7. Walk through MEGA

This chapter provides a tutorial for using MEGA through 6 examples. The data files for these examples can be found in the C:\MEGA\EXAMPLES directory. In these example files data are deliberately written in different input formats. We recommend that these examples be studied in the order presented because the techniques introduced in previous examples are used in the following ones.

In the following discussion, highlighted words indicate the keys to be pressed on the keyboard. If two keys are required to be pressed simultaneously, they are shown with a + sign between them (e.g., Alt + F3 means that the Alt and F3 keys should be pressed simultaneously). Italicized letters are used to mark the commands available in menus, submenus, and other options as they appear on the computer screen at various times. In every example, we discuss many procedures to introduce the techniques of analysis. For ease of reference in later examples, these procedures are arranged in steps that are numbered in the Ex u.v.w format, where u is the example number, v is the procedure number in the u-th example, and w is the step number in the procedure v. For instance, Ex 1.3.2 refers to the 2nd step of the 3rd procedure in example 1.

This example introduces procedures for changing the default directory, selecting options from menus, opening files in the read-only mode, activating a distance data file for analysis, and building trees from the distance data.

In this exercise the use of the Data|Data Presentation command for computing various statistical quantities of nucleotide sequences is illustrated. In addition, short-cuts for frequently used commands, method of accessing on-line helps, and the reason why some commands are enabled and others are disabled are explained.

In this example we compute various distances for the Adh sequences from 11 Drosophila species (Thomas and Hunt 1993). We used this data in the previous example to study various sequence statistics. In addition, you will be see how these distances can be written in a file in various formats through options for page size, precision, and relative placement of distances and their standard errors.

The CRAB.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 is 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 4.0.1

Go to the C:\MEGA directory first, type MEGA on the C:\MEGA> DOS prompt, and press Enter. Now again press Enter in the Welcome box that appears on the screen.

Now set C:\MEGA\EXAMPLES as the default directory using the File|Change Dir command, and examine the contents of the CRAB.MEG file (use hot-key F5). In this data file, note the comments starting on the third line. The comments indicate that the data are in the noninterleaved format and that '?' and '-' are used to designate missing-information and alignment gap sites. Close this file using Alt + F3. Let us activate the crab sequence data for analysis.

Ex 4.1.1	Select the Data|Open Data command and choose the DNA option. In the File Name dialog box, type CRAB.MEG and press Enter.
Ex 4.1.2	A dialog box will appear. Use the Tab key to move around in the dialog box but do not change anything. In this box the noninterleaved format is selected and the symbols used for missing-information data, identical sites, and alignment gap are '?', '.', and '-', respectively. So everything is fine. Just Enter (or click on the OK button).
Ex 4.1.3	A status report box informs that the data are being read. At this stage, the program inquires whether the nucleotide sequence data are from a coding or noncoding gene. Select the noncoding mode by pressing the N key. The Current Data and the Selection windows appear on the screen.

The use of Data|Data Presentation command was introduced in the second example. As an exercise, you may try to examine this data set on the screen by using that command. Just press F4, the hot-key for the Data Presentation command, and you will see the data on the screen. For help, press F1 any time.

Let us start by building a neighbor-joining tree. For this purpose, we need to specify a distance estimation method in the Distances menu and a tree building method in the Phylogeny menu. The Phylogeny|Construct Tree(s) command is then used for tree building.

Ex 4.2.1	Select the Distance|Nucleotide command. Choose the Jukes-Cantor Distance from the resultant submenu.
Ex 4.2.2	To use the neighbor-joining method for tree building, select the Phylogeny|Neighbor-Joining command.
Ex 4.2.3	Invoke the Phylogeny|Construct Tree(s) command. This brings a status report box with a message. The neighbor-joining tree will soon appear on the screen.
Ex 4.2.4	At this moment, you are automatically put into the phylogenetic-tree editor. This editor provides operations in two modes: view mode and edit mode. The edit mode can be recognized by the presence of a blinking cursor. By default you are placed in the view mode. Press E to enter the edit mode (a blinking cursor will appear).
Ex 4.2.5	Use the arrow keys ( ­, ¯, ®, ¬ ) to move to different branches on the tree and note the change in the branch length in the lower-left corner corresponding to the focused branch. Now, position your cursor on the far left corner of the screen.
Ex 4.2.6	At this time the cursor assumes a triangular shape instead of the diamond ( ¨ ). Press M, the mirror image of the original tree is displayed instantly. Press M again, the tree reverts to its original shape.
Ex 4.2.7	Press the Up arrow key ( ­ ) just once. The cursor moves upwards to the next branch. Press F, the Flip command. A mirror like effect is produced on the sub-tree anchored on the currently focused branch.
Ex 4.2.8	The Topology command is to display just the branching pattern of the tree. Press T, the Topology command, the branching pattern (without actual branch lengths) is displayed on the screen. Press T again, the actual NJ tree reappears.
Ex 4.2.9	Press F1 to examine the help for tree editor. Use the Tab key to get to the highlighted word Swap and press Enter. You will see information about the Swap command. This can be used for more commands. Press Esc to exit help.
Ex 4.2.10	DO NOT remove the tree from the screen. We shall use it for illustrating how a tree can be printed.

At this moment we have the NJ tree on the screen. In MEGA you can print this tree by using a printer. Let us see how.

Ex 4.3.1	You can print a tree in two ways. First, a tree can be written as an ASCII-text file. In this case, an exact replica of the tree displayed on the screen is written in the desired file. Since the NJ and UPGMA trees are shown with approximate branch lengths, this output does not reflect true branch lengths. By contrast, if you have a printer attached to your computer, you can print the tree with exact branch lengths.
Ex 4.3.2	Press P, the Print command. A dialog box with two options appears. If there is no printer attached to your computer, select the ASCII-Text file output option using the Tab and arrow keys, and then press Enter. For the output file name, type C:\TREE.NJ. If you have a printer attached to your computer, select the Printer option and press Enter. An dialog box appears on the screen. In this dialog box many options are available. (Press F1 to learn about them.)
Ex 4.3.3	Do not change anything in this dialog box, and just select the Preview command using the Tab key. A graphic image of the tree will be displayed on the screen. Press Enter, and you are back to the option box. Now go to Write information option, and select the Branch lengths. Again select the Preview command (you may press Alt + V). The tree is now drawn with branch lengths. Press Enter to come out of the graphics image.
Ex 4.3.4	To print the tree with a printer, select an appropriate printer using the Printer command.
Ex 4.3.5	Press Enter (or click on OK) to print the tree on the selected printer.
Ex 4.3.6	Press Esc to exit the phylogenetic-tree editor.

In MEGA, you can also construct maximum parsimony trees. Let us construct a maximum parsimony tree(s) by using the branch-and-bound search option.

Ex 4.4.1	Select the Phylogeny|Maximum Parsimony command. In the resultant submenu, choose the Branch-and-Bound Search option.
Ex 4.4.2	Invoke the Phylogeny|Construct Tree(s) command, and press Enter to accept default options in the dialog box produced. This brings a status report box. An MP tree appears on the screen as soon as the search is completed.
Ex 4.4.3	Note that no branch lengths are given for an MP tree in MEGA. Also that the Topology command is disabled because in this case only the branching pattern is available.
Ex 4.4.4	Now print this tree (See Ex 4.3.1 - 4.3.5). You do not have to specify the printer name again because MEGA remembers your selection.
Ex 4.4.5	Press Esc to exit the phylogenetic tree editor.
Ex 4.4.6	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 4.5.1	Select the Data|Select OTUs command. A Select OTUs list dialog box is displayed.
Ex 4.5.2	All the OTU labels are checked ( Ö ) in this box. This indicates that all OTUs are included in the current active data subset. To remove the first OTU from the data, press the Del key (or double click on the first OTU). The first OTU is no longer checked. Press Enter.
Ex 4.5.3	Note a change in the Used OTUs entry in the Current Data window. The number of OTUs used for analysis has been reduced by one.
Ex 4.5.4	Again use the Data|Data Presentation command (F4) to see the changes made.

Now, construct a neighbor-joining tree from this data set (Ex 4.2.3) that contains 12 OTUs instead of 13. Let us inactivate the currently used data set and end the current session of MEGA by pressing the hot-key Alt + X.

7.5 Tests of the Reliability of a Tree Obtained

In this example, we will conduct two different tests using mitochondrial 12S rRNA gene sequences from 12 flightless birds (ratites) and one related species (Cooper et al. 1992) and learn how to construct a condensed tree.

In this example, various analyses of protein-coding nucleotide sequences for five alleles from the human HLA-A locus (Nei and Hughes 1991) are presented.