KL_H02
DNA samples were prepared in accordance with the Illumina 16S Metagenomic Sequencing Library Preparation protocol. Amplification was directed at the V3V4 hypervariable regions of the 16S rRNA gene using the following primers: 16S Amplicon PCR Forward Primer (5 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCA-3) and 16S Amplicon PCR Reverse Primer (5-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATC-3). PCR amplification was performed on a 2720 thermal cycler (Life Technologies) using KAPA HiFi HotStart ReadyMix. Thermal cycling conditions comprised an initial denaturation step at 95C for 5 min, followed by 25 cycles of denaturation at 95C for 30 s, annealing at 55C for 30 s, and extension at 72C for 30 s, with a final extension at 72C for 5 min. Indexing of PCR products was carried out using the Illumina Index Primer set according to the manufacturers protocol. Amplicon generation was verified by electrophoresis on a 1.5% (w/v) agarose gel (Sigma-Merck, Germany) stained with SYBR Safe (Thermo Fisher Scientific, MA, USA). Amplified products were subsequently purified using the AMPure XP bead purification system (Beckman Coulter, CA, USA) following Illumina guidelines. Prior to library pooling, purified amplicons were quantified using the Qubit dsDNA High Sensitivity assay (Thermo Fisher Scientific, MA, USA) on a Qubit 3.0 fluorometer. Libraries were then normalised and pooled to a final concentration of 5 nM. Paired-end sequencing (2  300 bp) was performed on an Illumina NextSeq 2000 platform using the P1 600-cycle reagent kit (Illumina, San Diego, CA, USA). All sequencing procedures were carried out at the Teagasc Moorepark Research Centre Next Generation DNA Sequencing Facility.