華大基因 測序技術基礎原理.ppt

測序技術基礎,羅龍海 2009-03-02,sanger測序技術原理 第二代測序技術原理 第三代測序技術原理,1950,1960,1970,1980,1990,2000,2010,測序技術發(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 測序法原理,dr. fred sanger,illumina genome analyzer abi solid roche 454,第二代測序技術,二代測序技術,2005,2006,2007,年份,原理,pyrosequencing,邊合成邊測序,邊連接邊測序,454,solexa,solid,illumina solexa abi solid roche 454,第二代測序技術,可逆阻斷技術,illumina solexa flowcell,flowcell,一個 flowcell 包括8個lanes,lane 1,lane 8,each lane contains multiple tiles total 100 每個lane上有許多個tiles共計100個（見上圖） each tile is imaged four times per cycle one image per base 每個循環(huán)會對每個tile照4次相每個堿基都會成像,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,文庫構建,cluster 工作站,基因組測序,分析,* 生成“簇” * 5小時,* 開始測序 * 序列簇的豐度檢測 * 一個循環(huán)每個tile照4張照片 * 2-3天,* 圖像分析 * bustard: basecalling * gerald: 序列分析,?,樣品預處理 pcr成簇 測序 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片段的文庫構建,cluster generation序列簇的產生,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cluster上有1000個分子 1個tile上有20,000個cluster 每個實驗可完成3.2-4千萬個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 邊合成邊測序,1. incorporation 結合 2. scan 掃描拍照 3. cleavage 清洗,sbs cycle,first base incorporated 第一個堿基合成上,cycle 1: add sequencing reagents 加入合成所需反應物,detect signal 檢測熒光信號,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 雙末端測序、正反雙向測序,sample preparation 樣品前準備 cluster generation 分子簇的生成 sequence by synthesis 邊合成邊測序,（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) 尿嘧啶特異性識別位點-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個循環(huán),sequencing 測序,denaturation and hybridization sbs3,illumina solexa,形成dna簇； 可逆阻斷技術 邊合成邊測序（sbs）,40-50g / 10天 / 一個run,讀長：75bp （可雙向）,錯誤類型： 替換,illumina solexa abi solid roche 454,solid 測序技術,ab /solid workflow 工作流程,文庫制備 emulsion pcr beads enrichment 微珠沉積 連接測序 數據分析,文庫制備 emulsion pcr beads enrichment 微珠沉積 連接測序 數據分析,work flow：,序列可以用超聲波、機械剪切或酶解等方法，隨機或者定向的打斷成小片段,（大片段兩頭測序）,“mate-paired” 步驟：環(huán)化切割加接頭測序,酶切為粘性末端,對于復雜的分子環(huán)化，采用 低濃度的模板濃度進行連接,文庫制備 emulsion pcr 微珠富集 微珠沉積 連接測序 數據分析,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/ 每個bead 2 billion beads/ 每個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 連接測序 數據分析,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 連接測序 數據分析,work flow：,4. deposite beads,3-end modification,文庫制備 emulsion pcr 微珠富集 微珠沉積 連接測序 數據分析,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 邊連接邊測序（sbl）,50g / 大于10天 / 一個run,讀長：50bp （可雙向）,錯誤類型： 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 聚合測序,0.4-0.6g / 10h / 一個run,讀長：400bp max：800bp,錯誤類型： 插入&缺失,第二代測序技術小結,454測序儀： 454測序儀使用的方法，經微乳液pcr發(fā)擴增后，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序，每一輪測序反應都會摻入一個核苷酸，隨后加入反應試劑熒光素和腺苷酰硫酸。這樣在每一個小孔中每當有聚合酶將核苷酸摻入到模板上都會發(fā)光。最后用腺苷三磷酸雙磷酸酶洗滌去掉多余的核苷酸。 (對重復序列如poly a的測定不準確，因熒光信號具有累加效果) solexa測序儀：solexa測序儀使用橋式pcr直接在芯片進行模板擴增，然后同時加入四種經過修飾的脫氧核苷酸，每一個核苷酸都攜帶一種熒光集團和一個可被去除的終止基團。經過修飾的dna聚合酶催化引物延伸測序反應。采集圖像、然后切除熒光標記基團和終止基團，重復上述反應，完成測序。（邊合成邊測序） solid測序儀：solid測序儀使用微乳液pcr法擴增模板片段，然后吸附有大量擴增片段的直徑1um的磁珠倍制成高密度測序芯片，借助使用連接酶而不是聚合酶測序法完成測序。在solid測序一中，每一次反應都會在引物末端加上一個熒光標記的8bp的探針，在探針中央的兩個堿基上標記有熒光基團，探針被連接上之后發(fā)出熒光，隨后熒光基團部分被切除，重新系下一輪反應。（邊連接邊測序）,第一代測序技術 versus 第二代測序技術,current popular sequencing platform,第三代測序技術,heliscope單分子測序儀 -helicos biosciences,測序原理：邊合成邊測序 特點：無需對待測模板進行擴增，采用高靈敏度的熒光探測儀，直接對單鏈的dna模板進行合成測序，序列讀長為24到70個堿基，平均讀長為32個堿基。 流程： （1）待測文庫片段化； （2）3端加poly a尾，并與固定在芯片上的poly t進行雜交，將待測模板固定到芯片上，制成測序芯片。 （3）通過dna聚合酶將熒光標記的單核苷酸滲入到引物上，每一輪反應加入一種dntp。 （4）采集熒光信號，切除熒光標記集團，進行下一輪測序反應。,斯坦福大學的科學家最近利用helicos biosciences的heliscope單分子測序儀，對一名白人男子的基因組進