H1
Sequencing of the V3V4 region of the 16S rRNA gene in fish mucosal samples was performed using the Illumina MiSeq platform at the Unidad de Genmica del Parque Cientfico de Madrid (FPCM) following standard company procedures. The DNA concentration was measured with PicoGreen (Thermo Fisher Scientific) and 3 ng of each sample were used for the first PCR round, using the Q5 hot Start High-Fidelity DNA Polymerase (New England Biolabs) in a final reaction volume of 25 l. The primers used were: CS1-341F (5-ACACTGACGACATGGTTCTACACCTACGGGNGGCWGCAG-3) and CS2-805R (5- TACGGTAGCAGAGACTTGGTCTGACTACHVGGGTATCTAATCC-3). Cycling conditions were 98C 30 s; followed by 26 cycles of 98C 10 s, 50C 20 s, and 72C 20 s; with a final elongation step at 72C 2 min. A second PCR round was conducted using 1 l of the first PCR reaction in a 20 l reaction volume. The primers used were F: 5-AATGATACGGCGACCACCGAGATCTACACTGACGACATG GTTCTACA-3 and R: 5-CAAGCAGAAGACGGCATACGAGAT- [10 nucleotides barcode]-TACGGTAGCAGAGACTTGGTCT-3. Cycling conditions were the same as in the first round with the exception that only 12 cycles were used. PCR products were checked and quantified with Bioanalyzer (Agilent) and purified with AMPure Beads (Beckman Coulter). The amplicon pool was quantified by qPCR using Kapa-SYBR FAST qPCR kit for LightCycler480 and a reference library from the Unidad de Genmica FPCM. An equimolar pool of amplicons was sequenced following the manufacturers instructions and using the MiSeq Reagent Kit v3 600 Cycles.