Module 8: Phylogenetic Analysis

 

Exercise 8

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In this exercise, we will use a phenetic method (UPGMA) to analyze the evolutionary relationships between five related sequences of immunological significance.

I. Unweighted Pair Group Method with Arithmetic averages (UPGMA)

  • Link to the PHYLIP web server at the Pasteur Institute and click on the "protdist" link under the "Programs for molecular sequence data" section.
  • In the "Alignment File" box, copy and paste the five sequences from this file including the first line for each sequence indicating the FASTA format (essentially copy and paste the whole file). This file has the five sequences that we used for ClustalW alignment (exercise 5 in module 5) and they are (1) Mouse IL7 receptor (IL7R), (2) Human IL7 receptor, (3) Mouse IL21 receptor (IL21R), (4) Mouse Erythropoietin receptor (EPOR) and (5) Human class I cytokine receptor (CKR).
  • Enter your e-mail address and then hit the "Run protdist" button. In the ensuing window, choose 'neighbor', from the drop-down box, as the selected program to run on the "outfile". The outfile will show a distance matrix for the five sequences (not very useful). Hit the "Run the selected program on outfile" button.
  • Click the "Advanced neighbor form" button and then choose UPGMA as the distance method and hit the "Run neighbor" button. You can check out the "outfile" from this window to see the evolutionary distances separating the five proteins.
  • To view a graphical representation of the evolutionary relationships, select the 'drawgram' program from the drop-down menu and click the "Run the selected program on treefile" button. You have to change the following options in the next window to get a good graphical representation of the phylogenetic relationships between the five sequences.
    • Tree grows: Default
    • Tree style: Phenogram
    • Character ht / tip space: 0.1111
    • Ancestral nodes: Centered
    • Printer: Printer of your choice
  • Finally, to view the Drawgram plot, you have to save the 'plotfile' HTML document onto your local machine. Notice where in the Windows directory (or within a sub directory) that you are saving this plotfile. Go to the MS-DOS prompt and at the appropriate directory/subdirectory type "copy /b plotfile.html prn:". This will print a hard copy of a rooted evolutionary tree.
  • Notice that the murine sequences are closely related to each other compared to the human sequences. It is interesting to note that although the murine and human IL7 receptor molecules are very similar in their amino acid sequences they fall apart in evolutionary distance in this tree.
  • You may wish to analyze some of the sequences from your area of research. Please be aware that the various parameters for creating the distance matrix and then the distance tree may change based on the proteins or nucleotide sequences that you are studying.


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