Cited by 248Genomics and Phylogenetic StudiesMicrobial Community Ecology and PhysiologyEnvironmental DNA in Biodiversity Studies

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Metagenomic Sequencing of an In Vitro-Simulated Microbial Community

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Morgan, Jenna L., Darling, Aaron E., Eisen, Jonathan A.·2010·PLoS ONE

DOI 10.1371/journal.pone.0010209PMC PMC2855710

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Abstract

BACKGROUND: Microbial life dominates the earth, but many species are difficult or even impossible to study under laboratory conditions. Sequencing DNA directly from the environment, a technique commonly referred to as metagenomics, is an important tool for cataloging microbial life. This culture-independent approach involves collecting samples that include microbes in them, extracting DNA from the samples, and sequencing the DNA. A sample may contain many different microorganisms, macroorganisms, and even free-floating environmental DNA. A fundamental challenge in metagenomics has been estimating the abundance of organisms in a sample based on the frequency with which the organism's DNA was observed in reads generated via DNA sequencing. METHODOLOGY/PRINCIPAL FINDINGS: We created mixtures of ten microbial species for which genome sequences are known. Each mixture contained an equal number of cells of each species. We then extracted DNA from the mixtures, sequenced the DNA, and measured the frequency with which genomic regions from each organism was observed in the sequenced DNA. We found that the observed frequency of reads mapping to each organism did not reflect the equal numbers of cells that were known to be included in each mixture. The relative organism abundances varied significantly depending on the DNA extraction and sequencing protocol utilized. CONCLUSIONS/SIGNIFICANCE: We describe a new data resource for measuring the accuracy of metagenomic binning methods, created by in vitro-simulation of a metagenomic community. Our in vitro simulation can be used to complement previous in silico benchmark studies. In constructing a synthetic community and sequencing its metagenome, we encountered several sources of observation bias that likely affect most metagenomic experiments to date and present challenges for comparative metagenomic studies. DNA preparation methods have a particularly profound effect in our study, implying that samples prepared with different protocols are not suitable for comparative metagenomics.

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Authors: Morgan, J. L., Darling, A. E., & Eisen, J. A.
Affiliations: Joint Genome Institute (US)
University of California, Davis (US)
Corresponding author: Eisen, Jonathan A.
ORCID: Darling, Aaron E.
Eisen, Jonathan A.
Venue: PLoS ONE
Publisher: Public Library of Science (PLoS)
Published: 2010-04-16
Volume: 5
Issue: 4
Pages: e10209
ISSN: 1932-6203 (ISSN-L)
DOI: 10.1371/journal.pone.0010209
PMC: PMC2855710
OpenAlex: W2073909558
Cited by: 248
References: 54
Citations by year: 2026: 4 · 2025: 10 · 2024: 4 · 2023: 6 · 2022: 11 · 2021: 18 · 2020: 8 · 2019: 7 · 2018: 9 · 2017: 19 · 2016: 10 · 2015: 23 · 2014: 30 · 2013: 37 · 2012: 24
FWCI: 11.59
Type: journal article
Language: en
Open access: gold
doi.org
Licence: public-domain
creativecommons.org/publicdomain/zero/1.0/ (unspecified)
Locations: PLoS ONE (publishedVersion, public-domain)
PubMed Central (publishedVersion, public-domain)
PubMed (publishedVersion)
University of North Texas Digital Library (University of North Texas) (submittedVersion)
DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
eScholarship (California Digital Library) (submittedVersion)
UTS ePRESS (University of Technology Sydney) (submittedVersion)
OPUS - Open Publications of UTS Scholars (University of Technology Sydney) (submittedVersion)
OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion)
Lawrence Berkeley National Laboratory
Funders: National Science Foundation (DEAC02-05CH11231, 0630765)
U.S. Department of Energy (-AC02-05CH11231, Contract No. DE-AC02-05-CH11231, 05CH11231, No. DE-AC02-05-CH11231, DE AC02 05CH11231, DE-AC02-)
Joint Genome Institute (AC02-05CH11231, DE–AC02–05CH11231)
Office of Science (No. DE-AC02- 05-CH11231, DEAC02–05CH11231, AC02-05CH11231, DE-AC02, 05CH11231, Contract No. DE-AC02-05CH11231)
Lawrence Berkeley National Laboratory (AC02‐05CH11231, DE-AC02-05CH11231, No. DE-AC02-05CH11231, Contract No. DE-AC02-05CH11231, 05CH11231)
Topics: Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
Physical Sciences › Environmental Science › Ecology › Microbial Community Ecology and Physiology
Physical Sciences › Environmental Science › Ecology › Environmental DNA in Biodiversity Studies
Keywords: metagenomics, synthetic microbial communities, in vitro simulation, metagenome binning, relative abundance estimation, observation bias, DNA extraction methods, sequencing protocols
MeSH terms: Biometry, Computer Simulation, DNA, Bacterial, Genome, Bacterial, Sequence Analysis, DNA, Metagenomics

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