Transcription
Agilent NGS FFPE QC KitqPCR-based Quantificationand Qualification ofFFPE-derived DNA Samplesfor NGS Target EnrichmentProtocolVersion D0, December 2015For Research Use Only. Not for use in diagnosticprocedures.Agilent Technologies
Notices Agilent Technologies, Inc. 2015WarrantyNo part of this manual may be reproduced inany form or by any means (including electronic storage and retrieval or translationinto a foreign language) without prior agreement and written consent from AgilentTechnologies, Inc. as governed by UnitedStates and international copyright laws.The material contained in thisdocument is provided “as is,” andis subject to being changed, without notice, in future editions. Further, to the maximum extentpermitted by applicable law, Agilent disclaims all warranties,either express or implied, withregard to this manual and anyinformation contained herein,including but not limited to theimplied warranties of merchantability and fitness for a particularpurpose. Agilent shall not be liable for errors or for incidental orconsequential damages in connection with the furnishing, use,or performance of this documentor of any information containedherein. Should Agilent and theuser have a separate writtenagreement with warranty termscovering the material in this document that conflict with theseterms, the warranty terms in theseparate agreement shall control.Manual Part NumberG9700-90000EditionVersion D0, December 2015Printed in USAAgilent Technologies, Inc.5301 Stevens Creek BlvdSanta Clara, CA 95051 USATechnical SupportFor technical product support, contact yourlocal Agilent Support Services representative.For US and Canada, call (800) 227-9770(option 3,4,4). For other countries, find yoursupport center telephone numbers atwww.agilent.com/chem/contactus.Or send an e-mail to:[email protected] LicensesThe hardware and/or software described inthis document are furnished under a licenseand may be used or copied only in accordance with the terms of such license.Restricted Rights LegendU.S. Government Restricted Rights. Software and technical data rights granted tothe federal government include only thoserights customarily provided to end user customers. Agilent provides this customarycommercial license in Software and technical data pursuant to FAR 12.211 (TechnicalData) and 12.212 (Computer Software) and,for the Department of Defense, DFARS252.227-7015 (Technical Data - CommercialItems) and DFARS 227.7202-3 (Rights inCommercial Computer Software or Computer Software Documentation).2Agilent NGS FFPE QC Kit
Notices to PurchaserThis product is provided under an intellectual property license from Life Technologies Corporation. The purchase of this product conveys to thebuyer the non-transferable right to use the purchased product and components of the product only in research conducted by the buyer(whether the buyer is an academic or for-profit entity). The sale of this product is expressly conditioned on the buyer not using the product orits components for any Commercial Purposes. Commercial Purposes means any activity by the buyer to generate revenue, which may include,but is not limited to use of the product or its components: (1) in manufacturing or in quality assurance or quality control; (2) to provide a service, information, or data for a fee or other consideration; (3) for therapeutic or prophylactic purposes; (4) for diagnostic use; and (5) for resale,whether or not such items are resold for use in research. For information on purchasing a license to this product for purposes other thanresearch, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008 USA or outlicensing@lifetech. com.NOTICE TO PURCHASER: LIMITED LICENSE:Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 6,258,569,6,171,785, 6,127,155, 6,030,787, 5,994,056, 5,876,930, 5,804,375, 5,789,224, 5,773,258 (claims 1 and 6 only), 5,723,591, 5,677,152 (claims 1 to 23only), 5,618,711, 5,538,848, and claims outside the US corresponding to expired US Patent No. 5,079,352. The purchase of this product includesa limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser’s owninternal research. No right under any other patent claim and no right to perform commercial services of any kind, including without limitationreporting the results of purchaser’s activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel.This product is for research use only. Diagnostic uses under Roche patents require a separate license from Roche. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California94404, USA.SYBR is a registered trademark of Molecular Probes, Inc.Safety NoticesCA U T I O NA CAUTION notice denotes a hazard. It calls attention to an operating procedure, practice, or the like that, if notcorrectly performed or adhered to, could result in damage to the product or loss of important data. Do not proceedbeyond a CAUTION notice until the indicated conditions are fully understood and met.WARN I NGA WARNING notice denotes a hazard. It calls attention to an operating procedure, practice, or the like that, ifnot correctly performed or adhered to, could result in personal injury or death. Do not proceed beyond aWARNING notice until the indicated conditions are fully understood and met.Agilent NGS FFPE QC Kit3
In this Guide.This guide provides an optimized protocol for use of theAgilent NGS FFPE QC Kit to quantify and qualifyFFPE-derived DNA samples by qPCR prior to use in NGSapplications.1Before You BeginThis chapter contains information (such as procedural notes,safety information, required reagents and equipment andoverview of the workflow) that you should read andunderstand before you start an experiment.2ProceduresThis chapter describes the steps to qPCR-analyze theFFPE-derived DNA samples and provides guidelines forimplementing the findings in downstream NGS steps.3ReferenceThis chapter includes reference information, including kitcontents and troubleshooting.4Agilent NGS FFPE QC Kit
What’s New in Version D0 Support for use with HaloPlex HS Target EnrichmentSystem (see “Modifications for the HaloPlex HSWorkflow" on page 30)What’s New in Version C0 Updated guideline for acceptable amplification efficiencyto 85–110% (see page 26) New recommendation to limit freeze-thaw cycles to fivefor DNA Standards 1–5 and to aliquot the DNA Standardsolutions provided with 96-reaction kits (see page 14)What’s New in Version B0 Updated product labeling statementAgilent NGS FFPE QC Kit5
6Agilent NGS FFPE QC Kit
Contents1Before You Begin9Procedural NotesSafety Notes1010Required ReagentsRequired Equipment1111Overview of the Workflow12Preprotocol Considerations 14Preparing the FFPE-derived genomic DNA samples as templatePreventing template cross-contamination 14Limiting freeze-thaw cycles for the DNA Standards 14Recommended Plate Setup for qPCR 15Use of the Reference Dye 16Data acquisition with a real-time PCR instrument 16Dissociation Curve analysis 172Procedures1419Amplify FFPE DNA Samples using qPCR 20Step 1. Prepare the FFPE-derived genomic DNA samplesStep 2. Prepare the qPCR reactions 21Step 3. Run the qPCR amplification 2420Analyze the Data 26Step 1. Quantify the amplifiable DNA in FFPE samples using the StandardCurve 26Step 2. Determine DNA integrity scores for FFPE samples 27Implement NGS Target Enrichment and Downstream SequencingModifications 28Modifications for the SureSelect Workflow 28Modifications for the HaloPlex HS Workflow 30Agilent NGS FFPE QC Kit7
3Reference33Kit Contents 34Storage Conditions 34Troubleshooting Guide 358Agilent NGS FFPE QC Kit
Agilent NGS FFPE QC KitProtocol1Before You BeginProcedural Notes 10Safety Notes 10Required Reagents 11Required Equipment 11Overview of the Workflow 12Preprotocol Considerations 14Make sure you read and understand the information in this chapter andhave the necessary equipment and reagents listed before you start anexperiment.Agilent Technologies9
1Before You BeginProcedural NotesProcedural Notes For optimal consistency, mix gDNA sample solutions well on a vortexmixer then spin the sample tube briefly to collect the liquid. To prevent contamination of reagents by nucleases, always wearpowder-free laboratory gloves and use dedicated solutions and pipettorswith nuclease-free aerosol-resistant tips. Use best-practices to prevent PCR product contamination of samplesthroughout the workflow:1 Assign separate pre-PCR and post-PCR work areas and usededicated equipment, supplies, and reagents in each area. Inparticular, never use materials designated to post-PCR work areas forpre-PCR segments of the workflow.2 Maintain clean work areas. Clean pre-PCR surfaces that pose thehighest risk of contamination daily using a 10% bleach solution.3 Always use dedicated pre-PCR pipettors with nuclease-freeaerosol-resistant tips to pipette dedicated pre-PCR solutions.4 Wear powder-free gloves. Use good laboratory hygiene, includingchanging gloves after contact with any potentially-contaminatedsurfaces. When preparing frozen reagent stock solutions use the followingguidelines:1 Thaw buffer solutions at room temperature.Thaw all other reagents (such as enzymes, primers, and nucleotides)on ice.2 Mix briefly on a vortex mixer, then spin in a centrifuge for 5 to10 seconds to drive the contents off of walls and lid.3 Store all components on ice or in a cold block until use, unlessotherwise instructed in the protocol.Safety NotesCA U T I O N10 Wear appropriate personal protective equipment (PPE) when working in thelaboratory.Agilent NGS FFPE QC Kit
Before You BeginRequired Reagents1Required ReagentsTable 1Required ReagentsDescriptionVendor and part numberAgilent NGS FFPE QC KitAgilent16 reactions96 reactionsp/n G9700Ap/n G9700BQubit BR dsDNA Assay Kit100 assays500 assaysLife Technologiesp/n Q32850p/n Q32853Nuclease-free Water (not DEPC-treated)Ambion Cat #AM9930Required EquipmentTable 2Required EquipmentDescriptionVendor and part numberAriaMx Real-Time PCR System, or equivalentAgilent p/n G8830AAriaMx-compatible plasticware:96-well platesAgilent p/n 401490Optical strip capsAgilent p/n 401427Qubit FluorometerLife Technologies p/n Q32857Qubit Assay TubesLife Technologies p/n Q32856DNA LoBind Tubes, 1.5-ml PCR clean, 250 piecesEppendorf p/n 022431021 or equivalentMicrocentrifugeGeneral laboratory supplierP20, P200 and P1000 pipettesPipetman or equivalentVortex mixerIce bucketSterile, nuclease-free aerosol barrier pipette tipsAgilent NGS FFPE QC Kit11
1Before You BeginOverview of the WorkflowOverview of the WorkflowAgilent NGS FFPE QC Kit provides a qPCR-based system for determiningthe quality and amplifiable quantity of prepared genomic DNA samplesderived from formalin-fixed paraffin-embedded (FFPE) human tissues. TheAgilent NGS FFPE QC Kit sample quantification and qualificationworkflow is summarized in Figure 1.The Agilent NGS FFPE QC Kit is recommended for use in combinationwith Agilent’s NGS target enrichment systems including SureSelectXTTarget Enrichment kits (using the Low Input DNA protocol) and HaloPlexHS Target Enrichment kits. Both target enrichment systems are suitablefor FFPE samples after making minor protocol modifications based onsample quality, as outlined in this manual. Information on the SureSelectand HaloPlex HS platforms can be found at www.agilent.com/genomics.The kit helps optimize performance of FFPE-derived DNA samples in NGSapplications in the following ways: The population of DNA that is intact enough to allow amplification inthe FFPE-derived DNA sample is precisely quantified by qPCR.Quantification is done by standard curve analysis, using pre-dilutedDNA standards and Primer Set A, which targets a single-copy DNAregion of the human genome and produces a 42-bp amplicon. The quality of the DNA in the FFPE-derived sample is determined witha DNA integrity score. This analysis is done by comparing qPCR resultsusing Primer Set A to results using Primer Set B, which targets thesame DNA region but produces a 123-bp amplicon, resulting in a Cqintegrity score for the sample. This integrity score is normalized usingthe provided Human Reference DNA sample, to generate the normalizedDNA integrity score ( Cq). Guidelines are provided for how to modify Agilent’s SureSelectXT LowInput Target Enrichment protocol or the HaloPlex HS protocol accordingto the Cq value for each sample. Guidelines are also provided for recommended sequencing depthmodifications for FFPE-derived samples with various Cq values.12Agilent NGS FFPE QC Kit
Before You BeginOverview of the WorkflowFigure 1Agilent NGS FFPE QC Kit1Overall sample qualification workflow.13
1Before You BeginPreprotocol ConsiderationsPreprotocol ConsiderationsPreparing the FFPE-derived genomic DNA samples as templateBefore starting the protocol, purify gDNA samples from FFPE tissuesections. Agilent recommends the Qiagen QIAamp DNA FFPE Tissue Kit(Qiagen p/n 56404) and Qiagen Deparaffinization Solution (Qiagen p/n19093) for this step.DNA sample preparation instructions for qPCR (see page 20) begin withdetermination of initial sample concentration using the fluorometry-basedQubit dsDNA BR Assay. Using a fluorometry-based method to measureconcentration prior to preparing the qPCR samples is important to ensurethat the template concentration falls within the quantitative range of thestandard curve.High concentrations of FFPE DNA may be inhibitory to qPCR reactions.Be sure to dilute the FFPE DNA samples to 125 pg/µl, as directed onpage 20, for use as qPCR template.Preventing template cross-contaminationTake precautions to minimize the potential for carryover of nucleic acidsfrom one experiment to the next. Use separate work areas and pipettorsfor pre- and post-amplification steps. Use positive displacement pipets oraerosol-resistant pipet tips.Limiting freeze-thaw cycles for the DNA StandardsThe provided DNA Standards should be subjected to five or fewerfreeze-thaw cycles, to ensure optimal amplification efficiency duringstandard curve generation. When using a 96-reaction kit, after the firstthawing of DNA Standards 1–5, subaliquot each DNA Standard solutioninto single-use aliquots. The plate setup suggested on page 15 requires12 µl of each DNA Standard per run.14Agilent NGS FFPE QC Kit
Before You BeginRecommended Plate Setup for qPCR1Recommended Plate Setup for qPCRTo ensure accurate quantification while optimizing throughput, use thefollowing scheme (summarized in Table 3 on page 15) for the qPCR plate: Analyze all five of the provided DNA Standards 1–5 amplified withPrimer Set A (in triplicate) to generate a standard curve for each run.The range of DNA concentration covered by the standard curve is2500 pg/µl down to 9.77 pg/µl. Analyze 8 FFPE DNA samples per plate, with amplification usingPrimer Set A and Primer Set B in separate wells, performed intriplicate for each sample/primer combination. Analyze the provided Reference DNA with amplification using PrimerSet A and Primer Set B in separate wells, performed in triplicate. Include no-template control (NTC) reaction in triplicate for each PrimerSet A and B.Table 3ABCDEFGRecommended plate setup12Std 1Primers AStd 1Primers AStd 2Primers A3789101112Std 1FFPE 1FFPE 1FFPE 1Primers A Primers A Primers A Primers AFFPE 1Primers BFFPE 1Primers BFFPE 1Primers BRef DNAPrimers BRef DNAPrimers BRef DNAPrimers BStd 2Primers AStd 2FFPE 2FFPE 2FFPE 2Primers A Primers A Primers A Primers AFFPE 2Primers BFFPE 2Primers BFFPE 2Primers BNTCPrimers BNTCPrimers BNTCPrimers BStd 3Primers AStd 3Primers AStd 3FFPE 3FFPE 3FFPE 3Primers A Primers A Primers A Primers AFFPE 3Primers BFFPE 3Primers BFFPE 3Primers BNo ampNo amp No ampStd 4Primers AStd 4Primers AStd 4FFPE 4FFPE 4FFPE 4Primers A Primers A Primers A Primers AFFPE 4Primers BFFPE 4Primers BFFPE 4Primers BNo ampNo amp No ampStd 5Primers AStd 5Primers AStd 5FFPE 5FFPE 5FFPE 5Primers A Primers A Primers A Primers AFFPE 5Primers BFFPE 5Primers BFFPE 5Primers BNo ampNo amp No ampRef DNAPrimers ARef DNAPrimers ARef DNAFFPE 6FFPE 6FFPE 6Primers A Primers A Primers A Primers AFFPE 6Primers BFFPE 6Primers BFFPE 6Primers BNo ampNo amp No ampNTCPrimers ANTCPrimers ANTCFFPE 7FFPE 7FFPE 7Primers A Primers A Primers A Primers AFFPE 7Primers BFFPE 7Primers BFFPE 7Primers BNo ampNo amp No ampFFPE 8FFPE 8FFPE 8Primers A Primers A Primers AFFPE 8Primers BFFPE 8Primers BFFPE 8Primers BNo ampNo amp No ampHNo ampNo amp No ampAgilent NGS FFPE QC Kit45615
1Before You BeginUse of the Reference DyeUse of the Reference DyeA passive reference dye is included in this kit and may be added tocompensate for non-PCR related variations in fluorescence. Fluorescencefrom the passive reference dye does not change during the course of thePCR reaction but provides a stable baseline to which samples arenormalized. The excitation and emission wavelengths of the reference dyeare 584 nm and 612 nm, respectively. Although using the Reference Dye isoptional, it is required for optimal results with some instruments,including the recommended Agilent AriaMx instrument.The 1 mM stock of Reference Dye needs to be diluted using PCR-gradewater prior to adding it to the qPCR reactions. Use the dilution guidelinesbelow: If using an Agilent qPCR instrument (AriaMx, Mx3000P or Mx3005PReal-Time PCR system), dilute the Reference Dye 1:500 in nuclease-freewater to a concentration of 2 M. Using this dilution results in aReference Dye concentration of 30 nM in the final qPCR reactions. If using the ABI StepOnePlus instrument, dilute the Reference Dye 1:50in nuclease-free water for a final concentration of 20 M. Using thisdilution results in a Reference Dye concentration of 300 nM in the finalqPCR reactions. If using the Bio-Rad CFX96 instrument, omit the Reference Dye fromthe qPCR reactions.Keep all solutions containing the reference dye protected from light.The diluted reference dye, if stored in a light-protected tube at 4 C, canbe used within the same day for setting up additional assays.Data acquisition with a real-time PCR instrumentThe instrument should be set to collect SYBR Green I data in real-time atthe annealing/extension step of each cycle. Consult the manufacturer’sinstruction manual for the instrument and software version you are usingfor more information on setting up this mode of data acquisition.16Agilent NGS FFPE QC Kit
Before You BeginDissociation Curve analysis1Dissociation Curve analysisIncluding a dissociation segment in the qPCR thermal profile isrecommended but not required. When running the FFPE QC assays usingthe recommended Agilent AriaMx instrument and experiment subtypeQuantitative PCR-DNA Binding Dye Including Standard Melt, a dissociation segment isautomatically included in the thermal profile.Dissociation curves for samples analyzed using the AriaMx instrument areshown in Figure 2 below.Figure 2Dissociation curves for qPCR wells analyzed using the AriaMx platform.Agilent NGS FFPE QC Kit17
118Before You BeginDissociation Curve analysisAgilent NGS FFPE QC Kit
Agilent NGS FFPE QC KitProtocol2ProceduresAmplify FFPE DNA Samples using qPCR 20Step 1. Prepare the FFPE-derived genomic DNA samples 20Step 2. Prepare the qPCR reactions 21Step 3. Run the qPCR amplification 24Analyze the Data 26Step 1. Quantify the amplifiable DNA in FFPE samples using the StandardCurve 26Step 2. Determine DNA integrity scores for FFPE samples 27Implement NGS Target Enrichment and Downstream SequencingModifications 28Modifications for the SureSelect Workflow 28Modifications for the HaloPlex HS Workflow 30This chapter contains instructions for quantifying the concentration ofamplifiable DNA in FFPE-derived DNA samples and for determining thenormalized DNA integrity score for each sample.Agilent Technologies19
2ProceduresAmplify FFPE DNA Samples using qPCRAmplify FFPE DNA Samples using qPCRStep 1. Prepare the FFPE-derived genomic DNA samples1 Use the Qubit dsDNA BR Assay to estimate the initial concentration ofeach gDNA sample. Follow the instructions for the instrument.2 Dilute each gDNA sample with nuclease-free water to 2 ng/µl in a1.5-mL LoBind tube.Vortex the dilution tubes thoroughly then spin briefly to collect theliquid. Store on ice.3 In a fresh 1.5-mL LoBind tube, transfer 5 µl of each gDNA sampledilution from step 2 into 75 µl of nuclease-free water for a finalconcentration of 125 pg/µl.Vortex thoroughly then spin briefly to collect the liquid. Store on ice.4 Record the dilution factor used for each sample for future use. Thetotal dilution factor is product of the variable dilution factor used instep 2 16 (constant factor used in step 3).20Agilent NGS FFPE QC Kit
ProceduresStep 2. Prepare the qPCR reactions2Step 2. Prepare the qPCR reactions1 If using the Reference Dye, prepare a dilution of the 1 mM ReferenceDye stock using PCR-grade water according to Table 4, below.Table 4NOTEReference Dye DilutionqPCR PlatformDilution toPrepareVolume of Dilute DyeNeeded per 8-Sample PlateAgilent AriaMx, Mx3000P, or Mx3005P Instrument1:50023.7 lABI StepOne Plus Instrument1:5023.7 lKeep all solutions containing the reference dye protected from light. The dilutedreference dye, if stored in a light-protected tube at 4 C, can be used within the same day forsetting up additional assays.2 Thaw the 2 Brilliant III SYBR Green QPCR Master Mix and keep thetube on ice while setting up the reactions. SYBR Green is lightsensitive; solutions containing the master mix should be protectedfrom light whenever possible.3 Prepare the Primer Set A (Quantification) reaction mix by combiningthe components in Table 5 in order. Prepare a single reagent mixturefor all reactions (plus at least one reaction volume excess).Vortex the mixture well, then spin the tube briefly to collect the liquid.Keep on ice.Table 5Quantification Reaction Mix (Primer Set A)ReagentVolume for 1 reaction Volume for 8-sample plate(includes excess)2 Brilliant III SYBR Green QPCR Master Mix 10 µL470 µLPrimer Set A, 42 bp1 µL47 µLDiluted Reference Dye (from step 1)*0.3 µL14.1 µLNuclease-free, PCR-grade water4.7 µL220.9 µLTotal16 µL752 µL* If you are not using the Reference Dye, add an equivalent volume of nuclease-free water.Agilent NGS FFPE QC Kit21
2ProceduresStep 2. Prepare the qPCR reactions4 Place 16 l of the Primer Set A Reaction Mix prepared in step 3 intothe appropriate individual plate wells or reaction tubes. For 8-sampleruns using the recommended plate setup shown on page 15, place the16- l aliquots in the shaded wells of Column 1-6 shown below. Keep onice.5 Prepare the Primer Set B (Qualification) reaction mix by combining thecomponents in Table 6 in order. Prepare a single reagent mixture forall reactions (plus at least one reaction volume excess).Vortex the mixture well, then spin the tube briefly to collect the liquid.Keep on ice.Table 6Qualification Reaction Mix (Primer Set B)ReagentVolume for 1 reaction Volume for 8-sample plate(includes excess)2 Brilliant III SYBR Green QPCR Master Mix 10 µL320 µLPrimer Set B, 123 bp1 µL32 µLDiluted Reference Dye (from step 1)*0.3 µL9.6 µLNuclease-free, PCR-grade water4.7 µL150.4 µLTotal16 µL512 µL* If you are not using the Reference Dye, add an equivalent volume of nuclease-free water.22Agilent NGS FFPE QC Kit
ProceduresStep 2. Prepare the qPCR reactions26 Place 16 l of the Primer Set B Reaction Mix prepared in step 5 intoindividual plate wells or reaction tubes. For 8-sample runs using therecommended plate setup shown on page 15, place the 16- l aliquots inthe wells shown in the dark-shaded wells of Column 7-12 shown below.Keep on ice.7 Add 4 l of the appropriate DNA sample to each well to bring the finalreaction volume to 20 l in all wells. For the no-template control (NTC)reactions, add 4 l of nuclease-free water in place of the DNA. If usingthe plate setup in page 15, add the DNA samples as shown below.8 Seal the plate wells with strip caps, then vortex the plate to mix thereactions. Spin the plate briefly to collect the liquid and release anybubbles from the bottoms of the wells.Agilent NGS FFPE QC Kit23
2ProceduresStep 3. Run the qPCR amplificationStep 3. Run the qPCR amplification1 Set up the qPCR experiment in the qPCR system software. For Agilent’sAriaMx instrument, use experiment subtype Quantitative PCR-DNA Binding DyeIncluding Standard Melt. If using a different instrument, set up theappropriate experiment type for real-time detection of SYBR Greenfluorescence, reporting of quantification cycle (Cq) values, andsubsequent standard curve analysis.a Assign well information as needed to generate a standard curve fromthe wells containing the DNA Standards. Enter the amount oftemplate DNA in each Standard well, as shown in Table 7 below.Table 7Amount of template in standards wellsDNA DescriptionConcentrationAmount DNA/qPCR reactionDNA Standard 12500 pg/ l10,000 pgDNA Standard 2625 pg/ l2,500 pgDNA Standard 3156.25 pg/ l625 pgDNA Standard 439.06 pg/ l156.25 pgDNA Standard 59.77 pg/ l39.06 pgb Set up the thermal profile shown in Table 8 for Agilent AriaMx, ABIStepOne Plus, and BioRad CFX96 instruments or as shown inTable 9 for Agilent Mx3000P and Mx3005P instruments. Adissociation segment is automatically added to the thermal profilewhen using Agilent’s AriaMx instrument as directed.Set the instrument to report and detect fluorescence at each cycleduring the 63 C annealing/extension step.Table 824PCR Cycling Protocol for Agilent AriaMx, ABI StepOne Plus, or BioRad CFX96Number of CyclesTemperatureDuration195 C3 minutes4095 C10 seconds63 C20 secondsAgilent NGS FFPE QC Kit
ProceduresStep 3. Run the qPCR amplificationTable 92PCR Cycling Protocol for Agilent Mx3000P or Mx3005PNumber of CyclesTemperatureDuration195 C3 minutes4095 C10 seconds63 C25 seconds2 Place the plate in the qPCR instrument and run the PCR program.Agilent NGS FFPE QC Kit25
2ProceduresAnalyze the DataAnalyze the DataStep 1. Quantify the amplifiable DNA in FFPE samples using theStandard Curve1 In the qPCR analysis software, generate a standard curve (plot of initialDNA quantity versus Cq) using wells assigned to the provided DNAStandards. Verify that the standard curve has an R-squared value 0.98and an amplification efficiency between 85% and 110%. An example isshown below.2 From the standard curve analysis, get the amount of amplifiable DNA(nanograms) in each FFPE sample well.3 Calculate the concentration of amplifiable DNA (ng/ l) in each FFPEDNA sample:a Divide the amount determined using the standard curve by 4 l(volume added to each qPCR reaction).b Multiply by the dilution factor recorded for each sample in step 4 onpage 20.For FFPE samples with significantly-degraded DNA, thisqPCR-determined concentration of amplifiable DNA is used to calculatethe required DNA input volume for SureSelectXT or HaloPlex HSsample preparation.26Agilent NGS FFPE QC Kit
ProceduresStep 2. Determine DNA integrity scores for FFPE samples2Step 2. Determine DNA integrity scores for FFPE samplesDNA integrity scores are based on the relative amplification of DNA froma given sample using Primer Set A, which produces a 42-bp amplicon,compared to Primer Set B, which produces a 123-bp amplicon from thesame locus. The difference in quantification cycle number, or Cq whenamplifying using Primer Set B versus A, is used to assign a numericalvalue to the DNA integrity for each sample.1 Determine Cq results for the provided Reference DNA by subtractingthe Cq determined for Primer Set A from the Cq determined for PrimerSet B.NOTEIt is normal for the Reference DNA to have a slightly negative Cq. The larger amplicon maybind more of the SYBR Green dye and allow signal detection at an earlier cycle compared tothe smaller amplicon, even though the amplicon copy number is the same.2 Determine Cq results for each FFPE DNA sample by subtracting theCq determined for Primer Set A from the Cq determined for Primer SetB.3 Find the normalized DNA integrity score ( Cq) for each FFPE DNAsample by subtracting the Reference DNA Cq from the sample Cq.Use this Cq DNA integrity score to identify any NGS protocolmodifications appropriate for a given FFPE sample. See page 28through page 31 for recommended protocol modifications.Agilent NGS FFPE QC Kit27
2ProceduresImplement NGS Target Enrichment and Downstream Sequencing ModificationsImplement NGS Target Enrichment and Downstream SequencingModificationsFFPE-derived DNA samples may be used in Agilent’s NGS TargetEnrichment and downstream sequencing workflows after making minorprotocol modifications. The modifications appropriate for the SureSelectworkflow and for the HaloPlex workflow are described in separatesections below.Modifications for the SureSelect WorkflowStep 1. Implement SureSelect Protocol ModificationsFFPE-derived DNA samples may be used in the SureSelectXT LibraryPreparation and Target Enrichment protocols after making minor protocolmodifications. Protocol modifications that should be applied to all FFPEsamples are summarized in Table 10. Additional protocol modificationsmay be appropriate, depending on the quality of the FFPE sample DNA.Modifications based on the normalized integrity score ( Cq values) aresummarized in Table 11. For the complete SureSelectXT protocol, go towww.genomics.agilent.com and search for document part numberG7530-90000 (version B.2 or later). Use the low input (200 ng) librarypreparation protocol option.Table 10SureSelectXT protocol modifications for all FFPE samplesProtocol Step and ParameterCondition for non-FFPESamplesCondition for FFPE SamplesDuration of DNA Shearing6 minutes4 minutesDilution of SureSelect Adaptor Oligo Mixfor Ligation reactionUse 1:10 dilution
† Support for use with HaloPlex HS Target Enrichment System (see "Modifications for the HaloPlex HS . Qubit BR dsDNA Assay Kit 100 assays 500 assays Life Technologies p/n Q32850 . Qubit Assay Tubes Life Technologies p/n Q32856 DNA LoBind Tubes, 1.5-ml PCR clean, 250 pieces Eppendorf p/n 022431021 or equivalent .
