Development of Nanopore amplicon sequencing method for culture-free genotyping of Bacillus anthracis strains directly from environmental samples

Fast and accurate genetic subtyping of pathogens is required to respond appropriately to biological events caused by natural outbreaks or bioattacks involving anthrax. In this study, we developed and validated a culture-free genotyping method that combines a multiplex PCR-based amplicon sequencing method on the Nanopore platform with in silico multiple-locus variable-number tandem repeat analysis (MLVA) of 31 loci to identify an unknown Bacillus anthracis strain directly from environmental samples. The novel method accurately identified repeat numbers for all loci in 12 different MLVA genotype Bacillus anthracis strains analyzed in the study, matching 100% with the reference capillary electrophoresis and Sanger sequencing results. The detection limit of the method, at which all 31 variable-number tandem repeat loci were successfully identified, was found to be 10 4 CFU spores/sample for pure spore samples and at 10 6 CFU spores/sample for spiked environmental samples from three matrices (soil, swab, and muddy water). 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.