Quantifying and Understanding Well-to-Well Contamination in Microbiome Research

Area of Science:

• Microbiology
• Molecular Biology
• Bioinformatics

Background:

• Microbiome research is advancing rapidly, revealing microbial life in diverse environments.
• Contamination is a significant challenge, leading to the refutation of some findings and the establishment of strict validation requirements.
• Well-to-well contamination, a form of physical exchange between samples, has been poorly understood.

Purpose of the Study:

• To rigorously assess the behavior and sources of between-sample contamination in microbiome studies.
• To evaluate the impact of different DNA extraction methods and laboratory practices on well-to-well contamination.
• To identify strategies for minimizing contamination in microbiome data.

Main Methods:

• Utilized unique bacteria assigned to specific wells in a plate to track cross-contamination.
• Compared DNA extraction methods (plate-based vs. single-tube) across two laboratories.
• Included blanks, low-biomass, and high-biomass samples in a consistent plate layout.

Main Results:

• Well-to-well contamination primarily occurs during DNA extraction, with minor contributions from library preparation; barcode leakage was negligible.
• Contamination levels differed between laboratories and extraction methods; plate methods showed more well-to-well contamination, while single-tube methods had higher background contaminants.
• Contamination predominantly affected neighboring samples, was more frequent in low-biomass samples, and negatively impacted alpha and beta diversity metrics.

Conclusions:

• Sample contamination is a combination of cross-talk from adjacent samples and background contaminants.
• Effective strategies to reduce contamination include randomizing sample placement, processing similar biomasses together, and using manual single-tube extractions.
• Researchers should avoid oversimplified decontamination methods based solely on negative controls, as many "contaminants" may originate from other samples.