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Metagenomic Sequencing of an In Vitro-Simulated Microbial Community
Open accessAbstract
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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“Previous detailed investigations of specific variables in microbiome experiments such as DNA extraction method have, in common with the results we report, enriched for broad classes of Gram-negative rather than Gram-positive bacteria due to technical rather than biological variables9, 24, 27, 28.”
From Assessment of variation in microbial community amplicon sequencing by the Microbiome Quality Control (MBQC) project consortium (2017) · cites this paper as [28]
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Cites
Basic local alignment search tool
Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990) · Journal of Molecular Biology · doi:10.1006/jmbi.1990.9999 · Cited by 98,669
DNA sequencing with chain-terminating inhibitors
Sanger, F., Nicklen, S., & Coulson, A. R. (1977) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.74.12.5463 · Cited by 68,572
Base-Calling of Automated Sequencer Traces Using Phred. I. Accuracy AssessmentPreprint
Ewing, B., Hillier, L., Wendl, M. C., & Green, P. (1998) · Genome Research · doi:10.1101/gr.8.3.175 · Cited by 6,993
Base-Calling of Automated Sequencer Traces Using Phred. II. Error Probabilities
Ewing, B., & Green, P. (1998) · Genome Research · doi:10.1101/gr.8.3.186 · Cited by 5,439
Mauve: Multiple alignment of conserved genomic sequence with rearrangements
Darling, A. C. E., Mau, B., Blattner, F. R., & Perna, N. T. (2004) · Genome Research · doi:10.1101/gr.2289704 · Cited by 4,949
Self-citationEnvironmental genome shotgun sequencing of the sargasso sea
Venter, J. C., Remington, K., Heidelberg, J. F., Halpern, A. L., Rusch, D., Eisen, J. A., Wu, D., Paulsen, I., Nelson, K. E., Nelson, W., et al. (2004) · Science · doi:10.1126/science.1093857 · Cited by 4,376
Self-citationMEGAN analysis of metagenomic dataPreprint
Huson, D. H., Auch, A. F., Qi, J., & Schuster, S. C. (2007) · Genome Research · doi:10.1101/gr.5969107 · Cited by 3,294
Metagenomics: Application of Genomics to Uncultured Microorganisms
Handelsman, J. (2004) · Microbiology and Molecular Biology Reviews · doi:10.1128/mmbr.68.4.669-685.2004 · Cited by 2,528
Impact of Culture-Independent Studies on the Emerging Phylogenetic View of Bacterial Diversity
Hugenholtz, P., Goebel, B. M., & Pace, N. R. (1998) · Journal of Bacteriology · doi:10.1128/jb.180.18.4765-4774.1998 · Cited by 2,429
Community structure and metabolism through reconstruction of microbial genomes from the environment
Tyson, G. W., Chapman, J., Hugenholtz, P., Allen, E. E., Ram, R. J., Richardson, P. M., Solovyev, V. V., Rubin, E. M., Rokhsar, D. S., & Banfield, J. F. (2004) · Nature · doi:10.1038/nature02340 · Cited by 2,417
Comparative genomics of the lactic acid bacteria.
Makarova, K., Slesarev, A., Wolf, Y., Sorokin, A., Mirkin, B., Koonin, E., Pavlov, A., Pavlova, N., Karamychev, V., Polouchine, N., et al. (2006) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.0607117103 · Cited by 1,391
Accuracy and quality of massively parallel DNA pyrosequencing
Huse, S. M., Huber, J. A., Morrison, H. G., Sogin, M. L., & Welch, D. B. M. (2007) · Genome biology · doi:10.1186/gb-2007-8-7-r143 · Cited by 1,280
Chromosome replication and the division cycle of Escherichia coli
Cooper, S., & Helmstetter, C. E. (1968) · Journal of Molecular Biology · doi:10.1016/0022-2836(68)90425-7 · Cited by 1,195
Metagenomics: Genomic Analysis of Microbial Communities
Riesenfeld, C. S., Schloss, P. D., & Handelsman, J. (2004) · Annual Review of Genetics · doi:10.1146/annurev.genet.38.072902.091216 · Cited by 1,124
Pagani, I., Liolios, K., Jansson, J., Chen, I.-M. A., Smirnova, T., Nosrat, B., Markowitz, V. M., & Kyrpides, N. C. (2011) · Nucleic Acids Research · doi:10.1093/nar/gkr1100 · Cited by 1,066
Daniel, R. (2005) · Nature Reviews Microbiology · doi:10.1038/nrmicro1160 · Cited by 862
Pyrosequencing Sheds Light on DNA SequencingPreprint
Ronaghi, M. (2001) · Genome Research · doi:10.1101/gr.11.1.3 · Cited by 845
Genome sequence of Halobacterium species NRC-1
Ng, W. V., Kennedy, S., Mahairas, G. G., Berquist, B. R., Pan, M., Shukla, H. D., Lasky, S. R., Baliga, N. S., Thórsson, V., Sbrogna, J. L., et al. (2000) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.190337797 · Cited by 706
Liolios, K., Mavromatis, K., Tavernarakis, N., & Kyrpides, N. C. (2007) · Nucleic Acids Research · doi:10.1093/nar/gkm884 · Cited by 661
Accurate phylogenetic classification of variable-length DNA fragments
McHardy, A. C., Martín, H. G., Tsirigos, A., Hugenholtz, P., & Rigoutsos, I. (2006) · Nature Methods · doi:10.1038/nmeth976 · Cited by 564
Quantification of Bias Related to the Extraction of DNA Directly from Soils
Frostegård, Å., Courtois, S., Ramisse, V., Clerc, S., Bernillon, D., Gall, F. L., Jeannin, P., Nesme, X., & Simonet, P. (1999) · Applied and Environmental Microbiology · doi:10.1128/aem.65.12.5409-5420.1999 · Cited by 545
A Bioinformatician's Guide to Metagenomics
Kunin, V., Copeland, A., Lapidus, A. L., Mavromatis, K., & Hugenholtz, P. (2008) · Microbiology and Molecular Biology Reviews · doi:10.1128/mmbr.00009-08 · Cited by 499
Genome-Wide Experimental Determination of Barriers to Horizontal Gene Transfer
Sorek, R., Zhu, Y., Creevey, C. J., Francino, M. P., Bork, P., & Rubin, E. M. (2007) · Science · doi:10.1126/science.1147112 · Cited by 421
IMG/M: a data management and analysis system for metagenomes
Markowitz, V. M., Ivanova, N., Szeto, E., Palaniappan, K., Chu, K., Dalevi, D., Chen, I.-M. A., Grechkin, Y., Dubchak, I. L., Anderson, I., et al. (2007) · Nucleic Acids Research · doi:10.1093/nar/gkm869 · Cited by 392
The design, implementation, and evaluation of mpiBLAST
Darling, A. E., Carey, L. B., & Feng, W.-C. (2003) · OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) · Cited by 378
Self-citationUse of simulated data sets to evaluate the fidelity of metagenomic processing methods
Mavromatis, K., Ivanova, N., Barry, K., Shapiro, H., Goltsman, E., McHardy, A. C., Rigoutsos, I., Salamov, A. A., Korzeniewski, F., Land, M., et al. (2007) · Nature Methods · doi:10.1038/nmeth1043 · Cited by 345
A comparison of five methods for extraction of bacterial DNA from human faecal samples
McOrist, A. L., Jackson, M., & Bird, A. R. (2002) · Journal of Microbiological Methods · doi:10.1016/s0167-7012(02)00018-0 · Cited by 298
Comparison of different methods for the isolation and purification of total community DNA from soil
Krsek, M., & Wellington, E. M. H. (1999) · Journal of Microbiological Methods · doi:10.1016/s0167-7012(99)00093-7 · Cited by 283
Impact of Culture-Independent Studies on the Emerging Phylogenetic View of Bacterial Diversity
Hugenholtz, P., Goebel, B. M., & Pace, N. R. (1998) · Journal of Bacteriology · doi:10.1128/jb.180.24.6793-6793.1998 · Cited by 276
Comparison of six extraction techniques for isolation of DNA from filamentous fungi
van Burik, J. H., Schreckhise, R. W., White, T. C., Bowden, R. A., & Myerson, D. (1998) · Medical Mycology · doi:10.1080/02681219880000471 · Cited by 228
Metagenomics: Application of Genomics to Uncultured Microorganisms
Handelsman, J. (2005) · Microbiology and Molecular Biology Reviews · doi:10.1128/mmbr.69.1.195.2005 · Cited by 213
Yeast Protein Serine/Threonine Phosphatases: Multiple Roles and Diverse Regulation
Stark, M. J. R. (1996) · Yeast · doi:10.1002/(sici)1097-0061(199612)12:16 · Cited by 201
DNA extraction method affects microbial community profiles from soils and sediment
Carrigg, C., Rice, O., Kavanagh, S., Collins, G., & O’Flaherty, V. (2007) · Applied Microbiology and Biotechnology · doi:10.1007/s00253-007-1219-y · Cited by 196
Regulated Polyploidy in Halophilic Archaea
Breuert, S., Allers, T., Spohn, G., & Soppa, J. (2006) · PLoS ONE · doi:10.1371/journal.pone.0000092 · Cited by 192
Pyrosequencing Sheds Light on DNA SequencingPreprint
Ronaghi, M. (2001) · Genome Research · doi:10.1101/gr.150601 · Cited by 185
Metagenomics: Concept, methodology, ecological inference and recent advances
Singh, J., Behal, A., Singla, N., Joshi, A., Birbian, N., Singh, S., Bali, V., & Batra, N. (2009) · Biotechnology Journal · doi:10.1002/biot.200800201 · Cited by 161
Bertrand, H., Poly, F., Van, V. T., Lombard, N., Nalin, R., Vogel, T. M., & Simonet, P. (2005) · Journal of Microbiological Methods · doi:10.1016/j.mimet.2005.01.003 · Cited by 146
Barabote, R. D., Xie, G. G., Leu, D. H., Normand, P., Necşulea, A., Daubin, V., Médigue, C., Adney, W. S., Xu, X. C., Lapidus, A. L., et al. (2009) · Genome Research · doi:10.1101/gr.084848.108 · Cited by 139
Venkateswaran, K. J., Dollhopf, M. E., Aller, R. C., Stackebrandt, E. S., & Nealson, K. (1998) · International Journal of Systematic Bacteriology · doi:10.1099/00207713-48-3-965 · Cited by 130
Multiple pathways of selected gene amplification during adaptive mutation
Kugelberg, E., Kofoid, E., Reams, A. B., Andersson, D. I., & Roth, J. R. (2006) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.0608309103 · Cited by 129
Comparison of six extraction techniques for isolation of DNA from filamentous fungi
BURIK, V., SCHRECKHISE, WHITE, BOWDEN, & MYERSON (1998) · Medical Mycology · doi:10.1046/j.1365-280x.1998.00161.x · Cited by 120
What's in the mix: phylogenetic classification of metagenome sequence samples
McHardy, A. C., & Rigoutsos, I. (2007) · Current Opinion in Microbiology · doi:10.1016/j.mib.2007.08.004 · Cited by 112
Binning sequences using very sparse labels within a metagenome
Chan, C. K., Hsu, A. L., Halgamuge, S. K., & Tang, S. (2008) · BMC Bioinformatics · doi:10.1186/1471-2105-9-215 · Cited by 89
Palenik, B. P., Ren, Q., Tai, V., & Paulsen, I. T. (2008) · Environmental Microbiology · doi:10.1111/j.1462-2920.2008.01772.x · Cited by 88
Inference of population genetic parameters in metagenomics: A clean look at messy dataPreprint
Johnson, P. L. F., & Slatkin, M. (2006) · Genome Research · doi:10.1101/gr.5431206 · Cited by 75
Short-Insert Libraries as a Method of Problem Solving in Genome SequencingPreprint
McMurray, A. A., Sulston, J., & Quail, M. A. (1998) · Genome Research · doi:10.1101/gr.8.5.562 · Cited by 63
σ54Enhancer Binding Proteins andMyxococcus xanthusFruiting Body Development
Jakobsen, J. S., Jelsbak, L., Jelsbak, L., Welch, R. D., Cummings, C. A., Goldman, B. S., Stark, E., Slater, S., & Kaiser, D. (2004) · Journal of Bacteriology · doi:10.1128/jb.186.13.4361-4368.2004 · Cited by 62
Chan, C. K., Hsu, A. L., Tang, S., & Halgamuge, S. K. (2007) · BioMed Research International · doi:10.1155/2008/513701 · Cited by 61
Blow, N. S. (2008) · Nature · doi:10.1038/453687a · Cited by 44
Co‐ordinate regulation of the Escherichia coli cell cycle or The cloud of unknowing
Donachie, W. D. (2001) · Molecular Microbiology · doi:10.1046/j.1365-2958.2001.02439.x · Cited by 43
BioTorrents: A File Sharing Service for Scientific Data
Langille, M. G. I., & Eisen, J. A. (2010) · PLoS ONE · doi:10.1371/journal.pone.0010071 · Cited by 39
Self-citationTemperton, B., Field, D., Oliver, A., Tiwari, B., Mühling, M., Joint, I. R., & Gilbert, J. A. (2009) · The ISME Journal · doi:10.1038/ismej.2009.32 · Cited by 39
Binning Sequences Using Very Sparse Labels Within a Metagenome
Tseng, C., Chan, C. K., Hsu, A. L., Halgamuge, S. K., & Tang, S. (2015) · Springer eBooks · doi:10.1007/978-1-4899-7478-5_750 · Cited by 6
Xie, G. G. (2024) · OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) · Cited by 2
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Darling, A. E., Jospin, G., Lowe, E., Matsen, F. A., IV, Bik, H. M., & Eisen, J. A. (2014) · PeerJ · doi:10.7717/peerj.243 · Cited by 625
Self-citationThe Microbiome Quality Control Project Consortium, Sinha, R., Abu-Ali, G., Vogtmann, E., Fodor, A. A., Ren, B., Amir, A., Schwager, E., Crabtree, J., Ma, S., et al. (2017) · Nature Biotechnology · doi:10.1038/nbt.3981 · Cited by 587
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Metadata
- 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.
- Venue
- PLoS ONE
- Publisher
- Public Library of Science (PLoS)
- Published
- 2010-04-16
- Volume
- 5
- Issue
- 4
- Pages
- e10209
- ISSN
- 1932-6203 (ISSN-L)
- 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
- 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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