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


Seth-Smith, H M B; Harris, S R; Scott, P; Parmar, S; Marsh, P; Unemo, M; Clarke, I N; Parkhill, J; Thomson, N R (2013). Generating whole bacterial genome sequences of low-abundance species from complex samples with IMS-MDA. Nature Protocols, 8(12):2404-2412.

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 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 target genomic DNA for whole bacterial genome sequencing, from clinical samples of the difficult-to-culture, obligate intracellular pathogen Chlamydia trachomatis, without the need for culture. Our protocol combines Immunomagnetic Separation (IMS) for targeted bacterial enrichment with Multiple Displacement Amplification (MDA) for whole genome amplification, followed by high throughput sequencing. Compared to other techniques which might be used to generate such data, IMS-MDA is an inexpensive, low-technology and highly transferable process, which provides amplified genomic DNA for sequencing from target bacteria in under 5 hours, with little hands-on time.

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 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 target genomic DNA for whole bacterial genome sequencing, from clinical samples of the difficult-to-culture, obligate intracellular pathogen Chlamydia trachomatis, without the need for culture. Our protocol combines Immunomagnetic Separation (IMS) for targeted bacterial enrichment with Multiple Displacement Amplification (MDA) for whole genome amplification, followed by high throughput sequencing. Compared to other techniques which might be used to generate such data, IMS-MDA is an inexpensive, low-technology and highly transferable process, which provides amplified genomic DNA for sequencing from target bacteria in under 5 hours, with little hands-on time.

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16 citations in Web of Science®
16 citations in Scopus®
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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Functional Genomics Center Zurich
05 Vetsuisse Faculty > Institute of Veterinary Pathology
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Date:2013
Deposited On:12 Dec 2013 13:38
Last Modified:05 Apr 2016 17:14
Publisher:Nature Publishing Group
ISSN:1750-2799
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1038/nprot.2013.147
PubMed ID:24202554

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