Methods for evolutionary analysis: Difference between revisions

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The methods employed to obtain evolutionary data on selenium binding protein 2ece
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;


The aim to the evolutionary analysis is to find and categorize similar proteins to hint towards the proteins likely function and role, as well as giving solid evidence to base our conclusions on. A series of steps were taken in order to accomplish this goal, these are listed below.
[[Image:clus.jpg]]


'''Figure 1:''' Clustalx 1.83 alignment parameters [2]


== Psi Blast ==


The amino acid sequence of 2ece was put into a psi blast program using a non redundant database of proteins both the program and database was on the dvd. The amino acid sequence is below
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;


MAIVPFKRDPTFYPSPKMAMKAPPEDLAYVACLYTGTGINRADFIAVVDVNPKSETYSKIVHKVELPYINDELHHFGWNA CSSALCPNGKPNIERRFLIVPGLRSSRIYIIDTKPNPREPKIIKVIEPEEVKKVSGYSRLHTVHCGPDAIYISALGNEEG EGPGGILMLDHYSFEPLGKWEIDRGDQYLAYDFWWNLPNEVLVSSEWAVPNTIEDGLKLEHLKDRYGNRIHFWDLRKRKR IHSLTLGEENRMALELRPLHDPTKLMGFINMVVSLKDLSSSIWLWFYEDGKWNAEKVIEIPAEPLEGNLPEILKPFKAVP PLVTDIDISLDDKFLYLSLWGIGEVRQYDISNPFKPVLTGKVKLGGIFHRADHPAGHKLTGAPQMLEISRDGRRVYVTNS LYSTWDNQFYPEGLKGWMVKLNANPSGGLEIDKEFFVDFGEARSHQVRLSGGDASSDSYCYP
[[Image:mega.jpg]]


The output blast file from the psi blast program can be found here [[Media:blast.txt]]
'''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.

Latest revision as of 04:20, 15 June 2009

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;

Clus.jpg

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;

Mega.jpg

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.

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