Methods for evolutionary analysis

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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 [2] was then used to generate a multiple sequence alignment for the above sequences using the following parameters;


Figure 1: Clustalx 1.83 alignment parameters [2]

Following alignment; sequences were trimmed in Clustalx [2] 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 [3] using the program’s conversion to MEGA format tool. MEGA [3] was then used to construct a bootstrapped neighbour joining tree using the following parameters;


Figure 2: Mega alignment parameters [3], Gamma parameter was established using Quartet puzzling [1].

A bootstrapped tree representing radiation was then copied to Microsoft power point 2007 to emphasise key features and significant bootstrap values.