Direct RNA sequencing (original) (raw)

• Letter
• Published: 23 September 2009
• Adam R. Platt1,
• Dan R. Jones1,
• Jeffrey G. Reifenberger1,
• Lauryn E. Sass1,
• Peter McInerney1,
• John F. Thompson1,
• Jayson Bowers1,
• Mirna Jarosz1 &
• …
• Patrice M. Milos1

Nature volume 461, pages 814–818 (2009)Cite this article

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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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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.

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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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Authors and Affiliations

1. Helicos BioSciences Corporation, One Kendall Square, Cambridge, Massachusetts 02139, USA ,
   Fatih Ozsolak, Adam R. Platt, Dan R. Jones, Jeffrey G. Reifenberger, Lauryn E. Sass, Peter McInerney, John F. Thompson, Jayson Bowers, Mirna Jarosz & Patrice M. Milos

Authors

1. Fatih Ozsolak
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2. Adam R. Platt
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3. Dan R. Jones
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4. Jeffrey G. Reifenberger
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5. Lauryn E. Sass
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6. Peter McInerney
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7. John F. Thompson
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8. Jayson Bowers
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9. Mirna Jarosz
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10. Patrice M. Milos
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Correspondence toFatih 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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• Received: 18 May 2009
• Accepted: 05 August 2009
• Published: 23 September 2009
• Issue Date: 08 October 2009
• DOI: https://doi.org/10.1038/nature08390

Editorial Summary

A direct line to the transcriptome

Understanding the functional output of the genome — the sum total of messenger RNAs in a cell or cell population, known as the transcriptome — is an essential step on the way to understanding biology. Current methods for studying the transcriptome rely on microarray and sequencing approaches that require complementary DNA synthesis followed by multiple manipulations, which introduce biases and potential artefacts. Now a team based at Helicos BioSciences Corporation has developed a direct single-molecule RNA sequencing technique that when scaled-up promises a bias-free high-throughput transcriptome analysis.