Koichiro Tamura, Daniel Peterson, Nicholas Peterson, Glen Stecher, Masatoshi Nei, and Sudhir Kumar (2011) MEGA5: Molecular Evolutionary Genetics Analysis using Likelihood, Distance, and Parsimony methods. Molecular Biology and Evolution.
(to be submitted)
Kumar S, Dudley J, Nei M & Tamura K (2008) MEGA: A biologist-centric software for evolutionary analysis of DNA and protein sequences. Briefings in Bioinformatics 9: 299-306.
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Tamura K, Dudley J, Nei M & Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24: 1596-1599.
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Constructing Trees and Selecting OTUs
from Nucleotide Sequences

The Crab_rRNA.meg 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. Let us use this data file to illustrate the procedures of building trees and in-memory sequence data editing, using the commands present in the Data and Phylogeny menus.

Ex 3.0.1: Start MEGA by double-clicking on the MEGA desktop icon, or by using the Windows start-menu to click on the MEGA icon located in the programs folder.

Ex 3.1.1:  Activate the data file Crab_rRNA.meg using the instructions given in Ex 2.1.1 - Ex 2.1.3.

Let us start by building a neighbor-joining tree.

Ex 3.2.1:  Select the Phylogeny|Construct Phylogeny|Neighbor-Joining command to display the analysis preferences dialog box.

Ex 3.2.2:  In the Options Summary tab, click the Models pull-down (found in the Substitution Model section), and then select the Nucleotide|p-distance option.

Ex 3.2.3: Click “Compute” to accept the defaults for the rest of the options and begin the computations. A progress indicator will appear briefly before the tree displays in the Tree Explorer.

Ex 3.2.4: 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. To edit the OTU labels, double click on them.

Ex 3.2.5:  Change the branch style by using the View|Tree/Branch Style command from the Tree Explorer menu.

Ex 3.2.6:  Press the Up arrow key (↑) just once to move the cursor upwards to the next branch.

Ex 3.2.7:  Select the View|Topology Only command from the Tree Explorer menu to display the branching pattern (without actual branch lengths on the screen.

Ex 3.2.8:  Press F1 to examine the help for tree editor. Use this feature to become familiar with the many operations that Tree Explorer is capable of performing.

Ex 3.2.9:  DO NOT remove the tree from the screen. We shall use it for illustrating how a tree can be printed.

Now, you will print the NJ tree that you have on your screen in MEGA.

Ex 3.3.1:  Select the File|Print command from the Tree Explorer menu to bring up a standard Windows print dialog.

Ex 3.3.2:  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 menu.

Ex 3.3.3:  Select the File|Exit Tree Explorer (Ctrl-Q) command to exit the Tree Explorer. A warning box will inform you that your tree data has not been saved. Click the “OK” button to close Tree Explorer without saving the tree session.

In MEGA, you can also construct Maximum Parsimony (MP) trees. Let us construct a Maximum Parsimony tree(s) by using the branch-&-bound search option.

Ex 3.4.1:  Select the Phylogeny|Construct Phylogeny|Maximum Parsimony command. In the Analysis Preferences window, choose the Max-Mini Branch-&-Bound Search option in the MP Tree Search Options tab.

Ex 3.4.2:  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.

Ex 3.4.3:  Now print this tree (See Ex 3.3.1 - 3.3.2). You do not have to specify the printer name again, because MEGA remembers your selection.

Ex 3.4.4:  Select the File|Exit Tree Explorer (Ctrl-Q) command to exit the Tree Explorer. A warning box will inform you that your tree data has not been saved. Click “OK” to close Tree Explorer without saving the tree session.

Ex 3.4.5:  Compare the NJ and MP trees. For this data set, the branching pattern of these two trees is identical.

As an exercise, use the Heuristic Search for finding the MP tree. In this example, you will find the same tree as that obtained by the branch-and-bound method if you use the default option (search factor equal to 2 for all steps of OTU addition). However, the computational time will be much shorter. Actually, in this example, even a search factor equal to 0 will recover the MP tree.

We will now examine how some data editing features work in MEGA. For noncoding sequence data, OTUs as well as sites can be selected for analysis. Let us remove the first OTU from the current data set.

Ex 3.5.1:  Select the Data|Setup/Select Taxa & Groups command. A dialog box is displayed.

Ex 3.5.2:  All the OTU labels are checked ( ) in the left box. 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 OTU name in the left pane.

Ex 3.5.3: Now, from this data set, construct a neighbor-joining tree (Ex 3.2.1) that contains 12 OTUs instead of 13. To inactivate the operational data set and end the current session of MEGA, press the hot-key Alt + X.