Nearly finished genomes produced using gel microdroplet culturing reveal substantial intraspecies genomic diversity within the human microbiome.
Nearly finished genomes produced using gel microdroplet culturing reveal substantial intraspecies genomic diversity within the human microbiome.
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DOI：
10.1101/gr.142208.112
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发表时间：
2013-05
期刊：
影响因子：
7
通讯作者：
Han CS
中科院分区：
文献类型：
--
作者：
Fitzsimons MS;Novotny M;Lo CC;Dichosa AE;Yee-Greenbaum JL;Snook JP;Gu W;Chertkov O;Davenport KW;McMurry K;Reitenga KG;Daughton AR;He J;Johnson SL;Gleasner CD;Wills PL;Parson-Quintana B;Chain PS;Detter JC;Lasken RS;Han CS
大多数微生物基因组多样性尚未探索。这在很大程度上是由于我们无法隔离培养大多数微生物，而这是传统基因组测序的先决条件。单细胞测序使研究人员能够绕过这一限制。使用多重置换扩增（MDA）的全基因组扩增技术直接从单个细胞扩增DNA。然而，来自单个染色体拷贝的MDA遭受扩增偏差和来自甚至非常少量的DNA污染的大量特异性损失，这使得组装基因组困难并且完全完成基因组是不可能的，除非在特殊情况下。凝胶微滴培养允许培养不同的微生物群落，并提供数百至数千个遗传上相同的细胞作为MDA反应的输入。我们通过比较MDA后凝胶微滴和单细胞的测序结果证明了这种方法的实用性。MDA反应和基因组测序中的偏差降低，并且当使用凝胶微滴时，组装大大改善。我们从人类口腔和粪便微生物组样本中获得了两种细菌的多个近乎完整的基因组。发现了大量的基因组多样性，包括单核苷酸多态性和基因组重组。凝胶微滴提供了一种强大的高通量技术，用于从复杂样本中组装整个基因组以及探测自然发生群体的泛基因组。
The majority of microbial genomic diversity remains unexplored. This is largely due to our inability to culture most microorganisms in isolation, which is a prerequisite for traditional genome sequencing. Single-cell sequencing has allowed researchers to circumvent this limitation. DNA is amplified directly from a single cell using the whole-genome amplification technique of multiple displacement amplification (MDA). However, MDA from a single chromosome copy suffers from amplification bias and a large loss of specificity from even very small amounts of DNA contamination, which makes assembling a genome difficult and completely finishing a genome impossible except in extraordinary circumstances. Gel microdrop cultivation allows culturing of a diverse microbial community and provides hundreds to thousands of genetically identical cells as input for an MDA reaction. We demonstrate the utility of this approach by comparing sequencing results of gel microdroplets and single cells following MDA. Bias is reduced in the MDA reaction and genome sequencing, and assembly is greatly improved when using gel microdroplets. We acquired multiple near-complete genomes for two bacterial species from human oral and stool microbiome samples. A significant amount of genome diversity, including single nucleotide polymorphisms and genome recombination, is discovered. Gel microdroplets offer a powerful and high-throughput technology for assembling whole genomes from complex samples and for probing the pan-genome of naturally occurring populations.
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影响因子：
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