A multicenter study on accuracy and reproducibility of nanopore sequencing-based genotyping of bacterial pathogens.
Johanna Dabernig-Heinz, Mara Lohde, Martin H��lzer, Adriana Cabal, Rick Conzemius, Christian Brandt, Matthias Kohl, Sven Halbedel, Patrick Hyden, Martin A Fischer, Ariane Pietzka, Beatriz Daza, Evgeny A Idelevich, Anna St��ger, Karsten Becker, Stephan Fuchs, Werner Ruppitsch, Ivo Steinmetz, Christian Kohler, Gabriel E Wagner
Author Information
Johanna Dabernig-Heinz: Diagnostic and Research Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, Graz, Austria. ORCID
Mara Lohde: Institute for Infectious Diseases and Infection Control, Jena University Hospital, Jena, Germany.
Martin H��lzer: Genome Competence Center (MF1), Robert Koch Institute, Berlin, Germany.
Adriana Cabal: Austrian Agency for Health and Food Safety, Vienna, Austria.
Rick Conzemius: Ares Genetics GmbH, Vienna, Austria.
Christian Brandt: Institute for Infectious Diseases and Infection Control, Jena University Hospital, Jena, Germany.
Matthias Kohl: Medical and Life Sciences Faculty, Furtwangen University, Villingen-Schwenningen, Germany.
Sven Halbedel: Nosocomial Pathogens and Antibiotic Resistances (FG13), Robert Koch Institute, Wernigerode, Germany. ORCID
Patrick Hyden: Austrian Agency for Health and Food Safety, Vienna, Austria.
Martin A Fischer: Enteropathogenic bacteria and Legionella (FG11), Consultant Laboratory for Listeria, Robert Koch Institute, Wernigerode, Germany.
Ariane Pietzka: Austrian Agency for Health and Food Safety, Graz, Austria. ORCID
Beatriz Daza: Austrian Agency for Health and Food Safety, Vienna, Austria. ORCID
Evgeny A Idelevich: Friedrich Loeffler Institute for Medical Microbiology, F.-Sauerbruch-Str., Greifswald, Germany. ORCID
Anna St��ger: Austrian Agency for Health and Food Safety, Vienna, Austria.
Karsten Becker: Friedrich Loeffler Institute for Medical Microbiology, F.-Sauerbruch-Str., Greifswald, Germany. ORCID
Stephan Fuchs: Genome Competence Center (MF1), Robert Koch Institute, Berlin, Germany.
Werner Ruppitsch: Austrian Agency for Health and Food Safety, Vienna, Austria. ORCID
Ivo Steinmetz: Diagnostic and Research Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, Graz, Austria. ORCID
Christian Kohler: Friedrich Loeffler Institute for Medical Microbiology, F.-Sauerbruch-Str., Greifswald, Germany. ORCID
Gabriel E Wagner: Diagnostic and Research Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, Graz, Austria. ORCID
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.
