Computational protocol: A Phylogenetic analysis of Heparanase (HPSE) gene

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Protocol publication

[…] The GenBank database [] was queried to retrieve all available protein sequences of the heparanse gene. These sequences were retrieved and saved in FASTA sequence format. These sequences were then aligned using Clustal W [] algorithm using default parameters. The initial first-pass phylogenetic tree was constructed using Neighbour Joining [] method (maximum sequence difference of 0.85) using Basic Local Alignment Search Tool [BLAST] pairwise alignments between a query and the database sequences searched []. Evolutionary distance between two sequences modeled as expected fraction of amino acid substitutions per site given the fraction of mismatched amino acids in the aligned region was taken by the software using Grishin computation []. Using the results from BLAST [] we created a first-pass phylogenetic tree after which we used a purpose-built computational phylogenetic method using Phylogeny.fr software. Sequences were aligned with MUSCLE (v3.7) [] configured for highest accuracy (MUSCLE with default settings). After alignment, ambiguous regions (i.e. containing gaps and/or poorly aligned) were removed with Gblocks (v0.91b) [–]. 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 assuming an estimated proportion of invariant sites (of 0.108) and 4 gamma-distributed rate categories to account for rate heterogeneity across sites. The gamma shape parameter was estimated directly from the data (gamma=.163). 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) []. […]

Pipeline specifications

Software tools Clustal W, BLASTN, Phylogeny.fr, MUSCLE, Gblocks, PhyML, TreeDyn
Applications Phylogenetics, Nucleotide sequence alignment
Organisms Homo sapiens
Diseases Neoplasms