Thermo Fisher Scientific Oncomine Myeloid MRD Assay User guide

Brand
Thermo Fisher Scientific
Model
Oncomine Myeloid MRD Assay
Type
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.
©2022-2023 Thermo Fisher Scientific Inc. All rights reserved.
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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Thermo Fisher Scientific Oncomine Myeloid MRD Assay User guide

Brand
Thermo Fisher Scientific
Model
Oncomine Myeloid MRD Assay
Type
User guide
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