Methods for evolutionary analysis
Sequences for evolutionary analysis were gathered from NCBI using BLAST (refseq). These were selected from different phyla on the basis of sequence identity (>30%). More distantly related sequences were obtained using the Psi-BLAST algorithm available at Interpro. Selected sequences were gathered in FASTA format and input into a .txt file sequentially. ClustalX 1.83 [1] was then used to generate a multiple sequence alignment for the above sequences using the following parameters;
Figure 1: Clustalx 1.83 alignment parameters [1]
Following alignment; sequences were trimmed in Clustalx [1] from residues 31-184 to remove extending ‘tails’ for more accurate phylogenetic analysis. The alignment file was then saved as an .aln file and exported to MEGA 4.0 [2] using the program’s conversion to MEGA format tool. MEGA [2] was then used to construct a bootstrapped neighbour joining tree using the following parameters;
Figure 2: Mega alignment parameters [2], Gamma parameter was established using Quartet puzzling [3].
A bootstrapped tree representing radiation was then copied to Microsoft power point 2007 to emphasise key features and significant bootstrap values.