MIP assays do however allow for a focus on resolving branches of specific interest. Data from these assays then allows for targeted down selection of loci so that focal branches and isolates on them can be thoroughly interrogated using individual SNP assays. Identifying LCZ696 cost canonical SNPs and verifying their ability to differentiate clades by screening large numbers of isolates is the essential part of genotyping [17]. Less important is the type of assay used for SNP differentiation because it is highly dependent on the numbers of SNPs and samples one wants to screen. The MIP and CUMA SNP screening techniques are just two of many methods that can be used for SNP genotyping in Brucella and other bacteria. Conclusions

We developed SCH772984 molecular weight and evaluated two different see more SNP-based genotyping systems for three well studied species of Brucella: B. abortus, B. melitensis, and B. suis. The first genotyping approach, using Molecular Inversion Probes, divided the species into its three respective

groups and allowed for finer scale genetic resolution. Notably, this resolution occurred almost entirely within the lineages of the four strains that were used for SNP discovery: B. abortus 2308, B. abortus 9–941, B. melitensis 16 M, and B. suis 1330. This is to be expected since the choice of genomes for SNP discovery has a pervasive effect on the phylogenetic patterns that can be determined. We followed the MIP assay with development of Capillary electrophoresis Universal-tailed Mismatch Amplification mutation assays that targeted major

branch points in the MIP phylogeny. We then genotyped a large and diverse collection of isolates. The main result is the development of fine scale genotyping assays that target among the most important and widespread lineages of Brucella. Moreover, these and closely related isolates can be easily and quickly distinguished from all other Brucella isolates. Despite the era of whole genome sequencing being upon us, SNP-based genotyping and other targeted assays will remain relevant. Sequencing technology is advancing rapidly and costs per genome are quickly diminishing such that whole Liothyronine Sodium genome genotyping is the future of phylogenetics, forensics, and diagnostics. In fact, whole genome genotyping will soon be cost competitive with most other genotyping strategies and will have the advantage of capturing nearly all of the genetic variation with no issues of discovery bias. Nonetheless, targeted assays will remain a viable option for such goals as rapidly and easily characterizing large strain collections, clinical samples, and samples containing only trace amounts of DNA. Concerted efforts must be made to incorporate data from earlier genotyping strategies into genomic databases so this wealth of genetic information is not lost in the rush to sequence everything. Methods SNP selection SNPs were selected by comparisons of the four Brucella genomes that were available at the time of MIP development: B. melitensis 16 M [25], B.