DNA Extraction and Cecal Microbiota Analysis of Fecal Samples

Total microbial genomic DNA samples were extracted using the QIAamp DNA Stool Mini Kit (QIAGEN, Inc., Netherlands) and stored at −20°C prior to further analysis. The concentration and quality of the extracted DNAs were determined by the NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and agarose gel electrophoresis, respectively. The V3–V4 hypervariable region of the bacterial 16S rRNA gene was amplified by PCR with the forward primer 338F: 5′-ACTCCTACGGGAGGCAGCAG-3′ and the reverse primer 806R: 5′-GGAC- TACHVGGGTWTCTAAT-3′. Sample-specific 7-bp barcodes were incorporated into the primers for multiplex sequencing. The PCR components contained 5 μl of Q5 reaction buffer (5 ×), 5 μl of Q5 high-fidelity GC buffer (5 ×), 0.25 μl of Q5 high-fidelity DNA polymerase (5 U/μl), 2 μl (2.5 mM) of dNTPs, 1 μl (10 uM) of each forward and reverse primer, 2 μl of DNA template, and 8.75 μl of ddH₂O. Thermal cycling consisted of initial denaturation at 98°C for 2 min, followed by 25 cycles consisting of denaturation at 98°C for 15 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s, with a final extension of 5 min at 72°C. PCR amplicons were purified with Agencourt AMPure Beads (Beckman Coulter, Indianapolis, IN, USA) and quantified using the PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). After the individual quantification step, amplicons were pooled in equal amounts, and paired-end 2′ 300-bp sequencing was performed using the Illlumina MiSeq platform with MiSeq Reagent Kit v3 at the Shanghai Personal Biotechnology Co., Ltd. (Shanghai, China) (15, 16). The OTUs, alpha diversity, beta diversity analysis, and the microbiota structure analysis were done according to previously described procedures (17). OTU taxonomy was assigned by the Greengene database. For α-diversity analysis, Chao 1 index was calculated by Mothur, and the Shannon index was calculated by the R package “vegan” to estimate the bacterial community richness within each sample. β-diversity was assessed by MANOVA and principal coordinate analysis (PCoA).