Phylogenetic Assignment Tool FAQ:

1. Why can’t I log in?

- Login is case sensitive – make sure you are typing in the exact username and password that you selected when you registered.

2. I can’t select any digest files in Step 4.

- The digest files are fragment analysis files that you upload to our server. You should upload several files for analysis, one file containing the T-RFLP profiles for each of your restriction digests. If you used 3 restriction enzymes, you need to upload 3 separate files. If your raw data is in a single file, you must edit the results to generate several files. Profiles generated by different enzymes must be contained in separate text files.

- Upload the separate files from each restriction digest one at a time.

- Each file may contain several samples, but corresponding samples must have the same ID in each separate digest file. As these are text files, you may easily edit sample IDs if the raw output from the T-RFLP analysis did not result in give you the same IDs for corresponding samples in each digest.

- Try the analysis with our practice files: digest Hha, digest Msp, digest Rsa. You can look at these to see the file format that PAT expects. Note that each file contains several samples, and corresponding samples have the same sample ID in each digest. You can also upload these files in Step 4 to do a test analysis.

3. I loaded three digest files - why don’t I get any results?

- Check the file format, make sure it matches the format defined here

- Make sure that corresponding samples have the same sample ID.

- Try uploading our practice digest files: digest Hha, digest Msp, digest Rsa and use with the default database.

4. I want to use the default database. Why isn’t it available in Step 3?

- You need to indicate that you’d like to use the default database in Step 2 by clicking the “default database” link.

5. Steps 4 and 5 are missing.

- This may be a browser issue. PAT was optimized to work with Internet Explorer or Netscape. The website may not display properly in other browsers.

6. My MiCA output file does not work with PAT. What's wrong?

- The MiCA output contains more information than necessary for PAT, but can be edited for use with PAT.

To edit the MiCA output for PAT:

1. Remove any header information. The information that PAT uses includes the species name and the predicted T-RF from various restriction enzymes.The first column should be labeled "species" in the first row and include the species name and any other identifiers you wish to use (accession number, etc.). The second and subsequent columns should be labeled with a restriction enzyme name (e.g. HhaI) in the first row, and should include the predicted T-RF size produced by each enzyme.

2. MiCA output includes predicted T-RF sizes from both the 5' and 3' end of the sequence. Choose only one of these for use with PAT (the one that corresponds to your T-RFLP fingerprint data). Edit the MiCA output to include T-RFs from only one end of the T-RFLP template.

3. An example of the database format can be found here http://trflp.limnology.wisc.edu/defaultdb.jsp. You may also download our default database to examine the formatting while constructing a custom T-RF database.