Computational protocol: Molecular Taxonomic Evidence for Two Distinct Genotypes of Mycobacterium yongonense via Genome-Based Phylogenetic Analysis

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[…] For the phylogenetic analysis of the two INT-5 strains (MOTT-H4Y and MOTT-36Y), their genome sequences [MOTT-H4Y (Genbank accession No. AKIG00000000) and MOTT-36Y (Genbank accession No. NC_017904)] [, ] were compared with those of M. yongonense DSM 45126T (Genbank accession No. NC_021715) [], M. parascrofulaceum ATCC BAA-614T (Genbank accession No. ADNV00000000), and 3 strains belonging to M. intracellulare [one INT-1 strain: M. intracellulare MOTT-64 (GenBank accession No. NC_016948) and two INT-2 strains: M. intracellulare ATCC 13950T (GenBank accession No. NC_016946) and M. intracellulare MOTT-02 (GenBank accession No. NC_016947)] [–]. These genome sequences were subjected to whole-genome multiple sequence alignments using the neighbor-joining method [] by the Mauve Genome Alignments software ( A phylogenetic tree was generated using the aligned genome sequences and visualized by the TreeViewX program (; additionally, a Venn diagram was constructed by the BLASTCLUST program. The minimum length coverage and identity threshold in BLASTCLUST were 0.9 and 95%, respectively. [...] To analyze the sequence differences among the 3 M. intracellulare strains (M. intracellulare ATCC 13950T, M. intracellulare MOTT-02, and M. intracellulare MOTT-64), 2 INT-5 strains (MOTT-H4Y and MOTT-36Y) and M. yongonense DSM 45126T, the rpoB gene and 35 additional gene sequences were selected from the genome sequences. In the selected 35 genes, 10 genes (argH, cya, glpK, hsp65, murC, pta, recA, secA1 and sodA) [–] were included for mycobacterial MLST analysis, and other 25 genes were were randomly selected in the housekeeping genes without any standards. The list of chosen genes is as follows: adenylate kinase (adk), argininosuccinate lyase (argH), chorismate synthase (aroC), shikimate 5-dehydrogenase (aroE), F0F1 ATP synthase subunit beta (atpD), adenylate cyclase (cya), cytochrome b6 (cytB), ATP-dependent RNA helicase, dead/death box family protein (deaD), chromosomal replication initiation protein (dnaA), DNA primase (dnaG), molecular chaperone DnaK (dnaK), chaperone protein (dnaJ), 3-oxoacyl-(acyl-carrier-protein) reductase (fabG), cell division protein FtsZ (ftsZ), fumarate hydratase (fumC), malate synthase G (glcB), glutamine synthetase type I (glnA), glycerol kinase (glpK), fructose 1,6-bisphosphatase II (glpX), 6-phosphogluconate dehydrogenase (gnd), DNA gyrase subunit B (gyrB), heat-shock protein 65 kD (hsp65), myo-inositol-1-phosphate synthase (ino1), NAD-dependent DNA ligase LigA (ligA), ATP-dependent DNA ligase (ligB and ligC), UDP-N-acetylmuramate-L-alanine ligase (murC), endonuclease III (nth), glucose-6-phosphate isomerase (pgi), phosphoglycerate kinase (pgk), phosphate acetyltransferase (pta), recombinase A (recA), recombination protein F (recF), preprotein translocase subunit SecA (secA1), and [Mn]-superoxide dismutase (sodA) (). The retrieved rpoB gene or the concatenated 35 selected gene sequences were multiply aligned using the ClustalW method in the MEGA 4.0 software []. Using the multiple alignment matrix, phylogenetic trees were constructed using the neighbor-joining method [] in the MEGA 4.0 software []. The bootstrap values were calculated from 1,000 replications.SNPs were extracted from the multiple alignments of rpoB gene sequences and the 35 selected gene sequences from the 3 M. intracellulare strains (M. intracellulare ATCC 13950T, M. intracellulare MOTT-02, and M. intracellulare MOTT-64), 2 M. intracellulare INT-5 strains (MOTT-H4Y and MOTT-36Y), M. yongonense DSM 45126T and M. parascrofulaceum ATCC BAA-614T. Then, the patterns were compared. Additionally, the extracted SNP sequences were concatenated and used to construct a phylogenetic tree as described above. […]

Pipeline specifications

Software tools Mauve, TreeViewX, BLASTclust, Clustal W, MEGA
Applications Phylogenetics, Protein sequence analysis, Nucleotide sequence alignment
Organisms Mycobacterium intracellulare, Mycobacterium intracellulare subsp. yongonense, Mycobacterium intracellulare ATCC 13950, Mycobacterium avium