From the MCAN and MTBC ancestor alignment, we extracted those positions having one variant in all the MCAN strains and another variant in the M. tuberculosis ancestor. The divSNP frequency by nucleotide was calculated by dividing the total number of divSNPs (5688) by the total number of bases in the alignment. Next, the expected abundance of divSNPs for each gene was calculated by multiplying the nucleotide divSNP frequency by the number of nucleotides in each gene. From the expected and observed divSNP abundances, we used a Poisson distribution to calculate the probability of having the observed divSNPs by chance for each gene. We selected genes having a positive false discovery rate ≤0.01.

Complete mycobacterial genomes for reference strains (table S8) were downloaded from RefSeq and GenBank. The orthologous genes were obtained from the amino acid sequences and using the Proteinortho tool (57). A gene was considered as orthologous on the basis of reciprocal best hits in BLAST. BLAST analysis required a minimum identity of 25%, a query coverage of 50%, and a maximum e value of 1 × 10−5. The orthologous genes were aligned using Clustal, and the phylogenies were constructed using RAxML and applying the PROTCATIAUTO model. The reference phylogeny was constructed using only the core genome (proteins having orthologous genes in all the mycobacterial genomes downloaded) with RAxML using the same options as above. The reference and alternative phylogenies calculated with the orthologous genes for the divSNP-enriched genes were manually inspected to check for congruence.

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