Replicate reference dataset, and unknown outbreak datasets used to test OxBreaker
Real-time genomic surveillance is key to mitigate the increasing treatment costs of healthcare-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 au- tomated 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, and 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 SNPs for common species. We further validated the pipeline by accu- rately 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' facilitates the adoption of local, real-time genomic surveillance for frontline infection prevention and control.