A more flexible conducting multiple alignment and building a tree involves use of the 'Sequence analysis' (SA) window. This window enables you to add additional sequences before building aligning or building the tree. In this tutorial you will first build three trees to demonstrate how the SA window works.
Note: This tutorial assumes that you have already gone through the tutorial for multiple alignment and tree-building using only the 'Opened & processed' window. It assumes that you have already run PCR on the HIV sequences from that tutorial. |
Select Analyze -> Export -> Add selected files to contents of Export field, as shown at the bottom of the graphic below.
2. The above command causes the 'Sequence analysis' window to open automatically (on the right), and the selected PCR products are added to the Export field of this new window.
| Note: Clicking the blue 'Find FASTA' button in the upper-right hand corner of the Sequence analysis window allows you to quickly find the FASTA definition line of each sequence added to the window. |
3. Now click the Analyze menu (yellow menu on main screen below), and select Align / tree - > Build tree using sequences in Export Field as shown below.
4. If you are running security software, message boxes may appear asking if you really want to run the software. If they do, click the Run button on each message box.
5. The Case It message box that is shown below will also appear, and will close automatically after the alignment is finished. The large black box will also close automatically. Note that the second security message box is behind the Case It message box in the screen shot below, and the Run button on it (not shown) must also be clicked.
6. Mega4 will open and the tree will be displayed, along with the multiple alignment (performed by CLUSTALW).
Warning: You will probably have to move the MEGA windows around to uncover the ones with the multiple alignment and tree. When doing so, do not close any of the MEGA windows, especially the one that says 'MEGA 4.0.1' in its title bar. This is the main MEGA window, and closing it automatically closes all of the other windows. |
7. Close the MEGA4 windows by clicking the X on the "MEGA 4.01" window above right (this is the best way to do it, since closing this window automatically closes all other MEGA4 windows). This time, shift-click to highlight only the four PCR products shown below, and select Replace contents of Export field with selected file[s].
8. Use the Analyze menu and select Align / tree -> Build tree using sequences in Export field
9. A new alignment and tree will appear as shown below. Note that you may have to click 'Run' on security windows for this process to be completed.
10. Close the MEGA 4.0.1 window to close all of the other MEGA windows.