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

Panagiotis Tourlomousis, E. Katherine Kemsley, Karyn P. Ridgway, Michael J. Toscano, Thomas J. Humphrey, Arjan Narbad

Research output: Contribution to journalArticle

27 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)776-786
Number of pages11
JournalMicrobial Ecology
Volume59
Issue number4
Early online date3 Dec 2009
DOIs
Publication statusPublished - May 2010

Keywords

  • 16S RIBOSOMAL-RNA
  • MARINE BACTERIOPLANKTON COMMUNITIES
  • COMMERCIAL SOFTWARE PACKAGES
  • BACTERIAL COMMUNITIES
  • BROILER-CHICKENS
  • POLYMORPHISM PATTERNS
  • DNA
  • DIVERSITY
  • POPULATIONS
  • PROFILES

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