Supplementary data – Nanopore amplicon sequencing for culture-free genotyping of Bacillus anthracis – Nagy, 2026

Specificity tests yielded negative results for samples containing only non-Bacillus anthracis members of the Bacillus cereus group, which produced sequencing reads for 15 loci but were non-specific to Bacillus anthracis. To validate the method, we genotyped 11 Bacillus anthracis strains originating from a historical collection of Hungarian isolates. The MLVA31 typing scheme classified the strains into five groups, four of which fell into the A.Br.008/009 Trans-Eurasian (TEA) group within the clade A, and one into the B.Br.CNEVA group within the clade B. The largest group within clade A comprises six strains that are assumed to be members of the dominant Bacillus anthracis population in Hungary.

Our results demonstrate that PCR-based amplicon sequencing using the portable MinION device is highly effective for on-site genotyping of pathogens directly from environmental samples. This establishes the NGS-based MLVA genotyping as a valuable tool for biodefense laboratories in preliminary forensic investigations of bioterrorism-related anthrax outbreaks. Furthermore, our results provide new insights into the genetic diversity of Bacillus anthracis in a region (Hungary, Central Europe) that is underrepresented in research and has limited scientific data.