16S rDNA Sequencing

The ileal and cecal contents from piglets in the control (without Cu supplementation) group and the group receiving a pharmacological dose of Cu (200 mg Cu/kg feed, n = 6 per treatment) were collected for microbiota analysis, as described previously (Zhou et al., 2018; Xiong et al., 2019). Briefly, total bacterial DNA was extracted using a QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany), and the V3–V4 hypervariable region of the microbial 16S rRNA genes were sequenced using the primers, 341F: 5′-CCTAYGGGRBGCASCAG-3′ and 806R: 5′-GGACTACNNGGGTATCTAAT-3′; Illumina adaptors; and molecular barcodes. Libraries were prepared using a TruSeq DNA PCR-Free Sample Preparation Kit (Illumina Inc., San Diego, CA, United States) and were assessed using a Qubit 2.0 Fluorometer (Thermo Scientific, Madison, WI, United States) and a Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, United States). Libraries were sequenced on a HiSeq 2500 platform (Illumina Inc., San Diego, CA, United States). Raw 16S rDNA sequences were assembled using the QIIME (v1.9.0) and FLASH software packages. Operational taxonomic units (OTUs) were analyzed using UPARSE (v7.0.1001), and high-quality sequences were aligned against the SILVA reference database 1 and clustered into OTUs at a 97% similarity level using the UCLUST algorithm 2. Each OTU was assigned to a taxonomic level using the Ribosomal Database Project Classifier program v2.203. The assembled sequences were then submitted to the NCBI Sequence Read Archive (No. PRJNA551517) for open access.