華大基因 測(cè)序技術(shù)基礎(chǔ)原理.ppt

測(cè)序技術(shù)基礎(chǔ),羅龍海 2009-03-02,sanger測(cè)序技術(shù)原理 第二代測(cè)序技術(shù)原理 第三代測(cè)序技術(shù)原理,1950,1960,1970,1980,1990,2000,2010,測(cè)序技術(shù)發(fā)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 測(cè)序法原理,dr. fred sanger,illumina genome analyzer abi solid roche 454,第二代測(cè)序技術(shù),二代測(cè)序技術(shù),2005,2006,2007,年份,原理,pyrosequencing,邊合成邊測(cè)序,邊連接邊測(cè)序,454,solexa,solid,illumina solexa abi solid roche 454,第二代測(cè)序技術(shù),可逆阻斷技術(shù),illumina solexa flowcell,flowcell,一個(gè) flowcell 包括8個(gè)lanes,lane 1,lane 8,each lane contains multiple tiles total 100 每個(gè)lane上有許多個(gè)tiles共計(jì)100個(gè)（見上圖） each tile is imaged four times per cycle one image per base 每個(gè)循環(huán)會(huì)對(duì)每個(gè)tile照4次相每個(gè)堿基都會(huì)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,文庫構(gòu)建,cluster 工作站,基因組測(cè)序,分析,* 生成“簇” * 5小時(shí),* 開始測(cè)序 * 序列簇的豐度檢測(cè) * 一個(gè)循環(huán)每個(gè)tile照4張照片 * 2-3天,* 圖像分析 * bustard: basecalling * gerald: 序列分析,?,樣品預(yù)處理 pcr成簇 測(cè)序 拼接分析,5-6h 6-8d,（純化的基因組dna）,（基因組dna小片段 小于800bp）,（具有5-磷酸末端的粘性片段）,（去除未連接上的接頭）,（修飾末端）,（）,（基因組dna文庫）,（純化連接產(chǎn)物）,（）,（）,（）,（基因組dna小片段）,genomic dna,fragment library,mate-paired library,create library of dna fragments dna片段的文庫構(gòu)建,cluster generation序列簇的產(chǎn)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個(gè)cluster上有1000個(gè)分子 1個(gè)tile上有20,000個(gè)cluster 每個(gè)實(shí)驗(yàn)可完成3.2-4千萬個(gè)cluster),通過cluster將單分子放大、固定,oh,cluster generation: amplification,1st cycle annealing （no.1循環(huán)：退火）,diol,diol,cluster generation: amplification,blocking with ddntp () (ddntp阻斷末端),sequencing by synthesis 邊合成邊測(cè)序,1. incorporation 結(jié)合 2. scan 掃描拍照 3. cleavage 清洗,sbs cycle,first base incorporated 第一個(gè)堿基合成上,cycle 1: add sequencing reagents 加入合成所需反應(yīng)物,detect signal 檢測(cè)熒光信號(hào),cleave terminator and dye 去掉末端封閉，染色,cycle 2-n: add sequencing reagents and repeat,sequencing by synthesis (sbs),?,base calling 堿基識(shí)別,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 雙末端測(cè)序、正反雙向測(cè)序,sample preparation 樣品前準(zhǔn)備 cluster generation 分子簇的生成 sequence by synthesis 邊合成邊測(cè)序,（用于組裝較大的gene，一次最多讀100bp）,sample preparation,5,t,3,a,5,a,t,3,3,a,5,t,3,t,a,5,加雙末端,grafted flowcells,single read periodate linearization,paired end uracil specific excision reagent (user) 尿嘧啶特異性識(shí)別位點(diǎn)-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個(gè)循環(huán),sequencing 測(cè)序,denaturation and hybridization sbs3,illumina solexa,形成dna簇； 可逆阻斷技術(shù) 邊合成邊測(cè)序（sbs）,40-50g / 10天 / 一個(gè)run,讀長(zhǎng)：75bp （可雙向）,錯(cuò)誤類型： 替換,illumina solexa abi solid roche 454,solid 測(cè)序技術(shù),ab /solid workflow 工作流程,文庫制備 emulsion pcr beads enrichment 微珠沉積 連接測(cè)序 數(shù)據(jù)分析,文庫制備 emulsion pcr beads enrichment 微珠沉積 連接測(cè)序 數(shù)據(jù)分析,work flow：,序列可以用超聲波、機(jī)械剪切或酶解等方法，隨機(jī)或者定向的打斷成小片段,（大片段兩頭測(cè)序）,“mate-paired” 步驟：環(huán)化切割加接頭測(cè)序,酶切為粘性末端,對(duì)于復(fù)雜的分子環(huán)化，采用 低濃度的模板濃度進(jìn)行連接,文庫制備 emulsion pcr 微珠富集 微珠沉積 連接測(cè)序 數(shù)據(jù)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 位點(diǎn)/ 每個(gè)bead 2 billion beads/ 每個(gè)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,文庫制備 emulsion pcr 微珠富集 微珠沉積 連接測(cè)序 數(shù)據(jù)分析,work flow：,3. enrichment/微珠富集,centrifuge using a glycerol gradient 甘油梯度離心,captured beads (+ templates) in supernatant,uncaptured beads (no template) in pellet,文庫制備 emulsion pcr 微珠富集 微珠沉積 連接測(cè)序 數(shù)據(jù)分析,work flow：,4. deposite beads,3-end modification,文庫制備 emulsion pcr 微珠富集 微珠沉積 連接測(cè)序 數(shù)據(jù)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 邊連接邊測(cè)序（sbl）,50g / 大于10天 / 一個(gè)run,讀長(zhǎng)：50bp （可雙向）,錯(cuò)誤類型： 替換,illumina solexa abi solid roche 454,genome sequencer 20 syste（2005） genome sequencer flx syste（2006） gs flx titanium（2008）,發(fā)展歷程：,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 聚合測(cè)序,0.4-0.6g / 10h / 一個(gè)run,讀長(zhǎng)：400bp max：800bp,錯(cuò)誤類型： 插入&缺失,第二代測(cè)序技術(shù)小結(jié),454測(cè)序儀： 454測(cè)序儀使用的方法，經(jīng)微乳液pcr發(fā)擴(kuò)增后，攜帶有大量模板分子的微珠被放置到芯片上的微孔中。隨后使用焦磷酸法測(cè)序，每一輪測(cè)序反應(yīng)都會(huì)摻入一個(gè)核苷酸，隨后加入反應(yīng)試劑熒光素和腺苷酰硫酸。這樣在每一個(gè)小孔中每當(dāng)有聚合酶將核苷酸摻入到模板上都會(huì)發(fā)光。最后用腺苷三磷酸雙磷酸酶洗滌去掉多余的核苷酸。 (對(duì)重復(fù)序列如poly a的測(cè)定不準(zhǔn)確，因熒光信號(hào)具有累加效果) solexa測(cè)序儀：solexa測(cè)序儀使用橋式pcr直接在芯片進(jìn)行模板擴(kuò)增，然后同時(shí)加入四種經(jīng)過修飾的脫氧核苷酸，每一個(gè)核苷酸都攜帶一種熒光集團(tuán)和一個(gè)可被去除的終止基團(tuán)。經(jīng)過修飾的dna聚合酶催化引物延伸測(cè)序反應(yīng)。采集圖像、然后切除熒光標(biāo)記基團(tuán)和終止基團(tuán)，重復(fù)上述反應(yīng)，完成測(cè)序。（邊合成邊測(cè)序） solid測(cè)序儀：solid測(cè)序儀使用微乳液pcr法擴(kuò)增模板片段，然后吸附有大量擴(kuò)增片段的直徑1um的磁珠倍制成高密度測(cè)序芯片，借助使用連接酶而不是聚合酶測(cè)序法完成測(cè)序。在solid測(cè)序一中，每一次反應(yīng)都會(huì)在引物末端加上一個(gè)熒光標(biāo)記的8bp的探針，在探針中央的兩個(gè)堿基上標(biāo)記有熒光基團(tuán)，探針被連接上之后發(fā)出熒光，隨后熒光基團(tuán)部分被切除，重新系下一輪反應(yīng)。（邊連接邊測(cè)序）,第一代測(cè)序技術(shù) versus 第二代測(cè)序技術(shù),current popular sequencing platform,第三代測(cè)序技術(shù),heliscope單分子測(cè)序儀 -helicos biosciences,測(cè)序原理：邊合成邊測(cè)序 特點(diǎn)：無需對(duì)待測(cè)模板進(jìn)行擴(kuò)增，采用高靈敏度的熒光探測(cè)儀，直接對(duì)單鏈的dna模板進(jìn)行合成測(cè)序，序列讀長(zhǎng)為24到70個(gè)堿基，平均讀長(zhǎng)為32個(gè)堿基。 流程： （1）待測(cè)文庫片段化； （2）3端加poly a尾，并與固定在芯片上的poly t進(jìn)行雜交，將待測(cè)模板固定到芯片上，制成測(cè)序芯片。 （3）通過dna聚合酶將熒光標(biāo)記的單核苷酸滲入到引物上，每一輪反應(yīng)加入一種dntp。 （4）采集熒光信號(hào)，切除熒光標(biāo)記集團(tuán)，進(jìn)行下一輪測(cè)序反應(yīng)。,斯坦福大學(xué)的科學(xué)家最近利用helicos biosciences的heliscope單分子測(cè)序儀，對(duì)一名白人男子的基因組進(jìn)