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

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.

Flossing DNA in a Dual Nanopore Device.

Solid-state nanopores are a single-molecule technique that can provide access to biomolecular information that is otherwise masked by ensemble averaging. A promising application uses pores and barcoding chemistries to map molecular motifs along single DNA molecules. Despite recent research breakthroughs, however, it remains challenging to overcome molecular noise to fully exploit single-molecule data. Here, an active control technique termed "flossing" that uses a dual nanopore device is presented to trap a proteintagged DNA molecule and up to 100's of back-and-forth electrical scans of the molecule are performed in a few seconds. The protein motifs bound to 48.5 kb λ-DNA are used as detectable features for active triggering of the bidirectional control. Molecular noise is suppressed by averaging the multiscan data to produce averaged intertag distance estimates that are comparable to their known values. Since nanopore feature-mapping applications require DNA linearization when passing through the pore, a key advantage of flossing is that trans-pore linearization is increased to >98% by the second scan, compared to 35% for single nanopore passage of the same set of molecules. In concert with barcoding methods, the dual-pore flossing technique could enable genome mapping and structural variation applications, or mapping loci of epigenetic relevance.

A nanofluidic knot factory based on compression of single DNA in nanochannels.

Knots form when polymers self-entangle, a process enhanced by compaction with important implications in biological and artificial systems involving chain confinement. In particular, new experimental tools are needed to assess the impact of multiple variables influencing knotting probability. Here, we introduce a nanofluidic knot factory for efficient knot formation and detection. Knots are produced during hydrodynamic compression of single DNA molecules against barriers in a nanochannel; subsequent extension of the chain enables direct assessment of the number of independently evolving knots. Knotting probability increases with chain compression as well as with waiting time in the compressed state. Using a free energy derived from scaling arguments, we develop a knot-formation model that can quantify the effect of interactions and the breakdown of Poisson statistics at high compression. Our model suggests that highly compressed knotted states are stabilized by a decreased free energy as knotted contour contributes a lower self-exclusion derived free energy.

Hydrogel droplet single-cell processing: DNA purification, handling, release, and on-chip linearization.

The preparation and handling of mammalian single-cell genomic DNA is limited by the complexity bottleneck inherent to performing multi-step, multi-reagent operations in a microfluidic environment. We have developed a method for benchtop preparation of high-molecular weight, intact, single-cell genomes and demonstrate the extraction of long nucleic acid molecules in a microfluidic system. Lymphoblasts are encapsulated inside of alginate microparticles using a droplet microfluidics, and cells are lysed in bulk. The purified genomes are then delivered to and imaged on a dedicated microfluidic device. High-molecular weight DNA is protected from shear and retains its original cellular identity. Using this encapsulation protocol, we were able to extract individual nucleic acid strands on the millimeter scale inside of a microfluidic channel.

Dynamic compression of single nanochannel confined DNA via a nanodozer assay.

We show that a single DNA molecule confined and extended in a nanochannel can be dynamically compressed by sliding a permeable gasket at a fixed velocity relative to the stationary polymer. The gasket is realized experimentally by optically trapping a nanosphere inside a nanochannel. The trapped bead acts like a "nanodozer," directly applying compressive forces to the molecule without requirement of chemical attachment. Remarkably, these strongly nonequilibrium measurements can be quantified via a simple nonlinear convective-diffusion formalism and yield insights into the local blob statistics, allowing us to conclude that the compressed nanochannel-confined chain exhibits mean-field behavior.