高通量测序技术及原理介绍ppt课件

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tong.yigang,14,15,Illuminaworkflow,SamplepreparationShearing,ligateadapterClustergenerationBridgePCRSequencingonGenomeAnalyzerIIxRTA(RunTimeAnalysis)AnalysispipelineOfflineanalysis,alignment,SNPscalling,readscountingVisualizethedata,reportstheresults,Sequencingprocess,FragmentDNARepairends/AddAoverhangLigateadaptersSelectligatedDNAHybridizetoflowcellExtendhybridizedoligosPerformbridgeamplificationPerformsequencingonforwardstrandRe-generatereversestrand,PerformsequencingonreversestrandCONFIDENTIALDONOTDISTRIBUTE,1Libraryprep(6hrs)2AutomatedClusterGeneration(5hrs)1-8samples3Sequencing(46to120hrs)1-8samples,SamplePrep-ResequencingSurfaceboundadapter1SequencingprimerbindingsiteSurfaceboundadapter2CONFIDENTIALDONOTDISTRIBUTE,CONFIDENTIALDONOTDISTRIBUTE,Clonalclustersaregeneratedinacontainedenvironment(neednocleanrooms)Sequencingalsoperformedintheflowcellonthegeneratedclusters,Flowcell8channelsKeytothesimplifiedworkflow,Surfaceofflowcellcoatedwithalawnofoligopairs,Clustergeneration:Hybridizefragment&extendAdaptersequence50MsinglemoleculeshybridizetothelawnofprimersBoundmoleculesarethenextendedbypolymerases3extensionCONFIDENTIALDONOTDISTRIBUTE,Double-stranded,moleculeisdenatured.Originaltemplateiswashedaway.,Newlysynthesizedcovalentlyattachedtotheflowcellsurface.CONFIDENTIALDONOTDISTRIBUTE,Clustergeneration:Denaturedouble-strandedDNANewly,synthesized,strand,Originaltemplatediscard,Clustergeneration:CovalentlyboundspatiallyseparatedsinglemoleculesSinglemoleculesboundtoflowcellinarandompatternCONFIDENTIALDONOTDISTRIBUTE,Clustergeneration:BridgeamplificationSingle-strandflipsovertohybridizetoadjacentprimerstoformabridge.Hybridizedprimerisextendedbypolymerases.CONFIDENTIALDONOTDISTRIBUTE,Clustergeneration:Bridgeamplificationdouble-strandedbridgeisformed.CONFIDENTIALDONOTDISTRIBUTE,Clustergeneration:BridgeamplificationDouble-strandedbridgeisdenatured.Result:Twocopiesofcovalentlyboundsingle-strandedtemplates.CONFIDENTIALDONOTDISTRIBUTE,Clustergeneration:BridgeamplificationSingle-strandsflipovertohybridizetoadjacentprimerstoformbridges.Hybridizedprimerisextendedbypolymerase.CONFIDENTIALDONOTDISTRIBUTE,Clustergeneration:BridgeamplificationBridgeamplificationcyclerepeatedtillmultiplebridgesareformedCONFIDENTIALDONOTDISTRIBUTE,ClustergenerationdsDNAbridgesdenatured.Reversestrandscleavedandwashedaway.CONFIDENTIALDONOTDISTRIBUTE,Clustergenerationleavingaclusterwithforwardstrandsonly.CONFIDENTIALDONOTDISTRIBUTE,ClustergenerationFree3endsareblockedtopreventunwantedDNApriming.CONFIDENTIALDONOTDISTRIBUTE,CONFIDENTIALDONOTDISTRIBUTE,hybridizedtoadaptersequence.,SequencingSequencingprimeris,Sequencingprimer,Add4Fl-NTPs+Polymerase,IncorporatedFl-NTPisimaged,TerminatorandfluorescentdyearecleavedfromtheFl-NTP,X36CONFIDENTIALDONOTDISTRIBUTE,Sequencingprimer,FlowcellimagingTotalInternalReflectionFluorescenceFluidicsportFlowcellPrismFluidicsportCONFIDENTIALDONOTDISTRIBUTE,CONFIDENTIALDONOTDISTRIBUTE,PairedendsequencingSequenced,strandstrippedoff,3-endsunblocked,PairedendsequencingBridgeformation3extensionCONFIDENTIALDONOTDISTRIBUTE,PairedendsequencingDoublestrandedDNAisdenaturedCONFIDENTIALDONOTDISTRIBUTE,Pairedendsequencing3endsareblockedOriginalforwardstrandiscleavedCONFIDENTIALDONOTDISTRIBUTE,Add4Fl-NTPs+Polymerase,IncorporatedFl-NTPisimaged,TerminatorandfluorescentdyearecleavedfromtheFl-NTP,X36-50CONFIDENTIALDONOTDISTRIBUTE,SequencingreversestrandHybridizesequencingprimer,Solexa,FlowcellinGAIIx,CONFIDENTIALDONOTDISTRIBUTE,Imagere-analysispipleline,ImageAnalysisBasecallingSequenceAnalysis,GAAnalysisPipeline,InstrumentPC,AnalysisPC/cluster,datatransfer,Images(.tif)Lane1.8Cycle1.36Tile_Cycle_Image_a,Tile_Cycle_Image_c,Tile_Cycle_Image_g,Tile_Cycle_Image_t.paramsfile,Foreachtile:ClusterintensitiesClusternoiseForeachtile:CorrectedclusterintensitiesClustersequenceClusterprobabilitiesForalldata:QualityFilteringSequenceAlignmentRunStatisticsVisualization,CONFIDENTIALDONOTDISTRIBUTE,Bustard,Basewithhighestcorrectedintensityiscalled,A,C,G,T,C,Gerald,IAIA+IB,GEnerationof,RecursiveAnalysesLinkedby,Dependency,IAIB,FilteringremoveslowqualitybasecallsChastity:C=,Defaultvalue0.6Otherfiltersincludepurity,similarity,neighborandneighborhood.CONFIDENTIALDONOTDISTRIBUTE,Bustardoutput*_qseq.txt,Machinename,Runnumber,Lanenumber,Tilenumber,Xcoord,Ycoord,Sequence,Quality,PassedFilter,Index,Readformat,EAS189159111525AACCTT2TGACCAGCGTCAACCAGTACTACGTCTTTGTCGATAGaaaaa_V_OYOZZYUPJZRX1EAS189159111726AACCTT2TCTGGATGAAGAACGATCCGCTGCAGAGGTGCTGGCA_FNXXZWFZ_YYTYMUVBBBBBBBBBBB0EAS189159111860AACCTT2TATCGCGTAGTGTAGCACGGCCTTTTTTTCGTCCACCaaaXFUWQUHVN_ZRWZZXFWYFTX1EAS189159112377AACCTT2TTTTCTTCTCCTTCGCCATCAGCGACAAAATCAAGCAabbbabbbbbbaaaTaaaaaY_YNaZZ1EAS189159112538AACCTT2TGTGAATTAACAGTATTGGCGTAGTTACAGGCAGTGTaa_aabbaaa_aSYZYUBBBBB1EAS189159112576AACCTT2TCTCCTTCGTCTTCTTCCATCAGTTGTTCGACCGGCTGJRNGBBBBBBBBBBBBBBBBBBBBBBBBBBBBB0EAS189159112607AACCTT2TCCACCATCAACTGGTTGCCAGTGCGCGGGCAGTTAAaabaaaaaaX_YTTHTTZQYTX1EAS189159112255AACCTT2TGATGCTGATAAGCAGCGTGCTCACAACCCAGATTTGaaba_abaabbbbabababbbbb_aba_Zabbb1,Fastqformat,http:/www.bioinformatics.babraham.ac.uk/projects/fastqc/,GERALDsequence,Summary.html,(PF:passfilter),FastQC,Thirdpartsoftware,BriefBioinform.2011Jan18,NGS技术论坛,SEQwiki:,（荧光基团漂白）,捕获技术,Paired-endvs.Mate-Pair,SOLiDSystemMate-PairedLibraryPreparation,SolexaM