High-Fidelity Capture, Threading, and Infinite-Depth Sequencing of Single DNA Molecules with a Double-Nanopore System

Adnan Choudhary, Himanshu Joshi, Han-Yi Chou, Kumar Sarthak, James Wilson, Christopher Maffeo, and Aleksei Aksimentiev
ACS Nano 14 15566-15576 (2020)
DOI:10.1021/acsnano.0c06191  BibTex

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In this article, we describe a proof-of-principle demonstration of a new nanopore sequencing method that directly resolves the shortcomings of the current nanopore sequencing technology. Our method is built around a double-nanopore system that enables infinite-depth sequencing of individual DNA or RNA molecules by moving the same molecules repeatedly back and forth (‘flossing’) through the nanopores, a process that can be repeated as often as needed until the desired precision of nucleotide determination is reached (see SI Movie 1 and 2). In doing so, our approach removes the need for any error-prone enzyme for speed control and finally provides a feasible path for the development of a purely solid-state nanopore sequencing platform, a long-promised game-changer in the nanopore sequencing field. Our double-nanopore sequencing platform features a nanofluidic system for high-fidelity delivery and threading of native nucleic acids, enabling analysis of genomic-length DNA and RNA. The combination of high-fidelity loading and repeat sequencing of the same molecules allows for direct sequencing of DNA and RNA, with order of magnitude improvements in the performance over the current state-of-the-art nanopore sequencing with regard to ion-current signal and base calling at a truly single-molecule level.

Abstract

Nanopore sequencing of nucleic acids has an illustrious history of innovations that eventually made commercial nanopore sequencing possible. Nevertheless, the present nanopore sequencing technology leaves much room for improvement, especially with respect to accuracy of raw reads and detection of nucleotide modifications. Double-nanopore sequencing—an approach where a DNA molecule is pulled back and forth by a tug-of-war of two nanopores—could potentially improve single-molecule read accuracy and modification detection by offering multiple reads of the same DNA fragment. One principle difficulty in realizing such a technology is threading single-stranded DNA through both nanopores. Here, we describe and demonstrate through simulations a nanofluidic system for loading and threading DNA strands through a double-nanopore setup with nearly 100% fidelity. The high-efficiency loading is realized by using hourglass-shaped side channels that not only deliver the molecules to the nanopore but also retain molecules that missed the nanopore at the first passage to attempt the nanopore capture again. The second nanopore capture is facilitated by an orthogonal microfluidic flow that unravels the molecule captured by the first nanopore and delivers it to the capture volume of the second nanopore. We demonstrate the potential utility of our double-nanopore system for DNA sequencing by simulating repeat back-and-forth motion—flossing—of a DNA strand through the double-nanopore system. We show that repeat exposure of the same DNA fragments to the nanopore sensing volume considerably increases accuracy of the nucleotide sequence determination and that correlated displacement of ssDNA through the two nanopores may facilitate recognition of homopolymer fragments.

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Animation illustrating the capture, threading and sequencing of single-stranded DNA molecule in a nanofluidic double nanopore system.

Animation illustrating an all-atom MD simulation of repeat flossing of ssDNA through a double nanopore system. The bottom left and right movies show a cut-away, zoomed in view of individual nanopores of the double nanopore system shown at the top.