TOAST: a novel tool for designing targeted gene amplicons and an optimised set of primers for high-throughput sequencing in tuberculosis genomic studies.

BACKGROUND

Amplicon sequencing of Mycobacterium tuberculosis resistance-associated genes offers a cost-effective alternative to whole-genome sequencing for rapid profiling of infections and guiding clinical management. However, existing assays require frequent manual updates to accommodate emerging resistance mutations, limiting scalability and responsiveness.

RESULTS

We present TOAST (Tuberculosis Optimised Amplicon Sequencing Tool), a novel software tool that automates primer design by integrating mutation frequencies from a curated database of over 68,000 drug-resistant M. tuberculosis genomes. TOAST prioritises regions with the highest clinical relevance, accounting for single-nucleotide polymorphisms, insertions, and deletions. The software supports customisation of design parameters such as amplicon length, melting temperature, and GC content, while screening for undesirable primer properties, including self-dimers and off-target binding. Using TOAST, we designed a multiplex panel of 33 amplicons targeting mutations associated with resistance to 13 anti-TB drugs. These amplicons covered over 97% of resistance mutations in a 68 K isolate database and were validated using Oxford Nanopore sequencing of two clinical samples, achieving high uniform coverage with a minimum sequencing depth exceeding 50-fold across all targets.

CONCLUSIONS

TOAST represents a major advancement in targeted TB sequencing by integrating large-scale clinical genomic data directly into assay design. This enables rapid, high-coverage, and adaptable amplicon sequencing, enhancing diagnostic precision and surveillance capabilities for drug-resistant TB. TOAST’s framework is also extensible to other pathogens, supporting broader applications in infectious disease genomics.