. The protocol requires 10 ng of DNA isolated from unxed or formalin-xed

paran-embedded (FFPE) tissue per target amplication reaction (20 ng total).

This guide also provides optional protocols for normalizing library concentration

using 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 provides

Oncomine™ BRCA Research Assay Pools 1 and 2 at 2X concentration pre-measured in

barcoded Primer Pool tubes ready to load into an Ion AmpliSeq™ Chef Reagents DL8

cartridge.

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Oncomine

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BRCA Research Assay User Guide

5

Kit contents and storage

The Oncomine™ BRCA Research Assay (Cat. No. A32840) is a bundle of the

Oncomine™ BRCA Research Assay Manual Library Preparation panel and the Ion

AmpliSeq™ Library Kit Plus, sucient for manually preparing 24 libraries.

Component Amount Storage

Oncomine™ BRCA Research Assay Manual Library Preparation, 5X concentration (Part

No. 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 information

Kit contents and storage

1

6

Oncomine

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BRCA Research Assay User Guide

Ion Library Equalizer™ Kit

The Ion Library Equalizer™ Kit, ordered separately, provides a streamlined method

for normalizing library concentration without quantication.

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 dierent barcode adapters sucient for 640

total reactions.

Component Quantity Volume Storage

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

–30ºC to –10ºC

Ion 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 information

Ion Library Equalizer

™

Kit

1

Oncomine

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BRCA Research Assay User Guide

7

Required materials not supplied

Unless otherwise indicated, all materials are available through thermosher.com.

MLS: Fisher Scientic (sherscientic.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 Scientiﬁc

NC9959336, 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 Scientiﬁc

13-698-791

96-well plate centrifuge MLS

Chapter 1 Product information

Required materials not supplied

1

8

Oncomine

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BRCA Research Assay User Guide

Recommended materials and equipment (optional)

Unless otherwise indicated, all materials are available through thermosher.com.

MLS: Fisher Scientic (sherscientic.com) or other major laboratory supplier.

Item Source

Recommended additional equipment for qPCR

Real-time PCR instrument (for example, Applied

Biosystems™ 7900HT, 7500, StepOne™, StepOnePlus™,

ViiA™ 7, QuantStudio™ 3/5 Systems, or QuantStudio™ 12K

Flex 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 Library

Equalizer

™

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 information

Recommended materials and equipment (optional)

1

Oncomine

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BRCA Research Assay User Guide

9

Oncomine™ BRCA Research Assay, Chef-Ready Kit

The Oncomine™ BRCA Research Assay, Chef-Ready Kit (Cat. No. A32841, ordered

separately) provides Oncomine™ BRCA Research Assay Pools 1 and 2 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 sucient for preparing

32 libraries. See the Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User

Guide (Pub. No. MAN0013432) for detailed information on preparing Oncomine™

BRCA Research Assay libraries on the Ion Chef™ System.

Component Part No. Quantity per

kit 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 with

4 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 of

4 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 information

Oncomine

™

BRCA Research Assay, Chef-Ready Kit

1

10

Oncomine

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BRCA Research Assay User Guide

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, with

Sample 1 in wells A3 and A4, Sample 2 in wells B3 and B4, etc. After target

amplication in 10 µL reactions, Pool 1 and Pool 2 amplication reactions are

combined into new wells in the plate. After partial digestion of primers, ligation of

barcode adapters, and amplicon purication, barcoded libraries are equalized to

~100 pM concentration, or quantied and diluted to 100 pM concentration, and can be

combined before template preparation.

Chapter 1 Product information

Oncomine

™

BRCA Research Assay manual workﬂow

1

Oncomine

™

BRCA Research Assay User Guide

11

Prepare Oncomine™ BRCA Research

Assay libraries

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

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

■Combine the target amplication 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 amplied 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™ BRCA

Research Assay libraries. For procedures for using the Oncomine™ BRCA Research

Assay, Chef-Ready Kit to prepare libraries on the Ion Chef™ System, see the Ion

AmpliSeq™ 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 all

reagents for 5 seconds (EXCEPT for enzymes, which should be ick-mixed) and

pulse-centrifuge before use. A pulse-centrifugation is a 5-second centrifugation at

maximum 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 at

4°C for one year.

• Pipet viscous solutions slowly and ensure complete mixing by vigorous vortexing

or pipeing up and down 5 times.

2

12

Oncomine

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BRCA Research Assay User Guide

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 template

preparation. Always change pipee tips between samples.

• Before starting and after use, wash the working surface with 10% bleach followed

by two water rinses.

• Use inner wells of PCR plate if possible, and skip wells or columns to prevent

cross-contamination between samples.

• Use a calibrated thermal cycler that is specied in “Required materials not

supplied“ on page 8.

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

cycler.

• Store one 96-well cold block at –30°C to –10°C and one 96-well cold block at 2°C

to 8°C before use.

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

Pipeing recommendations:

• Use aerosol-barrier pipee tips. Change pipee 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 pipeing by keeping the pipee tip at the

boom of the solution in the wells.

• Set pipee to the recommended volume for up and down mixing and insert tip

into solution with pipee plunger depressed to avoid introducing air bubbles.

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

pipee.

• Touch tip to side of well and slowly dispense reagent on the side of the well to

form a droplet. This practice enables you both to pipet small volumes accurately

and to see that you added reagent to the well.

• Ensure that reagent is adequately dispensed from the pipee tip.

Guidelines for DNA isolation and quantication:

• For each target amplication reaction, use 3,000 copies (10 ng of genomic DNA in

≤6 µL) from peripheral blood, cell lines, or FFPE tissue. See “Required materials

not 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 ampliable human genomic DNA. The Qubit™ dsDNA HS Assay

Kit (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 DNA

and RNA and therefore are sensitive to small fragments of hydrolyzed RNA. Use

of densitometry can lead to overestimation of the concentration of sample DNA,

under-seeding of the target amplication reaction, low-quality libraries, and low

library yields.

Chapter 2 Prepare Oncomine™ BRCA Research Assay libraries

Procedural guidelines

2

Oncomine

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BRCA Research Assay User Guide

13

Amplify the targets

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

and 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 a

low volume pipeor, as described in the following table. See the plate layout

example following the table.

Note: If preparing multiple libraries, master mixes without DNA are

recommended. Add master mixes to the plate rst.

IMPORTANT! Ion AmpliSeq™ Sample ID Panel primer pairs are included in the

Oncomine™ BRCA Research Assay. Do not add additional Ion AmpliSeq™

Sample ID Panel primers to target amplication 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 Pool

1 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 libraries

Amplify the targets

2

14

Oncomine

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BRCA Research Assay User Guide

2. Mix thoroughly by pipeing up and down, then seal the plate with MicroAmp™

Adhesive Film. Alternatively, the plate can be vortexed after sealing, followed by

centrifugation at 100 × g for 30 seconds to collect volume at the boom of the

wells.

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 the

enzyme

99°C 2 minutes

Cycle (18 cycles for

non-FFPE DNA,

21 cycles for FFPE

DNA)

Denature 99°C 15 seconds

Anneal and Extend 60°C 4 minutes

Hold — 10°C Hold (16 hours

maximum)

Note: See the Ion AmpliSeq™ Library Kit 2.0 User Guide (Pub. No. MAN0006735)

for guidance on cycle number if your gDNA is <10 ng, or of questionable quality.

STOPPING POINT Amplication products can be stored at 10°C overnight

(12−16 hours) in the thermal cycler. For longer-term storage, store at –20°C.

Combine the target ampliﬁcation reactions

1. Centrifuge the plate at 100 × g for 30 seconds in a plate centrifuge to collect

contents at the boom of the wells.

2. Carefully remove the plate seal from the plate.

3. Combine the two 10-µL target amplication reactions for each sample by

transferring them to an empty well of a new column in the plate, or in a new

plate.

1 2

Primer Pool

A

B

C

D

E

F

G

H

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

Chapter 2 Prepare Oncomine™ BRCA Research Assay libraries

Combine the target ampliﬁcation reactions

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BRCA Research Assay User Guide

15

Partially digest primers

Note: FuPa Reagent is viscous. Flick to mix, then pulse-centrifuge. To avoid carrying

over 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 amplied sample. The total

volume of each reaction is now ~22 µL.

2. Seal the plate with MicroAmp™ Adhesive Film, vortex thoroughly, then

centrifuge at 100 × g for 30 seconds to collect droplets.

3. Place a MicroAmp™ Compression Pad on the plate, load the plate in the thermal

cycler, 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 Ion

Xpress™ Barcode X at a nal 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 barcode

adapter 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 and

dilute the

adapters

Chapter 2 Prepare Oncomine™ BRCA Research Assay libraries

Partially digest primers

2

16

Oncomine

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BRCA Research Assay User Guide

IMPORTANT! When handling barcode adapters, avoid cross-contamination by

changing gloves frequently and opening one tube at a time.

IMPORTANT! If there is visible precipitate in the Switch Solution, vortex or pipet up

and down at room temperature to dissolve. Ensure that there is no precipitate in the

tube cap.

1. Centrifuge the plate at 100 × g for 30 seconds in a plate centrifuge to collect

contents at the boom of the wells.

2. Carefully remove the plate seal, then add the following components in the order

indicated to each well containing digested sample. After adding each component,

mix by seing a pipee to 15 µL, then pipeing up and down slowly 5 times.

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

Order of

addition Component Volume

1 Switch Solution (yellow cap) 4 µL

2 Ion Xpress™ Barcode Adaptor/P1 Adaptor

mix[1]

2 µL

3 DNA Ligase (blue cap) 2 µL

— Total volume (including ~22 µL of digested

amplicons)

~30 µL

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

3. Seal the plate with a new MicroAmp™ Adhesive Film, vortex thoroughly, then

centrifuge at 100 × g for 30 seconds to collect droplets.

4. Place a MicroAmp™ Compression Pad on the plate, load the plate in the thermal

cycler, then run the following program:

Temperature Time

22°C 30 minutes

68°C 5 minutes

72°C 5 minutes

10°C Hold (for up to 24 hours)

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

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

Perform the

ligation reaction

Chapter 2 Prepare Oncomine™ BRCA Research Assay libraries

Ligate adapters to the amplicons and purify

2

Oncomine

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BRCA Research Assay User Guide

17

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

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

Do NOT substitute a Dynabeads™-based purication reagent for the AMPure™ XP

Reagent.

1. Prepare fresh 70% ethanol: Combine 230 µL of 100% ethanol with 100 µL of

Nuclease-free Water per sample.

2. Carefully remove the plate seal, then add 45 µL (1.5X sample volume) of

AMPure™ XP Reagent to each library. Pipet up and down 5 times to mix the bead

suspension 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 the

two positions of the magnet to wash the beads, then remove and discard the

supernatant 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 in

the magnet, air-dry the beads at room temperature for 5 minutes. Do not overdry.

IMPORTANT! Residual ethanol droplets inhibit library amplication. Ensure

that all ethanol droplets are removed from the wells. If residual ethanol is

present, place the plate back on the magnet for 2 minutes and use a pipee to

remove 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 amplied 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 libraries

Ligate adapters to the amplicons and purify

2

18

Oncomine

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BRCA Research Assay User Guide

Option 1: Equalize the library

IMPORTANT! We recommend the Ion Library Equalizer™ Kit when unamplied

library concentration is consistently 100−500 pM after elution in 50 µL. If sample

quality 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 normalizing

library concentration at ~100 pM without the need for special instrumentation for

quantication. First amplify the Oncomine™ BRCA Research Assay library, then

capture 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. Vortex

and centrifuge all reagents before use.

1. Prepare library amplication mix according to the following table. Prepare a

master mix for multiple reactions.

Order of

addition Component Volume per

reaction

Volume for 8

reactions[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 add

52 µL of library amplication mix to each well containing air-dried beads.

3. Seal the plate with a new MicroAmp™ Adhesive Film, vortex thoroughly, then

centrifuge at 100 × g for 30 seconds to collect droplets.

4. Place the plate back on the magnet for 2 minutes, then carefully transfer ~50 µL

of supernatant from each well to a new plate without disturbing the pellet.

5. Seal the plate with the adhesive lm, place a MicroAmp™ Compression Pad on

the 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 the

Equalizer™ Beads (if not previously performed)“ on page 20).

Before you begin

Amplify the library

Chapter 2 Prepare Oncomine™ BRCA Research Assay libraries

Option 1: Equalize the library

2

Oncomine

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BRCA Research Assay User Guide

19

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

Note: Beads for multiple reactions can be prepared in bulk, and stored in

Equalizer™ Wash Buer at 4°C for up to 12 months until use. After 12 months, re-

wash beads with an equal volume of Equalizer™ Wash Buer.

2. For each reaction, pipet 3 µL of beads into a clean 1.5-mL tube, then add

6 µL/reaction of Equalizer™ Wash Buer.

For example, if you have 4 reactions, add 12 µL of beads and 24 µL of wash

buer.

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™ Wash

Buer, then pipet up and down to resuspend.

1. Centrifuge the plate at 100 × g for 30 seconds in a plate centrifuge to collect

contents at the boom of the wells.

2. Carefully remove the seal from the plate, then add 10 µL of Equalizer™ Capture

to each library amplication reaction.

3. Seal the plate with a clear adhesive lm, vortex thoroughly, then centrifuge to

collect droplets. Alternatively, mix by pipeing at least half the total volume up

and 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 pipeing up and down.

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

3. Set the pipee volume to 40 µL, then pipet the mixture up and down at least

5 times to mix thoroughly.

4. Incubate at room temperature for 5 minutes. Briey centrifuge the plate to collect

all the liquid to the boom of the plate wells.

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

clear.

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, at surface, or briey

centrifuge to collect all the liquid to the boom of the plate wells.

Note: Save the supernatant for repeat analysis if needed.

7. Add 150 µL of Equalizer™ Wash Buer to each reaction.

Wash the

Equalizer™ Beads

(if not previously

performed)

Add Equalizer™

Capture to the

ampliﬁed library

Add Equalizer™

Beads and wash

Chapter 2 Prepare Oncomine™ BRCA Research Assay libraries

Option 1: Equalize the library

2

20

Oncomine

™

BRCA Research Assay User Guide

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Thermo Fisher Scientific Oncomine BRCA Research Assay User guide

Thermo Fisher Scientific Oncomine BRCA Research Assay User guide

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