dN/dS analysis on substitution in CenAsiaIII Shigella sonnei

For the 395 CenAsiaIII sequences, substitutions across the reference Ss046 were summarized and annotated using SnpEff v4.1b. Point mutations present within mobile, repetitive, or recombination elements were excluded, resulting in a set of 4,230 mutations available for examination. Due to the limited temporal sampling period, we assessed the dN/dS ratio (non-synonymous to synonymous substitution rate) to detect potential signals of selection, adopted from a previously recommended approach. In brief, a phylogenetic tree was constructed using these 4,230 SNPs in RAxML v8.1.3, and PAML was used for ancestral state reconstruction of all SNPs. Mutations were classified as intergenic, synonymous, or non-synonymous based on comparison of each SNP to its annotation and the reconstructed ancestral state. The dN/dS rate was adjusted for transition/transversion rate and codon frequencies under the NY98 model. Genes were determined to be under positive selection if their adjusted dN/dS ratio were >1.5 or if they possessed no synonymous and at least five non-synonymous mutations.