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. 2016 May 18:6:26276.
doi: 10.1038/srep26276.

Glycans affect DNA extraction and induce substantial differences in gut metagenomic studies

Emmanouil Angelakis  1 , Dipankar Bachar  1 , Bernard Henrissat  2 , Fabrice Armougom  1 , Gilles Audoly  1 , Jean-Christophe Lagier  1 , Catherine Robert  1 , Didier Raoult  1
Affiliations

Glycans affect DNA extraction and induce substantial differences in gut metagenomic studies

Emmanouil Angelakis et al. Sci Rep. .

Abstract

Exopolysaccharides produced by bacterial species and present in feces are extremely inhibitory to DNA restriction and can cause discrepancies in metagenomic studies. We determined the effects of different DNA extraction methods on the apparent composition of the gut microbiota using Illumina MiSeq deep sequencing technology. DNA was extracted from the stool from an obese female using 10 different methods and the choice of DNA extraction method affected the proportional abundance at the phylum level, species richness (Chao index, 227 to 2,714) and diversity (non parametric Shannon, 1.37 to 4.4). Moreover DNA was extracted from stools obtained from 83 different individuals by the fastest extraction assay and by an extraction assay that degradated exopolysaccharides. The fastest extraction method was able to detect 68% to 100% genera and 42% to 95% species whereas the glycan degradation extraction method was able to detect 56% to 93% genera and 25% to 87% species. To allow a good liberation of DNA from exopolysaccharides commonly presented in stools, we recommend the mechanical lysis of stools plus glycan degradation, used here for the first time. Caution must be taken in the interpretation of current metagenomic studies, as the efficiency of DNA extraction varies widely among stool samples.

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Figures

Figure 1
Figure 1. Rarefaction curves relating the sequencing effort to the number of putative species in the samples.
Figure 2
Figure 2. Consensus dendrogram illustrating the similarity of the microbial communities obtained using different DNA extraction methods.
Figure 3
Figure 3. Network of species only detected by each extraction method.
Figure 4
Figure 4. Correlation coefficient of species detected by DNA extraction methods 1 and 5 for 83 stool samples.
Amz, individuals from Amazone; Toua, Touareg individuals; Fr, French individuals; Mrm, Marasmus individuals; KW, kwashiorkor individuals; Ob, obese individuals Blue color, genera coefficient; red color, species coefficient. Correlation coefficient was estimated for each sample using the number of shared species between methods over the total number of species/genus identified.
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