Endangered wild salmon infected by newly discovered viruses

Mordecai, Gideon J; Miller, Kristina M; Di Cicco, Emiliano; Schulze, Angela D; Kaukinen, Karia H; Ming, Tobi J; Li, Shaorong; Tabata, Amy; Teffer, Amy; Patterson, David A; Ferguson, Hugh W; Suttle, Curtis A

Total RNA from the mixed tissue samples was evaluated for quality using the Total RNA Pico chip on the Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA) and quantified using the Qubit RNA Br kit (Invitrogen, Carlsbad, CA). A 1/100 dilution of the ERCC RNA Spike-In control mix 1 (Ambion, Carlsbad, CA) was added to each total RNA sample prior to ribosomal depletion and library preparation. The sequencing libraries and ribosomal removal were performed using the Epicentre ScriptSeq Complete Gold Kit (Epidemiology) (Illumina, San Diego, CA) according to manufacturer's instructions and included a positive control (Universal Human Reference RNA) (Agilent, Santa Clara, CA) and negative control (no total RNA). The rRNA depleted total RNA was purified using the Zymo RNA Clean and Concentrate-5 kit (Zymo Research, Irvine, CA) according to manufacturer's instructions and quantified using the Qubit RNA HS kit (Invitrogen, Carlsbad, CA). The ScriptSeq Index reverse primers were added to the cDNA during the final amplification step which involved 14 cycles. The 3'-terminal tagged cDNA and final amplified library were purified using the Agencourt AMPure XP system (Beckman Coulter, Brea, CA). The final library size was determined using the HS DNA chip on the Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA) and the concentration was determined using the Qubit dsDNA HS kit (Invitrogen, Carlsbad, CA). Sample libraries were normalised to 4 nM, pooled appropriately and denatured and diluted to obtain a final library of 17pM. Prior to loading into a v3 2 Â 300 bp kit (Illumina, San Diego, CA), 2% phiX was spiked in. Finally, a paired-end 251 bp sequencing run was performed on the Illumina MiSeq System (Illumina, San Diego, CA), with four samples barcoded and pooled for each run.