Quantifluor st blue fluorescence quantification system

Fifteen piglets (five piglets per group) were randomly selected for 16S rRNA MiSeq sequencing. Cecal genomic DNA was extracted from 50 mg of cecal samples using the E.Z.N.A. Soil DNA Kit (Omega Bio-tek, Norcross, GA, USA) following the manufacturer’s protocol. The concentration and purity of extracted bacterial DNA were determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). The forward (5′-ACTCCTACGGGAGGCAGCAG-3′) and reverse (5′-GGACTACHVGGGTWTCTAAT-3′) primers were used to amplify the highly variable V3–V4 region of the 16S rRNA gene. PCR was performed using TransGen AP221-02: TransStart FastPfu DNA Polymerase (Illumina, San Diego, CA, USA), with a 20 µL reaction system, and an ABI GeneAmp Model 9700 PCR thermal cycler.
DNA extraction, PCR amplification, and product purification and sequencing were performed with the assistance of Shanghai Meiji Biopharmaceutical Technology Co. Referring to the preliminary quantification results of electrophoresis, the PCR products were detected and quantified using the QuantiFluor™-ST Blue Fluorescence Quantification System (Promega, Beijing, China), and the TruSeqTM DNA Sample Prep Kit was selected for library construction. The purified amplicons were pooled at equimolar levels on the Illumina MiSeq PE300/NovaSeq PE250 platform (Illumina, San Diego, CA, USA) followed by paired-end sequencing.

Intestines (n = 4 repeats, 3 intestine samples pooled as one repeat) were used for 16S rRNA gene amplicons sequencing. Briefly, genomic DNA was extracted from 50 mg of tissue samples and detected through 2% agarose gel electrophoresis, amplified using an ABI GeneAmp^® 9700 PCR (ABI, Los Angeles, CA, USA), and the primer is 338F-806R. The PCR products were cut and recovered using the AxyPrepDNA gel recovery kit (AXYGEN, New York, USA), subsequently eluted by Tris_HCl [32 (link)]. The PCR products were quantified by the QuantiFluor™-ST Blue Fluorescence Quantification System (Promega, Madison, WI, USA). Miseq amplicon libraries were constructed and sequenced on the Illumina MiSeq-PE25 platform (Illumina, San Diego, CA, USA) in Majorbio Co. (Shanghai, China) [33 (link)]. The data were uploaded to the Majorbio Co., Cloud Platform (https://cloud.majorbio.com) for result analysis. All 16S rRNA sequence data can be downloaded from the National Center for Biotechnology Information (NCBI) under the project accession PRJNA1043446 (Submission ID: SUB13989547).

Specific primers with barcode were synthesized and the V3-V4 region of the 16 S rRNA gene was amplified using TransGen AP221-02. The PCR products were mixed and detected by 2% agarose gel electrophoresis. The products were purified by the AxyPrepDNA Gel Recovery Kit (AXYGEN, USA) and quantified using the QuantiFluor™ -ST Blue Fluorescence Quantification System (Promega, USA). Finally, sequencing was performed by Mejorbio Biopharmaceuticals on the llumina MiSeq platform (Illumina, USA).
UPARSE (version 7.0.1090 http://drive5.com/uparse/) was used for analysis of OTUs at 97% similarity. Alpha diversity indices (sobs, shannon, simpson, ace, chao, coverage) were calculated by Mothur (version 1.30.2 https://www.mothur.org/wiki/Download_mothur) to estimate community richness and diversity. The beta diversity of distance matrix was calculated by QIIME (version 1.9.1 http://qiime.org/install/index.html), the Non-metric multidimensional scaling (NMDS) analysis was performed by Vegan and differences between groups were analysed by Partial Least Squares Discriminant Analysis (PLS-DA). LEfSe analysis was performed to estimate differences of species abundance and the Kruskal-Wallis H test was used to assess the significance of the differences. Correlation coefficients of microbial communities were calculated using Spearman correlation algorithm and visualized by Cytoscape.