Next-Generation Sequencing Technologies

NHGRICurrentTopicsinGenomeAnalysis2012Week6:Next-GenerationSequencingTechnologiesFebruary22,2012ElaineMardis,Ph.D.1  theGenomeatWashingtonUniversityInstitute©ElaineR.MardisElaineR.MardisProfessorinGeneticsCo-director,TheGenomeInstituteNext-GenerationSequencingTechnologiesNHGRICurrentTopicsinGenomeAnalysis©ElaineR.MardisCurrentTopicsinGenomeAnalysis2012 ElaineMardis,Ph.D. NoRelevantFinancialRelationshipswithCommercialInterests NHGRICurrentTopicsinGenomeAnalysis2012Week6:Next-GenerationSequencingTechnologiesFebruary22,2012ElaineMardis,Ph.D.2  ©ElaineR.MardisOverview• Next-GenerationSequencing(NGS)Instruments• Roche/454• Illumina• LifeTechnologies• PacificBiosciences• IonTorrent• OxfordNanopore• NGSApplicationsacrossthespectrumofgenomics• Examplesfromourwork• FutureDirections©ElaineR.Mardis©ElaineR.MardisTheTrajectoryofThroughput:10yearsE.R.Mardis,Nature(2011)470:198-203NHGRICurrentTopicsinGenomeAnalysis2012Week6:Next-GenerationSequencingTechnologiesFebruary22,2012ElaineMardis,Ph.D.3  ©ElaineR.MardisCapillarytechnologyAppliedBiosystems3730xl(2004)$15,000,000Next-gentechnologyIlluminaHiSeq(2011)$10,000Comparativecosts:sequencingahumangenome©ElaineR.MardisNext-generationSequencerbasicsPlatformsandtheirattributesNHGRICurrentTopicsinGenomeAnalysis2012Week6:Next-GenerationSequencingTechnologiesFebruary22,2012ElaineMardis,Ph.D.4  ©ElaineR.MardisNext-generationDNAsequencinginstruments• Allcommercially-availablesequencershavethefollowingsharedattributes:• RandomfragmentationofstartingDNA,ligationwithcustomlinkers=“alibrary”• Libraryamplificationonasolidsurface(eitherbeadorglass)• Directstep-by-stepdetectionofeachnucleotidebaseincorporatedduringthesequencingreaction• Hundredsofthousandstohundredsofmillionsofreactionsimagedperinstrumentrun=“massivelyparallelsequencing”• Shorterreadlengthsthancapillarysequencers• A“digital”readtypethatenablesdirectquantitativecomparisons• Asequencingmechanismthatsamplesbothendsofeveryfragmentsequenced(“pairedend”reads)©ElaineR.MardisPaired-endreads• Allnext-genplatformsnowofferpairedendreadcapability,e.g.sequencescanbederivedfrombothendsofthelibraryfragments.• Differencesexistinthe_distance_betweenreadpairs,basedontheapproach/platform.• “pairedends”:linearfragmentsequencedatbothendsintwoseparatereactions• “matepairs”:circularizedfragmentof1kb,sequencedbyasinglereactionreadorbytwoseparateendreads(platformdependent)• Ingeneral,pairedendreadsofferadvantagesforsequencinglargeandcomplexgenomesbecausetheycanbemoreaccuratelyplaced(“mapped”)thancansingleendedshortreads.NHGRICurrentTopicsinGenomeAnalysis2012Week6:Next-GenerationSequencingTechnologiesFebruary22,2012ElaineMardis,Ph.D.5  ©ElaineR.MardisRoche/454LibraryConstructionandemPCRSingleStrandedAdapterLigatedLibrary• RandomFragmentation• AdapterLigation• emPCRforclonalamplificationonbead©ElaineR.MardisRoche/454PyrosequencingDNACaptureBeadContainingMillionsofCopiesofaSingleClonalFragmentAATCGGCATGCTAAAAGTCAAnnealPrimerTPPiATPLight+oxyluciferinSulfurylaseLuciferaseAPSluciferinCentrifugationLoadEnzymeBeadsLoadbeadsintoPicoTiter™PlateSequencingbySynthesisNHGRICurrentTopicsinGenomeAnalysis2012Week6:Next-GenerationSequencingTechnologiesFebruary22,2012ElaineMardis,Ph.D.6  ©ElaineR.Mardis454InstrumentationInstrumentRunTime(hr)ReadLength(bp)Yield(Mb/run)ErrorTypeErrorRate(%)PurchaseCost(x1000)454FLX+18-20700900Indel1$30A454FLXTitanium10400500Indel1$500454GSJr.Titanium1040050Indel1$108A–Requiresthe454FLXTitanium.Thisistheupgradecost.Notable:• Matepairpairedendreadsof3kb,8kband20kbseparationwithoutanincreaseinruntime.• Costperrunmakessequencinganentirehumangenomecost-prohibitiverelativetoothertechnologies(~$20/Mbp)• Greatplatformfortargetedvalidation©ElaineR.MardisIlluminaSequencing:LibraryPreparationNHGRICurrentTopicsinGenomeAnalysis2012Week6:Next-GenerationSequencingTechnologiesFebruary22,2012ElaineMardis,Ph.D.7  ©ElaineR.MardisIlluminaSequencingbySynthesisExcitationEmissionIncorporateDetectDe-blockCleavefluor©ElaineR.MardisIlluminaInstrumentationInstrumentRunTime(days)ReadLength(bp)Yield(Gb/run)ErrorTypeErrorRate(%)PurchaseCost(x1000)GAIIx14150x15096Sub0.1$525HiSeq20008100x100200x2Sub0.1$700HiSeq2000v310100x100600Sub0.1$700MiSeq1150x1502Sub0.1$125• 2010:HiSeq2000• Twoflowcellsperrun• 100Gbp/FCortwogenomeequivalentsperrun• Newscanningmechanics-scansbothsurfacesofFClanes• 2011:HiSeq2000• Improvedchemistry(v.3):increasedyieldandaccuracy• 2011:MiSeqNHGRICurrentTopicsinGenomeAnalysis2012Week6:Next-GenerationSequencingTechnologiesFebruary22,2012ElaineMardis,Ph.D.8  ©ElaineR.MardisLifeTechnologies:sequencingbyligation• customadapterlibrary• emPCRonmagneticbeads• sequencingbyligationusingfluorescentprobesfromacommonprimer• sequentialroundsofligationfromaseriesofprimers• fixed/knownnucleotidesforeachprobesetidentifytwobaseseachcycle,or“twobaseencoding”©ElaineR.MardisSOLiDInstrumentationInstrumentRunTime(days)ReadLength(bp)Yield(Gb/run)ErrorTypeErrorRate(%)PurchaseCost(x1000)SOLiD41250x35PE71A-TBias0.06$475SOLiD5500xl875x35PE60x60MP155A-TBias0.01$59