16s rRNA of gill from rainbow trout
Sequencing libraries were prepared from the purified amplicon libraries using a second PCR. Each PCR reaction (25L) contained PCRBIO HiFi buffer (1x), PCRBIO HiFi Polymerase (1 U/reaction) (PCRBiosystems, UK), adaptor mix(400 nM of each forward and reverse) and up to 10 ng of amplicon library template. PCR was conducted with thefollowing program: Initial denaturation at 95 C for 2 min, 8 cycles of amplification (95 C for 20 s, 55 C for 30 s,72 C for 60 s) and a final elongation at 72 C for 5 min. The resulting sequencing libraries were purified using thestandard protocol for Agencourt Ampure XP Beads (Beckman Coulter, USA) with a bead to sample ratio of 4:5. DNAwas eluted in 25 L of nuclease free water (Qiagen, Germany). DNA concentration was measured using Qubit dsDNAHS Assay kit (Thermo Fisher Scientific, USA). Gel electrophoresis using Tapestation 2200 and D1000/High sensitivityD1000 screentapes (Agilent, USA) was used to validate product size and purity of a subset of sequencing libraries.