Phusion enzyme

Rectal feces were collected from five groups of NOD mice with six samples in each group. DNA was extracted by TIANamp Stool DNA Kit (#DP328‐02; Tiangen) from fecal samples. PCR amplification and library construction were conducted by Phusion enzyme (K1031; APExBIO) and the V3‐V4 region primers (341F 5′‐CCTACGGGNGGCWGCAG‐3′ and 805R 5′‐GACTACHVGGGTATCTAATCC‐3′) of the 16S rRNA gene. An Illumina NovaSeq. 6000 instrument\s was used for paired‐end (PE250) sequencing to collect raw data. Qiime 2 (2020.2) was used to conduct data quality control, calculate the α diversity index, and measure relative abundance. Each amplicon sequence variant/operating taxonomic units (ASV/OTU) sequence was annotated by referring to the SilvA‐132‐99 database, and the corresponding species information and abundance distribution were obtained. R software (Venn Diagram package) and the Jvenn web page (http://www.bioinformatics.com.cn/static/others/jvenn/example.html) were used to analyze common and unique ASVs in groups. Linear discriminant analysis effect size analysis (LefSe, https://github.com/SegataLab/lefse) was applied to evaluate the differential microbiota.

According to the manufacturer's instructions, the fecal genome DNA extraction kit (CAT.#DP328-02, TIANGEN, China) was used to extract DNA from fecal samples. Qubit with dsDNA HS Assay Kit (CAT.12640ES76, YEASEN, China) was used for concentration detection. PCR amplification was performed using Phusion enzyme (K1031, APExBIO, USA) and bacterial primers targeting the V3-V4 region of the 16S rRNA gene (357F 5′-ACTCCTACGGRAGGCAGCAG-3′ and 806R 5′-GGACTACHVGGGTWTCTCATAT-3′), followed by adapter and library construction. The Illumina NovaSeq 6000 PE250 platform was utilized for multiplexed sequencing to generate raw sequencing data. The raw data were subjected to quality control using Qiime 2 (2020.2) analysis pipeline and DADA2 to obtain clean data. Species annotation of each ASV/OTU sequence for species abundance was conducted using the Silva-132-99 database. Qiime 2 software was employed to calculate the alpha diversity index for each sample, while the LDA effect size (LefSe, https://github.com/SegataLab/lefse) was used to assess the enrichment of functional pathways for intergroup differences in the analysis.