Bacterial DNA Contamination of Commercial PCR Enzymes: Considerations for Microbiome Protocols and Analysis

TL;DR

This study shows that commercial PCR enzymes can be contaminated with bacterial DNA, which can affect microbiome research results.

Contribution

The study demonstrates bacterial DNA contamination in PCR enzymes using accessible methods like endpoint PCR and Sanger sequencing.

Findings

Seven out of nine commercial PCR enzymes tested contained contaminating bacterial DNA.

Contaminating DNA came from multiple bacterial species.

Control reactions are needed to identify contamination in microbiome studies.

Abstract

The microbiome remains a top area of research, and it is now common to examine any organic and inorganic samples for bacterial colonization. However, due to the ubiquity of bacteria in the environment, separating the low-burden colonization of bacteria from the possible contamination of laboratory reagents remains problematic. When examining samples of expected low bacterial burden, it is common to first amplify any bacterial DNA present through PCR before sequencing. In this work, we examined nine different commercial PCR enzymes and their reaction components as possible sources of bacterial DNA contamination. We found contaminating bacterial DNA in seven of the nine reactions, and this DNA was shown to come from a variety of species. Importantly, we were able to perform these studies solely with endpoint PCR and Sanger sequencing, which are more accessible and affordable than high-throughput, short-read sequencing and real-time PCR. This work confirms that there needs to be an increased emphasis on including control reactions in microbiome studies so that contaminating DNA sequences can be identified and addressed, and that this can be achieved with minimal resources.

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