Electronic Mapping of a Bacterial Genome with Dual Solid-State Nanopores and Active Single-Molecule Control

Abstract

We present an electronic mapping of a bacterial genome using solid-state nanopore technology. A dual-nanopore architecture and active control logic are used to produce single-molecule data that enables estimation of distances between physical tags installed at sequence motifs within double-stranded DNA. Previously developed “DNA flossing” control logic generates multiple scans of each captured DNA. We extended this logic in two ways: first, to automate “zooming out” on each molecule to progressively increase the number of tags scanned during flossing, and second, to automate recapture of a molecule that exited flossing to enable interrogation of the same and/or different regions of the molecule. Custom analysis methods were developed to produce consensus alignments from each multiscan event. The combined multiscanning and multicapture method was applied to the challenge of mapping from a heterogeneous mixture of single-molecule fragments that make up the Escherichia coli (E. coli) chromosome. Coverage of 3.1× across 2355 resolvable sites of the E. coli genome was achieved after 5.6 h of recording time. The recapture method showed a 38% increase in the merged-event alignment length compared to single-scan alignments. The observed intertag resolution was 150 bp in engineered DNA molecules and 166 bp natively within fragments of E. coli DNA, with detection of 133 intersite intervals shorter than 200 bp in the E. coli reference map. We present results on estimating distances in repetitive regions of the E. coli genome. With an appropriately designed array, higher throughput implementations could enable human-sized genome and epigenome mapping applications.

DNA Carrier-Assisted Molecular Ping-Pong in an Asymmetric Nanopore

“…To date, most re-reading systems are based on symmetric nanopore structures, , where symmetric opening areas are at both sides of the membrane nanopore. This symmetric feature also exists in the two-nanopore system of the same size within the tug-of-war configuration. − This symmetry leads to the same recapture behavior for the forward and backward translocation. Our previous study using an asymmetric nanopore demonstrated that DNA polymers showed distinct translocation dynamics in opposite translocation directions, especially in the velocity profiles as a function of time.…”

“…Previous studies have shown that more recaptures can be achieved by minimizing the time interval between the end of the blockade event and the voltage reversal . Alternatively, a molecule may be trapped in a “tug-of-war” by two parallel nanopores − or using optical tweezers. − In each of these scenarios, the voltage difference between the two pores can be adjusted to shuttle the DNA molecule back and forth, facilitating multiple readings of the resultant signal. To date, most re-reading systems are based on symmetric nanopore structures, , where symmetric opening areas are at both sides of the membrane nanopore.…”

Solid-state nanopore detection of partially denatured dsDNA with single-strand binding protein: a preliminary analysis

“…Solid state nanopores, in contrast, have not achieved base-level sensitivity but offer much faster translocation rates and the capability of processing more complex biomolecules, such as higher-order protein and RNA structures and DNA-protein complexes [7]. There has been recent success in developing genomic mapping schemes using solid state nanopores, including through the use of CRISPR [8] and nick-bound oligonucleotide tags [9].…”

Discriminating protein tags on a dsDNA construct using a Dual Nanopore Device

“…If the molecule translocation is linear (i.e., no folds are present), the feature blockades can also provide information regarding the feature’s binding position with respect to the molecule’s underlying sequence, or the relative distance of the given feature from other features 7 , 10 . For example, molecular features that bind specifically to repetitive sequence motifs (for example, the recognition sequence of nicking endonucleases), produce a barcode that can then be aligned genome scale 15 . In proposed DNA information storage applications, molecular features can be used to represent the position of ‘1’ bits 11 .…”