HOLOTYPE HLA™ 24/7 A1 A2 U MHLA+24-7+U… · 2016 Increased volume of Enhancer 2, Enhancer 2 should now be saved after Combined DQB Enhancer is made, added new DRB4 Master Mix formulation,

HOLOTYPEHLA™24/7

A1&A2USERMANUAL

FORRESEARCHUSEONLY

V2.1

OmixonLimited2017

.

OmixonHolotypeHLA24/7UserManualv2.1.Forresearchuseonly.Copyright©2017,OmixonBiocomputingLtd.Confidential&Proprietary Page2│48

TableofContentsDOCUMENTHISTORY...........................................................................................................................................4THEPRINCIPLEOFTHEMETHOD:NGS-BASEDHLATYPINGFORTHEILLUMINAMISEQ.........................................6HOLOTYPEHLAPACKINGLIST...............................................................................................................................7

PRIMERCOMPONENTBOX..............................................................................................................................................7LIBRARYPREPARATIONREAGENTSCOMPONENTBOX...........................................................................................................796-WELLADAPTORPLATE...............................................................................................................................................7EXCELWORKBOOK........................................................................................................................................................7SOFTWARE–OMIXONHLATWIN....................................................................................................................................8

RECOMMENDATIONS...........................................................................................................................................9DNAEXTRACTIONRECOMMENDATIONS............................................................................................................................9TECHNICALANDEQUIPMENTRECOMMENDATIONS..............................................................................................................9ASSOCIATEDREAGENTRECOMMENDATIONS.......................................................................................................................9

MiSeqReagentKitcapacity...............................................................................................................................10RECOMMENDEDSUPPLIES.............................................................................................................................................10

LEGALNOTICE....................................................................................................................................................12SUMMARYOFSTEPS..........................................................................................................................................13GLOSSARY/DEFINITIONS.....................................................................................................................................15STEP0–GENOMICDNAPREPARATION..............................................................................................................16STEP1–HLAAMPLIFICATIONMASTERMIXPREPARATION................................................................................17

REAGENTLIST.............................................................................................................................................................17PROTOCOL.................................................................................................................................................................17

MasterMix:HLA-A,B,C,DRB1,DPB1,andDQA1.............................................................................................17MasterMix:HLA-DQB1Set3.............................................................................................................................18

STEP2–HLACLASSIANDIIAMPLIFICATION......................................................................................................19REAGENTLIST.............................................................................................................................................................19PROTOCOL.................................................................................................................................................................19

TaqPolymerase-LoadedMasterMix:HLA-DQB1set3......................................................................................20ClassIAmplification(HLA-A,BandC)...............................................................................................................20ExpectedAmpliconSizes....................................................................................................................................21

STEP3–AMPLICONQUANTITATIONANDNORMALIZATION(USINGAPLATEFLUOROMETER)...........................22REAGENTLIST.............................................................................................................................................................22PROTOCOL.................................................................................................................................................................22

Ampliconpooling...............................................................................................................................................23ExoSAP-ITExpressPCRPurification....................................................................................................................24

STEP4–LIBRARYPREPARATION........................................................................................................................25REAGENTLIST.............................................................................................................................................................25PROTOCOL.................................................................................................................................................................25

FragmentationMasterMix................................................................................................................................25FragmentationProgram....................................................................................................................................26EndRepairMasterMix......................................................................................................................................27EndRepairProgram...........................................................................................................................................27


LigationMasterMix...........................................................................................................................................28LigationProgram...............................................................................................................................................28Poolingandlibrarypurification.........................................................................................................................29

STEP5–LIBRARYSIZESELECTION.......................................................................................................................31REAGENTLIST.............................................................................................................................................................31PROTOCOL.................................................................................................................................................................31

STEP6–LIBRARYQUANTIFICATIONUSINGANINTERCALATINGDSDNAFLUORESCENTDYE...............................32REAGENTLIST.............................................................................................................................................................32PROTOCOL.................................................................................................................................................................32

STEP7–SEQUENCINGONILLUMINAMISEQ......................................................................................................34REAGENTLIST.............................................................................................................................................................34

MiSeqReagentKitcapacity...............................................................................................................................34PROTOCOL.................................................................................................................................................................34

STEP8–ANALYSISOFHLASEQUENCINGDATA..................................................................................................36AUTOMATEDPROTOCOL...............................................................................................................................................36

ITSetupandConfiguration................................................................................................................................36ProtocolperAnalysis.........................................................................................................................................36

MANUALSERVERPROTOCOL.........................................................................................................................................36ITSetupandConfiguration................................................................................................................................36ProtocolperAnalysis.........................................................................................................................................36

MANUALDESKTOPPROTOCOL.......................................................................................................................................36ITSetupandConfiguration................................................................................................................................36ProtocolperAnalysis.........................................................................................................................................37

TECHNICALASSISTANCE.....................................................................................................................................38PhoneSupport:..................................................................................................................................................38

SUPPLEMENTALFIGURES....................................................................................................................................39PLATEEXAMPLEFORAMPLICONPLATE,AMPLIFICATIONPLATE,DILUTIONPLATE,AMPLICONQUANTITATIONPLATE(FORANINDIVIDUALLOCUS)&REACTIONPLATE...........................................................................................................................39STANDARDSQUANTITATIONPLATEEXAMPLE....................................................................................................................40KAPALIBRARYQUANTITATIONQPCRPLATEEXAMPLE......................................................................................................40

APPENDIX1:PIPPINPREP...................................................................................................................................41PROGRAMMINGTHEPIPPINPREP...................................................................................................................................41RUNNINGTHEPIPPINPREP...........................................................................................................................................41

APPENDIX2:AMPLICONQUANTITATIONUSINGAQPCRINSTRUMENT..............................................................44REAGENTLIST.............................................................................................................................................................44PROTOCOL.................................................................................................................................................................44

APPENDIX3:LIBRARYQUANTITATIONUSINGAQPCRINSTRUMENT..................................................................46REAGENTLIST.............................................................................................................................................................46PROTOCOL.................................................................................................................................................................46

qPCRPrimerMix................................................................................................................................................46qPCRMasterMix...............................................................................................................................................47


DocumentHistoryVersion Date DescriptionofChanges ApprovalName

1.5 January,2015 InitialVersion PeterMeintjes

1.6 March,2015 DQB Enhancer combination, general edits,Appendix2:SampleSheet

PeterMeintjes

1.7 June,2015ExoSAP-ITStepChange,DQB1Set1&2pooling,DQB1 Set 1 optional, <16 samples/run, PippinPrep0.8

PeterMeintjes

1.7.1 June,2015DPB1capschangedfromCleartoPurple,DQA1caps changed from Clear to Brown. MiSeqReagentNanoKitv2requirementsupdated.

PeterMeintjes

1.7.2 July,2015 MiSeqReagentKitv2andMiSeqReagentNanoKitv2requirementsupdated.

PeterMeintjes

1.7.3 November,2015

Amplificationverificationandquantitationmaybe optional after sufficient and consistentexperience.

PeterMeintjes

1.7.4 February,2016

Increased volume of Enhancer 2, Enhancer 2should now be saved after Combined DQBEnhancerismade,addednewDRB4MasterMixformulation, increased the volume used inFragmentation and End Repair master mix forfull plate, and rounded some values to thenearesttenth.

EfiMelista

1.7.5 April,2016

Changed wording from “LR-PCR enzyme” to“Taq Polymerase” based on Qiagen’sdocumentation change. Removed X2 fromHolotype HLA product configuration name.Approval name has been added on documenthistory.

EfiMelista


1.7.6 April,2016 DQB1set1andset2statementupdate EfiMelista

1.7.7 July,2016 Updated library preparation reagent volumes,additionofadaptorplateA2andBinformation.

EfiMelista

2.0 January,2017

“DQB” removal from the DQB Enhancers, gelverification after LR-PCR made optional,amplicon quantitation simplification, per-samplepoolingvolumechangein11-locuskits,ExoSAP-IT replacement by ExoSAP-IT Express,Qubituseforlibraryquantitation.

EfiMelista

2.1 July,2017 DQB1 set 1 and set 2 primer mix replaced byDQB1set3,endrepairreactiontimedecrease,ligation reaction time decrease libraryquantitation method update, Sample sheetremovedfromAppendices


ThePrincipleoftheMethod:NGS-basedHLAtypingfortheIlluminaMiSeqFormany years theHLA community has beenworking toward amethod thatwill accuratelyidentifytheextensivepolymorphismoftheHLAgenesandoftheirgeneproducts.TheadventofPCR,combinedwithothertechnologies(Sangersequencing,SSOP,SSP,Luminex),providedaformula for significantly improving the detection of HLA polymorphisms albeit with severallimitationsthatcontinuetoinhibitourabilitytocomprehensivelycharacterizetheHLAgenes.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.MorespecificallytheHLAgenesA,B,C,DQA1andDQB1areamplifiedfortheirentirecodinglength,includingelementsofthe5’and3’untranslatedregions,whileDRB1isamplifiedfromintron 1 to intron 4 and DPB1 is amplified from intron 1 to the 3’ untranslated region. 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).TheuseoftwoindependentalgorithmsinHLATwinprovidesthehighestlevelofconfidencesothattheHLAgenotypingresultscanbereportedimmediatelywithoutfurtherattention.Sampleswithquestionableorambiguousgenotypingsareflaggedbythesoftwaretobeanalyzedmanually.


HolotypeHLAPackingList

PrimerComponentBoxPrimermix Rxns Vol/tube #Tubes ColorcodeHLA-A 24 60μL 1 YellowHLA-B 24 60μL 1 RedHLA-C 24 60μL 1 OrangeHLA-DRB1 24 60μL 1 GreenHLA-DPB1 24 60μL 1 PurpleHLA-DQA1 24 60μL 1 BrownHLA-DQB1(Set3) 24 60μL 1 BlueEnhancer1 96 1100μL 1 ClearEnhancer2 96 300μL 1 Clear

LibraryPreparationReagentsComponentBoxReagent 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

96-wellAdaptorPlateIndexedadaptorsina5μLsolutionforgenerating24individualsequencinglibraries.Two (2) Adaptor Plate configurations are available containing 24 individual indices. Adaptorplates,thatcontain24indicesonlyareavailableinthefollowingconfigurations:A1(i1-i24),A2(i25-i48).

ExcelWorkbookAnExcelWorkbookisprovidedtosupporttheHolotypeHLAprotocolwithvolumecalculations,platelayouts,reagenttraceability,recordkeepingandMiSeqSampleSheetgenerationofallofthesupportedadaptorplateconfigurations(A1,A2).Ifyoudonothaveacopy,[email protected].


Software–OmixonHLATwinContact [email protected] for Omixon HLA Twin license associated with your purchase ofHolotypeHLA.


Recommendations

DNAExtractionRecommendations§ HighqualitygenomicDNA(gDNA)extractedfromwholeblood,bloodcells(B-celllines,

buffycoats,cordbloodoranyfractionofwhitebloodcells),salivaandbuccalswabscanbeused.FortheamplificationofHLA-A,HLA-B,HLA-C,HLA-DRB1,HLA-DPB1,HLA-DQA1andHLA-DQB1,0.8-1.2µgofgDNAisrequired.

TechnicalandEquipmentRecommendations§ Thermalcyclerwith96-wellformat§ Platefluorometer(oranyinstrumentcapableoffluorescencedetectionin96-wellplate

format,forusewiththePromegaQuantiFluordsDNASystem)§ PippinPrep(Cat#PIP0001)orBluePippin(Cat#BLU0001)bySAGEScience§ Qubitfluorometer(Cat#Q33216,ThermoFisherScientific)§ qPCRinstrumentwith96or384-wellplateformat(optional)§ IlluminaMiSeq(Cat#SY-410-1003)§ 64-bitcomputerwithminimum4Coresand16GBofRAM§ Long-termdatastorage(approximately2TBofdataperMiSeqperyear)

AssociatedReagentRecommendations§ LongRangePCRkitsfromQiagen(Cat#206401,206402or206403)

§ Eachsamplerequires3.2μLofTaqPolymerase§ 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#Q32850contains100assays§ Cat#Q32853contains500assays

§ 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)§ MiSeqReagentKitfromIllumina

MiSeqReagentKitcapacityIlluminaMiSeqReagentKit

TimeHours

24/7Samples

24/11Samples

96/5Samples

96/7Samples

96/11Samples

Std300Cycle(MS-102-2002) ~24 24 24 96 96 72

Micro300Cycle(MS-103-1002) ~19 24 20 40 28 20

Nano300Cycle(MS-103-1001) ~17 6 4 8 6 4

Std500Cycle(MS-102-2003) ~39 24 24 96 96 96

Nano500Cycle(MS-103-1003) ~28 12 8 16 12 8

RecommendedSupplies§ 1.5mLmicrocentrifugetubes§ 1.5mLlow-bindmicrocentrifugetubes§ 2.0mLlow-bindmicrocentrifugetubes(EppendorfDNALoBindCat#022431048

recommended)§ 0.5mlthinwalltubesforQubitinstrument(QubitAssaytubesCat#Q32856

recommended)§ Adjustablevolumepipettes(1.0–1000μLcapacity)§ 8-channeladjustablevolumepipettes(1.0-100μLcapacity)§ 96-wellplatescompatiblewiththethermalcycler§ 96-wellopticalplatescompatiblewiththeplatefluorometer§ 96-wellplatescompatiblewiththeqPCRinstrument§ Platesealsforgeneraluse§ Platesealscompatiblewiththethermalcyclers(testedforlongrangePCR)§ OpticalplatesealscompatiblewiththeqPCRinstrument(optional)§ Magneticstandcompatiblewith2mLmicrocentrifugetubes§ 96-wellcoolerracks(2pieces)§ 50mLconicaltubes


§ 50mLreservoirs


LegalNoticeThe Holotype 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§ AmpliconQuantitationPlate–96-wellplate compatiblewith theplate fluorometeror

qPCRmachinewheretheampliconsarequantitated.§ 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§ Sample Library – A library prepared by combining (pooling) all HLA loci for a given

sample.§ PooledAmpliconsPlate–Platecontainingasamplelibrary(alllocicombined)perwell.§ StandardsQuantitationPlate–96-wellplatecompatiblewiththeplatefluorometeror

qPCRmachinewhereDNAstandardsareplacedtoallowforampliconquantitation.


Step0–GenomicDNAPreparationDuration:~45minutesIsolategDNAfromwholeblood,bloodcells(B-celllines,buffycoats,cordbloodoranyfractionofwhitebloodcells),salivaandbuccalswabs.gDNAshouldbedissolvedinwaterastheEDTAinTE buffer can inhibit long-range PCR reactions and its recommended concentration is 20-30ng/μl.Wehighlyrecommendtouseafluorescence-basedquantitationmethodtodeterminethegDNAconcentration.Itsquality,asevaluatedbyspectrophotometry,shouldbe:

1. A260nm/280nmabsorbanceratiobetween1.7and1.9.2. A260nm/230nmabsorbanceratioof1.7orgreater.3. Minimal degradation. DNA that is old or has gone through repeated

freeze/thawswillsufferfrommoredegradation.FortheamplificationofHLA-A,HLA-B,HLA-C,HLA-DRB1,HLA-DPB1,HLA-DQA1andHLA-DQB1,0.8-1.2µgofgDNAisrequiredforeachsample.


Step1–HLAAmplificationMasterMixPreparationDuration:~45minutesThepurposeofthisstepistopreparelocus-specificMasterMixestoamplifyeachtargetedHLAlocusindividually.ThelociamplifiedbythisprotocolareHLA-A,HLA-B,HLA-C,HLA-DRB1,HLA-DPB1,HLA-DQA1andHLA-DQB1,Note: TheMasterMixes prepared in this step include all reagents needed for amplificationexcepttheTaqPolymerase.

ReagentlistItem Storage SuppliedbyHLA-APrimerMix -20°C OmixonHLA-BPrimerMix -20°C OmixonHLA-CPrimerMix -20°C OmixonHLA-DRB1PrimerMix -20°C OmixonHLA-DPB1PrimerMix -20°C OmixonHLA-DQA1PrimerMix -20°C OmixonHLA-DQB1Set3PrimerMix -20°C OmixonEnhancer1 -20°C OmixonEnhancer2 -20°C OmixonLongRangePCRBuffer(10×) -20°C QiagendNTPs(10mMeach) -20°C QiagenMoleculargradeH2O -20°C Qiagen

Protocol1.1 - Removeall primermixes, Enhancer 1 and2, thedNTPs and the Long-RangePCRBuffer

(10×),fromstorageandthawatroomtemperature.

1.2 - Prepare the Combined Enhancer: add 132 μL of Enhancer 2 into the Enhancer 1 tube.RelabelEnhancer1tubeasCombinedEnhancer.

1.3 -PrepareaMasterMixforeachPrimerMixaccordingtothetablesbelow:

MasterMix:HLA-A,B,C,DRB1,DPB1,andDQA1Reagent Volume/sample/locus Volume/24samples/locusPrimerMix 2μL 51μLLongRangePCRBuffer(10×) 2.5μL 63.8μLdNTPMix(10mMeach) 1.25μL 31.9μLMoleculargradeH2O 13.85μL 353.2μL


TotalVolume 19.6μL 499.9μL

MasterMix:HLA-DQB1Set3

Reagent Volume/sample/locus Volume/24samples/locusPrimerMix 2μL 51μLLongRangePCRBuffer(10×) 2.5μL 63.8μLdNTPMix(10mMeach) 1.25μL 31.9μLCombinedEnhancer 5.6μL 142.8μLMoleculargradeH2O 7.85μL 200.2μLTotalVolume 19.2μL 489.7μL1.4 –VortexeachMasterMixandspinitdownfor1second.PlaceMasterMixesonice.

1.5 -DiluteallgDNAstoaconcentrationof20-30ng/μL(minimumvolumeis45μL).

Note: Holotype HLA includes sufficient reagents for 24 reactions plus additionalvolumeforpipettinglossandfailedamplification.


Step2–HLAClassIandIIAmplificationDuration:~6hours45minutesThepurposeof Step2 is to amplify theHLA loci.HLAClass I andClass II amplificationshavebeen optimized using two separate sets of PCR conditions. Once the PCR reactions arecompleted,amplificationisverifiedbyagarosegelelectrophoresis(optional).

Quicktip–Theagarosegelelectrophoresis,whilerecommended,isnotrequiredtosuccessfullycompletetheHolotypeHLAprotocol.Whenampliconsarequantitated(Step3),anyconcentrationabove50ng/μLisconsideredasuccessfulamplification.AgarosegelelectrophoresisisanimportantqualitycontrolstepandshouldnotbeskippedwithoutsufficientexperiencewiththecompleteHolotypeHLAprotocol.

ReagentlistItem Storage SuppliedbyHLA-AMasterMix -20°C Step1HLA-BMasterMix -20°C Step1HLA-CMasterMix -20°C Step1HLA-DRB1MasterMix -20°C Step1HLA-DPB1MasterMix -20°C Step1HLA-DQA1MasterMix -20°C Step1HLA-DQB1Set3MasterMix -20°C Step1TaqPolymerase -20°C QiagengDNA 4°C UserMoleculargradeH2O 20°C User

Protocol2.1–RemovetheTaqPolymerasefromstorage,spin itdown,andadd it toeachMasterMixaccordingtothetablesbelow,rinsingthepipettetipsthoroughlybypipetting:TaqPolymerase-LoadedMasterMix:HLA-A,B,C,DRB1,DPB1,andDQA1Reagent Volume/sample/locus Volume/24samples/locusMasterMixfromStep1 19.6μL 499.9μLTaqPolymerase 0.4μL 10.2μLTotal 20μL 510.1μL


TaqPolymerase-LoadedMasterMix:HLA-DQB1set3Reagent Volume/sample/locus Volume/24samples/locusMasterMixfromStep1 19.2μL 489.7μLTaqPolymerase 0.8μL 20.4μLTotal 20μL 510.1μL2.2 -Briefly vortexand spindownall TaqPolymerase-loadedMasterMixes.Aliquot20μLofeachTaqPolymerase-loadedMasterMixintoseparatewellsof96-wellPCRplates.

Note:Class IandClass IIamplificationhavebeenoptimizedusing twodifferentPCRconditions,soClassIandClassIIMasterMixesshouldnotbeinthesameplate.

2.3–Add5μLof eachdilutedgDNA into theappropriatewell of theplatesprepared in thepreviousstep.Mixbypipetting.Seal themwitha thermalsealandvisually inspecteachwell.Spin-downallAmplificationPlatesinacentrifuge.

2.4 -Place theAmplificationPlates into thermalcyclersandrun theprograms forClass IandClassIIamplificationaccordingtothetablesbelow:

ClassIAmplification(HLA-A,BandC)NumberofCycles Temperature Time

1 95°C 3minutes 95°C 15seconds

35 65°C 30seconds 68°C 5minutes1 68°C 10minutes1 4°C ∞

ClassIIAmplification(HLA-DRB1,DPB1,DQA1andDQB1)NumberofCycles Temperature Time

1 95°C 3minutes 93°C 15seconds

35 60°C 30seconds 68°C 9minutes1 68°C 10minutes1 4°C ∞

Note: Amplification success canbe verified by running 2 μL from each amplicon in astandard2%agarosegelat250Vfor30minutes.(Optional)


ExpectedAmpliconSizes

HLAlocus Expectedampliconsize(kb)HLA-A,BandC ~3HLA-DRB1 ~4.3HLA-DPB1 ~6.6HLA-DQA1 ~5.5

HLA-DQB1(Set3) ~6.5Safestoppingpoint.Ampliconscanbestoredat4°Covernightorat-20°Cforlonger.


Step 3 – Amplicon Quantitation and Normalization(usingaPlateFluorometer)Duration:~35minutesAmplicon 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 2 forInstructionsonhowtodotheAmpliconQuantitationusingaqPCRmachine.

Quick tip – The amplicon quantitation, while recommended, is not required tosuccessfullycompletetheHolotypeHLAprotocol.Ampliconnormalizationdoesnotrequireprecisemeasurementofampliconconcentration.Anestimateofampliconconcentrations based on experience or agarose gel electrophoresis can be usedinstead. Amplicon quantitation should not be skipped without consistentexperiencewiththecompleteHolotypeHLAprotocol.

ReagentlistItem Storage SuppliedbyClassIAmplificationPlate 4°C Step2ClassIIAmplificationPlate 4°C Step2LambdaDNAStandard(100ng/μL) 4°C PromegaQuantiFluordsDNADye(200×) 4°C Promega20×TEBuffer(pH7.5) 4°C PromegaMoleculargradeH2O 20°Cto25°C UserExoSAP-iTExpress -20°C Affymetrix

Protocol3.1 – 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/μLBlank Blank 0μL 250μL 0ng/μL


3.2 -PreparetheAmpliconQuantitationplates(seesupplementalfigures).Aliquot99μL1xTE

buffer to the wells of a 96-well optical plate for the total number of amplicons to bequantitated.

3.3 -Add1μLofampliconsfromcorrespondingwellsintheAmpliconPlatestoindividualwellsintheAmpliconQuantitationPlates.Mixbypipetting.

3.4 - Prepare 1× QuantiFluor Dye working solution using the following formula: 0.5 μL

QuantiFluor Dye (200X) + 99.5 μL 1× TE buffer. Prepare sufficient 1× QuantiFluor Dyeworkingsolutionsothateachsample(totalsamples inAmpliconPlates)andstandard(14total)willreceivea100μLaliquot.

3.5 -PrepareaStandardsQuantitationPlateandAmpliconQuantitationplates.Aliquot100μL

of1×QuantiFluorDyeworkingsolutiontowellsofthe96-wellopticalplatethatwillbetheStandardsQuantitationPlateandtotheAmpliconQuantitationPlatesfromStep3.2.

3.6 - Using the standards prepared above, add 100 μL of each standard, in duplicate, to

individualwellsintheStandardsQuantitationPlate(14wellstotal).Mixbypipetting.

3.7 -Vortexwelltomixandspindown.3.8 -RuntheStandardsQuantitationPlateontheplatefluorometerfollowedbytheAmplicon

QuantitationPlates.3.9 -Calculate theconcentrationofDNA in theAmpliconQuantitationPlatesusingRFUdata

generatedbytheplatefluorometer.RefertotheDilutionTabintheprovidedworkbookforassistancewithcalculations.

3.10 - Dilute DNA in the Amplicon Plates with molecular grade H2O so that the final

concentrationofDNAisapproximately67ng/μL.§ IfDNAconcentrationis150ng/μLorgreater:add25μLofH2O§ IfDNAconcentrationis100-150ng/μL:add10μLofH2O§ IfDNAconcentrationislessthan100ng/μL:donotaddanyH2O

Ampliconpooling3.11 - Pool all loci for each sample into a single Pooled Amplicons Plate. Combine the

volumesindicatedforeachlocusasinthefollowingtabletoobtainafinalvolumeof35μL:


HLAlocus Pooledvolume

A 5μLB 5μLC 5μL

DRB1 5μLDPB1 5μLDQA1 5μL

DQB1set3 5μL3.12 – Add 4 μL of ExoSAP-iT Express into each sample library. Rinse the pipette tips bypipetting.Sealtheplatewithathermalsealandspindown.3.13-PlacethePooledAmpliconsPlateintoathermalcyclerandrunthefollowingprogram:

ExoSAP-ITExpressPCRPurification

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

Safestoppingpoint.Ampliconscanbestoredat4°Covernightorat-20°Cforlonger.


Step4–LibraryPreparationDuration:~3hoursDuringthisstep,thepooledampliconsarepreparedforsequencingontheIlluminaMiSeq.Theampliconsareenzymatically fragmented, theendsare repairedandadenylated, and indexedadaptorsareligatedtotheends.ThelibrariesarethenpooledfollowedbyasinglecleanupandconcentrationstepperformedusingAMPureXPbeads.

Note:Omixonrecommendsvolumesgreaterthanisnecessaryfor24samplesbecausemanyoftheenzymesandbuffersareviscous,resultinginexcesspipettingloss.

ReagentlistItem Storage SuppliedbyPooledAmpliconsPlate 4°C Step3AdaptorPlate -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 UserMoleculargradeH2O 20°Cto25°C User

Protocol4.1-Turnonthethermalcycler.Verifythattheheatedlidiswarmingup.

Note:BesuretovortextheFragmentationEnzyme(A)thoroughlybeforeuse.4.2-PrepareFragmentationMasterMixaccordingtothetablebelow:

FragmentationMasterMix

Reagent Volumeperlibrary(μL)

Recommendedvolumesfor24libraries(μL)

Colorcode

FragmentationEnzyme(A) 2μL 55.2μL YellowFragmentationBuffer(B) 2μL 55.2μL RedTotalVolume 4μL 110.4μL


4.3-PrepareaReagentPlate:placeanew96-wellPCRplateonaPCRcoolerrackandaliquotandequalamountoftheFragmentationMasterMixintoeachwellofasinglecolumn.

Note:Thefragmentationreactionhasbeendesignedtoprovide ideallysizedDNAforsequencingonthe IlluminaMiSeq. It is important tokeepthereagentscolduntil thereaction is started in the thermal cycler to prevent excessive fragmentation. Use ofmulti-channel pipettes is recommended to minimize opportunities for excessivefragmentation.

4.4-CentrifugethePooledAmpliconsPlatefor10seconds,andplaceitoniceoracoldblock.4.5-PrepareaReactionPlate:placeafresh96-wellPCRplateonaPCRcoldblock.4.6-Add4μLofFragmentationMasterMixfromtheReagentPlateintowellsoftheReactionPlate, correspondingwith samples in thePooledAmpliconsPlate.Theuseofamulti-channelpipetteisrecommended.4.7 -Transfer16μLofeachamplicon fromthePooledAmpliconsPlate to thecorrespondingwellontheReactionPlateusingamulti-channelpipette.Mixbypipetting.4.8-CovertheReactionPlatewithathermalsealandcentrifugefor10seconds.4.9-IncubatetheReactionPlateinathermalcyclerwiththefollowingprogram:

FragmentationProgram


Safestoppingpoint.Librariescanbestoredat4°Covernightorat-20°Cforlonger.

4.10-PreparetheEndRepairMasterMixaccordingtothetablebelow:


EndRepairMasterMix

Reagent Volumeperlibrary(μL)

Recommendedvolumesfor24libraries(μL)

Colorcode

MoleculargradeH2O 1.25μL 34.8μL EndRepairEnzyme(C) 1.25μL 34.8μL GreenEndRepairBuffer(D) 2.5μL 69.6μL OrangeTotalVolume 5μL 139.2μL 4.11-AliquotanequalamountofEndRepairMasterMix intoasingleunusedcolumnoftheReagentPlate.4.12-CentrifugetheReactionPlate(containingthefragmentedSamples)for10seconds.Add5μLofEndRepairMasterMixfromtheReagentPlateintoeachwelloftheReactionPlate.Theuseofamulti-channelpipetteisrecommended.Mixbypipetting.4.13-CovertheReactionPlatewithathermalsealandcentrifugefor10seconds.4.14-IncubatetheReactionPlateinathermalcyclerwiththefollowingprogram:

EndRepairProgram



4.15-Removethe IndexedAdaptorsPlatefromstorageandthawatroomtemperatureafterthe End Repair Program starts in the thermal cycler. When the Adaptor Plate is at roomtemperature,centrifugeitfor3minutesat3000rpm.4.16-CarefullypullthesealoffoftheAdaptorPlate.DonotshaketheAdaptorPlateoncethesealisremovedtopreventcrosscontamination.4.17 – Transfer the entire volume of each well from the Reaction Plate (25 μL) to thecorrespondingwellintheAdaptorPlate.


Note: IftheentireAdaptorPlateisNOTgoingtobeused, it ispossibletouseonlythenecessarynumberofadaptors.Cuttheplatesealbetweenthewellstobeusedand thewells tobekept.Carefullypull the sealoff theAdaptorPlate, leaving thesealinplaceoverthewellstobekept.a. Transfer25μLfromeachend-repairedsampleoftheReactionPlatetoawellin

theAdaptorPlate,mixingwellwithapipette.

b. TransfertheentiretyofeachsamplefromtheAdaptorPlateintotheoriginalwelloftheReactionPlate.

c. ResealtheAdaptorPlateandreturnitto-20°C.UsetheReactionPlateinsteadoftheAdaptorPlatefortheremainingstepsinthemanual.

4.18-PreparetheLigationMasterMix.PrepareenoughLigationMasterMixforeachsample.

LigationMasterMix

Reagent Volume(μL) Recommendedvolumesfor24libraries(μL)

Colorcode

LigationEnzyme(E) 2.5μL 63.2μL BlueLigationBuffer(F) 30μL 757.5μL BlackTotalVolume 32.5 820.7μL

4.19-AliquottheLigationMasterMixintoanunusedcolumnoftheReagentPlate.Theuseofamulti-channelpipetteisrecommended.4.20 -Add32.5μLof LigationMasterMix intoeachwell of theReactionPlate. Theuseof amulti-channelpipetteisrecommended.Mixbypipetting.4.21-CovertheReactionPlatewithathermalsealandcentrifugefor10seconds.4.22-IncubatetheReactionPlateinthethermalcyclerwiththefollowingprogram:

LigationProgramNumberofCycles Temperature Time

1 25°C 10minutes1 70°C 10minutes1 4°C ∞



Poolingandlibrarypurification4.23-AllowAMPureXPbeadstocometoroomtemperature.Ensuretheyarehomogeneous(noclumpsorpellets).Preparefreshlymade5mLof80%ethanol(4mLEtOH+1mLH2O).4.24-CreatetheLibrarybycombininganaliquotfromeachpooledamplicon,nowasample-specificlibrary,intoasingle2.0mLlowbindmicrocentrifugetube.

I. For 16 ormore samples - Calculate the amount of each sample library to pooltogetherasasingleLibraryof900μLtotalvolume.Divide900μLbythenumberofsamplelibraries.ThisisthevolumeofaliquottobetakenfromeachsamplelibraryandpipettedintotheLibrary.

II. Forfewerthan16samples–Transfer60μLofeachsamplelibraryintoaLibrary.4.25 -Add900μLofAMPureXPbeads to theLibrary tube.Mix thoroughlybyvortexingandcentrifugebriefly.Donotallowthebeadstoseparate. IncubatetheLibrary for10minutesatroomtemperature.

Note: If there is less than 900 μL of library in the Final Pool, add an equivalentamountofAMPureXPbeads.Thereshouldbea1:1ratioofFinalPoolandAMPureXPbeads.

4.26-PlacetheLibrarytubeontoamagneticstandandincubatefor10minutes.4.27–Keepingthetubeonthemagneticstand,carefullyremoveanddiscardthesupernatantfromtheLibrarytube,withouttouchingthebeads.4.28-Keepingthetubeonthemagneticstand,add~1.5–2mLoffreshlyprepared80%ethanoltotheLibrarytube.Thevolumeofethanoladdedshouldbesufficienttocoverthebeads.

Note:Applytheethanoltothesideofthetubewithoutbeads.4.29 - Incubate the Library tube at room temperature for 30 seconds; afterwards, carefullyremoveanddiscardthesupernatant.4.30-Repeatsteps4.28and4.29.4.31-QuicklyspindowntheLibrarytubeandplaceitbackonthemagneticstandwiththelidopen.Removeresidualethanolwithapipette.Donottouchthebeads.

Note: Ensure the bead pellet does not contain residual ethanol. This may requirerotating the tubeon themagnetic stand to removeethanolwithoutdisturbing thebeadpellet.


4.32-Allowthebeadstoairdryfor5-8minutesonthemagneticstanduntilthebeadpelletisdry.4.33 - Remove the Library tube from the magnetic stand and elute the Library with 31 μLmoleculargradewater.Donotletthepipettetiptouchthebeads,astheywillsticktoit.4.34 - Vortex the Library to fully resuspend the beads. Centrifuge briefly if some dropletsremainonthesidewalls.Ensurethebeadsremaininsuspension.4.35-IncubatetheLibraryatroomtemperaturefor2minutes.4.36-PlacetheLibrarytubeonthemagneticstandfor2minutes.4.37-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 Step4Note:MarkerKisusedwiththePippinPrep.TheBluePippinusesMarkerR2.

Protocol5.1-BringtheMarkerKloadingsolutiontoroomtemperature.5.2-Combine31μLofthePoolwith10μLofMarkerKloadingsolution.5.3-Mixbyvortexingandspindown.5.4-ConfigurethePippinPreptocollectDNAfragmentsbetween650and1300bps.Loadthe40μLsampleintothesampleportandrun.Runtimeis45-50minutes.5.5-Collectthewholecontent(approximately40μL)withintheelutionportofthePippinPrepandtransferittoanew1.5mllowbindmicrocentrifugetube.Thisisthesize-selectedlibrary.

Safestoppingpoint.Librariescanbestoredat-20°Cforextendedperiodsoftime.


Step 6 – Library Quantification using an intercalatingdsDNAfluorescentdyeDuration:~15minutesIt isnecessarytoquantifythesize-selectedlibrary inordertooptimallyusetheoutputoftheIllumina MiSeq sequencer. The concentration of the size-selected library can be accuratelymeasured by an intercalating dsDNA fluorescent dye, such as SYBR green or equivalent.Commerciallyavailablekitsandinstrumentsforthispurposeinclude,butarenotlimitedto,theQubit reader by Thermo Fisher (uses the Qubit Broad-Range dsDNA assay kit), the QuantusreaderbyPromega(usestheQuantifluordsDNAfluorescentdye)andothers.Here,theQubitmethod isdescribedas it isthemostcommonlyused instrument. Incaseanother instrumentandkitisused,followthemanufacturer’sstandardinstructions.

Note: This dsDNA fluorometric method is a quick but accurate enough way todeterminetheconcentrationof thefinalsize-selected library. Itmeasuresallof thedsDNA that is present in the library.Optionally youmay use the KAPA BiosystemsLibraryQuantitationkitandqPCRmachine foramorespecificmeasurementof thelibraryconcentration.ForthisprotocolSeeAppendix3.

ReagentlistItem Storage SuppliedbyQubitdsDNABRAssayKit roomtemperature ThermoFisherQubitdsDNABRStandards 4°C ThermoFisherSizeSelectedLibrary 4°C Step5

Protocol6.1–BringtheQubitStandardstoroomtemperature.PrepareQubitassaytubes(500µL,thin-

walled) for your library in duplicate and the two standards. Vortex and centrifuge thestandardsandthelibrary.

6.2–Add995µL Bufferand5µLdyetoa1.5mlcentrifugetube.Vortexandspindown.

6.3–Transfer190µL fromthereagentmixtotheQubittubesforthetwostandards.Transfer198µL fromthereagentmixtothetwoQubittubesfortheduplicatesofthelibrary.

6.4–Add10µLfromstandard1tothecorrespondingQubittubeandvortexitfor2seconds.Repeatwithstandard2.

6.5–Add2µL fromthelibrarytothetwocorrespondingQubittubesandvortexfor2seconds.


6.6–IncubatetheQubittubesatroomtemperaturefor2minutes.

6.7–SwitchonQubitmachineandchooseBRprotocol.6.8–Putstandard1QubittubeinandpushGO.Repeatwithstandard2.6.9–PutthelibrarytubeinQubitandpushGO.Repeatforthereplicate.6.10 – To convert the Qubit result from ng/μL to nM concentration, enter the meanconcentration of the two library replicates in the Omixon Workbook tab called “LibraryQuantitation”.6.11–UsingtheresultsfromtheQubitmeasurement,dilute10μLoftheSizeSelectedLibrarytoaconcentrationof2nMwithsterileH2O ina fresh1.5-mL lowbindmicrocentrifuge tube.StoretheremainingSizeSelectedLibraryat-20°C.

Safestoppingpoint.Librariescanbestoredat-20°Cforextendedperiodsoftime.Incase of long-term storage, re-quantification of the library is highly recommendedbeforerunningitontheMiSeq.


Step7–SequencingonIlluminaMiSeqDuration:~24hoursTheIlluminaMiSeqisanautomatedNGSinstrumentthatcansequencethesize-selectedlibraryprepared intheprevioussteps.De-multiplexingofthe indexedsamples isdoneautomaticallyfollowingcompletionofthesequencingrun.

Quicktip–Youcanusea1%PhiXspike-inasanadditionalcontrol tomonitor thesequencing reaction. Refer to Illumina documentation on the PhiX control foradditionalinformation.

ReagentlistItem Storage SuppliedbyReagentCartridge -20°C IlluminaHT1 -20°C IlluminaPR2 4°C IlluminaMiSeqFlowCell 4°C IlluminaLibraryat2nM 4°C Step6NaOH1Nor2N 20°Cto25°C UserMoleculargradeH2O 20°Cto25°C User

MiSeqReagentKitcapacityIlluminaMiSeqReagentKit

TimeHours

24/7Samples

24/11Samples

96/5Samples

96/7Samples

96/11Samples

Std300Cycle(MS-102-2002) ~24 24 24 96 96 72

Micro300Cycle(MS-103-1002) ~19 24 20 40 28 20

Nano300Cycle(MS-103-1001) ~17 6 4 8 6 4

Std500Cycle(MS-102-2003) ~39 24 24 96 96 96

Nano500Cycle(MS-103-1003) ~28 12 8 16 12 8

Protocol7.1-PreparetheMiSeqaccordingtostandardIlluminaprotocols.7.2-Preparea1nMDenaturedLibrary:Combine10μLoffreshlyprepared0.2NNaOHand10μLof the2nMDilutedSizeSelectedLibrary inanew1.5mL lowbindmicrocentrifuge tube.


Vortex and spin down. Incubate this 1 nM Denatured Library at room temperature for 5minutes.7.3-Preparea20pMDenaturedLibrary:Add980μLofchilledHT1tothe20μLofthe1nMDenaturedLibrary.Vortexandspindown.7.4 -Preparea9pMDenaturedLibrary:Add550μLofchilledHT1and450μLof the20pMDenaturedLibrarytoafresh1.5mLlowbindmicrocentrifugetube.Vortexandspindown.7.5 – Transfer 600μLof the9pMDenatured Library into the Load Samples reservoir of theMiSeqreagentcartridge.

Quicktip–ItisadvisabletousetheprovidedworkbooktocreatetheSampleSheetthat is requiredby theMiseq.Ensure that thecorrectAdaptorPlate configuration(A1orA2) is selectedon theOmixonWorkbookSequencing tab that contains theappropriateindexedadaptorsequences.


Step8–AnalysisofHLASequencingDataTheIlluminaMiSeqwillprocessthe9pMPooledLibraryandgeneratesequencingdataasfastqfiles. Please refer to theHLATwinmanual for assistancewith the correct installationofHLATwinandforinformationoninterpretingthegenotypinganalysisofyoursequencingdata.Forimplementing the Automated Protocol and issues relating to installation or analyzing data,[email protected].

AutomatedProtocol

ITSetupandConfiguration1. InstallHLATwinServerontheServer.2. InstallHLATwinClientonaclientcomputer–multipleHLATwinClientsmayconnectto

theserver.3. ContactOmixonSupport([email protected])forcustominstallationinstructionsfor

Automation.

ProtocolperAnalysis1. LaunchHLATwinClientandlogin.2. Data is already processed or is being processed. Review the results using the Traffic

LightSysteminHLATwin.3. Exportthegenotypingresultsand/orconsensussequencesasrequired.

ManualServerProtocol

ITSetupandConfiguration1. InstallHLATwinServerontheServer.2. InstallHLATwinClientonaclientcomputer.

ProtocolperAnalysis1. LaunchHLATwinClientandlogin.2. SelecttheMiSeqdatainfastqorfastq.gzformatandstarttheHolotypeHLAtypingrun.3. After the Holotype HLA typing has finished, review the results using the Traffic Light

SysteminHLATwin.4. Exportthegenotypingresultsand/orconsensussequencesasrequired.

ManualDesktopProtocol

ITSetupandConfiguration1. InstallHLATwinDesktop.


ProtocolperAnalysis1. LaunchHLATwinandlogin.2. SelecttheMiSeqdatainfastqorfastq.gzformatandstarttheHolotypeHLAtypingrun.3. After the Holotype HLA typing has finished, review the results using the Traffic Light

SysteminHLATwin.4. Exportthegenotypingresultsand/orconsensussequencesasrequired.


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

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


SupplementalFigures

PlateexampleforAmpliconPlate,AmplificationPlate,DilutionPlate,AmpliconQuantitationPlate(foranindividuallocus)&ReactionPlate


StandardsQuantitationPlateExample

KAPALibraryQuantitationqPCRPlateExample


Appendix1:PippinPrep

ProgrammingthePippinPrep1. ClicktheProtocolEditorTabandclickthe“New”button.

2. ClickthefoldericonnexttotheCassettefieldandselect“1.5%DFMarkerK”forthePippinPrepor“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. Visually inspect thePippinPrep.Make sure the5 LEDsareonand that the insideof thedeviceiscleananddry.

3. Click on the Sage Science logo on the bottom right of the screen. Thiswill allow you toenterapassword.Thefactorydefaultpasswordis“pips”.

4. ClicktheFactorySetupTabandmakesuretheBase-to-Thresholdvalueissetto0.02.

5. PlacethecalibrationfixtureinsidethePippinPrep,makingsurethedarkstripisfacedownandovertheLEDlights.

6. IntheMaintab,clickthe“Calibration”button.

7. IntheCalibrationwindow,makesurethe“TargetIpHmA”fieldissetto0.80(0.60forBluePippin)andthenhitthe“Calibrate”button.

8. GototheProtocolsTab.Clickthe“Load”buttonandselecttheprogramforHolotypeHLAandthespecificlaneyouwillbeusing.Makesurethat:

a. Thecorrectlaneisturnedon

b. Broadspectrumselectionindicatorison

c. Thereferencelaneisthesamelanethatwillberunning.

9. GototheMainTab.Makesurethat:

a. Theprogramyouloadedistheonethatisselected.


b. Theappropriatereferencelaneisselectedandthatitisalsothelanethesamplewillberunin.

10. Inspect thecassette.Beforetakingoff thetapesealing thewells, looktosee if thereareany bubbles behind the elution port. If there are any bubbles behind the elution port,gentlytapandrollthecassetteinyourhandtoworkthebubblesout.

11. Placethecassette,withthetapestilloverthewells,insideofthePippinPrep.

12. Carefully peel off the tape,making sure to remove the tape from the clean side of thecassette (lane5) to theused sideof the cassette (lane1). Take carenot to splash liquidwhenthetapeisremovedtopreventcontamination.

13. Removetheentirevolumeofbufferfromtheelutionportofthelaneyouwillbeusingandadd40μLoffreshelectrophoresisbufferinthatelutionport.

14. Addathinstripoftapeovertheelutionports.

15. Any reservoirs that are less than 3/4ths full should be topped off with electrophoresisbuffer.Donotoverfillthewells!Theedgeofthebuffershould‘just’reachtheplasticnothighertopreventdraggingwhenthelidslides.Applybufferfromthecleanwells(Lane5)totheusedwells(Lane1).

16. Make sure each of the loadingwells (wells with agarose) are filledwith electrophoresisbuffer.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.Oncethecontinuity test completes, open the Pippin slowly. Make sure no fluid is getting pulledacrossthecassettebythelidofthePippinPrep.

19. Briefly vortex the Marker K loading solution and spin it down. Add 10 μL of Marker Kloadingsolutiontoyour~30μLoflibrary.

20. Brieflyvortexyourlibraryandspinitdown.

21. Remove40μLofbufferfromthesamplewellthatyouwillbeusing.

22. Add~40μLofyourlibraryloadedwithMarkerKtothesamplewellthatyouwillbeusing.

23. Markthelanethatyouareusingwiththetechnician’sinitialsandthedate.

24. Close the Pippin Prep and click the “Start” button.Make sure the appropriate lane hasbeenturnedon.Thesampleshouldrunforabout45minutes.

25. Aftertherunhascompleted,carefullyopenthePippinPrep.Watchtoseeiftheliddragsanyliquidacrossthecassette.

26. Removethetapeovertheelutionports,beingcarefulnottoflickanyliquid.

27. Transferallthevolumefromtheelutionportintoanew1.5mllowbindtube.


28. Coveralloftheopenwellswithtwopiecesofplatesealingtape.Remembertoleaveatabonthecleanside.Thiswillmakeitsimpletoremovethetapefromcleantoused.

29. Placethesealedcassetteintoitsbagandsetitaside.

30. TakethewashcassetteandfillitwithMiliQwater.GentlyclosethelidofthePippinPrep,watchingtoseeifyoupullanyliquidacrossthewashcassette.

32. LeavethePippinPrepclosedforseveralseconds.

33. OpenthePippinPrep,watchingtoseeifyoupullanyliquidacrossthewashcassette.

34. Removethewashcassette,emptyitofwater,andletitdry.

35. CleananywateroffofthePippinPrepandcloseitgently.

36. Selectthe“ShutDown”buttoninthePippinPrepmenu.


Appendix 2: Amplicon Quantitation using a qPCRinstrumentDuration:35min

ReagentlistItem Storage Suppliedby20×TEBuffer(pH7.5) 4°C PromegaLambdaDNAStandard(100ng/μL) 4°C Promega200×QuantiFluordsDNADye 4°C PromegaSterileH2O 20°Cto25°C UserClassIAmplificationPlate(s) 4°C Step2ClassIIAmplificationPlate(s) 4°C Step2

Protocol1. Createaserialdilutionusing1.5mLmicrocentrifugetubesandtheQuantiFluorLambda

DNAstandard(100ng/μL).Followthedilutiontablebelow:

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).Aliquot49.5μL1x

TEbuffertothewellsofaclean96-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-wellopticalplatesusingtheformatof 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


Appendix 3: Library Quantitation using a qPCRinstrumentDuration:~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 20°Cto25°C User1×TEBuffer(pH8.0) 20°Cto25°C 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 Setthe1:1000and1:2000triplicatesastargetedsamples,anddefinethestandards(points:

4, starting concentration: 20pM, dilution: 1:10). Run the following program on the qPCRmachine in order to determine the DNA concentration of the Size Selected Library:

NumberofCycles Temperature Time1 95°C 5minutes25

(nomeltcurve)95°C 30seconds60°C 90seconds(dataacquisition)

9 ToconverttheqPCRresultfrompMtonMconcentration,enterthe“Quantitymean”results

of the two library replicates in the Omixon Workbook tab called “Library Quantitation”.

10 - Using the volumes from the workbook, dilute 10 μL of the Size Selected Library to aconcentration of 2 nMwith sterile H2O in a fresh 1.5-mL low bindmicrocentrifuge tube.StoretheremainingSizeSelectedLibraryat-20°C.


Safestoppingpoint.Librariescanbestoredat-20°Cforextendedperiodsoftime.Incase of long-term storage, re-quantification of the library is highly recommendedbeforerunningitontheMiSeq.

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HOLOTYPE HLA™ 24/7 A1 A2 U MHLA+24-7+U… · 2016 Increased volume of Enhancer 2, Enhancer 2 should now be saved after Combined DQB Enhancer is made, added new DRB4 Master Mix formulation,