Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Recent advances in genomic DNA sequencing of microbial species from single cells

Nature Reviews Genetics volume 15, pages 577–584 (2014)Cite this article

Subjects

Abstract

The vast majority of microbial species remain uncultivated and, until recently, about half of all known bacterial phyla were identified only from their 16S ribosomal RNA gene sequence. With the advent of single-cell sequencing, genomes of uncultivated species are rapidly filling in unsequenced branches of the microbial phylogenetic tree. The wealth of new insights gained from these previously inaccessible groups is providing a deeper understanding of their basic biology, taxonomy and evolution, as well as their diverse roles in environmental ecosystems and human health.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Complementary methods used to investigate the genomics of uncultivated bacteria.
Figure 2: Filling in the bacterial tree of life.
Figure 3: Comparative genomics using single-cell DNA amplification.

References

  1. Dean, F. B. et al. Comprehensive human genome amplification using multiple displacement amplification. Proc. Natl Acad. Sci. USA 99, 5261–5266 (2002).

    Article  CAS  PubMed  Google Scholar 

  2. Dean, F. B., Nelson, J. R., Giesler, T. L. & Lasken, R. S. Rapid amplification of plasmid and phage DNA using Phi 29 DNA polymerase and multiply-primed rolling circle amplification. Genome Res. 11, 1095–1099 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Raghunathan, A. et al. Genomic DNA amplification from a single bacterium. Appl. Environ. Microbiol. 71, 3342–3347 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Chi, K. R. Singled out for sequencing. Nature Methods 11, 13–17 (2014).

    Article  CAS  PubMed  Google Scholar 

  5. Lasken, R. S. Genomic sequencing of uncultured microorganisms from single cells. Nature Rev. Microbiol. 10, 631–640 (2012).

    Article  CAS  Google Scholar 

  6. Marcy, Y. et al. Dissecting biological “dark matter” with single-cell genetic analysis of rare and uncultivated TM7 microbes from the human mouth. Proc. Natl Acad. Sci. USA 104, 11889–11894 (2007).

    Article  CAS  PubMed  Google Scholar 

  7. Podar, M. et al. Targeted access to the genomes of low-abundance organisms in complex microbial communities. Appl. Environ. Microbiol. 73, 3205–3214 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Campbell, J. H. et al. UGA is an additional glycine codon in uncultured SR1 bacteria from the human microbiota. Proc. Natl Acad. Sci. USA 110, 5540–5545 (2013).

    Article  CAS  PubMed  Google Scholar 

  9. McLean, J. S. et al. Candidate phylum TM6 genome recovered from a hospital sink biofilm provides genomic insights into this uncultivated phylum. Proc. Natl Acad. Sci. USA 110, E2390–E2399 (2013).

    Article  CAS  PubMed  Google Scholar 

  10. Youssef, N. H., Blainey, P. C., Quake, S. R. & Elshahed, M. S. Partial genome assembly for a candidate division OP11 single cell from an anoxic spring (Zodletone Spring, Oklahoma). Appl. Environ. Microbiol. 77, 7804–7814 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Dodsworth, J. A. et al. Single-cell and metagenomic analyses indicate a fermentative and saccharolytic lifestyle for members of the OP9 lineage. Nature Commun. 4, 1854 (2013).

    Article  Google Scholar 

  12. Wilson, M. C. et al. An environmental bacterial taxon with a large and distinct metabolic repertoire. Nature 506, 58–62 (2014).

    Article  CAS  PubMed  Google Scholar 

  13. Rinke, C. et al. Insights into the phylogeny and coding potential of microbial dark matter. Nature 499, 431–437 (2013).

    Article  CAS  PubMed  Google Scholar 

  14. Rinke, C. et al. Obtaining genomes from uncultivated environmental microorganisms using FACS-based single-cell genomics. Nature Protoc. 9, 1038–1048 (2014).

    Article  CAS  Google Scholar 

  15. Mussmann, M. et al. Insights into the genome of large sulfur bacteria revealed by analysis of single filaments. PLoS Biol. 5, e230 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  16. Siegl, A. et al. Single-cell genomics reveals the lifestyle of Poribacteria, a candidate phylum symbiotically associated with marine sponges. ISME J. 5, 61–70 (2011).

    Article  PubMed  Google Scholar 

  17. Kamke, J. et al. Single-cell genomics reveals complex carbohydrate degradation patterns in poribacterial symbionts of marine sponges. ISME J. 7, 2287–2300 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Mason, O. U. et al. Metagenome, metatranscriptome and single-cell sequencing reveal microbial response to Deepwater Horizon oil spill. ISME J. 6, 1715–1727 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Declerck, P. Biofilms: the environmental playground of Legionella pneumophila. Environ. Microbiol. 12, 557–566 (2010).

    Article  CAS  PubMed  Google Scholar 

  20. Shikuma, N. J. & Hadfield, M. G. Marine biofilms on submerged surfaces are a reservoir for Escherichia coli and Vibrio cholerae. Biofouling 26, 39–46 (2010).

    Article  CAS  PubMed  Google Scholar 

  21. Percival, S. L. & Thomas, J. G. Transmission of Helicobacter pylori and the role of water and biofilms. J. Water Health 7, 469–477 (2009).

    Article  PubMed  Google Scholar 

  22. Karch, H., Meyer, T., Russmann, H. & Heesemann, J. Frequent loss of Shiga-like toxin genes in clinical isolates of Escherichia coli upon subcultivation. Infect. Immun. 60, 3464–3467 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Brown, M. R. & Barker, J. Unexplored reservoirs of pathogenic bacteria: protozoa and biofilms. Trends Microbiol. 7, 46–50 (1999).

    Article  CAS  PubMed  Google Scholar 

  24. Horwitz, M. A. Formation of a novel phagosome by the Legionnaires' disease bacterium (Legionella pneumophila) in human monocytes. J. Exp. Med. 158, 1319–1331 (1983).

    Article  CAS  PubMed  Google Scholar 

  25. McLean, J. S. et al. Genome of the pathogen Porphyromonas gingivalis recovered from a biofilm in a hospital sink using a high-throughput single-cell genomics platform. Genome Res. 23, 867–877 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Bankevich, A. et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. 19, 455–477 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Zegans, M. E. et al. Interaction between bacteriophage DMS3 and host CRISPR region inhibits group behaviors of Pseudomonas aeruginosa. J. Bacteriol. 191, 210–219 (2009).

    Article  CAS  PubMed  Google Scholar 

  28. Wrighton, K. C. et al. Fermentation, hydrogen, and sulfur metabolism in multiple uncultivated bacterial phyla. Science 337, 1661–1665 (2012).

    Article  CAS  PubMed  Google Scholar 

  29. Kantor, R. S. et al. Small genomes and sparse metabolisms of sediment-associated bacteria from four candidate phyla. MBio 4, e00708–e00713 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  30. Nurk, S. et al. Assembling single-cell genomes and mini-metagenomes from chimeric MDA products. J. Comput. Biol. 20, 714–737 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Albertsen, M. et al. Genome sequences of rare, uncultured bacteria obtained by differential coverage binning of multiple metagenomes. Nature Biotech. 31, 533–538 (2013).

    Article  CAS  Google Scholar 

  32. Seth-Smith, H. M. et al. Whole-genome sequences of Chlamydia trachomatis directly from clinical samples without culture. Genome Res. 23, 855–866 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Hosono, S. et al. Unbiased whole-genome amplification directly from clinical samples. Genome Res. 13, 954–964 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Zhang, K. et al. Sequencing genomes from single cells by polymerase cloning. Nature Biotech. 24, 680–686 (2006).

    Article  CAS  Google Scholar 

  35. Fitzsimons, M. S. et al. Nearly finished genomes produced using gel microdroplet culturing reveal substantial intraspecies genomic diversity within the human microbiome. Genome Res. 23, 878–888 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Dichosa, A. E. et al. Artificial polyploidy improves bacterial single cell genome recovery. PLoS ONE 7, e37387 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Marcy, Y. et al. Nanoliter reactors improve multiple displacement amplification of genomes from single cells. PLoS Genet. 3, 1702–1708 (2007).

    Article  CAS  PubMed  Google Scholar 

  38. Gole, J. et al. Massively parallel polymerase cloning and genome sequencing of single cells using nanoliter microwells. Nature Biotech. 31, 1126–1132 (2013).

    Article  CAS  Google Scholar 

  39. Chitsaz, H. et al. Efficient de novo assembly of single-cell bacterial genomes from short-read data sets. Nature Biotech. 29, 915–921 (2011).

    Article  CAS  Google Scholar 

  40. Nurk, S. et al. in Research in Computational Molecular Biology 158–170 (Springer, 2013).

    Book  Google Scholar 

  41. Lasken, R. S. & Stockwell, T. B. Mechanism of chimera formation during the Multiple Displacement Amplification reaction. BMC Biotechnol. 7, 19 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  42. Peng, Y., Leung, H. C., Yiu, S. M. & Chin, F. Y. IDBA-UD: a de novo assembler for single-cell and metagenomic sequencing data with highly uneven depth. Bioinformatics 28, 1420–1428 (2012).

    Article  CAS  PubMed  Google Scholar 

  43. Shapiro, E., Biezuner, T. & Linnarsson, S. Single-cell sequencing-based technologies will revolutionize whole-organism science. Nature Rev. Genet. 14, 618–630 (2013).

    Article  CAS  PubMed  Google Scholar 

  44. McConnell, M. J. et al. Mosaic copy number variation in human neurons. Science 342, 632–637 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Fodor, A. A. et al. The “most wanted” taxa from the human microbiome for whole genome sequencing. PLoS ONE 7, e41294 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors acknowledge discussions with G. Tesler, S. Yooseph and J. Badger. They also acknowledge assistance with the phylogenetic tree from C. Rinke and T. Woyke. This work was supported by grants to R.S.L. from the Alfred P. Sloan Foundation (Sloan Foundation-2007-10-19) and the US National Institutes of Health (NIH 2R01 HG003647 and NIH-HHSN272200900007C), and by grants to J.S.M. from the US National Institute of General Medical Sciences (NIH 1R01GM095373).

Author information

Authors and Affiliations

  1. J. Craig Venter Institute, 4120 Capricorn Lane, La Jolla, 92037, California, USA

    Roger S. Lasken & Jeffrey S. McLean

  2. Department of Periodontics, School of Dentistry, University of Washington, Seattle, 98195, Washington, USA

    Jeffrey S. McLean

Authors
  1. Roger S. Lasken
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jeffrey S. McLean
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Roger S. Lasken.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Related links

PowerPoint slides

Glossary

16S ribosomal RNA gene PCR analysis

A method in which primers designed for highly conserved regions of the 16S rRNA gene enable PCR from most bacteria, and variable regions of the sequence can be used for taxonomic identification.

Amplification bias

Uneven representation of regions of the DNA template in amplified DNA.

Bacterial and archaeal tree of life

The phylogenetic tree of all known bacteria and archaea based on the 16S ribosomal RNA gene.

Biofilm

A layered aggregate of microorganisms. These adherent cells are frequently embedded within a self-produced extracellular matrix that is generally composed of DNA, proteins and polysaccharides.

Candidate phyla

Uncultivated microbial groups that branch independently from known sequences near the base of the bacterial clade.

Chimaera

A recombinant molecule of DNA composed of segments from more than one source; multiple displacement amplification (MDA) can generate chimaeras that are predominantly inversions through its branching mechanism of DNA replication.

Endosymbiont

An organism that lives within the body or cells of another organism; it can include facultative or obligate symbionts.

Metagenomics

The study of the collective genomes contained in environmental samples using shotgun sequencing of DNA extracted from such samples.

Metatranscriptomic data

The set of all mRNA molecules or transcripts produced in a population of cells; they are typically obtained by shotgun sequencing of cDNA from a mixed microbial community.

Multiple displacement amplification

(MDA). A whole-genome DNA amplification method in which a DNA polymerase (usually the highly processive, strand-displacing Φ29 DNA polymerase) extends random primers while concurrently displacing the older products of downstream priming, which results in an exponential branching mechanism of DNA replication.

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lasken, R., McLean, J. Recent advances in genomic DNA sequencing of microbial species from single cells. Nat Rev Genet 15, 577–584 (2014). https://doi.org/10.1038/nrg3785

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nrg3785

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing