Francisella tularensis, the causative agent of tularemia, is a highly infectious Category A pathogen that poses a serious threat to the United States due its potential use in bioterrorist attacks. Relative to other category A pathogens, Francisella is poorly characterized, due to the difficulty of culturing this organism. In the event of a bioterrorist attack, diagnostic tools that can rapidly and accurately differentiate F. tularensis strains would allow investigators to track the outbreak and identify its source. Due to the relatively low level of DMA variation in F. tularensis, only a genome wide analysis of nucleotide variation will allow us to discover patterns of nucleotide variation that will allow differentiation between individual strains (""""""""signature SNP patterns""""""""). To identify the signature SNPs we will design high-density oligonucleotide array-based SNP- discovery assays that interrogate the entire Francisella genome base by base, with multiple redundancy, using information derived from the genomes of a number of F. tularensis strains as well as strains of the closely related F. philomiragia. The entire genomes of 160 F. tularensis and 20 F. philomiragia strains will be interrogated using the SNP-discovery array to identify a comprehensive genome-wide set of SNPs. Such high resolution analysis of SNPs across the whole genome of multiple isolates is necessary for identifying the canonical SNPs involved in the discrimination of specific branch points for pathogens such as F. tularensis, due to their limited genetic variation. Informative SNPs discovered by this process will be genotyped in the same set of 180 Francisella strains using high-density oligonucleotide array based SNP-genotyping assays. This step will serve to verify the genotypes in these strains with very high confidence. Signature SNP patterns that allow differentiation between strains will be identified for each strain, and the pattern of signature SNPs unique to each strain will then be used for extremely high confidence strain identification. By choosing SNPs present in at least 4% of the strains, we will be able to use the resultant signature SNP patterns to classify new strains, including any previously unknown or bioengineered strains. These signature SNP patterns, which will be made available to the public, will be a valuable resource in the event of an outbreak, and also for the study of the evolution of this pathogen. These studies will enable the development of an inexpensive diagnostic test using 50 or less SNPs to discriminate rapidly between F. tularensis and diseases with similar clinical phenotypes, or between pathogenic and non-pathogenic Francisella strains. ? ? ?
