Tutorial: A Phylogenetic Tree with MEGA 5

Last Friday (September 27, 2013) instead of having an internship, I read the tutorial book my mentor gave me and made my first phylogenetic tree!

First of all, I had to download a program called MEGA 5, which allows us to align and compare related sequences with our sequence of interest. For this tutorial, we use a sequence of an alpha-glucuronidase gene from the bacterium Thermotoga petrophilia. We can obtained the sequence by clicking Do BLAST Search from the Align menu, and it will lead us to the following window:

MEGA 5 is linked to BLAST.

I entered the "accession numbers" and the "query subrange", which are given by the book, chose "Neucleotide collection" for the Database, and blastn for Program Selection.

The results showed up few seconds after I hit search. It gave all the sequences that produce similar alignment to our query sequence. The first entry was eventually my query sequence (because that's what I searched for!) Each entry consists of 8 elements: accession number, description, Max score, Total score, query coverage, E-value, and Max identity. For the sake of time, I will just briefly explained Max score, Total score, query coverage, and E-value. Based on the definitions given by the book,

• Max score - the score for the highest scoring segments of the subject sequence
• Total score - the sum of the scores for all the segments that aligned (including noncontiguous alignments)
• Query coverage (%) - shows how much of the query sequence aligns with the subject sequence.
• E-value - describes the number of hits with scores this high that one would "expect" to see by chance when searching a database of a particular size. The closer the E-value to 0 the better.

 The book asked us to include those subject sequences that have E-value <10^-3 and query coverage >60%. For that, I chose 9 sequences, and I clicked "Add to Alignment" for each (some sequenced require to be reversed before added to alignment).

Sequences of the nine samples

Then I clicked "Align DNA" and get:

This is the end portion of the sequences after being aligned

Finally, I construct a phylogenetic tree from my DNA alignment by choosing "Construct / Test Neighbor Joining Tree" and tada~

My first phylogenetic tree!

This Friday, I will discuss with Dr. Miller more about the assumptions of making phylogenetic tree and do more practices with MEGA 5. I can't wait!:)