Inherent bacterial DNA contamination of extraction and sequencing reagents may affect interpretation of microbiota in low bacterial biomass samples

Abstract

Background: The advent and use of highly sensitive molecular biology techniques to explore the microbiota and microbiome in environmental and tissue samples have detected the presence of contaminating microbial DNA within reagents. These microbial DNA contaminants may distort taxonomic distributions and relative frequencies in microbial datasets, as well as contribute to erroneous interpretations and identifications.

Results: We herein report on the occurrence of bacterial DNA contamination within commonly used DNA extraction kits and PCR reagents and the effect of these contaminates on data interpretation. When compared to previous reports, we identified an additional 88 bacterial genera as potential contaminants of molecular biology grade reagents, bringing the total number of known contaminating microbes to 181 genera. Many of the contaminants detected are considered normal inhabitants of the human gastrointestinal tract and the environment and are often indistinguishable from those genuinely present in the sample.

Conclusions: Laboratories working on bacterial populations need to define contaminants present in all extraction kits and reagents used in the processing of DNA. Any unusual and/or unexpected findings need to be viewed as possible contamination as opposed to unique findings.

Keywords: 16S sequencing; Contamination; Inflammatory bowel disease; Metagenomics; Microbiota; PCR.

Fig. 1

Contaminating endogenous DNA from DNA extraction and processing kits produced C_T values of 28–30 in no template controls using universal rRNA gene primers. Samples containing low bacterial biomass and high levels of competing human DNA, such as intestinal submucosal or peripheral blood samples, often produce C_T values greater than the no template controls. Bacteria are discernable from background only in samples containing a high bacterial biomass such as intestinal mucosal tissues. Reagent contamination interferes and prevents bacterial quantitation based on the rRNA genes in low bacterial biomass samples

Fig. 2

Background levels of bacterial genomes based on 16S rRNA gene universal primers suggest a level of at least 4 bacterial genomes per µl of qPCR reaction mixture. Although the true amount of contaminating bacterial DNA would be 4 + NTC, assuming the lowest possible figure, a standard 50 µl qPCR reaction would contain at least 200 E. coli-equivalent genomes or approximately 1400 rRNA gene copies contaminating the reaction