Amplicon sequencing protocol for MLVA genotyping of Bacillus anthracis strains on the Nanopore platform v2

Multiple-Locus Variable Number of Tandem Repeats (VNTR) Analysis (MLVA) is one of the gold standard strain-level subtyping methods for outbreak-related Bacillus anthracis strains. The repeat numbers of 31 VNTR loci can be determined by capillary electrophoresis of PCR amplicons spanning repeat regions or in silico analysis of whole genome sequencing (WGS) data. However, these methods require clear isolates of typeable strains and can be performed in fixed well-equipped high biosafety level laboratories. We developed field-applicable amplicon sequencing protocol for Bacillus anthracis MLVA typing directly from environmental samples without isolating clear cultures of Bacillus anthracis strains. 62 primers were used for generating PCR amplicons for 31 Bacillus anthracis VNTR loci, according to MLVA31 typing scheme described by Beyer et al. 2012. The primers generating amplicons longer than 200 bp were used from MLVA31 typing scheme (44 primers for 22 VNTR loci). For amplicons shorter than 200 bp, primers were redesigned to generate longer amplicons (between 300-700 bp) suitable for MinION sequencing. We optimized and tested this protocol on Hungarian virulent Bacillus anthracis strains, a 34F2 Bacillus anthracis vaccine strain, and on spiked environmental samples in the Hungarian Defence Forces field-deployable laboratory. This version of the protocol describes the preparation and native barcoding of PCR amplicons for 31 Bacillus anthracis VNTR loci using the Native Barcoding Kit 96 V14 (SQK-NBD114.96) with flow cell R10.4.1 (FLO-MIN114).