(A) High-content small interfering RNA (siRNA) cell-based screen using reverse transfection of the library in media containing serum for 72 hr, followed by 24 hr serum starvation, fixation and DAPI staining. Subsequent fluorescent imaging and algorithmic analysis performed for all pooled siRNAs. To assess ciliary candidates for the positive training, we used SYSCILIA gold standard (SCGSv1) and for the negative training the human metabolome database (HMDB 3.0) as well as a manually curated housekeeping gene data set. FDR, false discovery rate. (B) Segmentation algorithm for cytoplasm and cilia detection: (1) detected nuclei from DAPI channel, (2) nuclear automated segmentation, (3) cell outline automated using cytoplasm_detection_D of the program Acapella, and (4) cilia automated detection and segmentation. Images have been modified for illustration purposes. Scale bar: 10 μm. (C) Representative images of serum-starved SEMG cells without siRNA showing basal ciliation (small green rods in EGFP channel). Red (mCherry) marks cells in S/G2/M phase of the cycle, green (EGFP) marks cilia, blue (DAPI) marks nuclei. siRNAs used as positive controls: KIF3A interferes with ciliation but not cell cycle. ACTR3 shows increased length of cilia (Kim et al., 2010). CRNKL1 implicated in cell cycle progression (Zhang et al., 1991) and showed increased mCherry nuclei and reduced ciliation. Scale bar: 10 μm. (D) Receiver operating characteristic (ROC) for the classifier, which used features from three data sources. Dashed line: theoretical random classifier. (E) Precision-recall curve for the final classifier. (F) Median value (red center bar) and interquartile ranges (blue box) box plot of the classifier scores for the corresponding number of supporting number of evidences (NOEs) in Cildb and the genes used as negative and positive training examples. The indicated contrasts were found significant(*) with a highest value of p < 1.03 × 10−4 (one-tailed Wilcoxon's Rank sum test). (G) Same as (F), limited to the NOEs from humans only. The indicated contrasts were found significant(*) with a highest value of p < 1.43 × 10−10 (one-tailed Wilcoxon's Rank sum test). See Figure 1—figure supplement 1, 2 for the prediction score on the gold standard and candidates as well as the visible improvement of the ROC curve and precision–recall curve.