Computational protocol: Prevalence and Identification of Burkholderia pseudomallei and Near-Neighbor Species in the Malabar Coastal Region of India

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Protocol publication

[…] An initial in silico nucleotide search for highly conserved Burkholderia genus specific genes with marked level of interspecies variation was conducted using whole genome sequence of B. pseudomallei strain K96243 (accession no. BX571966) available on National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov) as reference. Care was taken to screen evolutionary conserved, fundamentally/metabolically important and versatility genes for this purpose. Accordingly, nucleotide sequences of oxidoreductases family, chaperones and structural protein genes were used as queries to determine specific sequences within the genus of Burkholderia. For each matching nucleotide sequence, in silico coverage analysis was done at two taxonomic levels of Burkholderia—genus and family Burkholderiaceae; using online BLASTN analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi). In this step, sequences showing near 100% coverage of query sequence and significant expect (e) value of < 10-100 were selected. Promising nucleotide sequences were further edited according to the BioEdit v7.2.5 (http://www.mbio.ncsu.edu/bioedit/bioedit.html) software default parameters and analyzed for the distribution of conserved regions and variation among the interspecies of the genus using the Entropy (Hx) plot option []. Of the sequences encountered, 3-hydroxybutyrate dehydrogenase (bdha) (BPSS0017) was selected as genus-specific gene target by analyzing for the unique detection specificity within the genus required for the present study. Pairwise progressive sequence alignment of bdha nucleotide sequence of Burkholderia and non-Burkholderia species, which were retrieved from NCBI, was performed using Clustal X2. Entropy plot for bdha was generated using BioEdit v7.2.5. The identity scores for the bdha sequence of Burkholderia species were obtained by the use of GAP program of GCG Wisconsin package (http://www.biology.wustl.edu/gcg/before_you_begin.html). [...] Bayesian analysis was conducted using the bdha, recA and 16S rDNA gene sequences to determine the rate of diversity within Burkholderia genus, as depicted by each gene. The nucleotide sequences of Burkholderia species of this study were also involved in this exercise (). MrBayes version v3.2 (MCMC package) was used for this purpose []. The diagnostic frequency was set for 5,000 generation. The default run length was 50,000 or until the standard deviation of split frequencies was below 0.05. The resultant phylogram of each gene examined was drawn using FigTree v.1.4.2 software [http://tree.bio.ed.ac.uk/software/figtree/]. […]

Pipeline specifications

Software tools BLASTN, BioEdit, Clustal W, MrBayes, FigTree
Applications Phylogenetics, WGS analysis
Organisms Burkholderia pseudomallei
Diseases Melioidosis