These ecological and reproductive differences which lead to genetic diversity make Francisella

an ideal choice for evaluation of diagnostic PCR-based DNA markers and developing sample sequencing methods for phylogenetic analyses. Over the last decade, PCR methods have been successfully applied for the rapid identification Tideglusib and classification of Francisella isolates [8]. An obvious drawback with DNA-based approaches is the possibility of cross-reactivity with non-pathogenic but closely related Francisella subspecies occurring naturally in the environment [3, 9, 10]. This could distract biological surveillance systems, such as the BioWatch Program [11], and give false-positive alarms find more [12, 13]. Therefore, primer pairs need to be defined so that an unknown isolate is identified and attributed to the correct species or subspecies. Previously published sequence markers designed for identification or detection of Francisella have been developed without taking into consideration the current knowledge of genetic diversity

of the genus, in particular the recently discovered species F. noatunensis and F. hispaniensis. The specificity of Francisella detection assays has often been controlled by testing reactivity with non-Francisella bacterial species. Typically, no other species besides F. selleck tularensis (including subspecies tularensis, mediasiatica and holarctica), F. novicida and F. philomiragia have been included as representatives of the Francisella genus [14–17]. As with PCR detection, current knowledge on the diversity of the Francisella genus affects the choice of genetic markers used for obtaining true phylogenetic trees by PCR-based

sequence-typing analysis. For F. tularensis, multi-locus typing schemes targeting overlapping, as well as separate, genes have been described [18, 19]. However, the resolution was limited, allowing discrimination of only the major genetic clades of the species. Recent advances in sequencing and the increased availability of publicly accessible genomic sequences have enabled phylogenetic trees obtained enough by analysing sequence markers to be evaluated. Whole-genome sequencing is not always desirable for large bacterial sample sets, as such analysis normally generates large amount of data which requires substantial increase in labour and time. Therefore, multiplexed target amplification of selected genomic regions using next generation sequencing (NGS) have recently been proposed [20, 21]. A considerable effort in the study of bacterial pathogens has been devoted to evaluating alternative evolutionary histories by comparing topologies [22–25]. In order to facilitate these comparisons, various topological distance metrics have been proposed, such as the Robinson-Foulds (RF) or symmetric distance [26], branch-score distance [27], path-length metrics [28] and nearest-neighbour interchanging [29].