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MONOTYPEHLA™HLA-B
24OR96SAMPLESUSERMANUAL
FORRESEARCHUSEONLY
V2.0
OmixonLimited
OmixonMonotypeHLA,HLA-BUserManualv2.0.Forresearchuseonly.Copyright©2017,OmixonBiocomputingLtd.Confidential&Proprietary Page2│46
TableofContentsDOCUMENTHISTORY...........................................................................................................................................5THEPRINCIPLEOFTHEMETHOD:NGS-BASEDHLATYPINGFORTHEILLUMINAMISEQ.........................................6MONOTYPEHLAPACKINGLIST.............................................................................................................................7
PRIMERCOMPONENTBOXFOR24SAMPLES.......................................................................................................................7PRIMERCOMPONENTBOXFOR96SAMPLES.......................................................................................................................7LIBRARYPREPARATIONREAGENTSCOMPONENTBOXFOR24SAMPLES....................................................................................7LIBRARYPREPARATIONREAGENTSCOMPONENTBOXFOR96SAMPLES....................................................................................796-WELLADAPTORPLATE...............................................................................................................................................7EXCELWORKBOOK........................................................................................................................................................8SOFTWARE–OMIXONHLATWIN....................................................................................................................................8
RECOMMENDATIONS...........................................................................................................................................9DNAEXTRACTIONRECOMMENDATIONS............................................................................................................................9TECHNICALANDEQUIPMENTRECOMMENDATIONS..............................................................................................................9ASSOCIATEDREAGENTRECOMMENDATIONS.......................................................................................................................9
MiSeqReagentKitcapacity...............................................................................................................................10RECOMMENDEDSUPPLIES.............................................................................................................................................10
LEGALNOTICE....................................................................................................................................................12SUMMARYOFSTEPS..........................................................................................................................................13GLOSSARY/DEFINITIONS.....................................................................................................................................14STEP0–GENOMICDNAPREPARATION..............................................................................................................15STEP1–HLAAMPLIFICATIONMASTERMIXPREPARATION................................................................................16
REAGENTLIST.............................................................................................................................................................16PROTOCOL.................................................................................................................................................................16
STEP2–HLA-BAMPLIFICATION..........................................................................................................................17REAGENTLIST.............................................................................................................................................................17PROTOCOL.................................................................................................................................................................17
EnzymeLoadedMasterMix:HLA-B...................................................................................................................17HLA-BAmplification...........................................................................................................................................18
STEP3–AMPLICONQUANTITATIONANDNORMALIZATION(USINGAPLATEFLUOROMETER)...........................19REAGENTLIST.............................................................................................................................................................19PROTOCOL.................................................................................................................................................................19
ExoSAP-ITPCRPurification.................................................................................................................................21STEP4–LIBRARYPREPARATION........................................................................................................................22
REAGENTLIST.............................................................................................................................................................22PROTOCOL.................................................................................................................................................................22
FragmentationMasterMix................................................................................................................................23FragmentationProgram....................................................................................................................................23EndRepairMasterMix......................................................................................................................................24EndRepairProgram...........................................................................................................................................24LigationMasterMix...........................................................................................................................................25LigationProgram...............................................................................................................................................25
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STEP5–LIBRARYSIZESELECTION.......................................................................................................................28REAGENTLIST.............................................................................................................................................................28PROTOCOL.................................................................................................................................................................28
STEP6–LIBRARYQUANTIFICATIONUSINGAQUBIT...........................................................................................29REAGENTLIST.............................................................................................................................................................29PROTOCOL.................................................................................................................................................................29
STEP7–SEQUENCINGONILLUMINAMISEQ......................................................................................................31REAGENTLIST.............................................................................................................................................................31
MiSeqReagentKitcapacity...............................................................................................................................31PROTOCOL.................................................................................................................................................................31
STEP8–ANALYSISOFHLASEQUENCINGDATA..................................................................................................33AUTOMATEDPROTOCOL...............................................................................................................................................33
ITSetupandConfiguration................................................................................................................................33ProtocolperAnalysis.........................................................................................................................................33
MANUALSERVERPROTOCOL.........................................................................................................................................33ITSetupandConfiguration................................................................................................................................33ProtocolperAnalysis.........................................................................................................................................33
MANUALDESKTOPPROTOCOL.......................................................................................................................................34ITSetupandConfiguration................................................................................................................................34ProtocolperAnalysis.........................................................................................................................................34
TECHNICALASSISTANCE.....................................................................................................................................35PhoneSupport:..................................................................................................................................................35
SUPPLEMENTALFIGURES....................................................................................................................................36PLATEEXAMPLEFORAMPLICONPLATE,AMPLIFICATIONPLATE,DILUTIONPLATE,AMPLICONQUANTIFICATIONPLATE&REACTIONPLATE.......................................................................................................................................................................36FOR24SAMPLES:........................................................................................................................................................36................................................................................................................................................................................36QUANTITATIONPLATEEXAMPLES....................................................................................................................................37
StandardsQuantitationPlate............................................................................................................................37...........................................................................................................................................................................37
QPCRPLATEEXAMPLE.................................................................................................................................................37................................................................................................................................................................................37
APPENDIX1:PIPPINPREP...................................................................................................................................38PROGRAMMINGTHEPIPPINPREP...................................................................................................................................385. CLICKTHE“SAVEAS”BUTTONANDNAMEYOURPROGRAM........................................................................................38RUNNINGTHEPIPPINPREP...........................................................................................................................................38
APPENDIX2:SAMPLESHEET...............................................................................................................................40APPENDIX3:AMPLICONQUANTITATIONUSINGAQPCRINSTRUMENT..............................................................43
REAGENTLIST.............................................................................................................................................................43PROTOCOL.................................................................................................................................................................43
APPENDIX4:LIBRARYQUANTIFICATIONUSINGQPCR........................................................................................45REAGENTLIST.............................................................................................................................................................45PROTOCOL.................................................................................................................................................................45
qPCRPrimerMix................................................................................................................................................45
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qPCRMasterMix...............................................................................................................................................46
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DocumentHistoryVersion Date DescriptionofChanges ApprovalName
1.0 June,2015 InitialVersion PeterMeintjes
1.1 November,2016 Increase of library preparation reagentvolumes
PeterMeintjes
2.0 April2017
“DQB”removal fromtheDQBEnhancers,gelverificationafterLR-PCRisoptional,ampliconquantitation simplification, per-samplepooling volume change in 11-locus kits,ExoSAP-ITreplacementbyExoSAP-ITExpress,Qubituseforlibraryquantitation.
PeterMeintjes
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ThePrincipleoftheMethod:NGS-basedHLAtypingfortheIlluminaMiSeqFormany years theHLA community has beenworking toward amethod thatwill accuratelyidentifytheextensivepolymorphismoftheHLAgenesandoftheirgeneproducts.TheadventofPCR,combinedwithothertechnologies(Sangersequencing,SSOP,SSP,Luminex),providedaformulaforsignificantlyimprovingthedetectionofHLApolymorphismsbutalbeitwithseverallimitationsthatcontinuetoinhibitourabilitytocomprehensivelycharacterizetheHLAgenes.Technologies developed over the last several years, cumulatively called Next GenerationSequencing (NGS),haveprovidednewopportunities thatallowthecompletecharacterizationoftheHLAgenesinhaploidfashion.NGShastwodistinctfeatures,1)clonalsequencingofDNAfragments, and 2) tremendously high throughput. NGS provides the capability to phasepolymorphismstherebyeliminatingallambiguitiesandprovidesHLAtypingatthethreetofourfieldlevelwithoutreflexivetesting,therebyintroducingapotentiallytotalsolutiontotheHLAtypingproblem.Theprotocoldescribedheretakesadvantageofthistechnologyandcombineslong-range PCR amplification of HLA geneswith sequencing on the IlluminaMiSeq platform.MorespecificallyHLA-Bisamplifiedforitsentirecodinglength,includingelementsofthe5’and3’enduntranslatedregions.Theampliconsarethenprocessedthroughaseriesofstepsthat:
1. FragmenttheampliconstoasizeappropriateforsequencingontheIlluminaplatform,
2. Blunt-endandadenylatetheendsofthefragmentedampliconsand
3. LigateadaptorsequencesthatareusedthroughouttheprocessontheMiSeqtocapture,amplify, and sequence the DNA. The adaptors also include an index which is a shortsequence, unique to each adaptor, which identifies the origins of the library(sample/locus).
Afterpoolingtheindexedlibraries,sizeselectionandquantitation,thesampleisloadedontheMiSeqforsequencing.Thewholeprocesstakes3-5daysdependingontheselectionoftheflowcellontheIlluminaplatform.ThegenerateddataareanalyzedusingtwodifferentalgorithmsinHLATwin™(www.omixon.com).TheuseoftwoindependentalgorithmsinHLATwinprovidesthehighest levelof confidence that theHLAgenotyping results canbe reported immediatelywithoutfurtherattention.Sampleswithquestionableorambiguousgenotypingsareflaggedbythesoftwaretobeanalyzedmanually.
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MonotypeHLAPackingList
PrimerComponentBoxfor24samplesPrimermix Samples Vol/tube #Tubes ColorcodeHLA-B 24 60μL 1 Red
PrimerComponentBoxfor96samplesPrimermix Samples Vol/tube #Tubes ColorcodeHLA-B 96 220μL 1 Red
LibraryPreparationReagentsComponentBoxfor24samplesReagent Rxns Vol/tube #Tubes ColorcodeFragmentationEnzyme(A) 24 70μL 1 YellowFragmentationBuffer(B) 24 70μL 1 RedEndRepairEnzyme(C) 24 41μL 1 GreenEndRepairBuffer(D) 24 82μL 1 OrangeLigationEnzyme(E) 24 81μL 1 BlueLigationBuffer(F) 24 900μL 2 Black
LibraryPreparationReagentsComponentBoxfor96samplesReagent Rxns Vol/tube #Tubes ColorcodeFragmentationEnzyme(A) 96 278μL 1 YellowFragmentationBuffer(B) 96 278μL 1 RedEndRepairEnzyme(C) 96 162μL 1 GreenEndRepairBuffer(D) 96 324μL 1 OrangeLigationEnzyme(E) 96 324μL 1 BlueLigationBuffer(F) 96 1800μL 2 Black
96-wellAdaptorPlateIndexedadaptorsina5μLsolutionforgenerating24or96individualsequencinglibraries.
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ExcelWorkbookAn Excel Workbook is provided to support the Monotype HLA protocol with volumecalculations, plate layouts, reagent traceability, record keeping and MiSeq Sample Sheetgenerationofallofthesupportedadaptorplateconfigurations(A,B,A1,A2).Ifyoudonothaveacopy,[email protected].
Software–OmixonHLATwinContact [email protected] for Omixon HLA Twin credits associated with your purchase ofMonotypeHLA.
OmixonMonotypeHLA,HLA-BUserManualv2.0.Forresearchuseonly.Copyright©2017,OmixonBiocomputingLtd.Confidential&Proprietary Page9│46
Recommendations
DNAExtractionRecommendations§ HighqualitygenomicDNA(gDNA)extractedfromwholeblood,bloodcells(B-celllines,
buffycoats,cordbloodoranyfractionofwhitebloodcells),salivaandbuccalswabscanbeused.ForamplificationofHLA-B100-150ngofgDNAarerequired.
TechnicalandEquipmentRecommendations§ Thermalcyclerwith96-wellformat§ Platefluorometer(oranyinstrumentcapableoffluorescencedetectionin96-wellplate
format,forusewiththePromegaQuantiFluordsDNASystem)§ PippinPrep(Cat#PIP0001)orBluePippin(Cat#BLU0001)bySAGEScience§ Qubitfluorometer(Cat#Q33216,ThermoFisherScientific)§ qPCRinstrument(optional)§ IlluminaMiSeq(Cat#SY-410-1003)§ 64-bitcomputerwithminimum4Coresand16GBofRAM§ Long-termdatastorage(approximately2TBofdataperMiSeqperyear)
AssociatedReagentRecommendations§ LongRangePCRkitsfromQiagen(Cat#206401,206402or206403)
§ Eachsamplerequires0.4μLofTaqPolymerase§ Cat#206401LongRangePCRkit(20)contains8μLofTaqPolymerase§ Cat#206402LongRangePCRkit(100)contains40μLofTaqPolymerase§ Cat#206403LongRangePCRkit(250)contains100μLofTaqPolymerase
§ ExoSAP-IT Express from Affymetrix (Cat#75001-200, 75001-1ML, 75001-4X-1ML or75001-10ML)
§ Eachpooledsamplerequires4μLofExoSAP-ITExpressenzyme§ Cat#75001-200contains200μLofExoSAP-ITExpressenzyme§ Cat#75001-1-MLcontains1mLofExoSAP-ITExpressenzyme§ Cat#75001-4X-1MLcontains4mLofExoSAP-ITExpressenzyme§ Cat#75001-10MLcontains10mLofExoSAP-ITExpressenzyme
§ QubitdsDNABRAssayKit(Cat#Q32850orQ32853)§ Cat#Q32850for100assays§ Cat#Q32853for500assays
§ Library Quantification Kit – Illumina/Universal from KAPA Biosystems (Cat# KK4824)(optionalifusingaqPCRinstrument)
§ QuantiFluordsDNASystemfromPromega(Cat#E2670)§ AgencourtAMPureXPbeadsfromBeckmanCoulter(Cat#A63880,A63881,orA63882)
§ EachHolotypeHLArunrequiresamaximumof900μLofAMpureXPbeads§ Cat#A63880contains5mLofAMPureXPbeads
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§ Cat#A63881contains60mLofAMPureXPbeads§ Cat#A63882contains450mLofAMPureXPbeads
§ Gelcassette,1.5%agarose,dyefreewithinternalstandard(MarkerK/R2),forthePippinPrep/BluePippin(Cat#CDF1510forPippinPrepandBDF1510forBluePippin)
§ Moleculargradeethanol(AnhydrousAlcohol)§ Moleculargradewater(DNaseandRNasefree)§ Sodiumhydroxide§ 1×TEbuffer(pH8.0)
MiSeqReagentKitcapacityIlluminaMiSeqReagentKit
TimeHours
#ofSamples
Std300Cycle(MS-102-2002) ~24 192
Micro300Cycle(MS-103-1002) ~19 192
Nano300Cycle(MS-103-1001) ~17 56
Std500Cycle(MS-102-2003) ~39 192
Nano500Cycle(MS-103-1003) ~28 80
RecommendedSupplies§ 1.5mLmicrocentrifugetubes§ 1.5mLlow-bindmicrocentrifugetubes§ 2.0mLlow-bindmicrocentrifugetubes(EppendorfDNALoBindCat#022431048
recommended)§ 0.5mlthinwalltubesforQubitinstrument(QubitAssaytubesCat#Q32856
recommended)§ Adjustablevolumepipettes(1.0–1000μLcapacity)§ 8-channeladjustablevolumepipettes(2.0-500μLcapacity)§ 96-wellplatescompatiblewiththethermalcycler§ 96-wellopticalplatescompatiblewiththeplatefluorometer§ 96-wellplatescompatiblewiththeqPCRinstrument(optional)§ Platesealsforgeneraluse§ Platesealscompatiblewiththethermalcyclers(testedforlongrangePCR)§ OpticalplatesealscompatiblewiththeqPCRinstrument(optional)§ Magneticstandcompatiblewith2mLmicrocentrifugetubes§ 96-wellcoolerracks(2pieces)§ 50mLconicaltubes
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§ 50mLreservoirs
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LegalNoticeThe Monotype HLA User Manual and all contents are proprietary to Omixon Biocomputing("Omixon"), and are intended solely for the contractual useby its customer in regard to theproduct(s)describedhereinandfornootherpurpose.Thisdocumentanditscontentsshallnotbe used or distributed for any other purpose and/or otherwise communicated, disclosed, orreproducedinanywaywhatsoeverwithoutthepriorwrittenconsentofOmixon.Omixondoesnotconveyanylicenseunderitspatent,trademark,copyright,orcommon-lawrightsnorsimilarrightsofanythirdpartiesbythisdocument.UseofOmixonHolotypeorMonotypeHLA(includingsoftwareandassay)isgovernedbyTermsandConditionsGoverningOmixonProducts(www.omixon.com/supply-agreement/),whichreferencestheOmixonHLATwinEULA(www.omixon.com/hla-twin-eula/).
UseofOmixonTargetsoftwareisgovernedbytheOmixonTargetEULA(www.omixon.com/omixon-target-eula/).
UseofOmixonHLATwinsoftwarealoneisgovernedbytheOmixonHLATwinEULA(www.omixon.com/hla-twin-eula/).
UseofOmixonHLAExploresoftwareisgovernedbytheOmixonHLAExploreEULA(www.omixon.com/hla-explore-eula/).
The instructions in this document must be strictly and explicitly followed by qualified andproperly trained personnel in order to ensure the proper and safe use of the product(s)describedherein.Allofthecontentsofthisdocumentmustbefullyreadandunderstoodpriortousingsuchproduct(s).FAILURE TO COMPLETELY READ AND EXPLICITLY FOLLOW ALL OF THE INSTRUCTIONSCONTAINED HEREIN MAY RESULT IN DAMAGE TO THE PRODUCT(S), INJURY TO PERSONS,INCLUDINGTOUSERSOROTHERS,ANDDAMAGETOOTHERPROPERTY.OMIXON DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THEPRODUCT(S)DESCRIBEDHEREIN(INCLUDINGPARTSTHEREOFORSOFTWARE)ORANYUSEOFSUCHPRODUCT(S)OUTSIDETHESCOPEOFTHEEXPRESSWRITTENLICENSESORPERMISSIONSGRANTED BY OMIXON IN CONNECTION WITH CUSTOMER'S ACQUISITION OF SUCHPRODUCT(S).FORRESEARCHUSEONLY©2017OmixonBiocomputingLtd.Allrightsreserved.
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SummaryofSteps
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Glossary/Definitions§ AmpliconPlate–AlternativenameforanAmplificationPlate; inthecaseofDQB1the
contentsofbothAmplificationPlates(Set1andSet2)arecombinedtofortheAmpliconPlate
§ AmpliconQuantitationPlate–96-wellplate compatiblewith theplate fluorometerorqPCRmachinewheretheampliconsarequantitated.
§ AmplificationPlate–96-wellPCRplateusedtoamplifytheHLAloci.§ Final Library – Library that includes all Sample Libraries ready to be sequenced in a
singleMiSeqrun.§ Reaction Plate – Platewhere the sequential reactions that fragment, end repair, and
ligatetheindexedadaptorstotheSampleLibrariesareperformed.§ ReagentPlate–PlateusedtoaliquotthevariousreagentsusedtopreparetheLibraries§ SampleLibrary–AlibrarypreparedforallHLAlociforagivensample.§ Sample Libraries Plate: Plate containing a sample library (all loci combined when
applicable)perwell.§ StandardsQuantitationPlate–96-wellplatecompatiblewiththeplatefluorometeror
qPCRmachinewhereDNAstandardsareplacedtoallowforampliconquantitation.
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Step0–GenomicDNAPreparationDuration:~upto45minutes(dependingonthenumberofsamplesused)Prepare genomic DNA in accordance with relevant DNA extraction/isolation protocols fromblood,saliva,buccalswabsorhematopoieticbloodcells(B-celllines,buffycoats,cordbloodorany fraction of white blood cells). 100 - 150 ng of input genomic DNA per amplification isrequired.
Note:Thesuccessof thisassaydependsonrobustPCRamplification,andhighqualitygenomicDNAisnecessaryforasuccessfulamplification.YourDNAshouldhave:1. A260nm/280nmabsorbanceratiobetween1.7and1.9.2. A260nm/230nmabsorbanceratioof1.7orgreater.3. Minimal degradation. DNA that is old or has gone through repeated
freeze/thawswillsufferfrommoredegradation.
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Step1–HLAAmplificationMasterMixPreparationDuration:~20minutesThe purpose of this step is to prepare locus-specific Master Mix to amplify HLA-B for eachsample. Note: TheMasterMix include all reagents needed for amplification except the TaqPolymeraseenzymemix.
ReagentlistItem Storage SuppliedbyHLA-BPrimerMix -20°C OmixonLongRangePCRBuffer(10×) -20°C Qiagen10mMdNTPs -20°C QiagenSterileH2O -20°C Qiagen
ProtocolRemoveHLA-BPrimerMix, LongRangePCRBuffer (10×), and10mMdNTPs fromstorageandthawatroomtemperature.1. CreateaMasterMixforeachPrimerMixaccordingtothetablesbelow:
MasterMix:HLA-BReagent Volumesfor
singlesampleVolumesfor24
samplesVolumesfor96
samplesPrimerMix 2μL 51μL 204μLLongRangePCRBuffer(10×) 2.5μL 63.8μL 255μLdNTPMix(10mMeach) 1.25μL 31.9μL 127.5μLSterileH2O 13.85μL 353.2μL 1412.7μLTotalVolume 19.6μL 499.9μL 1999.2μL2. VortextheMasterMixandspindownfor1second.PlaceMasterMixonice.3. Dilute5μLofgenomicDNAforeachsampletoaconcentrationof20-30ng/μL.
Note: Monotype HLA includes sufficient reagents for either 24 or 96 reactions(dependingonthekitsize)plusasmalladditionalvolumeforpipettinglossandfailedamplification.
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Step2–HLA-BAmplificationDuration:~4hours15minutesThepurposeofStep2istoamplifytheHLA-BlocususingoptimizedPCRconditions.OncethePCRreactionsarecompleted,amplificationisverifiedbyagarosegelelectrophoresis(optional).
Quicktip–Theagarosegelelectrophoresis(step2.5),whilerecommended, isnotrequiredtosuccessfullycompletetheMonotypeHLAprotocol.Whenampliconsarequantitated(Step3),anyconcentrationabove50ng/μL isconsideredasuccessfulamplification.Agarosegelelectrophoresis isanimportantqualitycontrolstepandshouldnotbeskippedwithoutsufficientexperiencewiththecompleteMonotypeHLAprotocol.
Reagentlist
Protocol1. PrepareanHLA-Bamplificationplate.Pipette5μLofeachdiluted sampleofgenomic
DNA into the designated wells of the locus-specific 96-well Amplification Plate (seesupplementalfigures).
2. AddTaqPolymeraseEnzymetoeachMasterMix.
EnzymeLoadedMasterMix:HLA-B
Reagent Volumesforsinglesample
Volumesfor24samples
Volumesfor96samples
MasterMixfromStep1 19.6μL 499.9μL 1999.2μLTaqPolymerase 0.4μL 10.2μL 40.8μLTotal 20μL 510.1μL 2040μL
3. BrieflyvortexeachenzymeloadedMasterMixandspindownfor1second.4. Aliquot20μLofenzymeloadedMasterMixineachsampleintheAmplificationPlate.5. SealtheAmplificationPlatewithathermalsealandcentrifugefor10seconds.
Item Storage SuppliedbyGenomicDNA 4°C UserTaqPolymerase -20°C QiagenMoleculargradeH2O 20°Cto25°C UserHLA-BMasterMix -20°C Step1
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6. Place theAmplificationPlate intoa thermalcyclerandrun the followingamplificationprogram:
HLA-BAmplificationNumberofCycles Temperature Time
1 95°C 3minutes 95°C 15seconds
35 65°C 30seconds 68°C 5minutes1 68°C 10minutes1 4°C ∞
Note: Amplification success canbe verified by running 2 μL from each amplicon in astandard 2% agarose gel at 250 V for 30 minutes. (Optional after sufficient andconsistentexperience)
ExpectedAmpliconSizes
HLAlocus Expectedampliconsize(kb)HLA-B ~3kb
Safestoppingpoint.Ampliconscanbestoredat4°Covernightorat-20°Cforlonger.
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Step 3 – Amplicon Quantitation and Normalization(usingaPlateFluorometer)Duration:~30hourAmplicon Quantitation and Normalization is recommended to ensure precise input into thelibrarypreparationstep(Optional).AmpliconconcentrationismeasuredusingtheQuantiFluordsDNA System that contains a fluorescent DNA-binding dye and DNA standard for sensitivequantitation of small amounts of double-stranded DNA (dsDNA). Refer to Appendix 3 forInstructionsonhowtodotheAmpliconQuantitationusingaqPCRmachine.
Quick tip – The amplicon quantitation, while recommended, is not required tosuccessfully complete theMonotype HLA protocol. Amplicon normalization doesnot require precise measurement of amplicon concentration. An estimate ofampliconconcentrationsbasedonexperienceoragarosegelelectrophoresiscanbeused instead. Amplicon quantitation should not be skipped without consistentexperiencewiththecompleteMonotypeHLAprotocol.
ReagentlistItem Storage SuppliedbyHLA-BAmplificationPlate(s) 4°C Step220×TEBuffer(pH7.5) 4°C PromegaLambdaDNAStandard(100ng/μL) 4°C Promega200×QuantiFluordsDNADye 4°C PromegaMoleculargradeH2O 20°Cto25°C User ExoSAP-iTExpress -20°C Affymetrix
Protocol1. Prepare DNA standards by serial dilution of the Lambda DNA standard (100 ng/μL)
providedintheQuantiFluorkitaccordingtothedilutiontablebelow:
Labelontube InputDNA VolumeDNA
(μL)Volume1xTE
(μL)FinalConc.ng/μL
Standard1 LambdaDNA 7.5μL 492.5μL 1.5ng/μLStandard2 Standard1 250μL 250μL 0.75ng/μLStandard3 Standard2 250μL 250μL 0.38ng/μLStandard4 Standard3 250μL 250μL 0.19ng/μLStandard5 Standard4 250μL 250μL 0.09ng/μLStandard6 Standard5 250μL 250μL 0.05ng/μL
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Blank Blank 0μL 250 μL 0ng/μL
2. PreparetheAmpliconQuantitationplates(seesupplemental figures).Aliquot99μL1x
TEbuffertothewellsofa96-wellopticalplateforthetotalnumberofampliconstobequantitated.
3. Add 1 μL of amplicons from correspondingwells in the Amplicon Plates to individualwellsintheAmpliconQuantitationPlates.Mixbypipetting
4. Prepare 1× QuantiFluor Dye working solution using the following formula: 0.5 μLQuantiFluorDye (200X) +99.5μL1×TEbuffer. Prepare sufficient 1×QuantiFluorDyeworkingsolutionso thateachsample (total samples inAmpliconPlates)andstandard(14total)willreceivea100μLaliquot.
5. PrepareaStandardsQuantitationPlateandAmpliconQuantitationplates.Aliquot100μLof1×QuantiFluorDyeworkingsolutiontowellsofthe96-wellopticalplatethatwillbetheStandardsQuantitationPlateandtotheAmpliconQuantitationPlatesfromStep3.2.
6. Using the standards prepared above, add 100 μL of each standard, in duplicate, toindividualwellsintheStandardsQuantitationPlate(14wellstotal).Mixbypipetting.
7. Vortexwelltomixandspindown.
8. Run the Standards Quantitation Plate on the plate fluorometer followed by theAmpliconQuantitationPlates.
9. CalculatetheconcentrationofDNAintheAmpliconQuantitationPlatesusingRFUdatageneratedbytheplatefluorometer.RefertotheDilutionTabintheprovidedworkbookforassistancewithcalculations.
10. DiluteDNAintheAmpliconPlatewithsterileH2OsothatthefinalconcentrationofDNAisapproximately67ng/μL.
§ IfDNAconcentrationis150ng/μLorgreater:add25μLofH2O
§ IfDNAconcentrationis100-150ng/μL:add10μLofH2O
§ IfDNAconcentrationislessthan100ng/μL:add0μLofH2O
11. Transfer20μLofthenormalizedampliconsinanew96-wellPCRplate.
12. Aliquot4μLofExoSAP-ITExpress (Affymetrix) intoeachnormalizedampliconandmixbypipetting.
13. SealthePlateswithathermalsealandcentrifugefor10seconds.
14. PlacePlatesintoathermalcyclerandrunthefollowingprogram:
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ExoSAP-ITPCRPurification
Safestoppingpoint.Ampliconscanbestoredat4°Covernightorat-20°Cforlonger.
NumberofCycles Temperature Time1 37°C 4minutes1 80°C 1minutes1 4°C ∞
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Step4–LibraryPreparationDuration:~3hours20minutesDuring this step, the amplicons are prepared for sequencing on the Illumina MiSeq. Theampliconsareenzymatically fragmented, theendsare repairedandadenylated, and indexedadaptorsareligatedtotheends.ThelibrariesarethenpooledfollowedbyasinglecleanupandconcentrationstepperformedusingAMPureXPbeads
Note: Omixon recommends volumes greater than is necessary for 24 or 96 samplesbecausemanyoftheenzymesandbuffersareviscous,resultinginexcesspipettingloss.
ReagentlistItem Storage SuppliedbyAmpliconPlate 4°C Step3AdaptorPlate(5μLperwell) -20°C OmixonFragmentationEnzyme(A) -20°C OmixonFragmentationBuffer(B) -20°C OmixonEndRepairEnzyme(C) -20°C OmixonEndRepairBuffer(D) -20°C OmixonLigationEnzyme(E) -20°C OmixonLigationBuffer(F) -20°C OmixonAMPureXPbeads 4°C BeckmanCoulter80%Ethanol(freshlyprepared) 20°Cto25°C UserSterileH2O 20°Cto25°C User
Protocol1. Turnonthethermalcycler.Verifythattheheatedlidiswarmingup.
2. Prepare enough FragmentationMasterMix for each amplicon in the Amplicon Plate,includingtheampliconpoolwells.
Note:BesuretovortextheFragmentationEnzyme(A)thoroughlybeforeuse.
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FragmentationMasterMix
Reagent Volumeperlibrary(μL)
Recommendedvolumesfor96libraries(μL)
Recommendedvolumesfor96libraries(μL)
Colorcode
FragmentationEnzyme(A) 2μL 55.2μL 220.8μL YellowFragmentationBuffer(B) 2μL 55.2μL 220.8μL RedTotalvolume 4μL 110.4μL 441.6μL
3. PrepareaReagentPlate:placeanew96-wellPCRplateonaPCRcoolerrackandaliquotandequalamountoftheFragmentationMasterMixintoeachwellofasinglecolumn.
Note: The fragmentation reactionhasbeendesigned toprovide ideally sizedDNA forsequencingon the IlluminaMiSeq. It is important to keep the reagents colduntil thereaction is started in the thermal cycler to prevent excessive fragmentation. Use ofmulti-channel pipettes is recommended to minimize opportunities for excessivefragmentation.
4. CentrifugetheAmpliconPlatefor10seconds,andplaceitoniceorcoldblock.
5. PrepareaReactionPlate:placeafresh96-wellPCRplateonaPCRcoldblock.
6. Add4μLofFragmentationMasterMixfromtheReagentPlateintowellsoftheReactionPlate, correspondingwith samples in the Amplicon Plate. The use of amulti-channelpipetteisrecommended.
7. Transfer16μLofeachampliconfromtheAmpliconsPlatetothecorrespondingwellontheReactionPlateusingamulti-channelpipette.Mixbypipetting.
8. CovertheReactionPlatewiththermalsealandcentrifugefor10seconds.
9. IncubatetheReactionPlateinathermalcyclerwiththefollowingprogram:
FragmentationProgram
NumberofCycles Temperature Time1 37°C 10minutes1 70°C 15minutes1 4°C ∞
Safestoppingpoint.Librarycanbestoredat4°Covernightorat-20°Cforlonger.
10. PreparetheEndRepairMasterMixaccordingtothetablebelow:
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EndRepairMasterMix
Reagent Volumeperlibrary(μL)
Recommendedvolumesfor24libraries(μL)
Recommendedvolumesfor96libraries(μL)
Colorcode
SterileH2O 1.25μL 34.8μL 139.2μL EndRepairEnzyme(C) 1.25μL 34.8μL 139.2μL GreenEndRepairBuffer(D) 2.5μL 69.6μL 278.4μL OrangeTotalvolume 5μL 139.2μL 556.8μL
11. AliquotanequalamountofEndRepairMasterMixintoasingleunusedcolumnoftheReagentPlate.
12. CentrifugetheReactionPlate(containingthefragmentedSamples)for10seconds.Add5μLofEndRepairMasterMix fromtheReagentPlate intoeachwellof theReactionPlate.Theuseofamulti-channelpipetteisrecommended.Mixbypipetting.
13. CovertheReactionPlatewithathermalsealandcentrifugefor10seconds.14. IncubatetheReactionPlateinathermalcyclerwiththefollowingprogram:
EndRepairProgramNumberofCycles Temperature Time
1 20°C 30minutes1 65°C 30minutes1 4°C ∞
Safestoppingpoint.Librarycanbestoredat4°Covernightorat-20°Cforlonger.
15. RemovetheIndexedAdaptorsPlatefromstorageandthawatroomtemperatureaftertheEndRepairProgramstartsinthethermalcycler.WhentheAdaptorPlateisatroomtemperature,centrifugeitfor3minutesat3000rpm.
16. CarefullypullthesealoffoftheAdaptorPlate.DonotshaketheAdaptorPlateoncethesealisremovedtopreventcrosscontamination.
17. Transfer theentirevolumeofeachwell fromtheReactionPlate to thecorrespondingwellintheAdaptorPlate.
Note:IftheentireAdaptorPlateisNOTgoingtobeused,itispossibletouseonlythenecessarynumberofadaptors.Cuttheplatesealbetweenthewellstobeusedandthewellstobekept.Carefullypull thesealofftheAdaptorPlate, leavingtheseal inplaceoverthewellstobekept.
i. Transfer25μLfromeachsampleintheReactionPlatetoanunusedwellintheAdaptorPlate,mixingwellwithapipette.
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ii. TransfertheentiretyofeachsamplefromtheAdaptorPlateintotheoriginalwelloftheReactionPlate.
iii. ResealtheAdaptorPlateandreturnitto-20°C.UsetheReactionPlateinsteadoftheAdaptorPlatefortheremainingstepsinthemanual.
18. PreparetheLigationMasterMix.PrepareenoughLigationMasterMixforeachsample.
LigationMasterMix
Reagent Volume(μL) Recommendedvolumesfor24libraries(μL)
Recommendedvolumesfor96libraries(μL)
Colorcode
LigationEnzyme(E) 2.5μL 63.2μL 252.5μL BlueLigationBuffer(F) 30μL 757.5μL 3030μL BlackTotalVolume 32.5μL 820.7μL 3282.5μL
19. AliquottheLigationMasterMixintoanunusedcolumnoftheReagentPlate.Theuseof
amulti-channelpipetteisrecommended.
20. Add32.5μLofLigationMasterMix intoeachwellof theReactionPlate.Theuseofamulti-channelpipetteisrecommended.Mixbypipetting.
21. CovertheAdaptorPlatewithathermalsealandcentrifugefor10seconds.
22. IncubatetheAdaptorPlateinthethermalcyclerwiththefollowingprogram:
LigationProgramNumberofCycles Temperature Time
1 25°C 10minutes1 65°C 20minutes1 4°C ∞
Safestoppingpoint.Librarycanbestoredat4°Covernightorat-20°Cforlonger.
23. AllowAMPureXPbeadstocometoroomtemperatureandprepare5mLof80%ethanol(4mLEtOH+1mLH2O).
24. CreatetheLibrarybycombininganaliquotfromeachpooledamplicon,nowasample-specificlibrary,intoasingle2.0mLlowbindmicrocentrifugetube.
i. For16ormoresamples -Calculate theamountofeachsample library topooltogetherasasingleLibraryof900μLtotalvolume.Divide900μLbythenumberofsample libraries.This isthevolumeofaliquottobetakenfromeachsamplelibraryandpipettedintotheLibrary.
ii. Forfewerthan16samples–Transfer60μLofeachsamplelibraryintoaLibrary.
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25. Add900μLofAMPureXPbeadstotheLibrarytube.Mixthoroughlybyvortexingand
centrifuge briefly. Do not allow the beads to separate. Incubate the Library for 10minutesatroomtemperature.
Note: If there is less than 900 μL of library in the Final Pool, add an equivalentamountofAMPureXPbeads.Thereshouldbea1:1ratioofFinalPoolandAMPureXPbeads.
26. PlacetheFinalPooltubeontoamagneticstandandincubatefor10minutes.
27. Keepingthetubeonthemagneticstand,carefullyremoveanddiscardthesupernatantfromtheLibrarytube,withouttouchingthebeads.
28. Keeping the tube on the magnetic stand, add ~1.5–2 mL of freshly prepared 80%ethanoltotheLibrarytube.Thevolumeofethanoladdedshouldbesufficienttocoverthebeads.
Note:Applytheethanoltothesideofthetubewithoutbeads.
29. IncubatetheFinalPooltubeatroomtemperaturefor30seconds;afterwards,carefullyremoveanddiscardthesupernatant.
30. Repeatsteps28and29.
31. QuicklyspindowntheLibrarytubeandplaceitbackonthemagneticstandwiththelidopen.Removeresidualethanolwithapipette.Donottouchthebeads.
Note: Ensure the bead pellet does not contain residual ethanol. This may requirerotating the tubeon themagnetic stand to removeethanolwithoutdisturbing thebeadpellet.
32. Allowthebeadstoairdryfor5-8minutesonthemagneticstanduntilthebeadpelletisdry.
33. Remove the Library tube from the magnetic stand and elute the Library with 31 μLmoleculargradewater.Donotletthepipettetiptouchthebeads,astheywillsticktoit.
34. Vortex the Library to fully resuspend the beads. Centrifuge briefly if some dropletsremainonthesidewalls.Ensurethebeadsremaininsuspension.
35. IncubatetheLibraryatroomtemperaturefor2minutes.
36. PlacetheLibrarytubeonthemagneticstandfor2minutes.
37. CollecttheLibrary:keepingtheFinalLibrarytubeinthemagneticstand,collect31μLofthesupernatantintoanew1.5mllowbindmicrocentrifugetube.
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Safestoppingpoint.Librariescanbestoredat-20°Cforextendedperiodsoftime
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Step5–LibrarySizeSelectionDuration:~1hourStep 5 takes the Library from Step 4 and performs size selection using the Pippin Prep. ThePippin Prep can automatically select a range of DNA fragment sizes and elute them into acollection chamber. Note: Blue Pippin may be used instead of the Pippin Prep. Refer toAppendix1forPippinPrepInstructionsforUse.
ReagentlistItem Storage Suppliedby1.5%AgaroseGelCassette,DyeFree 20°Cto25°C SageSciencePippinloadingsolution/markermix(labeledK)
4°C SageScience
PooledLibrary 4°C Step4
Note:MarkerKisusedwiththePippinPrep.TheBluePippinusesMarkerR2.
Protocol1. BringtheMarkerKloadingdyetoroomtemperature.
2. Combine31μLofthePoolwith10μLofMarkerKloadingsolution.
3. Mixbyvortexingandspindown.
4. ConfigurethePippinPreptocollectDNAfragmentsbetween650and1,300bp.Loadthe40μLsampleintothesampleportandrun.Runtimeis45-50minutes.
5. Collectthewholecontent(approximately40μL)fromtheelutionportofthePippinPrepandtransfer it toanew1.5ml lowbindmicrocentrifuge tube.This is thesize-selectedlibrary.
Safestoppingpoint.Librariescanbestoredat-20°Cforextendedperiodsoftime.
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Step6–LibraryQuantificationusingaQubitDuration:~15minutesItisnecessarytoquantifytheSize-selectedLibraryinordertooptimallyusetheoutputoftheIllumina MiSeq sequencer. The concentration of the Size-selected Library can be accuratelymeasuredbyQubitFluorometer.
Note: The Qubit Fluorometer is a quick and accurate way to determine theconcentrationofthefinalSizeSelectedLibrary. ItmeasuresallofthedsDNAthat ispresent in the library. Optionally you may use the KAPA Biosystems LibraryQuantitation kit andqPCRmachine for amore specificmeasurementof the libraryconcentration.ForthisprotocolSeeAppendix4.
ReagentlistItem Storage SuppliedbyQubitdsDNABRAssayKit -20°C ThermoFisherSizeSelectedLibrary 4°C Step5
Protocol1. PrepareQubitassaytubes(500microliter,thin-walled)foryour library induplicateandthe
twostandards.Vortexandcentrifugethestandardsandthesamples.
2. AddX*199µL fromBufferandX*1µL fromdyetoa10mlcentrifugetube.Vortexitfora
fewseconds.X=#ofsamples+2standards+1forpipettingloss.X>25notrecommended,becausethedyeislightsensitive.
3. Pipet190µL fromthemixtothetwostandards'Qubittubes.Pipet198µL fromthemixtothesamples'Qubittubes.
4. Pipet 10 µL from standard 1 to the Qubittube and vortex it for 2 seconds. Repeat withstandard2.
5. Pipet2µL fromthelibrarytotheirQubittubesandvortexfor2seconds.
6. Waitfor2mins.
7. SwitchonQubitmachineandchooseBRprotocol.
8. Putstandard1QubittubeinandpushGO.Repeatwithstandard2.
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9. PutthelibrarytubeinQubitandpushGO.Thenputthereplicate.10. ToconverttheQubitresultfromng/μLtonMconcentration,enterthemeanconcentration
ofthetwolibraryreplicatesintheOmixonWorkbooktabcalled“LibraryQuantitation”.
11. UsingtheresultsfromtheQubitmeasurement,dilute10μLoftheSizeSelectedLibrarytoa
concentrationof2nMwithsterileH2Oinafresh1.5-mLlowbindingmicrocentrifugetube.StoretheremainingSizeSelectedLibraryat-20°C.
Safestoppingpoint.Librariescanbestoredat-20°Cforextendedperiodsoftime.Incase of long-term storage, re-quantification of the library is highly recommendedbeforerunningitontheMiSeq.
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Step7–SequencingonIlluminaMiSeqDuration:~24hours45minutes(dependingonMiseqchemistryandflowcellsize)The Illumina MiSeq is an automated NGS instrument that can sequence the Size-selectedLibrary prepared in the previous steps. De-multiplexing of the indexed samples is doneautomaticallyfollowingcompletionofthesequencingrun.
Quicktip–Youcanusea1%PhiXspike-inasanadditionalcontrol tomonitor thesequencing reaction. Refer to Illumina documentation on the PhiX control foradditionalinformation
ReagentlistItem Storage SuppliedbyReagentCartridge -20°C IlluminaSterileH2O 20°Cto25°C UserHT1 -20°C IlluminaPR2 4°C IlluminaMiSeqFlowCell 4°C IlluminaDilutedPooledLibrary 4°C Step6NaOH,0.2N 20°Cto25°C User
MiSeqReagentKitcapacityIlluminaMiSeqReagentKit
TimeHours
#ofSamples
Std300Cycle(MS-102-2002) ~24 192
Micro300Cycle(MS-103-1002) ~19 192
Nano300Cycle(MS-103-1001) ~17 56
Std500Cycle(MS-102-2003) ~39 192
Nano500Cycle(MS-103-1003) ~28 80
Protocol1. PreparetheMiSeqaccordingtostandardIlluminaprotocols.
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2. Prepare a 1 nM Denatured Pooled Library: Combine 10 μL of freshly prepared 0.2 NNaOH and 10 μL of the 2 nM Diluted Pooled Library in a fresh 1.5 mL low bindingmicrocentrifugetube.Vortexandspindownfor1second.
3. Incubatethe1nMDenaturedPooledLibraryatroomtemperaturefor5minutes.
4. Preparea20pMDenaturedPooledLibrary:Add980μLofchilledHT1tothe20μLofthe1nMDenaturedPooledLibrary.Vortexandspindownfor1second.
5. Preparea9pMDenaturedPooledLibrary:Add550μLofchilledHT1and450μLofthe20pMDenaturedPooled Library to a fresh 1.5mL lowbindingmicrocentrifuge tube.Vortexandspindownfor1second.
6. Transfer600μLofthe9pMDenaturedPooledLibraryintotheLoadSamplesreservoiroftheMiSeqreagentcartridge.
Quicktip–ItisadvisabletousetheprovidedworkbooktocreatetheSampleSheetthatisrequiredbytheMiseq.
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Step8–AnalysisofHLASequencingDataThe IlluminaMiSeqwill process the 9 pMPooled Library and generate sequencingdata as afastQ file.Please refer to theHLATwinmanual forassistancewith the correct installationofHLATwinandforinformationoninterpretingthegenotypinganalysisofyoursequencingdata.ForimplementingtheAutomatedProtocolandissuesrelatingtoinstallationoranalyzingdata,[email protected].
AutomatedProtocol
ITSetupandConfiguration1. InstallHLATwinServerontheServer
2. InstallHLATwinClientonaclientcomputer–multipleHLATwinClientsmayconnecttotheserver
3. ContactOmixonSupport([email protected])forcustominstallationinstructionsforAutomation
ProtocolperAnalysis1. LaunchHLATwinClientandlogin
2. Data is already processed or is being processed. Review the results using the TrafficLightSysteminHLATwin
3. Exportthegenotypingresultsand/orconsensussequencesasrequired
ManualServerProtocol
ITSetupandConfiguration1. InstallHLATwinServerontheServer
2. InstallHLATwinClientonaclientcomputer
ProtocolperAnalysis1. LaunchHLATwinClientandlogin
2. Select theMiSeqdata in fastQor fastQ.gz formatandstart theMonotypeHLA typingrun
3. After theMonotypeHLA typinghas finished, review the resultsusing theTraffic LightSysteminHLATwin
4. Exportthegenotypingresultsand/orconsensussequencesasrequired
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ManualDesktopProtocol
ITSetupandConfiguration1. InstallHLATwinDesktop.
ProtocolperAnalysis1. LaunchHLATwinandlogin
2. Select theMiSeqdata in fastQor fastQ.gz formatandstart theMonotypeHLA typingrun
3. After theMonotypeHLA typinghas finished, review the resultsusing theTraffic LightSysteminHLATwin
4. Exportthegenotypingresultsand/orconsensussequencesasrequired
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TechnicalAssistanceForgeneralassistancewiththisprotocolcontactsupport@omixon.comSafetyDataSheetsareavailableatwww.omixon.com/holotype-hla-documentation/msds
PhoneSupport:UnitedStates|+1(617)500-0790Europe|+36705748001RestofWorld|+1(617)500-0790
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SupplementalFigures
PlateexampleforAmpliconPlate,AmplificationPlate,DilutionPlate,AmpliconQuantificationPlate&ReactionPlate
For24samples:
For96samples:
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QuantitationPlateexamples
StandardsQuantitationPlate
qPCRPlateexample
Appendix1:PippinPrep
ProgrammingthePippinPrep1. ClicktheProtocolEditorTabandclickthe“New”button.
2. Click the folder icon next to the Cassette field and select “1.5%DFMarker K” for thePippinPrepor“1.5%DFMarkerR2”fortheBluePippin.
3. Inthelanethatyouareprogramming:
a. Highlightthe“Range”field.b. Setthe“RefLane”tomatchthelanenumberyouareworkingin.c. Setthe“Start*”fieldto650.d. Setthe“End*”fieldto1300.
4. IntheReferenceLanefield,selectthelanethatyouareworkingin.
5. Clickthe“SaveAs”buttonandnameyourprogram.
RunningthePippinPrep1. TurnonthePippinPrepbypushingthepowerbuttoninthebackofthedevice.2. VisuallyinspectthePippinPrep.Makesurethe5LEDsareonandthattheinsideofthe
deviceiscleananddry.3. ClickontheSageSciencelogoonthebottomrightofthescreen.Thiswillallowyouto
enterapassword.Thefactorydefaultpasswordis“pips”.4. ClicktheFactorySetupTabandmakesuretheBase-to-Thresholdvalueissetto0.02.5. Place the calibration fixture inside the Pippin Prep,making sure the dark strip is face
downandovertheLEDlights.6. IntheMaintab,clickthe“Calibration”button.7. IntheCalibrationwindow,makesurethe“TargetIpHmA”fieldissetto0.80(0.60for
BluePippin)andthenhitthe“Calibrate”button.8. GototheProtocolsTab.Clickthe“Load”buttonandselecttheprogramforMonotype
HLAandthespecificlaneyouwillbeusing.Makesurethat:a. Thecorrectlaneisturnedon.b. Broadspectrumselectionindicatorisonc. Thereferencelaneisthesamelanethatwillberunning.
9. GototheMainTab.Makesurethat:a. Theprogramyouloadedistheonethatisselected.b. Theappropriatereferencelaneisselectedandthatitisalsothelanethesample
willberunin.10. Inspectthecassette.Beforetakingoffthetapesealingthewells,looktoseeifthereare
anybubblesbehindtheelutionport. If thereareanybubblesbehindtheelutionport,gentlytapandrollthecassetteinyourhandtoworkthebubblesout.
11. Placethecassette,withthetapestilloverthewells,insideofthePippinPrep.
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12. Carefullypeeloffthetape,makingsuretoremovethetapefromthecleansideofthecassette(lane5)totheusedsideofthecassette(lane1).Takecarenottosplashliquidwhenthetapeisremovedtopreventcontamination.
13. Removetheentirevolumeofbufferfromtheelutionportofthelaneyouwillbeusingandadd40μLoffreshelectrophoresisbufferinthatelutionport.
14. Addathinstripoftapeovertheelutionports.15. Anyreservoirs thatare lessthan3/4ths fullshouldbetoppedoffwithelectrophoresis
buffer.Donotoverfill thewells!Theedgeof thebuffer should ‘just’ reach theplasticnot higher to prevent draggingwhen the lid slides.Apply buffer from the cleanwells(Lane5)totheusedwells(Lane1).
16. Makesureeachoftheloadingwells(wellswithagarose)arefilledwithelectrophoresisbuffer.Thebuffershouldbe‘just’overtheagarose,appearingcompletelyflat.
17. ClosethePippinPrepslowly,watchingtomakesurenobufferistouchingthelidasyouareclosingthedevice.
18. Perform the continuity test.When the sensors dry out slightly, it is common for thecontinuitytesttofailonce.Ifthecontinuitytestfails,runthetestonemoretime.Oncethe continuity test completes, open the Pippin slowly. Make sure no fluid is gettingpulledacrossthecassettebythelidofthePippinPrep.
19. Brieflyvortex theMarkerK loading solutionandspin itdown.Add10μLofMarkerKloadingsolutiontoyour~30μLoflibrary.
20. Brieflyvortexyourlibraryandspinitdown.21. Remove40μLofbufferfromthesamplewellthatyouwillbeusing.22. Add ~40μL of your library loadedwithMarker K to the samplewell that youwill be
using.23. Markthelanethatyouareusingwiththetechnician’sinitialsandthedate.24. ClosethePippinPrepandclickthe“Start”button.Makesuretheappropriatelanehas
beenturnedon.Thesampleshouldrunforabout45minutes.25. After the run has completed, carefully open the Pippin Prep.Watch to see if the lid
dragsanyliquidacrossthecassette.26. Removethetapeovertheelutionports,beingcarefulnottoflickanyliquid.27. Transferallthevolumefromtheelutionportintoanew1.5mllowbindtube.28. Coveralloftheopenwellswithtwopiecesofplatesealingtape.Remembertoleavea
tabonthecleanside.Thiswillmakeitsimpletoremovethetapefromcleantoused.29. Placethesealedcassetteintoitsbagandsetitaside.30. Take thewash cassette and fill itwithMiliQwater.Gently close the lid of the Pippin
Prep,watchingtoseeifyoupullanyliquidacrossthewashcassette.32. LeavethePippinPrepclosedforseveralseconds.33. OpenthePippinPrep,watchingtoseeifyoupullanyliquidacrossthewashcassette.34. Removethewashcassette,emptyitofwater,andletitdry.35. CleananywateroffofthePippinPrepandcloseitgently.36. Selectthe“ShutDown”buttoninthePippinPrepmenu.
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Appendix2:SampleSheet[Header],,,,,,,IEMFileVersion,4,,,,,,InvestigatorName,RHP,,,,,,ExperimentName,030315S_RHP,,,,,,Date,3/3/15,,,,,,Workflow,GenerateFASTQ,,,,,,Application,FASTQOnly,,,,,,Assay,CHOPPCRFree,,,,,,Description,030315L_RHP,,,,,,Chemistry,Default,,,,,,,,,,,,,[Reads],,,,,,,251,,,,,,,251,,,,,,,,,,,,,,[Settings],,,,,,,Adapter,AGATCGGAAGAGCACACGTC,,,,,,AdapterRead2,AGATCGGAAGAGCGTCGTGT,,,,,,,,,,,,,[Data],,,,,,,Sample_ID,Sample_Name,Sample_Plate,Sample_Well,I7_Index_ID,index,Sample_Project,Description001_A_030315S_RHP_1,001_A_030315S_RHP_1,030315P_RHP,A1,1,TAAGTGATC,030315S_RHP,002_A_030315S_RHP_2,002_A_030315S_RHP_2,030315P_RHP,B1,2,GTGGTATTC,030315S_RHP,003_A_030315S_RHP_3,003_A_030315S_RHP_3,030315P_RHP,C1,3,ATAAGTACT,030315S_RHP,004_A_030315S_RHP_4,004_A_030315S_RHP_4,030315P_RHP,D1,4,TAGGATTCA,030315S_RHP,005_A_030315S_RHP_5,005_A_030315S_RHP_5,030315P_RHP,E1,5,AAGTGCATA,030315S_RHP,006_A_030315S_RHP_6,006_A_030315S_RHP_6,030315P_RHP,F1,6,TACACACAA,030315S_RHP,007_A_030315S_RHP_7,007_A_030315S_RHP_7,030315P_RHP,G1,7,TCTCCGAGC,030315S_RHP,008_A_030315S_RHP_8,008_A_030315S_RHP_8,030315P_RHP,H1,8,AGTAACCGC,030315S_RHP,009_A_030315S_RHP_9,009_A_030315S_RHP_9,030315P_RHP,A2,9,AGGCAAGTT,030315S_RHP,010_A_030315S_RHP_10,010_A_030315S_RHP_10,030315P_RHP,B2,10,CCGTACTAT,030315S_RHP,011_A_030315S_RHP_11,011_A_030315S_RHP_11,030315P_RHP,C2,11,GCCAGGTGC,030315S_RHP,012_A_030315S_RHP_12,012_A_030315S_RHP_12,030315P_RHP,D2,12,CAACATCAC,030315S_RHP,013_A_030315S_RHP_13,013_A_030315S_RHP_13,030315P_RHP,E2,13,GTCAGCACC,030315S_RHP,014_A_030315S_RHP_14,014_A_030315S_RHP_14,030315P_RHP,F2,14,CTTGGCTGT,030315S_RHP,015_A_030315S_RHP_15,015_A_030315S_RHP_15,030315P_RHP,G2,15,GTAGTACTA,030315S_RHP,016_A_030315S_RHP_16,016_A_030315S_RHP_16,030315P_RHP,H2,16,GCAAGTCAA,030315S_RHP,001_B_030315S_RHP_17,001_B_030315S_RHP_17,030315P_RHP,A3,17,ATTACTACC,030315S_RHP,002_B_030315S_RHP_18,002_B_030315S_RHP_18,030315P_RHP,B3,18,CGCTCTAAT,030315S_RHP,003_B_030315S_RHP_19,003_B_030315S_RHP_19,030315P_RHP,C3,19,ACTTCGGTC,030315S_RHP,004_B_030315S_RHP_20,004_B_030315S_RHP_20,030315P_RHP,D3,20,TGGTACGAC,030315S_RHP,005_B_030315S_RHP_21,005_B_030315S_RHP_21,030315P_RHP,E3,21,TGCATAATG,030315S_RHP,006_B_030315S_RHP_22,006_B_030315S_RHP_22,030315P_RHP,F3,22,CTGGTTGGC,030315S_RHP,007_B_030315S_RHP_23,007_B_030315S_RHP_23,030315P_RHP,G3,23,CTCTTATTC,030315S_RHP,008_B_030315S_RHP_24,008_B_030315S_RHP_24,030315P_RHP,H3,24,AGGACTCTC,030315S_RHP,009_B_030315S_RHP_25,009_B_030315S_RHP_25,030315P_RHP,A4,25,GCATTCCAA,030315S_RHP,010_B_030315S_RHP_26,010_B_030315S_RHP_26,030315P_RHP,B4,26,TCAAGGTCA,030315S_RHP,011_B_030315S_RHP_27,011_B_030315S_RHP_27,030315P_RHP,C4,27,CACTCCGTT,030315S_RHP,012_B_030315S_RHP_28,012_B_030315S_RHP_28,030315P_RHP,D4,28,CAGCCAGTT,030315S_RHP,013_B_030315S_RHP_29,013_B_030315S_RHP_29,030315P_RHP,E4,29,AGCAGCACA,030315S_RHP,
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014_B_030315S_RHP_30,014_B_030315S_RHP_30,030315P_RHP,F4,30,TTCCGTAAG,030315S_RHP,015_B_030315S_RHP_31,015_B_030315S_RHP_31,030315P_RHP,G4,31,TCCTGTGGA,030315S_RHP,016_B_030315S_RHP_32,016_B_030315S_RHP_32,030315P_RHP,H4,32,GTGATAGCC,030315S_RHP,001_C_030315S_RHP_33,001_C_030315S_RHP_33,030315P_RHP,A5,33,TAGCTCTGA,030315S_RHP,002_C_030315S_RHP_34,002_C_030315S_RHP_34,030315P_RHP,B5,34,GCGAGTTGA,030315S_RHP,003_C_030315S_RHP_35,003_C_030315S_RHP_35,030315P_RHP,C5,35,TAGGTAATG,030315S_RHP,004_C_030315S_RHP_36,004_C_030315S_RHP_36,030315P_RHP,D5,36,GTAACTATA,030315S_RHP,005_C_030315S_RHP_37,005_C_030315S_RHP_37,030315P_RHP,E5,37,CTCCAGCCG,030315S_RHP,006_C_030315S_RHP_38,006_C_030315S_RHP_38,030315P_RHP,F5,38,AATATACCG,030315S_RHP,007_C_030315S_RHP_39,007_C_030315S_RHP_39,030315P_RHP,G5,39,GCGAGACGT,030315S_RHP,008_C_030315S_RHP_40,008_C_030315S_RHP_40,030315P_RHP,H5,40,CCACCGGAT,030315S_RHP,009_C_030315S_RHP_41,009_C_030315S_RHP_41,030315P_RHP,A6,41,CGACGAGGT,030315S_RHP,010_C_030315S_RHP_42,010_C_030315S_RHP_42,030315P_RHP,B6,42,GCGCTGGCA,030315S_RHP,011_C_030315S_RHP_43,011_C_030315S_RHP_43,030315P_RHP,C6,43,GGCACTAGG,030315S_RHP,012_C_030315S_RHP_44,012_C_030315S_RHP_44,030315P_RHP,D6,44,CTGGAATCG,030315S_RHP,013_C_030315S_RHP_45,013_C_030315S_RHP_45,030315P_RHP,E6,45,CTTGTTATC,030315S_RHP,014_C_030315S_RHP_46,014_C_030315S_RHP_46,030315P_RHP,F6,46,AACCACTTA,030315S_RHP,015_C_030315S_RHP_47,015_C_030315S_RHP_47,030315P_RHP,G6,47,GGCGCTTCT,030315S_RHP,016_C_030315S_RHP_48,016_C_030315S_RHP_48,030315P_RHP,H6,48,CCGAGGCTT,030315S_RHP,001_DRB1_030315S_RHP_49,001_DRB1_030315S_RHP_49,030315P_RHP,A7,49,TCGATCGTT,030315S_RHP,002_DRB1_030315S_RHP_50,002_DRB1_030315S_RHP_50,030315P_RHP,B7,50,TTGGCTACG,030315S_RHP,003_DRB1_030315S_RHP_51,003_DRB1_030315S_RHP_51,030315P_RHP,C7,51,TATTGCGTC,030315S_RHP,004_DRB1_030315S_RHP_52,004_DRB1_030315S_RHP_52,030315P_RHP,D7,52,GATTCTAGG,030315S_RHP,005_DRB1_030315S_RHP_53,005_DRB1_030315S_RHP_53,030315P_RHP,E7,53,TCCAACACT,030315S_RHP,006_DRB1_030315S_RHP_54,006_DRB1_030315S_RHP_54,030315P_RHP,F7,54,TACCGAGGT,030315S_RHP,007_DRB1_030315S_RHP_55,007_DRB1_030315S_RHP_55,030315P_RHP,G7,55,TTGTACCGA,030315S_RHP,008_DRB1_030315S_RHP_56,008_DRB1_030315S_RHP_56,030315P_RHP,H7,56,TAGTGGAGG,030315S_RHP,009_DRB1_030315S_RHP_57,009_DRB1_030315S_RHP_57,030315P_RHP,A8,57,GACCTGTTA,030315S_RHP,010_DRB1_030315S_RHP_58,010_DRB1_030315S_RHP_58,030315P_RHP,B8,58,TTCTTCCTA,030315S_RHP,011_DRB1_030315S_RHP_59,011_DRB1_030315S_RHP_59,030315P_RHP,C8,59,ATTCCGGTT,030315S_RHP,012_DRB1_030315S_RHP_60,012_DRB1_030315S_RHP_60,030315P_RHP,D8,60,GTTAGGCTC,030315S_RHP,013_DRB1_030315S_RHP_61,013_DRB1_030315S_RHP_61,030315P_RHP,E8,61,AGTCTGACT,030315S_RHP,014_DRB1_030315S_RHP_62,014_DRB1_030315S_RHP_62,030315P_RHP,F8,62,ATACATAAC,030315S_RHP,015_DRB1_030315S_RHP_63,015_DRB1_030315S_RHP_63,030315P_RHP,G8,63,CGTTACGGC,030315S_RHP,016_DRB1_030315S_RHP_64,016_DRB1_030315S_RHP_64,030315P_RHP,H8,64,CCGCACTGG,030315S_RHP,001_DQB1_030315S_RHP_65,001_DQB1_030315S_RHP_65,030315P_RHP,A9,65,CACGTAGGC,030315S_RHP,002_DQB1_030315S_RHP_66,002_DQB1_030315S_RHP_66,030315P_RHP,B9,66,TAGAGCCAC,030315S_RHP,003_DQB1_030315S_RHP_67,003_DQB1_030315S_RHP_67,030315P_RHP,C9,67,TTGTCCAAT,030315S_RHP,004_DQB1_030315S_RHP_68,004_DQB1_030315S_RHP_68,030315P_RHP,D9,68,CCTGGTTCA,030315S_RHP,005_DQB1_030315S_RHP_69,005_DQB1_030315S_RHP_69,030315P_RHP,E9,69,ATCAATATG,030315S_RHP,006_DQB1_030315S_RHP_70,006_DQB1_030315S_RHP_70,030315P_RHP,F9,70,TGGTAACCG,030315S_RHP,007_DQB1_030315S_RHP_71,007_DQB1_030315S_RHP_71,030315P_RHP,G9,71,TTCTTGACG,030315S_RHP,008_DQB1_030315S_RHP_72,008_DQB1_030315S_RHP_72,030315P_RHP,H9,72,GTTCGTATC,030315S_RHP,009_DQB1_030315S_RHP_73,009_DQB1_030315S_RHP_73,030315P_RHP,A10,73,ATCCTAGGA,030315S_RHP,010_DQB1_030315S_RHP_74,010_DQB1_030315S_RHP_74,030315P_RHP,B10,74,GCATTATAT,030315S_RHP,011_DQB1_030315S_RHP_75,011_DQB1_030315S_RHP_75,030315P_RHP,C10,75,TGACGTCTA,030315S_RHP,012_DQB1_030315S_RHP_76,012_DQB1_030315S_RHP_76,030315P_RHP,D10,76,TCAGATCCA,030315S_RHP,013_DQB1_030315S_RHP_77,013_DQB1_030315S_RHP_77,030315P_RHP,E10,77,CTCTGTGGT,030315S_RHP,014_DQB1_030315S_RHP_78,014_DQB1_030315S_RHP_78,030315P_RHP,F10,78,AGCGAGCGC,030315S_RHP,015_DQB1_030315S_RHP_79,015_DQB1_030315S_RHP_79,030315P_RHP,G10,79,ACTCCTACG,030315S_RHP,016_DQB1_030315S_RHP_80,016_DQB1_030315S_RHP_80,030315P_RHP,H10,80,CGTAACATC,030315S_RHP,001_POOL_030315S_RHP_81,001_POOL_030315S_RHP_81,030315P_RHP,A11,81,CCGGTGTAT,030315S_RHP,002_POOL_030315S_RHP_82,002_POOL_030315S_RHP_82,030315P_RHP,B11,82,CGGATCCTG,030315S_RHP,
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003_POOL_030315S_RHP_83,003_POOL_030315S_RHP_83,030315P_RHP,C11,83,AGGCGCTAC,030315S_RHP,004_POOL_030315S_RHP_84,004_POOL_030315S_RHP_84,030315P_RHP,D11,84,ATTCTGCTA,030315S_RHP,005_POOL_030315S_RHP_85,005_POOL_030315S_RHP_85,030315P_RHP,E11,85,ACTTGTAGG,030315S_RHP,006_POOL_030315S_RHP_86,006_POOL_030315S_RHP_86,030315P_RHP,F11,86,GCCTGTTGT,030315S_RHP,007_POOL_030315S_RHP_87,007_POOL_030315S_RHP_87,030315P_RHP,G11,87,ACACGACCA,030315S_RHP,008_POOL_030315S_RHP_88,008_POOL_030315S_RHP_88,030315P_RHP,H11,88,CAGCTCGGT,030315S_RHP,009_POOL_030315S_RHP_89,009_POOL_030315S_RHP_89,030315P_RHP,A12,89,ATCAACCTA,030315S_RHP,010_POOL_030315S_RHP_90,010_POOL_030315S_RHP_90,030315P_RHP,B12,90,GCGGAAGCG,030315S_RHP,011_POOL_030315S_RHP_91,011_POOL_030315S_RHP_91,030315P_RHP,C12,91,CCTGTTAGG,030315S_RHP,012_POOL_030315S_RHP_92,012_POOL_030315S_RHP_92,030315P_RHP,D12,92,ATCTGAGCC,030315S_RHP,013_POOL_030315S_RHP_93,013_POOL_030315S_RHP_93,030315P_RHP,E12,93,ACCTCCTCA,030315S_RHP,014_POOL_030315S_RHP_94,014_POOL_030315S_RHP_94,030315P_RHP,F12,94,CGCGAAGAG,030315S_RHP,015_POOL_030315S_RHP_95,015_POOL_030315S_RHP_95,030315P_RHP,G12,95,GTTCTCCTG,030315S_RHP,016_POOL_030315S_RHP_96,016_POOL_030315S_RHP_96,030315P_RHP,H12,96,GAATGACCA,030315S_RHP,
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Appendix 3: Amplicon Quantitation using a qPCRinstrument
ReagentlistItem Storage Suppliedby20×TEBuffer(pH7.5) 4°C PromegaLambdaDNAStandard(100ng/μL) 4°C Promega200×QuantiFluordsDNADye 4°C PromegaSterileH2O 20°Cto25°C UserHLA-BAmplificationPlate(s) 4°C Step2
Protocol1. Createaserialdilutionusing1.5mLmicrocentrifugetubesandtheQuantiFluorLambda
DNAstandard(100ng/μL).Followthedilutiontablebelow:5.
Labelontube InputDNA VolumeDNA(μL)
Volume1xTE(μL)
FinalConc.(ng/μL)
Standard1 LambdaDNA 7.5μL 492.5μL 1.5ng/μLStandard2 Standard1 250μL 250μL 0.75ng/μLStandard3 Standard2 250μL 250μL 0.38ng/μLStandard4 Standard3 250μL 250μL 0.19ng/μLStandard5 Standard4 250μL 250μL 0.09ng/μLStandard6 Standard5 250μL 250μL 0.05ng/μLStandard7,Blank
Blank 0μL 250μL 0ng/μL
2. PreparetheAmpliconQuantitationplates(seesupplementalfigures).Aliquot49.5μL1xTEbuffertothewellsofaclean96-wellplateforthetotalnumberofampliconstobequantitated.
3. Add0.5μLofampliconsfromcorrespondingwells intheAmpliconPlatesto individualwellsintheAmpliconQuantitationPlates.Mixbypipetting.
4. Prepare 1× QuantiFluor Dye working solution using the following formula: 0.25 μLQuantiFluorDye(200X)+49.75μL1×TEbuffer.Preparesufficient1×QuantiFluorDyeworkingsolutionso thateachsample (total samples inAmpliconPlates)andstandard(14total)willreceivea50μLaliquot.
5. PrepareaStandardsQuantitationPlateandAmpliconQuantitationplates.Aliquot50μLof1×QuantiFluorDyeworkingsolutiontowellsofthe96-wellopticalplatesusingthe
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formatof theStandardsQuantitationPlateandtheAmpliconQuantitationPlates (seesupplementalfigures).
6. Using the standards prepared above, add 50 μL of each standard, in duplicate, toindividualwellsintheStandardsQuantitationPlate(14wellstotal).
7. Vortextomixthoroughlyandspindown.
8. PuteachQuantitationPlate in theqPCRmachineoneata timeandrun the followingprogram:NumberofCycles Temperature Time
1 25°C 10seconds 25°C 15seconds2 25°C 30seconds(dataacquisition)
9. CalculatetheconcentrationofDNA in theAmpliconQuantitationPlatesusingtherawRFUdatageneratedbytheqPCRinstrument.
10. DiluteDNA in the Amplicon Plateswith sterile H2O so that the final concentration ofDNAisapproximately67ng/μL.
§ IfDNAconcentrationis150ng/μLorgreater:add25μLofH2O
§ IfDNAconcentrationis100-150ng/μL:add10μLofH2O
§ IfDNAconcentrationislessthan100ng/μL:add0μLofH2O
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Appendix4:LibraryQuantificationusingqPCRDuration:~1hourItisnecessarytoquantifytheSize-selectedLibraryinordertooptimallyusetheoutputoftheIllumina MiSeq sequencer. The concentration of the Size-selected Library can be accuratelymeasuredbyqPCR.
ReagentlistItem Storage Suppliedby10×IlluminaPrimerPremix -20°C KAPABiosystems2×KAPASYBRFASTqPCRMasterMix -20°C KAPABiosystemsStd1(20.00pM) -20°C KAPABiosystemsStd2(2.00pM) -20°C KAPABiosystemsStd3(0.20pM) -20°C KAPABiosystemsStd4(0.02pM) -20°C KAPABiosystemsIlluminaDNAStandards -20°C KAPABiosystemsMoleculargradeH2O User1×TEBuffer(pH8.0) UserSizeSelectedLibrary 4°C Step5
Protocol1-PreparetheqPCRPrimerMixusingthe10×IlluminaPrimerPremixandthe2×KAPASYBRFASTqPCRMasterMix:
Note:TheKAPASYBRFASTqPCRkitreagents(qPCRMasterMix,PrimerPremixandROXsolutions)arecombinedduringthefirstuseofthekit.Thiscombinedsolutionisstableforatleast30freeze/thawcycles.FollowKAPAdocumentationtodetermineifROXisrecommendedforyourqPCRinstrument.
qPCRPrimerMix
Reagent Volume(mL)10×IlluminaPrimerPremix 1mL2×KAPASYBRFASTqPCRMasterMix 5mLTotalVolume 6mL2–PreparetheqPCRMasterMix.
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qPCRMasterMix
Reagent Volume(μL)qPCRPrimerMix 228μLMoleculargradeH2O 76μLTotalVolume 304μL3-PrepareaserialdilutionoftheSizeSelectedLibrary.
a. Preparea1:1000dilutionbyadding1μLofSizeSelectedLibrary to999μLof1×TEbuffer(pH8.0),thoroughlyrinsingthepipettetip.Vortexandspindown.
b. Prepare a 1:2000 dilution by adding 100 μL of the 1:1000 dilution to 100 μL 1× TEbuffer(pH8.0).Vortexandspindown.
4-PrepareaqPCRQuantitationPlateinafreshPCRplatecompatiblewithyourqPCRsystem.5-Aliquot16μLoftheqPCRMasterMixintriplicateforstandards1-4,the1:1000dilutionand
the1:2000dilution(seesupplementalfigures).6 - Aliquot 4 μL of standards 1-4, the 1:1000 dilution and the 1:2000 dilution into the
correspondingwells.7-SealtheqPCRQuantitationPlateandcentrifugeitfor10seconds.
Note: Avoid creating bubbles in the qPCRQuantitation Platewells. Centrifuge asneededtoeliminatebubbles.
8 -DeterminetheDNAconcentrationoftheSizeSelectedLibraryusingyourqPCRmachine.9 -UsingtheresultsfromtheqPCR,dilute10μLoftheSizeSelectedLibrarytoaconcentration
of 2 nM with sterile H2O in a fresh 1.5-mL low binding microcentrifuge tube. Store theremainingSizeSelectedLibraryat-20°C.
Safestoppingpoint.Librariescanbestoredat-20°Cforextendedperiodsoftime.Incase of long-term storage, re-quantification of the library is highly recommendedbeforerunningitontheMiSeq.