Whole-Genome Enrichment and Sequencing of Chlamydia trachomatis Directly from Patient Clinical Vaginal and Rectal Swabs

Abstract

Chlamydia trachomatis, an obligately intracellular bacterium, is the most prevalent cause of bacterial sexually transmitted infections (STIs) worldwide. Numbers of U.S. infections of the urogenital tract and rectum have increased annually. Because C. trachomatis is not easily cultured, comparative genomic studies are limited, restricting our understanding of strain diversity and emergence among populations globally. While Agilent SureSelect^XT target enrichment RNA bait libraries have been developed for whole-genome enrichment and sequencing of C. trachomatis directly from clinical urine, vaginal, conjunctival, and rectal samples, public access to these libraries is not available. We therefore designed an RNA bait library (34,795 120-mer probes based on 85 genomes, versus 33,619 probes using 74 genomes in a previous one) to augment organism sequencing from clinical samples that can be shared with the scientific community, enabling comparison studies. We describe the library and limit of detection for genome copy input, and we present results of 100% efficiency and high-resolution determination of recombination and identical genomes within vaginal-rectal specimen pairs in women. This workflow provides a robust approach for discerning genomic diversity and advancing our understanding of the molecular epidemiology of contemporary C. trachomatis STIs across sample types, geographic populations, sexual networks, and outbreaks associated with proctitis/proctocolitis among women and men who have sex with men.IMPORTANCEChlamydia trachomatis is an obligate intracellular bacterium that is not easily cultured, which limits our understanding of urogenital and rectal C. trachomatis transmission and impact on morbidity. To provide a publicly available workflow for whole-genome target enrichment and sequencing of C. trachomatis directly from clinical urine, vaginal, conjunctival, and rectal specimens, we developed and report on an RNA bait library to enrich the organism from clinical samples for sequencing. We demonstrate an increased efficiency in the percentage of reads mapping to C. trachomatis and identified recombinant and identical C. trachomatis genomes in paired vaginal-rectal samples from women. Our workflow provides a robust genomic epidemiologic approach to advance our understanding of C. trachomatis strains causing ocular, urogenital, and rectal infections and to explore geo-sexual networks, outbreaks of colorectal infections among women and men who have sex with men, and the role of these strains in morbidity.

Keywords: Chlamydia trachomatis; SNPs; recombination; whole-genome enrichment.

FIG 1

LOD of genome copies using the SureSelect^XT target enrichment workflow for spiked serial dilutions of reference strain D/UW-3/Cx gDNA. The data are the percent coverage of the reference genome for each serial dilution.

FIG 2

Percent coverage of reference genome and reads mapping to the respective reference genome for spiked mock samples of genotype L₂b and patient specimen sets. The percent coverage of the reference genome for each patient specimen is represented by bars, with bar coloring based on average mean read depth as indicated. R, rectal sample; V, vaginal sample. The orange line represents the percent down-selected Chlamydia spp. and C. trachomatis read pairs.

FIG 3

Global phylogeny of patient specimen sets and 94 C. trachomatis complete genomes. The four major lineages of C. trachomatis are highlighted with circular tip shapes in four distinct colors. The associated ompA genotypes for each genome derived from the whole-genome data are also shown with the color code on the right. Patient specimen sets are highlighted in yellow.