title: Distribution Analyzer, a methodology for identifying and clustering outlier conditions from single-cell distributions, and its application to a Nanog reporter RNAi screen.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: The Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA. julian.gingold@mssm.edu.|Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA. julian.gingold@mssm.edu.
authors.forename: Julian A
authors.initials: JA
authors.lastname: Gingold
authors.affiliation: Program in Applied Mathematics, Yale University, New Haven, CT, 06511, USA. ed.s.coakley@gmail.com.
authors.forename: Ed S
authors.initials: ES
authors.lastname: Coakley
authors.affiliation: The Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA. sujiepkubio@gmail.com.|Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA. sujiepkubio@gmail.com.|Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA. sujiepkubio@gmail.com.
authors.forename: Jie
authors.initials: J
authors.lastname: Su
authors.affiliation: The Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA. dung-fang.lee@mssm.edu.|Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA. dung-fang.lee@mssm.edu.
authors.forename: Dung-Fang
authors.initials: DF
authors.lastname: Lee
authors.affiliation: Integrated Screening Core, Experimental Therapeutics Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA. wingshan.lau@mssm.edu.
authors.forename: Zerlina
authors.initials: Z
authors.lastname: Lau
authors.affiliation: The Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA. hongwei.zhou@mssm.edu.|Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA. hongwei.zhou@mssm.edu.
authors.forename: Hongwei
authors.initials: H
authors.lastname: Zhou
authors.affiliation: The Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA. dan@felsenfeld.com.|Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA. dan@felsenfeld.com.|Integrated Screening Core, Experimental Therapeutics Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA. dan@felsenfeld.com.
authors.forename: Dan P
authors.initials: DP
authors.lastname: Felsenfeld
authors.affiliation: The Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA. christoph.schaniel@mssm.edu.|Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA. christoph.schaniel@mssm.edu.|Department of Pharmacology and Systems Therapeutics, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA. christoph.schaniel@mssm.edu.
authors.forename: Christoph
authors.initials: C
authors.lastname: Schaniel
authors.affiliation: The Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA. ihor.lemischka@mssm.edu.|Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA. ihor.lemischka@mssm.edu.|Department of Pharmacology and Systems Therapeutics, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA. ihor.lemischka@mssm.edu.
authors.forename: Ihor R
authors.initials: IR
authors.lastname: Lemischka
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D016000:Cluster Analysis; D019295:Computational Biology / Q000379:methods*; D017930:Genes, Reporter; D016678:Genome; D018398:Homeodomain Proteins / Q000235:genetics*; D056892:Mediator Complex / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D051379:Mice; D000066450:Mouse Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D000071317:Nanog Homeobox Protein; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D014212:Tretinoin / Q000494:pharmacology
chemical_list: D018398:Homeodomain Proteins; D056892:Mediator Complex; D000071317:Nanog Homeobox Protein; C475540:Nanog protein, mouse; D034741:RNA, Small Interfering; D014212:Tretinoin