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Length-independent DNA packing into nanopore zero-mode waveguides for low-input DNA sequencing

Abstract

Compared with conventional methods, single-molecule real-time (SMRT) DNA sequencing exhibits longer read lengths than conventional methods, less GC bias, and the ability to read DNA base modifications. However, reading DNA sequence from sub-nanogram quantities is impractical owing to inefficient delivery of DNA molecules into the confines of zero-mode waveguides—zeptolitre optical cavities in which DNA sequencing proceeds. Here, we show that the efficiency of voltage-induced DNA loading into waveguides equipped with nanopores at their floors is five orders of magnitude greater than existing methods. In addition, we find that DNA loading is nearly length-independent, unlike diffusive loading, which is biased towards shorter fragments. We demonstrate here loading and proof-of-principle four-colour sequence readout of a polymerase-bound 20,000-base-pair-long DNA template within seconds from a sub-nanogram input quantity, a step towards low-input DNA sequencing and mammalian epigenomic mapping of native DNA samples.

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Figure 1: NZMWs for DNA capture and sequencing.
Figure 2: DNA capture and packing into NZMWs.
Figure 3: High-yield DNA binding into NZMW arrays.
Figure 4: DNA capture and sequencing with NZMWs.

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References

  1. Eid, J. et al. Real-time DNA sequencing from single polymerase molecules. Science 323, 133–138 (2009).

    Article  CAS  Google Scholar 

  2. Flusberg, B. A. et al. Direct detection of DNA methylation during single-molecule, real-time sequencing. Nat. Methods 7, 461–465 (2010).

    Article  CAS  Google Scholar 

  3. Chaisson, M. J. P. et al. Resolving the complexity of the human genome using single-molecule sequencing. Nature 517, 608–611 (2014).

    Article  Google Scholar 

  4. Levene, M. J. et al. Zero-mode waveguides for single-molecule analysis at high concentrations. Science 299, 682–686 (2003).

    Article  CAS  Google Scholar 

  5. Berlin, K. et al. Assembling large genomes with single-molecule sequencing and locality-sensitive hashing. Nat. Biotechnol. 33, 623–630 (2015).

    Article  CAS  Google Scholar 

  6. Chaisson, M. J. P., Wilson, R. K. & Eichler, E. E. Genetic variation and the de novo assembly of human genomes. Nat. Rev. Genet. 16, 627–640 (2015).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  8. Jose, M. M.-M. et al. Cell investigation of nanostructures: zero-mode waveguides for plasma membrane studies with single molecule resolution. Nanotechnology 18, 195101 (2007).

    Article  Google Scholar 

  9. Miyake, T. et al. Real-time imaging of single-molecule fluorescence with a zero-mode waveguide for the analysis of protein−protein interaction. Anal. Chem. 80, 6018–6022 (2008).

    Article  CAS  Google Scholar 

  10. Uemura, S. et al. Real-time tRNA transit on single translating ribosomes at codon resolution. Nature 464, 1012–1017 (2010).

    Article  CAS  Google Scholar 

  11. Sandén, T. et al. A zeptoliter volume meter for analysis of single protein molecules. Nano Lett. 12, 370–375 (2012).

    Article  Google Scholar 

  12. Richards, C. I. et al. Live-cell imaging of single receptor composition using zero-mode waveguide nanostructures. Nano Lett. 12, 3690–3694 (2012).

    Article  CAS  Google Scholar 

  13. de Torres, J. et al. FRET enhancement in aluminum zero-mode waveguides. ChemPhysChem 16, 782–788 (2015).

    Article  CAS  Google Scholar 

  14. Robertson, R. M., Laib, S. & Smith, D. E. Diffusion of isolated DNA molecules: dependence on length and topology. Proc. Natl Acad. Sci. USA 103, 7310–7314 (2006).

    Article  CAS  Google Scholar 

  15. Pedone, D., Langecker, M., Abstreiter, G. & Rant, U. A pore−cavity−pore device to trap and investigate single nanoparticles and DNA molecules in a femtoliter compartment: confined diffusion and narrow escape. Nano Lett. 11, 1561–1567 (2011).

    Article  CAS  Google Scholar 

  16. Liu, X., Skanata, M. M. & Stein, D. Entropic cages for trapping DNA near a nanopore. Nat. Commun. 6, 6222 (2015).

    Article  CAS  Google Scholar 

  17. Han, J., Turner, S. W. & Craighead, H. G. Entropic trapping and escape of long DNA molecules at submicron size constriction. Phys. Rev. Lett. 83, 1688–1691 (1999).

    Article  CAS  Google Scholar 

  18. Coupland, P. et al. Direct sequencing of small genomes on the Pacific Biosciences RS without library preparation. BioTechniques 53, 365–372 (2012).

    Article  CAS  Google Scholar 

  19. Raley, C. et al. Preparation of next-generation DNA sequencing libraries from ultra-low amounts of input DNA: application to single-molecule, real-time (SMRT) sequencing on the Pacific Biosciences RS II. Preprint at bioRxivhttp://www.biorxiv.org/content/early/2014/03/25/003566 (2014).

  20. Larkin, J. et al. Reversible positioning of single molecules inside zero-mode waveguides. Nano Lett. 14, 6023–6029 (2014).

    Article  CAS  Google Scholar 

  21. Lundquist, P. M. et al. Parallel confocal detection of single molecules in real time. Opt. Lett. 33, 1026–1028 (2008).

    Article  Google Scholar 

  22. Assad, O. N., Di Fiori, N., Squires, A. H. & Meller, A. Two color DNA barcode detection in photoluminescence suppressed silicon nitride nanopores. Nano Lett. 15, 745–752 (2015).

    Article  CAS  Google Scholar 

  23. Sawafta, F. et al. Solid-state nanopores and nanopore arrays optimized for optical detection. Nanoscale 6, 6991–6996 (2014).

    Article  CAS  Google Scholar 

  24. Sun, S., Rao, V. B. & Rossmann, M. G. Genome packaging in viruses. Curr. Opin. Struct. Biol. 20, 114–120 (2010).

    Article  CAS  Google Scholar 

  25. Zhu, P. & Craighead, H. G. Zero-mode waveguides for single-molecule analysis. Annu. Rev. Biophys. 41, 269–293 (2012).

    Article  CAS  Google Scholar 

  26. Wulfmeyer, T. et al. Structural organization of DNA in chlorella viruses. PLoS ONE 7, e30133 (2012).

    Article  CAS  Google Scholar 

  27. Godfrey, J. E. & Eisenberg, H. The flexibility of low molecular weight double-stranded DNA as a function of length. II. Light scattering measurements and the estimation of persistence lengths from light scattering, sedimentation and viscosity. Biophys. Chem. 5, 301–318 (1976).

    Article  CAS  Google Scholar 

  28. Wanunu, M. et al. Electrostatic focusing of unlabelled DNA into nanoscale pores using a salt gradient. Nat. Nanotech. 5, 160–165 (2010).

    Article  CAS  Google Scholar 

  29. Bell, N. A. W., Muthukumar, M. & Keyser, U. F. Translocation frequency of double-stranded DNA through a solid-state nanopore. Phys. Rev. E 93, 022401 (2016).

    Article  Google Scholar 

  30. Freedman, K. J. et al. Nanopore sensing at ultra-low concentrations using single-molecule dielectrophoretic trapping. Nat. Commun. 7, 10217 (2016).

    Article  CAS  Google Scholar 

  31. Grosberg, A. Y. & Rabin, Y. DNA capture into a nanopore: interplay of diffusion and electrohydrodynamics. J. Chem. Phys. 133, 165102 (2010).

    Article  Google Scholar 

  32. Muthukumar, M. Theory of capture rate in polymer translocation. J. Chem. Phys. 132, 195101 (2010).

    Article  CAS  Google Scholar 

  33. Stellwagen, N. C., Gelfi, C. & Righetti, P. G. The free solution mobility of DNA. Biopolymers 42, 687–703 (1997).

    Article  CAS  Google Scholar 

  34. Wanunu, M. et al. DNA translocation governed by interactions with solid-state nanopores. Biophys. J. 95, 4716–4725 (2008).

    Article  CAS  Google Scholar 

  35. Wanunu, M. et al. Rapid electronic detection of probe-specific microRNAs using thin nanopore sensors. Nat. Nanotech. 5, 807–814 (2010).

    Article  CAS  Google Scholar 

  36. Zahid, O. K. et al. Sequence-specific recognition of microRNAs and other short nucleic acids with solid-state nanopores. Nano Lett. 16, 2033–2039 (2016).

    Article  CAS  Google Scholar 

  37. Kowalczyk, S. W. & Dekker, C. Measurement of the docking time of a DNA molecule onto a solid-state nanopore. Nano Lett. 12, 4159–4163 (2012).

    Article  CAS  Google Scholar 

  38. Yamazaki, H., Ito, S., Esashika, K. & Saiki, T. Optical observation of DNA motion during and immediately after nanopore translocation. Appl. Phys. Express 9, 017001 (2016).

    Article  Google Scholar 

  39. Berndsen, Z. T. et al. Nonequilibrium dynamics and ultraslow relaxation of confined DNA during viral packaging. Proc. Natl Acad. Sci. USA 111, 8345–8350 (2014).

    Article  CAS  Google Scholar 

  40. Wilchek, M. & Bayer, E. A. The avidin-biotin complex in bioanalytical applications. Anal. Biochem. 171, 1–32 (1988).

    Article  CAS  Google Scholar 

  41. Srisa-Art, M., Dyson, E. C., deMello, A. J. & Edel, J. B. Monitoring of real-time streptavidin−biotin binding kinetics using droplet microfluidics. Anal. Chem. 80, 7063–7067 (2008).

    Article  CAS  Google Scholar 

  42. Korlach, J. et al. Long, processive enzymatic DNA synthesis using 100% dye-labeled terminal phosphate-linked nucleotides. Nucleosides Nucleotides Nucleic Acids 27, 1072–1082 (2008).

    Article  CAS  Google Scholar 

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Acknowledgements

We acknowledge Y.-C. Tsai, I. Vilfan, J. Hanes, R. Lam and M. McCauley for aid in sample preparation, as well as J. Sutin for assistance with the multimode fibre setup on our microscope. This work was supported by funding from the National Institutes of Health (HG006873 and HG009186, to M.W. and J.K.). This work was performed in part at the Cornell Nanoscale Facility, a member of the National Nanotechnology Infrastructure Network (NNIN), which is supported by the National Science Foundation (grant ECCS-1542081).

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Authors

Contributions

J.L. and M.W. conceived and designed the experiments. J.L. and V.J. fabricated the NZMW devices. J.L., V.J. and R.Y.H. performed the experiments and analysed the data. R.Y.H. wrote the sequence analysis code. All authors wrote the manuscript.

Corresponding author

Correspondence to Meni Wanunu.

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Competing interests

J.K. is a full-time employee at Pacific Biosciences, a company developing sequencing technologies.

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Larkin, J., Henley, R., Jadhav, V. et al. Length-independent DNA packing into nanopore zero-mode waveguides for low-input DNA sequencing. Nature Nanotech 12, 1169–1175 (2017). https://doi.org/10.1038/nnano.2017.176

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