Fidelity of Select Restriction Endonucleases in Determining Microbial Diversity by Terminal-Restriction Fragment Length Polymorphism

Abstract

An evaluation of 18 DNA restriction endonucleases for use in terminal-restriction fragment length polymorphism (T-RFLP) analysis was performed by using richness and density indices in conjunction with computer simulations for 4,603 bacterial small-subunit rRNA gene sequences. T-RFLP analysis has become a commonly used method for screening environmental samples for precursory identification and community comparison studies due to its precision and high-throughput capability. The accuracy of T-RFLP analysis for describing a community has not yet been thoroughly evaluated. In this study, we attempted to classify restriction endonucleases based upon the ability to resolve unique terminal-restriction fragments (T-RFs) or operational taxonomic units (OTUs) from a database of gene sequences. Furthermore, we assessed the predictive accuracy of T-RFLP at fixed values of community richness (n ‫؍‬ 1, 5, 10, 50, and 100). Classification of restriction endonuclease fidelity was performed by measuring richness and density for the entire database of T-RFs. Further analysis of T-RFLP accuracy for determining richness was performed by iterative, random sampling from the derived database of T-RFs. It became apparent that two constraints were influential for measuring the fidelity of a given restriction endonuclease: (i) the ability to resolve unique sequence variants and (ii) the number of unique T-RFs that fell within a measurable size range. The latter constraint was found to be more significant for estimating restriction endonuclease fidelity. Of the 18 restriction endonucleases examined, BstUI, DdeI, Sau96I, and MspI had the highest frequency of resolving single populations in model communities. All restriction endonucleases used in this study detected <70% of the OTUs at richness values greater than 50 OTUs per modeled community. Based on the results of our in silico experiments, the most efficacious uses of T-RFLP for microbial diversity studies are those that address situations where there is low to intermediate species richness (e.g., colonization, early successional stages, biofilm formation).

Long-term experimental warming alters nitrogen-cycling communities but site factors remain the primary drivers of community structure in high arctic tundra soils

“…Assessments of microbial community structure based upon functional genes may track differences in potential gene function based upon larger scale environmental change, although T-RFLP is most effective where species diversity is low or moderate (Engebretson and Moyer, 2003). However, the link between shifts in microbial community structure (measured by changes in gene frequency, abundance and richness) and corresponding changes in the processes mediated by these communities has not been satisfactorily demonstrated.…”

Silica Biomineralization of Calothrix-Dominated Biofacies from Queen's Laundry Hot-Spring, Yellowstone National Park, USA

“…The methodology used for T-RFLP analysis has been reported in detail in previous publications (Davis and Moyer, 2008;Rassa et al, 2009;Fleming et al, 2013). This technique accurately resolves populations in microbial communities of low to intermediate richness (Engebretson and Moyer, 2003). Electropherograms are imported into the program BioNumerics (Applied Maths, Austin, TX) where community fingerprints were compared using average Pearson product moment correlation (Häne et al, 1993).…”

Use of Terminal Restriction Length Polymorphism (T-RFLP) Analysis to Evaluate Uncultivable Microbial Community Structure of Soil

“…This is due to the fact that different bacterial populations can share the same terminal restriction fragment length for a particular primer-enzyme combination but not others (Marsh et al, 2000). The ability of different restriction enzymes to resolve unique sequences has been examined in studies of gene sequence databases, communities with different richness, and iterative random sampling from a derived database of T-RFs (Engebretson and Moyer, 2003;Moyer et al, 1996). Engebretson and Moyer (2003) have evaluated 18 restriction enzymes and found that BstUI, DdeI, Sau961 and MspI most often resolved individual populations in their model communities.…”

“…Osborn et al (2000) pointed this out as discussed above, and it was examined again by Engebreston and Moyer (2003). In this study, the authors discussed that by choosing the correct enzymes and number of enzymes the resulting array of fragments would more accurately reflect the community being examined.…”

“…One was the ability of the enzyme to digest unique sequence variants and the other was the number of sequence variants that fell within a specific size range. Of the two constraints, the second was more important since the resolving power of the capillary system is potentially unable to detect multiple fragments that have nearly identical fragment sizes (Engebreston and Moyer, 2003).…”