Nanopore-based DNA Sequencing Research Articles

Discrimination of single‐stranded DNA homopolymers by sieving out G‐quadruplex using tiny solid‐state nanopores

Nanopore sensor has been developed as a promising technology for DNA sequencing at the single-base resolution. However, the discrimination of homopolymers composed of guanines from other nucleotides has not been clearly revealed due to the easily formed G-quadruplex in aqueous buffers. In this work, we report that a tiny silicon nitride nanopore was used to sieve out G tetramers to make sure only homopolymers composed of guanines could translocate through the nanopore, then the 20-nucleotide long ssDNA homopolymers could be identified and differentiated. It is found that the size of the nucleotide plays a major role in affecting the current blockade as well as the dwell time while DNA is translocating through the nanopore. By the comparison of translocation behavior of ssDNA homopolymers composed of nucleotides with different volumes, it is found that smaller nucleotides can lead to higher translocation speed and lower current blockage, which is also found and validated for the 105-nucleotide long homopolymers. The studies performed in this work will improve our understanding of nanopore-based DNA sequencing at single-base level.

Challenges of Single-Molecule DNA Sequencing with Solid-State Nanopores.

A powerful DNA sequencing tool with high accuracy, long read length and high-throughput would be required more and more for decoding the complicated genetic code. Solid-state nanopore has attracted many researchers for its promising future as a next-generation DNA sequencing platform due to the processability, the robustness and the large-scale integratability. While the diverse materials have been widely explored for a solid-state nanopore, silicon nitride (Si3N4) is especially preferable from the viewpoint of mass production based on semiconductor fabrication process. Here, as a nanopore sensing mechanism, we focused on the ionic blockade current method which is the most developed technique. We also highlight the main challenges of Si3N4 nanopore-based DNA sequencer that should be addressed: the fabrication of ultra-small nanopore and ultra-thin membrane, the modulation of DNA translocation speed and the detection of base-specific signals. In this chapter, we discuss the recent progress relating to solid-state nanopore DNA sequencing, which helps to provide a comprehensive information about the current technical situation.

Rapid preimplantation genetic screening using a handheld, nanopore-based DNA sequencer

To determine if a handheld, nanopore-based DNA sequencer can be used for rapid preimplantation genetic screening (PGS). Laboratory study. Academic medical center. Amplified genomic DNA from euploid and aneuploid trophectoderm biopsy samples (n=9) that was also tested using traditional next generation sequencing (NGS). Short-read DNA library preparation and nanopore-based sequencing using a hand-held MinION sequencer. Comparison of cytogenetic testing result from NGS and nanopore-based sequencing and the time required for library preparation and sequencing. Multiplexed short-read DNA library preparation was completed in 45minutes. Sequencing on a single sample was completed within 20minutes and 5 samples were simultaneously sequenced in under 2hours. Whole-chromosome aneuploidy screening results obtained from nanopore-based sequencing were identical to those obtained using NGS. Here we report the first application of nanopore-based sequencing for PGS on trophectoderm biopsy samples using a novel rapid multiplxed short-read nanopore sequencing library preparation protocol. Sequencing for aneuploidy screening could be performed on a single sample in 20minutes and on 5 samples, simultaneously, within 2hours. Overall, nanopore sequencing is a promising tool to perform rapid PGS onsite, enabling same day testing and embryo transfer, thus obviating the need for complex, large and expensive DNA sequencers or embryo freezing.

Molecular Dynamics Simulation Study of Electrokinetics Transport of Aqueous Solution through Functionalized Carbon Nanotubes: A New Membrane Model for Nanopours Based Biosensors.

DNA sequencing is an essential process for determining the nucleotides on a DNA strands and this process is of high importance in medicine and biomedical research. Nanopore based DNA sequencing has been widely researched and analyzed during last years and this is very important to improve all parameters involved in the process of DNA translocating through these nanopours. Ionic current resolution is a key parameter in designing these nanopours for better measuring of this infinitesimal current that is in order of nano A or even pico A. Here by means of molecular dynamics simulation we showed that using fluorinated carbon nanotubes while embedded inside silicon membrane results a higher ionic current compared to pristine carbon nanotubes. We simulated pristine carbon nanotubes (PCNT) and fluorinated carbon nanotubes (FCNT) with chirality of (8,8) and (10,10) which corresponds to diameter of 1.07 nm and 1.34 nm respectively. Ionic current analysis of these inorganic nanopours functionalized with carbon nanomaterials showed that it could double or triple the magnitude of ionic current. For FCNT (8,8) the magnitude of ionic current was 1.984 nA while it was 0.891 nA for PCNT (8,8) embedded in silicon nanopore. Our study introduces the fluorination of carbon nanotubes as potential method for designing high resolution DNA sequencing nanopore devices.

Nitrogen Doping of Carbon Nanoelectrodes for Enhanced Control of DNA Translocation Dynamics.

Controlling the dynamics of DNA translocation is a central issue in the emerging nanopore-based DNA sequencing. To address the potential of heteroatom doping of carbon nanostructures and for achieving this goal, herein, we carry out atomistic molecular dynamics simulations for single-stranded DNAs translocating between two pristine or doped carbon nanotube (CNT) electrodes. Specifically, we consider the substitutional nitrogen doping of capped CNT (capCNT) electrodes and perform two types of molecular dynamics simulations for the entrapped and translocating single-stranded DNAs. We find that the substitutional nitrogen doping of capCNTs facilitates and stabilizes the edge-on nucleobase configurations rather than the original face-on ones and slows down the DNA translocation speed by establishing hydrogen bonds between the N dopant atoms and nucleobases. Due to the enhanced interactions between DNAs and N-doped capCNTs, the duration time of nucleobases within the nanogap was extended by up to ∼300%. Given the possibility to be combined with the extrinsic light or gate voltage modulation methods, the current work demonstrates that the substitutional nitrogen doping is a promising direction for the control of DNA translocation dynamics through a nanopore or nanogap, based of carbon nanomaterials.

Rapid Multiplex Small DNA Sequencing on the MinION Nanopore Sequencing Platform.

Real-time sequencing of short DNA reads has a wide variety of clinical and research applications including screening for mutations, target sequences and aneuploidy. We recently demonstrated that MinION, a nanopore-based DNA sequencing device the size of a USB drive, could be used for short-read DNA sequencing. In this study, an ultra-rapid multiplex library preparation and sequencing method for the MinION is presented and applied to accurately test normal diploid and aneuploidy samples’ genomic DNA in under three hours, including library preparation and sequencing. This novel method shows great promise as a clinical diagnostic test for applications requiring rapid short-read DNA sequencing.

Quantitative Evaluation of Dielectric Breakdown of Silicon Micro- and Nanofluidic Devices for Electrophoretic Transport of a Single DNA Molecule.

In the present study, we quantitatively evaluated dielectric breakdown in silicon-based micro- and nanofluidic devices under practical electrophoretic conditions by changing the thickness of the insulating layer. At higher buffer concentration, a silicon nanofluidic device with a 100 nm or 250 nm silicon dioxide layer tolerated dielectric breakdown up to ca. 10 V/cm, thereby allowing successful electrophoretic migration of a single DNA molecule through a nanochannel. The observed DNA migration behavior suggested that parameters, such as thickness of the insulating layer, tolerance of dielectric breakdown, and bonding status of silicon and glass substrate, should be optimized to achieve successful electrophoretic transport of a DNA molecule through a nanopore for nanopore-based DNA sequencing applications.

Static charge outside chamber induces dielectric breakdown of solid-state nanopore membranes

Reducing device capacitance is effective for decreasing current noise observed in a solid-state nanopore-based DNA sequencer. On the other hand, we have recently found that voltage stress causes pinhole-like defects in such low-capacitance devices. The origin of voltage stress, however, has not been determined. In this research, we identified that a dominant origin is static charge on the outer surface of a flow cell. Even though the outer surface was not in direct contact with electrolytes in the flow cell, the charge induces high voltage stress on a membrane according to the capacitance coupling ratio of the flow cell to the membrane.

Topological Line Defects Around Graphene Nanopores for DNA Sequencing

Topological line defects in graphene represent an ideal way to produce highly controlled structures with reduced dimensionality that can be used in electronic devices. In this work, we propose using extended line defects in graphene to improve nucleobase selectivity in nanopore-based DNA sequencing devices. We use a combination of quantum mechanics/molecular mechanics and nonequilibrium Green’s function methods to investigate the conductance modulation, fully accounting for solvent effects. By sampling over a large number of different orientations generated from molecular dynamics simulations, we theoretically demonstrate that distinguishing between the four nucleobases using line defects in a graphene-based electronic device appears possible. The changes in conductance are associated with transport across specific molecular states near the Fermi level and their coupling to the pore. Through the application of a specifically tuned gate voltage, such a device would be able to discriminate the four types of...

Chasing a Protein’s Tail: Detection of Polypeptide Translocation through Nanopores

Chasing a Protein’s Tail: Detection of Polypeptide Translocation through Nanopores

Nanopore Fabrication in Ultrathin HFO2 Membranes for Nanopore-Based DNA Sequencing

Nanopore Fabrication in Ultrathin HFO2 Membranes for Nanopore-Based DNA Sequencing

Real-Time Nanopore-Based Recognition of Protein Translocation Success

A growing number of new technologies are supported by a single- or multi-nanopore architecture for capture, sensing, and delivery of polymeric biomolecules. Nanopore-based single-molecule DNA sequencing is the premier example. This method relies on the uniform linear charge density of DNA, so that each DNA strand is overwhelmingly likely to pass through the nanopore and across the separating membrane. For disordered peptides, folded proteins, or block copolymers with heterogeneous charge densities, by contrast, translocation is not assured, and additional strategies to monitor the progress of the polymer molecule through a nanopore are required. Here, we demonstrate a single-molecule method for direct, model-free, real-time monitoring of the translocation of a disordered, heterogeneously charged polypeptide through a nanopore. The crucial elements are two “selectivity tags”—regions of different but uniform charge density—at the ends of the polypeptide. These affect the selectivity of the nanopore differently and enable discrimination between polypeptide translocation and retraction. Our results demonstrate exquisite sensitivity of polypeptide translocation to applied transmembrane potential and prove the principle that nanopore selectivity reports on biopolymer substructure. We anticipate that the selectivity tag technique will be broadly applicable to nanopore-based protein detection, analysis, and separation technologies, and to the elucidation of protein translocation processes in normal cellular function and in disease.

Theoretical studies on key factors in DNA sequencing using atomically thin molybdenum disulfide nanopores.

Nanopore-based DNA sequencing is considered to be a low-cost, high resolution and superfast method. Solid state nanopores, especially MoS2 nanopores, have been considered to be a promising choice for DNA sequencing. However, researchers still have a very limited understanding of the effects of multiple factors on MoS2-based DNA sequencing. In this study, the effects of the applied voltage and the diameter of the MoS2 nanopore on the resolution of DNA sequencing were investigated. Our results demonstrate that the translocation time of DNA can increase with a decrease in the applied voltage. DNA can be stretched significantly to translocate a 2 nm nanopore under a high applied voltage (>400 mV nm-1). To achieve a 1 base per μs translocation speed (1 GHz bandwidth), we suggest that three methods could be applied, including a decrease in the applied voltage, a decrease in the diameter of the MoS2 nanopore or modification of the MoS2 nanopore. In addition, the size of the nanopore can severely affect the possibility of DNA entering the nanopore, and the translocation time of DNA could be significantly increased with a smaller MoS2 nanopore. These findings may help to design MoS2 nanopores with higher resolution for use in DNA sequencing.

DNA translocation through polyelectrolyte modified hairy nanopores

DNA translocation through nanopore has attracted researcher over the last decade due their immense potential for application in next generation sequencing technique. One of the major challenges of nanopore based DNA sequencing technique is to slow down the translocation speed for precise characterization. In this article, DNA translocation through nanopore functionalized with polyelectrolyte brush has been investigated theoretically. The effect of polyelectrolyte properties (its charge density and softness degree) on DNA translocation speed has been studied for different salt concentration and nanopore radius. The results show that, an increase in polyelectrolyte fixed charged density and softness degree helps in slowing down the DNA translocation speed. Even though a larger diameter nanopore can results in slight reduction in DNA translocation speed at higher salt concentration, it causes significant reduction in current blockade ratio and therefore a larger nanopore is not suitable for sequencing purpose. The results provide significant insight on DNA translocation through soft nanopore which can be applied in designing next generation DNA sequencing device.

Beyond Homopolymer Errors: a Systematic Investigation of Nanopore-based DNA Sequencing Characteristics Using HLA-DQA2

Electronic, nanopore based single molecule real-time DNA sequencing technology offers very long, albeit lower accuracy reads in sharp contrast to existing next-generation sequencing methods, which offer short, high-accuracy reads in abundance. We provide a systematic review of the error characteristics of this new sequencing platform, and demonstrate the most challenging aspects in the field of whole gene sequencing through the human HLA-DQA2 gene using long-range PCR products on multiplexed samples. We consider the limitations of these errors for the applications of this technology, and also indicate prospective improvements and expected thresholds with respect to these errors.

Graphene nanopores toward DNA sequencing: a review of experimental aspects

Nanopores for DNA sequencing have drawn much attention due to their potentials to achieve amplification-free, low-cost, and high-throughput analysis of nuclei acids. The material configuration and fabrication of the nanopore has become one important consideration in the nanopore based DNA sequencing research. Among various materials, the newly emerged graphene has brought more opportunities to the development of sequencing technology because of its unique structures and properties. This review mainly focuses on the experimental aspects of graphene nanopore research including the nanopore fabrication methods and processes. Meanwhile, the challenges in the present graphene nanopore research including hydrophobicity, translocation velocity and noise are also addressed and discussed.

Improving 3D Structure Prediction of Beta-Barrel Membrane Proteins

Beta-barrel membrane proteins are found in the outer membrane of gram-negative bacteria, mitochondria, and chloroplasts. They carry out diverse biological functions, including pore formation, membrane anchoring, enzyme activity, and bacterial virulence. In addition, beta-barrel membrane proteins increasingly serve as scaffolds for bacterial surface display and nanopore-based DNA sequencing. Due to difficulties in experimental structure determination, they are sparsely represented in the protein structure databank and computational methods are required to understand their biophysical principles. We have developed a novel 3D structural template for the construction of beta-barrel membrane proteins, which captures major geometric properties of these proteins. With the help of the prediction procedure of strand registers that we developed, we have achieved a high accuracy for the structure prediction of beta-barrel proteins. In addition, for the beta-barrel proteins with irregular barrel shape, we have further developed an elastic model, which characterizes bending and curvatures of the barrels, to improve the structure prediction of these proteins.

False positives and false negatives measure less than 0.001% in labeling ssDNA with osmium tetroxide 2,2'-bipyridine.

Osmium tetroxide 2,2’-bipyridine (OsBp) is known to react with pyrimidines in ssDNA and preferentially label deoxythymine (T) over deoxycytosine (C). The product, osmylated DNA, was proposed as a surrogate for nanopore-based DNA sequencing due to OsBp’s “perfect” label attributes. Osmylated deoxyoligos translocate unassisted and measurably slow via sub-2 nm SiN solid-state nanopores, as well as via the alpha-hemolysin (α-HL) pore. Both nanopores discriminate clearly between osmylated and intact nucleobase; α-HL was also shown to discriminate between osmylated T and osmylated C. Experiments presented here confirm that the kinetics of osmylation are comparable for short oligos and long ssDNA and show that pyrimidine osmylation is practically complete in two hours at room temperature with less than 15 mM OsBp. Under the proposed labeling conditions: deoxyoligo backbone degradation measures less than 1/1,000,000; false positives such as osmylated deoxyadenine (A) and osmylated deoxyguanine (G) measure less than 1/100,000; false negatives, i.e., unosmylated C measure less than 1/10,000; and unosmylated T must measure substantially lower than 1/10,000 due to the 27-fold higher reactivity of T compared to C. However, osmylated C undergoes degradation that amounts to about 1–2% for the duration of the labeling protocol. This degradation may be further characterized, possibly suppressed, and the properties of the degradation products via nanopore translocation can be evaluated to assure base calling quality in a DNA sequencing effort.

Improved 3D Structure Prediction of Beta-Barrel Membrane Proteins by using Evolutionary Coupling Constraints, Reduced State Space and an Empirical Potential Function

Beta-barrel membrane proteins are found in the outer membrane of gram-negative bacteria, mitochondria, and chloroplasts. They carry out diverse biological functions, including pore formation, membrane anchoring, enzyme activity, and bacterial virulence. In addition, beta-barrel membrane proteins increasingly serve as scaffolds for bacterial surface display and nanopore-based DNA sequencing. Due to difficulties in experimental structure determination, they are sparsely represented in the protein structure databank and computational methods can help to understand their biophysical principles. We have developed a novel computational method to predict the 3D structure of beta-barrel membrane proteins using evolutionary coupling (EC) constraints and a reduced state space. Combined with an empirical potential function, we can successfully predict strand register at >80% accuracy for a set of 49 non-homologous proteins with known structures. This is a significant improvement from previous results using EC alone (44%) [1] and using empirical potential function alone (73%) [2]. Our method is general and can be applied to genome-wide structural prediction.[1] Sikander Hayat, Chris Sander, Debora S. Marks, and Arne Elofsson. All-atom 3D structure prediction of transmembrane β-barrel proteins from sequences. PNAS 2015 112 (17) 5413-5418.[2] Hammad Naveed, Yun Xu, Ronald Jackups, Jr., and Jie Liang. Predicting Three-Dimensional Structures of Transmembrane Domains of β-Barrel Membrane Proteins. Journal of the American Chemical Society 2012 134 (3), 1775-1781.

Single pyrimidine discrimination during voltage-driven translocation of osmylated oligodeoxynucleotides via the α-hemolysin nanopore.

The influence of an electric field on an isolated channel or nanopore separating two compartments filled with electrolytes produces a constant ion flux through the pore. Nucleic acids added to one compartment traverse the pore, and modulate the current in a sequence-dependent manner. While translocation is faster than detection, the α-hemolysin nanopore (α-HL) successfully senses base modifications in ssDNA immobilized within the pore. With the assistance of a processing enzyme to slow down translocation, nanopore-based DNA sequencing is now a commercially available platform. However, accurate base calling is challenging because α-HL senses a sequence, and not a single nucleotide. Osmylated DNA was recently proposed as a surrogate for nanopore-based sequencing. Osmylation is the addition of osmium tetroxide 2,2’-bipyridine (OsBp) to the C5–C6 pyrimidine double bond. The process is simple, selective for deoxythymidine (dT) over deoxycytidine (dC), unreactive towards the purines, practically 100% effective, and strikingly independent of length, sequence, and composition. Translocation of an oligodeoxynucleotide (oligo) dA10XdA9 via α-HL is relatively slow, and exhibits distinct duration as well as distinct residual current when X = dA, dT(OsBp), or dC(OsBp). The data indicate that the α-HL constriction zone/β-barrel interacts strongly with both OsBp and the base. A 23 nucleotide long oligo with four dT(OsBp) traverses 18-times slower, and the same oligo with nine (dT+dC)(OsBp) moieties traverses 84-times slower compared to dA20, suggesting an average rate of 40 or 180 μs/base, respectively. These translocation speeds are well above detection limits, may be further optimized, and clear the way for nanopore-based sequencing using osmylated DNA.