第三代测序技术及其应用-张得芳

ChinaBiotechnology2013335125-131*12112＊＊12100372210037。5RNA。SMRTtSMSQ523+．82013-01-152013-02-27*20130410231270711＊＊[e-mail]。1。PCRDNADNA23。4-6PCR。HelicosbiosciencestSMSTMtruesinglemolecularsequencingPacificBiosciencesSMRTsinglemoleculereal-timeLifeTechnologiesFRETIonTorrent、OxfordNanoporeNechnologies。。11．1HelicosBiosciencestSMSTM7-8Braslavsky9。1013'polyA1aF1polyTDNApolyT1b1ctotalinternalreflectionmicroscopyTIRM1d1e。、10。Cy5-5-、647nm。DOI10.13523/j.cb.20130520ChinaBiotechnologyVol．33No．520131Helicos、Fig．1Helicossingle-moleculesequencingsamplepreparationandimagingofsingle-nucleotideincorporationaImageseriesillustratingtemplate-specificbaseadditionsuccessfulrinsingandsuccessfullinkercleavagebPCRRNA。HelicostSMSTMRNA。HeliScope22596480035bp≤97%111。1．2HelicosBiosciencesPacificBiosciencesDNASMRT12。SMRTSMRT。SMRTzero-modewaveguidesZMW100nmZMW2aDNA。DNAZMW。ZMWExcitationdNTPZMWdNTP13-14。SMRTDNA、、、15。621201333516。PacificBiosciencesC11300bp。C23000bp3～4。C2Long2500bp95%ile6500bpX-Long3000bp95%ile8500bp。C2SMRT90MMappabledata。12SMRT12×90=1080Mb。SMRT16-17。、。。Longreads1。、、18-19。SMRT。。。SMRT。1．3OxfordNanoporeTechnologies2bα2b。DNA“”DNADNADNA20-22。CCDRNA。1。1Table1ThedifferentparameterscomparisonofthethirdgenerationsequencingtechnologiesReads///HelicostSMS25～45bp97%108d$1DNAPacBioSMRT1000～4500bp87%99%3．5-7．50．5-1h$2OxfordNanopore100kb96%---FRET＞1500bp＞99%-20min---IonTorrent200bp＞98%＞5002h$11．4FRETfluorescenceresonanceenergytransferFRETVisiGenBiotechnologies。DNADNAγFRETDNA721ChinaBiotechnologyVol．33No．520132c23-24。。。1．5IonTorrent。EmusionPCR。2d。ionsensitivelayerionsensorDNAH+DNA2d。、。2aPacificBiosciencesSMRTDNA．bTheOxford．cLifeTechnologiesFRET．dTheionTorrent2、。2．1SMRTContig25。Christophern260．5×PacBioRS38×NGSPacbioRSContig10。DavidA．27PacBioRSC22900bp99．998%。。523。20022008ElTorO11991。NGS28。QuakeHelico。448000。90%282470bp32bp2805471SNPIlluminaHuman610-QuadSNPBeadArray99．8%29。HarrisHelicosM13150×100%。DNAM13DNA。DNA。、30。2．2SMRTDNA82120133351。5-mC5-hmC5-mC。5-mC5-hmC。SMRT5-hmC5-hmC。DNAN6-、5-mC5-hmC31-32。2．3SNPPCRSNP。FLT3AML。FLT3FLT3—AML。PacBio3000bp1%33。2．4RNARNA34。Helicos355062．4Reads。HelicosDNARNAPolydTRNARNARNAcDNA36。PacBioRNAUemura37mRNA。RNA。RNA38。PacBioRSATPolyARNAPolyARNA。2．5polyXCGGCGG5-44。FRM1FRM1200XCGG。Sanger。UCDavisPacBioFMR1CGG10kbCGG75039。2．6PacBioRS/。40。。300。1000。41。。、。34。2．7。Scaffolds42。GCGC。。3921ChinaBiotechnologyVol．33No．52013。。43。1PopMSalzburgSL．Bioinformaticschallengesofnewsequencingtechnology．TrendsGenet．2008243142-149．2AcinasSGSarnmRRKlepac-CerajVetal．PCRinducedsequenceartifactsandbiasinsightsfromcomparisonoftwo16SrRNAclonelibrariesconstructedfromthesamesample．ApplEnvironMicrobiol200571128966-8969．3TortesTTMettaMOttenwlderBetal．Geneexpressionprofilingbymassivelyparallelsequencing．GenomeRes．2008181172-177．4DerringtonIMButlerTZCollinsMD．etal．NanoporeDNAsequencingwithMsp．A．Proc．NatlAcad．Sci．USA20101076060-6065．5LuanBPengHPolonskySetal．Base-By-BaseratchetingofsinglestrandedDNAthroughasolid-statenanopore．Phys．Rev．Lett．2010104238103．6EidJFehrAGrayJetal．Real-timeDNAsequencingfromsinglepolymerasemolecules．Science2009323133-138．7BowersJMitchellJBeerEetal．Virtualterminatornucleotidesfornext-generationDNAsequencing．Nat．Methods20096593-595．8TesslerLAReifenbergerJGMitraRD．Proteinquantificationincomplexmixturesbysolidphasesingle-moleculecounting．AnalChem2009817141-7148．9BraslavskyIHebertBKartalovEetal．SequenceinformationcanbeobtainedfromsingleDNAmolecules．ProcNatlAcadSciUSA．200310073960-3964．10HarrisTDBuzbyPRBabcockHetal．Single-moleculeDNAsequencingofaviralgenome．Science20083205872106-109．11OzsolakFPlattARJonesDRetal．DirectRNAsequencing．Nature20094617265814-818．12PacificBPacBioMPCAUSAonWorldWideWebURLhttp//．pacificbiosciences．com．13StephenTurner．SingleMoleculeRealTimeSMRTTMDNASequencingpacificbiosciences1-12．14http//．pacificbiosciences．com/products/smrt-technology15TraversKJChinCSRankDRetal．AflexibleandefficienttemplateformatforcircularconsensussequencingandSNPdetection．NuclAcidsRes．20103815159．16EidJFehrAGrayJetal．Real-timeDNAsequencingfromsinglepolymerasemolecules．Science20093235910133-138．17LeveneMJKorlachJTurnerSWetal．Zero-modewaveguidesforsingle-moleculeanalysisathighconcentrations．Science20032995607682-686．18ShendureJAPorrecaGJChurchGMetal．OverviewofDNAsequencingstrategiescurrentprotocolsinmolecularbiology2011chapter77．1．1-1．1．23．19SchadtEETurnerSandKasarskisA．Awindowintothird-generationsequencinghumanmoleculargenetics2010192227-240．20ClarkeJWuHCJayasingheLetal．Continuousbaseidentificationforsingle-moleculenanoporeDNAsequencing．Nat．Nanotechnol20094265-270．21HoworkaSCheleySBayleyH．Sequence-specificdetectionofindividualDNAstrandsusingengineerednanopores．Nat．Biotechnol．200119636-639．22StoddartDHeronAJMikhailovaEetal．Single-nucleotidediscriminationinimmobilizedDNAoligonucleotideswithabiologicalnanopore．Proc．NatlAcad．SciUSA20091067702-7707．23RahulRoySungchulHTaekjipH．Apracticalguidetosingle-moleculeFRET．NatureMethods．20085507-516．24HardinSGaoXLBriggsJetal．AmericaMethodsforreal-timesinglemoleculesequencedetermination．US7329492