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1 HEREDITAS (Beijing) , 33(12): ISSN 综述 DOI: /SP.J 新一代测序技术在植物转录组研究中的应用梁烨, 陈双燕, 刘公社, 随着 DNA 测序技术的发展, 新一代测序技术以其高通量低成本的特点, 成为越来越多的生物学研究者在开展工作时的首选在所有的新一代测序技术中, 454 测序系统是最早实现商业化且发展相对成熟的一种, 目前被广泛的应用于各个领域的生物学研究中文章以 454 测序系统为例, 综述了新一代测序系统的原理优缺点, 及其在植物转录组研究中的应用, 并对其在植物研究领域中可能的发展应用方向进行了展望新一代测序平台 ; 454 测序技术 ; 植物转录组 ; EST; SNP Application of next generation sequencing techniques in plant transcriptome LIANG Ye, CHEN Shuang-Yan, LIU Gong-She The Research and Development Laboratory for Plant Resources, Institute of Bontany, Chinese Acdemy of Sciences, Beijing , China Abstract: With the development of DNA sequencing techniques, the next-generation sequencing (NGS) techniques with the characteristics of high-throughput and low cost have become the first choice for more and more researchers to carry out the biological researches. Among the next-generation sequencing techniques, the 454 sequencing platform is the first commercially available and relatively mature one and widely used in various fields of biological research. Taking 454 sequencing platform as an example, we illustrate the advantages and disadvantages of NGS technical principles, review their applications in plant transcriptome, and outlook their future development and applications in plant research field. Keywords: next generation sequencing techniques (NGS); 454 pyrosequencing technology; plant transcriptome; EST; SNP Sanger [1] DNA,, [2~4],,, [5], 收稿日期 : ; 修回日期 : 基金项目 : ( 973 )( 2007CB108905) 作者简介 :,, hawkliangsu27@sina.com 通讯作者 :,,, sychen@ibcas.ac.cn,,, liugs@ibcas.ac.cn 网络出版时间 : :52:38 URL:

2 1318 HEREDITAS (Beijing) ,, Illumina ABI Solexa SOLid,, 454 [6], ( ) [7],, SNP (454 Life Sciences) 2000, 2005 ( Roche Genome Sequencer 20 (GS20), GS20, Sanger, 2007 Genome Sequencer FLX System (GS FLX), GS FLX GS, Genome Sequencer FLX Titanium Series reagents, GS FLX Titanium, GS FLX, GS FLX,,, GS Junior System, - GS Junior Titanium, 400 bp, GS FLX 1.2 Illumina/Solexa Illumina Solexa Turcatti 4, Genome Analyzer HiSeq1000 HiSeq2000 MiSeq Genome Analyzer bp, 14 d Gb, HiSeq1000 HiSeq2000, TruSeq V3, 30 60, 270~300 Gb 540~600 Gb MiSeq,, 1 Gb, 1.3 SOLiD ABI SOLiD McKernan , SOLiD3, , SOLiD3 50Gb 2010, ABI SOLiD4, 100Gb, , ABI SOLiD5500xl SOLiD5500xl 30~45 Gb, , 300 Gb , 454, (Seqencing-by-synthesis, SBS) 4 DNA,, DNA, (NNN) n +NTP (NNN) n+1 +PPi, DNA, ATP, ATP, 4 NTP, NTP, DNA [6]

3 12 : Illumina/Solexa Illumina, PCR, PCR,, PCR, Bst, ,, ( ), DNA 4, 3, 4 4,, 2.3 SOLiD SOLiD, DNA empcr, 1 mol/l empcr,,,,,, 5,6,, 5 10,,,,, 2.4,,,,,,, 454GS FLX, Lundin [9], DNA DNA,, SOLiD, Farias-Hesson [10], 6 DNA, 17 h 32 empcr DNA DNA Illumina, Sengupta [11] 10 ng RNA mrna RNA,, Solexa, GS FlX [8] 单链 DNA 模板文库的建立 454 DNA PCR BACs cdna, RNA, cdna BACs, 300 bp 800 bp, PCR RNA,,, 3 5,

4 1320 HEREDITAS (Beijing) 对该文库固定并进行 empcr 扩增 DNA, ( ) DNA, PCR,, DNA empcr,, 通过 sequencing-by-synthesis 方法测序并生成数据 Bst, PicoTiter PicoTiter,, ( ATP ), FLX, ATP,, FLX CCD, 用各种生物信息学工具对数据进行分析 GS DNA XY GS FLX 10 h, GS Junior 10,, bp ; ; 2.6 Sanger, 1, SOLiD Illumina, 454 (de novo sequencing) % bp, , 40 GB, 454 [8],, 454, 454 Illumina SOLid,,, Illumina GA ;, Roche GS FLX 3 454,,,,,,, 454, (Expressed sequence tag, EST) EST cdna, 150~400 bp [13] EST, contig, cdna EST, 3.1,

5 12 : 1321 [12] 1 3 种新一代测序技术比较 /454 s GS FLX Titanium Illumina/Solexa s HiSeq2000 Life/APG s SOLiD5500xl /, MP/emPCR, MP/solid-phase, MP/emPCR (bp) 400~ (d) (Gb/ ) ~ ,,, ( 3 Mb), 16S, RNA, (, ), RNA,,, 454 [14, 15],, 454, (10%), 50% Weber [15] ESTs EST, 454, Cheung [16] GS20 (Medicago truncatula Gaertn.) EST, 454 EST, 454, Sato [17] Sanger 454 (Jatropha curcas L.) bp, Contig Singlet 95%, G+C 34.3% ,, 454 (Artemisia annua L.) [18] (Olea europaea L.) [19] (Zea mays L.) [20] (Eucalyptus grandis) [21] (Medicago sativa L.) [16] (Leymus chinensis (Trin.) Tzvel.) [22] (Paspalum dilatatum Poir) (Brachiaria brizantha Stapf.) [23], 454 [24] 454 GS FLX Titanium 2 (Salvia miltiorrhiza Bge.),, EST, 414 bp GenBank EST, unigene, EST BLAST Gene Ontology 27, 29, P450 70, , 454 [25] [26~28]

6 1322 HEREDITAS (Beijing) SNP, DNA (SSR) (SNP), SNP [13], SNP DNA, 454, SNP SSR Novaes [21] [25, 29, 30] (E. grandis) Cogan (Lolium perenne L.), Barbazuk [31] SNP Barbazuk, SNP B EST Mo EST SNP B , SNP SNP 85% Sanger 454 SNP, SNP Parchman [32] (Pinus Contorta Dougl.) SNP SSR, SSR PCR Guo [33] (Cucumis sativus L.) SSR SNP SSR, QDD [34],, /,, ( ),, , Schafleitner [35] Sanger 454 (Ipomoea batatas (L.) Lam), reads,, , 195 Shu [36] (Giycine max(l) Merrill) 454, SNP SNP,, Glyma07g UTR CAPS GS FLX (SAGE) SAGE 75 bp, SuperSAGE [37] EST SAGE EST, [38] Alagna [19] 2 (O. europaea), 3.4, EST,, 454,, Sun [39] 454 (Panax quinquefolius L.),, , 427, KEGG A

7 12 : 1323, 150 P450(CYP450) 235, 454, 454 EST, 黃 (A. annua) [18] (P. dilatatum) (B. brizantha) [23] (Epimedium sagittatum (Sieb. Et Zucc.) Maxim.) [40] (Papaver somniferum L.) [41] (Momordica charantia L.) [42] (Huperzia serrata (Thunb. ex Murray) Trev.) (Phlegmariurus carinatus (Desv. ex Poir.) Ching.) [43] (Glycyrrhiza uralensis Fisch.) [44] (Taxus cuspidata Sieb. et Zucc.) [45] 3.5,,,, Timme Delwiche [46] Sanger 454 (Coleochaete orbicularis L.) (Spirogyra pratensis L.), unigene, BLAST srna, srna srna, srna RNA, sirnas sirnas, srna, srna srna Chen [47] NGS 26 srna 24 srna srna, mirna, mir396, 454 [16, 31], [48], [21, 22, 31] 4,, EST, EST,, 2%~5% mrna cdna DNA,, 454, bp, bp,,,, 454,,,, SNP, 454,

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Lezione 11 La seconda fase: la scienza post-accademica Dal CUDOS al PLACE Dal PUS al marketing della scienza L economia della conoscenza e suoi proble

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