OxBreaker: species-agnostic pipeline for the analysis of outbreaks using nanopore sequencing

Abstract Real-time genomic surveillance may mitigate the spread of health-care-associated infections, but whole-genome sequencing costs and the need for specialised expertise constrain its wide implementation in public health. Here we present ‘ OxBreaker ’, an automated and species-agnostic pipeline optimised for the high-resolution analysis of bacterial and plasmid genomes sequenced via Oxford Nanopore Technologies (ONT). ‘ OxBreaker ’ streamlines the transition from raw reads to phylogenetic inference through automated reference selection and high-accuracy variant calling. It is accessible via a graphical user interface (GUI) that can be easily installed locally and operated by non-specialists. Benchmarking against technical and biological replicates of high-priority pathogens demonstrates high accuracy, with false positive variant rates reduced to 0–4 single-nucleotide polymorphisms (SNPs) for common species. We further validated the pipeline by accurately characterising previously published clonal and plasmid-mediated outbreaks, reproducing established phylogenies with improved accessibility. By providing a stable, scalable, open-source offline-compatible solution that matches the resolution of short-read platforms while maintaining the speed of long-read technology, ‘ OxBreaker ’ is designed to facilitate the adoption of local, real-time genomic surveillance for frontline infection prevention and control.