title: Exploiting single-cell expression to characterize co-expression replicability.
abstract: BACKGROUND
  Co-expression networks have been a useful tool for functional genomics, providing important clues about the cellular and biochemical mechanisms that are active in normal and disease processes. However, co-expression analysis is often treated as a black box with results being hard to trace to their basis in the data. Here, we use both published and novel single-cell RNA sequencing (RNA-seq) data to understand fundamental drivers of gene-gene connectivity and replicability in co-expression networks.
  
  
  RESULTS
  We perform the first major analysis of single-cell co-expression, sampling from 31 individual studies. Using neighbor voting in cross-validation, we find that single-cell network connectivity is less likely to overlap with known functions than co-expression derived from bulk data, with functional variation within cell types strongly resembling that also occurring across cell types. To identify features and analysis practices that contribute to this connectivity, we perform our own single-cell RNA-seq experiment of 126 cortical interneurons in an experimental design targeted to co-expression. By assessing network replicability, semantic similarity and overall functional connectivity, we identify technical factors influencing co-expression and suggest how they can be controlled for. Many of the technical effects we identify are expression-level dependent, making expression level itself highly predictive of network topology. We show this occurs generally through re-analysis of the BrainSpan RNA-seq data.
  
  
  CONCLUSIONS
  Technical properties of single-cell RNA-seq data create confounds in co-expression networks which can be identified and explicitly controlled for in any supervised analysis. This is useful both in improving co-expression performance and in characterizing single-cell data in generally applicable terms, permitting cross-laboratory comparison within a common framework.
authors.affiliation: Cold Spring Harbor Laboratory, One Bungtown Road, Cold Spring, Harbor, NY, 11724, USA.
authors.forename: Megan
authors.initials: M
authors.lastname: Crow
authors.affiliation: Cold Spring Harbor Laboratory, One Bungtown Road, Cold Spring, Harbor, NY, 11724, USA.
authors.forename: Anirban
authors.initials: A
authors.lastname: Paul
authors.affiliation: Cold Spring Harbor Laboratory, One Bungtown Road, Cold Spring, Harbor, NY, 11724, USA.
authors.forename: Sara
authors.initials: S
authors.lastname: Ballouz
authors.affiliation: Cold Spring Harbor Laboratory, One Bungtown Road, Cold Spring, Harbor, NY, 11724, USA.
authors.forename: Z Josh
authors.initials: ZJ
authors.lastname: Huang
authors.affiliation: Cold Spring Harbor Laboratory, One Bungtown Road, Cold Spring, Harbor, NY, 11724, USA. jgillis@cshl.edu.
authors.forename: Jesse
authors.initials: J
authors.lastname: Gillis
mesh_terms: D000818:Animals; D002469:Cell Separation / Q000379:methods; D020869:Gene Expression Profiling / Q000379:methods* / Q000592:standards; D053263:Gene Regulatory Networks; D051379:Mice; D015203:Reproducibility of Results; D017423:Sequence Analysis, RNA / Q000379:methods* / Q000592:standards; D059010:Single-Cell Analysis / Q000379:methods* / Q000592:standards
keywords: Autism; Brain; Co-expression; Interneuron; Meta-analysis; Network; Normalization; RNA-seq; Single cell