RNA m6A detection using raw current signals and basecalling errors from Nanopore direct RNA sequencing reads.

MOTIVATION: Nanopore direct RNA sequencing (DRS) enables the detection of RNA N6-methyladenosine (m6A) without extra laboratory techniques. A number of supervised or comparative approaches have been developed to identify m6A from Nanopore DRS reads. However, existing methods typically utilize either statistical features of the current signals or basecalling-error features, ignoring the richer information of the raw signals of DRS reads. RESULTS: Here, we propose RedNano, a deep-learning method designed to detect m6A from Nanopore DRS reads by utilizing both raw signals and basecalling errors. RedNano processes the raw-signal feature and basecalling-error feature through residual networks. We validated the effectiveness of RedNano using synthesized, Arabidopsis, and human DRS data. The results demonstrate that RedNano surpasses existing methods by achieving higher area under the ROC curve (AUC) and area under the precision-recall curve (AUPRs) in all three datasets. Furthermore, RedNano performs better in cross-species validation, demonstrating its robustness. Additionally, when detecting m6A from an independent dataset of Populus trichocarpa, RedNano achieves the highest AUC and AUPR, which are 3.8%-9.9% and 5.5%-13.8% higher than other methods, respectively. AVAILABILITY AND IMPLEMENTATION: The source code of RedNano is freely available at https://github.com/Derryxu/RedNano.

Nanopore-Enabled Microbiome Analysis: Investigating Environmental and Host-Associated Samples in Rainbow Trout Aquaculture.

Microbiome sequencing is at the forefront of health management development, and as such, it is becoming of great interest to monitor the microbiome in the aquaculture industry as well. Oxford Nanopore Technologies (ONT) platforms are gaining popularity to study microbial communities, enabling faster sequencing, extended read length, and therefore, improved taxonomic resolution. Despite this, there is a lack of clear guidelines to perform a metabarcoding study, especially when dealing with samples from non-mammalian species, such as aquaculture-related samples. In this article, we provide general guidelines for sampling, nucleic acid extraction, and ONT-based library preparation for both environmental (water, sediment) and host-associated (gill or skin mucus, skin, gut content, or gut mucosa) microbiome analysis. Our procedures focus specifically on rainbow trout (Oncorhynchus mykiss) reared in experimental facilities. However, these protocols can also be transferred to alternative types of samples, such as environmental DNA (eDNA) monitoring from alternative water sources, or to different fish species. The additional challenge posed by the low biomass and limited bacterial diversity inherent in fish-associated microbiomes is addressed through the implementation of troubleshooting solutions. Furthermore, we describe a bioinformatic pipeline starting from raw reads and leading to taxonomic abundance tables using currently available tools and software. Finally, we provide a set of specific guidelines and considerations related to the strategic planning of a microbiome study within the context of aquaculture. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Environmental sample collection Basic Protocol 2: Host-associated sample collection Alternate Protocol: Host-associated sample collection: Alternative sample types Basic Protocol 3: Sample pre-treatment and nucleic acid extraction Basic Protocol 4: Quality control and preparation for 16S rRNA gene sequencing Support Protocol 1: Assessment of inhibition by quantitative PCR Support Protocol 2: Bioinformatic analysis from raw files to taxonomic abundance tables.

Synthetic DNA nanopores for direct molecular transmission between lipid vesicles.

Lipid vesicles hold potential as artificial cells in bottom-up synthetic biology, and as tools in drug delivery and biosensing. Transmitting molecular signals is a key function for vesicle-based systems. One strategy to achieve this function is by releasing molecular signals from vesicles through nanopores. Nevertheless, in this strategy, an excess of molecular signals may be required to reach the targets, due to the dispersion of the signals during diffusion. The key to achieving the efficient utilization of signals is to shorten the distance between the sender vesicle and the target. Here, we present a pair of DNA nanopores that can connect and form a direct molecular pathway between vesicles. The nanopores are self-assembled from nine single DNA strands, including six 14-nucleotide single-stranded overhangs as sticky-end segments, enabling them to bind with each other. Incorporating nanopores shortens the distance between different populations of vesicles, allowing less diffusion of molecules into bulk solution. To further reduce the loss of molecules, a DNA nanocap is added to one of the nanopore's openings. The nanocap can be removed through the toehold-mediated DNA strand displacement when the nanopore meets its counterpart. Our DNA nanopores provide a novel molecular transmission tool to lipid vesicles-based systems.

Structural Determinants of Peptide Nanopore Formation.

We have evolved the nanopore-forming macrolittin peptides from the bee venom peptide melittin using successive generations of synthetic molecular evolution. Despite their sequence similarity to the broadly membrane permeabilizing cytolytic melittin, the macrolittins have potent membrane selectivity. They form nanopores in synthetic bilayers made from 1-palmitoyl, 2-oleoyl-phosphatidylcholine (POPC) at extremely low peptide concentrations and yet have essentially no cytolytic activity against any cell membrane, even at high concentration. Here, we explore the structural determinants of macrolittin nanopore stability in POPC bilayers using atomistic molecular dynamics simulations and experiments on macrolittins and single-site variants. Simulations of macrolittin nanopores in POPC bilayers show that they are stabilized by an extensive, cooperative hydrogen bond network comprised of the many charged and polar side chains interacting with each other via bridges of water molecules and lipid headgroups. Lipid molecules with unusual conformations participate in the H-bond network and are an integral part of the nanopore structure. To explore the role of this H-bond network on membrane selectivity, we swapped three critical polar residues with the nonpolar residues found in melittin. All variants have potency, membrane selectivity, and cytotoxicity that were intermediate between a cytotoxic melittin variant called MelP5 and the macrolittins. Simulations showed that the variants had less organized H-bond networks of waters and lipids with unusual structures. The membrane-spanning, cooperative H-bond network is a critical determinant of macrolittin nanopore stability and membrane selectivity. The results described here will help guide the future design and optimization of peptide nanopore-based applications.

Nanopore Translocation Reveals Electrophoretic Force on Noncanonical RNA:DNA Double Helix.

Electrophoretic transport plays a pivotal role in advancing sensing technologies. So far, systematic studies have focused on the translocation of canonical B-form or A-form nucleic acids, while direct RNA analysis is emerging as the new frontier for nanopore sensing and sequencing. Here, we compare the less-explored dynamics of noncanonical RNA:DNA hybrids in electrophoretic transport to the well-researched transport of B-form DNA. Using DNA/RNA nanotechnology and solid-state nanopores, the translocation of RNA:DNA (RD) and DNA:DNA (DD) duplexes was examined. Notably, RD duplexes were found to translocate through nanopores faster than DD duplexes, despite containing the same number of base pairs. Our experiments reveal that RD duplexes present a noncanonical helix, with distinct transport properties from B-form DD molecules. We find that RD and DD molecules, with the same contour length, move with comparable velocity through nanopores. We examined the physical characteristics of both duplex forms using atomic force microscopy, atomistic molecular dynamics simulations, agarose gel electrophoresis, and dynamic light scattering measurements. With the help of coarse-grained and molecular dynamics simulations, we find the effective force per unit length applied by the electric field to a fragment of RD or DD duplex in nanopores with various geometries or shapes to be approximately the same. Our results shed light on the significance of helical form in nucleic acid translocation, with implications for RNA sensing, sequencing, and the molecular understanding of electrophoretic transport.

Exploring the potential of Oxford Nanopore Technologies sequencing for Mycobacterium tuberculosis sequencing: An assessment of R10 flowcells and V14 chemistry.

Oxford Nanopore Technologies (ONT) sequencing is a promising technology. We assessed the performance of the new ONT R10 flowcells and V14 rapid sequencing chemistry for Mtb whole genome sequencing of Mycobacterium tuberculosis (Mtb) DNA extracted from clinical primary liquid cultures (CPLCs). Using the recommended protocols for MinION Mk1C, R10.4.1 MinION flowcells, and the ONT Rapid Sequencing Kit V14 on six CPLC samples, we obtained a pooled library yield of 10.9 ng/µl, generated 1.94 Gb of sequenced bases and 214k reads after 48h in a first sequencing run. Only half (49%) of all generated reads met the Phred Quality score threshold (>8). To assess if the low data output and sequence quality were due to impurities present in DNA extracted directly from CPLCs, we added a pre-library preparation bead-clean-up step and included purified DNA obtained from an Mtb subculture as a control sample in a second sequencing run. The library yield for DNA extracted from four CPLCs and one Mtb subculture (control) was similar (10.0 ng/µl), 2.38 Gb of bases and 822k reads were produced. The quality was slightly better with 66% of the produced reads having a Phred Quality >8. A third run of DNA from six CPLCs with bead clean-up pre-processing produced a low library yield (±1 Gb of bases, 166k reads) of low quality (51% of reads with a Phred Quality score >8). A median depth of coverage above 10× was only achieved for five of 17 (29%) sequenced libraries. Compared to Illumina WGS of the same samples, accurate lineage predictions and full drug resistance profiles from the generated ONT data could not be determined by TBProfiler. Further optimization of the V14 ONT rapid sequencing chemistry and library preparation protocol is needed for clinical Mtb WGS applications.

Submerged nanoporous anodized alumina structure for solar-powered desalination.

Development of nanoporous structures utilizing a single step of anodization technique is well recognized as a cost-effective and straightforward approach for several applications. In the current work, anodized alumina was developed with nanoporous structure by utilizing oxalic acid as an electrolyte with a continuous voltage of 40 V. The formed nanoporous structure was subjected to desalination application because of its high absorbance of broadband solar spectrum energy. The desalination setup consists of two solar stills namely conventional and modified. The developed structure is placed in the modified still to examine its performance. It was observed that the structure distributing heat to surrounding water by absorbing photon energy from the sun through the nanopores and giving an efficient pathway to the water vapours for developing effective desalination. The nanoporous structure having ~ 45 nm average diameter. Furthermore, the band gap energy of nanoporous structure was found to be ~ 2.5 eV (absorption spectrum fitting) and ~ 2.8 eV (Tauc plot). The nanoporous structure possess the visible light spectra in solar region which helps the band gaps of nanoporous structure to provide an additional supply of energy for generating more water to evaporate. Moreover, the Urbach energy of the structure is 0.5 eV which reveals less defects in the modified still. The overall distillate yield of modified still was increased to 21% in contrast to conventional. Water quality analysis was also carried out before and after the desalination experiments, and the results were within acceptable limits set by World Health Organization (WHO).

Nanoscale Quantitative Imaging of Single Nuclear Pore Complexes by Scanning Electrochemical Microscopy.

The nuclear pore complex (NPC) is a proteinaceous nanopore that solely and selectively regulates the molecular transport between the cytoplasm and nucleus of a eukaryotic cell. The ∼50 nm-diameter pore of the NPC perforates the double-membrane nuclear envelope to mediate both passive and facilitated molecular transport, thereby playing paramount biological and biomedical roles. Herein, we visualize single NPCs by scanning electrochemical microscopy (SECM). The high spatial resolution is accomplished by employing ∼25 nm-diameter ion-selective nanopipets to monitor the passive transport of tetrabutylammonium at individual NPCs. SECM images are quantitatively analyzed by employing the finite element method to confirm that this work represents the highest-resolution nanoscale SECM imaging of biological samples. Significantly, we apply the powerful imaging technique to address the long-debated origin of the central plug of the NPC. Nanoscale SECM imaging demonstrates that unplugged NPCs are more permeable to the small probe ion than are plugged NPCs. This result supports the hypothesis that the central plug is not an intrinsic transporter, but is an impermeable macromolecule, e.g., a ribonucleoprotein, trapped in the nanopore. Moreover, this result also supports the transport mechanism where the NPC is divided into the central pathway for RNA export and the peripheral pathway for protein import to efficiently mediate the bidirectional traffic.

Detection of aldehydes from degradation of lipid nanoparticle formulations using a hierarchically-organized nanopore electrochemical biosensor.

Degradation of ionizable lipids in mRNA-based vaccines was recently found to deactivate the payload, demanding rigorous monitoring of impurities in lipid nanoparticle (LNP) formulations. However, parallel screening for lipid degradation in customized delivery systems for next-generation therapeutics maintains a challenging and unsolved problem. Here, we describe a nanopore electrochemical sensor to detect ppb-levels of aldehydes arising from lipid degradation in LNP formulations that can be deployed in massively parallel fashion. Specifically, we combine nanopore electrodes with a block copolymer (BCP) membrane capable of hydrophobic gating of analyte transport between the bulk solution and the nanopore volume. By incorporating aldehyde dehydrogenase (ALDH), enzymatic oxidation of aldehydes generates NADH to enable ultrasensitive voltammetric detection with limits-of-detection (LOD) down to 1.2 ppb. Sensor utility was demonstrated by detecting degradation of N-oxidized SM-102, the ionizable lipid in Moderna's SpikeVax™ vaccine, in mRNA-1273 LNP formulation. This work should be of significant use in the pharmaceutical industry, paving the way for automated on-line quality assessments of next-generation therapeutics.

Functionally Active Synthetic α-Helical Pores.

ConspectusTransmembrane pores are currently at the forefront of nanobiotechnology, nanopore chemistry, and synthetic chemical biology research. Over the past few decades, significant studies in protein engineering have paved the way for redesigning membrane protein pores tailored for specific applications in nanobiotechnology. Most previous efforts predominantly centered on natural ß-barrel pores designed with atomic precision for nucleic acid sequencing and sensing of biomacromolecules, including protein fragments. The requirement for a more efficient single-molecule detection system has driven the development of synthetic nanopores. For example, engineering channels to conduct ions and biomolecules selectively could lead to sophisticated nanopore sensors. Also, there has been an increased interest in synthetic pores, which can be fabricated to provide more control in designing architecture and diameter for single-molecule sensing of complex biomacromolecules. There have been impressive advancements in developing synthetic DNA-based pores, although their application in nanopore technology is limited. This has prompted a significant shift toward building synthetic transmembrane α-helical pores, a relatively underexplored field offering novel opportunities. Recently, computational tools have been employed to design and construct α-helical barrels of defined structure and functionality.We focus on building synthetic α-helical pores using naturally occurring transmembrane motifs of membrane protein pores. Our laboratory has developed synthetic α-helical transmembrane pores based on the natural porin PorACj (Porin A derived from Corynebacterium jeikeium) that function as nanopore sensors for single-molecule sensing of cationic cyclodextrins and polypeptides. Our breakthrough lies in being the first to create a functional and large stable synthetic transmembrane pore composed of short synthetic α-helical peptides. The key highlight of our work is that these pores can be synthesized using easy chemical synthesis, which permits its easy modification to include a variety of functional groups to build charge-selective sophisticated pores. Additionally, we have demonstrated that stable functional pores can be constructed from D-amino acid peptides. The analysis of pores composed of D- and L-amino acids in the presence of protease showed that only the D pores are highly functional and stable. The structural models of these pores revealed distinct surface charge conformation and geometry. These new classes of synthetic α-helical pores are highly original systems of general interest due to their unique architecture, functionality, and potential applications in nanopore technology and chemical biology. We emphasize that these simplified transmembrane pores have the potential to be components of functional nanodevices and therapeutic tools. We also suggest that such designed peptides might be valuable as antimicrobial agents and can be targeted to cancer cells. This article will focus on the evolutions in assembling α-helical transmembrane pores and highlight their advantages, including structural and functional versatility.

Unraveling RNA Conformation Dynamics in Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like Episode Syndrome with Solid-State Nanopores.

This study investigates transfer ribonucleic acid (tRNA) conformational dynamics in the context of MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) using solid-state silicon nitride (SiN) nanopore technology. SiN nanopores in thin membranes with specific dimensions exhibit high signal resolution, enabling real-time and single-molecule electronic detection of tRNA conformational changes. We focus on human mitochondrial tRNALeu(UAA) (mt-Leu(UAA)) that decodes Leu codons UUA/UUG (UUR) during protein synthesis on the mt-ribosome. The single A14G substitution in mt-Leu(UAA) is the major cause of MELAS disease. Measurements of current blockades and dwell times reveal distinct conformational dynamics of the wild-type (WT) and the A14G variant of mt-Leu(UAA) in response to the conserved post-transcriptional m1G9 methylation. While the m1G9-modified WT transcript adopts a more stable structure relative to the unmodified transcript, the m1G9-modified MELAS transcript adopts a less stable structure relative to the unmodified transcript. Notably, these differential features were observed at 0.4 M KCl, but not at 3 M KCl, highlighting the importance of experimental settings that are closer to physiological conditions. This work demonstrates the feasibility of the nanopore platform to discern tRNA molecules that differ by a single-nucleotide substitution or by a single methylation event, providing an important step forward to explore changes in the conformational dynamics of other RNA molecules in human diseases.

Controlled synthesized of ternary Cu-Co-Ni-S sulfides nanoporous network structure on carbon fiber paper: a superior catalytic electrode for highly-sensitive glucose sensing.

BACKGROUND: Efficient monitoring of glucose concentration in the human body necessitates the utilization of electrochemically active sensing materials in nonenzymatic glucose sensors. However, prevailing limitations such as intricate fabrication processes, lower sensitivity, and instability impede their practical application. Herein, ternary Cu-Co-Ni-S sulfides nanoporous network structure was synthesized on carbon fiber paper (CP) by an ultrafast, facile, and controllable technique through on-step cyclic voltammetry, serving as a superior self-supporting catalytic electrode for the high-performance glucose sensor. RESULTS: The direct growth of free-standing Cu-Co-Ni-S on the interconnected three-dimensional (3D) network of CP boosted the active site of the composites, improved ion diffusion kinetics, and significantly promoted the electron transfer rate. The multiple oxidation states and synergistic effects among Co, Ni, Cu, and S further promoted glucose electrooxidation. The well-architected Cu-Co-Ni-S/CP presented exceptional electrocatalytic properties for glucose with satisfied linearity of a broad range from 0.3 to 16,000 µM and high sensitivity of 6829 µA mM- 1 cm- 2. Furthermore, the novel sensor demonstrated excellent selectivity and storage stability, which could successfully evaluate the glucose levels in human serum. Notably, the novel Cu-Co-Ni-S/CP showed favorable biocompatibility, proving its potential for in vivo glucose monitoring. CONCLUSION: The proposed 3D hierarchical morphology self-supported electrode sensor, which demonstrates appealing analysis behavior for glucose electrooxidation, holds great promise for the next generation of high-performance glucose sensors.

Sigmoni: classification of nanopore signal with a compressed pangenome index.

SUMMARY: Improvements in nanopore sequencing necessitate efficient classification methods, including pre-filtering and adaptive sampling algorithms that enrich for reads of interest. Signal-based approaches circumvent the computational bottleneck of basecalling. But past methods for signal-based classification do not scale efficiently to large, repetitive references like pangenomes, limiting their utility to partial references or individual genomes. We introduce Sigmoni: a rapid, multiclass classification method based on the r-index that scales to references of hundreds of Gbps. Sigmoni quantizes nanopore signal into a discrete alphabet of picoamp ranges. It performs rapid, approximate matching using matching statistics, classifying reads based on distributions of picoamp matching statistics and co-linearity statistics, all in linear query time without the need for seed-chain-extend. Sigmoni is 10-100× faster than previous methods for adaptive sampling in host depletion experiments with improved accuracy, and can query reads against large microbial or human pangenomes. Sigmoni is the first signal-based tool to scale to a complete human genome and pangenome while remaining fast enough for adaptive sampling applications. AVAILABILITY AND IMPLEMENTATION: Sigmoni is implemented in Python, and is available open-source at https://github.com/vshiv18/sigmoni.

Nanopores Reveal the Stoichiometry of Single Oligoadenylates Produced by Type III CRISPR-Cas.

Cyclic oligoadenylates (cOAs) are small second messenger molecules produced by the type III CRISPR-Cas system as part of the prokaryotic immune response. The role of cOAs is to allosterically activate downstream effector proteins that induce dormancy or cell death, and thus abort viral spread through the population. Interestingly, different type III systems have been reported to utilize different cOA stoichiometries (with 3 to 6 adenylate monophosphates). However, so far, their characterization has only been possible in bulk and with sophisticated equipment, while a portable assay with single-molecule resolution has been lacking. Here, we demonstrate the label-free detection of single cOA molecules using a simple protein nanopore assay. It sensitively identifies the stoichiometry of individual cOA molecules and their mixtures from synthetic and enzymatic origin. To achieve this, we trained a convolutional neural network (CNN) and validated it with a series of experiments on mono- and polydisperse cOA samples. Ultimately, we determined the stoichiometric composition of cOAs produced enzymatically by the CRISPR type III-A and III-B variants of Thermus thermophilus and confirmed the results by liquid chromatography-mass spectroscopy (LC-MS). Interestingly, both variants produce cOAs of nearly identical composition (within experimental uncertainties), and we discuss the biological implications of this finding. The presented nanopore-CNN workflow with single cOA resolution can be adapted to many other signaling molecules (including eukaryotic ones), and it may be integrated into portable handheld devices with potential point-of-care applications.

Identifying Viral Vector Characteristics by Nanopore Sensing.

Using viral vectors as gene delivery vehicles for gene therapy necessitates their quality control. Here, we report on nanopore sensing for nondestructively inspecting genomes inside the nanoscale cargoes at the single-molecule level. Using ionic current measurements, we motion-tracked the adeno-associated virus (AAV) vectors as they translocated through a solid-state nanopore. Considering the varying contributions of the electrophoretic forces from the negatively charged internal polynucleotides of different lengths, the nanocargoes carrying longer DNA moved more slowly in the nanochannel. Moreover, ion blockage characteristics revealed their larger volume by up to approximately 3600 nm3 in proportion to the length of single-stranded DNA packaged inside, thereby allowing electrical discriminations of AAV vectors by the gene-derived physical features. The present findings can be a promising tool for the enhanced quality control of AAV products by enabling the screening of empty and intermediate vectors at the single-particle level.

Effective training of nanopore callers for epigenetic marks with limited labelled data.

Nanopore sequencing platforms combined with supervised machine learning (ML) have been effective at detecting base modifications in DNA such as 5-methylcytosine (5mC) and N6-methyladenine (6mA). These ML-based nanopore callers have typically been trained on data that span all modifications on all possible DNA [Formula: see text]-mer backgrounds-a complete training dataset. However, as nanopore technology is pushed to more and more epigenetic modifications, such complete training data will not be feasible to obtain. Nanopore calling has historically been performed with hidden Markov models (HMMs) that cannot make successful calls for [Formula: see text]-mer contexts not seen during training because of their independent emission distributions. However, deep neural networks (DNNs), which share parameters across contexts, are increasingly being used as callers, often outperforming their HMM cousins. It stands to reason that a DNN approach should be able to better generalize to unseen [Formula: see text]-mer contexts. Indeed, herein we demonstrate that a common DNN approach (DeepSignal) outperforms a common HMM approach (Nanopolish) in the incomplete data setting. Furthermore, we propose a novel hybrid HMM-DNN approach, amortized-HMM, that outperforms both the pure HMM and DNN approaches on 5mC calling when the training data are incomplete. This type of approach is expected to be useful for calling other base modifications such as 5-hydroxymethylcytosine and for the simultaneous calling of different modifications, settings in which complete training data are not likely to be available.

How low can you go? Short-read polishing of Oxford Nanopore bacterial genome assemblies.

It is now possible to assemble near-perfect bacterial genomes using Oxford Nanopore Technologies (ONT) long reads, but short-read polishing is usually required for perfection. However, the effect of short-read depth on polishing performance is not well understood. Here, we introduce Pypolca (with default and careful parameters) and Polypolish v0.6.0 (with a new careful parameter). We then show that: (1) all polishers other than Pypolca-careful, Polypolish-default and Polypolish-careful commonly introduce false-positive errors at low read depth; (2) most of the benefit of short-read polishing occurs by 25× depth; (3) Polypolish-careful almost never introduces false-positive errors at any depth; and (4) Pypolca-careful is the single most effective polisher. Overall, we recommend the following polishing strategies: Polypolish-careful alone when depth is very low (<5×), Polypolish-careful and Pypolca-careful when depth is low (5-25×), and Polypolish-default and Pypolca-careful when depth is sufficient (>25×).

High-Resolution and Low-Noise Single-Molecule Sensing with Bio-Inspired Solid-State Nanopores.

Solid-state nanopores have been extensively explored as single-molecule sensors, bearing the potential for the sequencing of DNA. Although they offer advantages in terms of high mechanical robustness, tunable geometry, and compatibility with existing semiconductor fabrication techniques in comparison with their biological counterparts, efforts to sequence DNA with these nanopores have been hampered by insufficient spatial resolution and high noise in the measured ionic current signal. Here we show that these limitations can be overcome by the use of solid-state nanopores featuring a thin, narrow constriction as the sensing region, inspired by biological protein nanopores that have achieved notable success in DNA sequencing. Our extensive molecular dynamics simulations show that these bio-inspired nanopores can provide high spatial resolution equivalent to 2D material nanopores and, meanwhile, significantly inhibit noise levels. A theoretical model is also provided to assess the performance of the bio-inspired nanopore, which could guide its design and optimization.

Evaluation of the accuracy of bacterial genome reconstruction with Oxford Nanopore R10.4.1 long-read-only sequencing.

Whole-genome reconstruction of bacterial pathogens has become an important tool for tracking transmission and antimicrobial resistance gene spread, but highly accurate and complete assemblies have largely only historically been achievable using hybrid long- and short-read sequencing. We previously found the Oxford Nanopore Technologies (ONT) R10.4/kit12 flowcell/chemistry produced improved assemblies over the R9.4.1/kit10 combination, however long-read only assemblies contained more errors compared to Illumina-ONT hybrid assemblies. ONT have since released an R10.4.1/kit14 flowcell/chemistry upgrade and recommended the use of Bovine Serum Albumin (BSA) during library preparation, both of which reportedly increase accuracy and yield. They have also released updated basecallers trained using native bacterial DNA containing methylation sites intended to fix systematic basecalling errors, including common adenosine (A) to guanine (G) and cytosine (C) to thymine (T) substitutions. To evaluate these improvements, we successfully sequenced four bacterial reference strains, namely Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus, and nine genetically diverse E. coli bloodstream infection-associated isolates from different phylogroups and sequence types, both with and without BSA. These sequences were de novo assembled and compared against Illumina-corrected reference genomes. In this small evaluation of 13 isolates we found that nanopore long-read-only R10.4.1/kit 14 assemblies with updated basecallers trained using bacterial methylated DNA produce accurate assemblies with ≥40×depth, sufficient to be cost-effective compared with hybrid ONT/Illumina sequencing in our setting.

Salt gradient enhanced sensitivity in nanopores for intracellular calcium ion detection.

Intracellular calcium ion detection is of great significance for understanding the cell metabolism and signaling pathways. Most of the current ionic sensors either face the size issue or sensitivity limit for the intracellular solution with high background ion concentrations. In this paper, we proposed a calmodulin (CaM) functionalized nanopore for sensitive and selective Ca2+ detection inside living cells. A salt gradient was created when the nanopore sensor filled with a low concentration electrolyte was in contact with a high background concentration solution, which enhanced the surface charge-based detection sensitivity. The nanopore sensor showed a 10 × sensitivity enhancement by application of a 100-fold salt gradient, and a detection limit of sub nM. The sensor had a wide detection range from 1 nM to 1 mM, and allowed for quick calcium ion quantification in a few seconds. The sensor was demonstrated for intracellular Ca2+ detection in A549 cells in response to ionomycin.