Trends in Microbiology
OpinionContamination in Low Microbial Biomass Microbiome Studies: Issues and Recommendations
Section snippets
Prospects and Pitfalls of Microbiome Research
The completion of the Human Microbiome Project in 2017 [1] was a major landmark in microbiome (see Glossary) research. This research field has the potential to create novel therapies for human disease, aid in environmental conservation, improve agricultural outputs, understand the lifestyles of our ancestors, and identify criminals in forensic casework, among many other areas 2, 3, 4, 5, 6.
Amplification-based methods that target hypervariable regions (e.g., PCR amplification of the 16S rRNA
Contamination in Microbiome Studies
Two key types of contamination can arise in microbiome studies: contaminant DNA and cross-contamination. Contaminant DNA can originate from many sources despite utmost care in sample collection and preparation, including the sampling and laboratory environments 25, 26, 27, researchers, plastic consumables [28], nucleic acid extraction kits 5, 19, 23, 24, 29, 30, 31, 32, laboratory reagents including PCR mastermixes 16, 17, 18, 33, 34, 35, 36, and cross-contamination from other samples and
Sample Types Most Affected by Contamination
The impact of contaminant DNA and cross-contamination can vary between samples according to their levels of microbial biomass. The microbial biomass in a sample can be estimated by comparing the quantity of microbial DNA in samples (e.g., quantitative PCR of 16S rRNA amplicons) to that in DNA extraction blank controls [23]. Samples that typically contain high microbial biomass include feces and soil, and usually contain substantially more DNA than DNA extraction blank controls, while low
How Contaminant DNA Influences Microbiome Studies
The amount and composition of contaminant DNA and cross-contamination can vary through time and location, generating signals within low microbial biomass samples that can be easily perceived as biological; this concept is illustrated in Figure 1. Numerous studies have described contaminant DNA and have demonstrated how it can skew results, including those in published low microbial biomass studies 19, 23, 24. For example, >95% of the taxonomic composition in a Salmonella bongori culture diluted
How Has DNA Contamination Already Impacted on the Microbiome Research Field?
The failure to properly control for and assess DNA contaminants and cross-contamination has resulted in several controversial studies. For example, a recent study identified a distinct microbial community within human placenta without publishing appropriate controls [46]. Bacterial DNA contribution from maternal blood was raised as an issue [47], and no evidence for a distinct placental microbiota was found when placental samples were compared with blank controls in a follow-up study [23]. A
Mitigating the Impacts of Contaminant DNA
To control for contaminant DNA and cross-contamination in low microbial biomass microbiome studies, several measures need to be taken to (i) reduce all types of contamination and experimental bias, (ii) monitor and identify contaminant sources, and (iii) recognize and mitigate the effects of contaminant DNA and cross-contamination during analysis. In chronological order of how a study would be performed, we provide suggestions for each approach, and put forward minimum guidelines (the RIDE
Concluding Remarks
Microbiome research holds great promise for multiple fields, but methodological pitfalls can easily undermine the progress and reputation of this developing research area. Therefore, these pitfalls must be recognized and explicitly addressed at each phase of the scientific process by researchers, reviewers, and editors alike. We present here the RIDE checklist for contaminant assessment to be applied across a wide-range of disciplines interested in exploring the microbial communities in low
Acknowledgments
We would like to thank Alan W. Walker, Bastien Llamas, Jessica L. Metcalf, Kieren J. Mitchell, and Matilda Handsley-Davis for their feedback and suggestions. L.S.W. and R.E. were funded by a Discovery Early Career Researcher Award (DECRA; DE150101574) and a CABAH grant (CE170100015) from the ARC.
Glossary
- Contaminant DNA
- DNA from sources other than the sample(s) under study (e.g., DNA from reagents or researchers performing laboratory work).
- Contamination
- an umbrella term encompassing both contaminant DNA and cross-contamination (see below).
- Cross-contamination
- DNA exchange between samples within a study (e.g., accidental movement of DNA between different sample tubes during DNA extraction).
- DNA extraction blank control
- a negative control consisting of an empty tube/well that is processed alongside
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