Trends in Microbiology
Volume 27, Issue 2, February 2019, Pages 105-117
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Opinion
Contamination in Low Microbial Biomass Microbiome Studies: Issues and Recommendations

https://doi.org/10.1016/j.tim.2018.11.003Get rights and content

Highlights

There is increasing interest in applying metagenomic techniques to find correlations between microorganisms and disease.

Metagenomic techniques are highly sensitive and can detect contaminant DNA (DNA from sources other than the samples under study) and cross-contamination (DNA exchange between samples).

Recent studies have shown that contaminant DNA and cross-contamination can confound metagenomic studies, especially for sample types that have low microbial biomass.

There is an urgent need for the field to adopt authentication criteria to prevent future metagenomic studies from falling prey to the pitfalls of contaminant DNA and cross-contamination.

Next-generation sequencing approaches in microbiome research have allowed surveys of microbial communities, their genomes, and their functions with higher sensitivity than ever before. However, this sensitivity is a double-edged sword because these tools also efficiently detect contaminant DNA and cross-contamination, which can confound the interpretation of microbiome data. Therefore, there is an urgent need to integrate key controls into microbiome research to improve the integrity of microbiome studies. Here, we review how contaminant DNA and cross-contamination arise within microbiome studies and discuss their negative impacts, especially during the analysis of low microbial biomass samples. We then identify several key measures that researchers can implement to reduce the impact of contaminant DNA and cross-contamination during microbiome research. We put forward a set of minimal experimental criteria, the ‘RIDE’ checklist, to improve the validity of future low microbial biomass research.

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

References (84)

  • A. Sessitsch et al.

    21st century agriculture: integration of plant microbiomes for improved crop production and food security

    Microb. Biotechnol.

    (2015)
  • L.S. Weyrich

    Neanderthal behaviour, diet, and disease inferred from ancient DNA in dental calculus

    Nature

    (2017)
  • N. Fierer

    Forensic identification using skin bacterial communities

    Proc. Natl. Acad. Sci. U. S. A.

    (2010)
  • J.G. Caporaso

    Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms

    ISME J.

    (2012)
  • The Human Microbiome Project Consortium

    Structure, function and diversity of the healthy human microbiome

    Nature

    (2012)
  • B.C. Christner

    A microbial ecosystem beneath the West Antarctic ice sheet

    Nature

    (2014)
  • A. Checinska

    Microbiomes of the dust particles collected from the International Space Station and spacecraft assembly facilities

    Microbiome

    (2015)
  • K. Anantharaman

    Metagenomic resolution of microbial functions in deep-sea hydrothermal plumes across the Eastern Lau Spreading Center

    ISME J.

    (2016)
  • L.W. Kelly

    Local genomic adaptation of coral reef-associated microbiomes to gradients of natural variability and anthropogenic stressors

    Proc. Natl. Acad. Sci.

    (2014)
  • C. Lozupone et al.

    UniFrac: a new phylogenetic method for comparing microbial communities

    Appl. Environ. Microbiol.

    (2005)
  • D. Aird

    Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries

    Genome Biol.

    (2011)
  • D. Gagic

    Improving the genetic representation of rare taxa within complex microbial communities using DNA normalization methods

    Mol. Ecol. Resour.

    (2014)
  • M.A. Tanner

    Specific ribosomal DNA sequences from diverse environmental settings correlate with experimental contaminants

    Appl. Environ. Microbiol.

    (1998)
  • S.J. Salter

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

    BMC Biol.

    (2014)
  • R.W. Lusk

    Diverse and widespread contamination evident in the unmapped depths of high throughput sequencing data

    PLoS One

    (2014)
  • M. Laurence

    Common contaminants in next-generation sequencing that hinder discovery of low-abundance microbes

    PLoS One

    (2014)
  • R.I. Adams

    Microbiota of the indoor environment: a meta-analysis

    Microbiome

    (2015)
  • A.P. Lauder

    Comparison of placenta samples with contamination controls does not provide evidence for a distinct placenta microbiota

    Microbiome

    (2016)
  • A. Glassing

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

    Gut Pathog.

    (2016)
  • N. Witt

    An assessment of air as a source of DNA contamination encountered when performing PCR

    J. Biomol. Tech.

    (2009)
  • B. Llamas

    From the field to the laboratory: controlling DNA contamination in human ancient DNA research in the high-throughput sequencing era

    STAR Sci. Technol. Archaeol. Res.

    (2017)
  • S.T. Motley

    Improved multiple displacement amplification (iMDA) and ultraclean reagents

    BMC Genomics

    (2014)
  • N. Fierer

    The influence of sex, handedness, and washing on the diversity of hand surface bacteria

    Proc. Natl. Acad. Sci.

    (2008)
  • R.R. Dunn

    Home life: factors structuring the bacterial diversity found within and between homes

    PLoS One

    (2013)
  • S.N. Naccache

    The perils of pathogen discovery: origin of a novel parvovirus-like hybrid genome traced to nucleic acid extraction spin columns

    J. Virol.

    (2013)
  • R.I. Adams

    Airborne bacterial communities in Residences: similarities and differences with fungi

    PLoS One

    (2014)
  • G.A. McFeters

    Distribution of bacteria within operating laboratory water purification systems

    Appl. Environ. Microbiol.

    (1993)
  • T. Nogami

    Estimation of bacterial contamination in ultrapure water: application of the anti-DNA antibody

    Anal. Chem.

    (1998)
  • M.B. McAlister

    Survival and nutritional requirements of three bacteria isolated from ultrapure water

    J. Ind. Microbiol. Biotechnol.

    (2002)
  • V. Seitz

    A new method to prevent carry-over contaminations in two-step PCR NGS library preparations

    Nucleic Acids Res.

    (2015)
  • M. Ballenghien

    Patterns of cross-contamination in a multispecies population genomic project: detection, quantification, impact, and solutions

    BMC Biol.

    (2017)
  • L. Weyrich

    Laboratory contamination over time during low-biomass sample analysis

    bioRxiv

    (2018)
  • Cited by (556)

    View all citing articles on Scopus
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