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Cited by 685Microbial Community Ecology and PhysiologyEnvironmental DNA in Biodiversity StudiesGenomics and Phylogenetic Studies

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

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Poretsky, Rachel, Rodriguez-R, Luis M., Luo, Chengwei, Tsementzi, Despina, Konstantinidis, Konstantinos T.·2014·PLoS ONE
DOI 10.1371/journal.pone.0093827PMC PMC3979728
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Abstract

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.

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Figure 1 Diversity estimates for the four Lake Lanier timepoints . A) Alpha diversity based on observed species (97% OTUs) from 16S amplicons for each of the nine samples. Error bars represent the variation observed among duplicate sequencing runs. B) Redundancy curves of the metagenomes of the four timepoints using (see Methods for details). The curves show that NOV is a more diverse sample, e.g., with the same sequencing effort it results in a lower coverage.
Figure 1 Diversity estimates for the four Lake Lanier timepoints . A) Alpha diversity based on observed species (97% OTUs) from 16S amplicons for each of the nine samples. Error bars represent the variation observed among duplicate sequencing runs. B) Redundancy curves of the metagenomes of the four timepoints using (see Methods for details). The curves show that NOV is a more diverse sample, e.g., with the same sequencing effort it results in a lower coverage.CC BY · PubMed Central
Figure 2 Community composition shifts over time as revealed by 16S data. Taxonomic binning of 16S amplicon sequences for each of the 14 individual datasets at the phylum (top) and genus (middle) levels were based on the July 2011 version of the Greengenes database [31]. Freshwater lineages (bottom) were based on a freshwater database according to the taxonomy framework described in Newton et al., 2011. Datasets are ordered left to right by date, technical sequencing replicate (lane 1 and lane 2), and DNA replicate (A, B and C). Taxa identified as major drivers of the differences between timepoints (SIMPER analysis) are labeled (see figure key).
Figure 2 Community composition shifts over time as revealed by 16S data. Taxonomic binning of 16S amplicon sequences for each of the 14 individual datasets at the phylum (top) and genus (middle) levels were based on the July 2011 version of the Greengenes database [31]. Freshwater lineages (bottom) were based on a freshwater database according to the taxonomy framework described in Newton et al., 2011. Datasets are ordered left to right by date, technical sequencing replicate (lane 1 and lane 2), and DNA replicate (A, B and C). Taxa identified as major drivers of the differences between timepoints (SIMPER analysis) are labeled (see figure key).CC BY · PubMed Central
Figure 3 Sequence diversity of the samples used in this study . Chao1 diversity estimates of datasets based on phylum (A) and genus (B) level taxonomic classification are shown for all four metagenomic timepoints and seven selected 16S amplicon datasets.
Figure 3 Sequence diversity of the samples used in this study . Chao1 diversity estimates of datasets based on phylum (A) and genus (B) level taxonomic classification are shown for all four metagenomic timepoints and seven selected 16S amplicon datasets.CC BY · PubMed Central
Figure 4 Individual genera abundance shifts over time based on 16S and metagenomes. Genus-level taxonomic trends for a subset of genera identified within the metagenomic contigs (A) and 16S rRNA amplicon (B) datasets, based on NCBI taxonomy, are shown. The lines represent the general temporal trends of two genera, Synechococcus and Legionella, in each dataset.
Figure 4 Individual genera abundance shifts over time based on 16S and metagenomes. Genus-level taxonomic trends for a subset of genera identified within the metagenomic contigs (A) and 16S rRNA amplicon (B) datasets, based on NCBI taxonomy, are shown. The lines represent the general temporal trends of two genera, Synechococcus and Legionella, in each dataset.CC BY · PubMed Central

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Metadata

Authors
Poretsky, R., Rodriguez-R, L. M., Luo, C., Tsementzi, D., & Konstantinidis, K. T.
Affiliations
Georgia Institute of Technology (US)
Corresponding author
Poretsky, Rachel
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2014-04-08
Volume
9
Issue
4
Pages
e93827
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0093827
PMC
PMC3979728
OpenAlex
W2019163239
Cited by
684
References
63
Citations by year
2026: 50 · 2025: 70 · 2024: 45 · 2023: 78 · 2022: 71 · 2021: 83 · 2020: 69 · 2019: 67 · 2018: 62 · 2017: 37 · 2016: 29 · 2015: 20 · 2014: 2
FWCI
25.43
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • National Science Foundation (1241046)
  • U.S. Department of Energy (DESC0006662)
  • Emory University
Topics
  • Physical Sciences › Environmental Science › Ecology › Microbial Community Ecology and Physiology
  • Physical Sciences › Environmental Science › Ecology › Environmental DNA in Biodiversity Studies
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
Keywords
16S rRNA gene amplicon sequencing, metagenomics, planktonic microbial communities, freshwater microbial communities, Lake Lanier, community resilience, sequencing bias, PCR bias, seasonal mixing, storm disturbance, microbial succession, community diversity
MeSH terms
Bacteria, Fresh Water, Plankton, RNA, Ribosomal, 16S, Metagenome, Metagenomics

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