高通量测序技术及原理介绍

高通量测序技术及原理介绍童贻刚军事医学科学院微生物流行病研究所[e-mail]公司系统名测序长度优点缺点Roche/454FLXSystem200_700读长最长；通量高同聚性错误；仪器和试剂价格贵IlluminaHiSeq2000/miSeq2x150通量非常高价格贵；后期分析复杂ABI/SOLiD5500xlSOLiD25_35通量高；试剂消耗少读长太短HelicosHeliScope25_30通量高读长太短1415测序平台测序长度进化过程产出测序时间SOLiD30bp15bp30G10天SolexaHiseq2000150bpX230bp，50bp，75bp，100bp600G14天454750bp100bp，400bp0.7G7小时37301000bpX96300bp，600bp0.0001G2小时IlluminaworkflowSamplepreparationShearing,ligateadapterClustergenerationBridgePCRSequencingonGenomeAnalyzerIIxRTA(RunTimeAnalysis)AnalysispipelineOfflineanalysis,alignment,SNPscalling,readscountingVisualizethedata,reportstheresultsSequencingprocessFragmentDNARepairends/AddAoverhangLigateadaptersSelectligatedDNAHybridizetoflowcellExtendhybridizedoligosPerformbridgeamplificationPerformsequencingonforwardstrandRe-generatereversestrandPerformsequencingonreversestrandCONFIDENTIAL–DONOTDISTRIBUTE1Libraryprep(~6hrs)2AutomatedClusterGeneration(~5hrs)1-8samples3Sequencing(~46to120hrs)1-8samplesSamplePrep-ResequencingSurfaceboundadapter1SequencingprimerbindingsiteSurfaceboundadapter2CONFIDENTIAL–DONOTDISTRIBUTECONFIDENTIAL–DONOTDISTRIBUTE•Clonalclustersaregeneratedinacontainedenvironment(neednocleanrooms)•SequencingalsoperformedintheflowcellonthegeneratedclustersFlowcell8channelsKeytothesimplifiedworkflowSurfaceofflowcellcoatedwithalawnofoligopairsClustergeneration:Hybridizefragment&extendAdaptersequence50MsinglemoleculeshybridizetothelawnofprimersBoundmoleculesarethenextendedbypolymerases3’extensionCONFIDENTIAL–DONOTDISTRIBUTEDouble-strandedmoleculeisdenatured.Originaltemplateiswashedaway.Newlysynthesizedcovalentlyattachedtotheflowcellsurface.CONFIDENTIAL–DONOTDISTRIBUTEClustergeneration:Denaturedouble-strandedDNANewlysynthesizedstrandOriginaltemplatediscardClustergeneration:CovalentlyboundspatiallyseparatedsinglemoleculesSinglemoleculesboundtoflowcellinarandompatternCONFIDENTIAL–DONOTDISTRIBUTEClustergeneration:BridgeamplificationSingle-strandflipsovertohybridizetoadjacentprimerstoformabridge.Hybridizedprimerisextendedbypolymerases.CONFIDENTIAL–DONOTDISTRIBUTEClustergeneration:Bridgeamplificationdouble-strandedbridgeisformed.CONFIDENTIAL–DONOTDISTRIBUTEClustergeneration:BridgeamplificationDouble-strandedbridgeisdenatured.Result:Twocopiesofcovalentlyboundsingle-strandedtemplates.CONFIDENTIAL–DONOTDISTRIBUTEClustergeneration:BridgeamplificationSingle-strandsflipovertohybridizetoadjacentprimerstoformbridges.Hybridizedprimerisextendedbypolymerase.CONFIDENTIAL–DONOTDISTRIBUTEClustergeneration:BridgeamplificationBridgeamplificationcyclerepeatedtillmultiplebridgesareformedCONFIDENTIAL–DONOTDISTRIBUTEClustergenerationdsDNAbridgesdenatured.Reversestrandscleavedandwashedaway.CONFIDENTIAL–DONOTDISTRIBUTEClustergeneration…leavingaclusterwithforwardstrandsonly.CONFIDENTIAL–DONOTDISTRIBUTEClustergenerationFree3’endsareblockedtopreventunwantedDNApriming.CONFIDENTIAL–DONOTDISTRIBUTECONFIDENTIAL–DONOTDISTRIBUTEhybridizedtoadaptersequence.SequencingSequencingprimerisSequencingprimerAdd4Fl-NTP’s+PolymeraseIncorporatedFl-NTPisimagedTerminatorandfluorescentdyearecleavedfromtheFl-NTPX36CONFIDENTIAL–DONOTDISTRIBUTESequencingprimerFlowcellimagingTotalInternalReflectionFluorescenceFluidicsportFlowcellPrismFluidicsportCONFIDENTIAL–DONOTDISTRIBUTECONFIDENTIAL–DONOTDISTRIBUTEPairedendsequencingSequencedstrandstrippedoff3’-endsunblockedPairedendsequencingBridgeformation3’extensionCONFIDENTIAL–DONOTDISTRIBUTEPairedendsequencingDoublestrandedDNAisdenaturedCONFIDENTIAL–DONOTDISTRIBUTEPairedendsequencing3’endsareblockedOriginalforwardstrandiscleavedCONFIDENTIAL–DONOTDISTRIBUTEAdd4Fl-NTP’s+PolymeraseIncorporatedFl-NTPisimagedTerminatorandfluorescentdyearecleavedfromtheFl-NTPX36-50CONFIDENTIAL–DONOTDISTRIBUTESequencingreversestrandHybridizesequencingprimerSolexaFlowcellinGAIIxCONFIDENTIAL–DONOTDISTRIBUTEImagere-analysispiplelineImageAnalysisBasecallingSequenceAnalysisGAAnalysisPipelineInstrumentPCAnalysisPC/clusterdatatransferImages(.tif)Lane1..8Cycle1..36Tile_Cycle_Image_a,Tile_Cycle_Image_c,Tile_Cycle_Image_g,Tile_Cycle_Image_t.paramsfileForeachtile:•Clusterintensities•ClusternoiseForeachtile:•Correctedclusterintensities•Clustersequence•ClusterprobabilitiesForalldata:•QualityFiltering•SequenceAlignment•RunStatisticsVisualizationCONFIDENTIAL–DONOTDISTRIBUTEBustard•BasewithhighestcorrectedintensityiscalledACGTCGerald•IAIA+IBGEnerationofRecursiveAnalysesLinkedbyDependencyIAIBFilteringremoveslowqualitybasecallsChastity:C=Defaultvalue0.6Otherfiltersincludepurity,similarity,neighborandneighborhood.CONFIDENTIAL–DONOTDISTRIBUTEBustardoutput*_qseq.txtMachinenameRunnumberLanenumberTilenumberXcoordYcoordSequenceQualityPassedFilterIndexReadformatEAS189159111525AACCTT2TGACCAGCGTCAACCAGTACTACGTCTTTGTCGATAGaaaa`]`a`_`[V_\O[YO\^Z^\Z^^`YU[\PJZRX1EAS189159111726AACCTT2TCTGGATGAAGAACGATCCGCTGCAGAGGTGCTGGCA_`_]``^^FNXX^ZWFZ_]YYTYMUVBBBBBBBBBBB0EAS189159111860AACCTT2TATCGCGTAGTGTAGCACGGCCTTTTTTTCGTCCACCaa`a[`\XFU[WQ`UH[]VN]_ZR]W]ZZXFWYFTX]1EAS189159112377AACCTT2TTTTCTTCTCCTTCGCCATCAGCGACAAAATC