高通量测序技术简介.ppt

第五部分 其他类型的芯片 微缩芯片实验室 Lab on a chip 药物运输芯片生理功能辅助芯片纳米芯片 NextGenerationSequencing SamplefragmentationLibrarypreparationSequencingreactionDataanalysis Roche454焦磷酸测序PyrophosphateSequencing IlluminaSolexa合成测序SequencebySynthesize ABISOLiD连接法测序SequencebyLigation 第六部分高通量测序技术简介 Roche454焦磷酸测序PyrophosphateSequencing基本原理 454sequencing EmulsionPCR emPCR MixDNALibrary capturebeads limiteddilution Breakmicro reactors IsolateDNAcontainingbeads GenerationofmillionsofclonallyamplifiedtemplatesoneachbeadNocloningandcolonypicking Create Water in oil emulsion PCRReagents EmulsionOil PerformemulsionPCR AdaptercarryinglibraryDNA A B Micro reactors Adaptercomplement Enrich AnnealSeqprimer CentrifugeStep LoadEnzymeBeads 454sequencing DepositionofDNAbeadsintothePicoTiter Plate LoadbeadsintoPicoTiter Plate IlluminaSolexa合成测序SequencebySynthesize基本原理 ClonalSingleMoleculeArrays单分子克隆 1000moleculesper 1 mcluster 1000clustersper100 msquare 40millionclustersperexperiment PrepareDNAfragments Ligateadapters Attachsinglemoleculestosurface Amplifytoformclusters ReversibleTerminatorChemistry可逆终止反应 All4labellednucleotidesin1reaction Sequencing by Synthesis SBS Firstbaseincorporated Cycle1 Addsequencingreagents Removeunincorporatedbases Detectsignal Cycle2 n Addsequencingreagentsandrepeat 1 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 TTTTTTTGT 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 Basecallingfromtherawdata Solexa测序Workflow ABISOLiD连接法测序SequencebyLigation基本原理 文库制备 微珠单分子克隆 1024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 连接法测序 一 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 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 Advantage disadvantage 454sequencing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microRNAprofiling基于可逆反应 随反应轮数增加 效率降低 信号衰减 读取序列较短 给denovosequencing拼接带来困难SOLiDsequencing每个碱基读取两次非常高的准确性 特别是对于SNP的检测灵活的系统 完善的磁珠编码系统 可以进行样品的pooling 分割测序区域读取长度受连接反应的轮数限制 给denovosequencing拼接带来困难 高通量测序的应用 Denovo测序基因深度测序 genomere sequencing 转录组深度测序 transcriptomere sequencing DigitalexpressionprofilingChIP seqMethy seq Transcriptomeresequencing malignantpleuralmesotheliomas MPMs 恶性胸膜间皮瘤pulmonaryadenocarcinoma ADCA 肺腺癌 Transcriptomecharacteristics Solidline atleastonereadDashedline atleast20reads ExpressiondifferencebetweenMPMandADCAsamplecomparetoalungtissuecontrol Analysisofpercent ageofreadscontainingknowncodingregionSNVsinthesixtissuesamples SNV SingleNucleotideSubstitutionVariant Digitalexpressionprofiling 1 人大脑组织与UHR UniversalHumanReference 的表达差异 Digitalexpressionprofiling microRNAre sequencing hESC humanembryonicstemcellsEB embryoidbodies ChIP seq 1 人一号染色体DNA 蛋白相互作用 ChIP seq 2 Sequencedshortreads typically 25 50bp fromChIP Seqexperimentsare rstmappedontothereferencegenome Themappedreadsarethenusedtoestimatestatisticalparameters whichincludetheestimationoftheaveragelengthFofsequencedDNAfragments Methy seq 1 肿瘤和MCF7细胞系中BRCA 启动子区域的甲基化差异 Somehighlights CorrelationbetweenChIP SeqandhispriorSAGE likemethod calledGMAT hasr 0 906 HowevertheresolutionwithChIP Seqwasdramaticallyhigher Furthermore ChIP Seqwasmoresensitiveandgeneratedlessfalse negativeregions 12 726geneswhosetranscriptionlevelsareknowninCD4 T cellswerecorrelatedwiththehistonemodificationsand35 961PolIIbindingsite islands wereidentified Thiscost effectivemethodproducesdigital qualitydataandshouldfindbroadapplicationsinoureffortstounderstandthecontributionofthehumanepigenomesingeneexpressionandepigeneticinheritance Methy seq 2 部分参考文献阅读 Genomere sequencingvanOrsouwNJ HogersRC JanssenA etal Complexityreductionofpolymorphicsequences CRoPS anovelapproachforlarge scalepolymorphismdiscoveryincomplexgenomes PLoSONE 2007 2 11 e1172HillierLW MarthGT QuinlanAR etal Whole genomesequencingandvariantdiscoveryinC elegans NatMethods 2008 5 2 183 188Transcriptomere sequencingMortazaviA WilliamsBA McCueK etal MappingandquantifyingmammaliantranscriptomesbyRNA Seq NatMethods 2008 5 7 621 628SugarbakerDJ RichardsWG GordonGJ etal Transcriptomesequencingofmalignantpleuralmesotheliomatumors ProcNatlAcadSciUSA 2008 105 9 3521 3526DigitalexpressionprofilingRubyJG JanC PlayerC etal Large scalesequencingreveals21U RNAsandadditionalmicroRNAsandendogenoussiRNAsinC elegans Cell 2006 127 6 1193 1207MorinRD O ConnorMD GriffithM etal ApplicationofmassivelyparallelsequencingtomicroRNAprofilinganddiscoveryinhumanembryonicstemcells GenomeRes 2008 18 4 610 621ChIP seqJohnsonDS MortazaviA MyersRM etal Genome widemappingofinvivoprotein DNAinteractions Science 2007 316 5830 1497 1502RobertsonG HirstM BainbridgeM etal Genome wideprofilesofSTAT1DNAassociationusingchromatinimmunoprecipitationandmassivelyparallelsequencing NatMethods 2007 4 8 651 657 藤晓坤 肖华胜 基因芯片与高通量DNA测序技术前景分析 中国科学C辑 生命科学 2008 38 10 1 9 TengX XiaoH PerspectivesofDNAmicroarrayandnext generationDNAsequencingtechnologies SciChinaCLifeSci 2009 52 1 7 16 高通量测序技术虽然建立的时间不长 但是在基因组的各个研究领域都显示出其非凡的魅力 而且日益显示出其对基因芯片 取而