Protocol | Published:

Generating whole bacterial genome sequences of low-abundance species from complex samples with IMS-MDA

Nature Protocols volume 8, pages 24042412 (2013) | Download Citation

Abstract

The study of bacterial populations using whole-genome sequencing is of considerable scientific and clinical interest. However, obtaining bacterial genomic information is not always trivial: the target bacteria may be difficult to culture or uncultured, and they may be found within samples containing complex mixtures of other contaminating microbes and/or host cells, from which it is very difficult to derive robust sequencing data. Here we describe our procedure to generate sufficient DNA for whole-genome sequencing from clinical samples and without the need for culture, as successfully used on the difficult-to-culture, obligate intracellular pathogen Chlamydia trachomatis. Our protocol combines immunomagnetic separation (IMS) for targeted bacterial enrichment with multiple displacement amplification (MDA) for whole-genome amplification (WGA), which is followed by high-throughput sequencing. Compared with other techniques that might be used to generate such data, IMS-MDA is an inexpensive, low-technology and highly transferable process that provides amplified genomic DNA for sequencing from target bacteria in under 5 h, with little hands-on time.

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Acknowledgements

This work was supported by the Wellcome Trust (grant no. 098051). We thank L. Ellison for useful comments on the manuscript.

Author information

Author notes

    • Helena M B Seth-Smith

    Present address: Functional Genomics Center Zürich, Universität Zürich, Zürich, Switzerland.

Affiliations

  1. Pathogen Genomics, The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridgeshire, UK.

    • Helena M B Seth-Smith
    • , Simon R Harris
    • , Paul Scott
    • , Julian Parkhill
    •  & Nicholas R Thomson
  2. Clinical Microbiology and Public Health Laboratory, Health Protection Agency, Addenbrooke's Hospital, Cambridge, UK.

    • Surendra Parmar
  3. Public Health England Laboratory Southampton, Southampton General Hospital, Southampton, UK.

    • Peter Marsh
  4. World Health Organization (WHO) Collaborating Centre for Gonorrhoea and Other Sexually Transmitted Infections (STIs), National Reference Laboratory for Pathogenic Neisseria, Örebro University Hospital, Örebro, Sweden.

    • Magnus Unemo
  5. Molecular Microbiology Group, Faculty of Medicine, University of Southampton, Southampton General Hospital, Southampton, UK.

    • Ian N Clarke

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Contributions

N.R.T., P.M., I.N.C., H.M.B.S.-S. and S.R.H. conceived of the project. H.M.B.S.-S. designed and performed the experiments, researched and optimized the protocol, and wrote the manuscript. N.R.T. supervised the development of the protocol, analyzed the data and wrote the manuscript. S.R.H. performed key data analysis, helped in the development of the protocol and wrote the manuscript. P.S. and S.P. contributed valuable information to the development of the protocol. S.P. provided discarded clinical samples. P.M., M.U., S.P., P.S., I.N.C. and J.P. provided insight into the experimental design and progress of the protocol, and on the results and implications of the work.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Helena M B Seth-Smith or Nicholas R Thomson.

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DOI

https://doi.org/10.1038/nprot.2013.147

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