16S rRNA V4 amplicon sequencing of porcine gut microbiota (341F/805R)

Fecal samples were collected using fecal collection tubes and processed according to the standard 16S sequencing workflow (Illumina, San Diego, USA). Sequence data were analyzed with QIIME2. Raw reads were demultiplexed and quality-filtered using the DADA2 plugin in QIIME2, truncating reads with Phred scores below 20 and removing chimeric sequences. Taxonomy was assigned against the SILVA 138 reference database, and amplicon sequence variants were obtained from the resulting feature table.Alpha diversity metrics were calculated in QIIME2 to compare control and treatment groups. For beta diversity, Jaccard and Bray-Curtis distance matrices were computed, and permutational multivariate analysis of variance was used to assess significant differences in community composition.