Probiotic-Induced Gut Microbiota Modulation: A Comparative Analysis Using 16S rRNA V3-V4 and Targeted Sequencing

Area of Science:

• Microbiome research
• Gut microbiota analysis
• Probiotic efficacy studies

Background:

• Standard 16S rRNA V3-V4 sequencing has limitations in taxonomic resolution and primer mismatch.
• Accurate quantification of specific low-abundance taxa, such as probiotic strains, is challenging with current methods.
• Reliable assessment of probiotic efficacy requires precise microbial quantification.

Purpose of the Study:

• To compare the bias and reliability of standard 16S rRNA V3-V4 sequencing versus high-resolution targeted species sequencing (TSS).
• To establish reliability metrics for probiotic efficacy assessments using different sequencing methods.
• To evaluate the suitability of 16S rRNA V3-V4 sequencing for monitoring specific probiotic strains.

Main Methods:

• A longitudinal pilot study involving older participants receiving synbiotic supplementation.
• Collection and analysis of fecal samples over a nine-week period.
• Comparison of outcomes between standard 16S rRNA V3-V4 sequencing and high-resolution TSS.

Main Results:

• V3-V4 analysis revealed transient alpha-diversity reduction and genus-level fluctuations.
• Taxonomic overlap between V3-V4 and TSS was high at the phylum level but decreased to 6.7% at the species level.
• TSS successfully quantified administered *Bifidobacterium animalis*, unlike V3-V4 sequencing.

Conclusions:

• 16S rRNA V3-V4 sequencing introduces significant quantitative bias, limiting its use for specific probiotic strain monitoring.
• The reliability of clinical probiotic efficacy assessments is compromised by V3-V4 sequencing limitations.
• A dual-sequencing approach combining V3-V4 and TSS is recommended for rigorous probiotic efficacy evaluation.