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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

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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.

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Acknowledgment

This work was supported by the Department of Environment, Food and Rural Affairs (DEFRA), UK and the Higher Education Funding Council for England (Hefce) under the Veterinary Training and Research Initiative (VTRI).

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Authors and Affiliations

  1. Integrated Biology of GI Tract Programme, Institute of Food Research, Norwich Research Park, Colney, Norwich, NR4 7UA, UK

    Panagiotis Tourlomousis, Karyn P. Ridgway & Arjan Narbad

  2. Bioinformatics and Statistics Partnership, Institute of Food Research, Norwich Research Park, Colney, Norwich, NR4 7UA, UK

    E. Katherine Kemsley

  3. Department of Clinical Veterinary Sciences, University of Bristol, Langford, Bristol, BS40 5DU, UK

    Panagiotis Tourlomousis, Michael J. Toscano & Thomas J. Humphrey

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  1. Panagiotis Tourlomousis
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  2. E. Katherine Kemsley
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  6. Arjan Narbad
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Correspondence to Arjan Narbad.

Electronic Supplementary Material

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Figure S1

Raw images of all gels (except one which is shown in Fig. 1) used for the purpose of this study. (DOCX 1078 kb)

Figure S2

Matlab script (m-file) for importing DGGE data output from TL120 in Microsoft Excel format. The raw profiles are interpolated piecewise to common abscissae and collated into a matrix ready for multivariate analysis. The delimiters for each piece are defined in the TL120 alignment process and exported along with the raw profiles in the Excel spreadsheet. (DOCX 44 kb)

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Tourlomousis, P., Kemsley, E.K., Ridgway, K.P. et al. 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. Microb Ecol 59, 776–786 (2010). https://doi.org/10.1007/s00248-009-9613-x

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  • DOI: https://doi.org/10.1007/s00248-009-9613-x

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