title: DNA-Seq of porcine
design: Microbial DNA was extracted. Final DNA concentration and purity were determined using a NanoDrop 2000 UV-vis spectrophotometer (Thermo Scientific, Waltham, MA, USA), and DNA quality was determined using 1% agarose gel electrophoresis. The V3-V4 hypervariable regions of the bacterial 16S rRNA gene were amplified with primers 338F (5-ACTCCTACGGGAGGCAGCAG-3) and 806R (5-GGACTACHVGGGTWTCTAAT-3) using a thermocycler PCR system (GeneAmp 9700, Applied biosystems, Foster City, CA, USA). PCR was conducted as follows: 3 min of denaturation at 95 , 27 cycles: 30 s at 95 , 30 s of annealing at 55 , 45 s of elongation at 72 , and a final extension at 72  for 10 min. PCR was performed in triplicate in 20-L mixtures containing 4 L of 5FastPfu Buffer, 2 L of 2.5 mM dNTPs, 0.8 L of each primer (5 M), 0.4 L of FastPfu polymerase, and 10 ng of template DNA. The resulting PCR products were extracted from a 2% agarose gel and further purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and quantified using QuantiFluor-ST (Promega, Madison, WI, USA) according to the manufacturers protocol. Purified amplicons were pooled in equimolar concentrations and paired-end sequencing was performed (2  300) on an Illumina MiSeq platform (Illumina, San Diego, CA, USA) according to standard protocols.