Rapid genotyping of Salmonella using Oxford Nanopore-only sequencing for outbreak investigation

Aims Whole genome sequencing of Salmonella is a powerful tool for case identification during outbreak investigations. Long-read sequencing with Oxford Nanopore is a promising option due to its speed and cost-effectiveness, albeit with the downside of potential higher error rates. However, recent technological developments have greatly increased its accuracy. In this study, we tested the performance of Oxford Nanopore-only sequencing for core genome multilocus sequence typing (cgMLST) of Salmonella during outbreak investigations. Methods and Results We sequenced 40 Salmonella isolates from six different serovars from historical outbreak-associated and sporadic cases from Norway with Illumina and Oxford Nanopore technologies. We tested different basecalling, filtering, assemblers and polishing tools, and calculated allelic differences (ad) between Illumina and Oxford Nanopore assemblies. cgMLST typing of the Oxford Nanopore-only assemblies agreed with the Illumina references: all outbreak samples clustered appropriately, with an average ad of 0.10 between the two technologies. Basecalling and polishing steps had the greatest impact on Oxford Nanopore genotyping accuracy. We found accurate genotyping could be obtained in as early as five hours of sequencing, leading to a colony-to-result turnaround time of around 24 hours. However, accurate antimicrobial gene detection and plasmid reconstruction remained challenging. Conclusions As of R10.4.1 flowcells and v.4.3.0 basecalling models, Nanopore-only sequencing is a viable and rapid alternative to Illumina for Salmonella genotyping during outbreak investigations, offering comparable results in a faster turnaround time.