Phylogenetic Assignment Tool (PAT)

About PAT: Culture-independent DNA fingerprints are commonly used to assess the diversity of a microbial community. However, relating species composition to community profiles produced by community fingerprint methods such as ARISA, ARDRA or DGGE is not straightforward. Terminal restriction fragment length polymorphism (T-RFLP) is a community fingerprint method in which phylogenetic assignments may potentially be inferred from the terminal restriction fragment (T-RF) sizes through the use of web-based resources that predict T-RF sizes for known bacteria. The process quickly becomes computationally intensive due to the need to analyze profiles produced by multiple restriction digests and the complexity of profiles generated by natural microbial communities. We have developed this web-based tool to rapidly generate phylogenetic assignments from submitted T-RFLP profiles based on a database of fragments produced by known 16S rDNA sequences. Users have the option of submitting a customized database generated from unpublished sequences or from a gene other than the 16S rRNA gene.

The web interface for the PAT tool allows investigators to submit, process, and manage every aspect of the phylogenetic assignment. PAT allows for each user of the system to register and maintain an account for their data and configuration information. A user can upload digest and database files for use with their account. Users can also elect to use the default database of T-RFs produced by the in silico digestion of known 16S rDNA sequences by 27 separate restriction enzymes. The web interface provides management of a user's enzyme digests, known organism database files, and bin size configuration.

To generate phylogenetic assignments from the supplied data, the user selects a database file, associated digest files, and the bin sizes to use in the computation. The PAT program then prompts the user to assign a specific restriction enzyme present in the selected database to each of the digest files.

After the enzyme assignment, the program runs the PAT filtering algorithm to determine phylogenetic matches. The phylogenetic matches, matching statistics, and unmatched fragment information can then be downloaded from the results page.