A ‘dirty’ business: testing the limitations of terminal restriction fragment length polymorphism (TRFLP) analysis of soil fungi

Abstract

Terminal restriction fragment length polymorphism (TRFLP) is an increasingly popular method in molecular ecology. However, several key limitations of this method have not been fully examined especially when used to study fungi. We investigated the impact of spore contamination, intracollection ribosomal DNA internal transcribed spacer (ITS) region variation, and conserved restriction enzyme recognition loci on the results produced by TRFLP to characterize soil fungal communities. We find that (i) the potential for nontarget structures such as spores to contribute DNA to target sample extractions is high; (ii) multiple fragments (i.e. 'extra peaks') per PCR primer-restriction enzyme combination can be detected that are caused by restriction enzyme inefficiency and intracollection ribosomal DNA ITS variation; and (iii) restriction enzyme digestion in conserved vs. variable gene regions leads to different characterizations of community diversity. Based on these results, we suggest that studies employing TRFLP need to include information from known, identified fungi from sites within which studies take place and not to rely only on TRFLP profiles as a short cut to fungal community description.

Determining a minimum detection threshold in terminal restriction fragment length polymorphism analysis

“…Management of T-RFLP data also influences results because community diversity can be exaggerated when false peaks are not discarded or underestimated by strict minimum peak height thresholds (Avis et al 2006, Dickie et al 2002. By retaining all peak heights in this experiment, we found small but reproducible diagnostic peaks of rare taxa can fall within this range of false peaks and may be cut off by thresholds.…”

“…Field and greenhouse experiments, however, will not generate such clean, reproducible and distinguishable peak profiles (Avis et al 2006). When working with soil derived DNA, small peaks can be eliminated by the creation of a minimum detection threshold, which is often applied to clean up the data (Abdo et al 2006, Dunbar et al 2001, Osborne et al 2006.…”

“…The distinct cleavage patterns of different taxa can then be compiled into a reference database of diagnostic fragment sizes. Database T-RFLP is used to identify individual taxa within samples by comparing known species-specific electropherogram peaks against unknown samples for identification (Avis et al 2006, Dickie & FitzJohn 2007. Although T-RFLP is widely used to assess microbial community composition, taxa present in low concentrations may not be detected by T-RFLP analysis (Dickie et al 2002).…”

Technicalities and Glitches of Terminal Restriction Fragment Length Polymorphism (T-RFLP)

“…T-RFs obtained during different studies can be organized in the form of a database for cross referencing by other workers [34]. Availability of huge 16S rRNA gene sequence database is also another favorable factor for this technique [5,24]. Furthermore, phylogenetic inference obtained using T-RFLP has a greater resolution due to the use of superior capillary electrophoresis of automated DNA sequencer than other fingerprinting methods such as DGGE and SSCP.…”

“…It is an inexpensive, robust, reproducible, and rapid method for the study of microbial community structure. The potential of T-RFLP method for the study of community dynamics and structure is extensively studied for diverse habitats [5][6][7][8][9][10][11][12][13][14]. In addition to 16S rRNA gene, T-RFLP analysis also included genes representing a variety of functional groups [15][16][17].…”