Real-Time Nanopore Q20+ Sequencing Enables Extremely Fast and Accurate Core Genome MLST Typing and Democratizes Access to High-Resolution Bacterial Pathogen Surveillance.
Gabriel E Wagner, Johanna Dabernig-Heinz, Michaela Lipp, Adriana Cabal, Jonathan Simantzik, Matthias Kohl, Martina Scheiber, Sabine Lichtenegger, Ralf Ehricht, Eva Leitner, Werner Ruppitsch, Ivo Steinmetz
Author Information
Gabriel E Wagner: Diagnostic and Research Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, Graz, Austria. ORCID
Johanna Dabernig-Heinz: Diagnostic and Research Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, Graz, Austria.
Michaela Lipp: Diagnostic and Research Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, Graz, Austria.
Adriana Cabal: Austrian Agency for Health and Food Safety, Vienna, Austria. ORCID
Jonathan Simantzik: Medical and Life Sciences Faculty, Furtwangen University, Villingen-Schwenningen, Germany.
Matthias Kohl: Medical and Life Sciences Faculty, Furtwangen University, Villingen-Schwenningen, Germany.
Martina Scheiber: Diagnostic and Research Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, Graz, Austria.
Sabine Lichtenegger: Diagnostic and Research Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, Graz, Austria.
Ralf Ehricht: InfectoGnostics Research Campus, Centre for Applied Research, Jena, Germany.
Eva Leitner: Diagnostic and Research Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, Graz, Austria.
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
Next-generation whole-genome sequencing is essential for high-resolution surveillance of bacterial pathogens, for example, during outbreak investigations or for source tracking and escape variant analysis. However, current global sequencing and bioinformatic bottlenecks and a long time to result with standard technologies demand new approaches. In this study, we investigated whether novel nanopore Q20+ long-read chemistry enables standardized and easily accessible high-resolution typing combined with core genome multilocus sequence typing (cgMLST). We set high requirements for discriminatory power by using the slowly evolving bacterium Bordetella pertussis as a model pathogen. Our results show that the increased raw read accuracy enables the description of epidemiological scenarios and phylogenetic linkages at the level of gold-standard short reads. The same was true for our variant analysis of vaccine antigens, resistance genes, and virulence factors, demonstrating that nanopore sequencing is a legitimate competitor in the area of next-generation sequencing (NGS)-based high-resolution bacterial typing. Furthermore, we evaluated the parameters for the fastest possible analysis of the data. By combining the optimized processing pipeline with real-time basecalling, we established a workflow that allows for highly accurate and extremely fast high-resolution typing of bacterial pathogens while sequencing is still in progress. Along with advantages such as low costs and portability, the approach suggested here might democratize modern bacterial typing, enabling more efficient infection control globally.
