Abstract
Single-cell RNA sequencing (RNA-seq) is a powerful tool to reveal cellular heterogeneity, discover new cell types and characterize tumor microevolution. However, losses in cDNA synthesis and bias in cDNA amplification lead to severe quantitative errors. We show that molecular labels—random sequences that label individual molecules—can nearly eliminate amplification noise, and that microfluidic sample preparation and optimized reagents produce a fivefold improvement in mRNA capture efficiency.
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Mortazavi, A., Williams, B.A., McCue, K., Schaeffer, L. & Wold, B. Nat. Methods 5, 621–628 (2008).
Cloonan, N. et al. Nat. Methods 5, 613–619 (2008).
Wang, Y. et al. Nat. Genet. 40, 1478–1483 (2008).
Tang, F. et al. Nat. Methods 6, 377–382 (2009).
Islam, S. et al. Genome Res. 21, 1160–1167 (2011).
Ramsköld, D. et al. Nat. Biotechnol. 30, 777–782 (2012).
Hashimshony, T., Wagner, F., Sher, N. & Yanai, I. Cell Rep. 2, 666–673 (2012).
Kivioja, T. et al. Nat. Methods 9, 72–74 (2012).
Shiroguchi, K., Jia, T.Z., Sims, P.A. & Xie, X.S. Proc. Natl. Acad. Sci. USA 109, 1347–1352 (2012).
Fu, G.K., Hu, J., Wang, P.H. & Fodor, S.P. Proc. Natl. Acad. Sci. USA 108, 9026–9031 (2011).
Casbon, J.A., Osborne, R.J., Brenner, S. & Lichtenstein, C.P. Nucleic Acids Res. 39, e81 (2011).
Shalek, A.K. et al. Nature 498, 236–240 (2013).
Picelli, S. et al. Nat. Methods 10, 1096–1098 (2013).
Carter, M.G. et al. Gene Exp. Patterns 8, 181–198 (2008).
Kobayashi, T. et al. Genes Dev. 23, 1870–1875 (2009).
Chambers, I. et al. Nature 450, 1230–1234 (2007).
Galvin-Burgess, K.E., Travis, E.D., Pierson, K.E. & Vivian, J.L. Stem Cells 31, 48–58 (2013).
Maurer, J. et al. PLoS ONE 3, e3451 (2008).
Raj, A., Peskin, C.S., Tranchina, D., Vargas, D.Y. & Tyagi, S. PLoS Biol. 4, e309 (2006).
Dar, R.D. et al. Proc. Natl. Acad. Sci. USA 109, 17454–17459 (2012).
Islam, S. et al. Nat. Protoc. 7, 813–828 (2012).
Langmead, B., Trapnell, C., Pop, M. & Salzberg, S.L. Genome Biol. 10, R25 (2009).
Meyer, L.R. et al. Nucleic Acids Res. 41, D64–D69 (2013).
Huang, D.W. Nat. Protoc. 4, 44–57 (2009).
Acknowledgements
Illumina sequencing experiments were performed by A. Johnsson and A. Juréus (Karolinska Institutet). ES cells were generously provided by the Karolinska Center for Transgene Technologies. Recombinant Tn5 transposase was a generous gift from R. Sandberg (Karolinska Institutet). We are grateful for the advice and support of B. Jones and B. Fowler (Fluidigm). This work was supported by grants from the Swedish Research Council (STARGET) and the European Research Council (261063) to S.L. and from the Swedish Cancer Society, Swedish Research Council and Ragnar Söderberg Foundation to M.K.
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S.L. conceived the study. S.I., A.Z. and P.Z. developed the protocols and performed the RNA-seq experiments. S.J. and M.K. prepared cells. A.Z., G.L.M. and S.I. developed the tagmentation protocol. P.L. wrote the bioinformatics pipeline. P.L., S.L. and A.Z. analyzed data. S.L. and S.I. wrote the manuscript with support from all authors.
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Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–13 and Supplementary Note (PDF 1466 kb)
Supplementary Table 1
List of noisy genes (XLSX 42 kb)
Supplementary Table 2
List of custom oligonucleotides (XLSX 14 kb)
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Islam, S., Zeisel, A., Joost, S. et al. Quantitative single-cell RNA-seq with unique molecular identifiers. Nat Methods 11, 163–166 (2014). https://doi.org/10.1038/nmeth.2772
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DOI: https://doi.org/10.1038/nmeth.2772
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