Design and evaluation of probe-capture sequencing for clinical Neisseria gonorrhoeae specimens

Characterizing the molecular epidemiology of Neisseria gonorrhoeae is essential for understanding disease burden, elucidating transmission dynamics, and informing clinical decision-making and public health interventions. Given that routine, culture-independent diagnostics rapidly and sensitively detect this difficult-to-cultivate pathogen, aligning genomic surveillance with these workflows is necessary to understand its epidemiology and evolutionary dynamics. To address this challenge, we investigated the utility of probe-capture sequencing to selectively enrich for pathogen-specific DNA during next-generation sequencing. Custom biotinylated oligonucleotide baits were designed using genetic information from publicly available genomes hosted on Pathogenwatch. Of the 32,574 assemblies, 2,877 representative genomes were selected to inform probe generation using core genome amino acid distance clustering and representation of unique resistance-associated genotypes and predicted resistance phenotypes. The resulting probe set provided generous coverage of the reference genome, N. gonorrhoeae FA 1090, with 94.6% nucleotide coverage and 99.3% of annotated genes covered by at least one probe. Piloting this technology on 12 clinical urine specimens with variable relative pathogen abundances revealed that probe-capture sequencing substantially increased the relative abundance of N. gonorrhoeae sequence reads from a complex sample (fold-change range, 226 - 2046), enabling considerable genome coverage, the construction of high-quality genome assemblies, and the detection of resistance-associated genotypes. These findings demonstrate that probe-capture sequencing is a viable method for enriching N. gonorrhoeae genetic material from clinical samples, enabling culture-independent genomic investigations into the epidemiology of hard-to-culture pathogens and providing actionable insights to inform clinical and public health interventions.