In this tutorial, we will illustrate the procedures for building trees and in-memory sequence data editing, using the commands available in the Data and Phylogeny menus. We will be using the "Crab_rRNA.meg" file which can be found in the MEGA/Examples directory. This file contains nucleotide sequences for the large subunit mitochondrial rRNA gene from different crab species (Cunningham et al. 1992). Since the rRNA gene is transcribed, but not translated, it falls in the category of non-coding genes.
The Crab_rRNA.meg file used in this tutorial can be found in the MEGA/Examples folder (The default location for Windows users is C:\Program Files\MEGA\Examples. The default location for Mac users is $HOME/MEGA/Examples, where $HOME is the users home directory).
Building a Neighbor-Joining (NJ) Tree
In this example, we will illustrate the basics of phylogenetic tree re-construction using MEGA and become familiar with the Tree Explorer window.
Example 4.1:
Activate the "Crab_rRNA.meg" data file. If necessary, refer to Example 1.2 of the MEGA Basics tutorial.
From the main MEGA launch bar, select Phylogeny | Construct/Test Neighbor-Joining Tree menu option.
In the Analysis Preferences window select the p-distance option from the Model/Method drop-down.
Click Compute to accept the defaults for the rest of the options and begin the computation. A progress indicator will appear briefly before the tree displays in the Tree Explorer window.
To select a branch, click on it with the left mouse button. If you click on a branch with the right mouse button, you will get a small options menu that will let you flip the branch and perform various other operations on it.
Select a branch and then press the Up, Down, Left, and Right arrow keys to see how the cursor moves through the tree.
Change the branch style by selecting the View | Tree/Branch Style command from the Tree Explorer main menu.
Select the View | Topology Only command from the Tree Explorer main menu to display the branching pattern on the screen.
You can display the numerical branch lengths in the Topology Only option by selecting View | Options and clicking on the Branch tab. Check the box labeled Display Branch Length and click Ok.
Printing the NJ Tree (For Windows users)
Windows users can print directly from Tree Explorer.
Example 4.2a:
Select the File | Print option from the Tree Explorer main menu to bring up a standard Print window. This will print the tree full-sized and may take multiple sheets of paper. Press Cancel.
To restrict the size of the printed tree to a single sheet of paper, choose the File | Print in a Sheet command from the Tree Explorer main menu. Press Ok.
Select the File | Exit Tree Explorer command to exit the Tree Explorer. Click the OK button to close the Tree Explorer without saving the tree session.
Printing the NJ Tree (For Mac users)
MEGA does not support printing directly from Tree Explorer when running on a Mac system. To print a tree using a Mac, users can save the tree image to a PDF file and then print it by normal means.
Example 4.2b:
Select the Image | Save as PDF File option from the Tree Explorer main menu to bring up a standard Save window. Save the image to the desired location.
Once the document is saved, you can open it with your PDF reader and print the document in the same manner as any other PDF document.
Select the File | Exit Tree Explorer command to exit the Tree Explorer. Click the OK button to close the Tree Explorer without saving the tree session.
Construct a Maximum Parsimony (MP) Tree Using the Branch-&-Bound Search Option
Using MEGA, you can re-construct a phylogeny using Maximum Likelihood, Minimum Evolution, UPGMA, and Maximum Parsimony methods in addition to Neighbor-Joining. Here we re-construct the phylogeny for the Crab_rRNA.meg data using the Maximum Parsimony (MP) method.
Example 4.3
Select the Phylogeny | Construct/Test Maximum Parsimony Tree(s) menu option from the main MEGA launch bar. In the Analysis Preferences window, choose Max-mini Branch-&-bound for the MP Search Method option.
Click the Compute button to accept the defaults for the other options and begin the calculation. A progress window will appear briefly, and the tree will be displayed in Tree Explorer.
(Windows users) Now print this tree by selecting either of the Print options from the Tree Explorer's File menu.
(Mac users) Save the tree to a PDF file as described in Example 4.2b above.
Compare the NJ and MP trees. For this data set, the branching pattern of these two trees is identical.
Select the File | Exit Tree Explorer command to exit the Tree Explorer. Click OK to close Tree Explorer without saving the tree session.
Constructing a MP Tree using the Heuristic Search
For each method of phylogenetic inference, MEGA provides numerous options. In this example, we conduct MP analysis using the Min-Mini Heuristic search.
Example 4.4:
Follow the steps in Example 4.3 and instead of choosing Max-mini Branch-&-bound, choose Min-Mini Heuristic for MP Search Method. Change the MP Search Level to 2 and click Compute.
Note: In this example, the same tree is obtained by the Max-mini Branch-&-bound option as in the Min-Mini Heuristic option as long as the MP Search Level is set to 2. However, the computational time is much shorter for the Heuristic method.
Examining Data Editing Features
For noncoding sequence data, OTUs (Operational Taxonomic Units) as well as sites can be selected for analysis.
Example 4.5:
From the main MEGA window select the Data | Select Taxa and Groups option from the launch bar. A dialog box is displayed.
All the OTU labels are checked in the left panel. This indicates that all OTUs are included in the current active data subset. To remove the first OTU from the data, uncheck the checkbox next to the first OTU name in the left panel. Click the Close button.
Now, when you construct a neighbor-joining tree from this data set, it will contain 12 OTUs instead of 13. Close out of the Tree Explorer window by selecting File | Exit Tree Explorer and do not save. Deactivate the operational data set by selecting the Close Data icon from the main MEGA window.