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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 NextGenerationSequencingSamplefragmentationLibrarypreparationSequencingreactionDataanalysisRoche454焦磷酸测序PyrophosphateSequencingIlluminaSolexa合成测序SequencebySynthesizeABISOLiD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”IsolateDNAcontainingbeadsGenerationofmillionsofclonallyamplifiedtemplatesoneachbeadNocloningandcolonypickingCreate“Water-in-oil”emulsion+PCRReagents+EmulsionOilPerformemulsionPCRAdaptercarryinglibraryDNAABMicro-reactorsAdaptercomplementEnrichAnnealSeqprimer CentrifugeStepLoadEnzymeBeads454sequencing：DepositionofDNAbeadsintothePicoTiter™PlateLoadbeadsintoPicoTiter™Plate IlluminaSolexa合成测序SequencebySynthesize基本原理 ClonalSingleMoleculeArrays单分子克隆~1000moleculesper~1µmcluster ~1000clustersper100µmsquare~40millionclustersperexperimentPrepareDNAfragmentsLigateadaptersAttachsinglemoleculestosurfaceAmplifytoformclusters20micronsSequence ReversibleTerminatorChemistry可逆终止反应OPPPHNNOOcleavagesitefluor3’blockNextcycleIncorporationDetectionDeblock;fluorremovalODNAHNNOO3’O5’free3’endXOHAll4labellednucleotidesin1reaction Sequencing-by-Synthesis(SBS)5’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&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 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 TranscriptomecharacteristicsSolidline：atleastonereadDashedline：atleast20readsExpressiondifferencebetweenMPMandADCAsamplecomparetoalungtissuecontrolAnalysisofpercent-ageofreadscontainingknowncodingregionSNVsinthesixtissuesamples.SNV：SingleNucleotideSubstitutionVariant Digitalexpressionprofiling（1）： 人大脑组织与UHR（UniversalHumanReference）的表达差异 DigitalexpressionprofilingµRNAre-sequencing：hESC：humanembryonicstemcellsEB：embryoidbodies ChIP-seq（1）：人一号染色体DNA-蛋白相互作用 ChIP-seq（2）：Sequencedshortreads(typically25–50bp)fromChIP-Seqexperimentsarefirstmappedontothereferencegenome.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“取而代之”的咄咄态势。那么,基因芯片向何处去呢？基因芯片技术经过近15年的发展已经形成了一个系统的平台。深度测序要建立这样的一个体系同样需要若干年的完善。芯片杂交结果直观,分析快速,适合对大量生物学样,品进行已知信息的检测,同时芯片数据分析有成熟完整的理论,为后期数据分析提供强大的支持。基因芯片的缺点,就在于它是一个“封闭系统”,它只能检测人们已知序列的特征(或有限的变异)。而深度测序的强项,就在于它是一个“开放系统”,它的发现能力和寻找新的信息的能力,从本质上高于芯片技术。在一定程度上，这两种技术是优势互补，联合使用，将加快和加深我们的研究。 Thankyou
