Generating whole bacterial genome sequences of low-abundance species from complex samples with IMS-MDA
2013 (English)In: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 8, no 12, 2404-2412 p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
Nature Publishing Group, 2013. Vol. 8, no 12, 2404-2412 p.
Cell and Molecular Biology
IdentifiersURN: urn:nbn:se:oru:diva-56735DOI: 10.1038/nprot.2013.147ISI: 000328125800007PubMedID: 24202554OAI: oai:DiVA.org:oru-56735DiVA: diva2:1083949