Genome Science Programs, B-11 Bioenergy and Biome Sciences, Los Alamos National Laboratory, Los Alamos, New Mexico, USA.
1] Genome Science Programs, B-11 Bioenergy and Biome Sciences, Los Alamos National Laboratory, Los Alamos, New Mexico, USA. [2].
Nat Protoc. 2014 Mar;9(3):608-21. doi: 10.1038/nprot.2014.034. Epub 2014 Feb 13.
Assembling a complete genome from a single bacterial cell, termed single-cell genomics, is challenging with current technologies. Recovery rates of complete genomes from fragmented assemblies of single-cell templates significantly vary. Although increasing the amount of genomic template material by standard cultivation improves recovery, most bacteria are unfortunately not amenable to traditional cultivation, possibly owing to the lack of unidentified, yet necessary, growth signals and/or specific symbiotic influences. To overcome this limitation, we adopted and modified the method of cocultivation of single-captured bacterial cells in gel microdroplets (GMDs) to improve full genomic sequence recovery. By completing multiple genomes of two novel species derived from single cells, we demonstrated its efficacy on diverse bacterial species using human oral and gut microbiome samples. Here we describe a detailed protocol for capturing single bacterial cells, cocultivating them in medium and isolating microcolonies in GMDs with flow cytometry. Beginning with preliminary studies, obtaining GMDs with single microcolonies for whole-genome amplification may take ∼4 weeks.
从单个细菌细胞中组装完整的基因组，称为单细胞基因组学，这在当前技术下是具有挑战性的。从单细胞模板的碎片化组装中回收完整基因组的回收率差异很大。尽管通过标准培养增加基因组模板材料的数量可以提高回收率，但不幸的是，大多数细菌都不适合传统培养，这可能是由于缺乏未知但必要的生长信号和/或特定的共生影响。为了克服这一限制，我们采用并修改了在凝胶微滴（GMD）中捕获单个细菌细胞共培养的方法，以提高全基因组序列的回收。通过完成两个源自单细胞的新物种的多个基因组，我们使用人类口腔和肠道微生物组样本证明了其在多种细菌物种上的功效。在这里，我们描述了一种详细的捕获单个细菌细胞的方案，将它们在培养基中进行共培养，并通过流式细胞术在 GMD 中分离微集落。从初步研究开始，获得用于全基因组扩增的单个微集落的 GMD 可能需要大约 4 周的时间。
