Thermo Fisher Scientific Oncomine Myeloid MRD Assay User guide

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

Oncomine™ Myeloid MRD Assay (RUO)

USER GUIDE

Catalog NumbersA53837, A53838, A53839

Publication Number MAN0025670

Revision D.0

Life Technologies Corporation |

6055 Sunol Blvd |

Pleasanton, California 94566 USA

Products manufactured at this site:

•Oncomine™ Myeloid MRD Assay (RUO)

Life Technologies Corporation |

5781 Van Allen Way |

Carlsbad, California 92008 USA

Products manufactured at this site:

•Ion AmpliSeq™ HD Library Kit

•Ion AmpliSeq™ HD Dual Barcode Kit 1–24

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

Revision history: MAN0025670 D.0 (English)

Revision Date Description

D.0 21 June 2023 •Updated recommended materials to include AcroMetrix™ Custom MRD Control 0.1% VAF control.

•Added “DNA input requirements” on page33.

•Updated “Set up molecular tagging reactions—Oncomine™Myeloid MRD MHT panel libraries (concentrations

<20ng/µL)” on page43.

•Updated “Oncomine™ Myeloid MRD Assay (RUO) reference and BED ﬁles” on page90.

•Updated “Molecular tag sequencing technology—Important factors” on page92.

•Updated thermal cycler information.

•Content reorganization.

C.0 5 October 2022 Updated thermal cycler information.

B.0 12 July 2022 New product introduction. New publication documenting the use of the Oncomine™ Myeloid MRD Assay (RUO).

A.0 20 January 2022 Early access release for limited customer base documenting the use of the Oncomine™ Myeloid MRD Assay (RUO).

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. TaqMan is

a trademark of Roche Molecular Systems, Inc., used under permission and license. Bioanalyzer and Agilent are trademarks of Agilent

Technologies, Inc. Agencourt and AMPure are trademarks of Beckman Coulter, Inc. Eppendorf and Eppendorf LoBind are trademarks of

Eppendorf AG. Seraseq and SeraCare are trademarks of SeraCare Life Sciences, Inc.

Contents

■CHAPTER1Productinformation .................................................. 7

Productdescription ............................................................. 7

Ion GeneStudio™ S5 Series instrument reference ............................... 8

Contents and storage ............................................................ 8

Kit summary ............................................................... 8

Oncomine™ Myeloid MRD DNA Assay(RUO) ................................... 9

Oncomine™ Myeloid MRD RNA Assay(RUO) .................................. 10

Workﬂow overview ............................................................. 11

■CHAPTER2Reagents, supplies, and required materials ...................... 13

Required materials notsupplied ................................................. 13

Recommended materials andequipment ......................................... 14

■CHAPTER3Before youbegin .................................................... 16

Prerequisites .................................................................. 16

Proceduralguidelines ........................................................... 16

Before each use of thekit ....................................................... 17

Guidelines for preventingcontamination .......................................... 17

Guidelines for sample multiplexing and chipselection .............................. 18

■CHAPTER4Create a Planned Run in Torrent Suite™ Software ............... 19

Torrent Suite™ Software Planned Run templates ................................... 19

Create a PlannedRun .......................................................... 20

Create a Planned Run with SampleSets .......................................... 23

Microhaplotype analysis in Torrent Suite™ Software ................................. 27

Create a Planned Run to generate DRﬁles ........................................ 28

Download DRﬁles ............................................................. 29

Create a Planned Run for microhaplotypeanalysis ................................. 30

Oncomine™ Myeloid MRD Assay (RUO) User Guide 3

■CHAPTER5Prepare DNA molecular tagging reactions ....................... 32

Guidelines for DNA isolation, quantiﬁcation, andinput .............................. 32

DNA input requirements ........................................................ 33

Set up target ampliﬁcation reactions—Oncomine™ Myeloid MRD DNA Libraries ........ 33

Set up molecular tagging reactions—Oncomine™ Myeloid MRD DNA

Libraries (concentrations≥20ng/µL) ....................................... 33

Set up molecular tagging reactions—Oncomine™ Myeloid MRD DNA

Libraries (concentrations<20ng/µL) ....................................... 35

Set up target ampliﬁcation reactions—Oncomine™Myeloid MRD DNA with MHT

panel libraries ............................................................... 37

Prepare target ampliﬁcation reactions—Oncomine™Myeloid MRD DNA with

MHT panel libraries (concentrations≥20ng/µL) .............................. 37

Prepare target ampliﬁcation reactions—Oncomine™Myeloid MRD DNA with

MHT panel libraries (concentrations<20ng/µL) .............................. 39

Set up target ampliﬁcation reactions—Oncomine™Myeloid MRD MHT panel libraries ... 41

Set up molecular tagging reactions—Oncomine™Myeloid MRD MHT panel

libraries (concentrations≥20ng/µL) ........................................ 41

Set up molecular tagging reactions—Oncomine™Myeloid MRD MHT panel

libraries (concentrations<20ng/µL) ........................................ 43

■CHAPTER6Prepare Oncomine™ Myeloid MRD DNA libraries ................ 45

Amplify the targets ............................................................. 45

Combine target ampliﬁcation reactions ........................................... 46

Partially digest amplicons ...................................................... 46

Amplify the library with barcodedprimers ......................................... 47

Purify the library ............................................................... 48

Quantify library and calculate the dilution factor ................................... 50

■CHAPTER7Prepare Oncomine™ Myeloid MRD RNA libraries ................ 53

Guidelines for RNA isolation, quantiﬁcation, andinput .............................. 53

Reverse-transcribeRNA ........................................................ 53

Prepare cDNA target ampliﬁcation reactions ....................................... 55

Amplify the targets ............................................................. 56

Partially digestamplicons ....................................................... 56

Amplify the library with barcodedprimers ......................................... 57

Purify the library ............................................................... 59

Quantify library and calculate the dilution factor ................................... 61

■CHAPTER8Combining libraries, templating, andsequencing ............... 63

Combine libraries .............................................................. 63

Guidelines for templating andsequencing ......................................... 64

Guidelines for sample multiplexing and chipselection .............................. 65

Contents

4Oncomine™ Myeloid MRD Assay (RUO) User Guide

■CHAPTER9View microhaplotype results in Torrent Suite™ Software ........ 66

View a microhaplotype analysis report ............................................ 66

Example microhaplotype analysis report ...................................... 67

Run the microhaplotype_analysis plugin after the run is complete .................... 69

■CHAPTER10Review analysis results in Ion Reporter™ Software ............. 71

Ion Reporter™ Software Oncomine™ Myeloid MRD analysisworkﬂows ................ 72

Manually launch ananalysis ..................................................... 72

View Oncomine™ Myeloid MRD analysis results .................................... 73

The FLT3 Long ITDalgorithm ................................................ 74

Visualize Oncomine™ Myeloid MRD analysis results ................................ 74

Time seriesvisualizations ....................................................... 77

Create and view a time series graph .......................................... 78

Example time series graph .................................................. 81

Generate a report .............................................................. 81

Download variant ﬁles from Ion Reporter™ Software ................................ 83

■APPENDIXATips and troubleshooting ......................................... 84

Tips .......................................................................... 84

Troubleshooting ................................................................ 86

Library yield andquantiﬁcation .............................................. 86

Low amplicon uniformity (DNAonly) .......................................... 87

Other ..................................................................... 87

■APPENDIXBSupplementalinformation ........................................ 88

Digest total RNA with ezDNase™Enzyme ......................................... 88

Upload BEDﬁles ............................................................... 89

Oncomine™ Myeloid MRD Assay (RUO) reference and BEDﬁles ................. 90

Create a Sample Set by importing samples from a CSVﬁle .......................... 90

Conﬁgure the IonReporterUploader plugin in Torrent Suite™ Software ................. 91

■APPENDIXCLimit of Detection .................................................. 92

Molecular tag sequencing technology—Important factors ........................... 92

■APPENDIXDSafety ............................................................... 94

Chemicalsafety ................................................................ 95

Biological hazardsafety ......................................................... 96

Contents

Oncomine™ Myeloid MRD Assay (RUO) User Guide 5

■APPENDIXEDocumentation and support ...................................... 97

Relateddocumentation ......................................................... 97

Customer and technical support ................................................. 97

Contents

6Oncomine™ Myeloid MRD Assay (RUO) User Guide

Product information

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

in this document.

Product description

The Oncomine™ Myeloid MRD Assay (RUO) contains Ion AmpliSeq™ HD oligonucleotide primers and

associated reagents to generate amplicon libraries for next-generation sequencing (NGS) on an Ion

GeneStudio™ S5 Series system. The assay contains 3 panels, the Oncomine™ Myeloid MRD DNA

Panel, Oncomine™ Myeloid MRD RNA Panel, and the Oncomine™ Myeloid MRD MHT Panel. The

assay is designed to detect variants across 33 key genes and a broad range of fusions based on

43 fusion driver gene targets relevant in major myeloid disorders: acute myeloid leukemia (AML),

myeloid dysplastic syndrome (MDS), myeloproliferative neoplasms (MPN), chronic myeloid leukemia

(CML), chronic myelomonocytic leukemia (CMML), and juvenile myelomonocytic leukemia (JMML).

This highly sensitive method enables detection of low numbers of cancer cells known as measurable

residual disease (MRD). An optional microhaplotyping (MHT) panel also enables chimerism analysis of

donor/recipient DNA mixtures in post-transplantation samples.

Features include:

•Compatible with 60 ng/pool input DNA or 10 ng RNA per library from blood or bone marrow

samples

•Excellent coverage of challenging targets such as CEBPA and FLT3-ITDs

•Veriﬁed detection of somatic variants down to 0.1% allele frequency

Designed for research use only (RUO), the Oncomine™ Myeloid MRD Assay (RUO) is a comprehensive,

targeted NGS assay designed to help research scientists in the understanding of myeloid cancer. The

assay is a complete kit facilitating the ampliﬁcation of multiple gene targets from blood or bone marrow

samples with as little as 60 ng of input DNA per pool or 10 ng RNA. Leveraging the power of error-

correcting Ion AmpliSeq™ HD technology, this highly multiplexed NGS assay enables the generation

of results from multiple samples (4 DNA samples or 12 RNA samples on a single Ion 540™ Chip,

or 6 DNA samples or 18 RNA samples on an Ion 550™ Chip) with a single run. The DNA and RNA

assays can be run together or separately. The assay is aligned with bioinformatic analysis workﬂows

in Ion Reporter™ Software that utilize optimized variant-calling parameters for SNV, InDel, FLT3 internal

tandem duplication (ITD) detection, and microhaplotyping (MHT)-based chimerism analysis. For more

information, see “Molecular tag sequencing technology—Important factors” on page92. Torrent

Suite™ software is used to set up sequencing runs for primary analysis with plugins.

This guide covers library preparation from DNA and RNA using the following products:

•Oncomine™ Myeloid MRD DNA Assay (RUO) (Cat.No.A53838)

–Oncomine™ Myeloid MRD DNA Panel (2pools)

–Oncomine™ Myeloid MRD MHT Panel (1pool)

1

Oncomine™ Myeloid MRD Assay (RUO) User Guide 7

•Oncomine™ Myeloid MRD RNA Assay (RUO) (Cat.No.A53839)

–Oncomine™ Myeloid MRD RNA Panel (1pool)

•Ion AmpliSeq™ HD Library Kit (Cat. No.A37694)

•Ion AmpliSeq™ HD Dual Barcode Kit 1–24 (Cat. No.A37695)

•HD Enhancer (Cat.NoA53690)

The Ion AmpliSeq™ HD Library Kit (Cat. No.A37694) is designed for the preparation of amplicon

libraries as part of the Ion AmpliSeq™ HD workﬂow.

The Ion AmpliSeq™ HD Dual Barcode Kit 1–24 contains primers that are used to prepare dual‑barcoded

libraries, with barcodes placed at the 5′‑ end and the 3′‑end. The dual‑barcode design was speciﬁcally

developed to reduce the risk of cross‑barcode contamination.

The HD Enhancer reagent (Cat.NoA53690) is used with the Ion AmpliSeq™ HD Library Kit to optimize

library preparation. It is used instead of the CRC reagent, which is part of the Ion AmpliSeq™ HD Library

Kit.

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 Plus System (Cat. No. A38195)

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

Contents and storage

Kit summary

Two formats of the Oncomine™ Myeloid MRD Assay (RUO) are available.

•Oncomine™ Myeloid MRD DNA Assay (RUO) (Cat.No.A53838)

•Oncomine™ Myeloid MRD RNA Assay (RUO) (Cat.No.A53839)

Each kit contains sucient reagents for the rapid preparation of 24 barcoded DNA- or RNA libraries.

Component

Quantity per kit

A53838 A53839

20x Oncomine™ Myeloid MRD DNA Panel (PartNoA53688) 1 —

20X Oncomine™ Myeloid MRD RNA Panel (Part.NoA53689) — 1

20X Oncomine™ Myeloid MRD MHT Panel

(Packaged with the Oncomine™ Myeloid MRD DNA Panel)

1 —

Ion AmpliSeq™ HD Library Kit (Cat.NoA37694) 1 1

Ion AmpliSeq™ HD Dual Barcode Kit 1–24 (Cat.NoA37695) 1 1

Chapter1Product information

Contents and storage

1

8Oncomine™ Myeloid MRD Assay (RUO) User Guide

(continued)

Component

Quantity per kit

A53838 A53839

5X HD Enhancer (Cat.NoA53690) 1 1

Ion Torrent™ NGS Reverse Transcription Kit (Cat.NoA45003) NA 1

Oncomine™ Myeloid MRD DNA Assay (RUO)

Oncomine™ Myeloid MRD DNA Assay (RUO) (Cat.No.A53838) contains sucient reagents for the rapid

preparation of 24 barcoded DNA-libraries.

IMPORTANT! The HD Enhancer reagent is an essential component developed speciﬁcally for this

panel. Do not use the CRC reagent included in the Ion AmpliSeq™ HD Library Kit for this panel, use the

HD Enhancer instead.

Component Amount Storage

Oncomine™ Myeloid MRD DNA Panel

20X Oncomine™ Myeloid MRD DNA Panel (pool 1 of 2) 48µL

–30ºC to –10ºC20X Oncomine™ Myeloid MRD DNA Panel (pool 2 of 2) 48µL

20X Oncomine™ Myeloid MRD MHT Panel (single pool) 70µL

HD Enhancer

5X HD Enhancer 200µL –30ºC to –10ºC

Ion AmpliSeq™ HD Library Kit

4X Ion AmpliSeq™ HD Ampliﬁcation Mix (purple cap) 216µL

–30ºC to –10ºCIon AmpliSeq™ HD SUPA Reagent (green cap) 120µL

CRC (yellow cap) 220µL

Low TE (clear cap) 2 ×1.7mL each 15°C to 30°C[1]

Ion AmpliSeq™ HD Dual Barcode Kit 1–24

Ion AmpliSeq™ HD Dual Barcode Kit 1–24 (96‑well plate

format, liquid)

Single use reaction per well −30°C to −10°C

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

Chapter1Product information

Contents and storage 1

Oncomine™ Myeloid MRD Assay (RUO) User Guide 9

Oncomine™ Myeloid MRD RNA Assay (RUO)

Oncomine™ Myeloid MRD RNA Assay (RUO) (Cat.No.A53839) contains sucient reagents for the rapid

preparation of 24 barcoded libraries RNA-libraries).

IMPORTANT! The HD Enhancer reagent is an essential component developed speciﬁcally for this

panel. Do not use the CRC reagent included in the Ion AmpliSeq™ HD Library Kit for this panel, use the

HD Enhancer instead.

Component Amount Storage

Oncomine™ Myeloid MRD RNA Panel

20X Oncomine™ Myeloid MRD RNA Panel (pool 1 of 1) 70µL –30ºC to –10ºC

HD Enhancer

5X HD Enhancer 200µL –30ºC to –10ºC

Ion Torrent™ NGS Reverse Transcription Kit

5X Reaction Buer 64µL

–30ºC to –10ºC

10X RT Enzyme Mix 32µL

Ion AmpliSeq™ HD Library Kit

4X Ion AmpliSeq™ HD Ampliﬁcation Mix (purple cap) 216µL

–30ºC to –10ºC

Ion AmpliSeq™ HD SUPA Reagent (green cap) 120µL

CRC (yellow cap) 220µL

Low TE (clear cap) 2 ×1.7mL each 15°C to 30°C[1]

Ion AmpliSeq™ HD Dual Barcode Kit 1–24

Ion AmpliSeq™ HD Dual Barcode Kit 1–24 (96‑well plate

format, liquid)

Single use reaction per well −30°C to −10°C

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

Chapter1Product information

Contents and storage

1

10 Oncomine™ Myeloid MRD Assay (RUO) User Guide

Workﬂow overview

Note: The following workﬂow includes run planning in Torrent Suite™ Software for sequencing on an

Ion GeneStudio™ S5 Series Sequencer.

From library preparation to sequencing results report

Use a Planned Run template to create a Planned

Run in Torrent Suite™ Software (page19)

Use a pre-installed Planned Run template to create a Planned Run.

Prepare Oncomine™ Myeloid MRD DNA libraries

(page45)

OR

Prepare Oncomine™ Myeloid MRD RNA libraries

( page53)

Amplify the targets, partially digest the amplicons, amplify the libraries

with barcoded primers, then purify. Measure the concentration of libraries

using the Ion Library TaqMan™ Quantitation Kit.

Combine libraries (page63)

To sequence multiple barcoded libraries on a single chip, combine

libraries before template preparation. DNA and RNA libraries can be

sequenced separately or run together on the same chip.

Template preparation and sequencing (page64)

Perform template preparation and sequencing as described in the

instrument user guide.

Chapter1Product information

Workﬂow overview 1

Oncomine™ Myeloid MRD Assay (RUO) User Guide 11

From library preparation to sequencing results report

Review plugin results in Torrent Suite™ Software

(page66)

Review molecularCoverageAnalysis plugin results and view the donor to

recipient ratio in the microhaplotype analysis report.

Review Ion Reporter™ Software analysis results

(page71)

If results were uploaded to a conﬁgured Ion Reporter™ Software account

for analysis you can review results, visualize variants, and create time

series visualizations in Ion Reporter™ Software.

Generate a report (page81)

Generate a report in Ion Reporter™ Software.

Chapter1Product information

Workﬂow overview

1

12 Oncomine™ Myeloid MRD Assay (RUO) User Guide

Reagents, supplies, and required

materials

■ Required materials notsupplied ........................................................ 13

■ Recommended materials andequipment ................................................ 14

This chapter lists the reagents, supplies, and materials needed to operate the IonChef™ Instrument and

Ion GeneStudio™ S5 Series Sequencer, and provides consumables ordering and storage information.

Reagents and supplies can be ordered as kits and starter packs, but most consumables can also be

ordered individually as your needs require.

Note: Consumables that have catalog numbers are orderable. Components that have part numbers

cannot be ordered individually.

Required materials not supplied

In addition to a library kit, HD Enhancer, and panel, you need the following materials and equipment.

Unless otherwise indicated, all materials are available through thermoﬁsher.com.

Item Source

One of the following thermal cyclers:

•Veriti™ 96‑Well Thermal Cycler[1]

•Veriti™ Dx 96‑well Thermal Cycler, 0.2 mL

•VeritiPro™ 96-well Thermal Cycler

•SimpliAmp™ Thermal Cycler

•ProFlex™ Thermal Cycler

Various

Ion Library TaqMan™ Quantitation Kit and a real-time PCR instrument 4468802

One of the following:

•MicroAmp™ EnduraPlate™ Optical 96-Well Clear Reaction Plates with

Barcode

•MicroAmp™ Optical 96-Well Reaction Plate

•4483354

•N8010560

MicroAmp™ Optical Adhesive Film 4311971

MicroAmp™ Clear Adhesive Film 4306311

MicroAmp™ Optical Film Compression Pad 4312639

2

Oncomine™ Myeloid MRD Assay (RUO) User Guide 13

(continued)

Item Source

Eppendorf™ DNA LoBind™ Microcentrifuge Tubes, 1.5‑mL 13-698-791

(ﬁsherscientiﬁc.com)

Agencourt™ AMPure™ XP Kit NC9959336, NC9933872

(ﬁsherscientiﬁc.com)

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

Nuclease-Free Water (not DEPC-Treated) AM9932

Ethanol, Absolute, Molecular Biology Grade MLS

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

Template preparation and sequencing

One of the following:

•Ion 540™ Chip Kit and Ion 540™ Kit – Chef

•Ion 550™ Chip Kit and Ion 550™ Kit − Chef

•A27765 and A30011

•A34538 and A34541

[1] Supported but no longer available for purchase.

Recommended materials and equipment

Unless otherwise indicated, all materials are available through thermoﬁsher.com.

Item Source

Additional equipment

One of the following Applied Biosystems™ real-time PCR instruments:

•7500 Real-Time PCR System

•7900HT Fast Real‑Time PCR System[1]

•StepOne™ Real-Time PCR System

•StepOnePlus™ Real-Time PCR System

•ViiA™ 7 Real-Time PCR System

•QuantStudio™ Real‑Time PCR Systems

Various

Fisher Scientiﬁc™ Mini Plate Spinner Centrifuge,

or equivalent 96-well plate centrifuge

14-100-143

(ﬁsherscientiﬁc.com)

MicroAmp™ Adhesive Film Applicator 4333183

Nucleic acid isolation—DNA samples

MagMAX™ DNA Multi-Sample Ultra 2.0 Kit A36570

PureLink™ Genomic DNA Mini Kit K1820-01

Chapter2Reagents, supplies, and required materials

Recommended materials and equipment

2

14 Oncomine™ Myeloid MRD Assay (RUO) User Guide

(continued)

Item Source

Nucleic acid isolation—RNA samples

MagMAX™ mirVana™ Total RNA Isolation Kit A27828

PureLink™ Total RNA Blood Kit K156001

RNaseZap™ RNase Decontamination Solution AM9780

Nucleic acid quantiﬁcation

TaqMan™ RNase P Detection Reagents Kit[2] 4316831

Qubit™ 4 Fluorometer[3] Q33238

Qubit™ dsDNA HS Assay Kit (DNA) or Qubit™ RNA HS Assay Kit (RNA) Q32851 or Q32852

Recommended for library quantiﬁcation

Ion Library TaqMan™ Quantitation Kit 4468802

Controls

Ion S5™ Controls Kit Plus A30729

AcroMetrix™ Custom MRD Control 0.1% VAF[4] AMX7001

Seraseq™ Myeloid Fusion RNA Mix 0710-0407

(seracare.com)

AcroMetrix™ BCR-ABL Panel 956980TS

[1] Supported but no longer available for purchase.

[2] Other qPCR targets such as GUSB can be used for DNA and RNA quantification. Contact your local Field Application Scientist for

details.

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

[4] To purchase the AcroMetrix™ Custom MRD Control 0.1% VAF, go to https://www.thermofisher.com/us/en/home/global/forms/

clinical/acrometrix-molecular-controls-contact.html.

Chapter2Reagents, supplies, and required materials

Recommended materials and equipment 2

Oncomine™ Myeloid MRD Assay (RUO) User Guide 15

Before you begin

Prerequisites

IMPORTANT! This kit is compatible with Torrent Suite™ Software and Ion Reporter™ Software versions

5.18 or later. However, we recommend upgrading to Ion Reporter™ Software 5.20, as Ion Reporter™

Software 5.20 is required to enable the data analysis described. Results from runs can be automatically

uploaded to Ion Reporter™ Software, where you can view data and visualizations for further analysis.

Before proceeding, check for updates to the Torrent Suite™ Software, Ion Reporter™ Software, and

IonChef™ System instrument software, and install the updates when available. Contact a service

representative for help with software upgrades.

Users require a functional knowledge and understanding in the following areas:

•Ion Torrent™ sequencing chemistry and workﬂow (See “Related documentation” on page97 for

more information)

•Techniques for handling and preparing DNA and RNA libraries

Procedural guidelines

IMPORTANT! Do not combine FWD and REV primer subpools for storage. Primer subpools must

remain separate until they are combined during the reaction setup.

•Minimize freeze-thaw cycles of Oncomine™ Myeloid MRD Assay (RUO) panels by aliquoting primer

subpools for your experiments. Panels can be stored 4°C for 1 month. For longer term, store at

−20°C.

•Use a calibrated thermal cycler that is speciﬁed in “Required materials not supplied” on page13.

•Pipet viscous solutions, such as 4X Ion AmpliSeq™ HD Ampliﬁcation Mix, Ion AmpliSeq™ HD SUPA

Reagent, and HD Enhancer slowly. After thawing the capped tubes, ensure complete mixing by

vortexing for 10 seconds.

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

during puriﬁcation with the DynaMag™–96 Side Magnet or other plate magnet.

•Prepare fresh 70% ethanol each day.

•Use the default ramping rate when entering the cycling program for the following thermal cyclers.

–Veriti™ 96‑Well Thermal Cycler

–Veriti™ Dx 96‑well Thermal Cycler

–VeritiPro™ 96-well Thermal Cycler

–SimpliAmp™ Thermal Cycler

–ProFlex™ Thermal Cycler

3

16 Oncomine™ Myeloid MRD Assay (RUO) User Guide

Before each use of the kit

Thaw components that contain enzymes—such as 4X Ion AmpliSeq™ HD Ampliﬁcation Mix, and

Ion AmpliSeq™ HD SUPA Reagent—on ice, and keep them on ice during the procedure. All other

components, including primer subpools and 5X HD Enhancer, can be thawed at room temperature.

Gently vortex, then centrifuge each kit component to collect the contents before use.

Guidelines for preventing contamination

•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.

•When preparing libraries, we recommend alternating barcodes between consecutive sequencing

runs to prevent carryover contamination. The following table provides an example.

Library type

Barcode usage

System Run 1 System Run 2 System Run 3

Positive control 1 9 17

Sample 2 10 18

Sample 3 11 19

Sample 4 12 20

Sample 5 13 21

Sample 6 14 22

Sample 7 15 23

No‑template control (NTC) 8 16 24

Chapter3Before you begin

Before each use of the kit 3

Oncomine™ Myeloid MRD Assay (RUO) User Guide 17

Guidelines for sample multiplexing and chip selection

Use the following guidelines to determine how many libraries can be run on a chip with each panel

conﬁguration.

Note: If you are using an NTC library, the number of libraries per chip shown in the following table

include the NTC library. For example, for DNA libraries on an Ion 550™ Chip, either 6 positive libraries or

5 libraries + 1 NTC library can be loaded on the chip.

Chip

Panel(s)

DNA Panel +

RNA Panel

DNA Panel

only

RNA Panel

only

DNA with MHT

Panel +

RNA Panel

DNA with MHT

Panel[1]

MHT Panel

only

Ion 540™ Chip 2DNA + 2RNA 4DNA 12RNA 2DNA + 2RNA 3DNA 12DNA

Ion 550™ Chip 4DNA + 4RNA 6DNA 18RNA 3DNA + 3RNA 5DNA 18DNA

[1] Microhaplotyping (MHT) panel

Chapter3Before you begin

Guidelines for sample multiplexing and chip selection

3

18 Oncomine™ Myeloid MRD Assay (RUO) User Guide

Create a Planned Run in Torrent

Suite™ Software

IMPORTANT! This kit is compatible with Torrent Suite™ Software and Ion Reporter™ Software versions

5.18 or later. However, we recommend upgrading to Ion Reporter™ Software 5.20, as Ion Reporter™

Software 5.20 is required to enable the data analysis described. Results from runs can be automatically

uploaded to Ion Reporter™ Software, where you can view data and visualizations for further analysis.

Before proceeding, check for updates to the Torrent Suite™ Software, Ion Reporter™ Software, and

IonChef™ System instrument software, and install the updates when available. Contact a service

representative for help with software upgrades.

Torrent Suite™ Software Planned Run templates

Planned Run templates contain the settings that are used in a sequencing run, including the number of

ﬂows, kit types, barcodes, sample information, and any reference ﬁles. Planned Runs are used to track

samples, chips, and reagents throughout the sequencing workﬂow, from template preparation on the

IonChef™ Instrument through sequencing on the Ion GeneStudio™ S5 Series System and subsequent

data analysis. Each chip that is prepared in an IonChef™ Instrument run requires its own Planned Run.

Torrent Suite™ Software 5.18 or later contains 12 Planned Run templates for use with the Oncomine™

Myeloid MRD Assay (RUO).

•Six Planned Run templates are for variant detection only. The following templates are designed

to detect variants across 33 key DNA genes and a broad panel of 43 fusion driver gene targets

relevant in major myeloid disorders.

–Oncomine™ Myeloid MRD DNA for 540

–Oncomine™ Myeloid MRD DNA for 550

–Oncomine™ Myeloid MRD Fusions for 540

–Oncomine™ Myeloid MRD Fusions for 550

–Oncomine™ Myeloid MRD DNA and Fusions for 540

–Oncomine™ Myeloid MRD DNA and Fusions for 550

•Six templates are for microhaplotype analysis combined with variant detection. For more

information, see “Microhaplotype analysis in Torrent Suite™ Software” on page27.

4

Oncomine™ Myeloid MRD Assay (RUO) User Guide 19

Create a Planned Run

IMPORTANT! Before you create a Planned Run ensure that the Ion Reporter™ Software account is

conﬁgured, and that the most current Reference Library, Target Regions, and Hotspot Regions

BED ﬁles are uploaded. See “Upload BED ﬁles” on page89 for more information. Contact a service

representative to obtain the most current BED ﬁles.

1. Sign in to the Torrent Suite™ Software on a computer that is connected to the IonChef™

Instrument.

2. In the Plan tab, in the Templates screen, in the left navigation menu, select Oncology –

HemeOnc.

3. In the Template Name column, select one of the 12 Planned Run templates to use for your run.

The Create Plan workﬂow bar opens to the Create Plan step with preselected template settings.

4. Review the following information, make changes only if needed.

Table1The Create Plan step

Callout Field Action

1Run Plan Name Change the software-generated Run Plan name if needed.

2Analysis Parameters Ensure that Default (Recommended) is selected.

3Default Reference & BED

Files

The reference sequence ﬁles and BED ﬁles for the run are preselected. For

more information, see “Oncomine™ Myeloid MRD Assay (RUO) reference

and BED ﬁles” on page90.

4Use same reference & BED

ﬁles for all barcodes

Deselect if assigning dierent ﬁles to individual libraries.

5Same sample for DNA and

Fusions?

Deselect if using dierent samples for DNA and Fusions.

6Number of barcodes If needed, change the number of barcodes in the run, then click .

For more information about barcodes, see “Guidelines for sample

multiplexing and chip selection” on page18.

7Sample Tube Label Enter or scan the barcode of the IonChef™ Library Sample Tube used in the

run.

8Chip Barcode (Optional) Enter or scan the chip barcode.

Chapter4Create a Planned Run in Torrent Suite™ Software

Create a Planned Run

4

20 Oncomine™ Myeloid MRD Assay (RUO) User Guide

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