Abstract
Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is common, compromising gonorrhoea treatment internationally. Rapid characterisation of AMR strains could ensure appropriate and personalised treatment, and support identification and investigation of gonorrhoea outbreaks in nearly real-time. Whole-genome sequencing is ideal for investigation of emergence and dissemination of AMR determinants, predicting AMR, in the gonococcal population and spread of AMR strains in the human population. The novel, rapid and revolutionary long-read sequencer MinION is a small hand-held device that generates bacterial genomes within one day. However, accuracy of MinION reads has been suboptimal for many objectives and the MinION has not been evaluated for gonococci. In this first MinION study for gonococci, we show that MinION-derived sequences analysed with existing open-access, web-based sequence analysis tools are not sufficiently accurate to identify key gonococcal AMR determinants. Nevertheless, using an in house-developed CLC Genomics Workbench including de novo assembly and optimised BLAST algorithms, we show that 2D ONT-derived sequences can be used for accurate prediction of decreased susceptibility or resistance to recommended antimicrobials in gonococcal isolates. We also show that the 2D ONT-derived sequences are useful for rapid phylogenomic-based molecular epidemiological investigations, and, in hybrid assemblies with Illumina sequences, for producing contiguous assemblies and finished reference genomes.
Highlights
Gonorrhoea is a sexually transmitted infection caused by Neisseria gonorrhoeae
We evaluated the performance characteristics, ideal sequence analysis (tools and workflow for taxonomy, assembly, assembly improvement (“polishing”) and mapping), phylogenomic analysis, and prediction of decreased susceptibility or resistance to recommended therapeutic antimicrobials in N. gonorrhoeae isolates using the Oxford Nanopore MinION sequencer
The assemblies were evaluated against the PacBio RS II-sequenced 2016 World Health Organization (WHO) gonococcal reference genomes (Bioproject PRJEB14020)[40] for sequence variation and to assess whether the assemblies were of sufficient quality for characterisation of Antimicrobial resistance (AMR) determinants and phylogenomic-based molecular epidemiology of gonococci
Summary
Gonorrhoea is a sexually transmitted infection caused by Neisseria gonorrhoeae (gonococcus). Oxford Nanopore Technologies (ONT) (Oxford, UK) have introduced this approach with their single-molecule nanopore genome sequencing device MinION, a TGS platform with unique technology that was commercialized in mid-201526–30. When both strands are sequenced, a consensus sequence of the molecule can be produced; these consensus reads are termed two-directional reads (1D2 or 2D ONT reads) and generally have higher accuracy than reads from only a single pass of the molecule (1D ONT reads). The assemblies were evaluated against the PacBio RS II-sequenced 2016 WHO gonococcal reference genomes (Bioproject PRJEB14020)[40] for sequence variation and to assess whether the assemblies were of sufficient quality for characterisation of AMR determinants and phylogenomic-based molecular epidemiology of gonococci
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