Thermo Fisher Scientific Oncomine Focus Assay, Part I: Library Preparation User guide

Brand: Thermo Fisher Scientific

Model: Oncomine Focus Assay, Part I: Library Preparation

Type: User guide

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

Oncomine™ Focus Assay, Part I:

Library Preparation

USER GUIDE

for use with:

Oncomine™ Focus Assay, Select Library

Oncomine™ Focus Assay, 318 Solution

Catalog Numbers A29228, A28548

Publication Number MAN0013530

Revision D.0

Manufacturer:

Life Technologies Corporation |

6055 Sunol Blvd |

Pleasanton, CA 94566

Products:

Oncomine™ Focus DNA Assay

Oncomine™ 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 Kit

Ion 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 YOUR

USE OF IT.

Revision history: Revision history of MAN0013530

Revision Date Description

D.0 9 March 2018 Updated the cDNA ampliﬁcation protocol:

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

• updated thermal cycling parameters—decreased denaturation

temperature to 98°C

These changes to the protocol may improve the sequencing metrics and

potentially increase the calling rate of fusions by reducing false negatives.

C.0 4 February 2016 Updated the Oncomine™ Focus Assay documentation set with information

about 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 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. AMPure is a trademark 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.

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 ............................................... 13

Reverse transcribe RNA .................................................... 13

Amplify 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 ....................................... 16

Set up and perform ligation ................................................. 16

Purify the library .......................................................... 17

Equalize the library ............................................................ 18

Amplify the library ......................................................... 18

Prepare LIB beads ......................................................... 18

Add LIB Capture to the ampliﬁed library ...................................... 19

Add LIB beads and wash .................................................... 19

Elute Equalized library ..................................................... 20

Combine Equalized libraries ................................................ 20

Oncomine

™

Focus Assay, Part I: Library Preparation User Guide

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

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

Troubleshooting ............................................................... 23

Library yield and quantiﬁcation .............................................. 23

Other ..................................................................... 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

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 the

Oncomine™ Focus RNA Assay and Oncomine™ Focus DNA Assay panels, which are

included as part of the Oncomine™ Focus Assay, 318 Solution and Oncomine™ Focus

Assay, Select Library.

The library kit contains reagents for the rapid preparation of sample libraries from

DNA and RNA, which you can then combine and sequence together. The kit includes

16 unique barcode adapter mixes (BC 1–BC 16) that allows you to sequence multiple

sample libraries simultaneously. The kit requires 10 ng of DNA or RNA isolated from

normal or formalin-xed paran-embedded (FFPE) tissue per target amplication

reaction. The kit also provides a streamlined method for normalizing library

concentration without the need for quantication.

The Oncomine™ Focus Assay contains targeted, multi-biomarker panels that enable

simultaneous detection of thousands of variants across 52 genes relevant to solid

tumors. Designed for translational and clinical research, this assay includes solid

tumor genes targeted by on-market oncology drugs and published evidence. The

assay enables you to get results from up to 7 research samples + 1 DNA-NTC and 1

RNA-NTC per run for both DNA and RNA in a single workow.

System and software compatibility

The kits and components described in this guide are compatible with the following

instrument systems and software:

• The Ion PGM™ System with Torrent Suite™ Software version 5.0 or later. To

update the system software, see the Oncomine™ Focus Assay, Part IV: Sequencing

User Guide (Pub. No. MAN0013532).

• The Ion PGM™ Dx System with Torrent Suite™ Assay Development Software

version 5.0 or later, for Research Use Only experiments only.[1]

[1] The Ion PGM™ Dx System with Torrent Suite™ Assay Development Software is For Research

Use Only. Not for use in diagnostic procedures.

Oncomine

™

Focus Assay, Part I: Library Preparation User Guide

Contents and storage

The Oncomine™ Focus Assay, Select Library (Cat. No. A29228) includes the

Oncomine™ Focus Assay, Ion PGM™ Select Library Kit, and the Ion PGM™ Select

Library Equalizer Kit. Sucient regents are provided for the rapid preparation of 48

Oncomine™ Focus DNA Assay and 48 Oncomine™ Focus RNA Assay barcoded

libraries.

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 above

components 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 information

Contents and storage

Oncomine

™

Focus Assay, Part I: Library Preparation User Guide

Required materials and equipment (not provided)

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

One of the following:

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

Cycler

• 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 information

Required materials and equipment (not provided)

Oncomine

™

Focus Assay, Part I: Library Preparation User Guide

Item Source

Nucleic acid quantitation

TaqMan® RNase P Detection Reagents Kit

(Recommended

for 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 information

Required materials and equipment (not provided)

Oncomine

™

Focus Assay, Part I: Library Preparation User Guide

Methods

Procedure overview

“Prepare and amplify cDNA targets“ on page 13

“Amplify DNA targets“ on page 15

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

“Partially digest primers“ on page 16

“Set up and perform ligation“ on page 16

“Purify the library“ on page 17

“Amplify the library“ on page 18

“Prepare LIB beads“ on page 18

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

“Add LIB beads and wash“ on page 19

“Elute Equalized library“ on page 20

“Combine Equalized libraries“ on page 20

Oncomine

™

Focus Assay, Part I: Library Preparation User Guide

Workﬂow

Isolate and quantify

DNA & RNA

Reverse transcribe RNA

10 mL/well

Amplify cDNA (30 cycles)

20 mL/well

RNA Assay

Primer pool

Amplify DNA (20 cycles)

20 m L/well

DNA Assay

Transfer DNA to RNA plate

Digest primers, ligate adapters

& purify amplicons

Amplicons

Equalize libraries and combine

80:20 (DNA:RNA) in new plate

Libraries

Library

Combined

Library

DNA

RNA

DNA

RNA

DNA

RNA

RNA plate

1 2 34 5 6 7 8 9 10 11 12

RNA plate

1 2 34 5 6 7 8 9 10 11 12

X1X1X1X1X1X1

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

Combined plate

X X X X X X

Combined

Primer pool

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

DNA plate

1X1X1X1X1X1X

DNA

RNA

DNA

RNA

DNA

RNA

RNA plate

1 2 34 5 6 7 8 9 10 11 12

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 cycle

numbers. 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 RNA

libraries are Equalized and combined at a ratio of 80:20 (DNA:RNA).

Chapter 2 Methods

Workﬂow

Oncomine

™

Focus Assay, Part I: Library Preparation User Guide

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 pipeing 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 template

preparation. Always change pipee tips between samples.

• Use a calibrated thermal cycler specied in “Required materials and equipment

(not provided)“ on page 7.

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

cycler.

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

pipee.

• When pipeing into a plate well, touch the pipee tip to the side of the well and

slowly dispense reagent on the side of the well to form a droplet. This enables

you to both pipet small volumes accurately and see that you added reagent to the

well.

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

Chapter 2 Methods

Procedural guidelines

Oncomine

™

Focus Assay, Part I: Library Preparation User Guide

Before you begin

• Thaw components that contain enzymes—such as SuperScript™ Enzyme Mix, LIB

HiFi Mix, LIB FuPa, and LIB 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-spin before use. A pulse-spin is a 5-second centrifugation at maximum

speed.

•If there is visible precipitate in the 5X VILO™ Reaction Mix or the LIB Switch Soln

or the tube cap(s) after thawing, vortex or pipet up and down at room

temperature to resuspend.

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

followed by 75% ethanol.

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

to 8°C prior to use.

Input DNA requirements

For each target amplication reaction, use 3000 copies (10 ng of mammalian genomic

DNA) from normal or FFPE tissue.

See “Required materials and equipment (not provided)“ on page 7 for kits

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. We do not recommend methods such as

densitometry (e.g., NanoDrop™ Spectrophotometers), because they do not

discriminate between DNA and RNA and thus are extremely sensitive to small

fragments of hydrolyzed RNA. This can lead to gross overestimation of the

concentration of sample DNA, under-seeding of the target amplication reaction, low

quality libraries, and low library yields.

Input RNA requirements

In general, the library yield from high quality RNA is greater than from degraded

samples. Library yield is not indicative of sequencing performance. See “Required

materials and equipment (not provided)“ on page 7 for kits recommended for

isolating RNA. Each reverse transcription reaction requires 10 ng of DNase-treated

RNA (³ 1.4 ng/μL), prepared from normal or FFPE tissue.

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

RNA.

Chapter 2 Methods

Before you begin

Oncomine

™

Focus Assay, Part I: Library Preparation User Guide

Prepare and amplify cDNA targets

If RNA was prepared from FFPE tissue and not previously heat-treated, pre-heat at

80°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 or

pipet 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-well

PCR 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, then

centrifuge at 100 rcf for 30 seconds to collect droplets.

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

the thermal cycler, then run the following program to synthesize cDNA.

Temperature 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 the

thermal cycler. For longer periods, store at –20°C.

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

Reverse

transcribe RNA

Chapter 2 Methods

Prepare and amplify cDNA targets

Oncomine

™

Focus Assay, Part I: Library Preparation User Guide

IMPORTANT! Primer pool and LIB HiFi Mix are viscous. Pipet slowly. We

recommend PCR setup on ice or a cold block.

Note: Because the 5X Oncomine™ Focus RNA Assay (red cap) requires dierent

amplication conditions than the 5X Oncomine™ Focus DNA Assay (blue cap), each

must be run on its own separate plate.

1. Remove the seal from the plate with the cDNA synthesis reaction and add the

following 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, and

spin 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 thermal

cycler, and run the following program to amplify target cDNA regions.

Stage Step Temperature Time

Hold Activate the

enzyme

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) in

the thermal cycler. For longer periods, store at –20°C.

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

Amplify cDNA

targets

Chapter 2 Methods

Prepare and amplify cDNA targets

Oncomine

™

Focus Assay, Part I: Library Preparation User Guide

Amplify DNA targets

IMPORTANT! Primer pool and LIB HiFi Mix are viscous. Pipet slowly. We

recommend PCR setup on ice or a cold block.

Note: Because the 5X Oncomine™ Focus DNA Assay (blue cap) requires dierent

amplication conditions than the 5X Oncomine™ Focus RNA Assay (red cap), each

must be run on its own separate plate.

1. For each sample, add the following components. Prepare a master mix without

DNA 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

2. Seal the plate with a new MicroAmp™ Adhesive Film, vortex thoroughly, and

spin 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 thermal

cycler, and run the following program to amplify target DNA regions.

Stage Step Temperature Time

Hold Activate the

enzyme

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 is

questionable.

STOPPING POINT PCR products may be stored at 10°C overnight (12–16 hours) in

the thermal cycler. For longer periods, store at –20°C.

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

Transfer the DNA amplicons to the cDNA plate

1. Carefully remove the plate seals from the amplied DNA plate and the amplied

cDNA 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 “Workow“ on page 10).

Chapter 2 Methods

Amplify DNA targets

Oncomine

™

Focus Assay, Part I: Library Preparation User Guide

Partially digest primers

Note: LIB FuPa is highly viscous. Flick to mix, then pulse-spin. To avoid carrying

over excess enzyme, do not submerse the whole tip in the LIB FuPa solution; aspirate

solution from the surface.

1. Add 2 μL LIB FuPa (green 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, and spin

down 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)

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

Ligate adapters to the amplicons and purify

IMPORTANT! DNA and RNA fusion libraries must have dierent barcodes, even

when 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 up

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

tube cap.

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

containing digested sample in the order indicated in the following table (30-μL

total volume). After adding each component, mix by seing a pipee to 15 μL

and pipeing 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 and

perform ligation

Chapter 2 Methods

Partially digest primers

Oncomine

™

Focus Assay, Part I: Library Preparation User Guide

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

briey 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.

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

IMPORTANT! Bring the LIB AMPure™ 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 LIB AMPure™

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 and add 45 μL (1.5X sample volume) of LIB

AMPure™ Reagent to each library. Pipet up and down 5 times to thoroughly mix

the 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 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. 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 that

all the ethanol is removed.

IMPORTANT! 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. Residual ethanol droplets will inhibit library

amplication.

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

5 minutes.

Note: While the beads dry, prepare the library amplication mix described in

step 1 of “

Amplify the library“ on page 18.

Purify the library

Chapter 2 Methods

Ligate adapters to the amplicons and purify

Oncomine

™

Focus Assay, Part I: Library Preparation User Guide

Equalize the library

IMPORTANT! Before starting, warm all reagents in the Ion Library Equalizer™ Kit box

to room temperature. Vortex and centrifuge all reagents prior to use.

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

master mix for multiple reactions.

Order Component Volume per

reaction

Volume for 16

reactions[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 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, and

spin down in a centrifuge at 100 rcf 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 it in the thermal cycler, and run the following program:

Stage Temperature Time

Hold 98°C 2 min

7 cycles 98°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 LIB

beads“).

1. Bring the LIB Beads to room temperature and vortex for 10 seconds to

completely resuspend beads, then pulse spin.

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

6 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 LIB

Wash Soln at 4°C for up to 6 months. After 6 months, beads should be re-washed

with 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 Methods

Equalize the library

Oncomine

™

Focus Assay, Part I: Library Preparation User Guide

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 pipeing up and down, then set aside for later use.

1. The LIB Capture (violet cap) must be at room temperature. Vortex the LIB

Capture reagent for 5 seconds and pulse centrifuge.

2. After thermal cycling, carefully remove the adhesive plate seal and add 10 μL of

LIB Capture to each library amplication reaction.

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

spin 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 pipeing up and down 5 times, ensuring the

beads are resuspended.

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

3. Set the pipee to 40 μL and slowly pipet the mixture up and down 5 times to mix

thoroughly.

4. Incubate at room temperature for 5 minutes.

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

clear.

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 beads

for a total of 3 times back and forth (total of 6 washes), allowing the beads to

pellet for at least 5 seconds each time on each side.

Note: If your magnet does not have two positions for shifting the beads, remove

the plate from the magnet and gently pipet up and down 5 times with the pipee

set 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 the

supernatant 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 disturbing

the pellet. Remove excess LIB Wash Soln by pipeing extra times as needed.

Add LIB Capture

to the ampliﬁed

library

Add LIB beads and

wash

Chapter 2 Methods

Equalize the library

Oncomine

™

Focus Assay, Part I: Library Preparation User Guide

1. Remove the plate from the magnet and add 100 μL LIB Elution Soln to each

pellet. Seal the plate, vortex thoroughly, and spin down in a centrifuge at 100 rcf

for 30 seconds to collect droplets.

Note: Spin with enough force to collect droplets, but not pellet the beads. If the

beads are pelleted, set the pipee to 50 μL and resuspend the beads by pipeing

up and down 5 times.

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

5 minutes.

3. Place the plate in the magnet and incubate at room temperature for 5 minutes, or

until the solution is clear.

4. Transfer the supernatant containing the equalized library to a 1.5-mL

Eppendorf™ LoBind™ tube. The nal concentration of each equalized sample

library is ~100 pM.

STOPPING POINT The eluted sample libraries can be stored at –30°C to –10°C for

up to 30 days. If stored for longer than 30 days, prepare new libraries. Libraries

are now ready for sequencing. Refer to the following section for multiplexing

strategies.

We recommend sequencing up to 7 samples on a single Ion 318™ Select Chip, each

sample consisting of one DNA library and one RNA library, combined at an 80:20

ratio (8 μL of DNA library + 2 μL of RNA library). A typical Oncomine™ Focus Assay

sequencing run produces ~3.5 × 106 reads. To combine libraries in a dierent way, you

can determine the average number of reads per amplicon by doing a simple

calculation. 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 of

combining libraries at an 80:20 ratio (DNA:RNA).

DNA RNA

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

combined library

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

3. Average reads per amplicon: 400,000 reads per DNA

library/269 amplicons [2] = 1,487

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

combined library

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

3. Average reads per amplicon: 100,000 reads per RNA

library/13 amplicons[4]= 7,692

Note: If a fusion is detected, the total number of

amplicons 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 Equalized

library

Combine

Equalized

libraries

Chapter 2 Methods

Equalize the library

Oncomine

™

Focus Assay, Part I: Library Preparation User Guide

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Thermo Fisher Scientific Oncomine Focus Assay, Part I: Library Preparation User guide

Thermo Fisher Scientific Oncomine Focus Assay, Part I: Library Preparation User guide

Thermo Fisher Scientific Oncomine Focus Assay, Part I: Library Preparation User guide

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