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Applied and Environmental Microbiology, November 2003, p. 6768-6776, Vol. 69, No. 11
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.11.6768-6776.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Web-Based Phylogenetic Assignment Tool for Analysis of Terminal Restriction Fragment Length Polymorphism Profiles of Microbial Communities

Angela D. Kent,1 Dan J. Smith,1 Barbara J. Benson,1 and Eric W. Triplett1,2*

Microbial Observatory of the North Temperate Lakes Long-Term Ecological Research Site (NTL-LTER), Center for Limnology,1 Department of Agronomy, University of Wisconsin—Madison, Madison, Wisconsin 537062

Received 30 December 2002/ Accepted 29 August 2003

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 Users have the option of submitting a customized database generated from unpublished sequences or from a gene other than the 16S rRNA gene.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.

* Corresponding author. Present address: Department of Microbiology and Cell Science, University of Florida, P.O. Box 110700, Gainesville, FL 32611-0700. Phone: (352) 392-1906. Fax: (352) 392-5922. E-mail: ewt{at}ufl.edu.

Applied and Environmental Microbiology, November 2003, p. 6768-6776, Vol. 69, No. 11
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.11.6768-6776.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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