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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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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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.
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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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DOI: https://doi.org/10.1038/nnano.2017.176
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