16S rRNA sequencing of Oncorhynchus mykiss affected by RMS: positive control
Library preparation was performed using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, Cat. No. E7645) as follows: PCR amplification was performed using 15 L of Phusion High - Fidelity PCR Master Mix (New England Biolabs), 0.2 M of primers (forward and reverse), and approximately 10 ng template DNA, and thermal cycling conditions were set to 98 for 1 min for initial denaturation, followed by 30 cycles at 98C for 10 s, 50C for 30 s, 72C for 30 s, and final elongation at 72C for 5 min. The quality control of the PCR products was conducted with a capillary electrophoresis system (Agilent 5400). Amplicons were purified using magnetic beads. Equimolar amounts of PCR products from each sample were pooled, size-selected, end-repaired and A-tailed, and further ligated with Illumina adapters. Libraries were sequenced on the Illumina NovaSeq 6000 platform to generate 250bp paired-end raw reads, with a minimum of 30K reads/sample.