2.7. Gut Microbiota Analysis with 16S rRNA Gene Sequencing

Eight mice from each group were randomly selected for 16S rRNA gene sequencing. The total microbial DNA was isolated from weighed feces, which were collected at week 21 using the QIAamp DNA Stool Mini Kit (Qiagen, Dusseldorf, Germany) following the manufacturer’s protocols. The bacterial 16S rRNA genes, hypervariable regions V3–V4, were amplified with the forward primer and the reverse primer by the thermocycler PCR system (Gene Amp 9700, ABI, Waltham, MA, USA). The amplicons recovered from 2% agarose gel were purified with AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, America), quantified by QuantiFluor™-ST (Promega, Madison, WI, USA), and then paired-end sequenced (2 × 300) on an Illumina MiSeq platform (Illumina, San Diego, CA, USA) following the standard procedure by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). Then, raw reads from the original DNA fragments were quality-filtered and merged. Sequences were clustered into the same operational taxonomic units (OTUs) with the UPARSE software package. Then, the sequence was analyzed by the RDP Classifier algorithm against the Greengenes 16S rRNA bacteria database. The species abundance of each sample was counted at the phylum level as well as the family level and studied visually through the histogram visualization method. A heatmap based on the top 30 dominant genera was used to present the Community species composition and species abundance information. Linear discriminant analysis effect size (LEfSe) analysis was performed to analyze the species differences between the groups. Finally, the correlation heatmap was applied to visualize the relationship between different species in the sample and blood biochemical criterion and to evaluate the correlation between microbial classification and the blood biochemical criterion.