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Direct RNA sequencing

Abstract

Our understanding of human biology and disease is ultimately dependent on a complete understanding of the genome and its functions. The recent application of microarray and sequencing technologies to transcriptomics has changed the simplistic view of transcriptomes to a more complicated view of genome-wide transcription where a large fraction of transcripts emanates from unannotated parts of genomes1,2,3,4,5,6,7, and underlined our limited knowledge of the dynamic state of transcription. Most of this broad body of knowledge was obtained indirectly because current transcriptome analysis methods typically require RNA to be converted to complementary DNA (cDNA) before measurements, even though the cDNA synthesis step introduces multiple biases and artefacts that interfere with both the proper characterization and quantification of transcripts8,9,10,11,12,13,14,15,16,17,18. Furthermore, cDNA synthesis is not particularly suitable for the analysis of short, degraded and/or small quantity RNA samples. Here we report direct single molecule RNA sequencing without prior conversion of RNA to cDNA. We applied this technology to sequence femtomole quantities of poly(A)+ Saccharomyces cerevisiae RNA using a surface coated with poly(dT) oligonucleotides to capture the RNAs at their natural poly(A) tails and initiate sequencing by synthesis. We observed transcript 3′ end heterogeneity and polyadenylated small nucleolar RNAs. This study provides a path to high-throughput and low-cost direct RNA sequencing and achieving the ultimate goal of a comprehensive and bias-free understanding of transcriptomes.

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Figure 1: DRS chemistry and sequencing steps.
Figure 2: DRS sequencing read-length statistics.
Figure 3: DRS read distribution.
Figure 4: S. cerevisiae poly(A) + RNA DRS suggests overlapping transcription units and polyadenylated snoRNA and rRNA species.

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Accession codes

Data deposits

Sequencing data sets described in this study have been deposited at the National Center for Biotechnology Information (NCBI) Short Read Archive (SRA), accession no SRA 009023.

References

  1. Denoeud, F. et al. Annotating genomes with massive-scale RNA sequencing. Genome Biol. 9, R175 (2008)

    Article  Google Scholar 

  2. Kapranov, P., Willingham, A. T. & Gingeras, T. R. Genome-wide transcription and the implications for genomic organization. Nature Rev. Genet. 8, 413–423 (2007)

    Article  CAS  Google Scholar 

  3. Marioni, J. C., Mason, C. E., Mane, S. M., Stephens, M. & Gilad, Y. RNA-seq: an assessment of technical reproducibility and comparison with gene expression arrays. Genome Res. 18, 1509–1517 (2008)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  6. Sultan, M. et al. A global view of gene activity and alternative splicing by deep sequencing of the human transcriptome. Science 321, 956–960 (2008)

    Article  ADS  CAS  Google Scholar 

  7. Wilhelm, B. T. et al. Dynamic repertoire of a eukaryotic transcriptome surveyed at single-nucleotide resolution. Nature 453, 1239–1243 (2008)

    Article  ADS  CAS  Google Scholar 

  8. Cocquet, J., Chong, A., Zhang, G. & Veitia, R. A. Reverse transcriptase template switching and false alternative transcripts. Genomics 88, 127–131 (2006)

    Article  CAS  Google Scholar 

  9. Gubler, U. Second-strand cDNA synthesis: classical method. Methods Enzymol. 152, 325–329 (1987)

    Article  ADS  CAS  Google Scholar 

  10. Gubler, U. Second-strand cDNA synthesis: mRNA fragments as primers. Methods Enzymol. 152, 330–335 (1987)

    Article  CAS  Google Scholar 

  11. Haddad, F. et al. Regulation of antisense RNA expression during cardiac MHC gene switching in response to pressure overload. Am. J. Physiol. Heart Circ. Physiol. 290, H2351–H2361 (2006)

    Article  CAS  Google Scholar 

  12. Haddad, F., Qin, A. X., Giger, J. M., Guo, H. & Baldwin, K. M. Potential pitfalls in the accuracy of analysis of natural sense-antisense RNA pairs by reverse transcription-PCR. BMC Biotechnol. 7, 21 (2007)

    Article  Google Scholar 

  13. Mader, R. M. et al. Reverse transcriptase template switching during reverse transcriptase-polymerase chain reaction: artificial generation of deletions in ribonucleotide reductase mRNA. J. Lab. Clin. Med. 137, 422–428 (2001)

    Article  CAS  Google Scholar 

  14. Perocchi, F., Xu, Z., Clauder-Munster, S. & Steinmetz, L. M. Antisense artifacts in transcriptome microarray experiments are resolved by actinomycin D. Nucleic Acids Res. 35, e128 (2007)

    Article  Google Scholar 

  15. Roberts, J. D. et al. Fidelity of two retroviral reverse transcriptases during DNA-dependent DNA synthesis in vitro . Mol. Cell. Biol. 9, 469–476 (1989)

    Article  CAS  Google Scholar 

  16. Roy, S. W. & Irimia, M. When good transcripts go bad: artifactual RT-PCR ‘splicing’ and genome analysis. Bioessays 30, 601–605 (2008)

    Article  CAS  Google Scholar 

  17. Roy, S. W. & Irimia, M. Intron mis-splicing: no alternative? Genome Biol. 9, 208 (2008)

    Article  Google Scholar 

  18. Spiegelman, S. et al. DNA-directed DNA polymerase activity in oncogenic RNA viruses. Nature 227, 1029–1031 (1970)

    Article  ADS  CAS  Google Scholar 

  19. Varadaraj, K. & Skinner, D. M. Denaturants or cosolvents improve the specificity of PCR amplification of a G + C-rich DNA using genetically engineered DNA polymerases. Gene 140, 1–5 (1994)

    Article  CAS  Google Scholar 

  20. Wu, J. Q. et al. Systematic analysis of transcribed loci in ENCODE regions using RACE sequencing reveals extensive transcription in the human genome. Genome Biol. 9, R3 (2008)

    Article  Google Scholar 

  21. Braslavsky, I., Hebert, B., Kartalov, E. & Quake, S. R. Sequence information can be obtained from single DNA molecules. Proc. Natl Acad. Sci. USA 100, 3960–3964 (2003)

    Article  ADS  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  23. Karkas, J. D., Stavrianopoulos, J. G. & Chargaff, E. Action of DNA polymerase I of Escherichia coli with DNA-RNA hybrids as templates. Proc. Natl Acad. Sci. USA 69, 398–402 (1972)

    Article  ADS  CAS  Google Scholar 

  24. Rüttimann, C., Cotoras, M., Zaldívar, J. & Vicuna, R. DNA polymerases from the extremely thermophilic bacterium Thermus thermophilus HB-8. Eur. J. Biochem. 149, 41–46 (1985)

    Article  Google Scholar 

  25. Stenesh, J., Roe, B. A. & Snyder, T. L. Studies of the deoxyribonucleic acid from mesophilic and thermophilic bacteria. Biochim. Biophys. Acta 161, 442–454 (1968)

    Article  CAS  Google Scholar 

  26. Kent, W. J. BLAT—the BLAST-like alignment tool. Genome Res. 12, 656–664 (2002)

    Article  CAS  Google Scholar 

  27. Kim, M. et al. Distinct pathways for snoRNA and mRNA termination. Mol. Cell 24, 723–734 (2006)

    Article  CAS  Google Scholar 

  28. Grzechnik, P. & Kufel, J. Polyadenylation linked to transcription termination directs the processing of snoRNA precursors in yeast. Mol. Cell 32, 247–258 (2008)

    Article  CAS  Google Scholar 

  29. Slomovic, S., Laufer, D., Geiger, D. & Schuster, G. Polyadenylation of ribosomal RNA in human cells. Nucleic Acids Res. 34, 2966–2975 (2006)

    Article  CAS  Google Scholar 

  30. Lipson, D. et al. Quantification of the yeast transcriptome by single-molecule sequencing. Nature Biotechnol. 27, 652–658 (2009)

    Article  CAS  Google Scholar 

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Acknowledgements

We thank K. Kerouac, P. Kapranov, L. Kung, C. Hart and D. Lipson for technical assistance and discussions.

Author Contributions F.O. conceived the project, designed the experimental plan, coordinated the studies and analysed the data. F.O., J.B., L.E.S. and P.M. performed the enzyme kinetics assays. F.O., D.R.J., A.R.P. and J.G.R. did the sequencing experiments. J.F.T. and M.J. provided experimental reviews. F.O. and P.M.M. wrote the manuscript, which was reviewed by all authors.

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Correspondence to Fatih Ozsolak or Patrice M. Milos.

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All authors are or have been employees of Helicos BioSciences.

Supplementary information

Supplementary Information

This file contains Supplementary Methods, Supplementary Figures S1-S11 with Legends, Supplementary Tables S1-S3 and Supplementary References. (PDF 496 kb)

Supplementary Table 4

This zipped file contains aligned reads obtained with sequencing of poly-A+ S. cerevisiae RNA with DRS. (ZIP 2811 kb)

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Ozsolak, F., Platt, A., Jones, D. et al. Direct RNA sequencing. Nature 461, 814–818 (2009). https://doi.org/10.1038/nature08390

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