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1、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
2、 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 si
3、ngle 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 flowce
4、ll 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,*S
5、tart 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
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 surfa
7、ce,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 Generati
8、on: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 a
9、nd 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1
10、00bp）,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
11、: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
12、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 DN
13、A 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 followin
14、g 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
15、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 a
16、fter 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 a
17、s 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 neb
18、ulizationAdaptor 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,
19、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
20、+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 use
21、r 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
22、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
23、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
24、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
25、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
26、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。,
