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

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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Figure 1: Demuxlet: demultiplexing and doublet identification from single-cell data.
Figure 2: Performance of demuxlet.
Figure 3: Inter-individual variability in IFN-β response.
Figure 4: Genetic control over cell type proportion and gene expression (N = 23).

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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.

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Authors

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.

Corresponding authors

Correspondence to Hyun Min Kang or Chun Jimmie Ye.

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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.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–21 (PDF 3273 kb)

Life Sciences Reporting Summary (PDF 130 kb)

Supplementary Table 1

Cell type specific differentially expressed genes (XLSX 680 kb)

Supplementary Table 2

Pathway enrichment for differentially expressed genes (XLSX 97 kb)

Supplementary Table 3

Cell type specific eQTLs (PDF 369 kb)

Supplementary Table 4

Base Call Probabilities (XLSX 2726 kb)

Supplementary Code

Implementation of demuxlet (ZIP 4903 kb)

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Kang, H., Subramaniam, M., Targ, S. et al. Multiplexed droplet single-cell RNA-sequencing using natural genetic variation. Nat Biotechnol 36, 89–94 (2018). https://doi.org/10.1038/nbt.4042

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