2.5. Generation of spontaneous resistant mutants and whole genome sequencing

M. bovis BCG was cultured to mid-log phase (OD_600nm 0.4–0.8) and 1 × 10⁸ cells were plated on solid media containing 50× and 100× MIC HTB-03 and 20× MIC HTB-04 (based on MICs for M. tuberculosis H₃₇Rv). Potential resistant mutants were cultured and reselected on 100× MIC HTB-03 and 20× MIC HTB-04 to confirm the resistant phenotype. The genomic DNA (gDNA) from the mutants was purified following standard methods and the whole genome sequencing (WGS) and bioinformatics were performed by MicrobesNG, University of Birmingham. Briefly, barcoded DNA libraries were generated from genomic DNA, the fragments were purified and quantified before size evaluation and sequencing. High frequency, statistically relevant single nucleotide polymorphisms (SNPs) were identified by aligning the mutant sequence reads to the M. bovis BCG Pasteur 1173P2 (accession: {"type":"entrez-nucleotide","attrs":{"text":"NC_008769.1","term_id":"121635883","term_text":"NC_008769.1"}}NC_008769.1) genome sequence.