Paired-end sequencing of long-range DNA fragments for de novo assembly of large, complex Mammalian genomes by direct intra-molecule ligation.
Paired-end sequencing of long-range DNA fragments for de novo assembly of large, complex Mammalian genomes by direct intra-molecule ligation.
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DOI：
10.1371/journal.pone.0046211
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Zhang X
中科院分区：
文献类型：
--
作者：
Asan;Geng C;Chen Y;Wu K;Cai Q;Wang Y;Lang Y;Cao H;Yang H;Wang J;Zhang X
来自下一代测序（NGS）技术的相对短的读取长度仍然对复杂哺乳动物基因组的从头组装构成挑战。一个重要的解决方案是使用从长距离DNA片段（>1 kb）实验获得的配对末端（PE）序列信息。在这里，我们表征并扩展了一种基于NGS的直接分子内连接（或无分子接头环化）的远程PE文库构建方法。我们发现，该方法对于2-至5- kb DNA片段的PE测序表现稳定，并且可以延伸至10-20 kb（甚至在极端情况下，高达10 - 35 kb）。我们还表征了低质量输入DNA对该方法的影响，并开发了使用有限输入DNA（<1 μg）的基于全基因组扩增（WGA）的方案。使用这个PE数据集，我们准确地将第一个测序的亚洲基因组YanHuang（YH）基因组组装成大于2 Mb的支架N50大小，这比仅用小插入PE读取（17 kb）产生的初始大小大100倍以上。此外，我们映射两个7至8 kb的插入在YH基因组中使用较大的插入大小的远程PE数据。总之，我们在这里证明了这种长距离PE测序方法的有效性及其使用NGS短读段从头组装大型复杂基因组的用途。
The relatively short read lengths from next generation sequencing (NGS) technologies still pose a challenge for de novo assembly of complex mammal genomes. One important solution is to use paired-end (PE) sequence information experimentally obtained from long-range DNA fragments (>1 kb). Here, we characterize and extend a long-range PE library construction method based on direct intra-molecule ligation (or molecular linker-free circularization) for NGS. We found that the method performs stably for PE sequencing of 2- to 5- kb DNA fragments, and can be extended to 10–20 kb (and even in extremes, up to ∼35 kb). We also characterized the impact of low quality input DNA on the method, and develop a whole-genome amplification (WGA) based protocol using limited input DNA (<1 µg). Using this PE dataset, we accurately assembled the YanHuang (YH) genome, the first sequenced Asian genome, into a scaffold N50 size of >2 Mb, which is over100-times greater than the initial size produced with only small insert PE reads(17 kb). In addition, we mapped two 7- to 8- kb insertions in the YH genome using the larger insert sizes of the long-range PE data. In conclusion, we demonstrate here the effectiveness of this long-range PE sequencing method and its use for the de novo assembly of a large, complex genome using NGS short reads.
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影响因子：
1.7
作者：
Jurka, J;Kapitonov, VV;Walichiewicz, J
通讯作者：
Walichiewicz, J
影响因子：
7
作者：
Li, Ruiqiang;Zhu, Hongmei;Wang, Jun
通讯作者：
Wang, Jun
亚洲个体的二倍体基因组序列
影响因子：
64.8
作者：
Wang, Jun;Wang, Wei;Li, Ruiqiang;Li, Yingrui;Tian, Geng;Goodman, Laurie;Fan, Wei;Zhang, Junqing;Li, Jun;Zhang, Juanbin;Guo, Yiran;Feng, Binxiao;Li, Heng;Lu, Yao;Fang, Xiaodong;Liang, Huiqing;Du, Zhenglin;Li, Dong;Zhao, Yiqing;Hu, Yujie;Yang, Zhenzhen;Zheng, Hancheng;Hellmann, Ines;Inouye, Michael;Pool, John;Yi, Xin;Zhao, Jing;Duan, Jinjie;Zhou, Yan;Qin, Junjie;Ma, Lijia;Li, Guoqing;Yang, Zhentao;Zhang, Guojie;Yang, Bin;Yu, Chang;Liang, Fang;Li, Wenjie;Li, Shaochuan;Li, Dawei;Ni, Peixiang;Ruan, Jue;Li, Qibin;Zhu, Hongmei;Liu, Dongyuan;Lu, Zhike;Li, Ning;Guo, Guangwu;Zhang, Jianguo;Ye, Jia;Fang, Lin;Hao, Qin;Chen, Quan;Liang, Yu;Su, Yeyang;San, A.;Ping, Cuo;Yang, Shuang;Chen, Fang;Li, Li;Zhou, Ke;Zheng, Hongkun;Ren, Yuanyuan;Yang, Ling;Gao, Yang;Yang, Guohua;Li, Zhuo;Feng, Xiaoli;Kristiansen, Karsten;Wong, Gane Ka-Shu;Nielsen, Rasmus;Durbin, Richard;Bolund, Lars;Zhang, Xiuqing;Li, Songgang;Yang, Huanming;Wang, Jian
通讯作者：
Wang, Jian
影响因子：
3.1
作者：
Haussler, David;O'Brien, Stephen J.;Turner, Steve
通讯作者：
Turner, Steve
DOI：
10.1073/pnas.1232418100
发表时间：
2003-06-24
影响因子：
11.1
作者：
Volik, S;Zhao, SY;Collins, C
通讯作者：
Collins, C