Speed and accuracy of whole-genome nanopore sequencing for differential Neisseria gonorrhoeae strain detection in samples collected prospectively from a sexual health clinic

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Abstract
Background: Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a continuing global health challenge. Limitations to current national surveillance systems for reporting AMR trends, alongside reduction in culture-based diagnostics and susceptibility testing, has led to an increasing need for rapid diagnostics and identification of circulating N. gonorrhoeae strains. We investigated nanopore based sequencing time and depth needed to accurately identify closely related N. gonorrhoeae isolates, compared to Illumina MiSeq sequencing. Methods: N. gonorrhoeae strains prospectively collected from a London Sexual Health clinic were sequenced on both Illumina MiSeq and Oxford Nanopore Technologies (ONT) MinION platforms. Accuracy was determined by comparing variant calls at 68 nucleotide positions representing 37 pre-characterised resistance associated markers in N. gonorrhoeae . Accuracy at varying MinION sequencing depths were determined through retrospective analysis of time-stamped reads. Results: Of the 22 MinION-MiSeq sequence pairs that reached sufficient depth of coverage for comparison, overall agreement of variant call positions passing quality control criteria was 185/185 (95% CI: 98.0-100.0), 502/503 (95% CI: 98.9-99.9) and 564/565 (95% CI: 99.0-100.0) at 10x, 30x and 40x MinION depth, respectively. Isolates found to be genetically closely related by MiSeq, that is within one yearly evolutionary distance of ≤5 single nucleotide polymorphisms, were accurately identified as such via MinION. Conclusion: Nanopore based sequencing shows utility for use as a rapid surveillance tool to correctly detect closely related N. gonorrhoeae strains, with just 10x sequencing depth, taking a median sequencing time of 29 minutes. This highlights its potential utility for tracking local gonorrhoea transmission and AMR markers.

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