Comparison of Oxford Nanopore Technologies sequencing protocols using R9.4.1 and R10.4.1 flow cells under 20-hour and 48-hour conditions for genotyping methicillin methicillin-resistant Staphylococcus aureus (MRSA).

BACKGROUND

Rapid and accurate whole-genome sequencing (WGS) is increasingly important for detecting antimicrobial resistance and virulence determinants in clinical microbiology. Although short-read platforms remain widely used, long-read sequencing technologies have gained relevance due to their ability to resolve complex genomic regions.

METHODS AND

RESULTS

We analyzed 92 draft genomes generated from 23 clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates to compare Oxford Nanopore Technologies sequencing protocols using R9.4.1 (Kit V10) and R10.4.1 (Kit V14) flow cells under 20-hour and 48-hour conditions. The 20-hour R10.4.1 protocol (ONT20h R10) showed the best overall performance, with improved detection of antimicrobial resistance genes, virulence factors, plasmids, and mobile genetic elements. This protocol achieved perfect agreement with phenotypic antimicrobial susceptibility testing (accuracy 100%, κ = 1.0), supporting its suitability for rapid genomic analysis. Highly repetitive mobile genetic elements remained challenging to fully resolve, reflecting the technical difficulty of assembling complex repeats.

CONCLUSION

Overall, this study supports the use of rapid long-read sequencing as a complementary approach for clinical microbiology and genomic surveillance, particularly when timely genomic information is required.