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.

Figure 1

The effect of whole-genome enrichment on the proportion of reads mapping to C. trachomatis. The plot shows the proportion of reads mapping to a C. trachomatis reference (F/SW4) genome (% on-target reads) from a C. trachomatis cultured sample that was processed with and without whole-genome enrichment.

Figure 2

SNP profiles in a C. trachomatis sample processed with and without whole-genome enrichment. The plot illustrates SNP profiles in a cultured C. trachomatis sample processed with and without whole-genome enrichment compared to the GenBank reference strain isolate F/SW4 (Accession no. NC_017951.1). The two outer tracks shown in green illustrate the open reading frames (ORFs) annotated in the GenBank reference strain isolate F/SW4 with forward and reverse orientation. The red track shows the SNP differences found within coding regions between the cultured C. trachomatis sample processed without whole-genome enrichment and the reference strain. The blue track shows the SNP differences found within coding regions between the same cultured C. trachomatis sample processed with whole-genome enrichment and the reference strain. The SNPs are called at the consensus level.

Figure 3

Proportion of reads mapping to C. trachomatis reference genome in relation to total C. trachomatis genome copies input. Plot showing the relationship between the numbers of input C. trachomatis genomes calculated and the proportion of reads mapping to the C. trachomatis reference (F/SW4) genome (% on-target reads).

Figure 4

Coverage of reference genome and mean read depth in relation to total C. trachomatis genome copies input. The plot shows the amount of target DNA (C. trachomatis DNA) used in the library preparations and the percentage coverage of the reference genome obtained with the various target input. The bars (black, grey, and white) illustrate the minimum mean read depth obtained from each sample. The columns to the far left represent a sample which did not undergo whole-genome enrichment during library preparation (indicated with text - No enrichment).

Figure 5

Coverage of reference genome - consensus sequences obtained directly from clinical samples. The plot shows the coverage of the reference genome (%) found when aligning the consensus sequences generated from the eight clinical samples that produced good genomic data.

Figure 6

Neighbour-joining reconstruction of the phylogeny of C. trachomatis . The figure shows an un-rooted phylogenetic reconstruction of the alignment data including 8 cultured and 8 clinical C. trachomatis samples sequenced in this study and 23 C. trachomatis genomes obtained from GenBank. The Neighbour-joining tree was constructed with 500 bootstrap replicates using a Jukes Cantor model of evolution and gamma correction for among-site rate variation with four rate categories. The gap/missing data was treated with a site coverage cut-off of 90%. The blue clusters represent the ocular/urogenital biovar (dark blue T2 clade and light blue T1 clade) and the red cluster represents the LGV biovar. The ocular serovars A-C cluster within the dark blue T2 clade. The samples sequenced in this study are highlighted in colours (blue and red) and the GenBank strains are represented in black.