Article

Multiplexed droplet single-cell RNA-sequencing using natural genetic variation

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Abstract

Droplet single-cell RNA-sequencing (dscRNA-seq) has enabled rapid, massively parallel profiling of transcriptomes. However, assessing differential expression across multiple individuals has been hampered by inefficient sample processing and technical batch effects. Here we describe a computational tool, demuxlet, that harnesses natural genetic variation to determine the sample identity of each droplet containing a single cell (singlet) and detect droplets containing two cells (doublets). These capabilities enable multiplexed dscRNA-seq experiments in which cells from unrelated individuals are pooled and captured at higher throughput than in standard workflows. Using simulated data, we show that 50 single-nucleotide polymorphisms (SNPs) per cell are sufficient to assign 97% of singlets and identify 92% of doublets in pools of up to 64 individuals. Given genotyping data for each of eight pooled samples, demuxlet correctly recovers the sample identity of >99% of singlets and identifies doublets at rates consistent with previous estimates. We apply demuxlet to assess cell-type-specific changes in gene expression in 8 pooled lupus patient samples treated with interferon (IFN)-β and perform eQTL analysis on 23 pooled samples.

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Acknowledgements

M.S. and C.J.Y. are supported by NIH R01AR071522 and R21AI133337. S.T. is supported by NIH F30DK115167. H.M.K. is supported by U01HL137182. N.Z. is supported by NIH K25HL121295, R03DE025665, and Department of Defense W81WH-16-2-0018.

Author information

Author notes

    • Hyun Min Kang
    • , Meena Subramaniam
    •  & Sasha Targ

    These authors contributed equally to this work.

Affiliations

  1. Department of Biostatistics and Center for Statistical Genetics, University of Michigan School of Public Health, Ann Arbor, Michigan, USA.

    • Hyun Min Kang
  2. Biological and Medical Informatics Graduate Program, University of California, San Francisco, San Francisco, California, USA.

    • Meena Subramaniam
    • , Sasha Targ
    •  & Rachel E Gate
  3. Institute for Human Genetics (IHG), University of California, San Francisco, San Francisco, California, USA.

    • Meena Subramaniam
    • , Sasha Targ
    • , Lenka Maliskova
    • , Eunice Wan
    • , Simon Wong
    • , Rachel E Gate
    • , Noah Zaitlen
    • , Lindsey A Criswell
    •  & Chun Jimmie Ye
  4. Institute for Computational Health Sciences, University of California, San Francisco, San Francisco, California, USA.

    • Meena Subramaniam
    • , Sasha Targ
    • , Rachel E Gate
    •  & Chun Jimmie Ye
  5. Department of Epidemiology and Biostatistics, University of California, San Francisco, San Francisco, California, USA.

    • Meena Subramaniam
    • , Sasha Targ
    • , Rachel E Gate
    •  & Chun Jimmie Ye
  6. Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, California, USA.

    • Meena Subramaniam
    • , Sasha Targ
    • , Rachel E Gate
    •  & Chun Jimmie Ye
  7. Medical Scientist Training Program (MSTP), University of California, San Francisco, San Francisco, California, USA.

    • Sasha Targ
    •  & Elizabeth McCarthy
  8. Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, California, USA.

    • Michelle Nguyen
    •  & Alexander Marson
  9. Diabetes Center, University of California, San Francisco, San Francisco, California, USA.

    • Michelle Nguyen
    •  & Alexander Marson
  10. Innovative Genomics Institute, University of California, Berkeley, Berkeley, California, USA.

    • Michelle Nguyen
    •  & Alexander Marson
  11. Department of Neurology, University of California, San Francisco, San Francisco, California, USA.

    • Lenka Maliskova
  12. Developmental and Stem Cell Biology Graduate Program, University of California, San Francisco, San Francisco, California, USA.

    • Lauren Byrnes
  13. Department of Medicine, University of California, San Francisco, San Francisco, California, USA.

    • Cristina M Lanata
    • , Alexander Marson
    • , Noah Zaitlen
    •  & Lindsey A Criswell
  14. Rosalind Russell/Ephraim P Engleman Rheumatology Research Center, University of California, San Francisco, San Francisco, California, USA.

    • Cristina M Lanata
    •  & Lindsey A Criswell
  15. Department of Statistics, University of British Columbia, Vancouver, British Columbia, Canada.

    • Sara Mostafavi
  16. UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California, USA.

    • Alexander Marson
  17. Chan Zuckerberg Biohub, San Francisco, California, USA.

    • Alexander Marson
  18. Lung Biology Center, University of California, San Francisco, San Francisco, California, USA.

    • Noah Zaitlen
  19. Department of Orofacial Sciences, University of California, San Francisco, San Francisco, USA.

    • Lindsey A Criswell

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Contributions

H.M.K. and C.J.Y. conceived the project. M.S., S.T., L.M., R.G., L.B., E.W., S.W., and M.N. performed all experiments. H.M.K., M.S., S.T., E.M., S.M., and C.J.Y. analyzed the data. C.L. and L.A.C. provided the patient samples. N.Z. and A.M. provided helpful comments and discussion. H.M.K., M.S., S.T., and C.J.Y. wrote the manuscript.

Competing interests

A.M. is a founder of Spotlight Therapeutics and serves as an advisor to Juno Therapeutics and PACT Pharma; the Marson laboratory has received sponsored research support from Juno Therapeutics and Epinomics.

Corresponding authors

Correspondence to Hyun Min Kang or Chun Jimmie Ye.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–21

  2. 2.

    Life Sciences Reporting Summary

  3. 3.

    Supplementary Table 3

    Cell type specific eQTLs

Excel files

  1. 1.

    Supplementary Table 1

    Cell type specific differentially expressed genes

  2. 2.

    Supplementary Table 2

    Pathway enrichment for differentially expressed genes

  3. 3.

    Supplementary Table 4

    Base Call Probabilities

Zip files

  1. 1.

    Supplementary Code

    Implementation of demuxlet