Nanopore sequencing for accurate bacterial outbreak tracing

Abstract

AbstractBackgroundOur study investigated the effectiveness of Oxford Nanopore Technology for accurate outbreak tracing by resequencing a three-year-longKlebsiella pneumoniaeoutbreak with Illumina short-read sequencing data as the point of reference.ResultsWe detected considerable base errors through cgMLST and phylogenetic analysis of genomes sequenced with Nanopore compared to the Illumina data, leading to the false exclusion of some outbreak-related strains from the outbreak cluster. Nearby methylation sites cause these errors and can also be found in other species besidesK. pneumoniae. Based on this data, we explored PCR-based sequencing and a masking strategy, which both successfully addressed these inaccuracies and ensured accurate outbreak tracing. Additionally, we offer a bioinformatic workflow (MPOA is freely available on GitHub under the GNUv3 license: github.com/replikation/MPOA) to identify and mask problematic genome positions in a reference-free manner.ConclusionsOur research identified limitations in using Oxford Nanopore Technologies for sequencing prokaryotic organisms, especially for investing outbreaks. Without further technological developments, our study recommends either PCR-based sequencing or using our provided bioinformatic workflow to mask problematic genome positions.