Transposon sequencing library construction and analysis

The TnSeq libraries were constructed as previously described^{12 (link)}. The resultant TnSeq libraries were sequenced using a HiSeq. 2500HO, 125 bp PE run using v4 chemistry (Illumina). Sequence reads were analyzed as previously described^{12 (link)}. Briefly, we first trimmed sequence reads for transposon and adaptor sequences and then discarded the sequence reads that were shorter than 18 bp. We used the CutAdapt⁶⁷ default error rate of 0.1 for all trimming processes. The trimmed sequence reads were mapped (allowing 1 bp mismatch) to the M. intracellulare ATCC13950 genome (GenBank: NC_016946.1) and converted output to SAM format using Bowtie2^{68 (link)}. The numbers of sequence reads at each TA site were counted and converted to the wig format, the input file format for TRNSIT^{23 (link)} using the custom Python script^{12 (link)}. After averaging the obtained read counts between the three replicates in each experimental setting, statistical analysis for determination of essential genes and fitness for hypoxic growth was performed by the Hidden Markov Model (HMM) and resampling analysis on TRANSIT, respectively.