Evaluation of Methods for the Extraction and Purification of DNA from the Human Microbiome
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BACKGROUND: DNA extraction is an essential step in all cultivation-independent approaches to characterize microbial diversity, including that associated with the human body. A fundamental challenge in using these approaches has been to isolate DNA that is representative of the microbial community sampled. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we statistically evaluated six commonly used DNA extraction procedures using eleven human-associated bacterial species and a mock community that contained equal numbers of those eleven species. These methods were compared on the basis of DNA yield, DNA shearing, reproducibility, and most importantly representation of microbial diversity. The analysis of 16S rRNA gene sequences from a mock community showed that the observed species abundances were significantly different from the expected species abundances for all six DNA extraction methods used. CONCLUSIONS/SIGNIFICANCE: Protocols that included bead beating and/or mutanolysin produced significantly better bacterial community structure representation than methods without both of them. The reproducibility of all six methods was similar, and results from different experimenters and different times were in good agreement. Based on the evaluations done it appears that DNA extraction procedures for bacterial community analysis of human associated samples should include bead beating and/or mutanolysin to effectively lyse cells.
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Previous detailed investigations of variables such as DNA extraction method, like the present results, enriched for broad classes of Gram-negative rather than Gram-positive bacteria because of technical rather than biological variables.
“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 [27]
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- Authors
- Yuan, S., Cohen, D. B., Ravel, J., Abdo, Z., & Forney, L. J.
- Affiliations
- University of Idaho (US)
- University of Maryland, Baltimore (US)
- Corresponding author
- Forney, Larry J.
- Venue
- PLoS ONE
- Publisher
- Public Library of Science (PLoS)
- Published
- 2012-03-23
- Volume
- 7
- Issue
- 3
- Pages
- e33865
- ISSN
- 1932-6203 (ISSN-L)
- PMC
- PMC3311548
- OpenAlex
- W2090110425
- Cited by
- 511
- References
- 73
- Citations by year
- 2026: 16 · 2025: 19 · 2024: 24 · 2023: 29 · 2022: 24 · 2021: 57 · 2020: 50 · 2019: 50 · 2018: 63 · 2017: 60 · 2016: 37 · 2015: 28 · 2014: 32 · 2013: 20 · 2012: 2
- FWCI
- 23.16
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- PLoS ONE (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Figshare (submittedVersion, cc-by)
- Funders
- National Institutes of Health (5u01ai070921-07)
- National Institute of Allergy and Infectious Diseases (U01-AI070921)
- Topics
- Physical Sciences › Environmental Science › Ecology › Microbial Community Ecology and Physiology
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gut microbiota and health
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
- Keywords
- DNA extraction, human microbiome, mock communities, bead beating, mutanolysin, DNA shearing, 16S rRNA gene, microbial diversity, extraction method comparison, cell lysis
- MeSH terms
- Bacteria, DNA, Bacterial, Humans, Biodiversity
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
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