Assessment of Oxford Nanopore whole genome sequencing for large-scale genomic characterisation of Staphylococcus aureus

Haugan, I.; Flatby, H. M.; Lysvand, H.; Skei, N. V.; Zaragkoulias, K.; Solligard, E.; Ronning, T. G.; Olsen, L. C.; Damas, J. K.; Afset, J. E.; As, C. G.

doi:10.64898/2026.03.30.715209

bioRxiv · 10.64898/2026.03.30.715209

Assessment of Oxford Nanopore whole genome sequencing for large-scale genomic characterisation of Staphylococcus aureus

Haugan, I.·Flatby, H. M.·Lysvand, H.·Skei, N. V.·Zaragkoulias, K.·Solligard, E.·Ronning, T. G.·Olsen, L. C.·Damas, J. K.·Afset, J. E.·As, C. G.

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Abstract

Whole-genome sequencing (WGS) is increasingly being utilised in microbial diagnostics, surveillance, and research. In this paper we assess the performance of one leading long-read sequencing technology, Oxford Nanopore Technology (ONT), on 836 Staphylococcus aureus bacteraemia isolates. We compare the results to that of a leading short-read sequencing technology, Illumina. All isolates were sequenced using ONT MinION Mk1B and Illumina HiSeq or MiSeq. Libraries were prepared according to manufacturers instructions. Preprocessing and downstream bioinformatic analyses were performed using a combination of in-house pipelines and publicly available software tools. The average base substitution error rate in ONT assemblies was low but varied between sequence types, possibly due to lineage-specific methylation patterns. Multi locus sequence typing was similar between the technologies, while ONT assemblies allowed for better spa typing than Illumina assemblies. The reported detection rate was similar between ONT and Illumina assemblies for most virulence- and AMR-associated genes and variants. For 42 (22.2%) of 189 genes/variants, the two technologies disagreed in gene detection in 5 isolates or more, and in 39 (20.6.%) of these the highest detection rate was found with ONT. Discrepancies were mainly associated with low GC content, multiple repetitive segments, and small plasmids. Polishing of ONT data resulted in minor changes in gene/variant calling. Our study supports the use of ONT WGS for bacterial population genomic studies on a large collection of S. aureus isolates. While assembly of ONT reads may be affected by its own methodological limitations, it was superior to Illumina assemblies in detection of potentially clinically relevant genes and variants at a low read error rate. Understanding the advantages and limitations of WGS technologies is essential before undertaking studies involving such methods on large sets of bacteria. Author summaryIn this paper, we present a practical assessment of one important whole genome sequencing (WGS) method, Oxford Nanopore Technology (ONT), and compare its performance in bacterial population genomics to that of WGS with Illumina technology. Our goal was to investigate the usefulness of ONT in studies aiming to identify clinically relevant bacterial characteristics in large collections of bacteria, such as genotype-phenotype studies. We sequenced a large set of clinical S. aureus isolates from episodes of bloodstream infections using both ONT and Illumina technologies and performed analyses with widely used software and bioinformatic pipelines. We have elucidated inherent strengths and limitations of ONT and Illumina sequencing and report some of the practical consequences of these on bacterial typing and detection of clinically relevant genes. With this study, we present one of the most comprehensive assessments of long-read sequencing technology for the genomic characterisation of clinical bacterial isolates, and the findings provide guidance for researchers considering WGS in large-scale bacterial genomics.

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Haugan, I., Flatby, H. M., Lysvand, H., Skei, N. V., Zaragkoulias, K., Solligard, E., Ronning, T. G., Olsen, L. C., Damas, J. K., Afset, J. E., As, C. G.. 2026-04-01. Assessment of Oxford Nanopore whole genome sequencing for large-scale genomic characterisation of Staphylococcus aureus. https://doi.org/10.64898/2026.03.30.715209

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