in vitro fermentation pig feces
16S rRNA genes were amplified using a primer set corresponding to primers AGGATTAGATACCCTGGTA and CRRCACGAGCTGACGAC. These PCR primers target the V5 and V6 hypervariable 16S rRNA region. The forward primer contained the sequence of the Titanium A adaptor (5'-CCATCTCATCCCTGCGTGTCTCCGACTCAG-3') and a barcode sequence. The reverse primer contained the sequence of Titanium B adaptor (5’-CCTATCCCCTGTGTGCCTTGGCAGTCTCAG-3’). For each sample, a PCR mix of 100 µL was prepared containing 1 × PCR buffer, 2U of KAPA HiFi Hotstart polymerase blend and dNTPs (Kapabiosystems), 300 nM primers (Eurogentec, Liege, Belgium), and 60 ng gDNA. Thermal cycling consisted of initial denaturation at 95°C for 5 min, followed by 25 cycles of denaturation at 98°C for 20 s, annealing at 56°C for 40 s, and extension at 72°C for  20 s, with a ?nal extension of 5 min at 72°C. Amplicons were visualized on 1% agarose. Gels using GelGreen Nucleic Acid gel stain in 1x TAE (Biotium) and were cleaned using the Wizard SV Gel and PCR Clean-up System (Promega)  according to the manufacturer’s instructions. Amplicon DNA concentrations were determined using the Quant-iT PicoGreen dsDNA reagent and kit (Life Tech, Carlsbad, USA) following the manufacturer’s instructions. The final pool of DNA  was purified using Agencourt Ampure XP  Purification systems according to the manufacturer’s instructions (Agencourt Biosciences Corporation-Beckman coulter, USA) and then resuspended in 100µl of TE 1x. The  concentration of the purified pooled DNA was determined using  the Quant-iT PicoGreen dsDN reagent and kit (Life Tech, Carlsbad, USA) following the manufacturer’s instructions. Pyrosequencing was carried out using primer A on a 454 Life Sciences Genome Sequencer FLX instrument (Roche) following titanium chemistry. 