Cost-effective high-throughput single-haplotype iterative mapping and sequencing for complex genomic structures.
Cost-effective high-throughput single-haplotype iterative mapping and sequencing for complex genomic structures.
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DOI：
10.1038/nprot.2018.019
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发表时间：
2018-04
期刊：
影响因子：
14.8
通讯作者：
Page DC
中科院分区：
文献类型：
--
作者：
Bellott DW;Cho TJ;Hughes JF;Skaletsky H;Page DC
结构复杂区域的参考序列只能通过高精度的基于克隆的方法获得。我们和其他人已经成功地采用单单元型迭代作图和测序（SHIMS 1.0）来组装多个脊椎动物物种性染色体上结构复杂的区域，并对人类常染色体的参考序列进行有针对性的改进。然而，SHIMS 1.0 既昂贵又耗时，需要的资源只有基因组中心才能掌握。在这里，我们介绍 SHIMS 2.0，这是一种改进的 SHIMS 协议，即使是小型实验室也可以从复杂的基因组区域生成高质量的参考序列。使用简化和并行的文库制备方案，并利用高通量、廉价、短读长测序技术，一个具有分子生物学和生物信息学经验的小组可以在一周内测序和组装 192 个大插入 BAC 或 fosmid 克隆。与其他合并策略相比，每个克隆都使用独特的条形码进行测序，从而可以在单次测序运行中多重包含几乎相同序列的克隆并分别组装它们。相对于SHIMS 1.0，SHIMS 2.0将所需的成本和时间降低了两个数量级，同时保持了较高的测序准确性。该协议描述了 SHIMS 2.0，这是一种以经济高效、高通量的方式从结构复杂的基因组区域生成准确参考序列的方法。
The reference sequence of structurally complex regions can only be obtained through highly accurate clone-based approaches. We and others have successfully employed Single-Haplotype Iterative Mapping and Sequencing (SHIMS 1.0) to assemble structurally complex regions across the sex chromosomes of several vertebrate species and in targeted improvements to the reference sequences of human autosomes. However, SHIMS 1.0 was expensive and time consuming, requiring the resources that only a genome center could command. Here we introduce SHIMS 2.0, an improved SHIMS protocol to allow even a small laboratory to generate high-quality reference sequence from complex genomic regions. Using a streamlined and parallelized library preparation protocol, and taking advantage of high-throughput, inexpensive, short-read sequencing technologies, a small group with both molecular biology and bioinformatics experience can sequence and assemble 192 large-insert BAC or fosmid clones in one week. In contrast to other pooling strategies, each clone is sequenced with a unique barcode, making it possible to multiplex clones containing nearly identical sequences in a single sequencing run and assemble them separately. Relative to SHIMS 1.0, SHIMS 2.0 reduces the cost and time required by two orders of magnitude, while preserving high sequencing accuracy. This protocol describes SHIMS 2.0, a method to generate accurate reference sequences from structurally complex genomic regions in a cost-effective, high-throughput fashion.
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影响因子：
48
作者：
Langmead, Ben;Salzberg, Steven L.
通讯作者：
Salzberg, Steven L.
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.
DOI：
10.1126/science.aae0344
发表时间：
2016-04-01
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Gordon D;Huddleston J;Chaisson MJ;Hill CM;Kronenberg ZN;Munson KM;Malig M;Raja A;Fiddes I;Hillier LW;Dunn C;Baker C;Armstrong J;Diekhans M;Paten B;Shendure J;Wilson RK;Haussler D;Chin CS;Eichler EE
通讯作者：
Eichler EE
影响因子：
64.8
作者：
通讯作者：
--
影响因子：
1.7
作者：
Bankevich, Anton;Nurk, Sergey;Pevzner, Pavel A.
通讯作者：
Pevzner, Pavel A.