MacClade

MacClade is useful to modify your PAUP file, although this function is not the primary utility of this program

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1.  Open the file the alignment you exported from Sequencher in MacClade. You can do this by dragging the file icon into the MacClade icon, or by opening up MacClade, going to FILE, then OPEN FILE, and selecting you file.

2.  As many of the sequences have different lengths, use MacClade to trim the ends of the data file. Select the nucleotide position at top, and the whole row of nucleotides should be selected. By holding down on the shift key, you can select multiple positions. For example, if you want to delete characters 1-66, select characters 1-66 and hit delete. The program will ask if you want to delete 66 charactes. Say yes if you really want to do it.

3.  Go to FILE, OPTIONS FOR SAVING, and select NEXUS FORMAT. Remove the check on interleaved sequences and remove the value from the box for interleave XX characters per block. Select OK. This will remove the interleaved format that Sequencher gives you.

4.  Save the file.

5.  If you are working on a coding region, it is often useful to have MacClade set the base pair positions (e.g. 1st 2nd, or 3rd). To to this, first, under EDIT, go to SELECT, and then ALL to select all sequences.

6.  Under CHARACTERS, go to CODON POSITIONS. Here you can CALCULATE POSITIONS (by defining the first character as a 1st, 2nd, or 3rd position) or set as NON-CODING. As an exercise, select CALCULATE POSITIONS and set the position of the first base as a 2nd . Select OK.

7.  Under CHARACTERS, select GENETIC CODE

8.  Select the appropriate genetic code for your gene region. Select OK.

9.  In CHARACTERS select DATA FORMAT, then TRANSLATE TO PROTEIN. Select TRANSLATE.

10.  Scrolling along the AA sequence, look for stop codons (*). Check all three possible labelings of the fisrt base. The labeling that results in no stop codons is the one you want to use.

11.  IMPORTANT-DO NOT SAVE THE FILE AS PROTEIN OR ALL DNA SEQ DATA IS LOST

12.  Close file without saving. Open the file again.

13.  Define the first position defined as 1st, 2nd, or 3rd position, depending on which was the appropriate reading frame, then save your file.