Web-based phylogenetic assignment tool for analysis of terminal restriction fragment length polymorphism profiles of microbial communities

Abstract

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 is not straightforward. Terminal restriction fragment length polymorphism (T-RFLP) is a community fingerprint method in which phylogenetic assignments may 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. A web-based tool is described here that rapidly generates phylogenetic assignments from submitted community T-RFLP profiles based on a database of fragments produced by known 16S rRNA gene sequences. Users have the option of submitting a customized database generated from unpublished sequences or from a gene other than the 16S rRNA gene. This phylogenetic assignment tool allows users to employ T-RFLP to simultaneously analyze microbial community diversity and species composition. An analysis of the variability of bacterial species composition throughout the water column in a humic lake was carried out to demonstrate the functionality of the phylogenetic assignment tool. This method was validated by comparing the results generated by this program with results from a 16S rRNA gene clone library.

FIG. 1.

Cycle of the matching algorithm. For each digest, each individual fragment is assigned a collection of species from the database that are predicted to have a T-RF length that matches the observed fragment length (within the user-specified size tolerance) (step 1). Records that do not match fragments found in additional digests (steps 2 and 3) are discarded. Steps 1 through 3 are repeated for each digest file.

FIG. 2.

The PAT web interface allows users to manage uploaded digest files, known organism database files, and T-RF bin size configuration.

FIG. 3.

The PAT web interface prompts users to select the restriction enzymes used to generate their T-RF digest files from a list of enzymes found in the selected database.

FIG. 4.

Bacterial classes detected by T-RFLP in Devil's Lake (northern Wisconsin) at various depths in August 2002. The plots indicate the number of fragments in each sample that could be assigned to each indicated phylogenetic class.

FIG. 5.

Bacterial diversity of families within the γ-Proteobacteria in Devil's Lake (northern Wisconsin) throughout the water column in August 2002. The y axis plots the number of fragments in each sample that could be assigned to a particular phylogenetic group within the γ-Proteobacteria.