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Browsing Marine Biological Laboratory by Subject "16S"
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ArticleImproved bacterial 16S rRNA gene (V4 and V4-5) and fungal internal transcribed spacer marker gene primers for microbial community surveys(American Society for Microbiology, 2015-12-22) Walters, William ; Hyde, Embriette R. ; Berg-Lyons, Donna ; Ackermann, Gail ; Humphrey, Greg ; Parada, Alma ; Gilbert, Jack A. ; Jansson, Janet K. ; Caporaso, J. Gregory ; Fuhrman, Jed A. ; Apprill, Amy ; Knight, RobDesigning primers for PCR-based taxonomic surveys that amplify a broad range of phylotypes in varied community samples is a difficult challenge, and the comparability of data sets amplified with varied primers requires attention. Here, we examined the performance of modified 16S rRNA gene and internal transcribed spacer (ITS) primers for archaea/bacteria and fungi, respectively, with nonaquatic samples. We moved primer bar codes to the 5′ end, allowing for a range of different 3′ primer pairings, such as the 515f/926r primer pair, which amplifies variable regions 4 and 5 of the 16S rRNA gene. We additionally demonstrated that modifications to the 515f/806r (variable region 4) 16S primer pair, which improves detection of Thaumarchaeota and clade SAR11 in marine samples, do not degrade performance on taxa already amplified effectively by the original primer set. Alterations to the fungal ITS primers did result in differential but overall improved performance compared to the original primers. In both cases, the improved primers should be widely adopted for amplicon studies.
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ArticleOligotyping : differentiating between closely related microbial taxa using 16S rRNA gene data(John Wiley & Sons, 2013-10-23) Eren, A. Murat ; Maignien, Lois ; Sul, Woo Jun ; Murphy, Leslie G. ; Grim, Sharon L. ; Morrison, Hilary G. ; Sogin, Mitchell L.Bacteria comprise the most diverse domain of life on Earth, where they occupy nearly every possible ecological niche and play key roles in biological and chemical processes. Studying the composition and ecology of bacterial ecosystems and understanding their function are of prime importance. High-throughput sequencing technologies enable nearly comprehensive descriptions of bacterial diversity through 16S ribosomal RNA gene amplicons. Analyses of these communities generally rely upon taxonomic assignments through reference data bases or clustering approaches using de facto sequence similarity thresholds to identify operational taxonomic units. However, these methods often fail to resolve ecologically meaningful differences between closely related organisms in complex microbial data sets. In this paper, we describe oligotyping, a novel supervised computational method that allows researchers to investigate the diversity of closely related but distinct bacterial organisms in final operational taxonomic units identified in environmental data sets through 16S ribosomal RNA gene data by the canonical approaches. Our analysis of two data sets from two different environments demonstrates the capacity of oligotyping at discriminating distinct microbial populations of ecological importance. Oligotyping can resolve the distribution of closely related organisms across environments and unveil previously overlooked ecological patterns for microbial communities. The URL http://oligotyping.org offers an open-source software pipeline for oligotyping.