Gut Microbiota Analysis

Cecal microbiota DNA was extracted using a Stool DNA Kit (Guangzhou IGE Biotechnology, China) and applied to amplification of V3–V4 regions of 16S rDNA. Cecal microbiota composition was assessed using Illumina 2500 sequencing of 16S rDNA amplicon and QIIME-based microbiota analysis. High-quality reads for bioinformatics analysis were selected, and all of the effective reads from all samples were clustered into OTUs based on 99% sequence similarity according to QIIMEU clust. OTUs were annotated through the RDP Classifier (Version 2.2), with a confidence cutoff of 0.8 according to the Green Gene database, and the composition and abundance information of each sample at different classification levels were statistically summarized. Based on the OTU information from each sample, PCA was applied via R to examine the similarity between different samples; α-diversity analyses were performed via the R package phyloseq v.1.19.1 and vegan 2.4.2 packets to calculate the diversity index.