An open-source nanopore-only sequencing workflow for analysis of clonal outbreaks delivers short-read level accuracy

For the past decade, bacterial whole genome sequencing has been performed using high accuracy short-read sequencing. More recently, long-read sequencing with Oxford Nanopore Technologies (ONT) instruments has emerged as a potential alternative based on multiple advantages, including lower costs, portability, and speed. However, this platform has suffered from base call error rates that were too high for many applications in clinical microbiology, including outbreak tracing. With the release of new flow cell chemistries and base call algorithms, accuracy has improved dramatically, making this approach feasible for outbreak investigations. In this work, we optimize a streamlined nanopore-only workflow for epidemiologic analysis of bacterial pathogens. The workflow was validated with isolates from four previously identified clinical outbreaks with varying GC content and demonstrated fully concordant cgMLST clustering as compared to short-read references. This workflow will facilitate the broader implementation of inexpensive ONT-only genomes and cgMLST analysis to assist in hospital outbreaks worldwide.