Capturing and cultivating single bacterial cells in gel microdroplets to obtain near-complete genomes.
Nat Protoc
; 9(3): 608-21, 2014 Mar.
Article
en En
| MEDLINE
| ID: mdl-24525754
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 â¼4 weeks.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Bacterias
/
Genoma Bacteriano
/
Análisis de la Célula Individual
/
Geles
Límite:
Humans
Idioma:
En
Revista:
Nat Protoc
Año:
2014
Tipo del documento:
Article
País de afiliación:
Estados Unidos