Nanopore based bacterial genotyping/surveillance in multi-center performance study

Nanopore sequencing has shown the potential to democratize genomic surveillance of pathogens due to its ease-of-use and low entry cost. However, in genotyping there is a crucial need for validation before widespread adoption of Nanopore-only approaches, as recent studies show discrepant results compared to the established gold standard of short-read sequencing. In a unique performance study involving five participants sequencing four public health-relevant bacteria species with the latest R10.4.1 flow cells and sequencing kit (V14) we uncovered strain-specific typing errors in all species that make inter-laboratory comparisons infeasible. The investigation of the causative methylation errors, revealed a random, dichotomous allele assignment based on minimal differences in the reads. The resulting non-reproducible typings were noticeably improved by utilizing PCR barcoding and recent updates of basecalling models by ONT. Our study raises awareness that single nucleotide accuracy is critical and lays the foundation for future validation studies and computational approaches to reduce such errors. Our results highlight the potential for new errors with each new strain, flow cell, and basecalling model, necessitating a constant re-evaluation of established validation procedures - especially when each nucleotide matters.