Generating Whole Bacterial Genomes from Clinical Samples using a Target Enrichment Workflow

Obtaining complete genomes of bacterial sexually transmitted infections (STIs) directly from clinical samples is challenging due to the presence of human DNA, microbiota, and very low bacterial pathogen loads. Culture is often not an option, as these species are fastidious, and most samples are taken in transport media that lyse microorganisms, rendering them non-viable for culture. To address these issues, we used a probe panel designed across four species to generate whole-genome sequences of bacterial STIs and performed target enrichment, a two- to three-day hybridization procedure prior to genome sequencing. Our initial results produced excellent genomic data using samples for which direct sequencing was not successful. Target enrichment proved to be highly effective for sequencing bacterial STI genomes, particularly when pathogen load was high (Ct < 30). Tested samples containing Mycoplasma genitalium frequently had Ct values above 30, leading to lower genome recovery success rates; improvements have been observed after optimizing the hybridization temperature from 65 °C to 62.5 °C. In addition to enabling genomic surveillance, the availability of complete genomes can provide valuable insights into antimicrobial resistance, such as identifying acquired resistance determinants associated with Neisseria gonorrhoeae. This method has already led to the identification of a new lineage of Chlamydia trachomatis lymphogranuloma venereum in Buenos Aires, ompA-genotype L4, highlighting the potential of this approach for uncovering novel genomic diversity and improving STI surveillance.