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

HiSpoT, a one-tube dual-barcode PCR assay for Mycobacterium tuberculosis complex spoligotyping, multiplexes 96 samples and achieved 70% full concordance with WGS across 79 Israel (2020–2022) isolates.

• Why it matters: Existing spoligotyping workflows can be costly and slow, limiting rapid outbreak tracking and surveillance; a PCR-based, affordable NGS library method could improve throughput while staying comparable to legacy databases.
• What they did: The assay targets 43 conserved spacer oligonucleotides in the CRISPR direct repeat region using a newly designed dual-barcode primer set, then processes Illumina paired-end fastq with open-source Colab tools.
• The result: Disagreements versus WGS in the 79-isolate comparison were essentially all linked to single-spacer insertion-element disruptions at PCR primer sites, enabling rapid spoligotype reporting alongside higher-resolution WGS.

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