Site Preparation and Training Guide v2.1 - Amazon S3Preparation+and... · Omixon Holotype HLA Site...

HOLOTYPEHLA™SITEPREPARATIONGUIDE

FORRESEARCHUSEONLY

V2.1

OmixonLimited

CONFIDENTIAL&PROPRIETARY.ALLRIGHTSRESERVED.

OmixonHolotypeHLASitePreparationandTrainingGuidev2.1.Forresearchuseonly.Copyright©2017,OmixonBiocomputingLtd.Confidential&Proprietary Page2│25

TableofContentsRECOMMANDATIONS...........................................................................................................................................3

DNAEXTRACTIONRECOMMANDATIONS............................................................................................................................3TECHNICALANDEQUIPMENTRECOMMENDATIONS..............................................................................................................3ASSOCIATEDREAGENTRECOMMENDATIONS.......................................................................................................................3

MiSeqReagentKitcapacity.................................................................................................................................4RECOMMENDEDSUPPLIES...............................................................................................................................................4

DOCUMENTHISTORY...........................................................................................................................................6LEGALNOTICE......................................................................................................................................................7PROTOCOLOVERVIEW(24/7CONFIGURATION)...................................................................................................8AMPLIFICATIONSUMMARY..................................................................................................................................9

ExpectedAmpliconSizes......................................................................................................................................9STEP0–GENOMICDNAPREPARATION..............................................................................................................10STEP1–HLAAMPLIFICATIONMASTERMIXPREPARATION................................................................................11STEP2–HLACLASSIANDIIAMPLIFICATION......................................................................................................12

ClassIAmplification(HLA-A,BandC)...............................................................................................................12ClassIIAmplification(HLA-DRB1/3,DRB4,DRB5,DPA1/DPB1,DQA1,DQB1set1andDQB1set2)................12

STEP3–AMPLICONQUANTITATIONANDNORMALIZATION..............................................................................14STEP4–LIBRARYPREPARATION........................................................................................................................16

FragmentationProgram....................................................................................................................................16EndRepairProgram...........................................................................................................................................16LigationProgram...............................................................................................................................................16

STEP5–LIBRARYSIZESELECTION.......................................................................................................................18STEP6–LIBRARYQUANTIFICATION...................................................................................................................19STEP7–SEQUENCINGONILLUMINAMISEQ......................................................................................................20STEP8–ANALYSISOFHLASEQUENCINGDATA..................................................................................................21ONSITEANDREMOTETRAINING........................................................................................................................22TECHNICALASSISTANCE.....................................................................................................................................25

PhoneSupport:..................................................................................................................................................25


Recommandations

DNAExtractionRecommandations§ Genomic DNA (gDNA) extracted from blood, saliva, buccal swabs, and hematopoietic

blood cells (B-cell lines, buffy coats, cord blood or any fraction of white blood cells)sufficientfor150ngofstartinginputforeachlong-rangePCRreaction,a260nm/280nmODratioof1.7-1.9,anda260nm/230nmODratiogreaterthan1.7.Eachsampleshouldrequiretotalinputof0.8-1.2µggDNAfor7lociand1.0-1.5µgofgDNAfor11loci.gDNAshouldbedissolved inwater if possible. EDTA inTEbuffer can inhibit long-rangePCRreactions.

TechnicalandEquipmentRecommendations§ Thermalcyclerwith96-wellformat§ Platefluorometer(oranyinstrumentcapableoffluorescencedetectionin96-wellplate

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

AssociatedReagentRecommendations§ LongRangePCRkitsfromQiagen(Cat#206401,206402or206403)

§ Eachsamplerequires4.4μLofLongRangeenzymefor11loci§ Cat#206401LongRangePCRkit(20)contains8μLofenzyme§ Cat#206402LongRangePCRkit(100)contains40μLofenzyme§ Cat#206403LongRangePCRkit(250)contains100μLofenzyme

§ ExoSAP-IT Express from Affymetrix (Cat# 75001-200, 75001-1ML, 75001-4X-1ML or75001-10ML)

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

§ QubitdsDNABRAssayKit(Cat#Q32850orQ32853)§ Cat#Q32850for100assays§ Cat#Q32853for500assays

§ Library Quantification Kit – Illumina/Universal from KAPA Biosystems (Cat# KK4824) -optionalifusingqPCRforlibraryquantitation

§ 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 80 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(optional)§ Platesealsforgeneraluse§ Platesealscompatiblewiththethermalcyclers(testedforlongrangePCR)§ OpticalplatesealscompatiblewiththeqPCRinstrument(optional)§ Magneticstandcompatiblewith2mLmicrocentrifugetubes§ 96-wellcoolerracks(2pieces)


§ 50mLconicaltubes§ 50mLreservoirs


DocumentHistoryVersion Date DescriptionofChanges1.1 January,2015 InitialVersion1.2 March,2015 X2-24/7,X2-96/7added1.3 June,2015 ExoSAP-ITStepChange,AmplificationSummaryadded,

removedX2-96/5andX4-16/51.4 July,2015 1.5 March2016 DPA1,DRB3,DRB4andDRB5locusinformationadded,

Miseqreagentkitcapacitytableadded2.0 January2017 Substitution of ExoSAP-IT with ExoSAP-IT Express,

additionofQubitinstrumentandreagentinformation2.1 November2017 UpdateoflocusconfigurationsandupdateofEndrepair

and Ligation reaction times according to the updatedHolotypeHLAkit


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 andproperlytrainedpersonnelinordertoensuretheproperandsafeuseoftheproduct(s)describedherein.Allofthecontentsofthisdocumentmustbefullyreadandunderstoodpriortousingsuchproduct(s).FAILURETOCOMPLETELYREADANDEXPLICITLYFOLLOWALLOFTHEINSTRUCTIONSCONTAINEDHEREINMAY RESULT INDAMAGE TO THE PRODUCT(S), INJURY TO PERSONS, INCLUDING TOUSERSOROTHERS,ANDDAMAGETOOTHERPROPERTY.OMIXON DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THEPRODUCT(S)DESCRIBEDHEREIN(INCLUDINGPARTSTHEREOFORSOFTWARE)ORANYUSEOFSUCHPRODUCT(S)OUTSIDETHESCOPEOFTHEEXPRESSWRITTENLICENSESORPERMISSIONSGRANTEDBYOMIXONINCONNECTIONWITHCUSTOMER'SACQUISITIONOFSUCHPRODUCT(S).FORRESEARCHUSEONLY©2017OmixonBiocomputingLtd.Allrightsreserved.


ProtocolOverview(24/11configuration)


AmplificationSummary

ExpectedAmpliconSizes

HLAlocus Expectedampliconsize(kb)HLA-A,BandC ~3HLA-DRB1/3 ~4.3HLA-DRB4 ~4.2HLA-DRB5 ~5HLA-DPA1 ~5.5HLA-DPB1 ~6.6HLA-DQA1 ~5.5

HLA-DQB1(Set3) ~6.5


Step0–GenomicDNAPreparationDuration:~45minutesTheHolotypeHLAassayisdesignedtoprovideaccuratesequencingdataoftheHLAregionusingtheIlluminaMiSeq.ToachievethebestresultswithHolotypeHLA,itisimportantthathighqualitygenomicDNAisused.DNAcanbe isolatedfromnumeroussources,butat least0.8-1.2µgofgenomic DNA for 7 loci and 1.0-1.5 µg of genomic DNA for 11 loci with a spectroscopicallydetermined260nm/280nmpuritybetween1.7and1.9anda260nm/230nmratiogreaterthan1.7isrequiredforeachsample.TheDNAcanbesetasideuntilthelocusspecificmastermixesarereadyfortheamplificationstep.

Protocol sub-steps § ExtractgDNAfromblood,saliva,buccalswabsorhematopoieticbloodcells(B-celllines,

buffycoats,cordbloodoranyfractionofwhitebloodcells).§ 0.8-1.2µgofinputgenomicDNAwitha260nm/280nmODratiobetween1.7–1.9anda

260nm/230nmratiogreaterthan1.7persampleisrecommended.

Equipment/Supplies § 1.5mLmicrocentrifugetubes§ DeviceformeasuringconcentrationandpurityofgenomicDNA§ Singlechannelmicropipettes§ Vortex§ Microcentrifuge


Step1–HLAAmplificationMasterMixPreparationDuration:~50minutesOmixonprovidesmostofthereagentsnecessaryforalong-rangePCRreactionintheHolotypeHLAkit,andtheremainingreagentsareincludedwiththeQiagenLong-RangePCRkit.Thelocus-specific mixes contain the necessary primers for amplification. The dNTPs and buffers arecombinedwiththelocusspecificprimers,forminganAmplificationMasterMixforeachlocus.ThecombinedHLAAmplificationMasterMixcanthenbestoreduntiltheactualamplificationisperformed.

Protocol sub-steps § CreateLocus-SpecificMasterMixes

Reagents § HolotypeHLALocus-SpecificPrimermixes§ QiagenLong-RangePCRkit

Equipment/Supplies § Singlechannelmicropipettes§ 1.5mLmicrocentrifugetubes§ Vortex§ Microcentrifuge


Step2–HLAClassIandIIAmplificationDuration:~6hours45minutesTheHLA Class I and II Amplification takes the genomic DNA and locus-specificmastermixesprepared in earlier steps and begins the process of amplifying the HLA loci. Two separateamplificationconditions,forHLAClassIandIIrespectively,areconcurrentlyrun.Anagarosegelverificationcanbedoneoptionally.

Protocol sub-steps § PCRAmplifications

§ 7reactionspersampleinthe24/7and96/7kits§ 10reactionspersampleinthe24/11and96/11kits

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-DRB1/3,DRB4,DRB5,DPA1,DPB1,DQA1,DQB1set3)NumberofCycles Temperature Time

1 95°C 3minutes 93°C 15seconds

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

Reagents

HolotypeHLALocus-SpecificPrimerMasterMixesfromStep1

Equipment/Supplies § 96-wellPCRplates§ Singlechannelmicropipettes§ Multichannelmicropipettes§ Thermalseal§ Centrifugefor96-wellPCRplates§ Thermalcyclerswith96-wellplatecapacity


§ 3xthermalcyclersforthe24/7configuration§ 7xthermalcyclersforthe96/7configuration§ 3xthermalcyclersforthe24/11configuration§ 10xthermalcyclersforthe96/11configuration


Step3–AmpliconQuantitationandNormalizationDuration:~35minutesOncesampleshavebeensuccessfullyamplified,aninitialdilutionstepisnecessarytohaveanoptimumconcentrationofDNAforlibrarypreparation.TheconcentrationisdeterminedusingastandardplatefluorometerorqPCRmachineandtheQuantiFluordsDNAkitfromPromega.DNAis then diluted, pooled per sample, and then purified using the ExoSAP-IT Express kit fromAffymetrix.ThepooledDNAsamplesarethenreadyforlibrarypreparation.

Protocol sub-steps § Ampliconconsolidation§ AmpliconquantitationwithaplatefluorometerorqPCRmachine§ Amplicondilution§ Ampliconpooling§ Ampliconclean-upusingExoSAP-ITExpress

ExoSAP-ITExpressPCRPurification

Reagents § QuantiFluordsDNASystem§ 1xTEBuffer§ ExoSAP-ITExpress

Equipment/Supplies § Multichannelmicropipettes§ Singlechannelmicropipettes§ 1.5mlmicrocentrifugetubes§ Vortex§ Microcentrifuge§ Centrifugefor96-wellPCRplates§ Pipettingreservoir§ Serologicalpipettes§ 50-mlfalcontubes§ 96-wellopticalbottomplates(forplatefluorometer)§ qPCR96-wellPCRplates(forqPCRmachine)§ Opticalplateseals(forqPCRmachine)§ 96-wellstandardPCRplates

NumberofCycles Temperature Time1 37°C 4minutes1 80°C 1minute1 4°C ∞


§ Standardplateseals§ Platefluorometer§ qPCRinstrument§ Thermalcyclerswith96-wellplatecapacity


Step4–LibraryPreparationDuration:~3hours20minutesLibrarypreparationisasequenceofreactionsrunagainstthepooledamplicons.Fragmentationoftheampliconsisimmediatelyfollowedbyendrepair.Theampliconsarethentransferredintoa 96-well plate preloaded with adaptors compatible with the Illumina MiSeq system. Theampliconsandadaptorsarethenligatedtogether.Theligatedfragmentsarepooledtogether,purified,andconcentratedusingAMPureXPbeads.

Protocol sub-steps § Poolingsamplelocitogether§ Enzymaticfragmentationofamplicons§ Endrepairofamplicons§ Ligationofadaptorstotheamplicons§ Poolingoftheadaptorligatedampliconsintoalibrary§ AMPureXPbeadpurificationandconcentrationofthelibrary

FragmentationProgram

NumberofCycles Temperature Time1 37°C 10minutes1 70°C 15minutes1 4°C ∞

EndRepairProgram


LigationProgram


Reagents § HolotypeHLALibraryPreparationReagents§ AMPureXPbeads§ Ethanol


Equipment/Supplies§ Multichannelmicropipettes§ 96-wellPCRplates§ Thermalseal§ Thermalcycler§ 1.5mLmicrocentrifugetubes§ 1.5mLlow-bindmicrocentrifugetubes§ 2.0mLlow-bindmicrocentrifugetubes§ Vortex§ Microcentrifuge§ Centrifugefor96-wellPCRplates§ Singlechannelmicropipettes§ 2×frozenthermalrackscompatiblewith96-wellPCRplates§ Magnetictuberackforbeadseparation


Step5–LibrarySizeSelectionDuration:~1hourLibrarypreparationcangeneratenumerousfragmentsofvaryingsizes.ThePippinPrepfromSageScienceefficientlyselectsforfragmentsoftheappropriatesize,eliminatingtheneedforagelexcision and cleanup. The size-selected library can then be prepared for sequencing on theIlluminaMiSeq.

Protocol sub-steps § SizeselectionofthelibraryusingaPippinPreporBluePippin

Reagents § PippinPrepMarkerK(BluePippinMarkerR2)§ PippinPrepElectrophoresisBuffer

Equipment/Supplies§ Singlechannelmicropipettes§ PippinPrepwith1.5%dyefreeagarosecassette§ 1.5mLlow-bindmicrocentrifugetube§ Vortex§ Microcentrifuge


Step6–LibraryQuantificationDuration:~15minutesThefinalstepbeforerunningthesamplelibraryonanIlluminaMiSeqistodeterminethenumberofampliconssuccessfully ligatedtoadaptors.Theamountof ligatedadaptorsdeterminestheidealdilutionnecessaryforsequencingontheIlluminaMiSeq.Iftheconcentrationofampliconsistoolow,theremaybeallelicdropoutduringMiSeqanalysis.Iftheconcentrationofampliconsistoohigh,therewillbeexcessivenoiseintheMiSeqanalysis.LibraryquantificationcanbedoneusingaQubitandtheQubitBRAssaykit.Alternatively,qPCRwiththeKAPABiosystemsLibraryQuantificationkitcanbeused.

Protocol sub-steps § QubitBRassayreagentpreparation§ Librarydilution§ Qubitquantification

Reagents § QubitBRAssayKit

Equipment/Supplies§ QubitFluorometer§ Singlechannelmicropipettes§ 0.5mLmicrocentrifugetubes


Step7–SequencingonIlluminaMiSeqDuration:~19hoursTheIlluminaMiSeqhasbeendesignedtoautomatetheprocessofsequencinglargeamountsofDNAinarelativelyshortamountoftime.ThemostcriticalaspectsofoperatinganIlluminaMiSeqareperformingthenecessarywashesandmaintenancetokeepthedevicerunningingoodorder.ThenecessarystepsbeforeloadingyourlibraryontotheMiSeqinvolveaseriesofwashestoflushoutanyremainingcontaminationfromearlierruns,andmakingsurethattheflowcellisascleanaspossible.Once thesestepshavebeendone,a reagentcartridgewith thepreparedsamplelibraryisloadedandafreshlycleanedflowcellisinsertedintothedevice.

Protocol sub-steps § Librarydenaturationanddilution§ IlluminaMiSeqpreparation

Reagents § AnyIlluminaMiSeqReagentKitv2(dependingonsamplevolumeanddesiredTAT–300

cyclekitsrecommended)o 300-CycleNanoFlowCello 300-CycleMicroFlowCello 300-CycleStandardFlowCello 500-CycleNanoFlowCello 500-CycleStandardFlowCell

§ MiliQH2O§ Sodiumhydroxide

Equipment/Supplies§ Singlechannelmicropipettes§ Kimwipes§ Lenspaper§ IlluminaMiSeq§ 1.5mLlow-bindmicrocentrifugetubes§ Microcentrifuge


Step8–AnalysisofHLASequencingDataAfter sequencing theprovidedsamples, the IlluminaMiSeqautomatically createsa fastq file.When configured correctly by your bioinformaticians or IT admin, HLA Twin (the softwareincluded with Holotype HLA) automatically begins running the genotyping analysis with twoorthogonal algorithms. Please refer to the HLA Twinmanual for assistance with the correctinstallation of HLA Twin and for information on interpreting the genotyping analysis of yoursequencingdata.

Protocol sub-steps § DatatransferfromMiSeqtoanalysiscomputer§ AnalysisofdatausingHLATwin

Equipment/Supplies § 64-bitcomputerwithminimum4Coresand16GBofRAM§ Long-termstorageof2TBperMiSeqperyear


OnsiteandRemoteTrainingOmixonprovidesavarietyoftrainingoptionstousersofHolotypeHLAandHLATwin.AlltrainingoptionsinvolveaHolotypeHLAandHLATwintrainingseminar.Theseminarsareprovidedeitheron-site or remotely using aweb conference. Laboratories have different training needs, andOmixonprovidesa5,3,or1daytrainingtomeetthoseuniquerequirements.Beforetrainingcanbeginhowever,itisimportanttofollowoursitepreparationguidetooptimizeourtimetogetherwith you. The training schedule and site preparation guide are guidelines, andwe advise allcustomerstoworkwithsupport@omixon.cominordertotailortrainingtoyourlaboratory.

SitePreparationInadditiontohavingthelabsuppliesdescribedinSteps1through8onhand,yourlabshouldalsohavethefollowingprepared:

§ Makesureyourinstruments(platefluorometer,qPCRsystem,IlluminaMiSeq,fileserver)areconnectedtoalocalnetwork.

§ Necessaryinstrumentsareavailableandingoodworkingorder.§ Thermalcyclerprogramsareinputandsavedontothedevice.§ Quantifluortemplateshavebeencreatedforyourplatefluorometer.§ PippinPrepprogramsareinputandsavedontothedevice.§ KapalibrarypreparationkittemplateshavebeencreatedforyourqPCRsystem.§ TheMiSeqhasperformed2post-runwashes.§ HLATwinhasbeensuccessfullyinstalled.


5-day Training (highly recommended) Time Day1 Day2 Day3 Day4 Day58:00 Amplicon

quantitationSoftwaretraining

Ampliconquantitation

Softwaretraining

9:00 ExoSAPPurificationFragmentation

ExoSAPPurificationFragmentation

10:00 Pre-trainingpresentation

End-repair End-repair

11:00 Worksheetdemonstration

LigationandPooling

Questionandanswer

LigationandPooling

Questionandanswer

12:00 Lunchbreak AMPureXPconcentration

Lunchbreak AMPureXPconcentration

Lunchbreak

13:00 Equipmentandreagentcheck

PippinsizeselectionLunchbreak

PippinsizeselectionLunchbreak

Softwaretraining

14:00 gDNAdilutionandprimermixformulation

Qubit(orqPCR–optional)

gDNAdilutionandPrimerMixformulation

Qubit(orqPCR–optional)

15:00 LR-PCRamplification

LoadingMiSeq LR-PCRamplification

LoadingMiSeq

3-day Training (recommended) Time Day1 Day2 Day38:00 Ampliconquantitation Softwaretraining9:00 ExoSAPpurification

Fragmentation

10:00 Pre-trainingpresentation End-repair 11:00 Worksheetdemonstration Ligationandpooling Questionandanswer12:00 Lunchbreak AMPureXPconcentration Lunchbreak13:00 Equipmentandreagent

checkPippinSizeselectionLunchbreak

Softwaretraining

14:00 gDNAdilutionandprimermixformulation

Qubit(orqPCR-optional)

15:00 LR-PCRamplification LoadingMiSeq


1-day Training (by special request only) Time PrepDay Day18:00 Ampliconquantitation9:00 ExoSAPPurification,Fragmentation10:00 End-repair11:00 Ligationandpooling12:00 AMPureXPconcentration13:00 Pippinsizeselection

Lunchbreak14:00 Qubit(orqPCR–optional)15:00 gDNA dilution, primer mix formulation,

andLR-PCRamplificationLoadingMiSeq


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

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

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