Reagent and laboratory contamination can critically impact sequence-based microbiome analyses

TLDR

Culture‑independent methods such as 16S rRNA sequencing and metagenomics have transformed microbiome research, yet contamination from DNA extraction kits and laboratory reagents can confound these analyses. The authors demonstrate that contaminating DNA is ubiquitous in common kits and reagents, varies by kit and batch, and can severely bias 16S rRNA and shotgun metagenomic results from low‑biomass samples; they provide a list of potential contaminants, mitigation guidelines, and recommend sequencing negative controls.

Abstract

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. 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. 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.