Contamination in Low Microbial Biomass Microbiome Studies: Issues and Recommendations

Area of Science:

• Microbiology
• Genomics
• Bioinformatics

Background:

• Next-generation sequencing (NGS) offers unprecedented sensitivity in microbiome research, enabling detailed surveys of microbial communities, genomes, and functions.
• This high sensitivity, however, also leads to the detection of contaminant DNA and cross-contamination, potentially confounding study interpretations.
• The challenge is particularly acute in low microbial biomass samples, where contaminant DNA can dominate the signal.

Purpose of the Study:

• To review the sources and impacts of contaminant DNA and cross-contamination in microbiome studies.
• To identify strategies for mitigating these issues, particularly in low microbial biomass research.
• To propose a standardized checklist (RIDE) for improving the integrity of microbiome studies.

Main Methods:

• Literature review of contaminant DNA sources and impacts in microbiome research.
• Analysis of strategies for reducing contamination during sample collection, processing, and sequencing.
• Development and proposal of the RIDE checklist as a set of minimal experimental criteria.

Main Results:

• Contaminant DNA and cross-contamination can arise from various sources, including reagents, environment, and laboratory practices.
• These contaminants significantly impact data interpretation, especially in low microbial biomass samples, potentially leading to false positive or negative findings.
• Several practical measures and experimental controls can effectively reduce the influence of contaminants.

Conclusions:

• Addressing contaminant DNA and cross-contamination is essential for ensuring the reliability and validity of microbiome research.
• The proposed RIDE checklist provides a practical framework for researchers to implement necessary controls.
• Adoption of standardized controls will enhance the integrity and reproducibility of microbiome studies, particularly those involving low microbial biomass specimens.