Obtaining genomes from uncultivated environmental microorganisms using FACS–based single-cell genomics View Full Text


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2014-04-10

AUTHORS

Christian Rinke, Janey Lee, Nandita Nath, Danielle Goudeau, Brian Thompson, Nicole Poulton, Elizabeth Dmitrieff, Rex Malmstrom, Ramunas Stepanauskas, Tanja Woyke

ABSTRACT

Single-cell genomics is a powerful tool for exploring the genetic makeup of environmental microorganisms, the vast majority of which are difficult, if not impossible, to cultivate with current approaches. Here we present a comprehensive protocol for obtaining genomes from uncultivated environmental microbes via high-throughput single-cell isolation by FACS. The protocol encompasses the preservation and pretreatment of differing environmental samples, followed by the physical separation, lysis, whole-genome amplification and 16S rRNA–based identification of individual bacterial and archaeal cells. The described procedure can be performed with standard molecular biology equipment and a FACS machine. It takes <12 h of bench time over a 4-d time period, and it generates up to 1 μg of genomic DNA from an individual microbial cell, which is suitable for downstream applications such as PCR amplification and shotgun sequencing. The completeness of the recovered genomes varies, with an average of ∼50%. More... »

PAGES

1038-1048

Identifiers

URI

http://scigraph.springernature.com/pub.10.1038/nprot.2014.067

DOI

http://dx.doi.org/10.1038/nprot.2014.067

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1013309795

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/24722403


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