Generation of long insert pairs using a Cre-LoxP Inverse PCR approach.
Generation of long insert pairs using a Cre-LoxP Inverse PCR approach.
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DOI：
10.1371/journal.pone.0029437
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Chen F
中科院分区：
文献类型：
--
作者：
Peng Z;Zhao Z;Nath N;Froula JL;Clum A;Zhang T;Cheng JF;Copeland AC;Pennacchio LA;Chen F
大插入配对读段对从头组装的总体成功以及遗传和获得性结构变体的发现具有重大影响。配偶对读段的位置信息通常通过解析重复元件和/或排序重叠群来改善基因组组装。目前可用于构建此类文库的方法具有一个或多个限制，例如相对小的插入物尺寸;不能区分两端的连接;和/或低通量。我们开发了一种新的方法，Cre-LoxP反向PCR配对末端（CLIP-PE），该方法利用了（1）Cre-LoxP重组系统有效地环化大DNA片段，（2）反向PCR富集含有大DNA片段两端的所需产物，和（3）使用限制酶在连接片段末端之间引入可识别的连接位点并提高自身连接效率。我们已经成功地创建了高达22 kb的CLIP-PE文库，这些文库富含信息性读段对，并且小片段背景低。这些文库已经证明了改善基因组组装的能力。CLIP-PE方法可以与现有和未来的下一代测序平台一起实施。
Large insert mate pair reads have a major impact on the overall success of de novo assembly and the discovery of inherited and acquired structural variants. The positional information of mate pair reads generally improves genome assembly by resolving repeat elements and/or ordering contigs. Currently available methods for building such libraries have one or more of limitations, such as relatively small insert size; unable to distinguish the junction of two ends; and/or low throughput. We developed a new approach, Cre-LoxP Inverse PCR Paired-End (CLIP-PE), which exploits the advantages of (1) Cre-LoxP recombination system to efficiently circularize large DNA fragments, (2) inverse PCR to enrich for the desired products that contain both ends of the large DNA fragments, and (3) the use of restriction enzymes to introduce a recognizable junction site between ligated fragment ends and to improve the self-ligation efficiency. We have successfully created CLIP-PE libraries up to 22 kb that are rich in informative read pairs and low in small fragment background. These libraries have demonstrated the ability to improve genome assemblies. The CLIP-PE methodology can be implemented with existing and future next-generation sequencing platforms.
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影响因子：
14.9
作者：
Kelley, JM;Field, CE;Adams, MD
通讯作者：
Adams, MD
影响因子：
5.6
作者：
STERNBERG, N;HAMILTON, D;HOESS, R
通讯作者：
HOESS, R
DOI：
10.1073/pnas.2536670100
发表时间：
2003-12-23
影响因子：
11.1
作者：
Matsumura, H;Reich, S;Terauchi, R
通讯作者：
Terauchi, R
DOI：
10.1093/bioinformatics/btp352
发表时间：
2009-08-15
期刊：
Bioinformatics (Oxford, England)
影响因子：
--
作者：
Li H;Handsaker B;Wysoker A;Fennell T;Ruan J;Homer N;Marth G;Abecasis G;Durbin R;1000 Genome Project Data Processing Subgroup
通讯作者：
1000 Genome Project Data Processing Subgroup
影响因子：
7
作者：
Zerbino, Daniel R.;Birney, Ewan
通讯作者：
Birney, Ewan