Thermo Fisher Scientific Oncomine Focus Assay User guide

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

Oncomine™ Focus Assay

USER GUIDE

for use with the Oncomine™ Focus Assay, AmpliSeq™ Library or Oncomine™

Focus Assay, Chef‑Ready kits

Catalog NumbersA29230, A35957, A42008

Publication Number MAN0015819

Revision D.0

Life Technologies Corporation | 6055 Sunol Blvd | Pleasanton, California 94566 USA

For descriptions of symbols on product labels or product documents, go to thermoﬁsher.com/symbols-deﬁnition.

Revision history: MAN0015819 D.0 (English)

Revision Date Description

D.0 18 September

2023 Updated Ion AmpliSeq™ Kit for Chef DL8 (Cat. No. A29024) to include the PCR Plate Frame.

C.0 11 December

2018 •Three user guide set consolidated into a single user guide.

•Include instruction for library preparation with the IonChef™ System.

•Updated for use with Oncomine™ Reporter Software 4.1.

B.0 9 March 2018 Update to cDNA preparation protocol:

•Oncomine™ Focus RNA Assay used at 0.5X ﬁnal concentration when preparing libraries manually

•updated thermal cycling parameters—decreased denaturation temperature to 98°C for cDNA ampliﬁcation

These changes to the protocol may improve the sequencing metrics and potentially increase the calling rate of

fusions by reducing false negatives.

A.0 22 August 2016 Oncomine™ Focus Assay User Guide. Provides detailed, step-by-step instructions on how to create an Oncomine™

Focus Assay library.

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

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE

LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR

ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.

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. Agencourt

and Ampure are trademarks of Beckman Coulter, Inc. Agilent and Bioanalyzer are trademarks of Agilent Technologies, Inc. Eppendorf

and LoBind are trademarks of Eppendorf AG. TaqMan is a trademark of Roche Molecular Systems, Inc., used under permission and

license.

Contents

■CHAPTER1Productinformation .................................................. 6

Productdescription ............................................................. 6

Prerequisites ................................................................... 6

Contents and storage ............................................................ 7

Oncomine™ Focus Assay, AmpliSeq™ Library ................................... 7

Oncomine™ Focus Assay,Chef‑Ready ......................................... 8

Required materials notsupplied ................................................... 9

Recommended materials ....................................................... 10

Ion GeneStudio™ S5 Series instrument reference ................................... 11

■CHAPTER2Before youbegin .................................................... 12

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

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

Library preparation from genomic DNA orRNA .................................... 13

■CHAPTER3Manual library preparation ......................................... 14

Guidelines for RNA isolation andquantiﬁcation .................................... 14

Guidelines for DNA isolation andquantiﬁcation .................................... 14

Guidelines for the amount of DNA needed per target ampliﬁcation reaction ....... 15

RNA: Reverse transcribe and set up targetampliﬁcation ............................ 16

Reverse transcribeRNA .................................................... 16

Prepare cDNA target ampliﬁcation reactions .................................. 17

Amplify the cDNA targets ................................................... 17

Set up DNA target ampliﬁcation reactions ......................................... 19

Prepare DNA target ampliﬁcation reactions ................................... 19

Amplify the DNA targets .................................................... 20

Transfer the DNAamplicons ................................................. 20

Partially digestamplicons ....................................................... 21

Ligate adapters to the amplicons andpurify ....................................... 22

Ion Xpress™ Barcode Adapters only: Combine and dilute adapters ............... 22

Perform the ligation reaction ................................................ 22

Purify the library ........................................................... 23

Elute and dilute the library .................................................. 24

Oncomine™ Focus Assay User Guide 3

Quantify library by qPCR and calculate dilution factor ............................... 25

Combine DNA and RNA libraries to obtain dierent numbers of reads ................ 27

Examplecalculations ....................................................... 29

■CHAPTER4Chef Ready: Library preparation .................................. 31

Guidelines for RNA isolation andquantiﬁcation .................................... 31

Guidelines for DNA isolation andquantiﬁcation .................................... 31

Guidelines for the amount of DNA needed per target ampliﬁcation reaction ....... 31

Reverse transcribe RNA with the SuperScript™ VILO™ cDNA SynthesisKit ............ 32

Add DNA to an IonCode™ PCR plate ............................................. 33

IonChef™ Instrument setup information for Chef Ready kit users .................... 35

■CHAPTER5Create a PlannedRun .............................................. 36

About plannedruns ............................................................ 36

Create a custom Planned Run template ........................................... 37

Create a Planned Run from a template ............................................ 38

■CHAPTER6Guidelines for templating andsequencing ....................... 42

Ion GeneStudio™ S5 Series Systems ............................................. 42

■CHAPTER7Variantanalysis ..................................................... 43

Analysis workﬂows in Ion Reporter™ Software ..................................... 43

Manually launch ananalysis ..................................................... 44

View results ................................................................... 44

Generate a ﬁnal report .......................................................... 47

Download Ion Reporter™ annotation VCF or TSVﬁles ............................... 47

Oncomine™ Focus Assay annotations ........................................ 48

■CHAPTER8Generate an Oncomine™ Reporter report ........................ 50

Download the annotated VCF ﬁles from Ion Reporter™ Software ..................... 50

Upload Variant Call Format (VCF) ﬁles to Oncomine™ Reporter Software .............. 51

Create a report ................................................................ 52

Oncomine™ Reporter example report ........................................ 53

Contents

4Oncomine™ Focus Assay User Guide

■APPENDIXATips and troubleshooting ......................................... 54

Tips .......................................................................... 54

Troubleshooting ................................................................ 55

Low amplicon uniformity (DNAonly) .......................................... 55

Library yield andquantiﬁcation .............................................. 55

Other ..................................................................... 56

Chef-ready library preparation ............................................... 57

■APPENDIXBSupplementalinformation ........................................ 58

Conﬁgure the IonReporterUploader plugin in Torrent Suite™ Software ................. 58

Download and install BEDﬁles ................................................... 59

Make Oncomine™ Focus Assay templates available in Torrent Suite™ Software ......... 60

■APPENDIXCQuantify the ampliﬁed library with the Qubit™ Fluorometer .... 61

Amplify the library .............................................................. 61

Purify the ampliﬁed library ....................................................... 62

First-roundpuriﬁcation ..................................................... 62

Second-roundpuriﬁcation .................................................. 62

Qubit™ Fluorometer: Quantify the library and calculate the dilution factor .............. 64

Store libraries .................................................................. 64

■APPENDIXDSafety ............................................................... 65

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

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

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

Relateddocumentation ......................................................... 68

Obtain information from the Help system .......................................... 68

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

Limited product warranty ........................................................ 69

Contents

Oncomine™ Focus Assay User Guide 5

Product information

Product description

The Oncomine™ Focus Assay is a targeted multi-biomarker next-generation sequencing (NGS) assay

that enables the simultaneous detection of hundreds variants across 52 genes relevant to solid tumors.

This assays allows concurrent analysis of DNA and RNA to simultaneously detect multiple types of

variants, including hotspots, single nucleotide variants (SNVs), indels, copy number variants (CNVs) and

gene fusions, in a single workﬂow. Designed for translational and clinical research, this assay includes

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

This guide covers library preparation from DNA or RNA using the Ion AmpliSeq™ Library Kit 2.0 and the

Oncomine™ Focus DNA Assay and Oncomine™ Focus RNA Assay panels, which are included as part

of the Oncomine™ Focus Assay. The assay can be used with barcoded adapters so that up to seven

paired DNA and RNA samples plus DNA and RNA no template controls (NTCs) can be combined and

loaded onto a single Ion Chip in a single workﬂow to minimize the per-sample sequencing cost.

This guide covers the following products:

•Oncomine™ Focus Assay, AmpliSeq™ Library (Cat.Nos. A35957, A29230)

•Ion Xpress™ Barcode Adapters (various Cat. Nos.)

•IonCode™ Barcode Adapters (Cat. No. A29751)

•Ion AmpliSeq™ Sample ID Panel (Cat. No. 4479790)

•SuperScript™ VILO™ cDNA Synthesis Kit (Cat.No.11754050)

Note: The Oncomine™ Focus Assay, Chef‑Ready library preparation kit (Cat.No.A42008) is also

available for automated library preparation (see page8). The kit provides the Oncomine™ Focus

DNA Assay Panel (1-pool) and Oncomine™ Focus RNA Assay Panel (1-pool) at 2X concentration

premeasured in barcoded Primer Pool tubes ready to load into an Ion AmpliSeq™ Kit for Chef DL8

cartridge.

Prerequisites

This guide also assumes that you have:

•A general understanding of Ion Torrent™ sequencing chemistry and workﬂow

•Knowledge of techniques for handling and preparing DNA libraries

1

6Oncomine™ Focus Assay User Guide

Contents and storage

Oncomine™ Focus Assay, AmpliSeq™ Library

The 16‑reaction Oncomine™ Focus Assay, AmpliSeq™ Library (Cat.No.A35957) consists of the 5X

Oncomine™ Focus DNA Assay Panel and the 5X Oncomine™ Focus RNA Assay Panel, and 2 Ion

AmpliSeq™ Library Kit 2.0 (Cat.No.4475345) kits, for the rapid preparation of barcoded sample libraries

from DNA and RNA, which you can then combine and sequence simultaneously.

The 48‑reaction Oncomine™ Focus Assay, AmpliSeq™ Library (Cat.No.A29230) consists of the 5X

Oncomine™ Focus DNA Assay Panel and the 5X Oncomine™ Focus RNA Assay Panel, and the Ion

AmpliSeq™ Library Kit 2.0‑96LV (Cat.No.A26120) kit, for the rapid preparation of barcoded sample

libraries from DNA and RNA, which you can then combine and sequence simultaneously.

Contents Amount Storage

Oncomine™ Focus Assay,

AmpliSeq™ Library

Cat.No.A35957 (16

reactions)

Cat.No.A29230 (48

reactions)

5X Oncomine™ Focus DNA Assay 64µL 192 µL –30ºC to −10ºC

5X Oncomine™ Focus RNA Assay 64µL 192 µL

Ion AmpliSeq™ Library Kit 2.0 Cat.No.4475345 (8

libraries)

Cat.No.A26120 (96

libraries)

5X Ion AmpliSeq™ HiFi Mix

(redcap)

40 µL 480 µL –30ºC to −10ºC

FuPa Reagent (browncap) 16 µL 192 µL

Switch Solution (yellow cap) 32 µL 384 µL

DNA Ligase (blue cap) 16 µL 192 µL

Ion AmpliSeq™ Adapters

(greencap)

16 µL 192 µL

Platinum™ PCR SuperMix High

Fidelity (blackcap)

400 µL 3×1.6mL

Library Ampliﬁcation Primer Mix

(whitecap)

16 µL 192 µL

Low TE 6mL 12mL 15°C to 30°C[1]

[1] Can be stored at –30ºC to –10ºC for convenience.

Chapter1Product information

Contents and storage 1

Oncomine™ Focus Assay User Guide 7

Oncomine™ Focus Assay, Chef‑Ready

The Oncomine™ Focus Assay, Chef‑Ready Kit (Cat. No. A42008) provides the Oncomine™ Focus

DNA Assay Panel and the Oncomine™ Focus RNA Assay Panel at 2X concentration pre-measured

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

In addition, the kit provides all the reagents and supplies in an Ion AmpliSeq™ Kit for Chef DL8

(Cat.No.A29024) sucient for preparing 32 libraries. For more information about preparing Oncomine™

Focus Assay libraries on the IonChef™ System, see the Ion AmpliSeq™ Library Preparation on the

IonChef™ System User Guide (Pub. No. MAN0013432).

Component Amount Storage

Oncomine™ Focus DNA Assay, Chef‑Ready (32 reactions)

2X Oncomine™ Focus DNA Assay Panel 8×150µL –30°C to –10°C

Oncomine™ Focus RNA Assay, Chef‑Ready (32 reactions)

2X Oncomine™ Focus RNA Assay Panel 8×150µL –30°C to –10°C

Ion AmpliSeq™ Kit for Chef DL8 (Cat.No.A29024)

Ion AmpliSeq™ Chef Reagents DL8 2 × 4 cartridges –30°C to –10°C

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

Ion AmpliSeq™ Chef Supplies DL8 (per insert)

•Ion AmpliSeq™ Tip Cartridge L8

•PCR Frame Seal

•PCR Plate Frame

•Enrichment Cartridge

2 boxes 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)

2 sets 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

Contents and storage

1

8Oncomine™ Focus Assay User Guide

Required materials not supplied

Unless otherwise indicated, all materials are available through thermoﬁsher.com. "MLS" indicates that

the material is available from ﬁsherscientiﬁc.com or another major laboratory supplier.

Item Source

IonCode™ Barcode Adapters 1–384 Kit

OR

Ion Xpress™ Barcode Adapters 1–16 Kit

A29751



4471250, 4474009, 4474518, 4474519,

4474520, 4474521, or 4474517

Agencourt™ AMPure™ XP Kit Beckman Coulter, A63880 or A63881

(RNA only) SuperScript™ VILO™ cDNA Synthesis Kit 11754050

One of the following:

•GeneAmp™ PCR System 9700 or Dual 96-well Thermal Cycler

•Applied Biosystems™ 2720 Thermal Cycler

•96‑Well Thermal Cycler

•ProFlex™ 96‑well PCR System

See web product pages

MicroAmp™ Optical 96-Well Reaction Plate N8010560

4306737 (with barcode)

MicroAmp™ Fast Optical 96-Well Reaction Plate 4346907

MicroAmp™ Optical Adhesive Film 4311971

MicroAmp™ Clear Adhesive Film 4306311

MicroAmp™ Optical Film Compression Pad 4312639

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

Nuclease-Free Water (not DEPC-Treated) AM9932

Absolute ethanol MLS

Pipettors, 2–200 μL, and low-retention ﬁltered pipette tips MLS

Chapter1Product information

Required materials not supplied 1

Oncomine™ Focus Assay User Guide 9

Recommended materials

Unless otherwise indicated, all materials are available through thermoﬁsher.com. "MLS" indicates that

the material is available from ﬁsherscientiﬁc.com or another major laboratory supplier.

Item Source

Recommended additional equipment

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

Qubit™ 4 Fluorometer[1] Q33238

96-well plate centrifuge MLS

Recommended for nucleic acid isolation

RecoverAll™ Multi-Sample RNA/DNA Workﬂow A26069

RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE AM1975

MagMAX™ FFPE Total Nucleic Acid Isolation Kit 4463365

Recommended for nucleic acid quantiﬁcation

TaqMan™ RNase P Detection Reagents Kit 4316831

Qubit™ dsDNA HS Assay Kit (DNA) Q32851/ Q32854

Qubit™ RNA HS Assay Kit (RNA) Q32855

Recommended for library quantiﬁcation

Ion Library TaqMan™ Quantitation Kit 4468802

Recommended controls

Ion AmpliSeq™ Sample ID Panel 4479790

Horizon QMRS HD200

AcroMetrix™ Oncology Hotspot Control 969056

Biochain CancerSeq™ Plus T2235152-SC

lot # B906046

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

Chapter1Product information

Recommended materials

1

10 Oncomine™ Focus Assay User Guide

Ion GeneStudio™ S5 Series instrument reference

In this document, Ion GeneStudio™ S5 Series Sequencer or Ion GeneStudio™ S5 Series System refers

generically to the following systems, unless otherwise speciﬁed.

•Ion GeneStudio™ S5 System (Cat. No. A38194)

•Ion GeneStudio™ S5 Plus System (Cat. No. A38195)

•Ion GeneStudio™ S5 Prime System (Cat. No. A38196)

Chapter1Product information

Ion GeneStudio™ S5 Series instrument reference 1

Oncomine™ Focus Assay User Guide 11

Before you begin

Procedural guidelines

•Minimize freeze-thaw cycles of Oncomine™ Comprehensive Assay Plus panels by aliquoting as

needed for your experiments. Panels can be stored at 4°C for 1 year.

•Use good laboratory practices to minimize cross-contamination of products. If possible, perform

PCR setup in an area or room that is free of amplicon contamination. Always change pipette tips

between samples.

•Use a calibrated thermal cycler speciﬁed in “Required materials not supplied”.

•Pipet viscous solutions, such as 5X Ion AmpliSeq™ HiFi Mix, FuPa Reagent, Switch Solution, DNA

Ligase, and panels, slowly and ensure complete mixing by vortexing or pipetting up and down

several times.

•Arrange samples in alternating columns on the plate for easier pipetting with multichannel pipettes

during puriﬁcation with the DynaMag™ Side Magnet.

Before you begin

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

DNA Ligase, 10X SuperScript™ Enzyme Mix, and Platinum™ PCR SuperMix HiFi—on ice, and keep

on ice during procedure. All other components, including primer pools, may be thawed at room

temperature. Gently vortex and spin down before use.

•If there is visible precipitate in the 5X VILO™ Reaction Mix or Switch Solution after thawing, vortex

or pipet up and down at room temperature to resuspend.

2

12 Oncomine™ Focus Assay User Guide

Library preparation from genomic DNA or RNA

RNA

DNA or cDNA

P1

X

Barcode Adapters

Barcoded library

X

Primer pairs

Amplicons

Reverse transcribe

Amplify targets

Partially digest amplicons

Ligate adapters

Quantify libraries

Combine libraries (optional)

Isolate and quantify RNA

Isolate and quantify DNA

Chapter2Before you begin

Library preparation from genomic DNA or RNA 2

Oncomine™ Focus Assay User Guide 13

Manual library preparation

■ Guidelines for RNA isolation andquantiﬁcation ........................................... 14

■ Guidelines for DNA isolation andquantiﬁcation ........................................... 14

■ RNA: Reverse transcribe and set up targetampliﬁcation ................................... 16

■ Set up DNA target ampliﬁcation reactions ............................................... 19

■ Partially digestamplicons ............................................................. 21

■ Ligate adapters to the amplicons andpurify ............................................. 22

■ Quantify library by qPCR and calculate dilution factor ..................................... 25

■ Combine DNA and RNA libraries to obtain dierent numbers of reads ....................... 27

Guidelines for RNA isolation and quantiﬁcation

•Use kits in “Recommended materials” for isolating total RNA.

•We recommend the Qubit™ RNA HS Assay Kit (Cat. No. Q32855) for quantifying RNA.

•Each reverse transcription reaction requires 10 ng of DNase-treated total RNA (≥1.43ng/μL).

•In general, library yield from high quality RNA is greater than from degraded samples. Library yield

is not indicative of sequencing performance.

•Increasing the amount of RNA will usually result in higher quality libraries, especially when RNA

quality or quantity is unknown. We recommend using 1 ng total RNA only with high-quality, well-

quantiﬁed samples.

Guidelines for DNA isolation and quantiﬁcation

•See “Recommended materials” on page10 for isolating gDNA.

•We recommend the TaqMan™ RNase P Detection Reagents Kit (Cat. No. 4316831) or Qubit™

dsDNA HS Assay Kit (Cat. No. Q32851 or Q32854) for quantifying ampliﬁable human genomic

DNA.

•Spectrophotometric quantiﬁcation methods are not recommended, because they are not speciﬁc

for DNA. Use of these methods can lead to gross overestimation of the concentration of sample

DNA, under-seeding of the target ampliﬁcation reaction, and low library yields.

3

14 Oncomine™ Focus Assay User Guide

Guidelines for the amount of DNA needed per target ampliﬁcation reaction

•For each target ampliﬁcation reaction, use 300–30,000 copies of DNA (10ng of mammalian gDNA)

from normal or FFPE tissue.

•Increasing the amount of DNA results in higher-quality libraries, especially when DNA quality or

quantity is unknown. We recommend using 1ng gDNA (300copies) only with high-quality, well-

quantiﬁed samples.

Chapter3Manual library preparation

Guidelines for DNA isolation and quantiﬁcation 3

Oncomine™ Focus Assay User Guide 15

RNA: Reverse transcribe and set up target ampliﬁcation

Reverse transcribe RNA

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.

Chapter3Manual library preparation

RNA: Reverse transcribe and set up target ampliﬁcation

3

16 Oncomine™ Focus Assay User Guide

Prepare cDNA target ampliﬁcation reactions

IMPORTANT! Primer pools and HiFi Mix are viscous. Pipet slowly and mix thoroughly.

1. Place the 96-well plate in a pre-chilled cold block or on ice.

2. Thaw the 5X Ion AmpliSeq™ HiFi Mix on ice, gently vortex to mix, then centrifuge brieﬂy to collect.

3. Remove the seal from the plate, then add the following components to each cDNA synthesis

reaction. Prepare a master mix for multiple reactions.

Component Volume

5X Ion AmpliSeq™ HiFi Mix (red cap) 4 µL

5X Oncomine™ Focus RNA Assay[1] 2 µL

Nuclease-free Water 4 µL

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

[1] Updated manual library preparation cDNA amplification protocol uses the 5X Oncomine™ Focus RNA Assay primers at 0.5X

final concentration. Chef-ready library preparation does not require additional primer dilution.

4. Seal the plate with a new MicroAmp™ Adhesive Film, vortex thoroughly, then brieﬂy centrifuge to

collect droplets. Alternatively, mix by pipetting at least half the total volume up and down at least 5

times before sealing the plate.

Amplify the cDNA targets

1. Place a MicroAmp™ Compression Pad on the plate, then load the plate into the thermal cycler.

2. Run the following program to amplify the target regions.

Stage Step Temperature Time

Hold Activate enzyme 98°C 2min

Cycle; set number according to the following table Denature 98°C 15sec

Anneal and extend 60°C 4min

Hold — 10°C Hold

Input nucleic acid[1] Recommended cyclenumber (10ng)

Cycle number adjustment[2]

1ng RNA input 100ng RNA input

High quality RNA 27 +3 –3

FFPE RNA 30 +3 –3

[1] Cycle numbers can be increased when input material quality or quantity is questionable.

[2] The recommended cycle number is based on 10 ng RNA input. Adjust the cycle number for lower or higher RNA input.

Chapter3Manual library preparation

RNA: Reverse transcribe and set up target ampliﬁcation 3

Oncomine™ Focus Assay User Guide 17

IMPORTANT! When amplifying multiple samples in a single PCR plate, make sure that the input

across all samples is roughly equivalent so that the selected cycle number is optimal for all the

samples in the run.

STOPPING POINT Target ampliﬁcation reactions may be stored at 10°C overnight on the thermal

cycler. For longer periods, store at −20°C.

Chapter3Manual library preparation

RNA: Reverse transcribe and set up target ampliﬁcation

3

18 Oncomine™ Focus Assay User Guide

Set up DNA target ampliﬁcation reactions

Prepare DNA target ampliﬁcation reactions

IMPORTANT! Primer pools and HiFi Mix are viscous. Pipet slowly and mix thoroughly.

1. Place the 96-well plate in a pre-chilled cold block or on ice.

2. Thaw the 5X Ion AmpliSeq™ HiFi Mix on ice, gently vortex to mix, then centrifuge brieﬂy to collect.

3. Add the following components to a single well of a 96-well PCR plate. Prepare a master mix

without sample DNA for multiple reactions.

Component Volume

5X Ion AmpliSeq™ HiFi Mix (red cap) 4µL

5X Oncomine™ Focus DNA Assay 4µL

DNA (10ng)[1] ≤12µL

(Optional) Ion AmpliSeq™ Sample ID Panel 1µL

Nuclease-free Water to 20µL

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

DNA Assay

A

B

C

D

E

F

G

H

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

DNA plate

Sample DNA target ampliﬁcation reaction

No Template Control (NTC) target ampliﬁcation reaction

4. Seal the plate with a new MicroAmp™ Adhesive Film.

5. Vortex for 5 seconds to mix, then brieﬂy centrifuge to collect the contents. Alternatively, mix by

pipetting at least half the total volume up and down at least 5 times before sealing the plate.

Chapter3Manual library preparation

Set up DNA target ampliﬁcation reactions 3

Oncomine™ Focus Assay User Guide 19

Amplify the DNA targets

1. Place a MicroAmp™ Compression Pad on the plate, then load the plate into the thermal cycler.

2. Run the following program to amplify the target regions.

Stage Step Temperature Time

Hold Activate enzyme 99°C 2min

Cycle; set number according to the following table Denature 99°C 15sec

Anneal and extend 60°C 4min

Hold — 10°C Hold

Input nucleic acid[1] Recommended cyclenumber (10ng)

Cycle number adjustment[2]

1ng DNA input 100ng DNA input

High quality gDNA 17 +3 –3

FFPE DNA 20 +3 –3

[1] Cycle numbers can be increased when input material quality or quantity is questionable.

[2] The recommended cycle number is based on 10 ng DNA input. Adjust the cycle number for lower or higher DNA input.

Note: Addition of the Ion AmpliSeq™ Sample ID Panel does not require adjustment to cycle

number.

IMPORTANT! When amplifying multiple samples in a single PCR plate, make sure that the input

across all samples is roughly equivalent so that the selected cycle number is optimal for all the

samples in the run.

STOPPING POINT Target ampliﬁcation reactions may be stored at 10°C overnight on the thermal

cycler. For longer periods, store at −20°C.

Transfer the DNA amplicons

1. Remove the plate from the thermal cycler, then brieﬂy centrifuge to collect the contents.

2. Carefully remove the adhesive ﬁlm from the plate.

IMPORTANT! Be careful when removing the ﬁlm to minimize contamination.

Chapter3Manual library preparation

Set up DNA target ampliﬁcation reactions

3

20 Oncomine™ Focus Assay User Guide

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Thermo Fisher Scientific Oncomine Focus Assay User guide

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