Bacillus anthracis, a spore-forming pathogenic bacterium found throughout the world, is the causative agent of anthrax. In France, a few sporadic cases are recorded each year in areas where outbreaks had been reported previously, but large outbreaks could occur. Bacillus anthracis is one of the most feared microorganisms due to its high rate of infectivity and lethality, to its global dispersal pattern and to the extreme stability of its spores. It is considered as the main threat in what concerns bioterrorism. Different strategies exist to counter this threat. This project aims at improving forensics and bio-traceability of this potential biowarfare agent, thus discouraging its intentional use. B. anthracis is considered as an evolutionarily young species, recently emerged from the B. cereus group. It presents very low molecular diversity among strains. Due to this genetic homogeneity, only a combination of genotyping methods with high discrimination power (multiple-locus variable-number tandem repeat analysis (MLVA), single nucleotide repeat (SNR), genome-wide single nucleotide polymorphism (SNP) analyses or even whole-genome sequencing) has proved to be successful in differentiating strains. The recent and dramatic improvement of whole genome sequencing technology should allow its more frequent use for systematic characterization of B. anthracis. Our project aims at improving knowledge of the existing diversity in B. anthracis isolates in France in order to develop a highly precise and robust subtyping system and know-how for the primary investigation of strain traceability. High-resolution whole-genome sequencing and comparative genomics will be applied to 25 to 50 French strains from different geographic areas to identify their key genetic features. The clonal nature of B. anthracis makes SNPs, which are scattered throughout the whole genome, ideal signatures for subtyping this pathogen. Only rare diagnostic markers can provide the level of discrimination required for microbial forensics and for precise strain identification. As a consequence of this project, France will possess the required know-how to react rapidly when a strain identity needs to be established. High-Resolution Melting (HRM) will be used to validate these potentially diagnostic SNPs across a hundred of diverse strains. DNA signatures that discriminate lineages and strain groups, as well as specifically define a particular strain, will be selected. HRM capability to detect and distinguish SNRs will also be evaluated. Screening of a few SNR markers could add resolving power to the genotyping tool under development and might be part of the HRM assays.. Discriminative SNPs will be developed into a molecular typing assay and used to provide further data on the whole ANSES collection. Precise identification of B. anthracis isolates enables epidemiological analyses of natural anthrax cases or forensic investigations of criminal acts as part of the Biotox-Piratox network. Studies will be undertaken (1) to describe the historical, geographic and genetic diversity of the three lineages established in France (A.Br.008/009, A.Br.001/002 and B.Br.CNEVA), (2) to establish the phylogenetic relationships among B. anthracis isolates inside and outside France and (3) to investigate major epizootic recently occurred, such as those in Doubs in 2008 (involving strains affiliated with the A.Br.001/002 sub-group) and in Alps in 2009 (strains allocated into the B.Br.CNEVA sub-lineage). An extensive genotype data set will be created.