Abstract
BackgroundIn 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.ResultsIn 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.ConclusionsWhereas 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.
Highlights
In recent years, studies on the human intestinal microbiota have attracted tremendous attention
We observed significantly higher yields of DNA following extraction by the Metagenomics of the Human Intestinal Tract (MetaHIT) method compared with the Human Microbiome Project (HMP) method (P < 0.0001, Mann-Whitney test), which may in part be caused by a limited binding capacity of DNA in columns used in the HMP method
Distribution of taxa resulting from the two methods Depending on the applied DNA extraction method, we observed significant differences in numbers of raw sequencing reads mapped to known reference genomes
Summary
Studies on the human intestinal microbiota have attracted tremendous attention. Application of generation sequencing for mapping of bacterial phylogeny and function has opened new doors to this field of research. Little attention has been given to the effects of choice of methodology on the output resulting from such studies. The community structure of human intestinal bacteria has received tremendous attention. The option of generation sequencing for mapping of intestinal bacterial phylogeny and function has opened new doors to this field of research. Little attention has been paid to the effects of sampling procedure and choice of methodology on the output resulting from such studies. Several practical challenges are associated with the collection of fecal samples in large human studies. This normally cannot be achieved, and it is almost always necessary for microbiologists to base their studies
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