16S microbial community analysis of pig intestine: spiral colon
DNA extracted from digesta and mucosa was processed with an Illumina protocol for creating 16S rRNA sequencing libraries. Extracted genomic DNA was amplified with Illumina indexed adaptor primers (V4 Schloss primers). The PCR reaction contained 5 l of PCR buffer, 1 l of 10 mM dNTPs, 1 l of 25 mM of MgCl2, 2.5 of l of each primer, 0.25 l of Hot Start Taq (Qiagen Inc.), 32.8 l of molecular grade water, and 5 l of bacterial community DNA. Amplicons were purified with QIAquick PCR Purification kit (Qiagen Inc.) following the manufacturers recommendations. The effectiveness of the clean-up was checked by agarose gel electrophoresis, followed by quantification with a Qubit (Fisher Scientific, Ottawa, ON). Indexed DNA libraries were normalized to 1.5 ng l-1 and pooled together. A PhiX control (10%) was run with the normalized DNA library, and both were denatured and diluted to 8 pM prior to loading into the MiSeq Reagent Kit v2 500-cycle (Illumina, San Diego, CA).