Oncomine Focus Assay, Part I: Library Preparation · 2018. 9. 6. · For Research Use Only. Not for use in diagnostic procedures. Oncomine™ Focus Assay, Part I: Library Preparation

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

Oncomine™ Focus Assay, Part I:Library PreparationUSER GUIDE

for use with:Oncomine™ Focus Assay, Select LibraryOncomine™ Focus Assay, 318 Solution

Catalog Numbers A29228, A28548Publication Number MAN0013530

Revision D.0

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

Products:Oncomine™ Focus DNA AssayOncomine™ Focus RNA Assay

Manufacturer: Life Technologies Corporation |5781 Van Allen Way |Carlsbad, CA 92008

Products:SuperScript™ VILO™ cDNA Synthesis Kit

Manufacturer: Life Technologies Corporation |7335 Executive Way |Frederick, MD 21704 | USA

Products:Ion PGM™ Select Library KitIon PGM™ Select Library Equalizer Kit

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: Revision history of MAN0013530

Revision Date DescriptionD.0 9 March 2018 Updated the cDNA amplification protocol:

• Oncomine™ Focus RNA Assay used at 0.5X final concentration

• updated thermal cycling parameters—decreased denaturationtemperature to 98°C

These changes to the protocol may improve the sequencing metrics andpotentially increase the calling rate of fusions by reducing false negatives.

C.0 4 February 2016 Updated the Oncomine™ Focus Assay documentation set with informationabout Torrent Suite™ Assay Development Software.

B.0 July 22, 2015 Corrected minor typos and edited component names.

A.0 May 22, 2015 New guide for new product.

Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you acceptthe terms and conditions of all applicable Limited Use Label Licenses.Trademarks: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. AMPure is a trademark ofBeckman Coulter, Inc. Eppendorf and Eppendorf LoBind are trademarks of Eppendorf AG. TaqMan is a registered trademark of Roche MolecularSystems, Inc., used under permission and license.

©2018 Thermo Fisher Scientific Inc. All rights reserved.

Contents

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

Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

System and software compatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Required materials and equipment (not provided) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

■ CHAPTER 2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Procedure overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Procedural guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Input DNA requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Input RNA requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Prepare and amplify cDNA targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Reverse transcribe RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Amplify cDNA targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Amplify DNA targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Transfer the DNA amplicons to the cDNA plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

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

Ligate adapters to the amplicons and purify . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16Set up and perform ligation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16Purify the library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Equalize the library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18Amplify the library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18Prepare LIB beads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18Add LIB Capture to the amplified library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Add LIB beads and wash . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Elute Equalized library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20Combine Equalized libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Oncomine™ Focus Assay, Part I: Library Preparation User Guide 3

■ APPENDIX A Tips and troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Tips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23Library yield and quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23Other . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

■ APPENDIX B Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

■ Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Contents

4 Oncomine™ Focus Assay, Part I: Library Preparation User Guide

Product information

Product description

This guide covers library preparation using the Ion PGM™ Select Library Kit and theOncomine™ Focus RNA Assay and Oncomine™ Focus DNA Assay panels, which areincluded as part of the Oncomine™ Focus Assay, 318 Solution and Oncomine™ FocusAssay, Select Library.

The library kit contains reagents for the rapid preparation of sample libraries fromDNA and RNA, which you can then combine and sequence together. The kit includes16 unique barcode adapter mixes (BC 1–BC 16) that allows you to sequence multiplesample libraries simultaneously. The kit requires 10 ng of DNA or RNA isolated fromnormal or formalin-fixed paraffin-embedded (FFPE) tissue per target amplificationreaction. The kit also provides a streamlined method for normalizing libraryconcentration without the need for quantification.

The Oncomine™ Focus Assay contains targeted, multi-biomarker panels that enablesimultaneous detection of thousands of variants across 52 genes relevant to solidtumors. Designed for translational and clinical research, this assay includes solidtumor genes targeted by on-market oncology drugs and published evidence. Theassay enables you to get results from up to 7 research samples + 1 DNA-NTC and 1RNA-NTC per run for both DNA and RNA in a single workflow.

System and software compatibility

The kits and components described in this guide are compatible with the followinginstrument systems and software:

• The Ion PGM™ System with Torrent Suite™ Software version 5.0 or later. Toupdate the system software, see the Oncomine™ Focus Assay, Part IV: SequencingUser Guide (Pub. No. MAN0013532).

• The Ion PGM™ Dx System with Torrent Suite™ Assay Development Softwareversion 5.0 or later, for Research Use Only experiments only.[1]

1

[1] The Ion PGM™ Dx System with Torrent Suite™ Assay Development Software is For Research Use Only. Not for use in diagnostic procedures.


Contents and storage

The Oncomine™ Focus Assay, Select Library (Cat. No. A29228) includes theOncomine™ Focus Assay, Ion PGM™ Select Library Kit, and the Ion PGM™ SelectLibrary Equalizer Kit. Sufficient regents are provided for the rapid preparation of 48Oncomine™ Focus DNA Assay and 48 Oncomine™ Focus RNA Assay barcodedlibraries.

Oncomine™ Focus Assay (Cat. No. A28638)

Contents Amount Storage

Oncomine™ Focus DNA Assay (blue cap) 192 µL–30°C to –10°C

Oncomine™ Focus RNA Assay (red cap) 192 µL

Ion PGM™ Select Library Kit (Cat. No. A28394)

Component Amount Storage

Ion PGM™ Select Library Reagents (Part No. A28385)

LIB HiFi Mix (red cap) 6 x 252 µL

–30°C to –10°C

LIB FuPa (green cap) 6 x 32 µL

LIB Switch Soln (orange cap) 6 x 64 µL

LIB DNA Ligase (clear cap) 6 x 32 µL

BC 1 – BC 16 (white cap) 12 µL each

Ion PGM™ Select Library Equalizer (Part No. A28386)

LIB AMPure™ Reagent 4.4 mL

2°C to 8°C

LIB Beads 6 x 48 µL

LIB Primers 6 x 36 µL

LIB Capture 6 x 160 µL

LIB Wash Soln 30 mL

LIB Elution Soln 9.6 mL

The Oncomine™ Focus Assay, 318 Solution (Cat. No. A28548) includes all of the abovecomponents and:

• SuperScript™ VILO™ cDNA Synthesis Kit (Cat. No. 11754050)• Ion OneTouch™ Select Template Kit (Cat. No. A28395 )• Ion PGM™ Select Sequencing Kit (Cat. No. A28396)• Ion 318™ Select Chip Kit (Cat. No. A28384)

Chapter 1 Product informationContents and storage1


Required materials and equipment (not provided)

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

Item Source

One of the following:

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

• AB™ 2720 Thermal Cycler

• Veriti™ 96‑well Thermal Cycler

• ProFlex™ 96‑well PCR System

See web product pages

MicroAmp™ Optical 96-well Reaction Plates N8010560

4306737 (with barcode)

MicroAmp™ Adhesive Film 4306311

MicroAmp™ Compression Pad 4312639

DynaMag™-96 Side Magnet 12331D

DynaMag™-2 Side Magnet 12321D

Nuclease-free Water AM9932

Absolute ethanol MLS

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

Eppendorf™ LoBind™ Microcentrifuge Tubes, 1.5‑mL MLS

Additional equipment

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

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

See web product pages

96-well plate centrifuge MLS

Nucleic acid isolation

RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE AM1975

MagMAX™ FFPE Total Nucleic Acid Isolation Kit 4463365

PureLink™ Genomic DNA Mini Kit K182000

Chapter 1 Product informationRequired materials and equipment (not provided) 1


http://www.thermofisher.com

http://fisherscientific.com

Item Source

Nucleic acid quantitation

TaqMan® RNase P Detection Reagents Kit (Recommendedfor DNA)

4316831

Qubit™ 4 Fluorometer[1]

Qubit™ dsDNA HS Assay Kit (DNA), or

Qubit™ RNA HS Assay Kit (RNA)

Q33226

Q32851, Q32854

Q32852, Q32855

Controls

(Recommended) AcroMetrix™ Oncology Hotspot Control 969056

(Optional) AcroMetrix™ KRAS FFPE Process Controls 950450

(Optional) AcroMetrix™ MultiMix FFPE Controls 95‑7184 , 95‑7191

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

Chapter 1 Product informationRequired materials and equipment (not provided)1


Methods

Procedure overview

“Prepare and amplify cDNA targets“ on page 13

q

“Amplify DNA targets“ on page 15

q

“Transfer the DNA amplicons to the cDNA plate“ on page 15

q

“Partially digest primers“ on page 16

q

“Set up and perform ligation“ on page 16

q

“Purify the library“ on page 17

q

“Amplify the library“ on page 18

q

“Prepare LIB beads“ on page 18

q

“Add LIB Capture to the amplified library“ on page 19

q

“Add LIB beads and wash“ on page 19

q

“Elute Equalized library“ on page 20

q

“Combine Equalized libraries“ on page 20

2


Workflow

Isolate and quantifyDNA & RNA

Reverse transcribe RNA 10 mL/well

Amplify cDNA (30 cycles)20 mL/wellRNA Assay

Primer pool

Amplify DNA (20 cycles)20 m L/wellDNA Assay

Transfer DNA to RNA plate

Digest primers, ligate adapters & purify amplicons

Amplicons

Equalize libraries and combine80:20 (DNA:RNA) in new plate

Libraries

Library

CombinedLibrary

DN

AR

NA

RN

A

RN

A

RN

A

DN

A

DN

A

DN

A

DN

AR

NA

DN

AR

NA

RN

A p

late

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

B

C

D

EF

G

H

RN

A p

late

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

B

C

D

EF

G

H

X 1 X 1 X 1 X 1 X 1 X 1

A

B

C

D

EF

G

H

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

Com

bine

d pl

ate

X X X X X X

Com

bine

d

Com

bine

d

Com

bine

d

Com

bine

d

Com

bine

d

Com

bine

d

Primer pool

A

B

C

D

EF

G

H

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

DN

A p

late

1 X 1 X 1 X 1 X 1 X 1 X

DN

AR

NA

RN

A

RN

A

RN

A

DN

A

DN

A

DN

A

DN

AR

NA

DN

AR

NA

RN

A p

late

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

B

C

D

EF

G

H

Sample

NTC

Sample

NTC

Sample

NTC

Sample

NTC

Figure 1 Ion AmpliSeq™ DNA/RNA workflow for the Oncomine™ Focus Assay.A maximum of 48 DNA and RNA (cDNA) samples undergo target amplification on separate plates due to different cyclenumbers. Processing of 7 samples + 1 DNA‑ & RNA‑NTC (16 libraries) are shown. RNA libraries are prepared in even-numbered columns. After amplification, the DNA target amplification reactions are transferred to the RNA plate. Digestion,ligation, and purification steps are identical for all wells, with each well receiving a unique barcode. Lastly, DNA and RNAlibraries are Equalized and combined at a ratio of 80:20 (DNA:RNA).

Chapter 2 MethodsWorkflow2


Procedural guidelines

Reagent preparation:• Minimize freeze-thawing of LIB 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 pipetting up and down 5 times.

Laboratory and PCR set-up:• 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.

• Use a calibrated thermal cycler specified in “Required materials and equipment(not provided)“ on page 7.

• Verify that the correct cycling protocol is being used prior to starting the thermalcycler.

• 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, LIB Switch Soln) 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 volumes are equivalent if using a multi-channelpipette.

• When pipetting into a plate well, touch the pipette tip to the side of the well andslowly dispense reagent on the side of the well to form a droplet. This enablesyou to both pipet small volumes accurately and see that you added reagent to thewell.

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

Chapter 2 MethodsProcedural guidelines 2


Before you begin

• Thaw components that contain enzymes—such as SuperScript™ Enzyme Mix, LIBHiFi Mix, LIB FuPa, and LIB DNA Ligase—on ice, and keep on ice duringprocedure.

• Thaw kit components other than enzymes, including genomic DNA and primerpanels, 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-spin before use. A pulse-spin is a 5-second centrifugation at maximumspeed.

• If there is visible precipitate in the 5X VILO™ Reaction Mix or the LIB Switch Solnor the tube cap(s) after thawing, vortex or pipet up and down at roomtemperature to resuspend.

• Before beginning and after use, wash the working surface with 10% bleachfollowed by 75% ethanol.

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

Input DNA requirements

For each target amplification reaction, use 3000 copies (10 ng of mammalian genomicDNA) from normal or FFPE tissue.

See “Required materials and equipment (not provided)“ on page 7 for kitsrecommended for isolating DNA.

We recommend the TaqMan® RNase P Detection Reagents Kit (Cat. no. 4316831) forquantifying amplifiable human genomic DNA. The Qubit™ dsDNA HS Assay Kit(Cat. no. Q32851 or Q32854) can also be used. We do not recommend methods such asdensitometry (e.g., NanoDrop™ Spectrophotometers), because they do notdiscriminate between DNA and RNA and thus are extremely sensitive to smallfragments of hydrolyzed RNA. This can lead to gross overestimation of theconcentration of sample DNA, under-seeding of the target amplification reaction, lowquality libraries, and low library yields.

Input RNA requirements

In general, the library yield from high quality RNA is greater than from degradedsamples. Library yield is not indicative of sequencing performance. See “Requiredmaterials and equipment (not provided)“ on page 7 for kits recommended forisolating RNA. Each reverse transcription reaction requires 10 ng of DNase-treatedRNA (³ 1.4 ng/μL), prepared from normal or FFPE tissue.

We recommend the Qubit™ RNA HS Assay Kit (Cat. no. Q32855) for quantitatingRNA.

Chapter 2 MethodsBefore you begin2


Prepare and amplify cDNA targets

If RNA was prepared from FFPE tissue and not previously heat-treated, pre-heat at80°C for 10 minutes, then cool to room temperature.

Note: We recommend that PCR setup be done on ice or a cold block.

IMPORTANT! If there is visible precipitate in the 5X VILO™ Reaction Mix, vortex orpipet up and down to resuspend. Ensure that there is no precipitate in the tube cap.

1. For each sample, add the following components into a single well of a 96-wellPCR plate. Prepare a master mix without sample RNA for multiple reactions.

Component Volume

5X VILO™ Reaction Mix 2 µL

10X SuperScript™ Enzyme Mix 1 µL

Total RNA (10 ng)[1] ≤ 7 µL

Nuclease-free Water to 10 µL

Total volume per well 10 µL

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

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

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

Temperature Time

42°C 30 minutes

85°C 5 minutes

10°C Hold

STOPPING POINT Samples can be stored at 10°C overnight (12–16 hours) in thethermal cycler. For longer periods, store at –20°C.

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

Reversetranscribe RNA

Chapter 2 MethodsPrepare and amplify cDNA targets 2


IMPORTANT! Primer pool and LIB HiFi Mix are viscous. Pipet slowly. Werecommend PCR setup on ice or a cold block.

Note: Because the 5X Oncomine™ Focus RNA Assay (red cap) requires differentamplification conditions than the 5X Oncomine™ Focus DNA Assay (blue cap), eachmust be run on its own separate plate.

1. Remove the seal from the plate with the cDNA synthesis reaction and add thefollowing components to each well. Prepare a master mix for multiple reactions.

Component Volume

LIB HiFi Mix (red cap) 4 µL

5X Oncomine™ Focus RNA Assay[1] (red cap) 2 µL

Nuclease-free Water 4 µL

Total volume per well (includes 10 µL from cDNA synthesis) 20 µL

[1] Updated cDNA amplification protocol uses the 5X Oncomine™ Focus RNA Assay primers at 0.5X final concentration.

2. Seal the plate with a new MicroAmp™ Adhesive Film, vortex thoroughly, andspin down in a centrifuge at 100 rcf for 30 seconds to collect droplets.

3. Place a MicroAmp™ Compression Pad on the plate, load the plate in the thermalcycler, and run the following program to amplify target cDNA regions.

Stage Step Temperature Time

Hold Activate theenzyme

98°C 2 min

Cycle; (30 cycles ) Denature 98°C 15 sec

Anneal and extend 60°C 4 min

Hold — 10°C Hold

Note: Increase the cycle number to 33 if RNA quality or quantity is questionable.

STOPPING POINT PCR products may be stored at 10°C overnight (12–16 hours) inthe thermal cycler. For longer periods, store at –20°C.


Amplify cDNAtargets

Chapter 2 MethodsPrepare and amplify cDNA targets2


Amplify DNA targets

IMPORTANT! Primer pool and LIB HiFi Mix are viscous. Pipet slowly. Werecommend PCR setup on ice or a cold block.

Note: Because the 5X Oncomine™ Focus DNA Assay (blue cap) requires differentamplification conditions than the 5X Oncomine™ Focus RNA Assay (red cap), eachmust be run on its own separate plate.

1. For each sample, add the following components. Prepare a master mix withoutDNA for multiple reactions.

Component Volume

LIB HiFi Mix (red cap) 4 µL

5X Oncomine™ Focus DNA Assay 4 µL

DNA, 10 ng £ 12 µL

Nuclease-free Water to 20 µL


3. Place a MicroAmp™ Compression Pad on the plate, load the plate in the thermalcycler, and run the following program to amplify target DNA regions.

Stage Step Temperature Time

Hold Activate theenzyme

99°C 2 min

Cycle; (20 cycles ) Denature 99°C 15 sec

Anneal and extend 60°C 4 min

Hold — 10°C Hold

Note: Increase the cycle number to 23 if DNA quality or quantity isquestionable.

STOPPING POINT PCR products may be stored at 10°C overnight (12–16 hours) inthe thermal cycler. For longer periods, store at –20°C.


Transfer the DNA amplicons to the cDNA plate

1. Carefully remove the plate seals from the amplified DNA plate and the amplifiedcDNA plate.

2. Transfer each sample from the DNA plate to an empty well of the cDNA plate,adjacent to the amplicons from the same sample (see “Workflow“ on page 10).

Chapter 2 MethodsAmplify DNA targets 2


Partially digest primers

Note: LIB FuPa is highly viscous. Flick to mix, then pulse-spin. To avoid carryingover excess enzyme, do not submerse the whole tip in the LIB FuPa solution; aspiratesolution from the surface.

1. Add 2 μL LIB FuPa (green cap) to each amplified sample. The total volume ofeach reaction is now 22 μL.

2. Seal the plate with MicroAmp™ Adhesive Film, vortex thoroughly, and spindown in a centrifuge at 100 rcf for 30 seconds to collect droplets.

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

Temperature Time

50°C 10 min

55°C 10 min

60°C 20 min

10°C Hold (for up to 1 hour)


Ligate adapters to the amplicons and purify

IMPORTANT! DNA and RNA fusion libraries must have different barcodes, evenwhen they are derived from the same sample.

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

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

1. Carefully remove the plate seal, then add the following components to each wellcontaining digested sample in the order indicated in the following table (30-μLtotal volume). After adding each component, mix by setting a pipette to 15 μLand pipetting up and down slowly 5 times.

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

Order Component Volume

1 LIB Switch Soln (orange cap) 4 µL

2 Barcode Adapters (BC 1–16; white cap) 2 µL

3 LIB DNA Ligase (clear cap) 2 µL

Set up andperform ligation

Chapter 2 MethodsPartially digest primers2


2. Seal the plate with a new MicroAmp™ Adhesive Film, vortex thoroughly, thenbriefly centrifuge at 100 rcf for 30 seconds to collect droplets.

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

Temperature Time

22°C 30 min

72°C 10 min

10°C Hold (for up to 1 hour)

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


IMPORTANT! Bring the LIB AMPure™ 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 LIB AMPure™

reagent.

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 and add 45 μL (1.5X sample volume) of LIBAMPure™ Reagent to each library. Pipet up and down 5 times to thoroughly mixthe bead suspension with the DNA.

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

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

5. Add 150 μL of freshly prepared 70% ethanol and move the plate side-to-side inthe two 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. Remove the plate from the magnet and seal with a MicroAmp™ Adhesive Film.Spin the plate at 100 rcf for 30 seconds, and inspect each well to make sure thatall the ethanol is removed.

IMPORTANT! Ensure that all ethanol droplets are removed from the wells. Ifresidual ethanol is present, place the plate back on the magnet for 2 minutes anduse a pipette to remove the ethanol. Residual ethanol droplets will inhibit libraryamplification.

8. Place the plate back in the magnet and air-dry the beads at room temperature for5 minutes.

Note: While the beads dry, prepare the library amplification mix described instep 1 of “Amplify the library“ on page 18.

Purify the library

Chapter 2 MethodsLigate adapters to the amplicons and purify 2


Equalize the library

IMPORTANT! Before starting, warm all reagents in the Ion Library Equalizer™ Kit boxto room temperature. Vortex and centrifuge all reagents prior to use.

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

Order Component Volume perreaction

Volume for 16reactions[1]

1 Nuclease-free Water 40 µL 680 µL

2 LIB HiFi Mix (red cap) 10 µL 170 µL

3 LIB Primers (blue cap) 2 µL 34 µL

— Total 52 µL 884 µL

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

2. Remove the plate from the magnet (at step 8 of "Purify the library"), and add52 μL of library amplification mix to each well containing air-dried beads.


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 it in the thermal cycler, and run the following program:

Stage Temperature Time

Hold 98°C 2 min

7 cycles98°C 15 sec

60°C 1 min

Hold 10°C Hold (for up to 1 hour)

Note: During cycling, prepare the LIB Beads for later use (see “Prepare LIBbeads“).

1. Bring the LIB Beads to room temperature and vortex for 10 seconds tocompletely resuspend beads, then pulse spin.

2. For each reaction, pipet 3 μL of beads/reaction into a clean 1.5-mL tube and add6 μL/reaction of LIB Wash Soln. Vortex for 5 seconds, then pulse spin.

Note: Beads for multiple reactions may be prepared in bulk and stored in LIBWash Soln at 4°C for up to 6 months. After 6 months, beads should be re-washedwith an equal volume of LIB Wash Soln prior to use.

3. Place the tube in a DynaMag™-2 Side Magnet for 3 minutes.

Amplify the library

Prepare LIB beads

Chapter 2 MethodsEqualize the library2


4. Carefully remove and discard the supernatant without disturbing the pellet.

5. Remove the tube from the magnet, then add 6 μL/reaction of LIB Wash Soln.Resuspend by pipetting up and down, then set aside for later use.

1. The LIB Capture (violet cap) must be at room temperature. Vortex the LIBCapture reagent for 5 seconds and pulse centrifuge.

2. After thermal cycling, carefully remove the adhesive plate seal and add 10 μL ofLIB Capture to each library amplification reaction.

3. Seal the plate with a new MicroAmp™ Adhesive Film, vortex thoroughly, thenspin down in a centrifuge at 100 rcf for 30 seconds to collect droplets.

4. Incubate at room temperature for 5 minutes.

1. Mix the prepared LIB Beads by pipetting up and down 5 times, ensuring thebeads are resuspended.

2. Add 6 μL of prepared LIB Beads to each well containing a capture reaction.

3. Set the pipette to 40 μL and slowly pipet the mixture up and down 5 times to mixthoroughly.

4. Incubate at room temperature for 5 minutes.

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

6. Carefully remove and discard the supernatant without disturbing the pellet.

7. Add 150 μL LIB Wash Soln to each reaction.

8. Move the plate side-to-side in the two positions of the magnet to wash the beadsfor a total of 3 times back and forth (total of 6 washes), allowing the beads topellet for at least 5 seconds each time on each side.

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 the pipetteset to 75 μL. Then return the plate to the magnet and incubate for 2 minutes.

9. With the plate still in the magnetic rack, carefully remove and discard thesupernatant without disturbing the pellet.

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

IMPORTANT! Remove as much LIB Wash Soln as possible without disturbingthe pellet. Remove excess LIB Wash Soln by pipetting extra times as needed.

Add LIB Captureto the amplifiedlibrary

Add LIB beads andwash

Chapter 2 MethodsEqualize the library 2


1. Remove the plate from the magnet and add 100 μL LIB Elution Soln to eachpellet. Seal the plate, vortex thoroughly, and spin down in a centrifuge at 100 rcffor 30 seconds to collect droplets.

Note: Spin with enough force to collect droplets, but not pellet the beads. If thebeads are pelleted, set the pipette to 50 μL and resuspend the beads by pipettingup and down 5 times.

2. Elute the library by incubating the sample in a thermal cycler at 35°C for5 minutes.

3. Place the plate in the magnet and incubate at room temperature for 5 minutes, oruntil the solution is clear.

4. Transfer the supernatant containing the equalized library to a 1.5-mLEppendorf™ LoBind™ tube. The final concentration of each equalized samplelibrary is ~100 pM.

STOPPING POINT The eluted sample libraries can be stored at –30°C to –10°C forup to 30 days. If stored for longer than 30 days, prepare new libraries. Librariesare now ready for sequencing. Refer to the following section for multiplexingstrategies.

We recommend sequencing up to 7 samples on a single Ion 318™ Select Chip, eachsample consisting of one DNA library and one RNA library, combined at an 80:20ratio (8 μL of DNA library + 2 μL of RNA library). A typical Oncomine™ Focus Assaysequencing run produces ~3.5 × 106 reads. To combine libraries in a different way, youcan determine the average number of reads per amplicon by doing a simplecalculation. Four examples are provided.

Example 1:

You have 7 samples, each consisting of one DNA library and one RNA library. We recommend the standard method ofcombining libraries at an 80:20 ratio (DNA:RNA).

DNA RNA

1. 3.5 × 106 reads/7 samples = 500,000 reads percombined library

2. 0.8[1] × 500,000 = 400,000 reads per DNA library

3. Average reads per amplicon: 400,000 reads per DNAlibrary/269 amplicons [2] = 1,487

1. 3.5 × 106 reads/7 samples = 500,000 reads percombined library

2. 0.2[3] × 500,000 = 100,000 reads per RNA library

3. Average reads per amplicon: 100,000 reads per RNAlibrary/13 amplicons[4]= 7,692

Note: If a fusion is detected, the total number ofamplicons will be 14 instead of 13.

[1] Proportion of the mixed library that is made up of DNA.[2] Number of amplicons in Oncomine™ Focus DNA Assay[3] Proportion of the mixed library that is made up of RNA.[4] Number of amplicons in Oncomine™ Focus Fusion Assay

Elute Equalizedlibrary

CombineEqualizedlibraries

Chapter 2 MethodsEqualize the library2


Example 2:

You have four samples, each consisting of one DNA library and one RNA library, and an additional four RNA librariesthat you want to re-sequence. We recommend combining all the DNA libraries together, and all the RNA librariestogether, and finally combining the two at an 80:20 (DNA:RNA) ratio. You can then calculate the average number ofreads per amplicon.

DNA RNA

1. 3.5 × 106 reads × 0.8 = 2.8 × 106 reads for all DNAlibraries

2. 2.8 × 106 reads/4 DNA libraries = 700,000 reads perDNA library

3. Average reads per amplicon: 700,000 reads per DNAlibrary/269 amplicons = 2,602

1. 3.5 × 106 reads × 0.2 = 700,000 reads for all RNAlibraries

2. 700,000 reads/8 RNA libraries = 87,500 reads perRNA library

3. Average reads per amplicon: 87,500 reads per RNAlibrary/13 amplicons = 6,731

Note: If a fusion is detected, the total number ofamplicons will be 14 instead of 13.

To combine the DNA and RNA libraries to yield similar average reads per amplicon, you will need to combine them ata different ratio:

1. Determine proportion of DNA amplicons (RD)(RD = # DNA amplicons/Total number of amplicons)RD = (269 × 4)/((269 × 4) + (13 × 8)) = 0.91

2. 3.5 × 106 reads × RD = 3.185 × 106 reads for 4 DNAlibraries

3. 3.185 × 106 reads/(4 libraries × 269 amplicons) =2,960 average reads per amplicon

1. Determine proportion of RNA amplicons (RR)(RR = # RNA amplicons/Total number of amplicons)RR = (13 × 8)/((269 × 4) + (13 × 8)) = 0.09

2. 3.5 × 106 reads × RR = 3.15 × 105 reads for 8 RNAlibraries

3. 3.15 × 105 reads/(8 libraries × 13 amplicons) =3,029 average reads per amplicon

Example 3:

You have only RNA samples and want to determine how many libraries you can combine in a single sequencing runand still maintain a good average number of reads per amplicon.

RNA only

3.5 × 106 reads/100,000 reads per RNA library (calculated from Example 1) = 35 RNA libraries

Note: The limiting factor in combining libraries is the number of barcodes provided in each Ion PGM™ Select Kit. Thekit includes 16 barcodes, which limits the number of libraries that you can combine in a single sequencing run to 16.

Example 4:

You have only DNA samples and want to determine how many libraries you can combine in a single sequencing runand still maintain a good average number of reads per amplicon.

DNA only

3.5 × 106 reads/400,000 reads per DNA library (calculated from Example 1) = 8.75 DNA libraries

Note: We recommend that you combine no more than 7 DNA libraries for each sequencing run.

Chapter 2 MethodsEqualize the library 2


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 or RNA 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.

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 or RNA 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/RNA 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, see the “Documentation andSupport” section in this document.

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

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

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

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

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

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

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

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

handling, and disposal limitations may apply.

Appendix B SafetyChemical safetyB


Biological hazard safety

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

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

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

Appendix B SafetyBiological hazard safety B


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

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

Documentation and support

Customer and technical support

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

• Order and web support• Product documentation, including:

– User guides, manuals, and protocols– Certificates of Analysis– Safety Data Sheets (SDSs; also known as MSDSs)

Note: For SDSs for reagents and chemicals from other manufacturers,contact the manufacturer.

Limited product warranty

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Documents

Oncomine Focus Assay, Part I: Library Preparation · 2018. 9. 6. · For Research Use Only. Not for use in diagnostic procedures. Oncomine™ Focus Assay, Part I: Library Preparation