**2.4 Polymerase chain reaction amplicon production and high-throughput sequencing**

The variable regions V3-V4 of the 16S rDNA gene were amplified and sequenced. For the PCRs, 5 mM of each primer, 10 ng of DNA template, 4 liters of 1 FastPfu buffer, 2.5 mM dNTPs, and 0.4 liters of FastPfu polymerase in triplicate in a total volume of 20 liters were used (TransGen Biotech, Beijing, China). The PCR conditions were as follows: Denaturation at 95°C for 2 minutes, then 25 cycles of 30 seconds denaturation at 94°C, 30 seconds annealing at 55°C, and 30 seconds extension at 72°C, followed by a final extension at 72°C for 5 minutes. Amplicons produced from different intestinal luminal content samples were sent to a commercial company (BGI Genomic Lab, Tai Po Industrial Zone, New Territories, Hong Kong, China) for sequencing on the Illumina MiSequencing platform.
