Obtaining genomes from uncultivated environmental microorganisms using FACS–based single-cell genomics
2014; Nature Portfolio; Volume: 9; Issue: 5 Linguagem: Inglês
10.1038/nprot.2014.067
ISSN1754-2189
AutoresChristian Rinke, Janey Lee, Nandita Nath, Danielle Goudeau, Brian Thompson, Nicole Poulton, Elizabeth Dmitrieff, Rex R. Malmstrom, Ramûnas Stepanauskas, Tanja Woyke,
Tópico(s)Protist diversity and phylogeny
ResumoSingle-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%.
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