高通量测序简介

高通量测序技术2017年3月21号illumma9旬亟＞appliedbiosystemsThermoFisherSCIENTIFICMeItechnologies,主要内容、高通量测序简介Solexa和Semiconductor测序原理三、高通量测序部分应用、高通量测序简介■高通量测序(High・throughputsequencing)又称"下一代”测序(aNext-generation?sequencing),可一次性对几百万到十亿条DNA分子进行并行测序；■可以对一个物种的基因组和转录组进行深入、细致和全貌的分析，所以乂被称为深度测序(Deepsequencing)。高通量测序的发展o1992年LynxTherapeuticsMPSSo2003年PolonySequencing（哈佛）o2005年454Pyrosequencingo2006年SolexaSequencing-By-Synthesiso2007年ABISOLIDo2008年HelicostSMSSequencingo2009年IonTorrentSemiconductorSequencingo2011^PicficBiosciencesSMRTSequencing高通量测序的主要代表平台有：罗氏的(Roche)的454测序仪(RocheGSFLXsequencer);Illumina的Solexa基因组分析仪(IlluminaGenomeAnalyzer); 丿ABI的SOLiD测序仪(ABISOLiDsequencer);ThermoFisher的离子半导体测序仪(IonSemiconductor^sequencer).

二、Solexa和Semiconductor测序原理Illuminasolexa文库制备A>Ai.FragmentgenomicDNAAn.Double-strandedcDNA

(fromfiqure2B)A>Ai.FragmentgenomicDNAAn.Double-strandedcDNA

(fromfiqure2B)B.EndrepairandphosphorylateD.LigateindexadapterP5Rd1SPDNAInsertIndexA.>ARd2SP'P7'P5Rd1SPDNAInsertIndexA.>ARd2SP'P7'E.DenatureandamplifyforfinalproductC.A-tailingFlowcell□□□□口口口口口口口口口口口口口口口口口口口口口口口□□□+"□□3明口口口口口口口口口口口口口口口口口口口田口口口口41TileEachcolumncontains60tilesEachtileisimaged4timespercycle簇生成，模板变性OHP7flowcellTemplate

hybridizationInitialextensionDenaturation桥式扩增1st1stcycle

denaturation1stcycleannealing1stcycle 2ndcycleextension denaturation2ndcycleextension2ndcycleannealingNextCycleNextCycle并行合成测序法(SBS,Sequencing-By-Synthesis)和可逆性末端终止技术(ReversibleTerminatorChemistry)qCycle1:AddsequencingreagentsqFirstbaseincorporatedDetectSignalCleaveTerminatorandDyeCycle2-n:Addsequencingreagents

andrepeat

信号收集Clustersarebrightspotson

animageEachclusterrepresents

thousandsofcopiesofthe

sameDNAstrandina1-2

micronspot读取碱基序列在测序过程中，每种碱基被激发后都会发出特殊波长的荧光在每个循环过程中每一个簇只对应一张图像■嚙襲■■DOBTA3AT WWW.TTTTTTTGTIllumina测序平台展示MiSeqMiSeqNextSeq500测序通量读长测序时长数据量2xl50bp2x75bp~29h18h100-200GB50-80GB1x75bpUh25-30GB适用范围全基因组测序、全外显子测序、转录组测序特点高通量、灵活性、简便读长测序时长数据量2x75bp~24h3.3-3.8GB测序通量2xl50bp~24h4.5-5.1GB2x250bp~39h7.8-8.5GB2x300bp~55h13.2-15GB适用范围靶向测序、/」\基因组测序、靶向基因表达分析特点赢萨单次运行产生XOObP双部缽Hiseq4000测序通量读长测序时长 数据量2xl50bp1300-1500GB1x75bpl-3.5d 650-750GB1x50bp215-250GB适用范围全基因组测序、全夕卜显子测序、转录组测序特点最高的通量，每个样品的最低价格读长测序时长数据量测序通量2xl50bp<3d单流动槽 双流动槽8-9TB 16-18TB适用范围全基因组测序特点超高的通量，有望突破人类全基因组测序的$1000大美HiSeqXTen流动槽察S1S2S3S4lx50bp167GB280-333GBN/AbN/Ab<19h数据量a1x75bp333GB560-667GBN/AbN/Ab<29h2x150bp500GB850-1000GB2000GB3000GB<40h适用范围全基因组测序、膑重测序、转录组测序特点对几乎任何基因组、任何测序方法和规模的项目,量，灵活简便。都有可扩展的通所有产出和read数量规格都基于单个流动槽计算，NovaSeq6000系统可同时运行1个或2个流动槽。N/A:不适用。NovaSeq6000

IonSemiconductor测序流程Long/MidRangeAmp. 、BarcodedLibraryCreationLRFragmentsBarcodedLibraryLibraryPreparation； BCAmplicon P1SequenciSequenciClonalAmplificationontoIonSphereParticles(ISPs)IsolatePositiveISPs•LoadPGM™ChipandRunPGM™Sequencer'、、 •TorrentServerCollectsDataDuringSeq.Run•AnalyzewithTypeStreamPlug-In文库制备LibraryProparationBindngSeparationDNAQuantification:QuantificationofeachsampleisperformedusingtheQubit®InstrumenttopreparefordownstreamstepsoftheworkflowEthinolWnhButionBufferTrantferLibraryProparationBindngSeparationDNAQuantification:QuantificationofeachsampleisperformedusingtheQubit®InstrumenttopreparefordownstreamstepsoftheworkflowEthinolWnhButionBufferTrantferPCR产物纯化Purification:・RemovalofexcessPCRcomponentswiththegoalofisolatingthePCRproducts

usingAMPure®reagentfromBeckman-Coulter£AjercourtAMF\ireXP接头连接Fragmentation:AmpliconPCRProductsPre-FragmentabonAAdapterAmplicon:Fragmentation:AmpliconPCRProductsPre-FragmentabonAAdapterAmplicon:OutputofFragmentationreactionA-Adapter:ComplementarytoasequencingprimerAdaptorLigation:BarcodeP1AdapterBarcodeP1AdapterBarcode:Usedtotag/identifyaspecificDNAsampleP1Adapter:FacilitatesattachmenttotheIonSphere™Particles(ISPs)PCRProductsPost-FragmentationBarcodedLibraryBCPI片段筛选・SizeselectionisaccomplishedthroughtheuseofAMPure®reagentfromBeckman-

CoulterAlteringthebead:sampleratioallowsforselectionofthetargetsizedistribution样本混池QuantificationAllsamplesarequantifiedonefinaltimetoensureequalrepresentationusingtheAgilent.TapeStationInstrument:PoolingSample4BCAmpliconP1Sample4BCAmpliconP1克隆扩增Ibmplat©

PreparationIdealClonalAmplification:1IonSphere™Particle(ISP)+1BarcodedLibraryFragmentIsothermalAmplificatbn1IonSphere™Particle(ISP)+ClonallyAmplifiedBarcodedLibraryFragmentsAmplification不理想的扩增情况TemplatePreparationISP富集(IonSphereParticalEnrichment)EnrichmentStep1AddMagnetic

StreptavidinBeadEnrichmentStep2:ImmobilizetoMagnetandWashEnrichmentStep3:

DenatureISP

withNaOHTemplatePreparationReadsof

Interest

(1library

fragment/ISP)Polyclonal

ReadsProductof

ISPEnrichmentNon-

templated

ISP扩增引物的5'端标记了生物素,通过链霉亲和素标记的磁珠与标记了生物素的DNA结合.

测序4dNTPs自然情况下，DNA聚合酶将一个核昔酸渗入到DNA分子中就会释放出一个H+,导致局部可检验的PH值发生变化，被离子传感器检测到，从而转换为数字信号。碱基识别依次掺入4中碱基连续两个相同碱基结合IonTorrent仪器展示Ion314™Chip1millionwellsIon316™Chip6millionwellsIon318™Chip11millionwells1-2samples<24samples<48samplesIonTorrentPGMChip建PGM314PGM316PGM318传感器数1.3M6.3MUM| 数据量~100MB~1GB-2GB运行时间2.3/4.7h3.0/4.9h4.4/7.3h读长200/400bp2OO/4OObp2OO/4OObp应用氾围扩增子测序、靶向重测序、小基因组测序特点基于半导体技术测序I纏，快速“读"出碱DNA序列人基因组富高通量测序I湖全基因组测序动植物基因微生物基因咼通量测序（DNA外显子组测序甲基化测序扩增子测序向捕获测序全外显子纟ImRNA测序LncRNA测序RNA SmaHRNA测序CircularRNA测序全基因组测序♦全基因组重测序是对已知基因组序列的物种进行不同个体的基因组测序，通过序列比对，可以找到大量的单核昔酸多态性位点(SNP),插入缺失位点(InDei)、结构变异位点(SV)位点和拷贝数变异位点(CNV)。♦全基因组denovo测序是指对未知基因组序列的物种进行从头测序，用生物信息学分析方法进行拼接、组装，从而获得该物种的基因组序列图谱。pawed，EZJdGene&GcFsarsMaiemnd刀。gemrglCFoFsnrssenucngpaireQEnQ外显子组测序O外显子组虫一个物种基因组中全部外显子区域的总和；+人外显子组只占基因组的〜1%,约30Mb；4约85%的人类致病突变都位于这1%的蛋白质编码序列上。Day1OlagmentGenomicDNAReagents:50nggChW.ST2,FD,TDE2,Tris-HC110mM.pH85•CleanUpTagmenteoDNAReagents:RSB.SP3.Fresh80%EtOH文库构建AmplifyTagmentedDNAReagents:Irxlex1.Ind^x2.LAMSateStoppingPolniCleanupAmplifiedDNAReagents:RSB,SP3,Fresh60%EtOHSafeStoppingPoint[Recommended]Day2©HybridizeProbesRaagents:BLR.CEMEX,EHB1,EHB2,RSB,SPBOCaptureHybridizedProbesReagents：EE1,Er2,EEW,HP3.RSB.SMBSafeStoppingFConstructshotgunlibroryt■HybridizationGenomicDNA FragmentsCPerformSecondHybridizationWFReagents:BLR.CEX/EEX.EHB1.EHB2.SPB©PerformSecondCaptureRoagants:EE1,ET2.EEW,HP3SMRMapping,alignment,variantcallingAGGTCGTTACGTACGCTAC□ACCTACATCAGTACATAG<—GCATGACAAAGCTAC®TGTCleanUpCapturedLibraryRcagcntaRSB.SP9.Fresh60%EtOHSateStoppingPointOptiofialCXremiSafeStoppingPointNimblegen、AgilentAmplifyEnrichedLibraryReagouls:EAM,PPGCleanUpAmplifiedEnrichedLibraryReagents:RSB,SP9,Fresh80%EtOH(■Pre-Amp•Post-AmpIlluminamRNA测序omRNA测序(mRNAseq)是指利用高通量测序技术对cDNA进行测序从而得到一个样品中基因表达、cSNP、全新的转录、全新异构体、剪接位点、等位基因特异性表达和罕见转录等最全面的转录组信息。文库构建链特异性试剂盒建库 通过带oligoT的磁珠分离出mRNA度性RNA RNA片段 cDNA第一链 cDNA第二链 打断反转录dTTPBgy^dUTPcDNA第二链合成 加接头UNG觐理 扩增文库LncRNA(longnoncodingRNA)测序oLncRNA是一类长度超过200nt的长链非编码RNA,通过与DNA、RNA或蛋白质结合，在表观遗传、转录及转录后等水平调控基因表达。oIncRNA测序是研究已有参考基因组物种的特定组织或细胞在某个特定时期转录出的所有IncRNA和mRNAo文库构建gA提取针对不同的雜样品制定不同的提取方案总RN.A质尽检则 •浓度及纯度枪测誕性弃用田3专用席脂桶也冰女是入曲in21WBBjyzer险測rRNA去除通jgA去除试利盒去除收RNA片段化通过圈子打断的方式，播RNA打0F却統-粕此p片段通溯机引歯和逆转录髄作用合芯D\・A第一犠r台觎DM第二継时，注

锥特肄性發,将dggia^dUTP,大取提高结果的g性H大场3<iCH0bbpAstern21corm实2W枪測立旌大小,