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Quantitative assessment of single-cell RNA-sequencing methods

Nature Methods 11, 41–46 (2014) | Download Citation

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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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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).

Author information

    • Tomer Kalisky

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

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.

Competing interests

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

Corresponding author

Correspondence to Stephen R Quake.

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DOI

https://doi.org/10.1038/nmeth.2694