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

Christiansen, M., Brown, Amanda, Kundu, S., Tutill, H., Williams, Rachel, Brown, Julianne, Holdstock, J., Holland, M., Stevenson, S., Dave, J., Tong, W., Einer-Jensen, K., Depledge, D. and Breuer, J. (2014) Whole-genome enrichment and sequencing of Chlamydia trachomatis directly from clinical samples. BMC Infectious Diseases, 14 (591). ISSN 1471-2334.

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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.

Item Type: Article
Subjects: Q Science > QH Natural history > QH0301 Biology (General) > QH0426 Genetics
Q Science > QR Microbiology
Divisions: Faculty of Social and Applied Sciences > School of Human and Life Sciences
Depositing User: Samit Kundu
Date Deposited: 21 May 2015 15:57
Last Modified: 21 Jan 2016 16:39
URI: https://create.canterbury.ac.uk/id/eprint/13415

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Last edited: 29/06/2016 12:23:00