Besides SNPs, LD analysis, and Gubbins, RDP4 (51) was used to detect recombination signal in the MTBC dataset. To mark the regions reported by Gubbins as potentially recombinant, we required at least three of the methods implemented in RDP4 to agree in showing a significant signal.

Recombination was evaluated in the alignment containing 219 MTBC strains and 7 MCAN strains and in the one containing the MTBC ancestor and 7 MCAN strains. First, repetitive regions [i.e., PPE/PGRS(polymorphic GC-rich sequence)] were masked from both alignments, and second, recombination events were inferred using Gubbins (52), which identifies clusters of high SNP density as markers.

Gubbins identified 70 potential recombinant regions in the alignment containing the seven MCAN strains and the MTBC ancestor. Four of these regions were obviated because they fell in regions deleted in several MCAN strains. One more region was removed from the analysis because it was extremely short (41 bp), and we did not obtain reliable results in the subsequent analyses.

For the remaining 65 fragments, a phylogeny was calculated using RAxML (47) and applying the GTRCATI model. In addition, a reference phylogeny was calculated with the same method using the complete genomes after subtracting these 65 regions. This reference phylogeny had the same topology as the one obtained from the complete genomes. To test for phylogenetic incongruence between the putative recombination fragments and the genome phylogeny, we applied the Shimodaira-Hasegawa and expected likelihood weight tests implemented in TREE-PUZZLE (53).

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