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.
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.
Tourlomousis
Panagiotis
P
Integrated Biology of GI Tract Programme, Institute of Food Research, Norwich Research Park, Colney, Norwich, NR4 7UA, UK.
Kemsley
E Katherine
EK
Ridgway
Karyn P
KP
Toscano
Michael J
MJ
Humphrey
Thomas J
TJ
Narbad
Arjan
A
D000818:Animals; D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification; D001717:Birds / Q000382:microbiology; D004269:DNA, Bacterial / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005243:Feces / Q000382:microbiology; D007091:Image Processing, Computer-Assisted; D016133:Polymerase Chain Reaction / Q000379:methods*; D025341:Principal Component Analysis; D012336:RNA, Ribosomal, 16S / Q000032:analysis; D012015:Reference Standards; D012984:Software; D013552:Swine / Q000382:microbiology
D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S