Universal tails were added to the 5′ end of the allelic primers during primer synthesis. See Figures 1 and S1 for branch location
of SNPs in phylogeny. SNP positions are given for B. melitensis 16 M genome and all are on chromosome I except assays 6214 and 2995 are on chromosome II. SNPs used in the CUMA were randomly selected from the various options available on each branch, with fewer options possible with shorter branches. If development of the assay failed to produce effective primer pairs based on standard primer design parameters we simply selected a new SNP locus. Using the CUMA assays, we genotyped a diverse set of isolates (n = 340), which included TGF-beta inhibitor all recognized biovars and type strains (except B. microti and B. suis biovars 3 and 5), against 17 SNP assays for 10 branches. For each sample we determined if the SNP allele for each locus was ancestral or derived on the corresponding branch and then verified where the sample was placed on the tree. When possible, we selected two SNPs from each of the major branches. We generated amplicons for the SNP regions in four PCR reactions for each of the two multiplex PCRs and then pooled the PF-6463922 order PCR product in one capillary injection.
If the CUMA assay failed any locus in multiplex reactions, we reran that locus in singleplex, which generally allowed for determination of the SNP allele. Samples with singleplex failure largely
appeared to be of poor DNA quality since there were typically failures across several different CUMA Tacrolimus (FK506) assays (Additional file 4: Table S2). Acknowledgements We thank numerous contributors of DNA to our Brucella collection, including Brian Bell, Bryan Bellaire, Wally Buchholz, Robert Burgess, Barun De, Mike Dobson, Linda Getsinger, Ted Hadfield, and William Slanta. We thank Jim Schupp, Molly Matthews, and Jodi Beaudry for assistance with CUMA primer design and Ray Auerbach, Jolene Bowers, and Josh Colvin for help with data analysis and running samples. Recent whole genomes for comparisons were generated by the Broad Institute under the direction of David O’Callaghan, Adrian Whatmore, and Doyle Ward. Funding from the U.S. Department of Homeland Security (DHS) supported this work. Use of product or trade names does not constitute endorsement by the U.S. Government. Electronic supplementary material Additional file 1 Figure S1.: Brucella phylogeny using maximum selleck screening library parsimony developed using 777 single nucleotide polymorphisms. Letters on branches refer to phylogenetic locations of CUMA assays developed in this work. Stars on branches represent phylogenetic locations of species or clade specific assays from Foster et al. 2008. In this figure we rooted with B.