测序技术原理介绍ppt课件.ppt

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1、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）测序方法分类手工测序自动测序Sanger双脱氧链终止法Maxam-Gilbert化学降解法3100/3100-a3130/3130XL37003730/3730XLSolexa454焦磷酸法SOLiDGilbert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

2、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Chain-terminatorreactiondeoxynucleotides,dNTPdideoxynucleotides,ddNTPFrederickSangerOriginalSangersequencing1980-1990Radio–gelReadlength:~102bp~103bp/dayAutomatedSan

3、gersequencing1990-Fluorescent–capillaryReadlength:~103bp~106bp/dayABI3730DNAAnalyzer$376K/instrument650bp/read0.06Mb/run2h/run$10K/MbHierarchicalshotgun(map-based)methodLongfragmentBAClibraryPhysicalmapShotgunSequencingandassemblyHumanGenomeProject$3

4、billion,11yearsWhole-genomeshotgunmethodShortfragmentPlasmidlibraryMate-pairsequencing3years$300millionAssemblyContigScaffoldCraigVenterSangervsnext-generationsequencingNatBiotech26:1135(2008)Roche454Since2004GenomeSequencer/FLXTemplatepreparation-em

5、ulsionPCRTrendsinGenet24:133(2008)FragmentationLigationWater-in-oilemulsionMirco-reactoremPCRPicoTiterPlateloadingPyrosequencingSingledNTPtypeflowspercycleInorganicpyrophosphate(PPi)drivesvisiblelightthroughaseriesofreactionsRemoveunincorporatednucle

6、otideTrendsinGenet24:133(2008)BasecallingHomopolymererrorGV6330IlluminaSolexaSince2006GenomeAnalyzerTemplatepreparation-bridgeRCRAdaptorligationSurfaceattachmentBridgeamplificationDenaturationTrendsinGenet24:133(2008)CyclicreversibleterminationAllfou

7、rlabeledreversibleterminatorsareaddedpercycleRemoveunincorporatedbasesanddetectsignalRemovetheterminatinggroupandthefluorescentdyeTrendsinGenet24:133(2008)TerminatinggroupFluorophorecleavageNatRevGenet11:31(2010)BasecallingSolexa测序原理MainIlluminanoise

8、factors(a)Intheidealsituation,afterseveralcyclesthesignal(greenarrows)isstrong,coherentandcorrespondstotheinterrogatedposition.(b)Phasingnoiseintroduceslagging(bluearrows)andleading(redarrow)nascentstrands,whichtransmitamixtureofsignals.(c)Fadingisat