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 fluorescent sequencer (1985),invention of capillary sequencer (1996),invention of applied biosystems solid 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 analyzer abi solid roche 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 solexa abi solid roche 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 fragments dna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 tile 32-40 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 read periodate linearization,paired end uracil specific excision reagent (user) 尿嘧啶特异性识别位点-p5 formamidopyrimidine 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 hybridization sbs3,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 solexa abi solid roche 454,solid 测序技术,ab /solid workflow 工作流程,文库制备 emulsion pcr beads enrichment 微珠沉积 连接测序 数据分析,文库制备 emulsion pcr beads 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 bead start with 2 billion beads per emulsion,polymerase,100,000 p1 位点/ 每个bead 2 billion beads/ 每个emulsion,mix pcr aqueous phase into a water-in-oil emulsion 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,a 5,6. solid 4-color ligation visualization,c 20,t 15,g 25,a 5,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 bases to 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 solexa abi solid roche 454,genome sequencer 20 syste（2005） genome sequencer flx syste（2006） gs flx titanium（2008）,发展历程：,61,dna library preparation genome fragmented by nebulization adaptor ligation sstdna library created with adapters a/b fragments selected using avidin-biotin purification,emulsion pcr amplification anneal sstdna to an excess of dna capture beads emulsify beads and pcr reagents in water-in-oil microreactors clonal amplification occurs inside microreactors,sequencing by synthesis load beads into picotiter plate sequencing by synthesis photons generated are captured by camera sequencing image created,roche /454 gs flx workflow,1. dna library preparation,2.emulsion based clonal amplification,3.loading dna beads into the picotiterplate,dntp ppi ppi + aps atp atp + 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：400bp max：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