PCR and sequencing

The DNA isolated from fecal samples was used as the template for the amplification of the 16S rRNA V3-V4 region. The universal primers used were F (5’-NNNNNNN ACTCCTACGGGAGGCAGCA-3’) and R (5’-NNNNNNN GGACTACVSGGGTATCTAAT-3’), with the NNNNNNN being unique seven-base barcode used to tag each PCR product. The PCR reaction was performed according to the touchdown protocol[18] in a system of 25 μL containing 5.0 μL 5 × reaction buffer (TaKaRa, Dalian, China), 5.0 μL 5 × high GC buffer (TaKaRa, Dalian, China), 0.5 μL dNTPs (10 mmol/L) mixture , 1.0 µL forward primer (10 µmol/L), 1.0 μL reverse primer (10 µmol/L), 0.25 μL Q5 high-fidelity DNA polymerase (5 U/uL, TaKaRa, Dalian, China), and 1 μL DNA template. Each PCR product was purified by 2% agarose gel electrophoresis. DNA was isolated using the Axygen Axy Prep DNA Gel Extraction kit (Axygen, Shanghai, China). The sequencing was finished with the help of the Illumina Miseq System (Illumina).