Abstract
Single Nucleotide Polymorphisms (SNPs) are the most common and abundant genetic variation found in the genome of any living species, from bacteria to humans. In bacterial genotyping, these evolutionarily stable point mutations represent important DNA markers that can be used to elucidate deep phylogenetic relationships among worldwide strains, but also to discriminate closely related strains. With the advent of next generation sequencing (NGS) technologies, affordable solutions are now available to get access to the complete genome sequence of an organism. Sequencing efforts of an increasing number of strains provide an unprecedented opportunity to create comprehensive species phylogenies. In this study, a comparative analysis of 161 genomes of Bacillus anthracis has being conducted to discover new informative SNPs that further resolves B. anthracis SNP-based phylogenetic tree. Nine previously unpublished SNPs that define major groups or sub-groups within the B. anthracis species were selected and developed into SNP discriminative assays. We report here a cost-effective high-resolution melting-based genotyping method for the screening of 27 canonical SNPs that includes these new diagnostic markers. The present assays are useful to rapidly assign an isolate to one sub-lineages or sub-groups and determine its phylogenetic placement on the B. anthracis substructure population.
Highlights
Bacillus anthracis is a zoonotic pathogen that primarily affects grazing herbivores, but can be used as an agent ofHow to cite this paper: Girault, G., Thierry, S., Cherchame, E. and Derzelle, S. (2014) Application of High-Throughput Sequencing: Discovery of Informative SNPs to Subtype Bacillus anthracis
We have recently reported the high throughput sequencing and phylogeographical analysis of 122 strains of B. anthracis isolated in France [24]
Thirty-seven B. anthracis genomes available in public NCBI database or Sequence Read Archive (SRA) were used for this study [25] (Table 1)
Summary
Bacillus anthracis is a zoonotic pathogen that primarily affects grazing herbivores, but can be used as an agent ofHow to cite this paper: Girault, G., Thierry, S., Cherchame, E. and Derzelle, S. (2014) Application of High-Throughput Sequencing: Discovery of Informative SNPs to Subtype Bacillus anthracis. B. anthracis evolved as a highly fit clonal pathogen that has spread throughout the world and became ecologically established as local, genetically distinct populations [1]. In both microbiological forensics and epidemiological investigations, obtaining reliable information regarding the identification and source of a suspicious strain is often essential to set up effective responses, as highlighted by the Amerithrax investigations following the so-called 2001 “anthrax letter attacks” in the United States [2]. SNPs are generated by nucleotide substitutions, probably associated to errors during DNA replication that are not repaired These rare events occur at an estimated rate of approximately 10−10 changes per nucleotide per generation in the B. anthracis species [14]. This stability can be used for defining canonical markers representative of groups of related strains, such as phylogenetic sub-groups [9]
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