title: Generating whole bacterial genome sequences of low-abundance species from complex samples with IMS-MDA.
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.
authors.affiliation: 1] Pathogen Genomics, The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridgeshire, UK. [2].
authors.forename: Helena M B
authors.initials: HM
authors.lastname: Seth-Smith
authors.forename: Simon R
authors.initials: SR
authors.lastname: Harris
authors.forename: Paul
authors.initials: P
authors.lastname: Scott
authors.forename: Surendra
authors.initials: S
authors.lastname: Parmar
authors.forename: Peter
authors.initials: P
authors.lastname: Marsh
authors.forename: Magnus
authors.initials: M
authors.lastname: Unemo
authors.forename: Ian N
authors.initials: IN
authors.lastname: Clarke
authors.forename: Julian
authors.identifier: 0000000270695958
authors.initials: J
authors.lastname: Parkhill
authors.forename: Nicholas R
authors.initials: NR
authors.lastname: Thomson
mesh_terms: D001419:Bacteria / Q000235:genetics; D002692:Chlamydia trachomatis / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry*; D016680:Genome, Bacterial; D023281:Genomics; D018189:Immunomagnetic Separation / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods
chemical_list: D004269:DNA, Bacterial