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

Assembled viral sequence contigs from the appropriate sample were imported into Primer Express v3.0.1 software (Thermo Fisher Scientific, Waltham, MA) where qPCR Taqman assays were designed using default parameters (Supplementary file 1) . These assays were then tested using the Fluidigm BioMark microfluidics-based qPCR system following the same protocol as described below except with the new viral primer pairs included in the STA step and controls. From these initial screens, the most consistent assay was chosen and APC standards were constructed to include in future Fluidigm BioMark qPCR microbe panels. The assay-specific theoretical limit of detection was calculated as previously described (Miller et al., 2016) . The limit of detection was applied to categorise fish with amplifications above the 95% detection threshold that is the concentration of the analyte in the sample matrix that would be detected with high statistical certainty (95% of the time). Epidemiological maps were generated using these data with the limit of detection applied. The maps were created within R using ggplot2 (Wickham, 2016) and ggmap (Kahle and Wickham, 2013) .