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Applied and Environmental Microbiology, February 2006, p. 1270-1278, Vol. 72, No. 2
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.2.1270-1278.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

New Threshold and Confidence Estimates for Terminal Restriction Fragment Length Polymorphism Analysis of Complex Bacterial Communities{dagger}

Catherine A. Osborne,1 Gavin N. Rees,2 Yaniv Bernstein,3 and Peter H. Janssen1*

Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3010, Australia,1 Murray-Darling Freshwater Research Centre, P.O. Box 991, Wodonga, Victoria 3689, Australia,2 School of Computer Science and Information Technology, RMIT University, Melbourne, Victoria 3001, Australia3

Received 24 October 2005/ Accepted 6 December 2005

Terminal restriction fragment length polymorphism (T-RFLP) analysis has the potential to be useful for comparisons of complex bacterial communities, especially to detect changes in community structure in response to different variables. To do this successfully, systematic variations have to be detected above method-associated noise, by standardizing data sets and assigning confidence estimates to relationships detected. We investigated the use of different standardizing methods in T-RFLP analysis of PCR-amplified 16S rRNA genes to elucidate the similarities between the bacterial communities in 17 soil and sediment samples. We developed a robust method for standardizing data sets that appeared to allow detection of similarities between complex bacterial communities. We term this the variable percentage threshold method. We found that making conclusions about the similarities of complex bacterial communities from T-RFLP profiles generated by a single restriction enzyme (RE) may lead to erroneous conclusions. Instead, the use of multiple REs, each individually, to generate multiple data sets allowed us to determine a confidence estimate for groupings of apparently similar communities and at the same time minimized the effects of RE selection. In conjunction with the variable percentage threshold method, this allowed us to make confident conclusions about the similarities of the complex bacterial communities in the 17 different samples.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of Melbourne, Victoria 3010, Australia. Phone: 61 (3) 8344 5706. Fax: 61 (3) 9347 1540. E-mail: pjanssen{at}unimelb.edu.au.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, February 2006, p. 1270-1278, Vol. 72, No. 2
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.2.1270-1278.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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