m hla™ hla-b 24 96 m - amazon s3hla+hla-b+user... · long-range pcr amplification of hla genes...

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


FragmentationMasterMix................................................................................................................................23FragmentationProgram....................................................................................................................................23EndRepairMasterMix......................................................................................................................................24EndRepairProgram...........................................................................................................................................24LigationMasterMix...........................................................................................................................................25LigationProgram...............................................................................................................................................25


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


APPENDIX4:LIBRARYQUANTIFICATIONUSINGQPCR........................................................................................45REAGENTLIST.............................................................................................................................................................45PROTOCOL.................................................................................................................................................................45

qPCRPrimerMix................................................................................................................................................45


qPCRMasterMix...............................................................................................................................................46


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


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.


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.


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.


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


§ 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


§ 50mLreservoirs


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.


SummaryofSteps


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.


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.


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.


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


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.


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


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:


ExoSAP-ITPCRPurification

Safestoppingpoint.Ampliconscanbestoredat4°Covernightorat-20°Cforlonger.

NumberofCycles Temperature Time1 37°C 4minutes1 80°C 1minutes1 4°C ∞


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.


FragmentationMasterMix

Reagent Volumeperlibrary(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:


EndRepairMasterMix

Reagent Volumeperlibrary(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 ∞


15. RemovetheIndexedAdaptorsPlatefromstorageandthawatroomtemperatureaftertheEndRepairProgramstartsinthethermalcycler.WhentheAdaptorPlateisatroomtemperature,centrifugeitfor3minutesat3000rpm.

16. CarefullypullthesealoffoftheAdaptorPlate.DonotshaketheAdaptorPlateoncethesealisremovedtopreventcrosscontamination.

17. Transfer theentirevolumeofeachwell fromtheReactionPlate to thecorrespondingwellintheAdaptorPlate.

Note:IftheentireAdaptorPlateisNOTgoingtobeused,itispossibletouseonlythenecessarynumberofadaptors.Cuttheplatesealbetweenthewellstobeusedandthewellstobekept.Carefullypull thesealofftheAdaptorPlate, leavingtheseal inplaceoverthewellstobekept.

i. Transfer25μLfromeachsampleintheReactionPlatetoanunusedwellintheAdaptorPlate,mixingwellwithapipette.


ii. TransfertheentiretyofeachsamplefromtheAdaptorPlateintotheoriginalwelloftheReactionPlate.

iii. ResealtheAdaptorPlateandreturnitto-20°C.UsetheReactionPlateinsteadoftheAdaptorPlatefortheremainingstepsinthemanual.

18. PreparetheLigationMasterMix.PrepareenoughLigationMasterMixforeachsample.

LigationMasterMix

Reagent Volume(μL) Recommendedvolumesfor24libraries(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 ∞


23. AllowAMPureXPbeadstocometoroomtemperatureandprepare5mLof80%ethanol(4mLEtOH+1mLH2O).

24. CreatetheLibrarybycombininganaliquotfromeachpooledamplicon,nowasample-specificlibrary,intoasingle2.0mLlowbindmicrocentrifugetube.

i. For16ormoresamples -Calculate theamountofeachsample library topooltogetherasasingleLibraryof900μLtotalvolume.Divide900μLbythenumberofsample libraries.This isthevolumeofaliquottobetakenfromeachsamplelibraryandpipettedintotheLibrary.

ii. Forfewerthan16samples–Transfer60μLofeachsamplelibraryintoaLibrary.


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.


Safestoppingpoint.Librariescanbestoredat-20°Cforextendedperiodsoftime


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.


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.


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.


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.


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.


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



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



TechnicalAssistanceForgeneralassistancewiththisprotocolcontactsupport@omixon.comSafetyDataSheetsareavailableatwww.omixon.com/holotype-hla-documentation/msds

PhoneSupport:UnitedStates|+1(617)500-0790Europe|+36705748001RestofWorld|+1(617)500-0790


SupplementalFigures

PlateexampleforAmpliconPlate,AmplificationPlate,DilutionPlate,AmpliconQuantificationPlate&ReactionPlate

For24samples:

For96samples:


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.


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.


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,


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,


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,


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


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


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.


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.

m hla™ hla-b 24 96 m - amazon s3hla+hla-b+user... · long-range pcr amplification of hla genes...

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