Whole-genome enrichment and sequencing of Chlamydia trachomatis directly from clinical samples.
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.
Division of Infection and Immunity University College London (UCL), London, WC1E 6BT, UK. m.christiansen@ucl.ac.uk.
Mette T
MT
Christiansen
Oxford Gene Technology, Begbroke, Oxfordshire, OX5 1PF, UK. acb249@cornell.edu.|Present address: Department of Microbiology and Immunology, Cornell University, Ithaca, NY, 14853, USA. acb249@cornell.edu.
Amanda C
AC
Brown
Division of Infection and Immunity University College London (UCL), London, WC1E 6BT, UK. samit.kundu@ucl.ac.uk.|School of Human and Life Sciences, Canterbury Christchurch University, Canterbury, Kent, CT1 1QU, UK. samit.kundu@ucl.ac.uk.
Samit
S
Kundu
Division of Infection and Immunity University College London (UCL), London, WC1E 6BT, UK. h.tutill@ucl.ac.uk.
Helena J
HJ
Tutill
Division of Infection and Immunity University College London (UCL), London, WC1E 6BT, UK. rachel.williams@ucl.ac.uk.
Rachel
R
Williams
Great Ormond Street Hospital (GOSH), London, WC1N 3JH, UK. julianne.brown@nhs.net.
Julianne R
JR
Brown
Oxford Gene Technology, Begbroke, Oxfordshire, OX5 1PF, UK. Jolyon.Holdstock@ogt.com.
Jolyon
J
Holdstock
London School of Hygiene and Tropical Medicine (LSHTM), London, WC1E 7HT, UK. Martin.Holland@lshtm.ac.uk.
Martin J
MJ
Holland
University College London Hospital (UCLH), London, WC1E 6DE, UK. simon.stevenson@uclh.nhs.uk.
Simon
S
Stevenson
Barts Health NHS Trust, London, E1 2ES, UK. jayshree.dave@nhs.net.
Jayshree
J
Dave
Barts Health NHS Trust, London, E1 2ES, UK. William.Tong@bartshealth.nhs.uk.
C Y William
CY
Tong
QIAGEN-AAR, 8200, Aarhus N, Denmark. kjensen@clcbio.com.
Katja
K
Einer-Jensen
Division of Infection and Immunity University College London (UCL), London, WC1E 6BT, UK. d.depledge@ucl.ac.uk.
Daniel P
DP
Depledge
Division of Infection and Immunity University College London (UCL), London, WC1E 6BT, UK. j.breuer@ucl.ac.uk.
Judith
J
Breuer
D001483:Base Sequence; D002690:Chlamydia Infections / Q000382:microbiology*; D002692:Chlamydia trachomatis / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D010802:Phylogeny; D017422:Sequence Analysis, DNA