Title : Amplicon-sequencing method for strain-level subtyping of bacillus anthracis in field laboratory
Bacillus anthracis is the first ranked on the list of potential bioweapon agents. In case of biothreat emergency accurate and timely data resulted from on-site strain-level subtyping of the agent help to determine the origin of the used strain and provide the first bioforensic information for confirmation of deliberate misuse of pathogen. The study aimed to develop an amplicon-based targeted sequencing method on Nanopore platform combined with in-silico multiple-locus variable number tandem repeat analysis (MLVA) of 31 loci for strain-level subtyping of Bacillus anthracis directly from environmental samples.
We determined the genotype of 31 tandem repeat loci of 13 Bacillus anthracis strains with capillary electrophoresis and Sanger sequencing, and the results were used as reference. Performance and effectiveness of the newly developed method was evaluated by two approaches: (i) the number of correctly identified repeats and (ii) the number of consensus sequences identical to the reference. Performance evaluation included analysis of the 13 different MLVA genotype strains. Effectiveness was tested with Bacillus anthracis spore suspensions, and spiked environmental samples of three sample matrices (soil, surface, sludge) to determine the lowest spore concentration and DNA copy number that results in the correct identification of all 31 markers. Repeat numbers and sequences of the 31 loci were correctly determined and showed 100% agreement with the reference for all 13 strains. The concentration limit of correct identification of all 31 markers was 105 colony forming unit/sample in pure spore suspension, soil and surface samples. In sludge samples with this concentration 30 of 31 repeats were correctly typed. The lowest DNA copy number which resulted correct repeat typing was between 300 and 1000 copies depending on sample type.
Our results demonstrated that PCR-based amplicon sequencing with portable MinION device is a suitable method for on-site pathogen genotyping directly from low pathogen containing environmental samples.