Distribution Analyzer, a methodology for identifying and clustering outlier conditions from single-cell distributions, and its application to a Nanog reporter RNAi screen

Julian A. Gingold, Ed S. Coakley, Jie Su, Dung-Fang Lee, Zerlina Lau, Hongwei Zhou, Dan P. Felsenfeld, Christoph Schaniel, Ihor R. Lemischka

Chemical or small interfering (si) RNA screens measure the effects of many independent experimental conditions, each applied to a population of cells (e.g., all of the cells in a well). High-content screens permit a readout (e.g., fluorescence, luminescence, cell morphology) from each cell in the population. Most analysis approaches compare the average effect on each population, precluding identification of outliers that affect the distribution of the reporter in the population but not its average. Other approaches only measure changes to the distribution with a single parameter, precluding accurate distinction and clustering of interesting outlier distributions. We describe a methodology to identify outlier conditions by considering the cell-level measurements from each condition as a sample of an underlying distribution. With appropriate selection of a distance metric, all effects can be embedded in a fixed-dimensionality Euclidean basis, facilitating identification and clustering of biologically interesting outliers. We demonstrate that measurement of distances with the Hellinger distance metric offers substantial computational efficiencies over alternative metrics. We validate this methodology using an RNA interference (RNAi) screen in mouse embryonic stem cells (ESC) with a Nanog reporter. The methodology clusters effects of multiple control siRNAs into their true identities better than conventional approaches describing the median cell fluorescence or the commonly used Kolmogorov-Smirnov distance between the observed fluorescence distribution and the null distribution. It identifies outlier genes with effects on the reporter distribution that would have been missed by other methods. Among them, siRNA targeting Chek1 leads to a wider Nanog reporter fluorescence distribution. Similarly, siRNA targeting Med14 or Med27 leads to a narrower Nanog reporter fluorescence distribution. We confirm the roles of these three genes in regulating pluripotency by mRNA expression and alkaline phosphatase staining using independent short hairpin (sh) RNAs. Using our methodology, we describe each experimental condition by a probability distribution. Measuring distances between probability distributions permits a multivariate rather than univariate readout. Clustering points derived from these distances allows us to obtain greater biological insight than methods based solely on single parameters. We find several outliers from a mouse ESC RNAi screen that we confirm to be pluripotency regulators. Many of these outliers would have been missed by other analysis methods.