Reconstructing complex regions of genomes using long-read sequencing technology.
Reconstructing complex regions of genomes using long-read sequencing technology.
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DOI：
10.1101/gr.168450.113
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发表时间：
2014-04
期刊：
影响因子：
7
通讯作者：
Eichler EE
中科院分区：
文献类型：
--
作者：
Huddleston J;Ranade S;Malig M;Antonacci F;Chaisson M;Hon L;Sudmant PH;Graves TA;Alkan C;Dennis MY;Wilson RK;Turner SW;Korlach J;Eichler EE
获得高质量的序列连续性的复杂区域的最近节段复制仍然是完成基因组组装的主要挑战之一。在人类和小鼠基因组中，这是通过使用昂贵且费力的毛细管测序方法靶向大插入克隆来实现的。然而，克隆插入物的桑格鸟枪测序现在已经基本上被放弃，使得这些区域中的大多数在主要由下一代测序杂交方法产生的较新的基因组组装中未被解决。在这里，我们表明，有可能解决在全基因组范围内复杂但在隔离中简单的区域，其时间和成本仅为传统方法的一小部分，使用Pacific Biosciences（PacBio）的长读单分子实时（SMRT）测序和组装技术。我们测序和组装BAC克隆对应于染色体17 q21.31的1.3-Mbp的复合物区域，证明99.994%的同一性相同克隆的桑格组件。我们使用Illumina测序靶向44个差异，发现PacBio和桑格组装体共享相当数量的经验证变体，尽管具有不同的序列背景偏差。最后，我们针对黑猩猩基因组的一个组装不良的766-kbp重复区域，并解决了传统整理方法的成本和时间的一小部分的结构和组织。我们的数据表明了一条将基因组升级到更高质量成品状态的直接途径。
Obtaining high-quality sequence continuity of complex regions of recent segmental duplication remains one of the major challenges of finishing genome assemblies. In the human and mouse genomes, this was achieved by targeting large-insert clones using costly and laborious capillary-based sequencing approaches. Sanger shotgun sequencing of clone inserts, however, has now been largely abandoned, leaving most of these regions unresolved in newer genome assemblies generated primarily by next-generation sequencing hybrid approaches. Here we show that it is possible to resolve regions that are complex in a genome-wide context but simple in isolation for a fraction of the time and cost of traditional methods using long-read single molecule, real-time (SMRT) sequencing and assembly technology from Pacific Biosciences (PacBio). We sequenced and assembled BAC clones corresponding to a 1.3-Mbp complex region of chromosome 17q21.31, demonstrating 99.994% identity to Sanger assemblies of the same clones. We targeted 44 differences using Illumina sequencing and find that PacBio and Sanger assemblies share a comparable number of validated variants, albeit with different sequence context biases. Finally, we targeted a poorly assembled 766-kbp duplicated region of the chimpanzee genome and resolved the structure and organization for a fraction of the cost and time of traditional finishing approaches. Our data suggest a straightforward path for upgrading genomes to a higher quality finished state.
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影响因子：
64.5
作者：
Charrier C;Joshi K;Coutinho-Budd J;Kim JE;Lambert N;de Marchena J;Jin WL;Vanderhaeghen P;Ghosh A;Sassa T;Polleux F
通讯作者：
Polleux F
影响因子：
9.8
作者：
Church DM;Goodstadt L;Hillier LW;Zody MC;Goldstein S;She X;Bult CJ;Agarwala R;Cherry JL;DiCuccio M;Hlavina W;Kapustin Y;Meric P;Maglott D;Birtle Z;Marques AC;Graves T;Zhou S;Teague B;Potamousis K;Churas C;Place M;Herschleb J;Runnheim R;Forrest D;Amos-Landgraf J;Schwartz DC;Cheng Z;Lindblad-Toh K;Eichler EE;Ponting CP;Mouse Genome Sequencing Consortium
通讯作者：
Mouse Genome Sequencing Consortium
影响因子：
64.8
作者：
Hughes, Jennifer F.;Skaletsky, Helen;Brown, Laura G.;Pyntikova, Tatyana;Graves, Tina;Fulton, Robert S.;Dugan, Shannon;Ding, Yan;Buhay, Christian J.;Kremitzki, Colin;Wang, Qiaoyan;Shen, Hua;Holder, Michael;Villasana, Donna;Nazareth, Lynne V.;Cree, Andrew;Courtney, Laura;Veizer, Joelle;Kotkiewicz, Holland;Cho, Ting-Jan;Koutseva, Natalia;Rozen, Steve;Muzny, Donna M.;Warren, Wesley C.;Gibbs, Richard A.;Wilson, Richard K.;Page, David C.
通讯作者：
Page, David C.
影响因子：
12.3
作者：
Adey A;Morrison HG;Asan;Xun X;Kitzman JO;Turner EH;Stackhouse B;MacKenzie AP;Caruccio NC;Zhang X;Shendure J
通讯作者：
Shendure J
影响因子：
48
作者：
Chin, Chen-Shan;Alexander, David H.;Korlach, Jonas
通讯作者：
Korlach, Jonas