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

Seth-Smith, Helena M.B., Harris, Simon R., Scott, Paul, Parmar, Surendra, Marsh, Peter, Unemo, Magnus, Clarke, Ian N., Parkhill, Julian and Thomson, Nicholas R. (2013) Generating whole bacterial genome sequences of low-abundance species from complex samples with IMS-MDA. Nature Protocols, 8 (12), 2404-2412. (doi:10.1038/nprot.2013.147). (PMID:24202554)

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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Contributors

Author: Ian N. Clarke

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