HiSpoT: a novel one-tube dual-barcode PCR method for NGS spoligotyping amplicon library preparation in Mycobacterium tuberculosis complex

Tuberculosis remains one of the leading infectious causes of death globally, making rapid, accurate, and affordable strain typing essential for effective outbreak tracking and epidemiological surveillance. To address the limitations and high costs of current typing methods, we developed a novel one-tube, dual-barcode PCR method for preparing targeted next-generation sequencing (NGS) amplicon libraries for spoligotyping of Mycobacterium tuberculosis complex (MTBC), which we termed HiSpoT (High-throughput Spoligotyping by Targeted next generation sequencing). The assay targets 43 conserved spacer oligonucleotides in the CRISPR direct repeat (DR) region and is presented here together with the previously unpublished, difficult-to-design dual-barcode primer set needed to multiplex 96 samples in a single tube. HiSpoT is accurate, fast, high-throughput, and low-cost, eliminating the need for a commercial NGS library preparation kit. Because HiSpoT is PCR-based, the resulting spoligotypes remain directly comparable with the large PCR-based spoligotype databases accumulated over decades, enabling rapid outbreak tracking and biogeographic characterization of the tuberculosis burden of a country, while higher-resolution whole-genome sequencing (WGS) data are obtained. Analysis of 288 clinical MTBC isolates from Israel (2020-2022) demonstrated a broad range of genetic lineages, and comparison of 79 isolates analyzed by both HiSpoT and WGS showed 70% full concordance, with essentially all discrepancies attributable to single-spacer insertion-element disruptions of PCR primer sites. We also present two open-source Google Colab Python tools, Spoligotyping_MTBC_gcl and its condensed HiSpoT_Spoligotyping_v8_ready VIBE variant, freely available on GitHub, which automatically process Illumina paired-end fastq output from HiSpoT or WGS and report binary and octal spoligotypes, spoligo international types (SIT), and MTBC family assignments with accompanying quality-control reports.