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Changes in the Composition of Drinking Water Bacterial Clone Libraries Introduced by Using Two Different 16S rRNA Gene PCR Primers
HUMRIGHOUSE, B., R. P. REVETTA, B. IKER, J. W. SANTO-DOMINGO, AND D. B. OERTHER. Changes in the Composition of Drinking Water Bacterial Clone Libraries Introduced by Using Two Different 16S rRNA Gene PCR Primers . Presented at American Society for Microbiology, Philadelphia, PA, May 17 - 21, 2009.
To inform public
Sequence analysis of 16S rRNA gene clone libraries is a popular tool used to describe the composition of natural microbial communities. Commonly, clone libraries are developed by direct cloning of 16S rRNA gene PCR products. Different primers are often employed in the initial amplification (PCR) step but little is known about potential biases introduced by any given primer set. To understand some of these biases, we analyzed 328 clones from different drinking water clone libraries generated with two commonly used 16S rRNA gene eubacterial primer sets: 8F-685R and 8F-787R. Using 97% similarity as the operational taxonomic unit, the clones were grouped into 49 different contigs. Twelve of the contigs (which represented 25% of all contigs) contained clones from both clone libraries. Three of these contigs were primarily associated with 8F-685R derived clones (i.e., 81%), most of which were of cyanobacterial origin. Sequences closely related to Firmicutes and Mycobacterium spp. were also associated with these mixed contigs. Approximately 70% of the remaining contigs (i.e., 26 contigs) consisted of 8F-787R derived clones, many of which were associated with alpha-Proteobacteria. Pairwise comparison of clone libraries indicated that each primer set preferentially amplified different populations. Moreover, diversity analysis indicated that the 8F-787R primer set generated a more diverse pool of clones. The results from this study suggest that the comparison of microbial community structure of similar ecosystems may need to be restricted to studies using similar primer sets. However, the results also suggest that different primers should be used to better understand drinking water microbial community structure.