深度测序技术简介VIP专享VIP免费

SamplefragmentationLibrarypreparationSequencingreactionDataanalysishttp://www.gbio.cn绿宇生物科技专业建库测序QQ:110199525Roche454Roche454焦磷酸测序焦磷酸测序PyrophosphateSequencingPyrophosphateSequencingIlluminaSolexaIlluminaSolexa合成测序合成测序SequencebySynthesizeSequencebySynthesizeABISOLiDABISOLiD连接法测序连接法测序SequencebyLigationSequencebyLigation深度深度((高通量）测序技术简介高通量）测序技术简介http://www.gbio.cn绿宇生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:110199525MixDNALibrary&capturebeads(limiteddilution)“Breakmicro-reactors”IsolateDNAcontainingbeadsGenerationofmillionsofclonallyamplifiedtemplatesoneachbeadNocloningandcolonypickingCreate“Water-in-oil”emulsion+PCRReagents+EmulsionOilPerformemulsionPCRAdaptercarryinglibraryDNAABMicro-reactorsAdaptercomplementEnrichAnnealSeqprimerCentrifugeStepLoadEnzymeBeadshttp://www.gbio.cn绿宇生物科技专业建库测序QQ:110199525LoadbeadsintoPicoTiter™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http://www.gbio.cn绿宇生物科技专业建库测序QQ:110199525~1000moleculesper~1µmcluster~1000clustersper100µmsquare~40millionclustersperexperimentPrepareDNAfragmentsLigateadaptersAttachsinglemoleculestosurfaceAmplifytoformclusters20micronsSequencehttp://www.gbio.cn绿宇生物科技专业建库测序QQ:110199525OPPPHNNOOcleavagesitefluor3’blockNextcycleIncorporationDetectionDeblock;fluorremovalODNAHNNOO3’O5’free3’endXOH•All4labellednucleotidesin1reactionhttp://www.gbio.cn绿宇生物科技专业建库测序QQ:1101995255’GTCAGTCAGTCAGT3’5’CAGTCATCACCTAGCGTAFirstbaseincorporatedCycle1:AddsequencingreagentsRemoveunincorporatedbasesDetectsignalCycle2-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Basecallingfromtherawdatahttp://www.gbio.cn绿宇生物科技专业建库测序QQ:110199525Solexa测序Workflowhttp://www.gbio.cn绿宇生物科技专业建库测序QQ:110199525http://www.gbio.cn绿宇生物科技专业建库测序QQ:110199525ABISOLiD连接法测序SequencebyLigation基本原理文库制备：微珠单分子克隆http://www.gbio.cn绿宇生物科技专业建库测序QQ:1101995251024种8碱基探针4色荧光，4种双核苷酸，每色荧光有256个探针(4^6)SOLiD利用探针的连接反应读取模板的DNA序列http://www.gbio.cn绿宇生物科技专业建库测序QQ:110199525http://www.gbio.cn绿宇生物科技专业建库测序QQ:110199525每个探针进行检测的两个碱基后面有三个匹配碱基，因此一条测序引物读取的序列是不完整的测序引物与adapter退火探针连接，检测荧光切除荧光基团第二轮探针连接，检测荧光切除荧光基团连接法测序（二）测序引物沿着Adapter移动5次，确保每个位点都被检测http://www.gbio.cn绿宇生物科技专业建库测序QQ:110199525连接法测序（三）0位置是Adapter的最后一个碱基，因此只检测一次，该碱基是进行解码所必须的。http://www.gbio.cn绿宇生物科技专业建库测序QQ:110199525454sequencing读取长度大，400bp可以对未知基因组进行从头测序denovosequencing当遇到polymer时，如AAAAAA等，荧光强度和碱基个数不成线性关系，判定重复碱基个数有困难Solexasequencing高度自动化的系统读取片段多，适合进行大量小片段的测序，如microRNAp...