Computational protocol: A Metagenomic Study Highlights Phylogenetic Proximity of Quorum-Quenching and Xenobiotic-Degrading Amidases of the AS-Family

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Protocol publication

[…] Complete sequence of the p90H6 fosmid (46 kbp) harboring a 39 kbp insert was achieved by Sanger sequencing. To this end, a library of 2.0–2.5 kbp fragments was constructed, cloned and sequenced by GATC Biotech (Mulhouse, France). Assembling (7x in average coverage) was performed using DNASTAR Lasergene, and appropriate primers were designed for gap-closure. DNA map was generated by Geneious and orf determination by Bioedit softwares. For each of the putative proteins encoded by the p90H6 insert, 50 homologous sequences were retrieved from GenBank (http://www.ncbi.nlm.nih.gov) after identification by BLASTp. Multiple sequence alignments were carried out with ClustalW. The evolutionary history was inferred using the Maximum Likelihood method based on the JTT matrix-based model . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) was calculated . Initial tree(s) for the heuristic search were obtained automatically as follows. When the number of common sites was <100 or less than one fourth of the total number of sites, the maximum parsimony method was used; otherwise BIONJ method with MCL distance matrix was used. Evolutionary analyses were conducted inMEGA5 . […]

Pipeline specifications

Software tools DNASTAR Lasergene, Geneious, BioEdit, BLASTP, Clustal W
Applications Phylogenetics, Sanger sequencing
Organisms Escherichia coli