title: The effect of dietary supplementation with spent cider yeast on the swine distal gut microbiome- Day 21 Control
design: "There is an increasing need for alternatives to antibiotics for promoting animal health, given the increasing problems associated with antibiotic resistance. In this regard, we evaluated spent cider yeast as a potential probiotic for modifying the gut microbiota in weanling pigs using pyrosequencing of 16S rRNA gene libraries.                                                                                                                         Total metagenomic DNA was extracted from individual feacal samples using the QIAamp DNA Stool Mini Kit (Qiagen, West Sussex, UK) according to manufacturer?s instructions. The microbial composition of these samples was evaluated by pyrosequencing of 16S rRNA tags (V4 region: 239 nucleotide long) amplified using universal 16S primers predicted to bind 94% of all 16S genes that is the forward primer F1 (5'-AYTGGGYDTAAAGNG-3') and a combination of four reverse primers R1 (5'-TACCRGGGTHTCTAATCC-3'), R2 (5'- TACCAGAGTATCTAATTC-3'), R3 (5'-CTACDSRGGTMTCTAATC-3') and R4 (5'-TACNVGGGTATCTAATC-3'). The primers incorporated the proprietary 19-mer sequences at the 5'-end to allow emulsion-based clonal amplification for the 454 pyrosequencing system. Unique molecular identifier (MID) tags were incorporated between the adaptamer and the target-specific primer sequence, to allow identification of individual sequences from pooled amplicons. Amplicons were cleaned using the Qiagen PCR purification kit (Qiagen, West Sussex, UK) and sequenced on a 454 sequencer FLX Titanium platform (MWG, Ebersberg, Germany) according to 454 protocols."