Abstract

Sequencing the RNA in a biological sample can unlock a wealth of information, including the identity of bacteria and viruses, the nuances of alternative splicing or the transcriptional state of organisms. However, current methods have limitations due to short read lengths and reverse transcription or amplification biases. Here we demonstrate nanopore direct RNA-seq, a highly parallel, real-time, single-molecule method that circumvents reverse transcription or amplification steps. This method yields full-length, strand-specific RNA sequences and enables the direct detection of nucleotide analogs in RNA.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Primary accessions

BioProject

References

  1. 1.

    , & RNA-Seq: a revolutionary tool for transcriptomics. Nat. Rev. Genet. 10, 57–63 (2009).

  2. 2.

    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).

  3. 3.

    et al. Amplification-free Illumina sequencing-library preparation facilitates improved mapping and assembly of (G+C)-biased genomes. Nat. Methods 6, 291–295 (2009).

  4. 4.

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

  5. 5.

    et al. FRT-seq: amplification-free, strand-specific transcriptome sequencing. Nat. Methods 7, 130–132 (2010).

  6. 6.

    et al. Direct RNA sequencing. Nature 461, 814–818 (2009).

  7. 7.

    , , , & Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nat. Genet. 40, 1413–1415 (2008).

  8. 8.

    et al. Assessment of transcript reconstruction methods for RNA-seq. Nat. Methods 10, 1177–1184 (2013).

  9. 9.

    , , & Long-read sequencing of chicken transcripts and identification of new transcript isoforms. PLoS One 9, e94650 (2014).

  10. 10.

    et al. Analysis of RNA base modification and structural rearrangement by single-molecule real-time detection of reverse transcription. J. Nanobiotechnology 11, 8 (2013).

  11. 11.

    , , , & Detection of 3′-end RNA uridylation with a protein nanopore. ACS Nano 8, 1364–1374 (2014).

  12. 12.

    , , & Capture, unfolding, and detection of individual tRNA molecules using a nanopore device. Front. Bioeng. Biotechnol. 3, 91 (2015).

  13. 13.

    & GMAP: a genomic mapping and alignment program for mRNA and EST sequences. Bioinformatics 21, 1859–1875 (2005).

  14. 14.

    et al. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat. Biotechnol. 33, 290–295 (2015).

  15. 15.

    et al. Nanopore long-read RNAseq reveals widespread transcriptional variation among the surface receptors of individual B cells. Nat. Commun. 8, 16027 (2017).

  16. 16.

    , & Three decades of nanopore sequencing. Nat. Biotechnol. 34, 518–524 (2016).

  17. 17.

    Oxford Nanopore Technologies Ltd. Direct RNA sequencing (2016).

  18. 18.

    The HDF Group. Hierarchical data format, version 5, 1997–2017. .

  19. 19.

    & BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).

  20. 20.

    et al. Circos: an information aesthetic for comparative genomics. Genome Res. 19, 1639–1645 (2009).

  21. 21.

    et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007).

  22. 22.

    & Burrows–Wheeler Alignment Tool (2012).

  23. 23.

    , & A new decoding algorithm for hidden Markov models improves the prediction of the topology of all-beta membrane proteins. BMC Bioinformatics 6, S12 (2005).

Download references

Author information

Affiliations

  1. Oxford Nanopore Technologies Ltd., Oxford, UK.

    • Daniel R Garalde
    • , Elizabeth A Snell
    • , Daniel Jachimowicz
    • , Botond Sipos
    • , Joseph H Lloyd
    • , Mark Bruce
    • , Nadia Pantic
    • , Tigist Admassu
    • , Phillip James
    • , Anthony Warland
    • , Michael Jordan
    • , Jonah Ciccone
    • , Sabrina Serra
    • , Jemma Keenan
    • , Samuel Martin
    • , Luke McNeill
    • , E Jayne Wallace
    • , Lakmal Jayasinghe
    • , Chris Wright
    • , Javier Blasco
    • , Stephen Young
    • , Denise Brocklebank
    • , James Clarke
    • , Andrew J Heron
    •  & Daniel J Turner
  2. Oxford Nanopore Technologies Inc., New York, New York, USA.

    • Sissel Juul

Authors

  1. Search for Daniel R Garalde in:

  2. Search for Elizabeth A Snell in:

  3. Search for Daniel Jachimowicz in:

  4. Search for Botond Sipos in:

  5. Search for Joseph H Lloyd in:

  6. Search for Mark Bruce in:

  7. Search for Nadia Pantic in:

  8. Search for Tigist Admassu in:

  9. Search for Phillip James in:

  10. Search for Anthony Warland in:

  11. Search for Michael Jordan in:

  12. Search for Jonah Ciccone in:

  13. Search for Sabrina Serra in:

  14. Search for Jemma Keenan in:

  15. Search for Samuel Martin in:

  16. Search for Luke McNeill in:

  17. Search for E Jayne Wallace in:

  18. Search for Lakmal Jayasinghe in:

  19. Search for Chris Wright in:

  20. Search for Javier Blasco in:

  21. Search for Stephen Young in:

  22. Search for Denise Brocklebank in:

  23. Search for Sissel Juul in:

  24. Search for James Clarke in:

  25. Search for Andrew J Heron in:

  26. Search for Daniel J Turner in:

Contributions

D.R.G., A.J.H., J. Clarke and D.J.T. conceived the experiments. D.R.G. led the project. D.R.G., E.A.S., D.J., A.J.H., J.H.L., P.J., A.W., M.J., J.K., S.M. and L.M. designed and performed the experiments. J.H.L. tested, engineered and developed the motor protein. J.H.L., S.M., L.M., D.R.G., E.A.S., A.J.H., M.B., D.J., A.W. and E.J.W. designed or assessed motor protein mutations and the sequencing adaptor. D.J.T., D.R.G. and E.A.S. developed the library preparation. E.A.S. and J.K. created custom RNA templates. B.S. wrote custom analysis tools and performed analysis of all sequence data sets. N.P., T.A. and M.B. expressed and purified proteins. M.J., J. Ciccone and S.S. designed and prepared plasmids. M.J., E.J.W., L.J., S.Y., D.R.G., E.A.S., D.J., A.J.H., M.B., J.H.L. and D.B. assessed sequencing performance of buffers, voltages and pores. C.W. wrote squiggle-consensus algorithms. J.B., C.W., D.B., J.H.L., M.B. and S.Y. trained RNA basecallers or analyzed modified base data. D.J.T., B.S., D.R.G., S.J. and C.W. wrote the manuscript. A.J.H., S.Y. and P.J. contributed to the figures or to editing of the manuscript.

Competing interests

All authors are employees of Oxford Nanopore Technologies and are shareholders and/or share option holders.

Corresponding author

Correspondence to Daniel J Turner.

Integrated supplementary information

Supplementary information

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nmeth.4577