text: The result of a T-RFLP profiling is a graph called electropherogram which is an intensity plot representation of an electrophoresis experiment (gel or capillary). In an electropherogram the X-axis marks the sizes of the fragments while the Y-axis marks the fluorescence intensity of each fragment. Thus, what appears on an electrophoresis gel as a band appears as a peak on the electropherogram whose integral is its total fluorescence. In a T–RFLP profile each peak assumingly corresponds to one genetic variant in the original sample while its height or area corresponds to its relative abundance in the specific community. Both assumptions listed above, however, are not always met. Often, several different bacteria in a population might give a single peak on the electropherogram due to the presence of a restriction site for the particular restriction enzyme used in the experiment at the same position. To overcome this problem and to increase the resolving power of this technique a single sample can be digested in parallel by several enzymes (often three) resulting in three T-RFLP profiles per sample each resolving some variants while missing others. Another modification which is sometimes used is to fluorescently label the reverse primer as well using a different dye, again resulting in two parallel profiles per sample each resolving a different number of variants.
  In addition to convergence of two distinct genetic variants into a single peak artifacts might also appear, mainly in the form of false peaks. False peaks are generally of two types: background “noises” and “pseudo” TRFs. Background (noise) peaks are peaks resulting from the sensitivity of the detector in use. These peaks are often small in their intensity and usually form a problem in case the total intensity of the profile is low (i.e. low concentration of DNA). Because these peaks result from background noise they are normally irreproducible in replicate profiles, thus the problem can be tackled by producing a consensus profile from several replicates or by eliminating peaks below a certain threshold. Several other computational techniques were also introduced in order to deal with this problem. Pseudo TRFs, on the other hand, are reproducible peaks and are linear to the amount of DNA loaded. These peaks are thought to be the result of ssDNA annealing on to itself and creating double stranded random restriction sites which are later recognized by the restriction enzyme resulting in a terminal fragment which does not represent any genuine genetic variant. It has been suggested that applying a DNA exonuclease such as the Mung bean exonuclease prior to the digestion stage might eliminate such artifact.