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The Effect of DNA Extraction Methods on Observed Microbial Communities from Fibrous and Liquid Rumen Fractions of Dairy Cows - PubMed

  • ️Mon Jan 01 2018

The Effect of DNA Extraction Methods on Observed Microbial Communities from Fibrous and Liquid Rumen Fractions of Dairy Cows

Jueeli D Vaidya et al. Front Microbiol. 2018.

Abstract

DNA based methods have been widely used to study the complexity of the rumen microbiota, and it is well known that the method of DNA extraction is a critical step in enabling accurate assessment of this complexity. Rumen fluid (RF) and fibrous content (FC) fractions differ substantially in terms of their physical nature and associated microorganisms. The aim of this study was therefore to assess the effect of four DNA extraction methods (RBB, PBB, FDSS, PQIAmini) differing in cell lysis and/or DNA recovery methods on the observed microbial diversity in RF and FC fractions using samples from four rumen cannulated dairy cows fed 100% grass silage (GS100), 67% GS and 33% maize silage (GS67MS33), 33% GS and 67% MS (GS33MS67), or 100% MS (MS100). An ANOVA statistical test was applied on DNA quality and yield measurements, and it was found that the DNA yield was significantly affected by extraction method (p < 0.001) and fraction (p < 0.001). The 260/280 ratio was not affected by extraction (p = 0.08) but was affected by fraction (p = 0.03). On the other hand, the 260/230 ratio was affected by extraction method (p < 0.001) but not affected by fraction (p = 0.8). However, all four extraction procedures yielded DNA suitable for further analysis of bacterial, archaeal and anaerobic fungal communities using quantitative PCR and pyrosequencing of relevant taxonomic markers. Redundancy analysis (RDA) of bacterial 16S rRNA gene sequence data at the family level showed that there was a significant effect of rumen fraction (p = 0.012), and that PBB (p = 0.012) and FDSS (p = 0.024) also significantly contributed to explaining the observed variation in bacterial community composition. Whilst the DNA extraction method affected the apparent bacterial community composition, no single extraction method could be concluded to be ineffective. No obvious effect of DNA extraction method on the anaerobic fungi or archaea was observed, although fraction effects were evident for both. In summary, the comprehensive assessment of observed communities of bacteria, archaea and anaerobic fungi described here provides insight into a rational basis for selecting an optimal methodology to obtain a representative picture of the rumen microbiota.

Keywords: 454 pyrosequencing; DNA extraction methods; archaea; bacteria; fibrous content; fungi; qPCR; rumen fluid.

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Figures

FIGURE 1
FIGURE 1

(A) Principal component analysis (PCA) of the combined bacterial (16S rRNA gene), archaeal (16S rRNA gene) and anaerobic fungal (5.8S rRNA gene) qPCR data for rumen fluid (RF, Δ) and fibrous content (FC, aaa) samples. The GS100 diet has duplicate DNA extracts presented as individual datapoints. The percentages provided at the axes indicate the variation explained. (B) The corresponding loadings for the principal components indicate that anaerobic fungi are the major cause of sample separation in PC1, and archaea in PC2.

FIGURE 2
FIGURE 2

Redundancy analysis triplot (RDA) showing the relationship between the top five family level phylogenetic groupings, the variation of which is most strongly associated with DNA extraction methods and fractions. The canonical axes are labeled with percentage of total variance explained (%). Arrow length indicates the variance explained by extraction methods and fractions. The GS100 diet has duplicate DNA extracts presented as individual datapoints.

FIGURE 3
FIGURE 3

Bacterial family level composition of different DNA extracts obtained from rumen fluid (A) and fibrous content (B) samples from dairy cows each fed different ratios of grass silage (GS) to maize silage (MS), e.g., GS67MS33 is a diet containing 67% grass silage and 33% maize silage. All the stacked bars represent individual sample data except for GS100 which represents the mean of two different DNA extracts (error bars represent the standard deviation). All family level phylogenetic groupings > 1% are shown individually, with those <1% summed together and presented as minor families.

FIGURE 4
FIGURE 4

The effect of DNA extraction method (RBB, PDD, FDSS, and PQIAmini) on the relative abundance of the bacterial families Prevotellaceae (A), Fibrobacteraceae (B), Ruminococcaceae (C), and Lachnospiraceae (D) in rumen fluid (yellow) and fibrous content (green) samples. The boxplots represent the data from 5 observations per rumen fraction, and show the 25th, 50th and 75th percentiles, with whiskers showing the extremes of the data.

FIGURE 5
FIGURE 5

Chao1 richness index (A) and Shannon’s diversity index (B) values for all four DNA extraction methods (RBB, PBB, FDSS, and PQIAmini) applied to rumen fluid (yellow) and fibrous content (green) samples from four dairy cows each fed different ratios of grass silage (GS) to maize silage (MS), e.g., GS67MS33 is a diet containing 67% grass silage and 33% maize silage. The GS100 samples represent the mean of two different DNA extracts, and the error bars represent their standard deviation.

FIGURE 6
FIGURE 6

Relative abundance of archaeal taxa at genus level within rumen fluid (A) and fibrous content (B) samples from dairy cows each fed different ratios of grass silage (GS) to maize silage (MS), e.g., GS67MS33 is a diet containing 67% grass silage and 33% maize silage. All samples were subjected to each of the four different DNA extraction methods (RBB, PBB, FDSS, and PQIAmini). Missing bars indicate that it was not possible to generate an archaeal PCR product for sequencing. Error bars for the GS100 samples represent the standard deviation associated with two different extracts, except for the PQIAmini extracted GS100 rumen fluid sample (A) where n = 1.

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