Evaluating methods for genome sequencing of Chlamydia trachomatis and other sexually transmitted bacteria directly from clinical swabs

Rates of bacterial sexually transmitted infections (STIs) are rising, and accessing their genomes provides information on strain evolution, circulating strains and encoded antimicrobial resistance (AMR). Notable pathogens include Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Treponema pallidum (TP), globally the most common bacterial STIs. Mycoplasmoides (formerly Mycoplasma) genitalium (MG) is also a bacterial STI that is of concern due to AMR development. These bacteria are also fastidious or hard to culture, and standard sampling methods lyse bacteria, completely preventing pathogen culture. Clinical samples contain large amounts of human and other microbiota DNA. These factors hinder the sequencing of bacterial STI genomes. We aimed to overcome these challenges in obtaining whole- genome sequences and evaluated four approaches using clinical samples from Argentina (39), and Switzerland (14), and cultured samples from Finland (2) and Argentina (1). First, direct genome sequencing from swab samples was attempted through Illumina deep metagenomic sequencing, showing extremely low levels of target DNA, with under 0.01% of the sequenced reads being from the target pathogens. Second, host DNA depletion followed by Illumina sequencing was not found to produce enrichment in these very low- load samples. Third, we tried a selective long- read approach with the new adaptive sequencing from Oxford Nanopore Technologies, which also did not improve enrichment sufficiently to provide genomic information. Finally, target enrichment using a novel pan- genome set of custom SureSelect probes targeting CT, NG, TP and MG followed by Illumina sequencing was successful. We produced whole genomes from 64% of CT- positive samples, from 36% of NG- positive samples and 60% of TP- positive samples. Additionally, we enriched MG DNA to gain partial genomes from 60% of samples. This is the first publication to date to utilize a pan- genome STI panel in target enrichment. Target enrichment, though costly, proved essential for obtaining genomic data from clinical samples. These data can be utilized to examine circulating strains and genotypic resistance and guide public health strategies.