PCR-Denaturing Gradient Gel Electrophoresis of Complex Microbial Communities:A Two-Step Approach to Address the Effect of Gel-to-Gel Variation and Allow Valid Comparisons Across a Large Dataset

Tourlomousis, Panagiotis, Kemsley, E. Katherine, Ridgway, Karyn P., Toscano, Michael J., Humphrey, Thomas J. and Narbad, Arjan (2010) PCR-Denaturing Gradient Gel Electrophoresis of Complex Microbial Communities:A Two-Step Approach to Address the Effect of Gel-to-Gel Variation and Allow Valid Comparisons Across a Large Dataset. Microbial Ecology, 59 (4). pp. 776-786. ISSN 0095-3628

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Abstract

Denaturing gradient gel electrophoresis (DGGE) is widely used in microbial ecology to profile complex microbial communities over time and in response to different stimuli. However, inherent gel-to-gel variability has always been a barrier toward meaningful interpretation of DGGE profiles obtained from multiple gels. To address this problem, we developed a two-step methodology to align DGGE profiles across a large dataset. The use of appropriate inter-gel standards was of vital importance since they provided the basis for efficient within- and between-gel alignment and a reliable means to evaluate the final outcome of the process. Pretreatment of DGGE profiles by a commercially available image analysis software package (TL120 v2006, Phoretix 1D Advanced) followed by a simple interpolation step in Matlab minimized the effect of gel-to-gel variation, allowing for comparisons between large numbers of samples with a high degree of confidence. At the same time, data were obtained in the form of whole densitometric curves, rather than as band presence/absence or intensity information, and could be readily analyzed by a collection of well-established multivariate methods. This work clearly demonstrates that there is still room for significant improvements as to the way large DGGE datasets are processed and statistically interrogated.

Item Type: Article
Uncontrolled Keywords: 16s ribosomal-rna,marine bacterioplankton communities,commercial software packages,bacterial communities,broiler-chickens,polymorphism patterns,dna,diversity,populations,profiles
Faculty \ School: Faculty of Science > School of Chemistry
Faculty of Science > School of Biological Sciences
Depositing User: LivePure Connector
Date Deposited: 20 Jul 2018 15:33
Last Modified: 22 Apr 2020 06:51
URI: https://ueaeprints.uea.ac.uk/id/eprint/67735
DOI: 10.1007/s00248-009-9613-x

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