16S rRNA gut microbiome of juvenile Atlantic salmon
DNA from each gut sample was subjected to PCR of a region covering the V3-V4 region of the 16S rRNA gene using the universal bacterial primer pair S-D-Bact-0341-b-S-17 (5-CCTACGGGNGGCWGCAG-3) / S-D-Bact-0785-a-A-21 (5-GACTACHVGGGTATCTAATCC-3) (Klindworth et al., 2013). The PCR reactions were set up using 10 ng template DNA, 5 l 5X Phusion HF Buffer (New England BioLabs), 200 M dNTPs, 0.5 M of forward and 0.5 M reverse primers containing Illumina overhang adapters, 0.2 l Phusion High-Fidelity DNA polymerase and nuclease-free water to a total reaction volume of 25 l. Thermocycler conditions were set up with and initial denaturation step at 98C for 30 s followed by 30 cycles of: Denaturation at 98C for 10 s, annealing at 52C for 30 s and extension at 72C for 30 s. Final extension was set to 72C for 5 min. Libraries were multiplexed using Nextera XT v2 barcodes (Illumina), normalised on a Sequel-Prep Normalization Plates (ThermoFisher Scientific) and sequenced on a MiSeq desktop sequencer (Illumina) using v3 chemistry and 2 x 300 cycles.