深度测序技术简介

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1、NextGenerationSequencingSamplefragmentationLibrarypreparationSequencingreactionDataanalysishttp://www.gbio.cn绿宇生物科技专业建库测序QQ:110199525Roche454焦磷酸测序PyrophosphateSequencingIlluminaSolexa合成测序SequencebySynthesizeABISOLiD连接法测序SequencebyLigation深度(高通量）测序技术简介http://www.gbio.cn绿宇生

2、物科技专业建库测序QQ:110199525Roche454焦磷酸测序PyrophosphateSequencing基本原理http://www.gbio.cn绿宇生物科技专业建库测序QQ:110199525454sequencing：EmulsionPCR(emPCR)MixDNALibrary&capturebeads(limiteddilution)“Breakmicro-reactors”IsolateDNAcontainingbeadsGenerationofmillionsofclonallyamplifiedtemplates

3、oneachbeadNocloningandcolonypickingCreate“Water-in-oil”emulsion+PCRReagents+EmulsionOilPerformemulsionPCRAdaptercarryinglibraryDNAABMicro-reactorsAdaptercomplementEnrichAnnealSeqprimerCentrifugeStepLoadEnzymeBeadshttp://www.gbio.cn绿宇生物科技专业建库测序QQ:110199525454sequencing：Dep

4、ositionofDNAbeadsintothePicoTiter™PlateLoadbeadsintoPicoTiter™Platehttp://www.gbio.cn绿宇生物科技专业建库测序QQ:110199525IlluminaSolexa合成测序SequencebySynthesize基本原理ClonalSingleMoleculeArrays单分子克隆http://www.gbio.cn绿宇生物科技专业建库测序QQ:110199525~1000moleculesper~1µmcluster~1000clustersper100

5、µmsquare~40millionclustersperexperimentPrepareDNAfragmentsLigateadaptersAttachsinglemoleculestosurfaceAmplifytoformclusters20micronsSequenceReversibleTerminatorChemistry可逆终止反应http://www.gbio.cn绿宇生物科技专业建库测序QQ:110199525OPPPHNNOOcleavagesitefluor3’blockNextcycleIncorporation

6、DetectionDeblock;fluorremovalODNAHNNOO3’O5’free3’endXOHAll4labellednucleotidesin1reactionSequencing-by-Synthesis(SBS)http://www.gbio.cn绿宇生物科技专业建库测序QQ:1101995255’GTCAGTCAGTCAGT3’5’CAGTCATCACCTAGCGTAFirstbaseincorporatedCycle1:AddsequencingreagentsRemoveunincorporatedbasesD

7、etectsignalCycle2-n:Addsequencingreagentsandrepeat1、每轮测序反应加入四种带有荧光标记的dNTP，末端带有可以被去除的阻断基团2、每轮反应只能整合一个核苷酸，仪器读取相应的荧光信号3、信号读取结束，用化学方法去除阻断基团，进行下一轮测序反应123789456TTTTTTTGT…TGCTACGAT…Theidentityofeachbaseofaclusterisreadofffromsequentialimages根据每个点每轮反应读取的荧光信号序列，转换成相应的DNA序列Basecall

8、ingfromtherawdatahttp://www.gbio.cn绿宇生物科技专业建库测序QQ:110199525Solexa测序Workflowhttp://www.gbio.cn绿宇生