The goal of this section is to generate a multiple sequence alignment for use in detecting adaptive evolution.

Use either the ZP3.fas or the multiple fasta formated alignment you generated. Start VirtualPC and copy your alignment onto the PC desktop.

1. Start MEGA3 - go to "alignment" tab and select "alignment explorer/CLUSTAL". A dialog box will open, select "retreieve sequence from file" then select your multiple-fasta formated file. This will open as an unaligned file. To make the alignment, click on the "translated Protein Sequences" tab to work with translated sequences. Go to the "Alignment" tab and select "Align by ClustalW". This will prompt you to select alignment parameters, either go with default or try different penalties. This will make the first pass alignment. Now check the alignment by eye and make adjustments as needed, or export and use Se-Al to make adjustments. To export, return to "DNA" tab to view DNA sequences and export as FASTA file and MEGA file. Close the alignment browser.

2. Make a rough tree using MEGA. On the MEGA3 program, select "click me to activate a file" then select you aligned MEGA formated file. This will open the file in sequence data explorer. Confirm alignment is correct, then close the window. To compute tree, select the "phylogeny" tab and choose method (i.e. neighbor joining). Select parameters (for our purposes, use default) and then "compute". When tree is shown, export tree in Newick Standard format. Copy both Tree and alignment file in fasta format back to the Apple desktop.

3. Use Readseq to convert FASTA file to Phylip format. Then use BBedit to convert line breaks to Unix for use in PAML.