Seven MCAN draft genomes were aligned to each other and to the ancestor of MTBC using progressiveMauve (49). The segmented alignment obtained in XMFA (eXtended Multi-FastA) format was converted to a plain FASTA format using the MTBC ancestor as reordering reference with a custom Perl script. Positions with gaps in the reference sequence were removed from the final alignment; hence, the resulting aligned genomes had the same size as the reconstructed MTBC ancestor (4,411,532 Mb). The MTBC pseudogenomes reconstructed from mapping to the MTBC ancestor from the different datasets described above were concatenated to the MCAN alignment obtained in the previous step for further analyses.

From these alignments, homoplastic variants were identified using both parsimony and maximum likelihood approaches (50). Both approaches agreed in identifying the same homoplastic variants.

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