测序技术原理介绍

测序技术原理介绍化学测序Sanger测序NGS（二代测序）Formthenexttothethird（三代测序）Gilbert化学降解法1.目的DNA制备2.单侧末端标记待测DNA片段3.碱基的特异性修饰及化学降解4.聚丙烯酰胺凝胶电泳5.放射自显影6.阅读测序结果Sanger测序法1.单链DNA模板制备2.DNA模板与测序引物退火3.掺入法标记反应4.延伸－终止反应5.变性聚丙烯酰胺凝胶电泳6.放射自显影7.阅读测序结果Hierarchicalshotgun(map-based)methodLongfragmentBAClibraryPhysicalmapShotgunSequencingandassemblyHumanGenomeProject$3billion,11yearsWhole-genomeshotgunmethodShortfragmentPlasmidlibraryMate-pairsequencing3years$300millionAssemblyContigScaffoldCraigVenterSangervsnext-generationsequencingNatBiotech26:1135(2008)Roche454Since2004GenomeSequencer/FLXTemplatepreparation-emulsionPCRTrendsinGenet24:133(2008)FragmentationLigationWater-in-oilemulsionMirco-reactoremPCRPicoTiterPlateloadingPyrosequencingSingledNTPtypeflowspercycleInorganicpyrophosphate(PPi)drivesvisiblelightthroughaseriesofreactionsRemoveunincorporatednucleotideTrendsinGenet24:133(2008)Basecalling•HomopolymererrorGV6330IlluminaSolexaSince2006GenomeAnalyzerTemplatepreparation-bridgeRCRAdaptorligationSurfaceattachmentBridgeamplificationDenaturationTrendsinGenet24:133(2008)Cyclicreversibletermination•Allfourlabeledreversibleterminatorsareaddedpercycle•Removeunincorporatedbasesanddetectsignal•RemovetheterminatinggroupandthefluorescentdyeTrendsinGenet24:133(2008)TerminatinggroupFluorophorecleavageNatRevGenet11:31(2010)BasecallingSolexa测序原理ABISOLiDSince2007SOLiDAnalyzerTemplatepreparation-emulsionPCRNatureRevGenet11:31(2010)AnnuRevGenoHumGenet9:387(2008)SOLiDcolorspacefluorescentdyeN:DegeneratedbasesZ:UniversalbasesAnnuRevGenoHumGenet9:387(2008)SequencingbyligationPrimeandligate•Image•CleaveofffluorSequencingbyligationExtendsequence•Resetprimer•ExtendwithnewprimerAnnuRevGenoHumGenet9:387(2008)BasedecodingInstrumentcomparisonInstrument454GS-FLXTitaniumIlluminaHiSeq2000ABISOLiD5500xlAmplificationemPCRBridgePCRemPCRSequencingPyrosequencingReversibleterminationLigationPairedendsYesYesYesLength/read(bp)400-60010050-100Throughput/run(bp)500M200G~155GTime/run10h8days8daysReagentcost/Mb$12.4$0.10~$0.07Totaldirectcost/Mb$40$0.25~$0.11Purchasecost$500K$690K$595KJournalofGeneticsandGenomics38:95(2011)MolecularEcologyResources(2011)Pair-endsequencingRead1Read2KnownDistanceRepetitiveDNASinglereadmapstomultiplepositionsPairedreadmapsuniquelyMate-pairsequencingRead1Read2KnownDistanceMolecularEcologyResources(2011)Science327:1190(2010)NatureMethods8:41(2011)JonathanRothbergPersonalGenomeMachine$50K/instrument100bp/read1Gb/run2h/run$1.2/MbIonTorrentDetectH+releasedasavoltagechange—fastCommonmicrochipdesignstandards—low-costmanufacturingSequencingvolumeisincreasingSemiconductorsequencingHelicosBioSciences1.将待测DNA随机打成200bp左右的小片段，在每个小片段末端加上ploy-dA，最后一个A有荧光标记。2.与玻璃片上随机固定的多个poly-dT引物结合。3.激光刺激，标记荧光的A发光，确定位置。4.洗去荧光物质。HelicosBioSciences逐一加入荧光标记的末端终止子（这个终止子与Illumina终止子不一样，它所有的终止子都标有同一种单色染料），然后经过洗涤单色成像之后切开荧光染料和抑制基团，洗涤，加帽，允许下一个核酸进入。如此循环。Paired-EndReadsHybridize~25cycleToendPrimer~25cyclemeltPacificBiosciences模板制备将待测DNA随机打成250bp-6kb的片段，两端加上发卡状的引物序列。ZMW(zero-modewaveguide)短测序长测序讨论？？主要技术特点1.荧光标记到磷酸上，合成后自动脱落（传统的是标记到核苷酸的碱基上，大分子染料会干扰DNA合成酶的活性或者造成聚合反应提前终止）2.ZMW(zero-modewaveguide):十几纳米的小孔能让激光进入后迅速衰减，只有底部30纳米可见。排除背景影响。此外由于dNTP在荧光信号检测区停留的时间（毫秒级）与它进入和离开的时间（微秒级）相比会很长，所以信号强度会很大。3.荧光脉冲的到达时间和持续时间反映了聚合酶动力学信息，而各种修士对聚合酶动力学的影响不一，从而将它们（N6-甲基腺嘌呤、5-甲基胞嘧啶和5-羟甲基胞嘧啶等）区分开来。整个过程所用到的软件LifeTechnologies基于荧光共振能的单分子测序合成技术。（fluorescenceresonanceenergytransfer(FRET)-basedsingle-moleculesequencing-by-synthesistechnology）技术概要：量子点与聚合酶结合，当有带有荧光标记的核苷酸结合时，在激光的照射下，量子点与核苷酸相互作用，发出荧光，同时被检测到。OxfordNanoporeTechnologieOxfordNanoporeTechnologies公司正在研究的纳米孔单分子技术是一种基于电信号测序的技术。他们设计了一种以α-溶血素为材料制作的纳米孔，在孔内共价结合有分子接头环糊精。用核酸外切酶切割ssDNA时，被切下来的单个碱基会落入纳米孔，并和纳米孔内的环糊精相互作用，短暂地影响流过纳米孔的电流强度，这种电流强度的变化幅度就成为每种碱基的特征。碱基在纳米孔内的平均停留时间是毫秒级的，它的解离速率常数与电压有关，180mV的电压就能够保证在电信号记录后将碱基从纳米孔中清除。电信号成像Third-generationsequencingSinglemoleculartemplateReal-timesequencingLowcostLongreadsNatBiotech28:426(2010)