Whole-Genome Sequencing of Mycobacterium Tuberculosis

DNA libraries for whole-genome sequencing (WGS) were prepared with the Nextera (XT) kit from Illumina (San Diego, USA) according to the manufacturer’s instructions [21 (link)]. Pooled DNA libraries were then loaded into NextSeq Reagent cartridges for sequencing on a NextSeq system (Illumina, San Diego, CA, USA). Resulting sequencing reads were submitted to the European Nucleotide Archive under the project accession number PRJEB50767 and subsequently mapped to the H37Rv reference genome (GenBank ID: NC_000962.3) by Burrows–Wheeler alignment (BWA) tool aiming for a minimum of 50-fold average genome-wide coverage [22 (link),23 (link)]. We considered single nucleotide polymorphisms (SNPs) with at least 4 reads in both forward and reverse orientation, 4 reads calling the allele with at least a Phred score of 30, and 75% allele frequency for a concatenated sequence alignment. SNP positions that had reliable base call (as described above) in at least 95% of the strains were concatenated to a sequence alignment. SNPs from repetitive regions were excluded, including those which occurred within a window of 12 base pairs in neighboring strains [23 (link)].
A web tool was used for detecting isolates that harbored more than one phylogenetic lineage (i.e., mixed infections or laboratory contaminations) [24 (link)]. These samples/isolates were not considered in subsequent analysis.