Figure 3
Gene expression profiling of TDS between RNA-Seq and nCounter. (a) Of 222 genes identified as TDS by RNA-Seq, 97 showed a consistent direction of differential expression between tg-mel and wt in two separate tests. These 97 genes were weighted differently to reflect their expression patterns within the tg-mel and wt medaka populations and were retained as TDS genes for further test. Twenty three of these 97 genes were given a weight of two because their AUC values of ROC curve in each of the two tests were above 0.8. Weights of the remaining 74 genes were determined by the AUC values of the ROC curves. (b) Spearman ranking correlation analysis was performed on RNA-Seq data for ten tg-mel fish and ten wt individuals and two independent NanoString nCounter assessed medaka samples (a total of 35 wt and 35 tg-mel medaka). Genotypes (i.e., tg-mel or wt) clustered together independent of methodology. (c–f) The TDS gene expression pattern itself (i.e., Reference TDS) serves as a standard to calculate the TDS expression pattern; a reversed pattern of Reference TDS simulates a compound that can make each TDS gene return to a non-diseased expression level (i.e., Model compound). The TDS expression profile of each of 20 tg-mel individuals was compared to that of each of 20 wt medaka, resulting in 400 possible comparisons. Using the Log2FC values generated in these 400 comparisons, 400 incidences were simulated by randomly choosing Log2FC values to estimate the false positive and false negative rate of Z-score and Ks_drug score statistics. (c) Weighted TDS activation Z-scores were calculated for the Reference TDS, model compounds, 400 simulated datasets and 400 TDS expression patterns. (d) A Z-score of two resulted in 0% false negatives and 5% false positives in identifying TDS activation status. (e) The Ks_drug score was calculated for the Reference TDS, model compounds, 400 simulated datasets and 400 TDS expression patterns. (f) A Ks_drug score of 0.27 resulted in 0% false negatives and 5% false positives in identifying TDS activation pattern.
