Conventional sanger sequencing

Bisulfite-converted DNA from control and infected bAM was amplified in 25 µl reactions containing 0.2 µM primers, 1× buffer, 0.2 mM dNTPs, 2.5 U Platinum Taq DNA polymerase and 3 mM MgCl₂. Primer sequences are detailed in Table 1. PCR cycling conditions were as follows: 95 °C for 3 min followed by 35 cycles of 30 sec each at 95 °C; 58 °C, 72 °C for 30 sec, and a final elongation step of 5 min at 72 °C. PCR products were purified using the Wizard clean up kit (Promega) and cloned into the pJET1.2/blunt vector (Fermentas). Insertion of PCR products was verified by digestion with BglII and positive clones were sequenced using conventional Sanger sequencing (Eurofins Genomics). Combined bisulfite restriction analysis (COBRA) was carried out using TaqαI, and/or AciI as outlined in^{27 (link)}. Sequence analysis and alignment was performed using DNAStar EditSeq, MegAlign (www.dnastar.com) and BiQ Meth Analyzer (http://biq-analyzer.bioinf.mpi-inf.mpg.de). During this analysis, sequences with low C-T conversion rate (<95%) and with a high number of sequencing errors (sequence identity with genomic sequence less than 80%) were excluded from the alignment. Identical clones were also excluded from the analysis.

Mycobacterium tuberculosis BDQ resistant mutants were isolated by plating approximately 10⁸ and 10⁹ CFU from exponential growth phase cultures of IC1 and IC2 clinical isolates onto solid medium containing drug at concentrations exceeding the MIC (5X, 10X, 20X MIC). Following 6–8 weeks of incubation, BDQ resistant colonies were streaked onto 7H11 medium. At the same time, these colonies were streaked also onto 7H11 medium plus the same BDQ concentration used for mutant isolation to confirm the resistant phenotype. BDQ MIC values were also assessed by REMA. Genomic DNA was extracted from each mutant and Rv0678, atpE, and pepQ genes were amplified by PCR (oligonucleotides in Supplementary Table S1), purified using Wizard^® SV Gel and PCR Clean-Up System (Promega) and analyzed by conventional Sanger sequencing (Eurofins Genomics, Italy).