Oxford Nanopore Technologies R10 sequencing enables accurate cgMLST-based bacterial outbreak investigation of <i>Neisseria meningitidis</i> and <i>Salmonella enterica</i> when accounting for methylation-related errors

This study evaluates the suitability of Oxford Nanopore Technologies R10 sequencing for core-genome multi-locus sequence typing (cgMLST), a widely used method in (clinical) outbreak investigation and bacterial strain tracking. We have sequenced 24 <i>Neisseria meningitidis</i> and 24 <i>Salmonella enterica</i> strains, including confirmed outbreak cases, using Illumina and ONT R10 sequencing to evaluate the performance for cgMLST analysis. We used a PCR-based and native barcoding protocol for the ONT sequencing, which enabled us to demonstrate a substantial species-dependent impact of methylation-related errors on the performance. However, we demonstrate that when these errors are properly addressed, ONT R10 can be used for accurate cgMLST-based clustering, including integration with strains sequenced using Illumina. Our findings support the use of ONT R10 as an alternative to Illumina sequencing for cgMLST analysis in routine public health practice.