Abstract
Counting individual RNA or DNA molecules is difficult because they are hard to copy quantitatively for detection. To overcome this limitation, we applied unique molecular identifiers (UMIs), which make each molecule in a population distinct, to genome-scale human karyotyping and mRNA sequencing in Drosophila melanogaster. Use of this method can improve accuracy of almost any next-generation sequencing method, including chromatin immunoprecipitation–sequencing, genome assembly, diagnostics and manufacturing-process control and monitoring.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Ozsolak, F. et al. Nat. Methods 7, 619–621 (2010).
Lipson, D. et al. Nat. Biotechnol. 27, 652–658 (2009).
Ozsolak, F. et al. Nature 461, 814–818 (2009).
Vogelstein, B. & Kinzler, K.W. Proc. Natl. Acad. Sci. USA 96, 9236–9241 (1999).
Macevicz, S.C. US patent application 11/125,043 (2005).
Hug, H. & Schuler, R. J. Theor. Biol. 221, 615–624 (2003).
Konig, J. et al. Nat. Struct. Mol. Biol. 17, 909–915 (2010).
Wang, Z. et al. PLoS Biol. 8, e1000530 (2010).
Fu, G.K., Hu, J., Wang, P.H. & Fodor, S.P. Proc. Natl. Acad. Sci. USA 108, 9026–9031 (2011).
Kinde, I., Wu, J., Papadopoulos, N., Kinzler, K.W. & Vogelstein, B. Proc. Natl. Acad. Sci. USA 108, 9530–9535 (2011).
Casbon, J.A., Osborne, R.J., Brenner, S. & Lichtenstein, C.P. Nucleic Acids Res. 39, e81 (2011).
Chiu, R.W. et al. Proc. Natl. Acad. Sci. USA 105, 20458–20463 (2008).
Fan, H.C., Blumenfeld, Y.J., Chitkara, U., Hudgins, L. & Quake, S.R. Proc. Natl. Acad. Sci. USA 105, 16266–16271 (2008).
Anders, S. & Huber, W. Genome Biol. 11, R106 (2010).
Benita, Y., Oosting, R.S., Lok, M.C., Wise, M.J. & Humphery-Smith, I. Nucleic Acids Res. 31, e99 (2003).
Linnarsson, S. Exp. Cell Res. 316, 1339–1343 (2010).
Langmead, B., Trapnell, C., Pop, M. & Salzberg, S.L. Genome Biol. 10, R25 (2009).
Zhu, Y.Y., Machleder, E.M., Chenchik, A., Li, R. & Siebert, P.D. Biotechniques 30, 892–897 (2001).
Cloonan, N. et al. Nat. Methods 5, 613–619 (2008).
Levin, J.Z. et al. Nat. Methods 7, 709–715 (2010).
Stasinopoulos, D.M. & Rigby, R.A. J. Stat. Softw. 23, 1–46 (2007).
Li, H. & Durbin, R. Bioinformatics 25, 1754–1760 (2009).
Acknowledgements
We thank M. Taipale, H. Secher Lindroos, E. Ukkonen and T. Whitington for critical review of the manuscript, and E. Iwarsson (Karolinska University Hospital) for the trisomy-21 DNA. This work was supported by European Research Council project Growth Control, Academy of Finland postdoctoral researcher's projects 122197 and 134073, and the Swedish Foundation for Strategic Research grant MDB09-0052.
Author information
Authors and Affiliations
Contributions
S.L., J.T., A.V., T.K. and M.E. conceived and designed experiments. A.V., K.K. and M.B. performed biological experiments. S.L., J.T., A.V. and T.K. analyzed data. J.T., A.V., T.K., M.E. and S.L. wrote the paper.
Corresponding authors
Ethics declarations
Competing interests
S.L. and J.T. have submitted a patent application on the absolute molecule counting method (UK patent application 1016608.0).
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–7, Supplementary Table 1, Supplementary Note (PDF 1812 kb)
Rights and permissions
About this article
Cite this article
Kivioja, T., Vähärautio, A., Karlsson, K. et al. Counting absolute numbers of molecules using unique molecular identifiers. Nat Methods 9, 72–74 (2012). https://doi.org/10.1038/nmeth.1778
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nmeth.1778
This article is cited by
-
Benefits of applying molecular barcoding systems are not uniform across different genomic applications
Journal of Translational Medicine (2023)
-
Absolute quantification of single-base m6A methylation in the mammalian transcriptome using GLORI
Nature Biotechnology (2023)
-
Identification of TP53 mutations in circulating tumour DNA in high grade serous ovarian carcinoma using next generation sequencing technologies
Scientific Reports (2023)
-
Best Practices in Designing, Sequencing, and Identifying Random DNA Barcodes
Journal of Molecular Evolution (2023)
-
Prime-seq, efficient and powerful bulk RNA sequencing
Genome Biology (2022)