title: 16S rRNA gene amplicon sequencing of swine ileal digesta
design: After extraction, DNA was sent to the ISU DNA facility for sequencing using the Illumina MiSeq platform. Briefly, the genomic DNA from each sample was amplified using Platinum Taq DNA Polymerase (Thermo Fisher Scientific, Waltham, MA) with one replicate per sample using universal 16S rRNA gene bacterial primers [515F (5 GTGYCAGCMGCCGCGGTAA-3; (Parada et al., 2016)) and 806R (5- GGACTACNVGGGTWTCTAAT-3; (Apprill et al., 2015))] amplifying the variable region V4. All samples underwent PCR (QIAquick 96 PCR Purification Kit; Qiagen Sciences Inc, Germantown, MD) with an initial denaturation step at 94C for 3 min, followed by 45 s of denaturing at 94C, 20 s of annealing at 50C, and 90 s of extension at 72C. This was repeated for 35 total PCR cycles and finished with a 10 min extension at 72C. PCR bar-coded amplicons were mixed at equal molar ratios and used for Illumina MiSeq paired-end sequencing with 150 bp read length and cluster generation with 10% PhiX control DNA on an Illumina MiSeq platform (Illumina Inc., San Diego, CA).