Evaluation of an Oxford Nanopore sequencing workflow for mycobacteria from primary MGIT culture

Illumina sequencing of primary MGIT cultures is an established workflow in several reference mycobacteriology laboratories. Oxford Nanopore Technologies (ONT) provides real-time genetic sequencing yielding long reads which help resolve repetitive genomes and is being explored for in-house implementation within diagnostic laboratories. However, low DNA yields from primary MGIT cultures frequently limit the application of ONT workflows, due to high minimum DNA input requirements for library preparation. We evaluated a modified ONT workflow combining rapid, semi-automated DNA extraction from MGIT cultures with PCR-based whole-genome amplification, and compared its performance with Illumina sequencing for species identification and Mycobacterium tuberculosis complex (MTBC) single-nucleotide polymorphism (SNP) detection. A platformagnostic analysis pipeline enabled consistent human read removal, taxonomic assignment, and MTBC genomic characterisation. ONT sequencing data was subsampled at 1 h, 6 h, and 72 h to determine the earliest time point for reliable species identification. The concordance between sequencing platforms of species classification and MTBC lineage assignment was 95%. SNP agreement was high, with a mean of 1.0 and a median of 0 SNP differences between sequencing platforms after masking. These findings demonstrate the feasibility of PCR-amplified ONT sequencing as a reliable alternative for routine genomic characterisation of MGIT cultures. IMPORTANCERapid genomic characterisation of mycobacteria from primary MGIT cultures is valuable for timely and accurate clinical diagnosis. Although Illumina sequencing provides high sequence accuracy, its longer turnaround time and workflow complexity limit the rapid delivery of actionable results. Oxford Nanopore Technologies (ONT) sequencing enables continuous data generation and analysis, allowing real-time species identification and genomic characterisation. However, low DNA yield from primary MGIT cultures has limited the reliable application of ONT sequencing using standard extraction and PCR-free rapid library preparation methods. This study shows that combining semi-automated DNA extraction with PCR-based wholegenome amplification substantially improves ONT sequencing performance from primary MGIT cultures. The resulting increase in data yield enables more samples to be multiplexed and shorter sequencing run times while retaining comprehensive diagnostic information from a single whole-genome sequencing assay. Together, these improvements enable the practical implementation of ONT sequencing for routine mycobacterial diagnostics and may reduce both turnaround time and sequencing costs in clinical laboratories.