Database Creator

Coming Soon: Automatic Version!

Creating a database to use with Fragsort using your primer and restriction enzymes.

A database can be created in various ways to be used with Fragsort. As long as the file format is correct, the program can use any database of TRFs you may create.

The easiest way to create a database of bacterial 16S based TRFs to use with Fragsort is with the MiCA virtual digest program (http://mica.ibest.uidaho.edu/digest.php). Open the web page and choose (or enter) the forward and reverse primers you used for your TRFLP analysis. Then select the three restriction enzymes you used. Select the number of mismatches (usually 3) and the distance into the primer where the mismatches occur (usually 15). Choose bacteria. Choose to report “all fragments”. Sorting doesn’t matter. Click submit.

After a minute or two, the output will be displayed in an html file. Choose “CSV format (Spreadsheet)”. The file will open in your spreadsheet program in your browser, usually Excel. Open Excel separately then copy this data and paste it into a new excel file.

Create a new worksheet and format the data in the following format:

The first row is a list of enzymes used; there should be one enzyme in each cell.

The rest of the rows contain the data from MiCA. The first column is the Accession ID, the second column is the Organism Name, and any column after that contains the fragment size.

Create a second worksheet with the same format for the reverse data.

Save these files as “tab delineated text” files in the “data” folder in the Fragsort 5.0 program folder. (“C:\program files\FragSort\data”). Name them according to the primer pair used and whether the data are forward or reverse (eg.: forward(11f-926r).txt or reverse(11f-926r).txt)

Open the folder in windows and change these files file extension from, “*.txt” to “*.db”.

Run Fragsort 5.0 and when it asks to select the database, choose the file you just created.

Upload your TRFLP data and Fragsort it!