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Normalizing single-cell RNA sequencing data: challenges and opportunities

Nature Methods volume 14, pages 565–571 (2017)Cite this article

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Abstract

Single-cell transcriptomics is becoming an important component of the molecular biologist's toolkit. A critical step when analyzing data generated using this technology is normalization. However, normalization is typically performed using methods developed for bulk RNA sequencing or even microarray data, and the suitability of these methods for single-cell transcriptomics has not been assessed. We here discuss commonly used normalization approaches and illustrate how these can produce misleading results. Finally, we present alternative approaches and provide recommendations for single-cell RNA sequencing users.

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Figure 1: Cell- and gene-specific effects in RNA-seq experiments.
Figure 2: Comparison of bulk-based normalization methods in real and simulated data sets.
Figure 3: ERCC spike-ins can be used to estimate mRNA content.

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Acknowledgements

We thank several members of the Marioni laboratory (European Molecular Biology Laboratory - European Bioinformatics Institute, EMBL-EBI; Cancer Research UK - Cambridge Institute, CRUK-CI) for support and discussions throughout the preparation of this manuscript. In particular, we are grateful to A. Lun (CRUK-CI) for constructive comments on an earlier version of the manuscript. We are also grateful to UC Berkeley collaborator J. Ngai and his group members. C.A.V., A.S., and J.C.M. acknowledge core EMBL funding. C.A.V. was supported by core MRC funding (MRC MC UP 0801/1) and by The Alan Turing Institute under the EPSRC grant no. EP/N510129/1. J.C.M. acknowledges core support from CRUK. A.S. acknowledges funding from the Wellcome Trust Strategic Award 105031/D/14/Z, “Tracing early mammalian lineage decisions by single-cell genomics.” D.R. and S.D. are supported by the US National Institutes of Health BRAIN Initiative grant no. U01 MH105979 (PI, J. Ngai).

Author information

Author notes

  1. Davide Risso

    Present address: Present address: Division of Biostatistics and Epidemiology, Department of Healthcare Policy and Research, Weill Cornell Medicine, New York, New York, USA.,

  2. Catalina A Vallejos, Davide Risso and Antonio Scialdone: These authors contributed equally to this work.

Authors and Affiliations

  1. MRC Biostatistics Unit, Cambridge Institute of Public Health, Cambridge, UK

    Catalina A Vallejos

  2. EMBL-European Bioinformatics Institute, Wellcome Genome Campus, Cambridge, UK

    Catalina A Vallejos, Antonio Scialdone & John C Marioni

  3. The Alan Turing Institute, British Library, London, UK

    Catalina A Vallejos

  4. Department of Statistical Science, University College London, London, UK

    Catalina A Vallejos

  5. Division of Biostatistics, School of Public Health, University of California, Berkeley, Berkeley, California, USA.,

    Davide Risso & Sandrine Dudoit

  6. Department of Statistics, University of California, Berkeley, Berkeley, California, USA.,

    Sandrine Dudoit

  7. Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Cambridge, UK

    John C Marioni

  8. Wellcome Trust Sanger Institute, Wellcome Genome Campus, Cambridge, UK

    John C Marioni

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  1. Catalina A Vallejos
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Contributions

C.A.V., D.R., and A.S. performed analyses. C.A.V., D.R., A.S., S.D., and J.C.M. wrote the manuscript. S.D. and J.C.M. supervised the study.

Corresponding authors

Correspondence to Sandrine Dudoit or John C Marioni.

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Vallejos, C., Risso, D., Scialdone, A. et al. Normalizing single-cell RNA sequencing data: challenges and opportunities. Nat Methods 14, 565–571 (2017). https://doi.org/10.1038/nmeth.4292

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