Resolving a neonatal intensive care unit outbreak of methicillin-resistant Staphylococcus aureus to the SNV level using Oxford Nanopore simplex reads and HERRO error correction.

Background: Our laboratory began prospective genomic surveillance for healthcare-associated organisms in 2022 using Oxford Nanopore Technologies (ONT) as a standalone platform. While effective for early outbreak detection, the lower read accuracy compared to Illumina sequencing has limited single-nucleotide variant (SNV) analysis.

Aim: To determine whether Haplotype-aware ERRor cOrrection (HERRO) of ONT data could permit high-resolution comparison of outbreak isolates.

Methods: We used ONT simplex reads from isolates involved in a recent outbreak of methicillin-resistant Staphylococcus aureus (MRSA) in our neonatal unit. Raw data were basecalled and adapter-trimmed using Dorado v0.7.0, followed by HERRO correction. Genome assemblies and phylogenies were compared with previous analyses (using Dorado v0.3.4 no HERRO correction, and data generated by Illumina sequencing).

Findings: Five of nine outbreak isolates were analysed; four had insufficient read lengths (N50 values <10,000 bp) and did not achieve complete chromosome coverage post-HERRO correction. The average chromosome sequencing depth for nanopore data was 147× (range: 44-220×) with an average read N50 of 12,215 bp (interquartile range: 11,439-12,711 bp). The median pairwise SNV distance between outbreak isolates from the original investigation was 51 SNVs (range: 40-68), which decreased to 3 SNVs (range: 1-15) with HERRO correction. Illumina sequencing generated a median SNV distance of 2 (range: 0-13). The HERRO-corrected ONT phylogeny closely matched the Illumina-generated phylogeny.

Conclusion: HERRO correction enabled high-resolution analysis of MRSA outbreak isolates comparable to Illumina sequencing. ONT sequencing with HERRO correction represents a viable standalone option for detailed genomic analysis of hospital outbreaks, provided sufficient read lengths are achieved.