Computational protocol: Metabolic phenotype of clinical and environmental Mycobacterium avium subsp. hominissuis isolates

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Protocol publication

[…] The metabolic pathways of the two substrates of interest butyric and propionic acid have been further analyzed. Specifically, we extracted all sequences of the genes known to be associated with the pathways related to butyric and propionic acid from the KEGG pathway database (). We extracted the genes from all the M. avium subspecies (n = 8) present in the KEGG pathway database, namely: M. avium subsp. paratuberculosis K-10, M. avium subsp. paratuberculosis MAP4, M. avium subsp. paratuberculosis E1, M. avium subsp. paratuberculosis E93, M. avium subsp. avium DJO-44271, M. avium subsp. avium 2285 (R), M. avium subsp. avium 2285 (S) and the M. avium 104. The redundant genes have been excluded. Then we screened all such genes in genomes of our ten MAH isolates by performing a Custom BLAST analysis using Geneious version 9 (). The parameters for the screening that we used to determine if a gene was present or not were: sequence identity ≥90%, sequence coverage ≥90%, e value ≤0.01.In addition, we analyzed the number of Single Nucleotide polymorphisms (SNP)s (both synonymous and nonsynonymous) in the sequence of the genes detected in our MAH isolates. For each gene, we also constructed a phylogenetic tree using the nucleotide sequences to determine whether any SNP was associated with clinical or environmental source of the isolates based on the Tamura–Nei model using Geneious version 9. [...] Genomic DNAs were extracted from the MAH isolates as described previously (). Whole genome sequencing (WGS) was performed using Illumina MiSeq 300 bp paired-end sequencing, yielding a coverage that exceeded 100×. The NGS QC tool kit was used to assess the quality of the data reads, which was set as reads with a minimum of 70% of bases having a phred score greater than 20 (). De novo assembly of the resulting reads into multiple contigs was performed using CLC Genomics Workbench 8.0 (CLC bio, Aarhus, Denmark) and contigs annotation was done using RAST (). [...] We determined the maximum common genome (MCG), comprising those genes present in all of the ten MAH genomes, as reported previously (). All these genes were then extracted from all genomes, concatenated and aligned. The resulting alignment was used to generate a clustering tree using RAxML 8.1 ().For determination of the accessory genome we applied the PanGenome Pipeline –Roary. After determination of the accessory genome of the ten MAH genomes and its distribution within them, we separated those genes that are exclusively present only in either the environmental strains or the clinical strains (). […]

Pipeline specifications

Software tools Geneious, CLC Genomics Workbench, RAST, RAxML, Roary
Databases KEGG PATHWAY
Applications Genome annotation, Phylogenetics, WGS analysis
Organisms Mycobacterium avium, Homo sapiens
Diseases Infection, Lung Diseases, Lymphadenitis
Chemicals Carbon, Cysteine, Fatty Acids, Nitrogen, Butyric Acid