Mara Lohde1, Gabriel E. Wagner2, Johanna Dabernig-Heinz2, Adrian Viehweger3, Sascha D. Braun4,5, Stefan Monecke4,5, Celia Diezel4,5, Claudia Stein1, Mike Marquet1, Ralf Ehricht4,5,6, Mathias W. Pletz1,4 and Christian Brandt1,4 1Institute for Infectious Diseases and Infection Control, Jena University Hospital, 07747 Jena, Germany; 2Diagnostic and Research Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, 8010 Graz, Austria; 3Institute of Medical Microbiology and Virology, University Hospital Leipzig, 04103 Leipzig, Germany; 4InfectoGnostics Research Campus, Center for Applied Research, 07743 Jena, Germany; 5Leibniz-Institute of Photonic Technology (Leibniz-IPHT), 07745 Jena, Germany; 6Institute of Physical Chemistry, Friedrich-Schiller-University Jena, 07743 Jena, Germany Corresponding author: mara.lohdemed.uni-jena.de Abstract

Our study investigates the effectiveness of Oxford Nanopore Technologies for accurate outbreak tracing by resequencing 33 isolates of a 3-year-long Klebsiella pneumoniae outbreak with Illumina short-read sequencing data as the point of reference. We detect considerable base errors through cgMLST and phylogenetic analysis of genomes sequenced with Oxford Nanopore Technologies, 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 besides K. pneumoniae. Based on these data, we explore PCR-based sequencing and a masking strategy, which both successfully address these inaccuracies and ensure accurate outbreak tracing. We offer our masking strategy as a bioinformatic workflow (MPOA) to identify and mask problematic genome positions in a reference-free manner. Our research highlights limitations in using Oxford Nanopore Technologies for sequencing prokaryotic organisms, especially for investigating outbreaks. For time-critical projects that cannot wait for further technological developments by Oxford Nanopore Technologies, our study recommends either using PCR-based sequencing or using our provided bioinformatic workflow. We advise that read mapping–based quality control of genomes should be provided when publishing results.

Footnotes

[Supplemental material is available for this article.]

Article published online before print. Article, supplemental material, and publication date are at https://www.genome.org/cgi/doi/10.1101/gr.278848.123.

Freely available online through the Genome Research Open Access option.

Received December 18, 2023. Accepted May 7, 2024.