Abstract
BackgroundA low genetic diversity in Francisella tularensis has been documented. Current DNA based genotyping methods for typing F. tularensis offer a limited and varying degree of subspecies, clade and strain level discrimination power. Whole genome sequencing is the most accurate and reliable method to identify, type and determine phylogenetic relationships among strains of a species. However, lower cost typing schemes are necessary in order to enable typing of hundreds or even thousands of isolates.ResultsWe have generated a high-resolution phylogenetic tree from 40 Francisella isolates, including 13 F. tularensis subspecies holarctica (type B) strains, 26 F. tularensis subsp. tularensis (type A) strains and a single F. novicida strain. The tree was generated from global multi-strain single nucleotide polymorphism (SNP) data collected using a set of six Affymetrix GeneChip® resequencing arrays with the non-repetitive portion of LVS (type B) as the reference sequence complemented with unique sequences of SCHU S4 (type A). Global SNP based phylogenetic clustering was able to resolve all non-related strains. The phylogenetic tree was used to guide the selection of informative SNPs specific to major nodes in the tree for development of a genotyping assay for identification of F. tularensis subspecies and clades. We designed and validated an assay that uses these SNPs to accurately genotype 39 additional F. tularensis strains as type A (A1, A2, A1a or A1b) or type B (B1 or B2).ConclusionWhole-genome SNP based clustering was shown to accurately identify SNPs for differentiation of F. tularensis subspecies and clades, emphasizing the potential power and utility of this methodology for selecting SNPs for typing of F. tularensis to the strain level. Additionally, whole genome sequence based SNP information gained from a representative population of strains may be used to perform evolutionary or phylogenetic comparisons of strains, or selection of unique strains for whole-genome sequencing projects.
Highlights
IntroductionCurrent DNA based genotyping methods for typing F. tularensis offer a limited and varying degree of subspecies, clade and strain level discrimination power
A low genetic diversity in Francisella tularensis has been documented
Our whole-genome sequencing by hybridization approach made use of a set of bioinformatic filters to eliminate a majority of false positives and indicated a base call accuracy of 99.999% (Phred equivalent score 50) for type B strain live vaccine strain (LVS) [13]
Summary
Current DNA based genotyping methods for typing F. tularensis offer a limited and varying degree of subspecies, clade and strain level discrimination power. F. tularensis is divided into three subspecies (subsp.): tularensis (type A); holarctica (type B); and mediasiatica [1,2]. Tularemia caused by type A strains occurs only in North America, whereas tularemia caused by type B strains occurs throughout the northern hemisphere. Together these two species account for the majority of cases of tularemia worldwide. F. novicida has been suggested to be a subspecies of F. tularensis based on genetic similarity [4,5], but is still formally recognized as a distinct species. F. novicida has been isolated from North America and Australia, and rarely causes human disease even though it can cause a lethal infection in the murine model of disease [3,6]
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