Advantage of using inosine at the 3' termini of 16S rRNA gene universal primers for the study of microbial diversity

Abstract

To overcome the shortcomings of universal 16S rRNA gene primers 8F and 907R when studying the diversity of complex microbial communities, the 3' termini of both primers were replaced with inosine. A comparison of the clone libraries derived using both primer sets showed seven bacterial phyla amplified by the altered primer set (8F-I/907R-I) whereas the original set amplified sequences belonging almost exclusively to Proteobacteria (95.8%). Sequences belonging to Firmicutes (42.6%) and Thermotogae (9.3%) were more abundant in a library obtained by using 8F-I/907R-I at a PCR annealing temperature of 54 degrees C, while Proteobacteria sequences were more frequent (62.7%) in a library obtained at 50 degrees C, somewhat resembling the result obtained using the original primer set. The increased diversity revealed by using primers 8F-I/907R-I confirms the usefulness of primers with inosine at the 3' termini in studying the microbial diversity of environmental samples.

FIG. 1.

Estimation of microbial diversity in a wastewater environment as determined by using the 8F/907R primer set at an annealing temperature of 54°C (▵, dotted line) and the 8F-I/907R-I primer set at 54°C (○, dashed line) and 50°C (•, solid line).The cumulative number of identical 16S rRNA gene sequences is plotted against the total number of clones with either ≥97% (A) or ≥90% (B) identity. The regression lines were calculated using the modified hyperbolic equation y = x/(ax − b), where y is the cumulative number of different sequences, x is the total number of clones, and a and b are the coefficients proposed by Sekiguchi et al. (24). R² values are higher than 0.967 for all regression lines.

FIG. 2.

Phylogenetic tree based on 16S rRNA gene sequences that were retrieved from industrial wastewater by use of primer set 8F/907R at an annealing temperature of 54°C (blue circles) and primer set 8F-I/907R-I at 54°C (red triangles) and at 50°C (green triangles). The tree was constructed by the neighbor-joining method (21) with the MEGA package (12), using partial sequences of 16S rRNA genes. The bar represents two substitutions per 100 nucleotide positions. Bootstrap probabilities (6) are indicated at branch nodes.