华大基因 测序技术基础原理.ppt

测序技术基础,罗龙海2009-03-02,Sanger测序技术原理第二代测序技术原理第三代测序技术原理,1950,1960,1970,1980,1990,2000,2010,测序技术发展史,Development of Sanger Sequencing(1977),Invention of Automated FluorescentSequencer(1985),Invention of CapillarySequencer(1996),Invention of Applied BiosystemsSolid System(2007),Invention of Illumina Genome Analyzer System(2006),Invention of 454 GS 20 Sequencer(2005),chemical degradation method by Maxam-Gilbert method(1977),Chemical degradation method by Whitfield(1954),Invention of Heliscope single molecular sequencer,Invention of Single molecule real time(SMRT)DNA sequencing,Invention of Nanopore single molecular sequencing(Oxford Nanopore corporation),Sanger 测序法原理,Dr.Fred Sanger,Illumina Genome AnalyzerABI SOLiDRoche 454,第二代测序技术,二代测序技术,2005,2006,2007,年份,原理,Pyrosequencing,边合成边测序,边连接边测序,454,Solexa,SOLID,Illumina SolexaABI SOLiDRoche 454,第二代测序技术,可逆阻断技术,Illumina Solexa Flowcell,Flowcell,一个 flowcell 包括8个lanes,Lane 1,Lane 8,Each lane contains multiple tiles total 100 每个lane上有许多个tiles共计100个（见上图）Each tile is imaged four times per cycle one image per base 每个循环会对每个tile照4次相每个碱基都会成像,Image from 1 tile,Illumina/GA Workflow 工作流程,*Grow Clusters*5 hours*Or split process in stages*Safe stopping points,*Start Sequencing*Cluster Density Evaluation*4 images per tile per cycle*Run time:2-3 days,*Firecrest:Image Analysis*Bustard:Basecalling*Gerald:Sequence Alignment,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:序列分析,?,样品预处理 PCR成簇 测序 拼接分析,5-6h 6-8d,（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）,Genomic DNA,Fragment library,Mate-paired library,Create library of DNA fragmentsDNA片段的文库构建,Cluster Generation序列簇的产生,Prepare DNA fragments,Ligate adapters,Attach single molecules to surface,Amplify to form clusters,1000 molecules per 1 um cluster 20.000 clusters per tile32-40 million clusters per experiment（1个cluster上有1000个分子 1个tile上有20,000个cluster 每个实验可完成3.2-4千万个cluster),通过cluster将单分子放大、固定,OH,Cluster Generation:Amplification,1st cycle annealing（No.1循环：退火）,diol,diol,Cluster Generation:Amplification,Blocking with ddNTP()(ddNTP阻断末端),Sequencing By Synthesis边合成边测序,1.Incorporation 结合2.Scan 扫描拍照3.Cleavage 清洗,SBS Cycle,First base incorporated 第一个碱基合成上,Cycle 1:Add sequencing reagents 加入合成所需反应物,Detect Signal 检测荧光信号,Cleave Terminator and Dye 去掉末端封闭，染色,Cycle 2-n:Add sequencing reagents and repeat,Sequencing By Synthesis(SBS),?,Base Calling碱基识别,T T T T T T T G T,T G C T A C G A T,The identity of each base of a cluster is read off from sequential images,Paired End,Paired End 双末端测序、正反双向测序,Sample Preparation 样品前准备Cluster Generation 分子簇的生成Sequence By Synthesis 边合成边测序,（用于组装较大的Gene，一次最多读100bp）,Sample Preparation,5,T,3,A,5,A,T,3,3,A,5,T,3,T,A,5,加双末端,Grafted FlowCells,Single ReadPeriodate Linearization,Paired EndUracil Specific Excision Reagent(USER)尿嘧啶特异性识别位点-P5formamidopyrimidine glycosylase(fpg)糖基化酶-P3,8oxoG-P7 U-P5,OH,OH,U,8oxo-G,?,OH,OH,Grafted flowcell,U,P7 P5,Cluster Generation:Initial Extension,1st cycle annealing,Cluster Generation:Amplification 成簇,Cluster Amplification,25个循环,Sequencing 测序,Denaturation and HybridizationSBS3,Illumina Solexa,形成DNA簇；可逆阻断技术边合成边测序（SBS）,40-50G/10天/一个RUN,读长：75bp（可双向）,错误类型：替换,Illumina solexaABI SOLiDRoche 454,SOLID 测序技术,AB/SOLID Workflow 工作流程,文库制备Emulsion PCRBeads Enrichment微珠沉积连接测序数据分析,文库制备Emulsion PCRBeads Enrichment微珠沉积连接测序数据分析,Work Flow：,序列可以用超声波、机械剪切或酶解等方法，随机或者定向的打断成小片段,（大片段两头测序）,“Mate-paired”步骤：环化切割加接头测序,酶切为粘性末端,对于复杂的分子环化，采用低浓度的模板浓度进行连接,文库制备Emulsion PCR微珠富集微珠沉积连接测序数据分析,Work Flow：,2.Emulsion PCR,+,Templates,Enzyme+dNTPs,P1-coupled DNA beads 100,000 P1 sites per beadStart with 2 Billion beads per emulsion,Polymerase,100,000 P1 位点/每个bead2 Billion beads/每个emulsion,Mix PCR aqueous phase into a water-in-oilemulsion and carry out emulsion PCR,Reactor with template,bead and PCR reagents,Mineral oil+surfactants,Beads collected following emulsion PCR:,Beads with amplified product(40K PCR products per bead),Beads with no product,文库制备Emulsion PCR微珠富集微珠沉积连接测序数据分析,Work Flow：,3.Enrichment/微珠富集,Centrifuge using a Glycerol Gradient甘油梯度离心,Captured beads(+templates)in supernatant,Uncaptured beads(no template)in pellet,文库制备Emulsion PCR微珠富集微珠沉积连接测序数据分析,Work Flow：,4.Deposite beads,3-end modification,文库制备Emulsion PCR微珠富集微珠沉积连接测序数据分析,Work Flow：,5.SOLiD 4-color ligation reaction,5.SOLiD 4-color ligation reaction,A5,6.SOLiD 4-color ligation visualization,C20,T15,G25,A5,T 10,7.SOLiD 4-color ligation Reset,8.SOLiD 4-color ligation(1st cycle after reset),p5,Consequences of 2 Base Pair Encoding,Detecting a single color does not indicate a base Each reading contains information from two basesTo decode the bases you must know one of the bases in the sequence,If know first base is an A then immediately it decodes 2nd base.This must be an A as Blue translates 2nd base A if first base A,Example:,ABI SOLiD,Emulsion PCR边连接边测序（SBL）,50G/大于10天/一个RUN,读长：50bp（可双向）,错误类型：替换,Illumina solexaABI SOLiDRoche 454,Genome Sequencer 20 Syste（2005）Genome Sequencer FLX Syste（2006）GS FLX Titanium（2008）,发展历程：,61,DNA Library PreparationGenome fragmented by nebulizationAdaptor ligationsstDNA library created with adaptersA/B fragments selected using avidin-biotin purification,Emulsion PCR AmplificationAnneal sstDNA to an excess of DNA capture beadsEmulsify beads and PCR reagents in water-in-oil microreactorsClonal amplification occurs inside microreactors,Sequencing By SynthesisLoad beads into PicoTiter Plate Sequencing by synthesis Photons Generated are Captured by CameraSequencing Image Created,Roche/454 GS FLX Workflow,1.DNA library preparation,2.Emulsion Based Clonal Amplification,3.Loading DNA Beads into the PicoTiterPlate,dNTP PPiPPi+APS ATPATP+Luciferin luciferase Oxyluciferin+Light,4.Sequencing,The sequencing instrument consists of the following major subsystems:(a)a fluidic assembly，(b)a flow chamber that includes the well-containing fibre-optic slide，(c)a CCD camera-based imaging assembly，and a computer that provides the necessary user interface and instrument control.,Roche 454,微乳液 PCR聚合测序,0.4-0.6G/10h/一个RUN,读长：400bpMax：800bp,错误类型：插入&缺失,第二代测序技术小结,454测序仪：454测序仪使用的方法，经微乳液PCR发扩增后，携带有大量模板分子的微珠被放置到芯片上的微孔中。随后使用焦磷酸法测序，每一轮测序反应都会掺入一个核苷酸，随后加入反应试剂荧光素和腺苷酰硫酸。这样在每一个小孔中每当有聚合酶将核苷酸掺入到模板上都会发光。最后用腺苷三磷酸双磷酸酶洗涤去掉多余的核苷酸。(对重复序列如poly A的测定不准确，因荧光信号具有累加效果)Solexa测序仪：Solexa测序仪使用桥式PCR直接在芯片进行模板扩增，然后同时加入四种经过修饰的脱氧核苷酸，每一个核苷酸都携带一种荧光集团和一个可被去除的终止基团。经过修饰的DNA聚合酶催化引物延伸测序反应。采集图像、然后切除荧光标记基团和终止基团，重复上述反应，完成测序。（边合成边测序）Solid测序仪：Solid测序仪使用微乳液PCR法扩增模板片段，然后吸附有大量扩增片段的直径1um的磁珠倍制成高密度测序芯片，借助使用连接酶而不是聚合酶测序法完成测序。在solid测序一中，每一次反应都会在引物末端加上一个荧光标记的8bp的探针，在探针中央的两个碱基上标记有荧光基团，探针被连接上之后发出荧光，随后荧光基团部分被切除，重新系下一轮反应。（边连接边测序）,第一代测序技术 versus 第二代测序技术,Current popular sequencing platform,第三代测序技术,Heliscope单分子测序仪-Helicos Biosciences,测序原理：边合成边测序特点：无需对待测模板进行扩增，采用高灵敏度的荧光探测仪，直接对单链的DNA模板进行合成测序，序列读长为24到70个碱基，平均读长为32个碱基。流程：（1）待测文库片段化；（2）3端加poly A尾，并与固定在芯片上的poly T进行杂交，将待测模板固定到芯片上，制成测序芯片。（3）通过DNA聚合酶将荧光标记的单核苷酸渗入到引物上，每一轮反应加入一种dNTP。（4）采集荧光信号，切除荧光标记集团，进行下一轮测序反应。,斯坦福大学的科学家最近利用Helicos Biosciences的Heliscope单分子测序仪，对一名白人男子的基因组进行了测序，文章发表在最新一期的Nature Biotechnology在线版上。利用一台Heliscope测序仪和4次数据收集运行，完成了此次测序。研究人员报告称，他们产生了数十亿个Heliscope序列读取，覆盖了90%的人参考基因组，覆盖度达28倍。序列读长为24到70个碱基，平均读长为32个碱基。到目前为止，他们已经鉴定出280万个SNP和752个拷贝数变异。测序花了4个星期的时间，试剂花费为48000美元,Single molecule real time(SMRT)DNA测序技术Pacific Biosciences,（1）以SMRT芯片载体：带有3000个直径为70nm左右的纳米级小孔的金属片，将DNA聚合酶、待测序列和不同荧光标记的dNTP放入到ZMW孔中，进行合成反应。（2）SMRT技术的测序速度很快，可达到每秒大约10个dNTP。、它实现了DNA聚合酶内在自身的反应速度，一秒可以测10个碱基，测序速度是化学法测序的2万倍。（3）它实现了DNA聚合酶内在自身的processivity（延续性，也就是DNA聚合酶一次可以合成很长的片段），一个反应就可以测非常长的序列。二代测序现在可以测到上百个碱基，但是三代测序现在就可以测几千个碱基。这为基因组的重复序列的拼接提供了非常好的条件。（4）它的精度非常高，达到99.9999%。,纳米孔单分子技术-Oxford Nanopore公司,测序原理：不同碱基产生的电信号进行测序。步骤：特殊材料制成的纳米孔，孔内共价结合有分子接头环糊精，核酸外切酶切割单链DNA时，被切割下来的碱基落入纳米孔，并与环糊精相互作用，短暂影响流过纳米孔的电流强度，电流强度的变化幅度成为每种碱基的检测特征。碱基在纳米孔中的平均停留时间是毫秒级的，一定强度的电压可保证在电信号记录后将碱基从纳米孔中清除。独特特点：直接读取甲基化的胞嘧啶。,