华大基因 测序技术基础原理

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1、测序技术基础罗龙海2009-03-02Sanger测序技术原理第二代测序技术原理第三代测序技术原理1950196019701980199020002010测序技术发展史DevelopmentofSangerSequencing(1977)InventionofAutomatedFluorescentSequencer(1985)InventionofCapillarySequencer(1996)InventionofAppliedBiosystemsSolidSystem(2007)InventionofIlluminaGeno

2、meAnalyzerSystem(2006)Inventionof454GS20Sequencer(2005)chemicaldegradationmethodbyMaxam-Gilbertmethod(1977)ChemicaldegradationmethodbyWhitfield(1954)InventionofHeliscopesinglemolecularsequencerInventionofSinglemoleculerealtime(SMRT)DNAsequencingInventionofNanoporesing

3、lemolecularsequencing(OxfordNanoporecorporation)Sanger测序法原理Dr.FredSanger"dideoxy"sequencingtechnique(Sangeretal.,1977)DNA双脱氧链终止法测序FrederickSangerwasawardedtheprizeinboth1958and1980.HeisthefourthpersonintheworldtohavebeenawardedtwoNobelPrizesandtheonlypersontoreceivebo

4、thinchemistry.IlluminaGenomeAnalyzerABISOLiDRoche454第二代测序技术二代测序技术200520062007年份原理Pyrosequencing边合成边测序边连接边测序454SolexaSOLIDIlluminaSolexaABISOLiDRoche454第二代测序技术可逆阻断技术IlluminaSolexaFlowcellFlowcell一个flowcell包括8个lanesLane1Lane8Eachlanecontainsmultipletiles–total100每个lane上

5、有许多个tiles——共计100个（见上图）Eachtileisimagedfourtimespercycle–oneimageperbase每个循环会对每个tile照4次相——每个碱基都会成像Imagefrom1tileGenomeAnalyzerAnalysisPipelineLibraryPreparationClusterStationGenomicmRNASmallRNAChIP-SeqIllumina/GAWorkflow工作流程*GrowClusters*5hours*Orsplitprocessinstages*S

6、afestoppingpoints*StartSequencing*ClusterDensityEvaluation*4imagespertilepercycle*Runtime:2-3days*Firecrest:ImageAnalysis*Bustard:Basecalling*Gerald:SequenceAlignment文库构建Cluster工作站基因组测序分析*生成“簇”*5小时*开始测序*序列簇的丰度检测*一个循环每个tile照4张照片*2-3天*图像分析*Bustard:basecalling*Gerald:序列分

7、析?样品预处理PCR成簇测序拼接分析5-6h6-8d（纯化的基因组DNA）（基因组DNA小片段小于800bp）（具有5’-磷酸末端的粘性片段）（去除未连接上的接头）（修饰末端）（）（基因组DNA文库）（纯化连接产物）（）（）（）（基因组DNA小片段）27bp27bp27bp27bpGenomicDNAFragmentlibraryMate-pairedlibraryCreatelibraryofDNAfragmentsDNA片段的文库构建ClusterGeneration序列簇的产生PrepareDNAfragmentsLigat

8、eadaptersAttachsinglemoleculestosurfaceAmplifytoformclustersRandomarrayofclusters（Cluster的随机排列）100um~1000moleculesper~1umclu