New threshold and confidence estimates for terminal restriction fragment length polymorphism analysis of complex bacterial communities.
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.
Osborne
Catherine A
CA
Department of Microbiology and Immunology, University of Melbourne, Victoria 3010, Australia.
Rees
Gavin N
GN
Bernstein
Yaniv
Y
Janssen
Peter H
PH
D001419:Bacteria / Q000235:genetics*; D001483:Base Sequence; D016001:Confidence Intervals; D004269:DNA, Bacterial / Q000235:genetics; D017753:Ecosystem; D019015:Geologic Sediments / Q000382:microbiology; D010802:Phylogeny; D012150:Polymorphism, Restriction Fragment Length; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012988:Soil Microbiology
D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S