Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Digital RNA allelotyping reveals tissue-specific and allele-specific gene expression in human


We developed a digital RNA allelotyping method for quantitatively interrogating allele-specific gene expression. This method involves ultra-deep sequencing of padlock-captured single-nucleotide polymorphisms (SNPs) from the transcriptome. We characterized four cell lines established from two human subjects in the Personal Genome Project. Approximately 11–22% of the heterozygous mRNA-associated SNPs showed allele-specific expression in each cell line and 4.3–8.5% were tissue-specific, suggesting the presence of tissue-specific cis regulation. When we applied allelotyping to two pairs of sibling human embryonic stem cell lines, the sibling lines were more similar in allele-specific expression than were the genetically unrelated lines. We found that the variation of allelic ratios in gene expression among different cell lines was primarily explained by genetic variations, much more so than by specific tissue types or growth conditions. Comparison of expressed SNPs on the sense and antisense transcripts suggested that allelic ratios are primarily determined by cis-regulatory mechanisms on the sense transcripts.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Digital allelotyping with padlock probes.
Figure 2: ASE in human cell lines of various degrees of genetic and phenotypic similarities.
Figure 3: X-chromosome inactivation in female hESC lines.

Similar content being viewed by others


  1. Yan, H., Yuan, W., Velculescu, V.E., Vogelstein, B. & Kinzler, K.W. Allelic variation in human gene expression. Science 297, 1143 (2002).

    Article  CAS  Google Scholar 

  2. Pastinen, T. & Hudson, T.J. Cis-acting regulatory variation in the human genome. Science 306, 647–650 (2004).

    Article  CAS  Google Scholar 

  3. Lo, H.S. et al. Allelic variation in gene expression is common in the human genome. Genome Res. 13, 1855–1862 (2003).

    Article  CAS  Google Scholar 

  4. Knight, J.C., Keating, B.J., Rockett, K.A. & Kwiatkowski, D.P. In vivo characterization of regulatory polymorphisms by allele-specific quantification of RNA polymerase loading. Nat. Genet. 33, 469–475 (2003).

    Article  CAS  Google Scholar 

  5. Serre, D. et al. Differential allelic expression in the human genome: a robust approach to identify genetic and epigenetic cis-acting mechanisms regulating gene expression. PLoS Genet. 4, e1000006 (2008).

    Article  Google Scholar 

  6. Maynard, N.D., Chen, J., Stuart, R.K., Fan, J.B. & Ren, B. Genome-wide mapping of allele-specific protein-DNA interactions in human cells. Nat. Methods 5, 307–309 (2008).

    Article  CAS  Google Scholar 

  7. Pant, P.V. et al. Analysis of allelic differential expression in human white blood cells. Genome Res. 16, 331–339 (2006).

    Article  CAS  Google Scholar 

  8. Milani, L. et al. Allelic imbalance in gene expression as a guide to cis-acting regulatory single nucleotide polymorphisms in cancer cells. Nucleic Acids Res. 35, e34 (2007).

    Article  Google Scholar 

  9. Cloonan, N. et al. Stem cell transcriptome profiling via massive-scale mRNA sequencing. Nat. Methods 5, 613–619 (2008).

    Article  CAS  Google Scholar 

  10. Mortazavi, A., Williams, B.A., McCue, K., Schaeffer, L. & Wold, B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat. Methods 5, 621–628 (2008).

    Article  CAS  Google Scholar 

  11. Nilsson, M. et al. Padlock probes: circularizing oligonucleotides for localized DNA detection. Science 265, 2085–2088 (1994).

    Article  CAS  Google Scholar 

  12. Hardenbol, P. et al. Multiplexed genotyping with sequence-tagged molecular inversion probes. Nat. Biotechnol. 21, 673–678 (2003).

    Article  CAS  Google Scholar 

  13. Hardenbol, P. et al. Highly multiplexed molecular inversion probe genotyping: over 10,000 targeted SNPs genotyped in a single tube assay. Genome Res. 15, 269–275 (2005).

    Article  CAS  Google Scholar 

  14. Porreca, G.J. et al. Multiplex amplification of large sets of human exons. Nat. Methods 4, 931–936 (2007).

    Article  CAS  Google Scholar 

  15. Deng, J. et al. Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming. Nat. Biotechnol. 27, 353–360 (2009).

    Article  CAS  Google Scholar 

  16. Ge, B. et al. Survey of allelic expression using EST mining. Genome Res. 15, 1584–1591 (2005).

    Article  CAS  Google Scholar 

  17. Emilsson, V. et al. Genetics of gene expression and its effect on disease. Nature 452, 423–428 (2008).

    Article  CAS  Google Scholar 

  18. Chen, A.E. et al. Optimal timing of inner cell mass isolation increases the efficiency of human embryonic stem cell derivation and allows generation of sibling cell lines. Cell Stem Cell 4, 103–106 (2009).

    Article  CAS  Google Scholar 

  19. He, Y., Vogelstein, B., Velculescu, V.E., Papadopoulos, N. & Kinzler, K.W. The antisense transcriptomes of human cells. Science 322, 1855–1857 (2008).

    Article  CAS  Google Scholar 

  20. Hoffman, L.M. et al. X-inactivation status varies in human embryonic stem cell lines. Stem Cells 23, 1468–1478 (2005).

    Article  CAS  Google Scholar 

  21. Shen, Y. et al. X-inactivation in female human embryonic stem cells is in a nonrandom pattern and prone to epigenetic alterations. Proc. Natl. Acad. Sci. USA 105, 4709–4714 (2008).

    Article  CAS  Google Scholar 

  22. Silva, S.S., Rowntree, R.K., Mekhoubad, S. & Lee, J.T. X-chromosome inactivation and epigenetic fluidity in human embryonic stem cells. Proc. Natl. Acad. Sci. USA 105, 4820–4825 (2008).

    Article  CAS  Google Scholar 

  23. Ng, P.C. et al. Genetic variation in an individual human exome. PLoS Genet. 4, e1000160 (2008).

    Article  Google Scholar 

  24. Bjornsson, H.T. et al. SNP-specific array-based allele-specific expression analysis. Genome Res. 18, 771–779 (2008).

    Article  CAS  Google Scholar 

  25. Milani, L. et al. Allele-specific gene expression patterns in primary leukemic cells reveal regulation of gene expression by CpG site methylation. Genome Res. 19, 1–11 (2009).

    Article  CAS  Google Scholar 

  26. Gimelbrant, A., Hutchinson, J.N., Thompson, B.R. & Chess, A. Widespread monoallelic expression on human autosomes. Science 318, 1136–1140 (2007).

    Article  CAS  Google Scholar 

  27. Dixon, A.L. et al. A genome-wide association study of global gene expression. Nat. Genet. 39, 1202–1207 (2007).

    Article  CAS  Google Scholar 

  28. Stranger, B.E. et al. Population genomics of human gene expression. Nat. Genet. 39, 1217–1224 (2007).

    Article  CAS  Google Scholar 

  29. Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).

    Article  CAS  Google Scholar 

  30. Dimos, J.T. et al. Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science 321, 1218–1221 (2008).

    Article  CAS  Google Scholar 

Download references


We thank C. Ludka and Narimene Lekmine for assistance for Illumina sequencing. This study was supported by National Human Genome Research Institute (P50-HG003170); National Heart, Lung and Blood Institute (R01-HL094963); the Broad Institute and Personal Genome Project donations (to G.M.C.); and the new faculty startup fund from the University of California at San Diego (to K.Z.). J.D. was sponsored by a California Institute of Regenerative Medicine postdoctoral fellowship.

Author information

Authors and Affiliations



K.Z. and J.B.L. developed and optimized the digital allelotyping method; J.D., Z.L. and J.L. participated in the experiments; D.E. and K.E. provided DNA/RNA of hESCs; E.M.L. provided oligonucleotide libraries; Y.G. and B.X. performed Illumina sequencing. K.Z., J.B.L. and J.A. performed data analysis; and K.Z. and G.M.C. oversaw the project.

Corresponding authors

Correspondence to Kun Zhang or George M Church.

Ethics declarations

Competing interests

G.M.C. has advisory roles, royalties and/or equity holding in several next-generation sequencing companies. He donates these funds to

E.M.L. is an employee of Agilent Technologies Inc.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Table 1 and Supplementary Note (PDF 593 kb)

Supplementary Table 2

Detailed information of the CES27k probe set. (XLS 11282 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zhang, K., Li, J., Gao, Y. et al. Digital RNA allelotyping reveals tissue-specific and allele-specific gene expression in human. Nat Methods 6, 613–618 (2009).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing