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Quantification of the yeast transcriptome by single-molecule sequencing

Nature Biotechnology volume 27, pages 652–658 (2009)Cite this article

Abstract

We present single-molecule sequencing digital gene expression (smsDGE), a high-throughput, amplification-free method for accurate quantification of the full range of cellular polyadenylated RNA transcripts using a Helicos Genetic Analysis system. smsDGE involves a reverse-transcription and polyA-tailing sample preparation procedure followed by sequencing that generates a single read per transcript. We applied smsDGE to the transcriptome of Saccharomyces cerevisiae strain DBY746, using 6 of the available 50 channels in a single sequencing run, yielding on average 12 million aligned reads per channel. Using spiked-in RNA, accurate quantitative measurements were obtained over four orders of magnitude. High correlation was demonstrated across independent flow-cell channels, instrument runs and sample preparations. Transcript counting in smsDGE is highly efficient due to the representation of each transcript molecule by a single read. This efficiency, coupled with the high throughput enabled by the single-molecule sequencing platform, provides an alternative method for expression profiling.

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Figure 1: Sample preparation, sequencing and analysis workflow.
Figure 2: Data description.
Figure 3: Reproducibility and counting accuracy.
Figure 4: Count reproducibility.
Figure 5: TSS mapping.
Figure 6: Sequence information.

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References

  1. Lockhart, D.J. & Winzeler, E.A. Genomics, gene expression and DNA arrays. Nature 405, 827–836 (2000).

    Article  CAS  Google Scholar 

  2. Churchill, G.A. Fundamentals of experimental design for cDNA microarrays. Nat. Genet. 32 Suppl, 490–495 (2002).

    Article  CAS  Google Scholar 

  3. Velculescu, V.E., Zhang, L., Vogelstein, B. & Kinzler, K.W. Serial analysis of gene expression. Science 270, 484–487 (1995).

    Article  CAS  Google Scholar 

  4. Brenner, S. et al. Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays. Nat. Biotechnol. 18, 630–634 (2000).

    Article  CAS  Google Scholar 

  5. Saha, S. et al. Using the transcriptome to annotate the genome. Nat. Biotechnol. 20, 508–512 (2002).

    Article  CAS  Google Scholar 

  6. Shiraki, T. et al. Cap analysis gene expression for high-throughput analysis of transcriptional starting point and identification of promoter usage. Proc. Natl. Acad. Sci. USA 100, 15776–15781 (2003).

    Article  CAS  Google Scholar 

  7. Hashimoto, S. et al. 5′-end SAGE for the analysis of transcriptional start sites. Nat. Biotechnol. 22, 1146–1149 (2004).

    Article  CAS  Google Scholar 

  8. Kim, J.B. et al. Polony multiplex analysis of gene expression (PMAGE) in mouse hypertrophic cardiomyopathy. Science 316, 1481–1484 (2007).

    Article  CAS  Google Scholar 

  9. Chen, J. & Rattray, M. Analysis of tag-position bias in MPSS technology. BMC Genomics 7, 77 (2006).

    Article  CAS  Google Scholar 

  10. Siddiqui, A.S. et al. Sequence biases in large scale gene expression profiling data. Nucleic Acids Res. 34, e83 (2006).

    Article  Google Scholar 

  11. Gilchrist, M.A., Qin, H. & Zaretzki, R. Modeling SAGE tag formation and its effects on data interpretation within a Bayesian framework. BMC Bioinformatics 8, 403 (2007).

    Article  Google Scholar 

  12. Hene, L. et al. Deep analysis of cellular transcriptomes - LongSAGE versus classic MPSS. BMC Genomics 8, 333 (2007).

    Article  Google Scholar 

  13. So, A.P., Turner, R.F. & Haynes, C.A. Minimizing loss of sequence information in SAGE ditags by modulating the temperature dependent 3′ → 5′ exonuclease activity of DNA polymerases on 3′-terminal isoheptyl amino groups. Biotechnol. Bioeng. 94, 54–65 (2006).

    Article  CAS  Google Scholar 

  14. Nagalakshmi, U. et al. The transcriptional landscape of the yeast genome defined by RNA sequencing. Science 320, 1344–1349 (2008).

    Article  CAS  Google Scholar 

  15. 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 

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

    Article  CAS  Google Scholar 

  17. Oshlack, A. & Wakefield, M.J. Transcript length bias in RNA-seq data confounds systems biology. Biol. Direct 4, 14 (2009).

    Article  Google Scholar 

  18. Harris, T.D. et al. Single-molecule DNA sequencing of a viral genome. Science 320, 106–109 (2008).

    Article  CAS  Google Scholar 

  19. Bowers, J. et al. Novel virtual terminator nucleotides for next generation DNA sequencing. Nat. Methods (in the press).

  20. Fisk, D.G. et al. Saccharomyces cerevisiae S288C genome annotation: a working hypothesis. Yeast 23, 857–865 (2006).

    Article  CAS  Google Scholar 

  21. Zhang, Z. & Dietrich, F.S. Mapping of transcription start sites in Saccharomyces cerevisiae using 5′ SAGE. Nucleic Acids Res. 33, 2838–2851 (2005).

    Article  CAS  Google Scholar 

  22. Miura, F. et al. A large-scale full-length cDNA analysis to explore the budding yeast transcriptome. Proc. Natl. Acad. Sci. USA 103, 17846–17851 (2006).

    Article  CAS  Google Scholar 

  23. Kent, W.J. et al. The human genome browser at UCSC. Genome Res. 12, 996–1006 (2002).

    Article  CAS  Google Scholar 

  24. Juneau, K., Palm, C., Miranda, M. & Davis, R.W. High-density yeast-tiling array reveals previously undiscovered introns and extensive regulation of meiotic splicing. Proc. Natl. Acad. Sci. USA 104, 1522–1527 (2007).

    Article  CAS  Google Scholar 

  25. Holstege, F.C. et al. Dissecting the regulatory circuitry of a eukaryotic genome. Cell 95, 717–728 (1998).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank all of the past and present colleagues at Helicos who have contributed to this work.

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Author notes

  1. Doron Lipson and Tal Raz: These authors contributed equally to this work.

Authors and Affiliations

  1. Helicos Biosciences Corporation, Cambridge, Massachusetts, USA

    Doron Lipson, Tal Raz, Alix Kieu, Daniel R Jones, Eldar Giladi, Edward Thayer, John F Thompson, Stan Letovsky, Patrice Milos & Marie Causey

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  1. Doron Lipson
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  2. Tal Raz
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  3. Alix Kieu
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  4. Daniel R Jones
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Correspondence to Tal Raz.

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All of the authors are or have been employees of Helicos Biosciences.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3, Supplementary Tables 4 and 6, and Supplementary Methods (PDF 369 kb)

Supplementary Table 1

Transcript counts (XLS 1784 kb)

Supplementary Table 2

qPCR measurements (XLS 25 kb)

Supplementary Table 3

Detected sequence variants (XLS 498 kb)

Supplementary Table 5

Coverage peaks in yeast genome. (XLS 106 kb)

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Lipson, D., Raz, T., Kieu, A. et al. Quantification of the yeast transcriptome by single-molecule sequencing. Nat Biotechnol 27, 652–658 (2009). https://doi.org/10.1038/nbt.1551

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