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Quantitative assessment of single-cell RNA-sequencing methods

Nature Methods volume 11, pages 41–46 (2014)Cite this article

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Abstract

Interest in single-cell whole-transcriptome analysis is growing rapidly, especially for profiling rare or heterogeneous populations of cells. We compared commercially available single-cell RNA amplification methods with both microliter and nanoliter volumes, using sequence from bulk total RNA and multiplexed quantitative PCR as benchmarks to systematically evaluate the sensitivity and accuracy of various single-cell RNA-seq approaches. We show that single-cell RNA-seq can be used to perform accurate quantitative transcriptome measurement in individual cells with a relatively small number of sequencing reads and that sequencing large numbers of single cells can recapitulate bulk transcriptome complexity.

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Figure 1: Initial validation of single-cell RNA-seq methods.
Figure 2: Correlation between single-cell RNA-seq and single-cell multiplexed qPCR for each sample preparation method.
Figure 3: Comparison of gene expression distributions for 40 genes between samples prepared in microliter and nanoliter volumes.
Figure 4: Merging many single-cell transcriptomes quantitatively recreates the bulk measurement.
Figure 5: Saturation curves for the different sample preparation methods.

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Acknowledgements

The authors would like to acknowledge W. Pan for helping with part of the sample preparation, W. Koh and B. Passarelli for help and discussions regarding bioinformatics pipelines and statistical analysis, and I. de Vlaminck for critical reading of the manuscript. A.R.W. was supported by US National Institutes of Health (NIH) U01HL099999-01 and RC4NS073015. N.F.N. was supported by NIH U01HL099999-01 and U01CA154209. T.K. was supported by P01CA139490. B.T. was supported by U01HL099995-01. F.M.M. was sponsored by the Stanford Institute of Medicine Summer Research Program. P.D. was supported by a scholarship from the Thomas and Stacey Siebel Foundation and by a BD Biosciences Stem Cell Research Grant (Summer 2011).

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  1. Tomer Kalisky

    Present address: Present address: Faculty of Engineering, Bar-Ilan University, Ramat Gan, Israel.,

Authors and Affiliations

  1. Department of Bioengineering, Stanford University, Stanford, California, USA

    Angela R Wu, Norma F Neff, Tomer Kalisky, Barbara Treutlein, Francis M Mburu, Gary L Mantalas & Stephen R Quake

  2. Department of Medicine, Division of Oncology, Stanford University Medical Center, Stanford, California, USA

    Piero Dalerba & Michael F Clarke

  3. Institute for Stem Cell Biology and Regenerative Medicine, Stanford University Medical Center, Stanford, California, USA

    Piero Dalerba, Sopheak Sim & Michael F Clarke

  4. The Ludwig Cancer Center, Stanford University Medical Center, Stanford, California, USA

    Piero Dalerba & Michael F Clarke

  5. Department of Medicine, Division of Gastroenterology and Hepatology, Stanford University Medical Center, Stanford, California, USA

    Michael E Rothenberg

  6. Howard Hughes Medical Institute, Stanford, California, USA

    Francis M Mburu & Stephen R Quake

  7. Department of Applied Physics, Stanford University, Stanford, California, USA

    Stephen R Quake

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Contributions

A.R.W., N.F.N., T.K., P.D., M.E.R., M.F.C. and S.R.Q. conceived of the study and designed the experiments. A.R.W., N.F.N., T.K., P.D., M.E.R., B.T., F.M.M., G.L.M. and S.S. performed the experiments. A.R.W., N.F.N., T.K., P.D., M.E.R., B.T., M.F.C. and S.R.Q. analyzed the data and/or provided intellectual guidance in their interpretation. P.D., M.E.R. and M.F.C. provided samples and reagents. A.R.W., N.F.N, B.T. and S.R.Q. wrote the paper.

Corresponding author

Correspondence to Stephen R Quake.

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Competing interests

S.R.Q. is a founder and consultant for Fluidigm Corporation.

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Supplementary Figures 1–7, Supplementary Tables 1 and 2 and Supplementary Note 1 (PDF 3157 kb)

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Wu, A., Neff, N., Kalisky, T. et al. Quantitative assessment of single-cell RNA-sequencing methods. Nat Methods 11, 41–46 (2014). https://doi.org/10.1038/nmeth.2694

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