高通量測序技術(shù)簡介

1、第五部分：其他類型的芯片,微縮芯片實驗室（Lab-on-a-chip) 藥物運輸芯片 生理功能輔助芯片 納米芯片,Next Generation Sequencing,Sample fragmentationLibrary preparationSequencing reactionData analysis,Roche 454 焦磷酸測序Pyrophosphate Sequencing,Illumina Sole

2、xa 合成測序Sequence by Synthesize,ABI SOLiD 連接法測序Sequence by Ligation,,第六部分 高通量測序技術(shù)簡介,,Roche 454 焦磷酸測序Pyrophosphate Sequencing基本原理,454 sequencing： Emulsion PCR (emPCR),Mix DNA Library & capture beads(limited diluti

3、on),,“Break micro-reactors”Isolate DNA containing beads,Generation of millions of clonally amplified templates on each beadNo cloning and colony picking,Create “Water-in-oil” emulsion,+ PCR Reagents,+ Emulsion Oil,P

4、erform emulsion PCR,,,,,Adapter carrying library DNA,A,B,Micro-reactors,,,,,,Adapter complement,,Enrich,Anneal Seqprimer,,Centrifuge Step,Load Enzyme Beads,454 sequencing： Deposition of DNA beads into the PicoTiter

5、?Plate,,Load beads into PicoTiter?Plate,,Illumina Solexa 合成測序Sequence by Synthesize基本原理,Clonal Single Molecule Arrays單分子克隆,~1000 molecules per ~ 1 µm cluster ~1000 clusters per 100 µm square~40 million clu

6、sters per experiment,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,Prepare DNA fragments,,,Ligate adapters,Attach single molecules to surface,,Amplify to form clusters,Reversible Terminator Chemistry可逆終止反應(yīng),All 4 labelled nucleotides i

7、n 1 reaction,Sequencing-by-Synthesis (SBS),First base incorporated,Cycle 1: Add sequencing reagents,Remove unincorporated bases,Detect signal,Cycle 2-n: Add sequencing reagents and repeat,1、每輪測序反應(yīng)加入四種帶有熒光標(biāo)記的dNTP，末端帶有可以

8、被去除的阻斷基團(tuán)2、每輪反應(yīng)只能整合一個核苷酸，儀器讀取相應(yīng)的熒光信號3、信號讀取結(jié)束，用化學(xué)方法去除阻斷基團(tuán)，進(jìn)行下一輪測序反應(yīng),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根據(jù)每個點每輪反應(yīng)讀取的熒光信號序列，轉(zhuǎn)換成相應(yīng)的DNA序列,Base ca

9、lling from the raw data,Solexa 測序 Workflow,,ABI SOLiD 連接法測序Sequence by Ligation基本原理,文庫制備：微珠單分子克隆,1024種8堿基探針4色熒光，4種雙核苷酸，每色熒光有256個探針(4^6),SOLiD 利用探針的連接反應(yīng)讀取模板的DNA序列,連接法測序（一）,每個探針進(jìn)行檢測的兩個堿基后面有三個匹配堿基，因此一條測序引物讀取的序列是不完整的,,測序

10、引物與adapter退火,探針連接，檢測熒光,切除熒光基團(tuán),第二輪探針連接，檢測熒光,切除熒光基團(tuán),連接法測序（二）,測序引物沿著Adapter移動5次，確保每個位點都被檢測,連接法測序（三）,0位置是Adapter的最后一個堿基，因此只檢測一次，該堿基是進(jìn)行解碼所必須的。,Advantage & disadvantage,454 sequencing讀取長度大，400bp可以對未知基因組進(jìn)行從頭測序de novo s

11、equencing當(dāng)遇到polymer時，如AAAAAA等，熒光強(qiáng)度和堿基個數(shù)不成線性關(guān)系，判定重復(fù)堿基個數(shù)有困難Solexa sequencing高度自動化的系統(tǒng)讀取片段多，適合進(jìn)行大量小片段的測序，如microRNA profiling基于可逆反應(yīng)，隨反應(yīng)輪數(shù)增加，效率降低，信號衰減，讀取序列較短，給de novo sequencing 拼接帶來困難SOLiD sequencing每個堿基讀取兩次非常高的準(zhǔn)確性，

12、特別是對于SNP的檢測靈活的系統(tǒng)，完善的磁珠編碼系統(tǒng)，可以進(jìn)行樣品的pooling，分割測序區(qū)域讀取長度受連接反應(yīng)的輪數(shù)限制，給de novo sequencing 拼接帶來困難,高通量測序的應(yīng)用,De novo 測序基因深度測序（genome re-sequencing）轉(zhuǎn)錄組深度測序（transcriptome re-sequencing）Digital expression profilingChIP-seqMeth

13、y-seq,Transcriptome resequencing：,malignant pleural mesotheliomas (MPMs) ：惡性胸膜間皮瘤pulmonary adenocarcinoma (ADCA)：肺腺癌,Transcriptome characteristics,Solid line： at least one readDashed line：at least 20 reads,Expression d

14、ifference between MPM and ADCA sample compare to a lung tissue control,Analysis of percent-age of reads containing known coding region SNVs in the six tissue samples.,SNV： Single Nucleotide Substitution Variant,,,Digi

15、tal expression profiling（1）：人大腦組織與UHR（Universal Human Reference）的表達(dá)差異,Digital expression profiling &microRNA re-sequencing：,hESC： human embryonic stem cellsEB： embryoid bodies,,ChIP-seq（1）：人一號染色體DNA-蛋白相互作用,Ch

16、IP-seq（2）：,Sequenced short reads (typically
25–50 bp) from ChIP-Seq experiments are ?rst mapped onto the reference genome. The mapped reads are then used to estimate statistical parameters, which include the estimation

17、of the average length Fof sequenced DNA fragments.,Methy-seq（1）：腫瘤和MCF7細(xì)胞系中 BRCA!啟動子區(qū)域的甲基化差異,Some highlights:Correlation between ChIP-Seq and his prior SAGE-like method (called GMAT) has r=0.906‘However the resolut

18、ion with ChIP-Seq was dramatically higher. Furthermore, ChIP-Seq was more sensitive and generated less false-negative regions’12,726 genes whose transcription levels are known in CD4+ T-cells were correlated with the h

19、istone modifications and 35,961 Pol II binding site ‘islands’ were identified‘This cost-effective method produces digital-quality data and should find broad applications in our efforts to understand the contribution of

20、 the human epigenomes in gene expression and epigenetic inheritance’,Methy-seq（2）：,部分參考文獻(xiàn)閱讀,Genome re-sequencing van Orsouw N J, Hogers R C, Janssen A, et al. Complexity reduction of polymorphic sequences (CRoPS): a nov

21、el approach for large-scale polymorphism discovery in complex genomes. PLoS ONE, 2007, 2(11): e1172Hillier L W, Marth G T, Quinlan A R, et al. Whole-genome sequencing and variant discovery in C. elegans. Nat Methods, 20

22、08, 5(2): 183—188Transcriptome re-sequencingMortazavi A, Williams B A, McCue K, et al. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods, 2008, 5(7): 621—628Sugarbaker D J, Richards W G, Gordon

23、G J, et al. Transcriptome sequencing of malignant pleural mesothelioma tumors. Proc Natl Acad Sci USA, 2008, 105(9): 3521—3526 Digital expression profilingRuby J G, Jan C, Player C, et al. Large-scale sequencing reveal

24、s 21U-RNAs and additional microRNAs and endogenous siRNAs in C. elegans. Cell, 2006, 127(6): 1193—1207Morin R D, O'Connor M D, Griffith M, et al. Application of massively parallel sequencing to microRNA profiling an

25、d discovery in human embryonic stem cells. Genome Res, 2008, 18(4): 610—621ChIP-seqJohnson D S, Mortazavi A, Myers R M, et al. Genome-wide mapping of in vivo protein-DNA interactions. Science, 2007, 316(5830): 1497—150

26、2Robertson G, Hirst M, Bainbridge M, et al. Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing. Nat Methods, 2007, 4(8): 651—657,藤曉坤，肖華勝*，基因芯片與高通量DNA測序技術(shù)前

27、景分析。中國科學(xué)C輯：生命科學(xué)。 2008：（38）10：1-9。Teng X, Xiao H. Perspectives of DNA microarray and next-generation DNA sequencing technologies. Sci China C Life Sci. 2009 ;52(1):7-16,高通量測序技術(shù)雖然建立的時間不長, 但是在基因組的各個研究領(lǐng)域都顯示出其非凡的魅力, 而且日益顯示出其

28、對基因芯片“取而代之”的咄咄態(tài)勢。 那么, 基因芯片向何處去呢？ 基因芯片技術(shù)經(jīng)過近15年的發(fā)展已經(jīng)形成了一個系統(tǒng)的平臺。 深度測序要建立這樣的一個體系同樣需要若干年的完善。芯片雜交結(jié)果直觀, 分析快速, 適合對大量生物學(xué)樣,品進(jìn)行已知信息的檢測, 同時芯片數(shù)據(jù)分析有成熟完整的理論, 為后期數(shù)據(jù)分析提供強(qiáng)大的支持。 基因芯片的缺點, 就在于它是一個“封閉系統(tǒng)”, 它只能檢測人們已知序列的特征(或有限的變異)。 而深度測序的強(qiáng)項