A multicenter study on accuracy and reproducibility of nanopore sequencing-based genotyping of bacterial pathogens.

Autor:	Dabernig-Heinz J; Diagnostic and Research Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, Graz, Austria., Lohde M; Institute for Infectious Diseases and Infection Control, Jena University Hospital, Jena, Germany., Hölzer M; Genome Competence Center (MF1), Robert Koch Institute, Berlin, Germany., Cabal A; Austrian Agency for Health and Food Safety, Vienna, Austria., Conzemius R; Ares Genetics GmbH, Vienna, Austria., Brandt C; Institute for Infectious Diseases and Infection Control, Jena University Hospital, Jena, Germany., Kohl M; Medical and Life Sciences Faculty, Furtwangen University, Villingen-Schwenningen, Germany., Halbedel S; Nosocomial Pathogens and Antibiotic Resistances (FG13), Robert Koch Institute, Wernigerode, Germany.; Institute for Medical Microbiology and Hospital Hygiene, Otto von Guericke University Magdeburg, Magdeburg, Germany., Hyden P; Austrian Agency for Health and Food Safety, Vienna, Austria., Fischer MA; Enteropathogenic bacteria and Legionella (FG11), Consultant Laboratory for Listeria, Robert Koch Institute, Wernigerode, Germany., Pietzka A; Austrian Agency for Health and Food Safety, Graz, Austria., Daza B; Austrian Agency for Health and Food Safety, Vienna, Austria., Idelevich EA; Friedrich Loeffler Institute for Medical Microbiology, F.-Sauerbruch-Str., Greifswald, Germany., Stöger A; Austrian Agency for Health and Food Safety, Vienna, Austria., Becker K; Friedrich Loeffler Institute for Medical Microbiology, F.-Sauerbruch-Str., Greifswald, Germany., Fuchs S; Genome Competence Center (MF1), Robert Koch Institute, Berlin, Germany., Ruppitsch W; Austrian Agency for Health and Food Safety, Vienna, Austria., Steinmetz I; Diagnostic and Research Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, Graz, Austria., Kohler C; Friedrich Loeffler Institute for Medical Microbiology, F.-Sauerbruch-Str., Greifswald, Germany., Wagner GE; Diagnostic and Research Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, Graz, Austria.
Jazyk:	angličtina
Zdroj:	Journal of clinical microbiology [J Clin Microbiol] 2024 Sep 11; Vol. 62 (9), pp. e0062824. Date of Electronic Publication: 2024 Aug 19.
DOI:	10.1128/jcm.00628-24
Abstrakt:	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. Competing Interests: R.C. was an employee of the company Ares Genetics. This does not affect the authors' adherence to all the journal's policies on sharing data and materials. Twenty flow cells were provided free of charge by Oxford Nanopore Technologies. However, the manufacturer did not participate in the study's design, data collection, interpretation, or any other aspects of the research.
Databáze:	MEDLINE
Externí odkaz:	Zobrazit plný text záznamu