Computational protocol: Massive dispersal of Coxiella burnetii among cattle across the United States

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Protocol publication

[…] DNA libraries were prepared for multiplexed, paired-end sequencing on GAIIx sequencing platforms (Illumina). Sequence data generated have been submitted to the Sequence Read Archive (CMSC1, SRR3347458; CMCA1c1, SRR3347491; and ESFL1, SRR3347474). SNP detection among the three whole-genome sequences presented here and the three whole-genome sequences of ST20 from NCBI (Table S1, available in the online Supplementry Material) was performed using the Northern Arizona SNP Pipeline (NASP) (http://tgennorth.github.io/NASP/) and the In Silico Genotyper pipeline () with RSA 493 (NC_002971.3) as the reference. All SNPs (excluding those that were autapomorphic for the reference) are given in Table S2 available in the online supplementary material. [...] We used parsimony criteria and a heuristic search with default options using PAUP 4.0b10 (). As a measure of accuracy, we report homoplasies using the consistency index (); bootstrapping is a poor method for measuring accuracy for trees with little homoplasy () such are recovered when using SNPs from clonal organisms like C. burnetii (). The phylogeny was rooted using RSA493 which was previously established as an outgroup to ST20 (). Phylogenetic branches were named according to precedent (), assigned to SNPs (Table S2), and designated on trees (Fig. S1, available in the online Supplementary Material). In short, each branch contains a prefix ‘Br.III’ to indicate the genomic group, followed by an assigned number based upon the order of branch discovery within the clade. For SNPs that are specific to a single genome, the genome name, rather than a branch number is used. This method maintains the branch name from previous publications and allows for the identification of novel branches. These SNPs (Table S2) can therefore serve as a resource for others looking to differentiate among isolates in this clade. Novel genome sequences will cause existing branches to be split, requiring additional branch names and updating the branch designation of these SNPs. For evolutionarily stable characters such as SNPs found in clonal organisms like C. burnetii, a single locus can define a branch and thus serve as a ‘canonical SNP’ (). As such, the character states of only a small number of SNP loci need to be interrogated in order to place an unknown strain into the established phylogenetic order. […]

Pipeline specifications

Software tools NASP, ISG, PAUP*
Databases SRA
Applications Phylogenetics, WGS analysis
Organisms Coxiella burnetii, Bos taurus