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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.

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.

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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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