Whole-genome enrichment and sequencing of Chlamydia trachomatis directly from clinical samples.

Abstract:

BACKGROUND:Chlamydia trachomatis is a pathogen of worldwide importance, causing more than 100 million cases of sexually transmitted infections annually. Whole-genome sequencing is a powerful high resolution tool that can be used to generate accurate data on bacterial population structure, phylogeography and mutations associated with antimicrobial resistance. The objective of this study was to perform whole-genome enrichment and sequencing of C. trachomatis directly from clinical samples. METHODS:C. trachomatis positive samples comprising seven vaginal swabs and three urine samples were sequenced without prior in vitro culture in addition to nine cultured C. trachomatis samples, representing different serovars. A custom capture RNA bait set, that captures all known diversity amongst C. trachomatis genomes, was used in a whole-genome enrichment step during library preparation to enrich for C. trachomatis DNA. All samples were sequenced on the MiSeq platform. RESULTS:Full length C. trachomatis genomes (>95-100% coverage of a reference genome) were successfully generated for eight of ten clinical samples and for all cultured samples. The proportion of reads mapping to C. trachomatis and the mean read depth across each genome were strongly linked to the number of bacterial copies within the original sample. Phylogenetic analysis confirmed the known population structure and the data showed potential for identification of minority variants and mutations associated with antimicrobial resistance. The sensitivity of the method was >10-fold higher than other reported methodologies. CONCLUSIONS:The combination of whole-genome enrichment and deep sequencing has proven to be a non-mutagenic approach, capturing all known variation found within C. trachomatis genomes. The method is a consistent and sensitive tool that enables rapid whole-genome sequencing of C. trachomatis directly from clinical samples and has the potential to be adapted to other pathogens with a similar clonal nature.

journal_name

BMC Infect Dis

journal_title

BMC infectious diseases

authors

Christiansen MT,Brown AC,Kundu S,Tutill HJ,Williams R,Brown JR,Holdstock J,Holland MJ,Stevenson S,Dave J,Tong CY,Einer-Jensen K,Depledge DP,Breuer J

doi

10.1186/s12879-014-0591-3

subject

Has Abstract

pub_date

2014-11-12 00:00:00

pages

591

issn

1471-2334

pii

s12879-014-0591-3

journal_volume

14

pub_type

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