Impact of Preservation Method and 16S rRNA Hypervariable Region on Gut Microbiota Profiling

Area of Science:

• Microbiology
• Bioinformatics
• Genetics

Background:

• Accurate gut microbiome profiling is crucial for understanding human health and disease.
• Self-collected stool samples require preservation methods that maintain microbial integrity during transport when cold chain is not feasible.
• Minimizing batch biases from sample collection and processing is essential for large-scale microbiota studies.

Purpose of the Study:

• To evaluate the efficacy of six preservation solutions in maintaining human stool microbial community composition and inhibiting bacterial growth at room temperature.
• To assess the impact of different bacterial 16S rRNA gene hypervariable regions and primer pairs on gut microbiota profiling.

Main Methods:

• Human stool samples were preserved using six solutions (Norgen, OMNI, RNAlater, CURNA, HEMA, Shield) and stored at room temperature for 7 days.
• Bacterial 16S rRNA gene amplicon sequencing was performed on three hypervariable regions (V1-V2, V3-V4, V4).
• Microbial community composition was compared to -80°C standards, and bacterial growth inhibition was assessed.

Main Results:

• Norgen and OMNI preservatives demonstrated the least microbial community shift and effectively inhibited aerobic and anaerobic bacterial growth.
• RNAlater showed significant bacterial activity and larger community shifts compared to Norgen and OMNI.
• Different 16S rRNA gene primer pairs exhibited varying sensitivity in detecting microbial communities and specific taxa, with the V4 region showing higher alpha diversity.

Conclusions:

• The choice of preservation solution and 16S rRNA gene primer pair significantly impacts gut microbiota profiling results.
• Norgen and OMNI are recommended for stool sample preservation when cold chain transport is unavailable.
• Consistent methodologies in sample preservation and sequencing are critical for minimizing biases and enabling reliable comparisons in microbiota research.