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.