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

Standard 16S rRNA V3-V4 sequencing encounters primer mismatch issues and insufficient taxonomic resolution, hindering the accurate quantification of specific, low-abundance taxa, such as administered probiotic strains. Therefore, we empirically compared outcomes between the standard V3-V4 method and high-resolution targeted species sequencing (TSS) to assess bias and establish reliability metrics for probiotic efficacy assessments. A longitudinal pilot study was conducted over nine weeks in older participants receiving synbiotic supplementation; their fecal samples were collected and analyzed. V3-V4 analysis successfully captured a significant transient reduction in alpha-diversity with multidirectional genus-level fluctuations. However, taxonomic overlap between these two methods was high at the phylum level and sharply declined to 6.7% at the species level. Notably, compared with V3-V4 sequencing, TSS could successfully quantify the abundance of administered Bifidobacterium animalis. This study empirically demonstrated that 16S rRNA V3-V4 sequencing introduces substantial quantitative bias, limiting its suitability for monitoring specific probiotic strains and compromising the reliability of clinical efficacy assessments. Therefore, we recommend a dual-sequencing framework that integrates the broad ecological screening capabilities of V3-V4 with the precise species-level quantification of TSS to establish the necessary scientific rigor for assessing probiotic efficacy.