Choice of bacterial DNA extraction method from fecal material influences community structure as evaluated by metagenomic analysis
Agata Wesolowska-Andersen1, Martin Iain Bahl2, Vera Carvalho2, Karsten Kristiansen3, Thomas Sicheritz-Pontén1, Ramneek Gupta1* and Tine Rask Licht2*
1Center for Biological Sequence analysis, Technical University of Denmark, Lyngby DK-2800, Denmark
2National Food Institute, Technical University of Denmark, Søborg DK-2860, Denmark
3Department of Biology, University of Copenhagen, Copenhagen N DK-2200, Denmark
Abstract
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.