Transcription
Oncomine Tumor Mutation Load AssayUSER GUIDECatalog Numbers A37909, A37910Publication Number MAN0017042Revision C.0For Research Use Only. Not for use in diagnostic procedures.
Manufacturer: Life Technologies Corporation 6055 Sunol Blvd Pleasanton, CA 94566The 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.Revision history: Pub. No. MAN0017042RevisionDateC.04 October 2018B.031 August 2018Description Include troubleshooting information for FFPE DNA samples withhigh levels of deamination Include Uracil DNA Glycosylase (UDG) treatment supplementalprotocol Updated Mutation Load calculation Updated to include information for use with the Ion 550 Chip A.020 December 2017 Updated for use with Ion Reporter Software 5.10 Oncomine Tumor Mutation Load Assay User Guide, provides instructionfor library preparation, templating, sequencing, and results analysis of the Oncomine Tumor Mutation Load Assay.Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you acceptthe terms and conditions of all applicable Limited Use Label Licenses. TRADEMARKS: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Microsoft and Excel aretrademarks of Microsoft Corporation. 2018 Thermo Fisher Scientific Inc. All rights reserved.
Contents CHAPTER 1 Product information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Definition of Mutation Load . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Oncomine Tumor Mutation Load Assay – Manual Library Preparation Kit . . . . . . . . . 9 Oncomine Tumor Mutation Load Assay – Chef‑Ready Library Preparation . . . . . . . 10Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11Recommended materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12Sequencer compatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 CHAPTER 2 Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Procedural guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Before each use of the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 CHAPTER 3 Library preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Ion Chef Instrument setup information for Chef Ready kit users . . . . . . . . . . . . . . . . . . . . 14Set up DNA target amplification reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Guidelines for DNA isolation and quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Guidelines for the amount of DNA needed per target amplification reaction . . . . . . . 14Prepare DNA target amplification reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15Amplify the targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16Combine target amplification reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16Partially digest the amplicons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Ligate adapters to the amplicons and purify . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ion Xpress adapters only: Combine and dilute adapters . . . . . . . . . . . . . . . . . . . . . . .Perform the ligation reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Purify the library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Elute the library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1718181920Quantify library by qPCR and calculate the dilution factor . . . . . . . . . . . . . . . . . . . . . . . . . . . 21Combine libraries for equal depth of coverage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23Guidelines for templating and sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24Oncomine Tumor Mutation Load Assay User Guide3
Contents CHAPTER 4 Create an assay-specific Planned Run . . . . . . . . . . . . . . . . . 25About Planned Runs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25Create a custom Planned Run template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26Create a Planned Run from a template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 CHAPTER 5 Mutation load analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31Review sequencing run results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Ion Reporter analysis workflows . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32View results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32Generate an Analysis Results Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35View the Mutation Load statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36Visualize Mutation Load analysis results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37Sample Results tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38Sample QC tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 APPENDIX A Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42Tips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 APPENDIX B Quantify the amplified library with the Qubit Fluorometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44Amplify the library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44Purify the amplified library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45First-round purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45Second-round purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 Qubit Fluorometer: Quantify the library and calculate the dilution factor . . . . . . . . . . . . . 47Store libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 APPENDIX C Supplemental information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 Torrent Suite Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Install the IonReporter Uploader plugin on your Torrent Server . . . . . . . . . . . . . . . . . .Configure the IonReporter Uploader plugin in the Torrent Browser . . . . . . . . . . . . . . .Download and install BED files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ion Reporter Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oncomine Tumor Mutation Load Assay filter chain criteria . . . . . . . . . . . . . . . . . . . . .Enable Mutation Load calculation in existing workflows . . . . . . . . . . . . . . . . . . . . . . . . Manually launch an analysis in Ion Reporter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Customize the Ion Reporter workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .484849495050515253Remove deaminated bases from FFPE DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 554Oncomine Tumor Mutation Load Assay User Guide
Contents APPENDIX D Example reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57Example Analysis Results Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 Oncomine Tumor Mutation Load Assay report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 APPENDIX E Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65Oncomine Tumor Mutation Load Assay User Guide5
1Product informationDefinition of Mutation LoadMutation Load, also known as Tumor Mutational Burden (TMB), as determined bythe Oncomine Tumor Mutation Load Assay is defined as the number ofnonsynonymous variants (missense and nonsense single nucleotide variants (SNVs)),plus insertion and deletion variants (INDELs) detected per megabase (Mb) of exonicsequence. The Ion Reporter Software TMB algorithm filters out germline mutationsusing the Mutation Load Calculation Filter Chain when calculating the Mutation Loadresult.Table 1 Mutation Load algorithm versions and descriptionsVersion[1]TMB algorithm v2.5DescriptionUsed in the Oncomine Tumor Mutation Load - w2.0 - DNA - Single Sample workflow. TheMutation Load Calculation Filter Chain parameter Mutation Load (Somatic Mutations) bydefault.precalibration Mutation Load (Nonsynonymous Somatic Mutations x 106)Total Exonic Bases with Sufficient CoverageIF precalibration Mutation Load 25, THEN Mutation Load (precalibration Mutation Load 25) x Calibration Slope 25IF precalibration Mutation Load 25, THEN no calibration is requiredMutation Load precalibration Mutation Load x 1.0 Includes exonic INDELs in the Mutation Load calculation. Calibration factor applied to the Mutation Load calculation.Note: To enable other workflows to apply the TMB algorithm v2.5 you must copy then editthe Oncomine Tumor Mutation Load - w2.0 - DNA - Single Sample workflow to match theworkflow of choice (for example, Oncomine Comprehensive v3 - w3.2 - DNA and Fusions Single Sample). For more information, see “Enable Mutation Load calculation in existingworkflows“ on page 51.6Oncomine Tumor Mutation Load Assay User Guide
Chapter 1 Product informationDefinition of Mutation LoadVersion[1]TMB algorithm v2.01DescriptionUsed in workflows enabled by the user to include the mutation load calculation by selecting aMutation Load Calculation Filter Chain parameter[2].Mutation Load (Nonsynonymous Somatic Mutations x 106)Total Exonic Bases with Sufficient Coverage Includes only exonic nonsynonymous somatic SNVs in the Mutation Load calculation.INDELs are not included in the Mutation Load calculation even if the selected filter chainfiltered‑in INDELs. The Mutation Load Calculation Filter Chain parameter can be applied to any "DNA Single Sample" or "DNA and Fusions - Single Sample" workflow.TMB algorithm v1.0[3] Used in the Oncomine Tumor Mutation Load - w1.0 - DNA - Single Sample workflow.Includes all exonic and intronic synonymous and nonsynonymous somatic SNVs in theMutation Load calculation.[1][2][3]To determine the TMB algorithm version used for analysis of a particular sample, look in the statistic.txt file. For more information,see page 36.We recommend setting the Mutation Load Calculation Filter Chain parameter to "Mutation Load (Somatic SNVs)" filter chain. For moreinformation, see the Ion Reporter Software online help.Hidden from view in Ion Reporter Software 5.6.Note: Reanalysis of samples previously run with TMB algorithm v1.0 (or v2.0) withthe new Ion Reporter Software 5.10 TMB algorithm v2.5 may result in a differentMutation Load result due to changes in the algorithm.Oncomine Tumor Mutation Load Assay User Guide7
1Chapter 1 Product informationProduct descriptionProduct descriptionThe Oncomine Tumor Mutation Load Assay is a targeted next-generationsequencing (NGS) assay that is designed for tumor profiling by annotating cancerdriver variants and provides an accurate assessment of Mutation Load(mutations/Mb). The assay detects and annotates low frequency somatic variants(SNPs and INDELs) from 409 genes, spanning 1.7 Mb of genomic space,encompassing 1.2 Mb of exonic sequence. This assay is designed to facilitatesuccessful selection and identification of samples most likely to derive responses fromtargeted therapies to immunotherapies in cancer research.This guide covers library preparation from formalin-fixed paraffin-embedded (FFPE)tumor samples—10 ng of DNA per primer pool—using the Oncomine TumorMutation Load Assay library preparation kits (manual and Chef-Ready), throughresults analysis. The assay is used with barcoded adapters so that multiple librariescan be combined and loaded onto a single chip to minimize the per-samplesequencing cost.Barcoded libraries per chip[1][2]Ion 540 ChipIon 550 Chip[1]2–8[2]2–16[2]The Ion 550 Chip is only compatible with the Ion GeneStudio S5 Plus Sequencer, Ion GeneStudio S5 PrimeSequencer, or Ion S5 XL Sequencer. The Ion 550 Chip cannot be sequenced on the Ion S5 Sequencer,orbase model Ion GeneStudio S5 Sequencer.Calculation of the Mutation Load alone does not require as many reads as variant calling. We recommendmultiplex sequencing of no more than 4 libraries (Ion 540 Chip)—6 libraries (Ion 550 Chip)— to achievesufficient read depth for variant calls at a 5% allele frequency.The Ion Reporter Software 5.10 analysis workflow uses a custom variant calling andgermline variant filtering algorithm to accurately estimate somatic variants in cancerresearch samples, with no matched normal sample required. The software provides adetailed report, feature-rich visualization, annotation of low frequency cancer drivervariants (SNVs and INDELs), and the normalized Mutation Load (mutations/Mb).Included in the results are the percentage of mutations consistent with UV damage,tobacco smoke damage, deamination, and specific substitutions.This guide covers the following products: Oncomine Tumor Mutation Load Assay – Manual primer panel (2-pools)(Cat. No. A37907) Ion AmpliSeq Library Kit Plus (Cat. No. 4488990) Ion Xpress Barcode Adapters (various Cat. Nos.) IonCode Barcode Adapters (Cat. No. A29751)Note: Oncomine Tumor Mutation Load Assay – Chef-Ready Library Preparation(Cat. No. A37910) is also available for automated library preparation (see page 10).The kit provides the Oncomine Tumor Mutation Load Assay – Chef-Ready primerpanel (2-pools) at 2X concentration pre-measured in barcoded Primer Pool tubesready to load into an Ion AmpliSeq Chef Reagents DL8 cartridge.8Oncomine Tumor Mutation Load Assay User Guide
Chapter 1 Product informationContents and storage1Contents and storageOncomine TumorMutation LoadAssay – ManualLibraryPreparation KitThe Oncomine Tumor Mutation Load Assay – Manual Library Preparation Kit(Cat. No. A37909) consists of the Oncomine Tumor Mutation Load Assay – ManualLibrary Preparation primer panel (2-pools), and the Ion AmpliSeq Library Kit Plus.Sufficient reagents are provided for the rapid preparation of 24 barcoded samplelibraries from DNA.ContentsAmountStorageOncomine Tumor Mutation Load Assay – Manual Library Preparation primer panel(Part No. A37907, 24 reactions)Oncomine Tumor Mutation Load Assay (2X)(blue cap) (pool 1 of 2)120 µLOncomine Tumor Mutation Load Assay (2X)(blue cap) (pool 2 of 2)120 µL–30ºC to –10ºCIon AmpliSeq Library Kit Plus (Part No. 4488990)5X Ion AmpliSeq HiFi Mix (red cap)120 µLFuPa Reagent (brown cap)48 µLSwitch Solution (yellow cap)96 µLDNA Ligase (blue cap)48 µL25X Library Amp Primers (pink cap)48 µL1X Library Amp Mix (black cap)1.2 mLLow TE[1]6 mL–30ºC to –10ºC15 C to 30 C[1]Can be stored at –30ºC to –10ºC for convenience.Oncomine Tumor Mutation Load Assay User Guide9
1Chapter 1 Product informationContents and storageOncomine TumorMutation LoadAssay –Chef‑ReadyLibraryPreparationThe Oncomine Tumor Mutation Load Assay – Chef-Ready Library Preparation Kit(Cat. No. A37910) provides the Oncomine Tumor Mutation Load Assay –Chef-Ready Library Preparation primer panel at 2X concentration pre-measured inbarcoded Primer Pool tubes ready to load into an Ion AmpliSeq Chef Reagents DL8cartridge. In addition, the kit provides all the reagents and supplies in an IonAmpliSeq Kit for Chef DL8 sufficient for preparing 32 libraries. See the IonAmpliSeq Library Preparation on the Ion Chef System User Guide (Pub. No.MAN0013432) for detailed information on preparing Oncomine Tumor MutationLoad Assay libraries on the Ion Chef System.ComponentAmountStorageOncomine Tumor Mutation Load Assay – Chef‑Ready (Part No. A37908, 32 reactions)Oncomine Tumor Mutation Load Assay – Manual (2X)(pool 1 of 2)4 150 µLOncomine Tumor Mutation Load Assay – Manual (2X)(pool 2 of 2)4 150 µL–30 C to –10 CIon AmpliSeq Kit for Chef DL8 (Cat. No. A29024)Ion AmpliSeq Chef Reagents DL8 (Part No. A29025)4 cartridges–30 C to –10 CIon AmpliSeq Chef Solutions DL8 (Part No. A29026)4 cartridges2 C to 8 C[1]1 box with4 inserts15 C to 30 C1 set of4 plates15 C to 30 CIon AmpliSeq Chef Supplies DL8 (per insert)(Part No. A29027) Ion AmpliSeq Tip Cartridge L8 PCR Frame Seal Enrichment CartridgeIonCode 0101–0132 in 96 Well PCR Plates (dried)(Part No. A29028)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)[1]10Ion AmpliSeq Chef Solutions DL8 cartridges are shipped at ambient temperature, but need to be stored at2 C to 8 C upon arrival.Oncomine Tumor Mutation Load Assay User Guide
Chapter 1 Product informationRequired materials not supplied1Required materials not suppliedUnless otherwise indicated, all materials are available through thermofisher.com.MLS: Fisher Scientific (fisherscientific.com) or other major laboratory supplier.ItemIonCode Barcode Adapters 1–384 KitSourceA29751ORIon Xpress Barcode Adapters4471250, 4474009, 4474518,4474519, 4474520, 4474521,or 4474517Agencourt AMPure XP KitBeckman Coulter, A63880 orA63881One of the following:See web product pages GeneAmp PCR System 9700 or Dual 96-well ThermalCycler AB 2720 Thermal Cycler Veriti 96-well Thermal Cycler ProFlex 96‑well PCR SystemMicroAmp Optical 96-well Reaction PlateN80105604306737 (with barcode)MicroAmp Fast Optical 96-Well Reaction Plate4346907MicroAmp Optical Adhesive Film4311971MicroAmp Adhesive Film4306311MicroAmp Compression Pad4312639DynaMag -96 Side Magnet, or other plate magnet12331DEppendorf DNA LoBind Microcentrifuge Tubes, 1.5 mL13-698-791fisherscientific.comNuclease-free WaterAM9932Absolute ethanolMLSPipettors, 2–200 μL, and low-retention filtered pipette tipsMLSOncomine Tumor Mutation Load Assay User Guide11
1Chapter 1 Product informationRecommended materialsRecommended materialsUnless otherwise indicated, all materials are available through thermofisher.com.MLS: Fisher Scientific (fisherscientific.com) or other major laboratory supplier.ItemSourceRecommended additional equipmentReal-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 pages96-well plate centrifugeMLSQubit 4 Fluorometer[1]Q33226Recommended for nucleic acid isolationRecoverAll Multi-Sample RNA/DNA WorkflowA26069RecoverAll Total Nucleic Acid Isolation Kit for FFPEAM1975MagMAX FFPE Total Nucleic Acid Isolation Kit4463365Recommended for nucleic acid quantificationQubit dsDNA HS Assay Kit (DNA)Q32851/Q32854Recommended for library quantificationIon Library TaqMan Quantitation Kit4468802Recommended for Uracil-DNA Glycosylase treatmentUracil-DNA Glycosylase (UDG), heat labile[1]78310100UNQubit 2.0 & Qubit 3.0 Fluorometers are supported but no longer available for purchase.Sequencer compatibilityIon GeneStudio S5 Series System refers generically to the three Ion GeneStudio S5Systems, 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)12Oncomine Tumor Mutation Load Assay User Guide
2Before you beginProcedural guidelines Minimize freeze-thaw cycles of Oncomine Tumor Mutation Load Assay panelsby aliquoting as needed for your experiments. Panels can be stored at 4 C for oneyear. Use good laboratory practices to minimize cross-contamination of products. Ifpossible, perform PCR setup in an area or room that is free of ampliconcontamination. 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 byvortexing or pipetting up and down several times. Arrange samples in alternating columns on the plate for easier pipetting withmultichannel pipettes during purification with the DynaMag Side Magnet.Before each use of the kit Thaw components that contain enzymes—such as 5X Ion AmpliSeq HiFi Mix,FuPa Reagent, DNA Ligase, and 1X Library Amp Mix—on ice, and keep on iceduring procedure. All other components, including primer pools, can be thawedat room temperature. Gently vortex and centrifuge before use. If there is visible precipitate in the Switch Solution after thawing, vortex or pipetup and down at room temperature to resuspend.Oncomine Tumor Mutation Load Assay User Guide13
3Library preparationIMPORTANT! Oncomine Tumor Mutation Load Assay – Chef-Ready LibraryPreparation Kit (Cat. No. A37910) users.· For instructions to prepare Oncomine Tumor Mutation Load Assay libraries on·the Ion Chef System, see the Ion AmpliSeq Library Preparation on the Ion Chef System User Guide (Pub. No. MAN0013432).When templating the combined libraries, use at 50 pM concentration.Ion Chef Instrument setup information for Chef Ready kit usersDuring Ion Chef Instrument setup, enter the following parameters when prompted.Stating material# of primer poolsTarget amplificationcyclesAnneal & extensiontimeFFPE DNA21616 minutesSet up DNA target amplification reactionsGuidelines forDNA isolation andquantification We recommend the RecoverAll Multi-Sample RNA/DNA Workflow(Cat. No. A26069) for isolating gDNA. We recommend the TaqMan RNase P Detection Reagents Kit (Cat. No. 4316831)for quantifying amplifiable human genomic DNA (see Demonstrated Protocol:Sample Quantification for Ion AmpliSeq Library Preparation Using the TaqMan RNAse P Detection Reagents Kit (Pub. No. MAN0007732) available atthermofisher.com). The Qubit dsDNA HS Assay Kit (Cat. No. Q32851 or Q32854) can also be usedfor quantification, particularly for FFPE DNA, and highly degraded DNAsamples. Quantification methods such as densitometry (for example, using a NanoDrop spectrophotometer) are not recommended, because they are not specific for DNA.Use of these methods can lead to gross overestimation of the concentration ofsample DNA, under-seeding of the target amplification reaction, low libraryyields, and poor chip loading.Guidelines for theamount of DNAneeded per targetamplificationreaction14 For each target amplification reaction, use 300–30,000 copies of DNA (10 ng ofmammalian gDNA) from normal or FFPE tissue. Increasing the amount of DNA results in higher-quality libraries, especially whenDNA quality or quantity is unknown. We recommend using 1 ng gDNA(300 copies) only with high-quality, well-quantified samples.Oncomine Tumor Mutation Load Assay User Guide
Chapter 3 Library preparationPrepare DNA target amplification reactions3Prepare DNA target amplification reactionsIMPORTANT! Primer pools and 5X Ion AmpliSeq HiFi Mix are viscous. Pipet slowlyand mix thoroughly.1. Place a 1.5-mL tube and 96-well plate on ice or in a pre-chilled 4 C cold block.2. For each sample, prepare a target amplification master mix without primers in a1.5-mL tube on ice.ComponentVolume 5X Ion AmpliSeq HiFi Mix (red cap)DNA (205 µLng)[1] 7.5 µLNuclease-free Water[1]to 12.5 µLSubstitute 5 µL AcroMetrix Oncology Hotspot Control to prepare a control library.3. Mix thoroughly by pipetting up anddown 5 times, then transfer 5 µL ofeach sample-specific master mix to 2wells of a 96-well PCR plate on ice orin a pre-chilled 4 C cold block.4. Add 5 µL of Oncomine TumorMutation Load Assay (2X) manuallibrary preparation primer pool 1into the first well, and 5 µL of primerpool 2 to the second well.5 μL5 μLSampleMasterMix5 μL2X primerpool 15 μL2X primerpool 25. Seal the plate with a MicroAmp Adhesive Film.6. 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 least5 times before sealing the plate.Oncomine Tumor Mutation Load Assay User Guide15
3Chapter 3 Library preparationAmplify the targetsAmplify the targetsIMPORTANT! When amplifying multiple samples in a single PCR plate, make surethat the input across all samples is roughly equivalent so that the selected cyclenumber is optimal for all the samples in the run.1. Place a MicroAmp Compression Pad on the plate, then load the plate into thethermal cycler.2. Run the following program to amplify the target regions.StageHold15 CyclesStepTemperatureTimeActivate theenzyme99 C2 minDenature99 C15 secAnneal and extend60 C16 min—10 CHoldHoldSTOPPING POINT Target amplification reactions may be stored at 10 C overnighton the thermal cycler. For longer periods, store at 20 C.Combine target amplification reactionsNote: Perform the following steps on ice or in a pre-chilled 4 C cold block.1. Remove the plate from the thermal cycler.2. Centrifuge briefly to collect the contents, then carefully remove the plate seal.3. For each sample, combine both 10-µL target amplification reactions into a singlewell.IMPORTANT! Accurate volume transfer in this step is critical. We recommendusing a single-channel pipettor. If you are using a multi-channel pipettor, visuallycheck pipette tips to ensure that volumes are equivalent.1234567819 10 11 12BBCCDDEEFFGGHH11223456789 10 11 12AAPool 1Pool 2233Combined poolsThe total volume for each sample should be 20 µL.16Oncomine Tumor Mutation Load Assay User Guide
Chapter 3 Library preparationPartially digest the amplicons3Partially digest the ampliconsIMPORTANT! Keep the plate on ice or in a pre-chilled 4 C cold block while preparingthe reactions.1. Thaw the FuPa Reagent (brown cap) on ice, gently vortex to mix, then centrifugebriefly to collect.2. Add 2 µL of FuPa Reagent to each amplified sample. The total volume is 22 µL.3. Seal the plate with a clear adhesive film, vortex thoroughly, then centrifugebriefly to collect droplets. Alternatively, mix by pipetting at least half the totalvolume up and down at least 5 times before sealing the plate.4. Place a compression pad on the plate, load in the thermal cycler, then run thefollowing program:TemperatureTime50 C20 minutes55 C20 minutes60 C20 minutes10 CHold (for up to 1 hour)STOPPING POINT Store plate at –20 C for longer periods.Ligate adapters to the amplicons and purifyWhen sequencing multiple libraries on a single chip, you must ligate a differentbarcode adapter to each library. DNA and RNA libraries from the same sample alsorequire different barcodes.IonCode Adapters are provided at the appropriate concentration and includeforward and reverse adapters in a single well. No further handling is necessary.Ion X
This guide covers library preparation from formalin-fixed paraffin-embedded (FFPE) tumor samples—10 ng of DNA per primer pool—using the Oncomine Tumor Mutation Load Assay library preparation kits (manual and Chef-Ready), through results analysis. The assay is used with barcoded adapters so that multiple libraries
