Obtaining genomes from uncultivated environmental microorganisms using FACS-based single-cell genomics.
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%.
Department of Energy (DOE) Joint Genome Institute, Walnut Creek, California, USA.
Christian
C
Rinke
Department of Energy (DOE) Joint Genome Institute, Walnut Creek, California, USA.
Janey
J
Lee
Department of Energy (DOE) Joint Genome Institute, Walnut Creek, California, USA.
Nandita
N
Nath
Department of Energy (DOE) Joint Genome Institute, Walnut Creek, California, USA.
Danielle
D
Goudeau
Bigelow Laboratory for Ocean Sciences, East Boothbay, Maine, USA.
Brian
B
Thompson
Bigelow Laboratory for Ocean Sciences, East Boothbay, Maine, USA.
Nicole
N
Poulton
Bigelow Laboratory for Ocean Sciences, East Boothbay, Maine, USA.
Elizabeth
E
Dmitrieff
Department of Energy (DOE) Joint Genome Institute, Walnut Creek, California, USA.
Rex
R
Malmstrom
Bigelow Laboratory for Ocean Sciences, East Boothbay, Maine, USA.
Ramunas
R
Stepanauskas
Department of Energy (DOE) Joint Genome Institute, Walnut Creek, California, USA.
Tanja
T
Woyke
D002469:Cell Separation / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D023281:Genomics / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing; D064307:Microbiota / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D059010:Single-Cell Analysis / Q000379:methods*
D012336:RNA, Ribosomal, 16S