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Increasing the accuracy of nanopore DNA sequencing using a time-varying cross membrane voltage

Abstract

Nanopore DNA sequencing is limited by low base-calling accuracy. Improved base-calling accuracy has so far relied on specialized base-calling algorithms, different nanopores and motor enzymes, or biochemical methods to re-read DNA molecules. Two primary error modes hamper sequencing accuracy: enzyme mis-steps and sequences with indistinguishable signals. We vary the driving voltage from 100 to 200 mV, with a frequency of 200 Hz, across a Mycobacterium smegmatis porin A (MspA) nanopore, thus changing how the DNA strand moves through the nanopore. A DNA helicase moves the DNA through the nanopore in discrete steps, and the variable voltage moves the DNA continuously between these steps. The electronic signal produced with variable voltage is used to overcome the primary error modes in base calling. We found that single-passage de novo base-calling accuracy of 62.7 ± 0.5% with a constant driving voltage improves to 79.3 ± 0.3% with a variable driving voltage. The variable-voltage sequencing mode is complementary to other methods to boost the accuracy of nanopore sequencing and could be incorporated into any enzyme-actuated nanopore sequencing device.

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Data availability

Data for the main text figures as well as for all the constant- and variable-voltage sequencing reads used for the validation study can be found on figshare at https://doi.org/10.6084/m9.figshare.7723214. This also contains all of the Matlab code and supporting files that are necessary to replicate thesequencing analysis for both constant- and variable-voltage, as well as the scripts to generate the main text figures from their underlying data.

Code availability

Code and supporting files for constant- and variable-voltage sequencing analysis, as well as for main text figure generation can be found on github at https://github.com/uwnanopore/variable-voltage-sequencing.git.

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Competing interests

The authors J.H.G., M.T.N., and H.B., along with the University of Washington, have filed provisional patent applications covering the methods presented in this work. The patent has been filed under application number 62/805,870 by the University of Washington CoMotion.

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Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Acknowledgements

This work was supported by the National Human Genome Research Institute grant R01HG005115 (to J.H.G.).

Author information

Competing interests

The authors J.H.G., M.T.N., and H.B., along with the University of Washington, have filed provisional patent applications covering the methods presented in this work. The patent has been filed under application number 62/805,870 by the University of Washington CoMotion.

Correspondence to Jens H. Gundlach.

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Supplementary Information

Supplementary Figures 1–19, Supplementary Tables 1–3 and Supplementary Notes 1–15

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Fig. 1: Basic principles of variable-voltage nanopore sequencing.
Fig. 2: Error correction in variable-voltage sequencing.
Fig. 3: Performance using constant- and variable-voltage sequencing.