Department of Microbiology and Immunology, The University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia
Department of Microbiology and Immunology, The University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia; Centre for Pathogen Genomics, The University of Melbourne, Melbourne, Australia
Department of Microbiology and Immunology, The University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia; Centre for Pathogen Genomics, The University of Melbourne, Melbourne, Australia
Department of Microbiology and Immunology, The University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia
Eike J Steinig
Department of Microbiology and Immunology, The University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia
Department of Infectious Diseases, The University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia; Monash Infectious Diseases, Monash Health, Melbourne, Australia
Mark Davies
Department of Microbiology and Immunology, The University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia
Torsten Seemann
Department of Microbiology and Immunology, The University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia; Centre for Pathogen Genomics, The University of Melbourne, Melbourne, Australia
Department of Microbiology and Immunology, The University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia; Centre for Pathogen Genomics, The University of Melbourne, Melbourne, Australia
Department of Microbiology and Immunology, The University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia
Variant calling is fundamental in bacterial genomics, underpinning the identification of disease transmission clusters, the construction of phylogenetic trees, and antimicrobial resistance detection. This study presents a comprehensive benchmarking of variant calling accuracy in bacterial genomes using Oxford Nanopore Technologies (ONT) sequencing data. We evaluated three ONT basecalling models and both simplex (single-strand) and duplex (dual-strand) read types across 14 diverse bacterial species. Our findings reveal that deep learning-based variant callers, particularly Clair3 and DeepVariant, significantly outperform traditional methods and even exceed the accuracy of Illumina sequencing, especially when applied to ONT’s super-high accuracy model. ONT’s superior performance is attributed to its ability to overcome Illumina’s errors, which often arise from difficulties in aligning reads in repetitive and variant-dense genomic regions. Moreover, the use of high-performing variant callers with ONT’s super-high accuracy data mitigates ONT’s traditional errors in homopolymers. We also investigated the impact of read depth on variant calling, demonstrating that 10× depth of ONT super-accuracy data can achieve precision and recall comparable to, or better than, full-depth Illumina sequencing. These results underscore the potential of ONT sequencing, combined with advanced variant calling algorithms, to replace traditional short-read sequencing methods in bacterial genomics, particularly in resource-limited settings.
