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. Mycoplasma genitalium (MG) is also a bacterial STI which 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), 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 (ONT), 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 from 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. This data can be utilized to examine circulating strains, genotypic resistance, and guide public health strategies. Graphical Impact statement: Genome data on circulating sexually transmitted infections (STIs) is important to better understand transmission networks, antimicrobial resistance and to guide treatment decisions. For many bacterial STIs, this information is difficult to obtain, as the bacteria are fastidious, in some cases intracellular, and often recalcitrant to culture. We have developed and tested a target enrichment STI panel of baits to capture whole genomes of Chlamydia trachomatis , Neisseria gonorrhoeae , Treponema pallidum, and Mycoplasma genitalium with approximately 50% success in genome sequencing for the first three pathogens. We compare this against other sequencing and enrichment methods, which did not provide sufficient data for genome analysis. This panel approach shows potential for clinical samples carrying these pathogens and can potentially also be developed for further pathogen groups. Data summary: All illumina sequence data, with human read data removed using Hostile (1) and KrakenTools ( https://github.com/jenniferlu717/KrakenTools ), is deposited with the European Nucleotide Archive (ENA) under project number PRJEB72167.

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