title: Culture-independent sequence analysis of Chlamydia trachomatis in urogenital specimens identifies regions of recombination and in-patient sequence mutations.
abstract: A culture-independent genome sequencing approach was developed and used to examine genomic variability in Chlamydia trachomatis-positive specimens that were collected from patients in the Seattle, WA, USA, area. The procedure is based on an immunomagnetic separation approach with chlamydial LPS-specific mAbs, followed by DNA purification and total DNA amplification, and subsequent Illumina-based sequence analysis. Quality of genome sequencing was independent of the total number of inclusion-forming units determined for the sample and the amount of non-chlamydial DNA in the Illumina libraries. A geographically and temporally linked clade of isolates was identified with evidence of several different regions of recombination and variable ompA sequence types, suggesting that recombination is common within outbreaks. Culture-independent sequence analysis revealed a linkage pattern at two nucleotide positions that was unique to the genomes of isolates from patients, but not in C. trachomatis recombinants generated in vitro. These data demonstrated that culture-independent sequence analysis can be used to rapidly and inexpensively collect genome data from patients infected by C. trachomatis, and that this approach can be used to examine genomic variation within this species.
authors.affiliation: Molecular and Cellular Biology Program, Oregon State University, Corvallis, OR, USA.
authors.forename: Timothy E
authors.initials: TE
authors.lastname: Putman
authors.affiliation: Division of Allergy and Infectious Disease, Department of Medicine, University of Washington, Seattle, WA, USA.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Suchland
authors.affiliation: Microbiology Program, Oregon State University, Corvallis, OR, USA.
authors.forename: John D
authors.initials: JD
authors.lastname: Ivanovitch
authors.affiliation: Department of Biomedical Sciences, Oregon State University, Corvallis, OR, USA.
authors.forename: Daniel D
authors.initials: DD
authors.lastname: Rockey
mesh_terms: D001431:Bacteriological Techniques / Q000379:methods*; D002690:Chlamydia Infections / Q000382:microbiology*; D002692:Chlamydia trachomatis / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014644:Genetic Variation; D005835:Genitalia / Q000382:microbiology*; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods; D008967:Molecular Biology / Q000379:methods; D008969:Molecular Sequence Data; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D014481:United States
chemical_list: D004269:DNA, Bacterial