Many M.tb AMR mutations are known, but the contribution to the resistance phenotype and penetrance remains unclear for a substantial fraction of variants. We therefore included only high-likelihood AMR mutations relevant for the MDR and XDR definitions. All loci were selected a priori for their perceived relevance and strength of association to AMR. For INH, we included the katG S315T mutation and any nonsense or frameshift mutations in the gene plus the classical −15 and −8 inhA promoter mutations. For RIF, we included all nonsynonymous mutations in the resistance-determining region (amino acids 426 to 450) of rpoB. For kanamycin/amikacin/capreomycin, we included eis promoter mutations in positions −14, −12, and −10 (35, 45), as well as mutation in position 1401 of rrs. For FLQ, we restricted the analysis to a manually curated collection of high-likelihood mutations, namely, gyrB mutations leading to amino acid substitution at positions 461, 499 to 501, and 642, as well as gyrA mutations resulting in amino acid substitutions at positions 88 to 94 and 288 (46, 47).

Resistance-associated loci were extracted from the whole-genome alignment using EMBOSS v6.6.0.0 (48) using their H37Rv coordinates. The loci were translated to protein, and sequences were sorted by alleles. Mutations were manually annotated on the phylogenetic tree using simple parsimony (e.g., an internal node was inferred to have allele A if all descendent nodes had allele A.). The figure was drawn using iTOL (49).

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