title: Data exploration, quality control and testing in single-cell qPCR-based gene expression experiments.
abstract: BACKGROUND
  Cell populations are never truly homogeneous; individual cells exist in biochemical states that define functional differences between them. New technology based on microfluidic arrays combined with multiplexed quantitative polymerase chain reactions now enables high-throughput single-cell gene expression measurement, allowing assessment of cellular heterogeneity. However, few analytic tools have been developed specifically for the statistical and analytical challenges of single-cell quantitative polymerase chain reactions data.
  
  
  RESULTS
  We present a statistical framework for the exploration, quality control and analysis of single-cell gene expression data from microfluidic arrays. We assess accuracy and within-sample heterogeneity of single-cell expression and develop quality control criteria to filter unreliable cell measurements. We propose a statistical model accounting for the fact that genes at the single-cell level can be on (and a continuous expression measure is recorded) or dichotomously off (and the recorded expression is zero). Based on this model, we derive a combined likelihood ratio test for differential expression that incorporates both the discrete and continuous components. Using an experiment that examines treatment-specific changes in expression, we show that this combined test is more powerful than either the continuous or dichotomous component in isolation, or a t-test on the zero-inflated data. Although developed for measurements from a specific platform (Fluidigm), these tools are generalizable to other multi-parametric measures over large numbers of events.
  
  
  BACKGROUND
  All results presented here were obtained using the SingleCellAssay R package available on GitHub (http://github.com/RGLab/SingleCellAssay).
authors.affiliation: Department of Statistics, University of Washington, Seattle, WA 98195, USA.
authors.forename: Andrew
authors.initials: A
authors.lastname: McDavid
authors.forename: Greg
authors.initials: G
authors.lastname: Finak
authors.forename: Pratip K
authors.initials: PK
authors.lastname: Chattopadyay
authors.forename: Maria
authors.initials: M
authors.lastname: Dominguez
authors.forename: Laurie
authors.initials: L
authors.lastname: Lamoreaux
authors.forename: Steven S
authors.initials: SS
authors.lastname: Ma
authors.forename: Mario
authors.initials: M
authors.lastname: Roederer
authors.forename: Raphael
authors.initials: R
authors.lastname: Gottardo
mesh_terms: D020869:Gene Expression Profiling / Q000379:methods* / Q000592:standards; D006801:Humans; D046210:Microfluidic Analytical Techniques; D015233:Models, Statistical; D011786:Quality Control; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D059010:Single-Cell Analysis