DNA capture and amplicon enrichment approaches for next-generation sequencing of Mycoplasma genitalium directly from clinical samples

Direct genome sequencing of Mycoplasma genitalium from clinical specimens is challenging due to the organisms low bacterial load. We developed and compared two DNA enrichment strategies--amplicon-based and hybridisation capture-based--coupled with next-generation sequencing, and assessed the suitability of DNA capture for whole-genome sequencing and high-resolution molecular typing. The enrichment approaches, RNA bait hybridisation and targeted sequence amplification, were combined with paired-end sequencing on the Illumina iSeq 100. Method performance was evaluated in 89 M. genitalium-positive specimens across five genomic loci: 23S and 16S rRNA, and parC and gyrA for macrolide, tetracycline, and fluoroquinolone resistance, respectively, and mgpB for phylogenetic analysis. Regarding antimicrobial resistance (AMR) determinants, the multiplex amplicon sequencing method demonstrated the highest sensitivity, at 93.3-98.9%. Although < 50% of samples were characterised using DNA capture, concordance between the two methods was excellent. Using DNA capture, 24.7% of specimens achieved a minimum coverage of 95% at 1x depth. Sequencing success was inversely correlated with human DNA contamination and the presence of low-quality reads. Whole-genome single-nucleotide polymorphism analysis provided higher discriminatory power than multi-locus sequence typing schemes and confirmed the clonality of multidrug-resistant M. genitalium strains belonging to the mgpB genotype 159. In conclusion, targeted amplicon-based enrichment is the most accurate and reliable approach for epidemiological studies focused on AMR and mgpB typing, whereas DNA capture is valuable for generating comprehensive genomic data from selected M. genitalium-positive specimens. Impact statementCulturing Mycoplasma genitalium from clinical specimens is challenging; consequently, epidemiological studies rely on molecular techniques. Whole-genome sequences have been obtained from a few M. genitalium clinical isolates, and direct sequencing from clinical specimens requires enrichment strategies to overcome the organisms low bacterial load. Consequently, published data on antimicrobial resistance (AMR) mechanisms and phylogenetic relationships among circulating strains remain incomplete. We demonstrated that targeted amplicon-based enrichment provided high-resolution AMR detection and highly sensitive mgpB genotyping, enabling the identification of minority variants. In contrast, hybridisation capture-based enrichment showed lower sensitivity but permitted successful WGS in a subset of M. genitalium-positive specimens and supported the development of new typing schemes. Although amplicon-based enrichment remains a reliable approach for epidemiological studies, strategic application of DNA capture facilitates the generation of comprehensive genomic data even though performance is reduced in the context of low bacterial loads, high host DNA contamination or suboptimal DNA quality.