Phylogenetic specificity and reproducibility and new method for analysis of terminal restriction fragment profiles of 16S rRNA genes from bacterial communities

Terminal restriction fragment (TRF) analysis of 16S rRNA genes is an increa singly popular method for rapid comparison of microbial communities, but an alysis of the data is still in a developmental stage. We assessed the phylo genetic resolution and reproducibility of TRF profiles in order to evaluate the limitations of the method, and we developed an essential analysis tech nique to improve the interpretation of TRF data. The theoretical phylogenet ic resolution of TRF profiles was determined based on the specificity of TR Fs predicted from 3,908 16S rRNA gene sequences. With sequences from the Pr oteobacteria or gram-positive division, as much as 73% of the TRFs were phy logenetically specific (representing strains from at most two general. Howe ver, the fraction decreased when sequences from the two divisions were comb ined. The data show that phylogenetic inference sill be most effective if T RF profiles represent only a single bacterial division or smaller group. Th e analytical precision of the TRF method was assessed by comparing nine rep licate profiles of a single soil DNA sample. Despite meticulous care in pro ducing the replicates, numerous small, irreproducible peaks were observed. As many as 85% of the 169 distinct TRFs found among the profiles were irrep roducible (i.e., not present in all nine replicates). Substantial variation also occurred in the height of synonymous peaks. To make comparisons of mi crobial communities more reliable, we developed an analytical procedure tha t reduces variation and extracts a reproducible subset of data from replica te TRF profiles. The procedure can also be used with other DNA fingerprinti ng techniques for microbial communities or microbial genomes.