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

Abstract

Terminal restriction fragment (TRF) analysis of 16S rRNA genes is an increasingly popular method for rapid comparison of microbial communities, but analysis of the data is still in a developmental stage. We assessed the phylogenetic resolution and reproducibility of TRF profiles in order to evaluate the limitations of the method, and we developed an essential analysis technique to improve the interpretation of TRF data. The theoretical phylogenetic resolution of TRF profiles was determined based on the specificity of TRFs predicted from 3,908 16S rRNA gene sequences. With sequences from the Proteobacteria or gram-positive division, as much as 73% of the TRFs were phylogenetically specific (representing strains from at most two genera). However, the fraction decreased when sequences from the two divisions were combined. The data show that phylogenetic inference will be most effective if TRF profiles represent only a single bacterial division or smaller group. The analytical precision of the TRF method was assessed by comparing nine replicate profiles of a single soil DNA sample. Despite meticulous care in producing the replicates, numerous small, irreproducible peaks were observed. As many as 85% of the 169 distinct TRFs found among the profiles were irreproducible (i.e., not present in all nine replicates). Substantial variation also occurred in the height of synonymous peaks. To make comparisons of microbial communities more reliable, we developed an analytical procedure that reduces variation and extracts a reproducible subset of data from replicate TRF profiles. The procedure can also be used with other DNA fingerprinting techniques for microbial communities or microbial genomes.

FIG. 1

Effects of fluorescence standardization on variation in the number of TRFs observed in each of nine replicate RsaI TRF profiles of 16S rDNA amplified from a single soil DNA sample.

FIG. 2

Effects of fluorescence standardization on variation in the height of TRFs observed in nine replicate RsaI TRF profiles of 16S rDNA amplified from a soil DNA sample. Each bar represents the SD of the mean height of a given peak.

FIG. 3

Composite profile showing the average abundance of all the TRFs (169 in total) observed among nine replicate, standardized RsaI TRF profiles of 16S rDNA amplified from a soil DNA sample. The values along the x axis indicate the number of replicates in which each TRF was observed. Error bars are SDs of the mean height of each TRF.

FIG. 4

Dendrograms showing the similarity of three theoretical samples before and after standardization of the sample profiles. The aligned TRF profile for each sample is shown in parentheses and lists the peak heights for seven theoretical TRFs.