TRUFFLER: programs to study microbial community composition and flux from fluorescent DNA fingerprinting data

Abstract

Terminal-restriction fragment length polymorphism (T-RFLP) and length heterogeneity-polymerase chain reaction (LH-PCR) are DNA fingerprinting technologies which use PCR amplification of a gene of interest e.g. small subunit rRNA gene, to study microbial community structure and dynamics. Either one or both of the forward and reverse strand primers used to amplify the gene are fluorescently labelled. The products of restriction endonuclease digestion are electrophoresed with an automated sequencer that detects only the terminal (labelled) restriction fragments (T-RFs). In LH-PCR, products are electrophoresed without digestion, with different fragment lengths being due to inherent variation in the amplified sequence. A novel software system, TRUFFLER, has been developed to mimic this process in silico allowing comparison of experimental data against databases of theoretically determined T-RFs. As a given combination of forward and reverse primers and restriction endonuclease can yield identical T-RFs across a number of species, combinations of different endonucleases (and/or primers) are typically used In addition to fragment length data, data on fluorescence levels is also available. Computationally, this can be viewed as a constraint satisfaction problem which can be solved to allow identification of the dominant members of the microbial community, often down to individual species or at least genus level, and their relative proportions.