title: Whole-genome sequences of Chlamydia trachomatis directly from clinical samples without culture.
abstract: The use of whole-genome sequencing as a tool for the study of infectious bacteria is of growing clinical interest. Chlamydia trachomatis is responsible for sexually transmitted infections and the blinding disease trachoma, which affect hundreds of millions of people worldwide. Recombination is widespread within the genome of C. trachomatis, thus whole-genome sequencing is necessary to understand the evolution, diversity, and epidemiology of this pathogen. Culture of C. trachomatis has, until now, been a prerequisite to obtain DNA for whole-genome sequencing; however, as C. trachomatis is an obligate intracellular pathogen, this procedure is technically demanding and time consuming. Discarded clinical samples represent a large resource for sequencing the genomes of pathogens, yet clinical swabs frequently contain very low levels of C. trachomatis DNA and large amounts of contaminating microbial and human DNA. To determine whether it is possible to obtain whole-genome sequences from bacteria without the need for culture, we have devised an approach that combines immunomagnetic separation (IMS) for targeted bacterial enrichment with multiple displacement amplification (MDA) for whole-genome amplification. Using IMS-MDA in conjunction with high-throughput multiplexed Illumina sequencing, we have produced the first whole bacterial genome sequences direct from clinical samples. We also show that this method can be used to generate genome data from nonviable archived samples. This method will prove a useful tool in answering questions relating to the biology of many difficult-to-culture or fastidious bacteria of clinical concern.
authors.affiliation: Pathogen Genomics, The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire CB10 1SA, United Kingdom. hss@sanger.ac.uk
authors.forename: Helena M B
authors.initials: HM
authors.lastname: Seth-Smith
authors.forename: Simon R
authors.initials: SR
authors.lastname: Harris
authors.forename: Rachel J
authors.initials: RJ
authors.lastname: Skilton
authors.forename: Frans M
authors.initials: FM
authors.lastname: Radebe
authors.forename: Daniel
authors.initials: D
authors.lastname: Golparian
authors.forename: Elena
authors.initials: E
authors.lastname: Shipitsyna
authors.forename: Pham Thanh
authors.initials: PT
authors.lastname: Duy
authors.forename: Paul
authors.initials: P
authors.lastname: Scott
authors.forename: Lesley T
authors.initials: LT
authors.lastname: Cutcliffe
authors.forename: Colette
authors.initials: C
authors.lastname: O'Neill
authors.forename: Surendra
authors.initials: S
authors.lastname: Parmar
authors.forename: Rachel
authors.initials: R
authors.lastname: Pitt
authors.forename: Stephen
authors.initials: S
authors.lastname: Baker
authors.forename: Catherine A
authors.initials: CA
authors.lastname: Ison
authors.forename: Peter
authors.initials: P
authors.lastname: Marsh
authors.forename: Hamid
authors.initials: H
authors.lastname: Jalal
authors.forename: David A
authors.initials: DA
authors.lastname: Lewis
authors.forename: Magnus
authors.initials: M
authors.lastname: Unemo
authors.forename: Ian N
authors.initials: IN
authors.lastname: Clarke
authors.forename: Julian
authors.initials: J
authors.lastname: Parkhill
authors.forename: Nicholas R
authors.initials: NR
authors.lastname: Thomson
mesh_terms: D001483:Base Sequence; D002690:Chlamydia Infections / Q000235:genetics / Q000382:microbiology; D002692:Chlamydia trachomatis / Q000235:genetics* / Q000472:pathogenicity; D016680:Genome, Bacterial; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans