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

Nature Protocols volume 9, pages 1038–1048 (2014)Cite this article

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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%.

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Figure 1: Single-cell whole-genome amplification workflow.
Figure 2: High-throughput single-cell sorting.
Figure 3: Real-time whole-genome amplification and 16S rRNA gene PCR.

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Acknowledgements

The work conducted by the US DOE Joint Genome Institute is supported by the Office of Science of the US Department of Energy under contract no. DE-AC02-05CH11231. Work conducted by Bigelow Laboratory for Ocean Sciences is supported by National Science Foundation grants OCE-1232982, OCE-821374, EF-0633142, EF-826924 and MCB-738232.

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Authors and Affiliations

  1. Department of Energy (DOE) Joint Genome Institute, Walnut Creek, California, USA

    Christian Rinke, Janey Lee, Nandita Nath, Danielle Goudeau, Rex Malmstrom & Tanja Woyke

  2. Bigelow Laboratory for Ocean Sciences, East Boothbay, Maine, USA

    Brian Thompson, Nicole Poulton, Elizabeth Dmitrieff & Ramunas Stepanauskas

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  1. Christian Rinke
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Contributions

C.R., T.W., R.M. and R.S. conceived the strategies. C.R., J.L., N.N., D.G., B.T., N.P. and E.D. performed the experiments and analyzed the data. C.R. wrote the paper with significant input from T.W., R.M. and R.S.

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Correspondence to Tanja Woyke.

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The authors declare no competing financial interests.

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Rinke, C., Lee, J., Nath, N. et al. Obtaining genomes from uncultivated environmental microorganisms using FACS–based single-cell genomics. Nat Protoc 9, 1038–1048 (2014). https://doi.org/10.1038/nprot.2014.067

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