P059

1:Statens Serum Institut, Copenhagen, Denmark; 2:Jessenius Faculty of Medicine in Martin, Comenius University, Slovakia

Sequencing-based approaches are increasingly used in the identification and characterization of mycobacteria, including both Mycobacterium tuberculosis and nontuberculous mycobacteria (NTM). These pathogens are surrounded by a complex, lipid-rich cell-wall, which challenges the outcome and quality of the DNA-extraction step required prior to sequencing. This study aimed to compare the sensitivity of an Illumina-based purification strategy with an optimised method designed for Oxford Nanopore Technologies (ONT). Ten diverse clinical isolates were included: three M. tuberculosis, three M. avium, one M. intracellulare, one M. heraklionense, one M. porcinum (fortuitum group), and an unclassified member of the M. avium complex (MAC).

Illumina sequencing typically requires lower amounts of input genomic DNA for library preparation (1–100 ng depending on the protocol), and the resulting libraries are normalized to very low concentrations (typically 0.2–0.4 ng/μl) prior to sequencing. In contrast, ONT protocols such as Rapid Barcoding require a higher total input amount of 200–400 ng per sample. Although the required DNA input differed significantly between DNA-extraction methods, both yielded usable sequence data for all samples for Illumina sequencing, but only the Oxford Nanopore DNA-extraction was useable for ONT. Preliminary analysis indicated that ONT sequencing provided better genome coverage. The use of 400 ng of input DNA per sample further enhanced sequencing depth and coverage uniformity. In contrast, Illumina sequencing demonstrated greater base-calling consistency and lowered error rates. These complementary strengths highlight the advantages of integrating both technologies for comprehensive genomic analysis of mycobacteria.