Choice of bacterial DNA extraction method from fecal material influences community structure as evaluated by metagenomic analysis
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BACKGROUND: In recent years, studies on the human intestinal microbiota have attracted tremendous attention. Application of next generation sequencing for mapping of bacterial phylogeny and function has opened new doors to this field of research. However, little attention has been given to the effects of choice of methodology on the output resulting from such studies. RESULTS: IN THIS STUDY WE CONDUCTED A SYSTEMATIC COMPARISON OF THE DNA EXTRACTION METHODS USED BY THE TWO MAJOR COLLABORATIVE EFFORTS: The European MetaHIT and the American Human Microbiome Project (HMP). Additionally, effects of homogenizing the samples before extraction were addressed. We observed significant differences in distribution of bacterial taxa depending on the method. While eukaryotic DNA was most efficiently extracted by the MetaHIT protocol, DNA from bacteria within the Bacteroidetes phylum was most efficiently extracted by the HMP protocol. CONCLUSIONS: Whereas it is comforting that the inter-individual variation clearly exceeded the variation resulting from choice of extraction method, our data highlight the challenge of comparing data across studies applying different methodologies.
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Comparisons of results from the Human Microbiome Project (HMP) with the MetaHIT project showed stark differences in microbiome profiles, and indicated that the HMP protocol was less effective at extracting DNA from eukaryotes and Gram-positive bacterial lineages.
“comparisons of results from the Human Microbiome Project (HMP) with the MetaHIT project showed stark differences in microbiome profiles, and indicated that the HMP protocol was less effective at extracting DNA from eukaryotes and Gram-positive bacterial lineages 35 .”
From Human microbiome myths and misconceptions (2023) · cites this paper as [35]
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Experimental — accuracy not yet measuredPrevious 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 [24]
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- Authors
- Wesolowska-Andersen, A., Bahl, M. I., Carvalho, V., Kristiansen, K., Sicheritz-Pontén, T., Gupta, R., & Licht, T. R.
- Affiliations
- Technical University of Denmark (DK)
- University of Copenhagen (DK)
- Corresponding author
- Gupta, Ramneek
- Licht, Tine Rask
- Venue
- Microbiome
- Published
- 2014-06-05
- Volume
- 2
- Issue
- 1
- Pages
- 19-19
- ISSN
- 2049-2618 (ISSN-L)
- PMC
- PMC4063427
- OpenAlex
- W2161919313
- Cited by
- 277
- References
- 28
- Citations by year
- 2026: 19 · 2025: 19 · 2024: 15 · 2023: 19 · 2022: 17 · 2021: 31 · 2020: 26 · 2019: 23 · 2018: 29 · 2017: 32 · 2016: 30 · 2015: 15 · 2014: 1
- FWCI
- 10.65
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- cc-by
- Locations
- Microbiome (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Technical University of Denmark, DTU Orbit (Technical University of Denmark, DTU) (submittedVersion, other-oa)
- Funders
- Strategic Research Council
- Topics
- 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
- Physical Sciences › Environmental Science › Ecology › Microbial Community Ecology and Physiology
- Keywords
- bacterial DNA extraction, fecal microbiota, human gut microbiome, extraction method comparison, sample homogenization, Bacteroidetes, bacterial community structure, inter-study comparability
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