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Characterizing transcriptional heterogeneity through pathway and gene set overdispersion analysis

Nature Methods volume 13, pages 241–244 (2016)Cite this article

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Abstract

The transcriptional state of a cell reflects a variety of biological factors, from cell-type-specific features to transient processes such as the cell cycle, all of which may be of interest. However, identifying such aspects from noisy single-cell RNA-seq data remains challenging. We developed pathway and gene set overdispersion analysis (PAGODA) to resolve multiple, potentially overlapping aspects of transcriptional heterogeneity by testing gene sets for coordinated variability among measured cells.

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Figure 1: Overview of PAGODA.
Figure 2: PAGODA analysis of data from 3,005 mouse cortical and hippocampal cells5.
Figure 3: Transcriptional heterogeneity of 65 NPCs in embryonic mouse cortex.

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Gene Expression Omnibus

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Acknowledgements

We thank D. Usoskin, P. Ernfors and S. Linnarsson for helpful comments on the analysis approach. This work was supported by an Ellison Medical Foundation award and a US National Science Foundation (NSF) CAREER award (NSF-14-532) to P.V.K., an NSF graduate research fellowship (DGE1144152) to J.F., and US National Institutes of Health (NIH) grants U01 MH098977 (to K.Z. and J.C.) and NIH R01 NS084398 (to J.C.). G.E.K. was supported by NIH grant T32 AG00216.

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Author notes

  1. Jian-Bing Fan

    Present address: Present address: AnchorDx Corporation, International Biotech Island, Guangzhou, Guangdong, China.,

Authors and Affiliations

  1. Department of Biomedical Informatics, Harvard Medical School, Boston, Massachusetts, USA

    Jean Fan, Joseph L Herman & Peter V Kharchenko

  2. Illumina Inc., San Diego, California, USA

    Neeraj Salathia, Fiona Kaper & Jian-Bing Fan

  3. Department of Bioengineering, University of California, San Diego, California, USA

    Rui Liu & Kun Zhang

  4. Department of Molecular and Cellular Neuroscience, Dorris Neuroscience Center, The Scripps Research Institute, La Jolla, California, USA

    Gwendolyn E Kaeser, Yun C Yung & Jerold Chun

  5. Harvard Stem Cell Institute, Cambridge, Massachusetts, USA.,

    Peter V Kharchenko

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Contributions

K.Z., J.C. and P.V.K. conceived the study. N.S., R.L., G.E.K., Y.C.Y., F.K. and J.-B.F. carried out the single-cell purification and RNA-seq measurements. G.E.K. and J.C. carried out RNAscope in situ validation. J.F. and P.V.K. designed and implemented the statistical analysis approach, with the help of J.L.H. P.V.K. and J.F. wrote the manuscript with the help of J.C. and K.Z.

Corresponding author

Correspondence to Peter V Kharchenko.

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Competing interests

N.S. and F.K. are a current employees and shareholders of Illumina, Inc.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Notes 1–3 (PDF 9354 kb)

Supplementary Software

Source code: SCDE R Package (ZIP 1862 kb)

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Fan, J., Salathia, N., Liu, R. et al. Characterizing transcriptional heterogeneity through pathway and gene set overdispersion analysis. Nat Methods 13, 241–244 (2016). https://doi.org/10.1038/nmeth.3734

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