Computational protocol: Phylogenetic analysis of cubilin (CUBN) gene

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Protocol publication

[…] We queried the GenBank database [] for all available protein sequences of the CUBN. The retrieved sequences were saved in FASTA format. An initial first-pass phylogenetic tree was constructed using Neighbour Joining method [] (maximum sequence difference of 0.85) using Domain Enhanced Lookup Time Accelerated Basic Local Alignment Search Tool [DELTA BLAST] pairwise alignments between the query and the database sequences searched []. Grishin computation was used by the software to calculate the evolutionary distance between any two sequences. This was modeled as expected fraction of amino acid substitutions per site [].The list of sequences obtained after DELTA BLAST were narrowed down to delete sequences which are not CUBN and which were hypothetical. The concerned retrieved sequences were then aligned using Kalign software 2.03 version [] with the results being produced in the Clustal W [] format and employing a gap extension penalty of 0.85. The phylogenetic tree was reconstructed using the maximum likelihood method implemented in the PhyML program (v3.0 aLRT) [, ]. The Jones-Taylor-Thornton (JTT) substitution model was selected along with gamma-distributed rate categories to account for rate heterogeneity across sites. The reliability for internal branch was assessed using the aLRT test (SH-Like). The graphical representation and edition of the phylogenetic tree were performed with TreeDyn (v198.3) []. The RAxML (Randomized Axelerated Maximum Likelihood) program version 7.2.8 was then used for sequential and parallel Maximum Likelihood of the sequences []. An additional analysis of the presence of horizontal gene transfer was performed using the T-REX online software []. […]

Pipeline specifications

Software tools BLASTN, DELTA-BLAST, Kalign, Clustal W, PhyML, TreeDyn, RAxML, T-REX
Applications Phylogenetics, Amino acid sequence alignment
Organisms Homo sapiens, Saccharomyces cerevisiae
Diseases Anemia
Chemicals Vitamin B 12