DNA reference reagents isolate biases in microbiome profiling: a global multi-lab study.

Area of Science:

• Microbiology
• Genomics
• Bioinformatics

Background:

• Technical biases in human gut microbiome analysis hinder translational research and data comparability.
• Variability in sequencing and bioinformatics approaches impacts the reliability of microbiome profiling.

Purpose of the Study:

• To analyze sequencing and bioinformatic biases in human gut microbiome profiling.
• To establish standardized DNA reference reagents (RRs) and minimum quality criteria (MQC) for accurate microbiome analysis.

Main Methods:

• Global study involving 23 labs analyzing shotgun and 16S rRNA gene amplicon sequencing.
• Evaluation of wet and dry lab biases including sequencing depth, primer/database choice, rarefaction, and 16S copy number adjustment.
• Development and application of minimum quality criteria (MQC) for performance evaluation.

Main Results:

• Shotgun sequencing data exhibited greater variability than 16S rRNA gene amplicon sequencing.
• Identified specific sources of bias in both wet lab (e.g., sequencing depth, primers) and dry lab (e.g., database choice, rarefaction) procedures.
• Established well-defined RRs and MQC to assess and mitigate technical bias.

Conclusions:

• Standardized reference reagents and quality criteria are crucial for combating technical bias in microbiome research.
• This study provides a benchmark for evaluating microbiome profiling methodologies, promoting reliable and comparable research.
• Workflow optimization based on identified biases will accelerate innovation and translational applications in microbiome science.