Tweak your DNA Matrix for better Align To Folder searches with primers

You can use the Database | Align To Folder function as your own “personal BLAST search”, comparing a sequence to all of the sequences in a target folder hierarchy. The files in the folder can be in any format MacVector recognizes, including fasta and fastq formatted multiple sequence files.

Many users take this approach to scan a sequence against a “database” of primers maintained in a fasta file. One problem you may encounter using this approach is that MacVector fails to find matches to perfect primers, particularly if they are short (20nt or less). The reason for this is due to settings in the matrix file used for the search. If you want to find matches to short sequences, such as primers, you need to make some changes to the data in the .nmat file you use for your search. Open the .nmat file you are using for the search and click on the appropriately named “Tweak” toolbar item;


This will bring up the “Tweak Editor” dialog;


The key here is to change the p4 setting. It represents the “minimum” score that should be exceeded to consider something a match. The default is 80. Note how the scores for an A vs A or G vs G match in the matrix is 4. That means that a perfect match of a 20 nt primer against a sequence will give a score of 20 x 4 = 80. The default settings require the score to be GREATER THAN the p4 threshold, so a perfect match of a 20 nt primer would be rejected. If you changed p4 to be 79, then those would be accepted.

Now consider what would happen if you had a 19 out of 20 match to a 20 nt primer. The score would be (19 x 4) – 2 = 74 because mismatches score “-2” in the matrix. So you would need to drop p4 to be 73 or less to identify those matches.

If you drop it too low, like the “60” indicated here, you may get many thousands of spurious matches. But the best ones will always be shown at the top of the list.

This entry was posted in Tips and tagged , . Bookmark the permalink. Both comments and trackbacks are currently closed.