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Capturing and cultivating single bacterial cells in gel microdroplets to obtain near-complete genomes

Nature Protocols volume 9, pages 608–621 (2014)Cite this article

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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 ∼4 weeks.

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Figure 1: Workflow of cell capture in GMDs for MDA and genome recovery.
Figure 2: Bacterial cell extraction with Nycodenz solution (Step 1A(x)).
Figure 3: Estimation of GMD cell occupancy.
Figure 4: E.coli microcolony growing in a GMD.
Figure 5: FACS analysis of GMDs.
Figure 6: GMD occupancy.

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Acknowledgements

This work was supported by Los Alamos National Laboratory through a Directed Research program with project code 20110034DR.

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Author notes

  1. Michael S Fitzsimons

    Present address: Present address: Nugen Technologies, San Carlos, California, USA.,

Authors and Affiliations

  1. Genome Science Programs, B-11 Bioenergy and Biome Sciences, Los Alamos National Laboratory, Los Alamos, New Mexico, USA

    Armand E K Dichosa, Ashlynn R Daughton, Krista G Reitenga, Michael S Fitzsimons & Cliff S Han

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  1. Armand E K Dichosa
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  2. Ashlynn R Daughton
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  3. Krista G Reitenga
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  4. Michael S Fitzsimons
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Contributions

C.S.H., M.S.F. and A.E.K.D. conceived the GMD-SCG strategies. M.S.F., A.E.K.D., A.R.D. and K.G.R. performed the experiments. The authors analyzed the data. A.E.K.D., A.R.D., K.G.R. and C.S.H. wrote the paper.

Corresponding author

Correspondence to Cliff S Han.

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The authors declare no competing financial interests.

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Dichosa, A., Daughton, A., Reitenga, K. et al. Capturing and cultivating single bacterial cells in gel microdroplets to obtain near-complete genomes. Nat Protoc 9, 608–621 (2014). https://doi.org/10.1038/nprot.2014.034

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