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.