Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Three decades of nanopore sequencing

A long-held goal in sequencing has been to use a voltage-biased nanoscale pore in a membrane to measure the passage of a linear, single-stranded (ss) DNA or RNA molecule through that pore. With the development of enzyme-based methods that ratchet polynucleotides through the nanopore, nucleobase-by-nucleobase, measurements of changes in the current through the pore can now be decoded into a DNA sequence using an algorithm. In this Historical Perspective, we describe the key steps in nanopore strand-sequencing, from its earliest conceptualization more than 25 years ago to its recent commercialization and application.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Nanopore sequencing.
Figure 2

Marina Corral Spence/Nature Publishing Group

Figure 3: D.D.'s notebook.
Figure 4: Sensing regions in MspA and α-hemolysin.
Figure 5: Nanopore researchers at the Advances in Genome Biology and Technology Meeting, Marco Island, Florida, 2012.
Figure 6

References

  1. Walker, B., Kasianowicz, J., Krishnasastry, M. & Bayley, H. A pore-forming protein with a metal-actuated switch. Protein Eng. 7, 655–662 (1994).

    Article  CAS  Google Scholar 

  2. Menestrina, G. Ionic channels formed by Staphylococcus aureus alpha-toxin: voltage-dependent inhibition by divalent and trivalent cations. J. Membr. Biol. 90, 177–190 (1986).

    Article  CAS  Google Scholar 

  3. Bezrukov, S.M. & Kasianowicz, J.J. Current noise reveals protonation kinetics and number of ionizable sites in an open protein ion channel. Phys. Rev. Lett. 70, 2352–2355 (1993).

    Article  CAS  Google Scholar 

  4. Bezrukov, S.M., Vodyanoy, I., Brutyan, R.A. & Kasianowicz, J.J. Dynamics and free energy of polymers partitioning into a nanoscale pore. Macromolecules 29, 8517–8522 (1996).

    Article  CAS  Google Scholar 

  5. Song, L. et al. Structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore. Science 274, 1859–1866 (1996).

    Article  CAS  Google Scholar 

  6. Kasianowicz, J.J., Brandin, E., Branton, D. & Deamer, D.W. Characterization of individual polynucleotide molecules using a membrane channel. Proc. Natl. Acad. Sci. USA 93, 13770–13773 (1996).

    Article  CAS  Google Scholar 

  7. Akeson, M., Branton, D., Kasianowicz, J.J., Brandin, E. & Deamer, D.W. Microsecond time-scale discrimination among polycytidylic acid, polyadenylic acid, and polyuridylic acid as homopolymers or as segments within single RNA molecules. Biophys. J. 77, 3227–3233 (1999).

    Article  CAS  Google Scholar 

  8. Meller, A., Nivon, L., Brandin, E., Golovchenko, J. & Branton, D. Rapid nanopore discrimination between single polynucleotide molecules. Proc. Natl. Acad. Sci. USA 97, 1079–1084 (2000).

    Article  CAS  Google Scholar 

  9. Wang, H., Dunning, J.E., Huang, A.P.-H., Nyamwanda, J.A. & Branton, D. DNA heterogeneity and phosphorylation unveiled by single-molecule electrophoresis. Proc. Natl. Acad. Sci. USA 101, 13472–13477 (2004).

    Article  CAS  Google Scholar 

  10. Mathé, J., Aksimentiev, A., Nelson, D.R., Schulten, K. & Meller, A. Orientation discrimination of single-stranded DNA inside the alpha-hemolysin membrane channel. Proc. Natl. Acad. Sci. USA 102, 12377–12382 (2005).

    Article  Google Scholar 

  11. Butler, T.Z., Gundlach, J.H. & Troll, M.A. Determination of RNA orientation during translocation through a biological nanopore. Biophys. J. 90, 190–199 (2006).

    Article  CAS  Google Scholar 

  12. Ashkenasy, N., Sánchez-Quesada, J., Bayley, H. & Ghadiri, M.R. Recognizing a single base in an individual DNA strand: a step toward DNA sequencing in nanopores. Angew. Chem. Int. Ed. 44, 1401–1404 (2005).

    Article  CAS  Google Scholar 

  13. Stoddart, D., Heron, A.J., Mikhailova, E., Maglia, G. & Bayley, H. Single-nucleotide discrimination in immobilized DNA oligonucleotides with a biological nanopore. Proc. Natl. Acad. Sci. USA 106, 7702–7707 (2009).

    Article  CAS  Google Scholar 

  14. Stoddart, D. et al. Nucleobase recognition in ssDNA at the central constriction of the alpha-hemolysin pore. Nano Lett. 10, 3633–3637 (2010).

    Article  CAS  Google Scholar 

  15. Stoddart, D., Maglia, G., Mikhailova, E., Heron, A.J. & Bayley, H. Multiple base-recognition sites in a biological nanopore: two heads are better than one. Angew. Chem. Int. Ed. 49, 556–559 (2010).

    Article  CAS  Google Scholar 

  16. Church, G., Deamer, D., Branton, D., Baldarelli, R. & Kasianowicz, J. Characterization of individual polymer molecules based on monomer-interface interactions. US patent 5,795,782 (1998).

  17. Benner, S. et al. Sequence-specific detection of individual DNA polymerase complexes in real time using a nanopore. Nat. Nanotechnol. 2, 718–724 (2007).

    Article  CAS  Google Scholar 

  18. Hornblower, B. et al. Single-molecule analysis of DNA-protein complexes using nanopores. Nat. Methods 4, 315–317 (2007).

    Article  CAS  Google Scholar 

  19. Cockroft, S.L., Chu, J., Amorin, M. & Ghadiri, M.R. A single-molecule nanopore device detects DNA polymerase activity with single-nucleotide resolution. J. Am. Chem. Soc. 130, 818–820 (2008).

    Article  CAS  Google Scholar 

  20. Olasagasti, F. et al. Replication of individual DNA molecules under electronic control using a protein nanopore. Nat. Nanotechnol. 5, 798–806 (2010).

    Article  CAS  Google Scholar 

  21. Chu, J., González-López, M., Cockroft, S.L., Amorin, M. & Ghadiri, M.R. Real-time monitoring of DNA polymerase function and stepwise single-nucleotide DNA strand translocation through a protein nanopore. Angew. Chem. 49, 10106–10109 (2010).

    Article  CAS  Google Scholar 

  22. Lieberman, K.R. et al. Processive replication of single DNA molecules in a nanopore catalyzed by phi29 DNA polymerase. J. Am. Chem. Soc. 132, 17961–17972 (2010).

    Article  CAS  Google Scholar 

  23. Cherf, G.M. et al. Automated forward and reverse ratcheting of DNA in a nanopore at 5-Å precision. Nat. Biotechnol. 30, 344–348 (2012).

    Article  CAS  Google Scholar 

  24. Manrao, E.A. et al. Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase. Nat. Biotechnol. 30, 349–353 (2012).

    Article  CAS  Google Scholar 

  25. Laszlo, A.H. et al. Detection and mapping of 5-methylcytosine and 5-hydroxymethylcytosine with nanopore MspA. Proc. Natl. Acad. Sci. USA 110, 18904–18909 (2013).

    Article  CAS  Google Scholar 

  26. Schreiber, J. et al. Error rates for nanopore discrimination among cytosine, methylcytosine, and hydroxymethylcytosine along individual DNA strands. Proc. Natl. Acad. Sci. USA 110, 18910–18915 (2013).

    Article  CAS  Google Scholar 

  27. Laszlo, A.H. et al. Decoding long nanopore sequencing reads of natural DNA. Nat. Biotechnol. 32, 829–833 (2014).

    Article  CAS  Google Scholar 

  28. Wescoe, Z.L., Schreiber, J. & Akeson, M. Nanopores discriminate among five C5-cytosine variants in DNA. J. Am. Chem. Soc. 136, 16582–16587 (2014).

    Article  CAS  Google Scholar 

  29. Meller, A., Nivon, L. & Branton, D. Voltage-driven DNA translocations through a nanopore. Phys. Rev. Lett. 86, 3435–3438 (2001).

    Article  CAS  Google Scholar 

  30. Niederweis, M. et al. Cloning of the mspA gene encoding a porin from Mycobacterium smegmatis. Mol. Microbiol. 33, 933–945 (1999).

    Article  CAS  Google Scholar 

  31. Trias, J. & Benz, R. Permeability of the cell wall of Mycobacterium smegmatis. Mol. Microbiol. 14, 283–290 (1994).

    Article  CAS  Google Scholar 

  32. Faller, M., Niederweis, M. & Schulz, G.E. The structure of a mycobacterial outer-membrane channel. Science 303, 1189–1192 (2004).

    Article  CAS  Google Scholar 

  33. Butler, T.Z., Pavlenok, M., Derrington, I.M., Niederweis, M. & Gundlach, J.H. Single-molecule DNA detection with an engineered MspA protein nanopore. Proc. Natl. Acad. Sci. USA 105, 20647–20652 (2008).

    Article  CAS  Google Scholar 

  34. Derrington, I.M. et al. Nanopore DNA sequencing with MspA. Proc. Natl. Acad. Sci. USA 107, 16060–16065 (2010).

    Article  CAS  Google Scholar 

  35. Jain, M. et al. Improved data analysis for the MinION nanopore sequencer. Nat. Methods 12, 351–356 (2015).

    Article  CAS  Google Scholar 

  36. Ip, C.L.C. et al. MinION Analysis and Reference Consortium: phase 1 data release and analysis. F1000Res. 4, 1075 (2015).

    Article  Google Scholar 

  37. Loman, N.J., Quick, J. & Simpson, J.T. A complete bacterial genome assembled de novo using only nanopore sequencing data. Nat. Methods 12, 733–735 (2015).

    Article  CAS  Google Scholar 

  38. Madoui, M.-A. et al. Genome assembly using Nanopore-guided long and error-free DNA reads. BMC Genomics 16, 327 (2015).

    Article  Google Scholar 

  39. Szalay, T. & Golovchenko, J.A. De novo sequencing and variant calling with nanopores using PoreSeq. Nat. Biotechnol. 33, 1087–1091 (2015).

    Article  CAS  Google Scholar 

  40. Ashton, P.M. et al. MinION nanopore sequencing identifies the position and structure of a bacterial antibiotic resistance island. Nat. Biotechnol. 33, 296–300 (2015).

    Article  CAS  Google Scholar 

  41. Quick, J., Quinlan, A.R. & Loman, N.J. A reference bacterial genome dataset generated on the MinION portable single-molecule nanopore sequencer. Gigascience 3, 22 (2014).

    Article  Google Scholar 

  42. Bolisetty, M.T., Rajadinakaran, G. & Graveley, B.R. Determining exon connectivity in complex mRNAs by nanopore sequencing. Genome Biol. 16, 204 (2015).

    Article  Google Scholar 

  43. Norris, A.L., Workman, R.E., Fan, Y., Eshleman, J.R. & Timp, W. Nanopore sequencing detects structural variants in cancer. Cancer Biol. Ther. 10.1080/15384047.2016.1139236 (19 January 2016).

  44. Peters, B.A. et al. Accurate whole-genome sequencing and haplotyping from 10 to 20 human cells. Nature 487, 190–195 (2012).

    Article  CAS  Google Scholar 

  45. Bhattacharya, S. et al. Molecular dynamics study of MspA arginine mutants predicts slow DNA translocations and ion current blockades indicative of DNA sequence. ACS Nano 6, 6960–6968 (2012).

    Article  CAS  Google Scholar 

  46. Manrao, E.A., Derrington, I.M., Pavlenok, M., Niederweis, M. & Gundlach, J.H. Nucleotide discrimination with DNA immobilized in the MspA nanopore. PLoS One 6, e25723 (2011).

    Article  CAS  Google Scholar 

  47. Viterbi, A.J. Error bounds for convolutional codes and an asymptotically optimum decoding algorithm. IEEE Trans. Inf. Theory 13, 260–269 (1967).

    Article  Google Scholar 

  48. Timp, W., Comer, J. & Aksimentiev, A. DNA base-calling from a nanopore using a Viterbi algorithm. Biophys. J. 102, L37–L39 (2012).

    Article  CAS  Google Scholar 

  49. Kuan, A.T., Lu, B., Xie, P., Szalay, T. & Golovchenko, J.A. Electrical pulse fabrication of graphene nanopores in electrolyte solution. Appl. Phys. Lett. 106, 203109 (2015).

    Article  Google Scholar 

  50. Garaj, S., Liu, S., Golovchenko, J.A. & Branton, D. Molecule-hugging graphene nanopores. Proc. Natl. Acad. Sci. USA 110, 12192–12196 (2013).

    Article  CAS  Google Scholar 

  51. Gershow, M. & Golovchenko, J.A. Recapturing and trapping single molecules with a solid-state nanopore. Nat. Nanotechnol. 2, 775–779 (2007).

    Article  CAS  Google Scholar 

  52. Goyal, P. et al. Structural and mechanistic insights into the bacterial amyloid secretion channel CsgG. Nature 516, 250–253 (2014).

    Article  CAS  Google Scholar 

  53. Hoenen, T. et al. Nanopore sequencing as a rapidly deployable Ebola outbreak tool. Emerg. Infect. Dis. 22, 331–334 (2016).

    Article  CAS  Google Scholar 

  54. Quick, J. et al. Real-time, portable genome sequencing for Ebola surveillance. Nature 10.1038/nature16996 (3 February 2016).

Download references

Acknowledgements

The authors are funded by the National Human Genome Research Institute of the National Institutes of Health award numbers HG006321, HG007827 and HG003703.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to David Deamer.

Ethics declarations

Competing interests

All of the authors are members of the Technology Advisory Board of Oxford Nanopore Technologies, Oxford, UK, for which they receive compensation, including stock options of unknown value that may exceed $10,000. They are inventors on the following nanopore sequencing–related US patents owned by their respective universities: 5,795,782; 6,015,714; 6,267,193; 6,267,872; 6,362,002; 6,464,842; 6,428,959; 6,673,615; 6,746,594; 6,783,643; 6,936,433; 7,118,657; 7,189,503; 7,238,485; 7,258,838; 7,253,434; 7,466,069; 7,435,353; 7,468,271; 7,582,490; 7,803,607; 7,846,738; 7,993,538; 7,969,079; 8,092,697; 8,726,465.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Deamer, D., Akeson, M. & Branton, D. Three decades of nanopore sequencing. Nat Biotechnol 34, 518–524 (2016). https://doi.org/10.1038/nbt.3423

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.3423

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing