代测序实验与测序原理

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1、二代测序的建库与测序原理何有裕yyhe@sibs.ac.cnyyhe@biosino.com.cn上海生物信息技术研究中心上海众信生物技术有限公司苏州众信生物技术有限公司内容样本处理与测序原理简介罗氏454Illuminasolexa原始数据质量控制TruSeqRNAandDNASamplePreparationClusterGenerationOverview~1000-6000moleculesperclusterOHOHflowcelldiolP7P5ClusterGeneration,TemplateHybr

2、idizationdioldiolTemplatehybridizationdioldiolInitialextensiondioldiolDenaturationdioldiol1stcycledenaturation1stcycleannealingdioldioln=251stcycleextensiondioldioldioldiol2ndcycledenaturation2ndcycleannealingdioldioldiolClusterGeneration,BridgePCRdioldioldiol2

3、ndcycleextensionTEMPLATEPREPARATION-BRIDGERCRAdaptorligationSurfaceattachmentBridgeamplificationDenaturationTrendsinGenet24:133(2008)CAGTCATCACCTAGCGTA5’GTCAGTCAGTCAGT3’5’FirstbaseincorporatedCycle1:AddsequencingreagentsDetectSignalCleaveTerminatorandDyeCycle2-

4、n:AddsequencingreagentsandrepeatSequencingbySynthesisOverviewCYCLICREVERSIBLETERMINATIONAllfourlabeledreversibleterminatorsareaddedpercycleRemoveunincorporatedbasesanddetectsignalRemovetheterminatinggroupandthefluorescentdyeTrendsinGenet24:133(2008)Terminatingg

5、roupFluorophorecleavageNatRevGenet11:31(2010)BASECALLINGFlowcelllayoutonGAIIAflowcellcontains8lanesLane1Lane2Lane8...Column1Column2TileEachlanecontains2columnsEachcolumncontains60tilesEachtileisimaged4timespercyclePrimaryDataAnalysisByFirecrestandBustardinRTA/O

6、LBtiffimagefileIntensityfileFirecrestBustardX,YACGTCycle1Cycle2PositionTile#Lane#ACGTX,YLane#SequenceSequencefileOHdioldiolOHClusterGeneration,SequencingPrimerHybridization（Single测序方式处理步骤）LinearizationOHBlockingwithddNTP()DenatureandHybridizationSBS3OHSEQUENCE

7、MULTIPLESAMPLESINTHESAMELANESDNAinsertRead1IndexReadRead2DNAinsertIndexIndexSPRd2SPRd1SPMultiplexing–multiplesamplesinthesamelanesPAIR-END测序优势Read1Read2KnownDistanceRepetitiveDNASinglereadmapstomultiplepositionsPairedreadmapsuniquelyMATE-PAIR建库和测序Read1Read2Know

8、nDistanceMolecularEcologyResources(2011)TEMPLATEPREPARATION-EMULSIONPCRTrendsinGenet24:133(2008)FragmentationLigationWater-in-oilemulsionMirco-reactoremPCRPicoTiterPlateload