Whole genome amplification and de novo assembly of single bacterial cells.
Whole genome amplification and de novo assembly of single bacterial cells.
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DOI：
10.1371/journal.pone.0006864
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发表时间：
2009-09-02
期刊：
影响因子：
3.7
通讯作者：
Chisholm SW
中科院分区：
文献类型：
--
作者：
Rodrigue S;Malmstrom RR;Berlin AM;Birren BW;Henn MR;Chisholm SW
单细胞基因组测序有可能允许深入探索在未培养的微生物中发现的巨大遗传多样性。我们使用海洋蓝藻原绿球藻作为模型系统来解决高通量全基因组扩增（WGA）和单个细胞全基因组测序面临的重要挑战。我们描述了一个管道，使单细胞WGA在数百个细胞的时间，同时几乎消除非靶DNA的反应。我们进一步开发了一种扩增后归一化程序，以减轻与多重位移扩增（MDA）相关的测序覆盖率的极端变化，并证明该程序提高了测序效率并促进了基因组组装。我们报道基因组恢复高达99.6%的参考引导组装，95%的从头组装从单细胞开始。我们还分析了MDA过程中嵌合体形成对从头组装的影响，并讨论了减少嵌合体中不正确连接区域存在的策略。本文描述的方法将有助于从各种样品中测序单个细胞的基因组。
Single-cell genome sequencing has the potential to allow the in-depth exploration of the vast genetic diversity found in uncultured microbes. We used the marine cyanobacterium Prochlorococcus as a model system for addressing important challenges facing high-throughput whole genome amplification (WGA) and complete genome sequencing of individual cells. We describe a pipeline that enables single-cell WGA on hundreds of cells at a time while virtually eliminating non-target DNA from the reactions. We further developed a post-amplification normalization procedure that mitigates extreme variations in sequencing coverage associated with multiple displacement amplification (MDA), and demonstrated that the procedure increased sequencing efficiency and facilitated genome assembly. We report genome recovery as high as 99.6% with reference-guided assembly, and 95% with de novo assembly starting from a single cell. We also analyzed the impact of chimera formation during MDA on de novo assembly, and discuss strategies to minimize the presence of incorrectly joined regions in contigs. The methods describe in this paper will be useful for sequencing genomes of individual cells from a variety of samples.
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影响因子：
4.5
作者：
Marcy, Yann;Ishoey, Thomas;Lasken, Roger S.;Stockwell, Timothy B.;Walenz, Brian P.;Halpern, Aaron L.;Beeson, Karen Y.;Goldberg, Susanne M. D.;Quake, Stephen R.
通讯作者：
Quake, Stephen R.
DOI：
10.1007/10_2007_073
发表时间：
2007-01-01
期刊：
CELL SEPARATION: FUNDAMENTALS, ANALYTICAL AND PREPARATIVE METHODS
影响因子：
--
作者：
Ibrahim, Sherrif F.;van den Engh, Ger
通讯作者：
van den Engh, Ger
影响因子：
4.4
作者：
Raghunathan, A;Ferguson, HR;Lasken, RS
通讯作者：
Lasken, RS
DOI：
10.1073/pnas.0808028105
发表时间：
2008-10-07
影响因子：
11.1
作者：
Pan, Xinghua;Urban, Alexander Eckehart;Weissman, Sherman M.
通讯作者：
Weissman, Sherman M.
DOI：
10.1073/pnas.0700496104
发表时间：
2007-05-22
影响因子：
11.1
作者：
Stepanauskas, Ramunas;Sieracki, Michael E.
通讯作者：
Sieracki, Michael E.