第二代高通量测序技术的简介

第二代高通量测序技术的简介第1页/共30页Roche454焦磷酸测序PyrophosphateSequencing基本原理第2页/共30页454sequencing：EmulsionPCR(emPCR)MixDNALibrary&capturebeads(limiteddilution)“Breakmicro-reactors”IsolateDNAcontainingbeadsGenerationofmillionsofclonallyamplifiedtemplatesoneachbeadNocloningandcolonypickingCreate“Water-in-oil”emulsion+PCRReagents+EmulsionOilPerformemulsionPCRAdaptercarryinglibraryDNAABMicro-reactorsAdaptercomplementEnrichAnnealSeqprimer第3页/共30页CentrifugeStepLoadEnzymeBeads454sequencing：

DepositionofDNAbeadsinto thePicoTiter™PlateLoadbeadsintoPicoTiter™Plate第4页/共30页IlluminaSolexa合成测序SequencebySynthesize基本原理第5页/共30页ClonalSingleMoleculeArrays

单分子克隆~1000moleculesper~1µmcluster

~1000clustersper100µmsquare~40millionclustersperexperimentPrepareDNAfragmentsLigateadaptersAttachsinglemoleculestosurfaceAmplifytoformclusters20micronsSequence第6页/共30页ReversibleTerminatorChemistry

可逆终止反应OPPPHNNOOcleavagesitefluor3’blockNextcycleIncorporationDetectionDeblock;fluorremovalODNAHNNOO3’O5’free3’endXOHAll4labellednucleotidesin1reaction第7页/共30页Sequencing-by-Synthesis(SBS)

5’GTCAGTCAGTCAGT3’5’CAGTCATCACCTAGCGTA FirstbaseincorporatedCycle1: Addsequencingreagents Removeunincorporatedbases DetectsignalCycle2-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第8页/共30页123789456TTTTTTT

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T…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第9页/共30页Solexa测序Workflow第10页/共30页ABISOLiD连接法测序SequencebyLigation基本原理第11页/共30页文库制备：微珠单分子克隆第12页/共30页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序列第13页/共30页连接法测序

（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第14页/共30页连接法测序

（二）测序引物沿着Adapter移动5次，确保每个位点都被检测第15页/共30页连接法测序

（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。第16页/共30页Advantage&disadvantage454sequencing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拼接带来困难第17页/共30页高通量测序的应用Denovo

测序基因深度测序（genomere-sequencing）转录组深度测序（transcriptomere-sequencing）DigitalexpressionprofilingChIP-seqMethy-seq第18页/共30页Transcriptomeresequencing：malignantpleuralmesotheliomas(MPMs)：恶性胸膜间皮瘤pulmonaryadenocarcinoma(ADCA)：肺腺癌第19页/共30页TranscriptomecharacteristicsSolidline：atleastonereadDashedline：atleast20readsExpressiondifferencebetweenMPMandADCAsamplecomparetoalungtissuecontrolAnalysisofpercent-ageofreadscontainingknowncodingregionSNVsinthesixtissuesamples.SNV：SingleNucleotideSubstitutionVariant第20页/共30页Digitalexpressionprofiling（1）：

人大脑组织与UHR（UniversalHumanReference）的表达差异第21页/共30页DigitalexpressionprofilingµRNAre-sequencing：hESC：humanembryonicstemcellsEB：embryoidbodies第22页/共30页ChIP-seq（1）：人一号染色体DNA-蛋白相互作用第23页/共30页ChIP-seq（2）：Sequencedshortreads(typically25–50bp)fromChIP-Seqexperimentsarefirstmappedontothereferencegenome.Themappedreadsarethenusedtoestimatestatisticalparameters,whichincludetheestimationoftheaveragelengthFofsequencedDNAfragments.第24页/共30页Methy-seq（1）：肿瘤和MCF7细胞系中BRCA!启动子区域的甲基化差异第25页/共30页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）：第26页/共30页部分参考文献阅读Genomere-sequencing

vanOrsouwNJ,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第27页/共