Fecal samples were homogenized thoroughly before DNA extraction. Genomic DNA was extracted from a 1-ml mixture of 0.211–0.299 mg of feces and VXL buffer using a bead beater (Mini-bead Beater, Bio Spec Products, Bartlesville, UK). DNA was then extracted by using the QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany). The DNA quantity and quality were checked using NanoDrop 2000 (Thermo Scientific, Wilmington, USA), and DNA samples were diluted to 80 ng/μl before being subjected to PCR amplification.
The PCR amplification of bacterial V3–V4 hypervariable region of 16S rRNA gene was performed with universal primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) (16 (link)). The PCR amplification conditions used were as follows: initial denaturation at 94°C for 3 min, followed by 30 cycles of 94°C for 40 s, 56°C for 60 s, and 72°C for 60 s, and a final extension at 72°C for 10 min. The PCR products were gel purified using GeneJET Gel Recovery Kit (Thermo Scientific, USA) according to the manufacturer's instructions. The purified amplicons were used for the library construction, and sequencing was performed by using an Illumina MiSeq system with the MiSeq Reagent Kit v2 2 × 250 bp (Illumina, San Diego, USA).
The PCR amplification of bacterial V3–V4 hypervariable region of 16S rRNA gene was performed with universal primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) (16 (link)). The PCR amplification conditions used were as follows: initial denaturation at 94°C for 3 min, followed by 30 cycles of 94°C for 40 s, 56°C for 60 s, and 72°C for 60 s, and a final extension at 72°C for 10 min. The PCR products were gel purified using GeneJET Gel Recovery Kit (Thermo Scientific, USA) according to the manufacturer's instructions. The purified amplicons were used for the library construction, and sequencing was performed by using an Illumina MiSeq system with the MiSeq Reagent Kit v2 2 × 250 bp (Illumina, San Diego, USA).