Metagenomics of pig feces
DNA was extracted from the faecal samples taken at BL, T24, T48, T72, 3d PT and 10d PT. 16S rRNA gene amplicons (V3V4 region) were generated and sequenced using the Illumina MiSeq platform. The V3-V4 variable region of the 16S rRNA gene was amplified from 240 faecal DNA samples using the 16S metagenomic sequencing library protocol (Illumina San Diego, CA). Samples were quantified and libraries were prepared for sequencing as described previously (31). In brief, the 16S rRNA amplicon sequencing libraries were prepared as follows: two PCR reactions were completed on the template DNA. DNA was firstly amplified with primers specific to the V3V4 region of the 16S rRNA gene, which also incorporate the Illumina overhang adaptor (forward primer 5TCGT CGGC AGCG TCAG ATGT GTATAAGA GACA GCCT ACGG GNGG CWGCAG; reverse primer 5GTCT CGTG GGCTCGGA GATG TGTA TAAG AGAC AGGA CTAC HVGG GTAT CTAATCC). Following this, a second PCR reaction was performed with two indexing primers (Illumina Nextera XT indexing primers) added to allow for demultiplexing. Samples were analysed on the Illumina MiSeq at the Teagasc Sequencing Centre, Moorepark, Fermoy, Co. Cork, Ireland using the Miseq 600 cycle v2 kit and following standard Illumina sequencing protocols. Following sequencing, the data was analysed to establish the effect of nisin treatment on the composition of the gut microbiota. The average number of OTUs per sample was 123,814 +/- 34,649 with a range between 24,578 and 231,778.