Whole genome shotgun (WGS) metagenomic approaches provide robust estimates of microbial community composition and diversity without the need to target and amplify a specific gene. Metagenome approaches are commonly used to describe microbial communities in different systems, e.g., –, without the biases inherent to PCR amplification of a single gene, although it remains a challenge to accurately infer taxonomic origin from metagenomic reads. Here, we coupled detailed analyses of replicate 16S rRNA gene datasets to comparisons with companion community shotgun metagenomics data from the same samples. Recently, 16S rRNA gene amplicon sequencing was compared to metagenomic data from synthetic communities, but to our knowledge, there has been no systematic evaluation of high-throughput 16S rRNA gene sequencing involving multiple sequencing and PCR replicates from natural microbial communities. Furthermore, the use of a single marker gene to assess diversity is challenging, given the prevalence of horizontal gene transfer and the difficulty inherent in defining bacterial species – as well as the limited resolution of the 16S rRNA gene among closely related species. Massively parallel sequencing methods are increasingly being applied to the characterization of microbial communities based on amplification of this gene and have led to a better appreciation of extant biodiversity however, the 16S rRNA -based techniques are known to be limited by the short read lengths obtained, sequencing errors, , differences arising from the different regions chosen, and difficulties in assessing operational taxonomic units (OTUs). To date, most studies of microbial communities in systems ranging from the open ocean to soil to the human gut have depended on a single gene, the 16S small subunit ribosomal RNA (rRNA) gene –. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ĭompeting interests: The authors have declared that no competing interests exist.Ī key step in understanding microbial community structure, dynamics, and how organisms might influence or be influenced by their surroundings is to classify DNA sequences taxonomically or phylogenetically. National Science Foundation (Award 1241046). Department of Energy (Award DE-SC0006662) and by U. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.įunding: This research was supported in part by the U.S. Received: JanuAccepted: FebruPublished: April 8, 2014Ĭopyright: © 2014 Poretsky et al. PLoS ONE 9(4):Įditor: Francisco Rodriguez-Valera, Universidad Miguel Hernandez, Spain This study provides insights into freshwater microbial communities and advances the approaches for assessing community diversity and dynamics in situ.Ĭitation: Poretsky R, Rodriguez-R LM, Luo C, Tsementzi D, Konstantinidis KT (2014) Strengths and Limitations of 16S rRNA Gene Amplicon Sequencing in Revealing Temporal Microbial Community Dynamics. A strong summer storm had less of an effect on community composition than did seasonal mixing, which revealed a distinct succession of organisms. We also identified OTUs that showed marked shifts in abundance over four close timepoints separated by perturbations and tracked these taxa in the metagenome vs. Our analyses showed that the 16S rRNA gene captures broad shifts in community diversity over time, but with limited resolution and lower sensitivity compared to metagenomic data. In comparisons with companion whole-community shotgun metagenome datasets, the estimated number of OTUs at each timepoint was concordant, but 1.5 times and ∼10 times as many phyla and genera, respectively, were identified in the metagenomes. Replicates obtained from the same biological sample clustered together but several biases were observed, linked to either the PCR or sequencing-preparation steps. 16S rRNA gene amplicons generated from four temporally discrete samples were sequenced with 454 GS-FLX-Ti yielding ∼40,000 rRNA gene sequences from each sample and representing ∼300 observed OTUs. This study explored the short-term planktonic microbial community structure and resilience in Lake Lanier (GA, USA) while simultaneously evaluating the technical aspects of identifying taxa via 16S rRNA gene amplicon and metagenomic sequence data.

16s rdna sequence analysis