Computational protocol: Polymorphism, selection and tandem duplication of transferrin genes in Atlantic cod (Gadus morhua) - Conserved synteny between fish monolobal and tetrapod bilobal transferrin loci

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Protocol publication

[…] All computational experiments were conducted on a Hewlett-Packard xw8600 workstation running Red Hat Enterprise Linux 5. Sequence analysis was performed using BLAST [] through the Protein Data Bank (BLOSUM62 matrix). Human serum transferrin (Protein Data Bank ID 2HAU) [] was selected as the most appropriate template to generate comparative models and showed sequence identities with cod Tf1-NE, Tf1-NW, Tf2, MTf and OMP of 45%, 45%, 44%, 40% and 35%, respectively. The template structure was then aligned against the cod sequences using ClustalW program []. Each sequence alignment was then checked to ensure that (i) all the secondary structural elements had a minimum number of insertions or deletions within them, and (ii) Cys residues forming consensus disulfide bridges were conserved []. Once an acceptable alignment had been produced, an ensemble of 50 models of the five different cod proteins were built using MODELLER v. 9.7 [] as implemented in Discovery Studio (Accelrys Inc., San Diego, CA, USA) and ranked using the MODELLER objective function, which is highly efficient in ranking different models calculated from the same alignment. It should be noted that the disulfide bridges between conserved Cys were not included as a restraint in the modeling process, but that the relative position of two Cys side chains in the resulting (unrestrained) model led us to suggest the existence of a disulfide. The stereochemical quality of the structures was assessed by PROCHECK [] supplemented by the profile programs VERIFY3D [] and ProSA-web []. In order to assess the reliability of each model, the corresponding energy graphs were compared with the template 2HAU structure. [...] Database searches were used to identify in total 40 amino acid sequences from teleost species, Ciona intestinalis and man (Additional file : Table S4). The C-terminal domain of Tf and MTf sequences was aligned with OMP sequences using ClustalX [] and by manual editing. The final alignment consisted of 40 taxa and 330 characters. The best evolution model based on the sequence alignment was determined using ProtTest []. The sequences were used to infer the phylogeny in a Bayesian framework applying the program MrBayes v3.1.2 []. The Bayesian inferences were done as follows: two independent runs, each with three cold and one heated MCMC (Markov Chain Monte Carlo) chains were started from a random starting tree. The two runs lasted for 5,000,000 generations. The covarion (COV) model was used together with the WAG+G+I to accommodate for different substitution rates across sites (G + proportion of invariable sites (I)) and across sequences (COV). The maximum likelihood (ML) tree was estimated using the program RAxML v.6 []. The topology with the highest likelihood score out of 100 heuristic searches, each from a random starting tree, was selected, and bootstrapping was done with 100 pseudoreplicates and one heuristic search per replicate. In the ML analyses, the WAG model with a gamma-distributed rate of variation across sites (G) was employed. All phylogenetic analyses were done on the freely available Bioportal at University of Oslo The Nei-Gojobori method [] implemented in DNAsp 5.0 was used to calculate rates of synonymous and non-synonymous substitutions in the identified Norwegian Tf1 cDNA sequence compared to the Canadian sequence []. We then used the z-test in MEGA (4.0) to test for positive selection. [...] SNP heterozygosities and population differentiation (FST) were calculated using Genepop 4.0.10 []. Statistical significance of population differentiation was assessed using Fishers exact test implemented in Genepop. […]

Pipeline specifications

Software tools Clustal W, ProtTest, MrBayes, RAxML, DnaSP, Genepop
Databases BioPortal
Applications Phylogenetics, Population genetic analysis
Organisms Gadus morhua, Gallus gallus
Chemicals Iron