Strengths and Limitations of 16S rRNA Gene Amplicon Sequencing in Revealing Temporal Microbial Community Dynamics

By Rachel S. Poretsky, L. Rodriguez-R., Chengwei Luo, D. Tsementzi, Konstantinos T. Konstantinidis. PLoS ONE, 2014

629 citations recorded by the source corpus.

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 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. Replicates obtained from the same biological sample clustered together but several biases were observed, linked to either the PCR or sequencing-preparation steps. 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. 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. 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. 16S rRNA amplicon data. A strong summer storm had less of an effect on community composition than did seasonal mixing, which revealed a distinct succession of organisms. This study provides insights into freshwater microbial communities and advances the approaches for assessing community diversity and dynamics in situ.

Subfields: Ecology, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/b916f001cccdbb154ade6797cab2ff99b5e8f63c

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Citations in the corpus

Biological, physicochemical and plant health responses in lettuce and strawberry in soil or peat amended with biochar (2016, positive): “This combination is quite new in this research area and provides several bene ﬁ ts. NGS is known to give reliable information for the taxonomy of the sequences, given as OTUs, especially for higher order identi ﬁ cation (Poretsky et al., 2014).”
Effects of rising CO₂ on the harmful cyanobacterium Microcystis (2016, background): “Alternatively, amplicon sequencing can be used to quantify different cyanobacterial strains (Shokralla et al. , 2012; Poretsky et al. , 2014).”
What is the Healthy Gut Microbiota Composition? A Changing Ecosystem across Age, Environment, Diet, and Diseases (2019, background): “The majority of these techniques are based on the extraction of DNA and the amplification of the 16S ribosomal RNA gene (rRNA) [5,6].”
The Road to Metagenomics: From Microbiology to DNA Sequencing Technologies and Bioinformatics (2015, background): “However, amplicon-dependent techniques are prone to sequencing errors, such as result discrepancy from using diﬀerent ribosomal variable regions, primers bias, and OTU assignment errors (Fox et al., 1992; Logares et al., 2012; Poretsky et al., 2014 ).”