Effect of DNA extraction methods and sampling techniques on the apparent structure of cow and sheep rumen microbial communities
Open accessAbstract
Molecular microbial ecology techniques are widely used to study the composition of the rumen microbiota and to increase understanding of the roles they play. Therefore, sampling and DNA extraction methods that result in adequate yields of microbial DNA that also accurately represents the microbial community are crucial. Fifteen different methods were used to extract DNA from cow and sheep rumen samples. The DNA yield and quality, and its suitability for downstream PCR amplifications varied considerably, depending on the DNA extraction method used. DNA extracts from nine extraction methods that passed these first quality criteria were evaluated further by quantitative PCR enumeration of microbial marker loci. Absolute microbial numbers, determined on the same rumen samples, differed by more than 100-fold, depending on the DNA extraction method used. The apparent compositions of the archaeal, bacterial, ciliate protozoal, and fungal communities in identical rumen samples were assessed using 454 Titanium pyrosequencing. Significant differences in microbial community composition were observed between extraction methods, for example in the relative abundances of members of the phyla Bacteroidetes and Firmicutes. Microbial communities in parallel samples collected from cows by oral stomach-tubing or through a rumen fistula, and in liquid and solid rumen digesta fractions, were compared using one of the DNA extraction methods. Community representations were generally similar, regardless of the rumen sampling technique used, but significant differences in the abundances of some microbial taxa such as the Clostridiales and the Methanobrevibacter ruminantium clade were observed. The apparent microbial community composition differed between rumen sample fractions, and Prevotellaceae were most abundant in the liquid fraction. DNA extraction methods that involved phenol-chloroform extraction and mechanical lysis steps tended to be more comparable. However, comparison of data from studies in which different sampling techniques, different rumen sample fractions or different DNA extraction methods were used should be avoided.
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The diet fed to ruminants is the primary determinant of bacterial community structure.
“The diet fed to ruminants is the primary determinant of bacterial community structure (95–97).”
From Phytogenic Additives Can Modulate Rumen Microbiome to Mediate Fermentation Kinetics and Methanogenesis Through Exploiting Diet–Microbe Interaction (2020) · cites this paper as [95]
The Introduction says that changes in diet can produce shifts in rumen microbial communities. It does not say that diet is the primary determinant of bacterial community structure. The Results and Discussion instead compare differences associated with sampling and DNA extraction methods; no passage provided ranks diet against other determinants. (LLM, conf=0.83)
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- Authors
- Henderson, G., Cox, F., Kittelmann, S., Miri, V. H., Zethof, M., Noel, S. J., Waghorn, G. C., & Janssen, P. H.
- Affiliations
- AgResearch (NZ)
- Massey University (NZ)
- DairyNZ (NZ)
- Corresponding author
- Henderson, Gemma
- ORCID
- Venue
- PLoS ONE
- Publisher
- Public Library of Science (PLoS)
- Published
- 2013-09-11
- Volume
- 8
- Issue
- 9
- Pages
- e74787
- ISSN
- 1932-6203 (ISSN-L)
- PMC
- PMC3770609
- OpenAlex
- W1994441172
- Cited by
- 328
- References
- 35
- Citations by year
- 2026: 13 · 2025: 7 · 2024: 13 · 2023: 17 · 2022: 32 · 2021: 42 · 2020: 30 · 2019: 34 · 2018: 32 · 2017: 39 · 2016: 21 · 2015: 26 · 2014: 21
- FWCI
- 48.17
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- PLoS ONE (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, other-oa)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Figshare (submittedVersion, cc-by)
- Funders
- AgResearch
- DairyNZ
- Fonterra Co-Operative Group
- Ministerio de Ciencia e Innovación
- Topics
- Life Sciences › Agricultural and Biological Sciences › Agronomy and Crop Science › Ruminant Nutrition and Digestive Physiology
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gut microbiota and health
- Physical Sciences › Engineering › Building and Construction › Anaerobic Digestion and Biogas Production
- Keywords
- rumen microbiota, DNA extraction methods, sampling methods, rumen fistula, microbial community composition, archaea, rumen ciliates, rumen fungi, Bacteroidetes, Firmicutes, Clostridiales, Prevotellaceae, cattle, sheep, phenol-chloroform extraction
- MeSH terms
- Animals, Cattle, DNA, Ecology, Phylogeny, Rumen, Sheep, Polymerase Chain Reaction, Sequence Analysis, DNA, Principal Component Analysis, Microbial Consortia
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