Background: The study of microbial communities has been revolutionised in recent years by the widespread adoption of culture independent analytical techniques such as 16S rRNA gene sequencing and metagenomics. One potential confounder of these sequence-based approaches is the presence of contamination in DNA extraction kits and other laboratory reagents. Results: In this study we demonstrate that contaminating DNA is ubiquitous in commonly used DNA extraction kits and other laboratory reagents, varies greatly in composition between different kits and kit batches, and that this contamination critically impacts results obtained from samples containing a low microbial biomass. Contamination impacts both PCR-based 16S rRNA gene surveys and shotgun metagenomics. We provide an extensive list of potential contaminating genera, and guidelines on how to mitigate the effects of contamination. Conclusions: These results suggest that caution should be advised when applying sequence-based techniques to the study of microbiota present in low biomass environments. Concurrent sequencing of negative control samples is strongly advised.
Bibliographical noteFunding Information:
SJS, JP, AWW and sequencing costs were supported by the Wellcome Trust (grant number 098051). MJC was supported by a Wellcome Trust Centre for Respiratory Infection Basic Science Fellowship. STC is funded by the National Institute for Health Research (NIHR). WOC and MFM are supported by a Wellcome Trust Joint Senior Investigator’s Award, which also supports EMT. PT was supported by a Wellcome Trust Clinical Training Fellowship (grant number 083735/Z/07/Z). NJL is supported by a Medical Research Council Special Training Fellowship in Biomedical Informatics. AWW and The Rowett Institute of Nutrition and Health, University of Aberdeen receive core funding support from the Scottish Government Rural and Environmental Science and Analysis Service (RESAS). The views expressed are those of the authors and not necessarily those of the Wellcome Trust, the NHS, the NIHR or the Department of Health. We would like to thank the Wellcome Trust Sanger Institute’s core sequencing team, Paul Scott for his assistance in the laboratory and for providing a list of contaminants derived from multiple displacement amplification kits, Phil James for assistance with qPCR, and Christine Boinett for R advice.
© 2014 Salter et al.
- 16S rRNA