Fecal genome dna extraction kit

DNA was extracted from fecal samples using the fecal genome DNA extraction kit (TIANGEN, #DP328-02). The Phusion enzyme (APExBIO K1031) and bacterial primers for the V3-V4 region of 16S rRNA gene (357F 5’-ACTCCTACGGRAGGCAGCAG-3’ and 806R5’-GGACTACHVGGGTWTCTCATAT-3’) were used for PCR amplification, insertion and library construction. Illumina novaseq6000 PE250 was used for mixed sequencing to obtain raw data. Qiime 2 (2020.2) analysis process was applied to call DADA2 for quality control to get clean data. Species annotations were made for each ASV/OTU sequence using the silva-132-99 database. Qiime 2 software calculates the Alpha and beta diversity indices (Principal component analysis and Anosim analysis) for each sample, as well as the relative abundance of species. Principal component analysis (PCA) analyzes the community distribution of different samples to reflect the similarities and differences between samples [21 (link)]. The closer two samples are, the more similar their species composition. R software (VennDiagram package) and jvenn (http://www.bioinformatics.com.cn/static/others/jvenn/example.html) page were used for sample or between groups of common and unique ASVs visualization. LDA Effect Size (LefSe, https://github.com/SegataLab/lefse) was applied to analyze the different 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.