holotype hla™ 24/11 a1, a2, a3, a4 u m€¦ · sequencing (ngs), have provided new opportunities...

HOLOTYPEHLA™24/11

A1,A2,A3,A4USERMANUAL

FORRESEARCHUSEONLY

Version3.0

OmixonBiocomputingLimited10/22/2018

.

OmixonHolotypeHLA24/11UserManualv3.0Forresearchuseonly.Copyright©2018,OmixonBiocomputingLtd.Confidential&Proprietary Page2│52

TableofContentsDOCUMENTHISTORY...........................................................................................................................................5THEPRINCIPLEOFTHEMETHOD:NGS-BASEDHLATYPINGFORTHEILLUMINAMISEQ.........................................7HOLOTYPEHLAPACKINGLIST...............................................................................................................................8

PRIMERCOMPONENTBOX..............................................................................................................................................8LIBRARYPREPARATIONREAGENTSCOMPONENTBOX...........................................................................................................896-WELLADAPTORPLATE...............................................................................................................................................8EXCELWORKBOOK........................................................................................................................................................8SOFTWARE–OMIXONHLATWIN....................................................................................................................................9PRODUCTKNOWNLIMITATIONS.......................................................................................................................................9

RECOMMENDATIONS.........................................................................................................................................10DNAEXTRACTIONRECOMMENDATIONS..........................................................................................................................10TECHNICALANDEQUIPMENTRECOMMENDATIONS............................................................................................................10ASSOCIATEDREAGENTRECOMMENDATIONS.....................................................................................................................10

MiSeqReagentKitcapacity...............................................................................................................................11RECOMMENDEDSUPPLIES.............................................................................................................................................11

LEGALNOTICE....................................................................................................................................................13SUMMARYOFSTEPS..........................................................................................................................................14GLOSSARY/DEFINITIONS.....................................................................................................................................15STEP0–GENOMICDNAPREPARATION..............................................................................................................16STEP1–HLAAMPLIFICATIONMASTERMIXPREPARATION................................................................................17

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

MasterMix:HLA-A,B,C,DRB1/DRB3,DRB5,DPA1,DPB1andDQA1...............................................................18MasterMix:HLA-DQB1set3.............................................................................................................................18MasterMix:HLA-DRB4......................................................................................................................................18

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

ClassIAmplification(HLA-A,BandC)...............................................................................................................20ClassIIAmplification(HLA-DRB1/DRB3,DRB4,DRB5,DPA1,DPB1,DQA1andDQB1).....................................20ExpectedAmpliconSizes....................................................................................................................................20

STEP3–AMPLICONQUANTITATIONANDNORMALIZATION(USINGAPLATEFLUOROMETER)...........................21REAGENTLIST.............................................................................................................................................................21PROTOCOL.................................................................................................................................................................21

AmpliconpoolingandExoSAP-ITExpresspurification.......................................................................................23ExoSAP-ITExpressPCRPurification....................................................................................................................23

STEP4–LIBRARYPREPARATION........................................................................................................................24REAGENTLIST.............................................................................................................................................................24PROTOCOL.................................................................................................................................................................24

FragmentationMasterMix................................................................................................................................25FragmentationProgram....................................................................................................................................25


EndRepairMasterMix......................................................................................................................................26EndRepairProgram...........................................................................................................................................26LigationMasterMix...........................................................................................................................................27LigationProgram...............................................................................................................................................27LibraryPooling...................................................................................................................................................28

STEP5–BEAD-BASEDLIBRARYSIZESELECTION..................................................................................................29REAGENTLIST.............................................................................................................................................................29PROTOCOL.................................................................................................................................................................29

STEP6–LIBRARYQUANTITATIONUSINGANINTERCALATINGDSDNAFLUORESCENTDYE..................................33REAGENTLIST.............................................................................................................................................................33PROTOCOL.................................................................................................................................................................33

STEP7–SEQUENCINGONILLUMINAMISEQ......................................................................................................35REAGENTLIST.............................................................................................................................................................35

MiSeqReagentKitcapacity...............................................................................................................................35PROTOCOL.................................................................................................................................................................36

STEP8–ANALYSISOFHLASEQUENCINGDATA..................................................................................................37AUTOMATEDPROTOCOL...............................................................................................................................................37

ITSetupandConfiguration................................................................................................................................37ProtocolperAnalysis.........................................................................................................................................37

MANUALSERVERPROTOCOL.........................................................................................................................................37ITSetupandConfiguration................................................................................................................................37ProtocolperAnalysis.........................................................................................................................................37

MANUALDESKTOPPROTOCOL.......................................................................................................................................37ITSetupandConfiguration................................................................................................................................37ProtocolperAnalysis.........................................................................................................................................38

TECHNICALASSISTANCE.....................................................................................................................................39PhoneSupport:..................................................................................................................................................39

SUPPLEMENTALFIGURES....................................................................................................................................40PLATEEXAMPLEFORAMPLICONPLATE,AMPLIFICATIONPLATE,DILUTIONPLATE,AMPLICONQUANTITATIONPLATE(FORANINDIVIDUALLOCUS)&REACTIONPLATE...........................................................................................................................40STANDARDSQUANTITATIONPLATEEXAMPLE....................................................................................................................41KAPALIBRARYQUANTITATIONQPCRPLATEEXAMPLE......................................................................................................41

APPENDIX1:AMPLICONQUANTITATIONUSINGAQPCRINSTRUMENT..............................................................42REAGENTLIST.............................................................................................................................................................42PROTOCOL.................................................................................................................................................................42

APPENDIX2:LIBRARYSIZESELECTIONUSINGPIPPINPREP.................................................................................44REAGENTLIST.............................................................................................................................................................44PROTOCOL.................................................................................................................................................................44LIBRARYPURIFICATION.................................................................................................................................................44LIBRARYSIZESELECTION...............................................................................................................................................46PROGRAMMINGTHEPIPPINPREP...................................................................................................................................47RUNNINGTHEPIPPINPREP...........................................................................................................................................47

APPENDIX3:LIBRARYQUANTITATIONUSINGAQPCRINSTRUMENT..................................................................50


REAGENTLIST.............................................................................................................................................................50PROTOCOL.................................................................................................................................................................50

qPCRPrimerMix................................................................................................................................................50qPCRMasterMix...............................................................................................................................................51


DocumentHistoryVersion Date DescriptionofChanges Approval

Name

1.5 January,2015 InitialVersion PeterMeintjes

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

PeterMeintjes

1.7 June,2015ExoSAP-IT StepChange,DQB1Set 1&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,2015Amplification verification and quantitationmaybe 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 EfiMelista1.7.7 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,ampliconquantitationsimplification,per-samplepoolingvolumechangein11-locuskits,ExoSAP-ITreplacementbyExoSAP-ITExpress,Qubituseforlibraryquantitation.

EfiMelista

2.1 July,2017 DQB1 set 1 and set 2 primer mix replaced byDQB1 set 3, end repair reaction timedecrease,ligation reaction time decrease libraryquantitation method update, Sample sheetremovedfromAppendices

EfiMelista

2.2 November,2017 Bead based size selection introduced in coremanual, Pippin size selection moved toAppendix, Pippin size range modified to 500-1500bp,Pippinelutionmodifiedto50ul,1:4000dilution introduced in qPCR-based libraryquantitation,librarydenaturationconcentrationchangedto4nM,knownlimitationsadded

EfiMelista

2.2.1 May,2018 Pippinsizeselectionrangerevertedto650-1300bp, Pippin elution volume modified to 40 ul,transferofindexedadaptorsfromAdaptorPlateaddedtoligationstep

EfiMelista

3.0 October,2018 Decreased input DNA, Long range polymerasechanged to Promega GoTaq, Enhancer 2removed from protocol, new AmplificationMasterMixvolumes,modifiedAmpliconPoolingvolumes, new Fragmentation Master Mixvolumes, specified storage times, wordingchanges

EfiMelista


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.Morespecifically theHLAgenesA,B,C,DPA1,DQA1andDQB1areamplified for theirentirecodinglength,includingelementsofthe5’and3’untranslatedregions,whileDRB1,DRB3andDRB4areamplifiedfromintron1tointron4,andDRB5andDPB1areamplifiedfromintron1tothe3’untranslatedregion.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.Thewholeprocesstakeslessthan48hoursdependingontheselectionoftheflowcellontheIlluminaplatform.Thegenerateddataareanalyzedusingtwodifferentalgorithms inHLATwin™ (www.omixon.com).Theuseof two independentalgorithms inHLATwin provides the highest level of confidence so that the HLA genotyping results can bereported immediately without further attention. Samples with questionable or ambiguousgenotypingsareflaggedbythesoftwaretobeanalyzedmanually.


HolotypeHLAPackingList

PrimerComponentBoxPrimermix Rxns Vol/tube #Tubes ColorcodeHLA-A 24 60μL 1 YellowHLA-B 24 60μL 1 RedHLA-C 24 60μL 1 OrangeHLA-DRB1/DRB3 24 60μL 1 GreenHLA-DRB4 24 60μL 1 WhiteHLA-DRB5 24 60μL 1 PinkHLA-DPA1 24 60μL 1 BlackHLA-DPB1 24 60μL 1 PurpleHLA-DQA1 24 60μL 1 BrownHLA-DQB1(Set3) 24 60μL 1 BlueEnhancer1 96 1100μL 1 Clear

LibraryPreparationReagentsComponentBoxReagent Rxns Vol/tube #Tubes ColorcodeFragmentationEnzyme(A) 24 85μL 1 YellowFragmentationBuffer(B) 24 85μL 1 RedEndRepairEnzyme(C) 24 50μL 1 GreenEndRepairBuffer(D) 24 95μL 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),A3(i49-i72)andA4(i73-i96).

ExcelWorkbookAnExcelWorkbookisprovidedtosupporttheHolotypeHLAprotocolwithvolumecalculations,platelayouts,reagenttraceability,recordkeepingandMiSeqSampleSheetgenerationofallofthesupportedadaptorplateconfigurations(A1,A2,A3,A4).TheWorkbookversioncompatiblewiththisUserManualisv3.0.Ifyoudonothaveacopy,[email protected].


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

ProductKnownLimitationsPlease refer to the Product Known Limitations Guide document for known ambiguities andknown software limitation of the Holotype HLA product. The Guide may be found on theOmixonwebsiteunderMyHolotype/SupportandServices/HLATwin Instructions forUse,or [email protected].


Recommendations

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

buffycoats,cordbloodoranyfractionofwhitebloodcells),salivaandbuccalswabscanbe used. For the amplification of HLA-A, HLA-B, HLA-C, HLA-DRB1/DRB3, HLA-DRB4,HLA-DRB5, HLA-DPA1, HLA-DPB1, HLA-DQA1 and HLA-DQB1, 0.6-1.4 µg of gDNA isrequired.

TechnicalandEquipmentRecommendations§ Thermalcyclerwith96-wellformat§ Platefluorometer(oranyinstrumentcapableoffluorescencedetectionin96-wellplate

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

AssociatedReagentRecommendations§ PromegaGoTaq2XLongPCRMasterMix(Cat#M402A)

§ Eachsamplerequires73μLofPromegaGoTaqMasterMixfor11loci§ QuantiFluordsDNASystemfromPromega(Cat#E2670)§ ExoSAP-IT Express fromThermoFisher (Cat#75001-200, 75001-1ML, 75001-4X-1MLor

75001-10ML)§ Eachpooledsamplerequires4μLofExoSAP-ITExpressenzyme§ Cat#75001-200contains200μLofExoSAP-ITExpressenzyme§ Cat#75001-1-MLcontains1mLofExoSAP-ITExpressenzyme§ Cat#75001-4X-1MLcontains4mLofExoSAP-ITExpressenzyme§ Cat#75001-10MLcontains10mLofExoSAP-ITExpressenzyme

§ AgencourtAMPureXPbeadsfromBeckmanCoulter(Cat#A63880,A63881,orA63882)§ EachHolotypeHLArunrequiresamaximumof1000μLofAMpureXPbeads§ Cat#A63880contains5mLofAMPureXPbeads§ Cat#A63881contains60mLofAMPureXPbeads§ Cat#A63882contains450mLofAMPureXPbeads

§ QubitdsDNABRAssayKit(Cat#Q32850orQ32853)§ Cat#Q32850contains100assays§ Cat#Q32853contains500assays


§ 1XTris-EDTA(pH8.0)§ Tween20§ Moleculargradeethanol(AnhydrousAlcohol)§ Moleculargradewater(DNaseandRNasefree)§ 10NSodiumhydroxide§ MiSeqReagentKitfromIllumina§ Gelcassette,1.5%agarose,dyefreewithinternalstandard(MarkerK/R2),forthePippin

Prep/BluePippin(Cat#CDF1510forPippinPrepandBDF1510forBluePippin)-optional§ Library Quantification Kit – Illumina/Universal from KAPA Biosystems (Cat# KK4824) -

optional

MiSeqReagentKitcapacityIlluminaMiSeqReagentKit

TimeHours

24/7Samples

24/11Samples

96/5Samples

96/7Samples

96/11Samples

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

Micro300Cycle(MS-103-1002) ~19 24 24 48 32 24

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§ 2.0 mL low-bind microcentrifuge tubes (Eppendorf DNA LoBind Cat# 022431048

recommended)§ 0.5 ml thin wall tubes for Qubit instrument (Qubit Assay tubes Cat# Q32856

recommended)§ Adjustablevolumepipettes(1.0–1000μLcapacity)§ 8-channeladjustablevolumepipettes(1.0-100μLcapacity)§ 96-wellplatescompatiblewiththethermalcycler§ 96-well optical plates compatible with the plate fluorometer or with the qPCR

instrument§ 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/).

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©2018OmixonBiocomputingLtd.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§ ExoSAP-edAmpliconsPlate–PlatecontainingalldilutedampliconsafteraddingExoSAP-

ITExpressreagent§ StandardsQuantitationPlate–96-wellplatecompatiblewiththeplatefluorometeror

qPCRmachinewhereDNAstandardsareplacedtoallowforampliconquantitation


Step0–GenomicDNAPreparationDuration:~20minutes

RefertoWorkbook:gDNApreparationtabIsolategDNAfromwholeblood,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.For the amplification of HLA-A, HLA-B, HLA-C, HLA-DRB1/DRB3, HLA-DRB4, HLA-DRB5, HLA-DPA1,HLA-DPB1,HLA-DQA1andHLA-DQB1,0.6–1.4µgofgDNAisrequiredforeachsample.


Step1–HLAAmplificationMasterMixPreparationDuration:~15minutes

RefertoWorkbook:PCRpreparationtab

Thepurposeofthisstepistopreparelocus-specificMasterMixestoamplifyeachtargetedHLAlocusindividually.ThelociamplifiedbythisprotocolareHLA-A,HLA-B,HLA-C,HLA-DRB1/DRB3,HLA-DRB4,HLA-DRB5,HLA-DPA1,HLA-DPB1,HLA-DQA1andHLA-DQB1.

ReagentlistItem Storage SuppliedbyHLA-APrimerMix -20°C OmixonHLA-BPrimerMix -20°C OmixonHLA-CPrimerMix -20°C OmixonHLA-DRB1/3PrimerMix -20°C OmixonHLA-DRB4PrimerMix -20°C OmixonHLA-DRB5PrimerMix -20°C OmixonHLA-DPA1PrimerMix -20°C OmixonHLA-DPB1PrimerMix -20°C OmixonHLA-DQA1PrimerMix -20°C OmixonHLA-DQB1Set3PrimerMix -20°C OmixonEnhancer1 -20°C OmixonPromegaGoTaq2XLongPCRMasterMix -20°C PromegaMoleculargradeH2O Roomtemperature User

Protocol1.1 -RemoveallprimermixesandthePromegaGoTaq2XLongPCRMasterMixesfromstorage

andthawatroomtemperature.

Note:TheTaqpolymeraseinthePromegaGoTaq2XLongPCRMasterMixisahotstartenzyme,thereforeitisnotrequiredtokeeptheMasterMixonice.

1.2 -VortexallprimermixesandthePromegaGoTaq2XPCRMasterMixesfor3seconds,thenspin them down for 10 seconds (quick spin). Prepare aMasterMix for each PrimerMixaccording to the tables below (for less, than 24 samples use the Excel Workbook tocalculatethevolumes):


MasterMix:HLA-A,B,C,DRB1/DRB3,DRB5,DPA1,DPB1andDQA1Reagent Volume/sample/locus Volume/24samples/locusMoleculargradeH2O 7.5μL 195μLPromegaGoTaq2XPCRMasterMix 7μL 182μLPrimerMix 1.5μL 39μLTotalVolume 16μL 416μL

MasterMix:HLA-DQB1set3

Reagent Volume/sample/locus Volume/24samples/locusMoleculargradeH2O 5.5μL 143μLPromegaGoTaq2XPCRMasterMix 7μL 182μLEnhancer1 2μL 52μLPrimerMix 1.5μL 39μLTotalVolume 16μL 416μL

MasterMix:HLA-DRB4

Reagent Volume/sample/locus Volume/24samples/locusMoleculargradeH2O 4.5μL 117μLPromegaGoTaq2XPCRMasterMix 10μL 260μLPrimerMix 1.5μL 39μLTotalVolume 16μL 416μL1.3 –VortexeachMasterMixfor5secondsandspinthemdownfor10seconds(quickspin).

1.4 -DiluteallgDNAstoaconcentrationof20-30ng/μL(minimumvolumeis44μLpersample).

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


Step2–HLAClassIandIIAmplificationDuration:~6hours30minutes

RefertoWorkbook:PlatemapstabThepurposeof 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-DRB1/DRB3MasterMix -20°C Step1HLA-DRB4MasterMix -20°C Step1HLA-DRB5MasterMix -20°C Step1HLA-DPA1MasterMix -20°C Step1HLA-DPB1MasterMix -20°C Step1HLA-DQA1MasterMix -20°C Step1HLA-DQB1Set3MasterMix -20°C Step1gDNA 4°C User

Protocol2.1–Aliquot16μLofeachMasterMixintoseparatewellsof96-wellPCRplates.

Note:Class IandClass IIamplificationhavebeenoptimizedusing twodifferentPCRconditions,soClassIandClassIIMasterMixesshouldnotbeinthesameplate.

2.2–Add4μLof eachdilutedgDNA into theappropriatewell of theplatesprepared in the

previousstep.Mixbypipetting.


2.3 – Seal the plate with a thermal seal and visually inspect each well. Spin-down allAmplificationPlatesinacentrifugefor10seconds(quickspin).

2.4–PlacetheAmplificationPlates intothermalcyclersandruntheprogramsforClass IandClassIIamplificationaccordingtothetablesbelow:

ClassIAmplification(HLA-A,BandC)NumberofCycles Temperature Time

1 95°C 2minutes 95°C 15seconds

40 65°C 30seconds 68°C 4minutes1 68°C 10minutes1 4°C ∞

Class II Amplification (HLA-DRB1/DRB3, DRB4, DRB5, DPA1, DPB1, DQA1 andDQB1)

NumberofCycles Temperature Time1 95°C 2minutes 95°C 15seconds

40 65°C 30seconds 68°C 8minutes1 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/DRB3 ~4.3

HLA-DRB4 ~4.2HLA-DRB5 ~5HLA-DPA1 ~5.5HLA-DPB1 ~6.6HLA-DQA1 ~5.5

HLA-DQB1(Set3) ~6.5Safe stoppingpoint.Amplicons canbe storedat4°Covernightorat -20°C forup to3months.


Step 3 – Amplicon Quantitation and Normalization(usingaPlateFluorometer)Duration:~40minutes

RefertoWorkbook:AmpliconquantitationtabAmplicon 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 1 forInstructionsonhowtodotheAmpliconQuantitationusingaqPCRmachine.

Quicktip–Theampliconquantitation,whilerecommended,isnotrequiredtosuccessfullycomplete the Holotype HLA protocol. Amplicon normalization does not require precisemeasurement of amplicon concentration. An estimate of amplicon concentrations basedonexperienceoragarosegelelectrophoresis canbeused instead.Ampliconquantitationshould not be skipped without consistent experience with the complete Holotype HLAprotocol.

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 Lambda

DNA7.5μL 492.5μL 1.5ng/μL

Standard2 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/μL3.2 -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 –Vortexwelltomix(5seconds)andspindown(for10seconds).3.8 –RuntheStandardsQuantitationPlateontheplatefluorometerfollowedbytheAmplicon

QuantitationPlates.3.9 –CalculatetheconcentrationofDNAintheAmpliconQuantitationPlatesusingRFUdata

generatedbytheplatefluorometer.RefertotheAmpliconQuantitationTabintheprovidedworkbookforassistancewithcalculations.

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

concentrationofDNAisinarangeof50-100ng/μL.§ IfDNAconcentrationisgreaterthan180ng/μL:add20μLofH2O§ IfDNAconcentrationis115-180ng/μL:add10μLofH2O§ IfDNAconcentrationislessthan115ng/μL:donotaddanyH2O


AmpliconpoolingandExoSAP-ITExpresspurification

RefertoWorkbook:Librarypreparationtab

3.11 – Pool all loci for each sample into a single Pooled Amplicons Plate. Combine the

volumesindicatedforeachlocusasinthefollowingtabletoobtainafinalvolumeof38μL:

HLAlocus PooledvolumeA 2μLB 2μLC 2μL

DPA1 4μLDQA1 4μLDRB4 4μLDRB5 4μLDPB1 5μL

DQB1set3 5μLDRB1/3 6μL

3.12 – Add 4 μL of ExoSAP-iT Express into each sample library. Rinse the pipette tips by

pipetting.Sealtheplatewithathermalsealandspindownfor10seconds(quickspin).3.13–PlacethePooledAmpliconsPlateintoathermalcyclerandrunthefollowingprogram:

ExoSAP-ITExpressPCRPurification

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

Safe stoppingpoint.Amplicons canbe storedat4°Covernightorat -20°C forup to3months.


Step4–LibraryPreparationDuration:~1hour40minutes

RefertoWorkbook:LibrarypreparationtabDuringthisstep,thepooledampliconsarepreparedforsequencingontheIlluminaMiSeq.Theampliconsareenzymaticallyfragmented,theendsarerepairedandadenylated,then indexedadaptorsareligatedtotheends.

Note:Omixonrecommendsvolumesgreaterthanisnecessaryfor24samplesbecausemanyof theenzymesandbuffers are viscous, resulting in excesspipetting loss. It isalso recommended touseaReagentplate formore than8 samples:placea96-wellplate on a PCR cooler block and aliquote the Master Mix into a clean column fordispensingwithamultichannelpipette.

ReagentlistItem Storage SuppliedbyPooledExoSAP-edAmpliconsPlate 4°C Step3MoleculargradeH2O Roomtemperature UserFragmentationEnzyme(A) -20°C OmixonFragmentationBuffer(B) -20°C OmixonEndRepairEnzyme(C) -20°C OmixonEndRepairBuffer(D) -20°C OmixonLigationEnzyme(E) -20°C OmixonLigationBuffer(F) -20°C OmixonAdaptorPlate -20°C Omixon

Protocol4.1-Turnonthethermalcycler.Verifythattheheatedlidiswarmingup.4.2-ThawtheFragmentationBuffertoroomtemperatureandkeepitonice.

Note:BesuretovortextheFragmentationEnzymethoroughlybeforeuse.Spinitquickly(10seconds),thenkeepitonice.

4.3-PrepareFragmentationMasterMixaccordingtothetablebelow(forless,than24samplesusetheExcelWorkbooktocalculatethevolumes):


FragmentationMasterMix

Reagent Volumeperlibrary(μL)

Recommendedvolumesfor24libraries(μL)

Colorcode

MoleculargradeH2O 2μL 56μL FragmentationBuffer(B) 2μL 56μL RedFragmentationEnzyme(A) 1μL 28μL YellowTotalVolume 5μL 140μL 4.4-VortextheFragmentationMasterMixthoroughly(for10seconds)andspindownfor10

seconds(quickspin).PlacetheFragmentationMasterMixonice.

Note:Thefragmentationreactionhasbeendesignedtoprovide ideallysizedDNAforsequencingonthe IlluminaMiSeq. It is important tokeepthereagentscolduntil thereaction is started in the thermal cycler to prevent excessive fragmentation. Use ofmulti-channelpipettesandthecoolerracksisrecommendedtominimizeopportunitiesforexcessivefragmentation.

4.5-CentrifugetheExoSAP-edPooledAmpliconsPlatefor10seconds(quickspin),andplaceitoniceoracoldblock.

4.6-PrepareaReactionPlate:placeafresh96-wellPCRplateonaPCRcoldblock.4.7 - Add 5 μL of Fragmentation Master Mix into the empty wells of the Reaction Plate,

corresponding with samples in the Pooled Amplicons Plate. The use of a multi-channelpipetteisrecommended.

4.8 -Transfer15μLofeachamplicon fromthePooledAmpliconsPlate to thecorresponding

well on the Reaction Plate using a multi-channel pipette. Mix by pipetting. Keep theReactionPlateonthecoldblock.

4.9-CovertheReactionPlatewithathermalsealandcentrifugefor10seconds(quickspin).4.10-IncubatetheReactionPlateinathermalcyclerwiththefollowingprogram:

FragmentationProgramNumberofCycles Temperature Time

1 37°C 10minutes1 70°C 15minutes1 4°C ∞

Safestoppingpoint.Librariescanbestoredat4°Covernightorat-20°Cforlonger.


4.11 - Prepare the End Repair Master Mix according to the table below (for less, than 24samplesusetheExcelWorkbooktocalculatethevolumes):

EndRepairMasterMix

Reagent Volumeperlibrary(μL)

Recommendedvolumesfor24libraries(μL)

Colorcode

MoleculargradeH2O 1.25μL 35μL EndRepairBuffer(D) 2.5μL 70μL OrangeEndRepairEnzyme(C) 1.25μL 35μL GreenTotalVolume 5μL 140μL 4.12-VortextheEndRepairMasterMixandspindownfor10seconds(quickspin).4.13-CentrifugetheReactionPlate(containingthefragmentedSamples)for10seconds.Add5

μLofEndRepairMasterMix intoeachwellof theReactionPlateandmixbypipetting.Theuseofamulti-channelpipetteisrecommended.

4.14-CovertheReactionPlatewithathermalsealandcentrifugefor10seconds(quickspin).4.15-IncubatetheReactionPlateinathermalcyclerwiththefollowingprogram:

EndRepairProgram

NumberofCycles Temperature Time1 20°C 30minutes1 70°C 5minutes1 4°C ∞

Safestoppingpoint.Librariescanbestoredat4°Covernightorat-20°Cforlonger.

4.16-Removethe IndexedAdaptorsPlatefromstorageandthawatroomtemperatureafter

theEndRepairProgramstarts inthethermalcycler.WhentheAdaptorPlateisatroomtemperature,centrifugeitfor1minuteat3000rpm.

4.17-CarefullypullthesealofftheAdaptorPlate.DonotshaketheAdaptorPlateoncetheseal

isremovedtopreventcrosscontamination.4.18 – Transfer 5 μL from eachwell of the Adaptor Plate to the corresponding well on the

ReactionPlate.Mixwellbypipetting.UsetheReactionPlateinsteadoftheAdaptorPlatefortheremainingstepsinthemanual.


Note: IftheentireAdaptorPlateisNOTgoingtobeused, it ispossibletouseonlythenecessarynumberofadaptors.Cuttheplatesealbetweenthewellstobeusedand thewells tobekept.Carefullypull the sealoff theAdaptorPlate, leaving thesealinplaceoverthewellstobekept.a. Transfer5μLfromeachwelloftheAdaptorPlatetothecorrespondingwellon

theReactionplate,andmixwellbypipetting.

b. ResealtheAdaptorPlateandreturnitto-20°C.UsetheReactionPlateinsteadoftheAdaptorPlatefortheremainingstepsinthemanual.

4.19-PreparetheLigationMasterMixaccordingtothetablebelow(forless,than24samples

usetheExcelWorkbooktocalculatethevolumes):

LigationMasterMix

Reagent Volume(μL) Recommendedvolumesfor24libraries(μL)

Colorcode

LigationEnzyme(E) 2.5μL 70μL BlueLigationBuffer(F) 30μL 840μL BlackTotalVolume 32.5μL 910μL

4.20-VortextheLigationMasterMixthoroughly(for5seconds)andspindownfor10seconds

(quickspin).4.21-Aliquote32.5μLofLigationMasterMixintoeachwelloftheReactionPlate.Theuseofa

multi-channelpipetteisrecommended.Mixbypipetting.4.22-CovertheReactionPlatewithathermalsealandcentrifugefor10seconds(quickspin).4.23-IncubatetheReactionPlateinthethermalcyclerwiththefollowingprogram:

LigationProgramNumberofCycles Temperature Time

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

Safe stopping point. Libraries can be stored at 4°C overnight or at -20°C for up to 3months.


LibraryPooling4.24-CreatetheLibrarybycombininganaliquotfromeachpooledamplicon,nowasample-specificlibrary,intoasingle2.0mLlowbindmicrocentrifugetube.

I. For 12 ormore samples - Calculate the amount of each sample library to pooltogetherasasingleLibraryof900μLtotalvolume.Divide900μLbythenumberofsamplelibraries.ThisisthevolumeofaliquottobetakenfromeachsamplelibraryandpipettedintotheLibrary.

II. Forfewerthan12samples–Transfer60μLofeachsamplelibraryintoa2.0mLlowbindtubethatwillcontainthepooledLibrary.

To perform Size Selection using AMPure XPmagnetic beads, please proceed to Step 5. ToperformSizeSelectionusingthePippinPrep,pleaseproceedtoAppendix2.


Step5–Bead-basedLibrarySizeSelectionDuration:~1hour20minutes

RefertoWorkbook:Librarypreparationtab

Step 5 describes the protocol for size selection of a single pool of indexed libraries usingAMPpure XP magnetic beads. Agencourt AMPure XP beads use solid phase reversibleimmobilizationchemistry tobind specific sizesofDNA to thebeadsbasedonabead/sample(v/v) ratio. Lower ratios of beads (0.2X- 0.8X) bind larger fragments of DNA leaving smallerfragments in solution. Increasing the bead/sample ratio (1X – 2X) allows the beads to bindsmallerfragmentsaswell.

ReagentlistItem Storage SuppliedbyPooledLibrary 4°C Step4AMPureXPbeads 4°C BeckmanCoulter1XTris-EDTA 20°Cto25°C UserTween20 20°Cto25°C User80%ethanol(freshlyprepared) 20°Cto25°C UserMoleculargradeWater 20°Cto25°C User

Note:Thefollowingprotocoliswrittenforlibrariescontainingatleast12samples.Ifyour Pooled Library is less than 700μL, use the ExcelWorkbook for calculating thevolumesorusethefollowingratios:

Library:BeadsratioFirstSelection 1:0.2SecondSelection 1:1ThirdSelection Alwaysfixed

ProtocolReagentPreparation:5.1 PreparetheBeadWashBufferaccordingtothetablebelow:

Reagent Volume1XTris-EDTA 40mLTween20 20μLTotalVolume 40.02mL


Note:TheBeadWashBuffercanbestoredatroomtemperatureforupto3months.

5.2 Make5mlof80%ethanol(4mlEtOH+1mlH2O)

5.3 VortexAMPureXPbeadsthoroughlytoresuspend(for20seconds).

5.4 Aliquot840μLofAMPureXPbeadstoa2mllowbindtubeandbringtoroomtemp.KeepthestockAMpurebeadsatroomtemperatureforuseinSelection3stepbelow.

5.5 Add840μLofbeadwashbuffertothealiquotedbeads.Mixthoroughlyonavortexmixer.Spinfor5seconds(quickspin).

5.6 Incubatefor1minuteatroomtemperature.

5.7 Putthetubeonthemagneticstandfor2minutesoruntilsolutionisclear(typicallynomorethan4minutes).

5.8 Carefullyremoveanddiscardthesupernatant.Becarefulnottodisturbthebeads.

5.9 Add840μLofbeadwashbuffertothebeads.Mixthoroughlyonavortexmixer.Spinfor5seconds(quickspin).

5.10 Incubatefor1minuteatroomtemperature.

5.11 Vortexbeadsfor5secondstoresuspendandaliquot140μLtoaclean2mllowbindtube.Ifusingfewerthan12samples,seeExcelWorkbookforthevolumeofbeadstouse.

5.12 KEEPremaining700μLofbeadsinbeadwashbufferasideforthe2ndselectionstep.1stSelection:

5.13 Placethetubecontaining140μLofbeadsinbeadwashbufferonamagneticstandfor2minutes.Forless,than12samplesseeExcelWorkbookforthevolumeofbeadstouse.

5.14 Carefullyremoveanddiscardsupernatant.Removethetubefromthemagnet.

5.15 Add700μLofthepooledlibrarytothebeads.Vortexbeadsfor5secondstoresuspendandspinfor5seconds(quickspin).Ifusingfewerthan12samplesseeExcelWorkbookforthevolumeofPooledLibrarytouse.

5.16 Incubateatroomtemperaturefor5minutes.

5.17 Placethetubebackonthemagneticstandfor4minutestopelletthebeads.

5.18 COLLECTtheeluate(library)intoafresh1.5mllowbindtube.


2ndSelection:

5.19 Placethetubecontaining700μLofbeadsinbeadwashbufferonamagnettopelletthebeadsfor2minutes.Carefullyremoveanddiscardthesupernatant.Ifusingfewerthan12samplesseeExcelWorkbookforthevolumeofbeadstouse.

5.20 Removethetubecontainingthebeadsfromthemagnet.

5.21 Addthelibraryeluatecollectedfromthe1stselectionstep5.18tothebeads.

5.22 Mixthoroughlybyvortexingfor10secondstoresuspendbeads.Spinfor5seconds(quickspin).



5.25 Carefullyremoveanddiscardthesupernatant.

5.26 Add~0.7-1mLof80%freshlypreparedethanoltothetubewhilestillonthemagneticstand.Volumeshouldbesufficienttocoverthebeads.

5.27 Incubateonthemagnetatroomtemperaturefor30seconds,andthencarefullyremoveanddiscardthesupernatant.

5.28 Repeatsteps5.26and5.27once.

5.29 Spintubefor3secondsincentrifuge(quickspin),quicklyplacebackonthemagnet,andremoveresidualethanolwithapipette.

5.30 Leavetubeonmagnetwithlidopenfor5minutestoairdrythebeadpellet.

5.31 RemovetubefrommagnetandeluteDNAtargetbyadding200μLsterilewatertothebeads.



5.34 Placethetubeonthemagneticstandfor2minutes.

5.35 TRANSFERthe200μLofeluate(library)intoafresh2mllowbindtube.3rdSelection:

5.36 Add160μLofthoroughlyvortexed(for20seconds),roomtemperatureAMPureXPbeadsfromthestockbottletothe200μLoflibraryfromstep5.35ofthe2ndselection.





5.40 Carefullyremoveanddiscardthesupernatant.

5.41 Add~0.5-1mLof80%freshlypreparedethanoltothetubewhilestillonthemagneticstand.Volumeshouldbesufficienttocoverthebeads.

5.42 Incubateonthemagnetatroomtemperaturefor30seconds,andthencarefullyremoveanddiscardthesupernatant.

5.43 Repeatsteps5.41and5.42once.

5.44 Spintubefor5secondsincentrifuge(quickspin),quicklyplacebackonthemagnet,andremoveresidualethanolwithapipette.

5.45 Leavetubeonmagnetwithlidopenfor5minutestoairdrybeads.

5.46 Removetubefrommagnetandelutelibrarybyadding50μLsterilewatertothebeads.



5.49 Placethetubeonthemagneticstandfor2minutes.

5.50 TRANSFERthe50μLofeluate(library)intoafresh1.5mllowbindtube.Safestoppingpoint.Librariescanbestoredat-20°Cforupto3months.


Step 6 – Library Quantitation using an intercalatingdsDNAfluorescentdyeDuration:~15minutes

RefertoWorkbook:LibraryquantitationtabIt 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)foryourlibraryinduplicateandthetwostandards.Vortexthestandardsandthelibraryfor5secondsandspinthemfor5seconds(quickspin).

6.2–Add995µL Bufferand5µLdyetoa1.5mlcentrifugetube.Vortexfor2secondsandspin

downfor5seconds(quickspin).

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,dilute10μLoftheSizeSelectedLibrarytoaconcentrationof4nMwithsterileH2O ina fresh1.5-mL lowbindmicrocentrifuge tube.StoretheremainingSizeSelectedLibraryat-20°C.

Safestoppingpoint.Librariescanbestoredat-20°Cforto3months.Incaseoflong-termstorage,re-quantificationofthelibraryishighlyrecommendedbeforerunningitontheMiSeq.


Step7–SequencingonIlluminaMiSeqDuration:~24hours

RefertoWorkbook:V3SequencingtabandV3SampleSheettabTheIlluminaMiSeqisanautomatedNGSinstrumentthatcansequencethesize-selectedlibraryprepared intheprevioussteps.De-multiplexingofthe indexedsamples isdoneautomaticallyfollowingcompletionofthesequencingrun.

Quick tip–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 IlluminaLibraryat4nM 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 96

Micro300Cycle(MS-103-1002) ~19 24 24 48 32 24

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-PreparetheMiSeqmachineaccordingtostandardIlluminaprotocols.7.2-Denaturethe4nMLibrary:Combine5μLoffreshlyprepared0.2NNaOHand5μLofthe4

nMDilutedSizeSelectedLibrary inanew1.5mLlowbindmicrocentrifugetube.Vortexfor 5 seconds and spindown for 5 seconds (quick spin). Incubate this 2nMDenaturedLibraryatroomtemperaturefor5minutes.

7.3 -Preparea20pMDenaturedLibrary:Add990μLofchilledHT1tothe10μLofthe2nM

DenaturedLibrary.Vortexfor5secondsandspindownfor5seconds(quickspin).7.4 -Preparea9pMDenaturedLibrary:Add550μLofchilledHT1and450μLof the20pM

DenaturedLibrarytoafresh1.5mLlowbindmicrocentrifugetube.Vortexfor5secondsandspindownfor5seconds(quickspin).

7.5 – Transfer 600μLof the9pMDenatured Library into the Load Samples reservoir of the

MiSeqreagentcartridge.

Quick tip– It isadvisable touse theprovidedworkbooktocreate theSampleSheet that is required by the Miseq. Ensure that the correct Adaptor Plateconfiguration(A1,A2,A3orA4)isselectedontheSequencingtabthatcontainstheappropriateindexedadaptorsequences.


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

1:4000 1:4000 1:4000


Appendix 1: Amplicon Quantitation using a qPCRinstrumentDuration:~40minutes

RefertoWorkbook:Ampliconquantitationtab

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×QuantiFluorDye


workingsolutionso 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. Vortexfor10secondstomixthoroughlyandspindownfor5seconds(quickspin).

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. Dilute DNA in the Amplicon Plates with molecular grade H2O so that the finalconcentrationofDNAisinarangeof50-100ng/μL.

§ IfDNAconcentrationisgreaterthan180ng/μL:add20μLofH2O§ IfDNAconcentrationis115-180ng/μL:add10μLofH2O§ IfDNAconcentrationislessthan115ng/μL:donotaddanyH2O


Appendix2:LibrarySizeSelectionUsingPippinPrepDuration:~1hour

RefertoWorkbook:LibrarypreparationtabThissteptakestheLibraryfromStep4,performspurificationwithAMPureXPbeadsandsizeselection using the Pippin Prep. The Pippin Prep can automatically select a range of DNAfragment sizes and elute them into a collection chamber. Note: Blue Pippin may be usedinsteadofthePippinPrep.

Quicktip–ForinstructionsonhowtosetupthePippinPrep,refertopage47.

ReagentlistItem Storage SuppliedbyPooledLibrary 4°C Step4AMPureXPbeads 4°C BeckmanCoulter80%Ethanol(freshlyprepared) 20°Cto25°C UserMoleculargradeH2O 20°Cto25°C User1.5%AgaroseGelCassette,DyeFree 20°Cto25°C SageSciencePippinloadingsolution/markermix(labeledK)

4°C SageScience

PooledLibrary 4°C Step4Note:MarkerKisusedwiththePippinPrep.TheBluePippinusesMarkerR2.

Protocol

LibraryPurification1.-AllowAMPureXPbeadstocometoroomtemperature.Ensuretheyarehomogeneous(noclumpsorpellets)byvortexingfor20seconds.2.-Preparefreshlymade5mLof80%ethanol(4mLEtOH+1mLH2O).3. -Add900μLofAMPureXPbeads to theLibrary tube.Mix thoroughlybyvortexing for10secondsandcentrifugefor5seconds(quickspin).Donotallowthebeadstoseparate.IncubatetheLibraryfor10minutesatroomtemperature.


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

4.-PlacetheLibrarytubeontoamagneticstandandincubatefor10minutes.5. – Keeping the tube on themagnetic stand, carefully remove and discard the supernatantfromtheLibrarytube,withouttouchingthebeads.6.-Keepingthetubeonthemagneticstand,add~1.5–2mLoffreshlyprepared80%ethanoltotheLibrarytube.Thevolumeofethanoladdedshouldbesufficienttocoverthebeads.

Note:Applytheethanoltothesideofthetubewithoutbeads.7. - Incubate the Library tube at room temperature for 30 seconds; afterwards, carefullyremoveanddiscardthesupernatant.8.-Repeatsteps6and7above.9.-Quicklyspindown(for2seconds)theLibrarytubeandplaceitbackonthemagneticstandwiththelidopen.Removeresidualethanolwithapipette.Donottouchthebeads.

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

10.-Allowthebeadstoairdryfor5-8minutesonthemagneticstanduntilthebeadpelletisdry.11. - Remove the Library tube from the magnetic stand and elute the Library with 31 μLmoleculargradewater.Donotletthepipettetiptouchthebeads,astheywillsticktoit.12.-VortextheLibraryfor20secondstofullyresuspendthebeads.Centrifugefor5seconds(quickspin)ifsomedropletsremainonthesidewalls.Ensurethebeadsremaininsuspension.13.-IncubatetheLibraryatroomtemperaturefor2minutes.14.-PlacetheLibrarytubeonthemagneticstandfor2minutes.15.-CollecttheLibrary:keepingtheFinalLibrarytubeinthemagneticstand,collect31μLofthesupernatantintoanew1.5mllowbindmicrocentrifugetube.


Safestoppingpoint.Librariescanbestoredat-20°Cforupto3months.

LibrarySizeSelection16.-BringtheMarkerKloadingsolutiontoroomtemperature.17.-Combine31μLofthePoolwith10μLofMarkerKloadingsolution.18.-Mixbyvortexingfor5secondsandspindownfor5seconds(quickspin).19.-ConfigurethePippinPreptocollectDNAfragmentsbetween650and1300bps.Loadthe40μLsampleintothesampleportandrun.Runtimeis45-50minutes.20.-Collectthewholecontent(approximately40μL)fromtheelutionportofthePippinPrepandtransferittoanew1.5mllowbindmicrocentrifugetube.Thisisthesize-selectedlibrary.

Safestoppingpoint.Librariescanbestoredat-20°Cforupto3months.


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 3: Library Quantitation using a qPCRinstrumentDuration:~1hour

RefertoWorkbook:LibraryquantitationtabItisnecessarytoquantifytheSize-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 6mL


2–PreparetheqPCRMasterMix.

qPCRMasterMix

Reagent Volume(μL)qPCRPrimerMix 264μLMoleculargradeH2O 88μLTotalVolume 352μL3-PrepareaserialdilutionoftheSizeSelectedLibrary.

a. Preparea1:1000dilutionbyadding1μLofSizeSelectedLibrary to999μLof1×TEbuffer(pH8.0),thoroughlyrinsingthepipettetip.Vortexfor5secondsandspindownfor5seconds(quickspin).

b. Prepare a 1:2000 dilution by adding 100 μL of the 1:1000 dilution to 100 μL 1× TEbuffer(pH8.0).Vortexfor5secondsandspindownfor5seconds(quickspin).

c. Preparea1:4000dilutionbyadding20μLofthe1:2000dilutionto20μL1×TEbuffer(pH8.0).Vortexfor5secondsandspindownfor5seconds(quickspin).

4-PrepareaqPCRQuantitationPlateinafreshPCRplatecompatiblewithyourqPCRsystem.5-Aliquot16μLoftheqPCRMasterMixintriplicateforstandards1-4,the1:1000dilution,the

1:2000andthe1:4000dilution(seesupplementalfigures).6 - Aliquot 4 μL of standards 1-4, the 1:1000 dilution, the 1:2000 dilution and the 1:4000

dilutionintothecorrespondingwells.7-SealtheqPCRQuantitationPlateandcentrifugeitfor10seconds.

Note: Avoid creating bubbles in the qPCRQuantitation Platewells. Centrifuge asneededtoeliminatebubbles.

8 Set the 1:1000, the 1:2000 and 1:4000 triplicates as targeted samples, and define the

standards (points: 4, starting concentration: 20pM, dilution: 1:10). Run the followingprogram on the qPCRmachine in order to determine the DNA concentration of the SizeSelectedLibrary:

NumberofCycles Temperature Time

1 95°C 5minutes25

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


9 - To convert the qPCR result from pM to nM concentration, enter the “Quantity mean”

resultsof the library replicates in theOmixonWorkbook tabcalled“LibraryQuantitation”.

10 - Using the volumes from the workbook, dilute 10 μL of the Size Selected Library to aconcentration of 4 nMwith sterile H2O in a fresh 1.5-mL low bindmicrocentrifuge tube.StoretheremainingSizeSelectedLibraryat-20°C.

Safestoppingpoint. Librariescanbestoredat -20°C forup to3months. Incaseoflong-term storage, re-quantification of the library is highly recommended beforerunningitontheMiSeq.

holotype hla™ 24/11 a1, a2, a3, a4 u m€¦ · sequencing (ngs), have provided new opportunities...

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