Computational protocol: Functional Desaturase Fads1 (Δ5) and Fads2 (Δ6) Orthologues Evolved before the Origin of Jawed Vertebrates

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Protocol publication

[…] The Fads gene portfolio was identified in release 63 of the Ensembl database (www.ensembl.org) and Genbank from the following species: Homo sapiens (human-placental mammal), Macaca mullata (Rhesus macaque-placental mammal), Mus musculus (mouse-placental mammal), Canis familiaris (dog-placental mammal), Oryctolagus cuniculus (rabbit-placental mammal), Monodelphis domestica (opossum-marsupial), Ornithorhynchus anatinus (platypus-monotreme), Anolis carolinensis (green anole-reptile), Gallus gallus (chicken-bird), Xenopus tropicalis (western clawed frog-amphibian), D. rerio (teleost), Gasterosteus aculeatus (three-spined stickleback-teleost), Oryzias latipes (medaka-teleost), T. nigroviridis (green-spotted pufferfish-teleost), T. rubripes (‘Fugu’ pufferfish-teleost), and Branchiostoma floridae (amphioxus-cephalochordate). The search of the cartilaginous fish Callorhinchus milii (elephant shark-chondrichthyes) genome was performed using the human FADS1 and FADS2 sequences with TBLASTN at http://esharkgenome.imcb.a-star.edu.sg/Blast/ .To identify sequences that were poorly annotated or non-annotated in databases we also performed a BLAST search Blastp (PSI-BLAST). Some sequences were manually curated using the human gene FADS1 or FADS2 gene structure as a model.Protein sequences from Fads genes were aligned using MAFFT (http://mafft.cbrc.jp/alignment/server/) with the L-INS-i method . The alignment was further adjusted manually with gap removal. The final dataset had a total of 55 sequences and 297 characters. For tree reconstruction, we first applied ProtTest to estimate the optimal model of amino acid substitution (JTT+I+G+F). A maximum likelihood tree was constructed using PHYML (online) with the JTT model. The amino acid frequency (equilibrium frequency), proportion of invariable sites and gamma-shape (4 rate substitution categories) for the amino acid substitution rate heterogeneity parameters were estimated from the dataset. Confidence in each node was assessed by aLRT non-parametric branch support (SH-like) . The phylogenetic tree was rooted with the amphioxus sequence using NJPlot (version 2.3) (pbil.univ-lyon1.fr/software/njplot.html) .Genomic regions containing Fads genes were identified in Ensembl and Genbank databases. The two closest gene families flanking Fads genes were identified. […]

Pipeline specifications

Software tools TBLASTN, BLASTP, MAFFT, ProtTest, PhyML, NJplot
Applications Phylogenetics, Amino acid sequence alignment
Organisms Scyliorhinus canicula, Homo sapiens
Chemicals Fatty Acids, Fatty Acids, Unsaturated