Validation of an optimized Oxford Nanopore sequencing workflow versus Illumina for mycobacteria from primary MGIT culture

ABSTRACT Illumina short-read sequencing of primary Mycobacterial Growth Indicator Tube (MGIT) cultures is an established approach for mycobacterial genomic characterization but relies on labor-intensive workflows and centralized sequencing facilities. Oxford Nanopore Technologies (ONT) offers long-read sequencing and is increasingly being explored for in-house implementation in diagnostic laboratories; however, low DNA yields from primary MGIT cultures frequently limit standard PCR-free ONT workflows, restricting routine implementation. We developed and evaluated a semi-automated DNA extraction and Rapid PCR Barcoding workflow for ONT sequencing and compared its performance with Illumina for species identification and Mycobacterium tuberculosis complex (MTBC) single-nucleotide polymorphism (SNP) detection. A platform-agnostic bioinformatics pipeline was used for human read removal, taxonomic assignment, and MTBC genomic characterization. The time required to achieve reliable species identification was assessed by subsampling ONT data at 1, 6, and 72 h. Concordance between ONT and Illumina for species classification was 98.3% (95% CI: 95.8%–99.5%), with all discordant classifications attributable to potential mixed infections. MTBC SNP concordance was high, with a mean of 0.3 and a median of 0 SNP differences between platforms after masking. Reliable species identification was achieved for 233/237 (98.3%) samples within 6 h of sequencing, with no incorrect primary species assignments. These findings demonstrate that an optimized PCR-amplified ONT workflow provides a practical approach for routine in-house genomic characterization of primary MGIT cultures, achieving high concordance with Illumina while addressing a major barrier to ONT implementation in diagnostic laboratories. IMPORTANCE Rapid genomic characterization of mycobacterial infections is important for timely patient management, infection control, and public health surveillance. However, many diagnostic laboratories rely on referral of positive cultures to regional reference centers for sequencing, introducing delays associated with transport, batching, and processing. Although Oxford Nanopore Technologies (ONT) can support local sequencing, implementation has been limited by the low DNA yields typically obtained from primary Mycobacterial Growth Indicator Tube (MGIT) cultures and the absence of practical workflows suitable for routine diagnostic laboratories. In this study, we developed and validated a semi-automated DNA extraction and PCR-based ONT workflow designed to support implementation in routine diagnostic laboratories. The workflow generated genomic results highly concordant with Illumina sequencing while overcoming a major barrier to ONT implementation by enabling reliable sequencing from low-yield MGIT-derived DNA. This approach supports routine in-house genomic characterization of mycobacterial cultures and has the potential to reduce turnaround times and dependence on external sequencing services.