The impact of storage buffer, DNA extraction method, and polymerase on microbial analysis

Area of Science:

• Microbiology
• Bioinformatics
• Genomics

Background:

• Next-generation sequencing (NGS) is crucial for microbial community analysis.
• Technical limitations in sample handling can introduce significant data bias.
• Standardized protocols are needed to minimize bias across diverse sample types.

Purpose of the Study:

• To evaluate the impact of different sample handling conditions on microbial community profiling using NGS.
• To identify optimal protocols for swab-based sample processing to minimize bias.
• To establish a foundation for standardizing microbial community analysis protocols.

Main Methods:

• Utilized a mock microbial community of 22 bacterial strains at even concentrations.
• Investigated various combinations of storage buffers, DNA extraction kits, and 16S rRNA gene amplification methods.
• Analyzed bacterial composition and abundance using sequencing data.

Main Results:

• The choice of storage buffer and extraction kit significantly altered the detected bacterial composition.
• Different 16S rRNA amplification methods had a minimal effect on the overall profile.
• All bacterial genera were detected with low contamination, but abundance profiles deviated from expected values across all conditions.

Conclusions:

• Sample handling protocols, particularly storage buffer and extraction kit selection, critically influence microbial community profiling.
• Observed deviations in abundance highlight the need for rigorous validation of protocols using mock communities and controls.
• Standardization of sample handling is essential to reduce variability and improve the reliability of NGS-based microbial studies.