A multicenter study on accuracy and reproducibility of nanopore sequencing-based genotyping of bacterial pathogens
Nanopore sequencing has shown the potential to democratize genomic pathogen surveillance due to its ease of use and low entry cost. However, recent genotyping studies showed discrepant results compared to gold-standard short-read sequencing. Furthermore, although essential for widespread application, the reproducibility of nanopore-only genotyping remains largely unresolved. In our multicenter performance study involving five laboratories, four public health-relevant bacterial species were sequenced with the latest R10.4.1 flow cells and V14 chemistry. Core genome MLST analysis of over 500 data sets revealed highly strain-specific typing errors in all species in each laboratory. Investigation of the methylation-related errors revealed consistent DNA motifs at error-prone sites across participants at read level. Depending on the frequency of incorrect target reads, this either leads to correct or incorrect typing, whereby only minimal frequency deviations can randomly determine the final result. PCR preamplification, recent basecalling model updates and an optimized polishing strategy notably diminished the non-reproducible typing. Our study highlights the potential for new errors to appear with each newly sequenced strain and lays the foundation for computational approaches to reduce such typing errors. In conclusion, our multicenter study shows the necessity for a new validation concept for nanopore sequencing-based, standardized bacterial typing, where single nucleotide accuracy is critical.
Authors
- Johanna Dabernig‐Heinz (ORCID: https://orcid.org/0009-0007-2800-3659)
- Patrick Hyden
- Gabriel E. Wagner (ORCID: https://orcid.org/0000-0002-5704-3955)
- Martin A. Fischer (ORCID: https://orcid.org/0000-0002-9319-3862)
- Christian Brandt (ORCID: https://orcid.org/0000-0002-7199-3957)
- Beatriz Daza Prieto (ORCID: https://orcid.org/0000-0002-7138-5441)
- Rick Conzemius (ORCID: https://orcid.org/0000-0002-7116-8180)
- Adriana Cabal Rosel (ORCID: https://orcid.org/0000-0001-9346-1612)
- Ariane Pietzka (ORCID: https://orcid.org/0000-0003-2987-2905)
- Martin Hölzer (ORCID: https://orcid.org/0000-0001-7090-8717)
- Sven Halbedel (ORCID: https://orcid.org/0000-0002-5575-8973)
- Stephan Fuchs (ORCID: https://orcid.org/0000-0002-1338-2699)
- Anna Stöger
- Matthias Kohl (ORCID: https://orcid.org/0000-0001-9514-8910)
- Karsten Becker (ORCID: https://orcid.org/0000-0002-6391-1341)
- Evgeny A. Idelevich (ORCID: https://orcid.org/0009-0009-4207-5290)
- Werner Ruppitsch (ORCID: https://orcid.org/0000-0001-9940-3333)
- Ivo Steinmetz (ORCID: https://orcid.org/0000-0003-0510-7336)
- Mara Lohde (ORCID: https://orcid.org/0009-0009-1718-7231)
- Christian Köhler (ORCID: https://orcid.org/0000-0003-3921-6776)
Institutions
- Friedrich-Loeffler-Institut (DE)
- Medical University of Graz (AT)
- Robert Koch Institute (DE)
- Jena University Hospital (DE)
- Austrian Agency for Health and Food Safety (AT)
- Furtwangen University (DE)
- Otto-von-Guericke-Universität Magdeburg (DE)
Publication Details
- Journal
- Journal of Clinical Microbiology
- Published
- 2024-08-19
- DOI
- https://doi.org/10.1128/jcm.00628-24
- Citations
- 25
- Primary Topic
- Bacterial Identification and Susceptibility Testing
- Type
- article
- Field-Weighted Citation Impact
- 9.53
Funders
- Oxford Nanopore Technologies
- Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology
- Thüringer Ministerium für Wirtschaft, Wissenschaft und Digitale Gesellschaft
- Robert Koch Institut