高通量測(cè)序技術(shù)簡(jiǎn)介

1、next generation sequencingsample fragmentationlibrary preparationsequencing reactiondata analysisroche 454 焦磷酸測(cè)序pyrophosphate sequencing基本原理454 sequencing： emulsion pcr (empcr)mix dna library & capture beads(limited dilution)“break micro-reactors”isolate dna containing beads generation of millio

2、ns of clonally amplified templates on each bead no cloning and colony pickingcreate “water-in-oil” emulsion + pcr reagents + emulsion oil perform emulsion pcr adapter carrying library dnaabmicro-reactors adapter complementenrichanneal seqprimercentrifuge stepload enzyme beads454 sequencing： depositi

3、on of dna beads into the picotiterplateload beads into picotiterplate illumina solexa 合成測(cè)序sequence by synthesize基本原理clonal single molecule arrays單分子克隆1000 molecules per 1 m cluster 1000 clusters per 100 m square40 million clusters per experimentprepare dna fragmentsligate adaptersattach single molec

4、ules to surfaceamplify to form clusters20 micronssequencereversible terminator chemistry可逆終止反應(yīng)o ppphnnoocleavagesitefluor3blocknext cycleincorporationdetectiondeblock; fluor removalodnahnnoo3o5free 3 endxohall 4 labelled nucleotides in 1 reactionsequencing-by-synthesis (sbs)5gtcagtcagtcagt35cagtcatc

5、acctagcgtafirst base incorporatedcycle 1: add sequencing reagentsremove unincorporated basesdetect signalcycle 2-n: add sequencing reagents and repeat1、每輪測(cè)序反應(yīng)加入四種帶有熒光標(biāo)記的dntp，末端帶有可以被去除的阻斷基團(tuán)2、每輪反應(yīng)只能整合一個(gè)核苷酸，儀器讀取相應(yīng)的熒光信號(hào)3、信號(hào)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(cè)序反應(yīng)123789456t t t t t t t g t t g c t a c g a t the identi

6、ty of each base of a cluster is read off from sequential images根據(jù)每個(gè)點(diǎn)每輪反應(yīng)讀取的熒光信號(hào)序列，轉(zhuǎn)換成相根據(jù)每個(gè)點(diǎn)每輪反應(yīng)讀取的熒光信號(hào)序列，轉(zhuǎn)換成相應(yīng)的應(yīng)的dna序列序列base calling from the raw datasolexa 測(cè)序測(cè)序 workflowabi solid 連接法測(cè)序sequence by ligation基本原理文庫(kù)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(gè)探針(46)solid 利用探針的連接反應(yīng)讀取模板的利用探針的連接反應(yīng)讀取模板的dna序列序列連

7、接法測(cè)序（一）每個(gè)探針進(jìn)行檢測(cè)的兩個(gè)堿基后面有三個(gè)匹配堿基，因此一條測(cè)序引物讀取的序列是不完整的每個(gè)探針進(jìn)行檢測(cè)的兩個(gè)堿基后面有三個(gè)匹配堿基，因此一條測(cè)序引物讀取的序列是不完整的測(cè)序引物與測(cè)序引物與adapteradapter退火退火探針連接，檢測(cè)熒光探針連接，檢測(cè)熒光切除熒光基團(tuán)切除熒光基團(tuán)第二輪探針連接，檢測(cè)熒光第二輪探針連接，檢測(cè)熒光切除熒光基團(tuán)切除熒光基團(tuán)連接法測(cè)序（二）測(cè)序引物沿著測(cè)序引物沿著adapter移動(dòng)移動(dòng)5次，確保每個(gè)位點(diǎn)都被檢測(cè)次，確保每個(gè)位點(diǎn)都被檢測(cè)連接法測(cè)序（三）0位置是位置是adapter的最后一個(gè)堿基，因此只檢測(cè)一次，的最后一個(gè)堿基，因此只檢測(cè)一次，該堿基是進(jìn)行解

8、碼所必須的。該堿基是進(jìn)行解碼所必須的。advantage & disadvantage454 sequencing讀取長(zhǎng)度大，400bp可以對(duì)未知基因組進(jìn)行從頭測(cè)序de novo sequencing當(dāng)遇到polymer時(shí)，如aaaaaa等，熒光強(qiáng)度和堿基個(gè)數(shù)不成線性關(guān)系，判定重復(fù)堿基個(gè)數(shù)有困難solexa sequencing高度自動(dòng)化的系統(tǒng)讀取片段多，適合進(jìn)行大量小片段的測(cè)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(hào)衰減，讀取序列較短，給de novo sequencing 拼接帶來(lái)困難solid sequencing每個(gè)堿基讀取兩次非常高

9、的準(zhǔn)確性，特別是對(duì)于snp的檢測(cè)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(cè)序區(qū)域讀取長(zhǎng)度受連接反應(yīng)的輪數(shù)限制，給de novo sequencing 拼接帶來(lái)困難高通量測(cè)序的應(yīng)用de novo 測(cè)序基因深度測(cè)序（genome re-sequencing）轉(zhuǎn)錄組深度測(cè)序（transcriptome re-sequencing）digital expression profilingchip-seqmethy-seqtranscriptome resequencing：malignant pleural mesotheliomas (mpms) ：惡性胸膜間皮瘤pulmo

10、nary adenocarcinoma (adca)：肺腺癌transcriptome characteristicssolid line： at least one readdashed line：at least 20 readsexpression difference between mpm and adca sample compare to a lung tissue controlanalysis of percent-age of reads containing known coding region snvs in the six tissue samples.snv： s

11、ingle nucleotide substitution variant tag sequencebrainuhrsymbol gene descriptiongatcaaaccaaggcccaggc118861rpl29ribosomal protein l29gatcactgttaatgatttgc162163prdx2peroxiredoxin 2gatcagtgtcttttcagcac8535pfn2profilin 2gatcatcatgaccaatgaaa730stmn4stathmin-like 4gatcatgctggctgcaaaga9196cox5bcytochrome

12、c oxidase subunit vbgatccaaacccaagtcttga480gabra1gamma-aminobutyric acid (gaba) a receptor, alpha 1gatccaagataaagaaggca266271ubbubiquitin bgatcccagactggttcttga2171538ring1ring finger protein 1gatccccaattgactcagag910diras2diras family, gtp-binding ras-like 2gatccggggctgcaggcttg1010phf20phd finger pro

13、tein 20gatcctacagaagtggagct0113igjimmunoglobulin j polypeptidegatcctagtaattgcctaga46799fn1fibronectin 1gatcctgcgggagtctcccg4112nfixnuclear factor i/x (ccaat-binding transcription factor)gatcctgtgaaggcctggaa042top2atopoisomerase (dna) ii alpha 170kdagatcgagacacgtgatggga8346krt8keratin 8gatcgaggacagtg

14、caacca059itgb7integrin, beta 7gatctcaatgccaatcctcc24081basp1brain abundant, membrane attached signal protein 1gatctgcacgccgctgaccc510drd1ipdopamine receptor d1 interacting proteingatctgtgcccagagatggg7160gfapglial fibrillary acidic proteingatctgtggagaatgtacac13269aplp2amyloid beta (a4) precursor-like

15、 protein 2digital 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 bodieschip-seq（1）：人一號(hào)染色體dna-蛋白相互作用chip-seq（2）： sequenced short reads (typically 2550 bp) from chip-seq experiments

16、are first mapped onto the reference genome. the mapped reads are then used to estimate statistical parameters, which include the estimation of the average length fof sequenced dna fragments.methy-seq（1）：腫瘤和mcf7細(xì)胞系中 brca!啟動(dòng)子區(qū)域的甲基化差異some highlights:correlation between chip-seq and his prior sage-like

17、method (called gmat) has r=0.906however the resolution with chip-seq was dramatically higher. furthermore, chip-seq was more sensitive and generated less false-negative regions12,726 genes whose transcription levels are known in cd4+ t-cells were correlated with the histone modifications and 35,961

18、pol ii binding site islands were identifiedthis cost-effective method produces digital-quality data and should find broad applications in our efforts to understand the contribution of the human epigenomes in gene expression and epigenetic inheritancemethy-seq（2）：部分參考文獻(xiàn)閱讀genome re-sequencing van orso

19、uw n j, hogers r c, janssen a, et al. complexity reduction of polymorphic sequences (crops): a novel 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

20、. nat methods, 2008, 5(2): 183188transcriptome re-sequencingmortazavi a, williams b a, mccue k, et al. mapping and quantifying mammalian transcriptomes by rna-seq. nat methods, 2008, 5(7): 621628sugarbaker d j, richards w g, gordon g j, et al. transcriptome sequencing of malignant pleural mesothelioma tumors. proc natl acad sci usa, 2008, 105(9): 35213526 digital expression profilingruby j