Faster TB genetics: PCR-amplified Nanopore works on primary MGIT™ cultures

Rapid genetic information about tuberculosis bacteria can change how quickly patients are diagnosed and treated, but getting that information from the first bacterial growth bottles can be technically hard. Illumina sequencing of primary MGIT™ cultures is already an established workflow in several reference mycobacteriology laboratories, but it can take longer to deliver results. Oxford Nanopore Technologies (ONT) offers a different approach: real-time sequencing that produces long reads and can be analysed as the data arrive. A practical obstacle has been that primary MGIT™ cultures often yield only small amounts of DNA, and standard ONT library methods usually need more input DNA than those cultures provide. Derrick W. Crook and colleagues set out to test whether a modified ONT workflow could overcome that limit. They combined a rapid, semi-automated DNA extraction from primary MGIT™ cultures with PCR-based whole-genome amplification to increase the amount of DNA available for sequencing. The team then compared the ONT results to Illumina sequencing for species identification and Mycobacterium tuberculosis complex (MTBC) single-nucleotide polymorphism (SNP) detection, using a platformagnostic analysis pipeline to handle human read removal, taxonomic assignment, and MTBC genomic characterisation.

The study evaluated a workflow that paired semi-automated extraction from MGIT™ cultures with PCR-based whole-genome amplification before Oxford Nanopore Technologies (ONT) sequencing. ONT data were analysed alongside Illumina sequencing using a platformagnostic analysis pipeline to ensure consistent human read removal, taxonomic assignment, and MTBC genomic characterisation. To test how quickly useful information appeared, ONT sequencing data were subsampled at 1 h, 6 h, and 72 h to find the earliest time point for reliable species identification. The comparison between platforms showed 95% concordance in species classification and Mycobacterium tuberculosis complex (MTBC) lineage assignment. For SNP detection, agreement between ONT and Illumina was high: after masking, the mean SNP difference was 1.0 and the median was 0 SNP differences. These results demonstrate that PCR-amplified ONT sequencing from primary MGIT™ cultures produced enough data for accurate species calls, lineage assignment, and close agreement on SNPs compared with Illumina.

Taken together, the findings suggest a feasible alternative for routine genomic characterisation of MGIT™ cultures using Oxford Nanopore Technologies with PCR amplification. By boosting DNA yield before sequencing, the modified workflow produced enough sequence data to multiplex more samples and to shorten sequencing runs while keeping comprehensive diagnostic information from a single whole-genome sequencing assay. Because ONT produces data continuously, laboratories could potentially obtain species identification and genomic characterisation earlier in a run, and the semi-automated extraction plus PCR step addresses the main barrier of low DNA yield from primary MGIT™ cultures. The study supports the practical implementation of ONT sequencing for routine mycobacterial diagnostics, with the potential to reduce both turnaround time and sequencing costs in clinical laboratories while retaining the genomic detail clinicians and public-health teams need.