T-RFPred: a nucleotide sequence size prediction tool for microbial community description based on terminal-restriction fragment length polymorphism chromatograms

To show the accuracy of the program, T-RFLP profiles of a marine bacterial community were described using artificial bacterioplankton clone libraries of sequences obtained from public databases. For all valid chromatogram peaks, a phylogenetic group could be assigned.T-RFPred offers enhanced functionality of T-RFLP profile analysis over current available programs. In particular, it circumvents the need for full-length 16S rRNA gene sequences during taxonomic assignments of T-RF peaks. Thus, large 16S rRNA gene datasets from environmental studies, including metagenomes, or public databases can be used as the reference set. Furthermore, T-RFPred is useful in experimental design for the selection of primers as well as the type and number of restriction enzymes that will yield informative chromatograms from natural microbial communities.Terminal-Restriction Fragment Length Polymorphism (T-RFLP) analysis of 16S rRNA gene amplicons is a rapid fingerprinting method for characterization of microbial communities [1,2]. It is based on the restriction endonuclease digestion profile of fluorescently end-labeled PCR products. The digested products are separated by capillary gel electrophoresis, detected and registered on an automated sequence analyzer. Each T-RF is represented by a peak in the output chromatogram and corresponds to members of the community that share a given terminal fragment size. Peak area is proportional to the abundance of the T-RF in the PCR amplicon pool, which can be used as a proxy for relative abundance in natural populations [3]. This method is rapid, relatively inexpensive and provides distinct profiles that reflect the taxonomic composition of sampled communities. Although it has extensively been used for comparative purposes, a T-RFLP fingerprint alone does not allow for conclusive taxonomic identification of individual phylotypes because it is technically challenging to recover terminal fragments for direct sequencing. However, when coupled with se