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Nat Protoc. 2014 Mar;9(3):608-21. doi: 10.1038/nprot.2014.034. Epub 2014 Feb 13.

Capturing and cultivating single bacterial cells in gel microdroplets to obtain near-complete genomes.

Author information

1
Genome Science Programs, B-11 Bioenergy and Biome Sciences, Los Alamos National Laboratory, Los Alamos, New Mexico, USA.
2
1] Genome Science Programs, B-11 Bioenergy and Biome Sciences, Los Alamos National Laboratory, Los Alamos, New Mexico, USA. [2].

Abstract

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.

PMID:
24525754
DOI:
10.1038/nprot.2014.034
[Indexed for MEDLINE]

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