Sybr egel

Polymerase chain reaction (PCR) and sequencing were performed by the Australian Genome Research Facility. PCR amplicons were generated using the primers 341F (5′ CCTAYGGGRBGCASCAG 3′) and 806R (5′ GGACTACNNGGGTATCTAAT 3′) (Yu et al., 2005 (link)). Thermocycling was completed with an Applied Biosystem 384 Veriti and using AmpliTaq Gold 360 master mix (Life Technologies, Australia) for the primary PCR. The first stage PCR was cleaned using magnetic beads, and samples were visualized on 2% Sybr Egel (Thermo-Fisher). A secondary PCR to index the amplicons was performed with TaKaRa Taq DNA Polymerase (Clontech). The resulting amplicons were cleaned again using magnetic beads, quantified by fluorometry (Promega Quantifluor) and normalized. The equimolar pool was cleaned a final time using magnetic beads to concentrate the pool and then measured using a High-Sensitivity D1000 Tape on an Agilent 2200 TapeStation. The pool was diluted to 5 nM and molarity was confirmed again using a High-Sensitivity D1000 Tape. This was followed by sequencing on an Illumina MiSeq instrument with a V3 (600 cycles) kit (Illumina).

DNA from microbial samples was isolated using the DNeasy PowerLyzer PowerSoil Kit (Qiagen). PCR amplification and sequencing were performed at the Australian Genome Research Facility using the primers and conditions outlined in S1 Table. Thermocycling was completed with an Applied Biosystem 384 Veriti and using Platinum SuperFi mastermix (Life Technologies, Australia) for the primary PCR. The first stage PCR was cleaned using magnetic beads, and samples were visualised on 2% Sybr Egel (Thermo-Fisher). A secondary PCR to index the amplicons was performed with TaKaRa PrimeStar Max DNA Polymerase (Clontech). The equimolar pool was cleaned a final time using magnetic beads to concentrate the pool and then measured using a High-Sensitivity D1000 Tape on an Agilent 2200 TapeStation. The pool was diluted to 5nM and molarity was confirmed again using a Qubit High Sensitivity dsDNA assay (ThermoFisher). Amplicons were quantified with a dsDNA fluorometry assay (Promega Quantifluor) after two rounds of PCR. Sequencing took place on an Illumina MiSeq (San Diego, CA, USA) with a V3, 600 cycle kit (2 x 300 base pairs paired-end) and a 25% PhiX spike-in to improve nucleotide diversity. A variation of the Illumina 16S metagenomics sequencing protocol was utilized for this purpose.

Fecal samples, collected on day 20, from three pigs in each group were sequenced for diversity profiling to compare the microbiome between the two groups. Polymerase chain reaction (PCR) amplification and sequencing were performed by the Australian Genome Research Facility (Melbourne, VIC). PCR amplicons were generated using the 341F and 806R primers to amplify the V3-V4 region of the 16S gene. Thermocycling was completed with an Applied Biosystem 384 Veriti and using Platinum SuperFi mastermix (Life Technologies, Australia) for the primary PCR. The first stage PCR was cleaned using magnetic beads, and samples were visualized on 2% Sybr Egel (Thermo-Fisher). A secondary PCR to index the amplicons was performed with TaKaRa Taq DNA Polymerase (Clontech). The resulting amplicons were cleaned again using magnetic beads, quantified by fluorometry (Promega Quantifluor) and normalized. The eqimolar pool was cleaned a final time using magnetic beads to concentrate the pool and then measured using a High-Sensitivity D1000 Tape on an Agilent 2200 TapeStation. The pool was diluted to 5 nM and molarity was confirmed again using a High-Sensitivity D1000 Tape. This was followed by sequencing on an Illumina MiSeq (San Diego, CA, USA) with a V3, 600 cycle kit (2 × 300 base pairs paired-end).