Quantitative analysis of C. elegans transcripts by Nanopore direct-cDNA sequencing reveals terminal hairpins in non trans-spliced mRNAs.

In nematodes and kinetoplastids, mRNA processing involves a trans-splicing step through which a short sequence from a snRNP replaces the original 5' end of the primary transcript. It has long been held that 70% of C. elegans mRNAs are submitted to trans-splicing. Our recent work suggested that the mechanism is more pervasive but not fully captured by mainstream transcriptome sequencing methods. Here we use Oxford Nanopore's long-read amplification-free sequencing technology to perform a comprehensive analysis of trans-splicing in worms. We demonstrate that spliced leader (SL) sequences at the 5' end of the mRNAs affect library preparation and generate sequencing artefacts due to their self-complementarity. Consistent with our previous observations, we find evidence of trans-splicing for most genes. However, a subset of genes appears to be only marginally trans-spliced. These mRNAs all share the capacity to generate a 5' terminal hairpin structure mimicking the SL structure and offering a mechanistic explanation for their non conformity. Altogether, our data provide a comprehensive quantitative analysis of SL usage in C. elegans.

Nanopore Detection Using Supercharged Polypeptide Molecular Carriers.

The analysis at the single-molecule level of proteins and their interactions can provide critical information for understanding biological processes and diseases, particularly for proteins present in biological samples with low copy numbers. Nanopore sensing is an analytical technique that allows label-free detection of single proteins in solution and is ideally suited to applications, such as studying protein-protein interactions, biomarker screening, drug discovery, and even protein sequencing. However, given the current spatiotemporal limitations in protein nanopore sensing, challenges remain in controlling protein translocation through a nanopore and relating protein structures and functions with nanopore readouts. Here, we demonstrate that supercharged unstructured polypeptides (SUPs) can be genetically fused with proteins of interest and used as molecular carriers to facilitate nanopore detection of proteins. We show that cationic SUPs can substantially slow down the translocation of target proteins due to their electrostatic interactions with the nanopore surface. This approach enables the differentiation of individual proteins with different sizes and shapes via characteristic subpeaks in the nanopore current, thus facilitating a viable route to use polypeptide molecular carriers to control molecular transport and as a potential system to study protein-protein interactions at the single-molecule level.

Gold Nanotriangle-Assembled Nanoporous Structures for Electric Field-Assisted Surface-Enhanced Raman Scattering Detection of Adenosine Triphosphate.

A reliable, rapid, cost-effective, and simple method for the detection of biomolecules would greatly promote the research of analytical detection of single molecules. A nanopore-based analytical technique is promising for detecting biomolecules. Conventional electrochemical nanopores cannot distinguish biomolecules precisely because of their fast translocation speed and limited electrochemical information. Therefore, it is highly desirable to develop electrochemical surface-enhanced Raman scattering (SERS) nanopores to obtain multidimensional information. Herein, we designed and fabricated gold nanotriangle (AuNT)-assembled porous structures at the tip of a glass capillary using dithiol adenosine triphosphate (ATP) aptamers as cross-linking molecules. The AuNTs exhibited an edge length of 57.3 ± 6.2 nm and thickness of about 15 nm. The gold nanoporous structure (GPS) showed a strong ion rectification even at a high concentration of electrolyte (2 M) and a high SERS activity. Based on these designed structures, SERS and electrochemistry techniques were combined to control the rapid movement of ATP to the vicinity of the GPS by an applied potential of +1 V, where ATP was concentrated by ATP aptamers and the molecular signals were amplified by SERS. As a result, the GPS successfully detected ATP at a concentration as low as 10-7 M.

Nanopore translocation of topologically linked DNA catenanes.

The electrical signal associated with a biopolymer translocating through a nanoscale pore depends on the size, topology, and configuration of each molecule. Building upon recent interest in using solid-state nanopores for studying the topology of knotted and supercoiled DNA, we present experimental observations of topologically linked catenanes translocating through a solid-state nanopore. Using restriction enzymes, linked circular molecules were isolated from the mitochondrial DNA of Crithidia fasciculata, a structure known as a kinetoplast that comprises thousands of topologically interlocked minicircles. Digested kinetoplasts produce a spectrum of catenane topologies, which are identified from their nanopore translocation signals by spikes in the blockade current associated with the topological linkages. We attribute the different patterns of the measured electrical signals to 2-catenanes, linear and triangular 3-catenanes, and several types of 4- and 5-catenanes as well as more complex structures. Measurements of the translocation time of signals consistent with 2- and 3-catenanes suggest that topological friction between the linkages and the pore slows the translocation time of these structures, as predicted in recent simulations.

Characterization and simulation of metagenomic nanopore sequencing data with Meta-NanoSim.

BACKGROUND: Nanopore sequencing is crucial to metagenomic studies as its kilobase-long reads can contribute to resolving genomic structural differences among microbes. However, sequencing platform-specific challenges, including high base-call error rate, nonuniform read lengths, and the presence of chimeric artifacts, necessitate specifically designed analytical algorithms. The use of simulated datasets with characteristics that are true to the sequencing platform under evaluation is a cost-effective way to assess the performance of bioinformatics tools with the ground truth in a controlled environment. RESULTS: Here, we present Meta-NanoSim, a fast and versatile utility that characterizes and simulates the unique properties of nanopore metagenomic reads. It improves upon state-of-the-art methods on microbial abundance estimation through a base-level quantification algorithm. Meta-NanoSim can simulate complex microbial communities composed of both linear and circular genomes and can stream reference genomes from online servers directly. Simulated datasets showed high congruence with experimental data in terms of read length, error profiles, and abundance levels. We demonstrate that Meta-NanoSim simulated data can facilitate the development of metagenomic algorithms and guide experimental design through a metagenome assembly benchmarking task. CONCLUSIONS: The Meta-NanoSim characterization module investigates read features, including chimeric information and abundance levels, while the simulation module simulates large and complex multisample microbial communities with different abundance profiles. All trained models and the software are freely accessible at GitHub: https://github.com/bcgsc/NanoSim.

A generalizable nanopore sensor for highly specific protein detection at single-molecule precision.

Protein detection has wide-ranging implications in molecular diagnostics. Substantial progress has been made in protein analytics using nanopores and the resistive-pulse technique. Yet, a long-standing challenge is implementing specific interfaces for detecting proteins without the steric hindrance of the pore interior. Here, we formulate a class of sensing elements made of a programmable antibody-mimetic binder fused to a monomeric protein nanopore. This way, such a modular design significantly expands the utility of nanopore sensors to numerous proteins while preserving their architecture, specificity, and sensitivity. We prove the power of this approach by developing and validating nanopore sensors for protein analytes that drastically vary in size, charge, and structural complexity. These analytes produce unique electrical signatures that depend on their identity and quantity and the binder-analyte assembly at the nanopore tip. The outcomes of this work could impact biomedical diagnostics by providing a fundamental basis for biomarker detection in biofluids.

Nanopore Sequencing Discloses Compositional Quality of Commercial Probiotic Feed Supplements.

The market for the application of probiotics as a livestock health improvement supplement has increased in recent years. However, most of the available products are quality-controlled using low-resolution techniques and un-curated databases, resulting in misidentification and incorrect product labels. In this work, we deployed two workflows and compared results obtained by full-length 16S rRNA genes (16S) and metagenomic (Meta) data to investigate their reliability for the microbial composition of both liquid and solid forms of animal probiotic products using Oxford Nanopore long-read-only (without short-read). Our result revealed that 16S amplicon data permits to detect the bacterial microbiota even with the low abundance in the samples. Moreover, the 16S approach has the potential to provide species-level resolution for prokaryotes but not for assessing yeast communities. Whereas, Meta data has more power to recover of high-quality metagenome-assembled genomes that enables detailed exploration of both bacterial and yeast populations, as well as antimicrobial resistance genes, and functional genes in the population. Our findings clearly demonstrate that implementing these workflows with long-read-only monitoring could be applied to assessing the quality and safety of probiotic products for animals and evaluating the quality of probiotic products on the market. This would benefit the sustained growth of the livestock probiotic industry.

Transcriptome dataset from Solanum lycopersicum L. cv. Micro-Tom; wild type and two mutants of INDOLE-ACETIC-ACID (SlIAA9) using long-reads sequencing oxford nanopore technologies.

OBJECTIVE: Tomatoes are the most widely consumed fruit vegetable and are relatively easy to cultivate. However, an increase in temperature causes some plants to respond with a decrease in fruit production. So, it is necessary to develop plants resistant to extreme temperature changes. The tomato cv. Micro-Tom has genetic variations in the gene of INDOLE-ACETIC-ACID, namely SlIAA9-3 and SlIAA9-5. However, the genetic information regarding the full-length transcript of the gene from this type of tomato plant is unknown. Therefore, this study aimed to determine the full-length transcript of the genes of these three types of tomatoes using long-reads sequencing technology from Oxford Nanopore. DATA DESCRIPTION: The total RNA from three types of Micro-Tom was isolated with the RNeasy PowerPlant Kit. Then, the RNA sequencing process used PCR-cDNA Barcoding kit - SQK-PCB109 and continued with the processing of raw reads based on the protocol from microbepore protocol ( https://github.com/felixgrunberger/microbepore ). The resulting raw reads were 578 374, 409 905, and 851 948 for wildtype, iaa9-3, and iaa9-5, respectively. After obtaining cleaned reads, each sample was mapped to the tomato reference genome (S. lycopersicum ITAG4.0) with the Minimap2 program. In particular, 965 genes were expressed only in the iaa9-3 mutant, and 2332 genes were expressed only in the iaa9-5 mutant. Whereas in the wild type, 1536 genes are specifically expressed. In cluster analysis using the heatmap analysis, separate groups were obtained between the wild type and the two mutants. This proves an overall difference in transcript levels between the wild type and the mutants.

Polishing De Novo Nanopore Assemblies of Bacteria and Eukaryotes With FMLRC2.

As the accuracy and throughput of nanopore sequencing improve, it is increasingly common to perform long-read first de novo genome assemblies followed by polishing with accurate short reads. We briefly introduce FMLRC2, the successor to the original FM-index Long Read Corrector (FMLRC), and illustrate its performance as a fast and accurate de novo assembly polisher for both bacterial and eukaryotic genomes.

Promotion of Wound Healing Using Nanoporous Silk Fibroin Sponges.

Wound dressings are important for wound repair. The morphology of the biomaterials used in these dressings, and in particular, the pore structure affects tissue regeneration by facilitating attachment and proliferation of cells due to the hierarchical multiscale, water absorbance, and nutrient transport. In the present study, silk fibroin (SF) sponges with walls containing nanopores (SFNS) were prepared from SF nanoparticles generated during the autoclaving of SF solutions, followed by leaching the SF nanoparticles from the freeze-dried sponges of SF. The nano/microporous structure, biofluid absorbance, and porosity of the SF sponges with and without nanopores were characterized. In vitro cell proliferation, in vivo biocompatibility, and wound healing were evaluated with the sponges. The results demonstrated that SFNS had significantly increased porosity and water permeability, as well as cell attachment and proliferation when compared with SF sponges without the nanopores (SFS). Wound dressings were assessed in a rat skin wound model, and SFNS was superior to SFS in accelerating wound healing, supported by vascularization, deposition of collagen, and increased epidermal thickness over 21 days. Hence, such a dressing material with a hierarchical multiscale pore structure could promote cell migration, vascularization, and tissue regeneration independently without adding any growth factor, which would offer a new strategy to design and engineer better-performed wound dressing.

Calling and Phasing of Single-Nucleotide and Structural Variants of the LDLR Gene Using Oxford Nanopore MinION.

The LDLR locus has clinical significance for lipid metabolism, Mendelian familial hypercholesterolemia (FH), and common lipid metabolism-related diseases (coronary artery disease and Alzheimer's disease), but its intronic and structural variants are underinvestigated. The aim of this study was to design and validate a method for nearly complete sequencing of the LDLR gene using long-read Oxford Nanopore sequencing technology (ONT). Five PCR amplicons from LDLR of three patients with compound heterozygous FH were analyzed. We used standard workflows of EPI2ME Labs for variant calling. All rare missense and small deletion variants detected previously by massively parallel sequencing and Sanger sequencing were identified using ONT. One patient had a 6976 bp deletion (exons 15 and 16) that was detected by ONT with precisely located breakpoints between AluY and AluSx1. Trans-heterozygous associations between mutation c.530C>T and c.1054T>C, c.2141-966_2390-330del, and c.1327T>C, and between mutations c.1246C>T and c.940+3_940+6del of LDLR, were confirmed. We demonstrated the ability of ONT to phase variants, thereby enabling haplotype assignment for LDLR with personalized resolution. The ONT-based method was able to detect exonic variants with the additional benefit of intronic analysis in one run. This method can serve as an efficient and cost-effective tool for diagnosing FH and conducting research on extended LDLR haplotype reconstruction.

Assembling the perfect bacterial genome using Oxford Nanopore and Illumina sequencing.

A perfect bacterial genome assembly is one where the assembled sequence is an exact match for the organism's genome-each replicon sequence is complete and contains no errors. While this has been difficult to achieve in the past, improvements in long-read sequencing, assemblers, and polishers have brought perfect assemblies within reach. Here, we describe our recommended approach for assembling a bacterial genome to perfection using a combination of Oxford Nanopore Technologies long reads and Illumina short reads: Trycycler long-read assembly, Medaka long-read polishing, Polypolish short-read polishing, followed by other short-read polishing tools and manual curation. We also discuss potential pitfalls one might encounter when assembling challenging genomes, and we provide an online tutorial with sample data (github.com/rrwick/perfect-bacterial-genome-tutorial).

Hybrid Genome Assembly of Short and Long Reads in Galaxy.

Galaxy is a web browser-based data analysis platform that is widely used in biology. Public Galaxy instances allow the analysis of data and interpretation of results without requiring software installation. NanoGalaxy is a public Galaxy instance with tools and workflows for nanopore data analysis. This chapter describes the steps involved in performing genome assembly using short and long reads in NanoGalaxy.

Microbial Genome Sequencing and Assembly Using Nanopore Sequencers.

Microbial genomes are typically several million base pairs in length and are relatively easy to sequence and assemble into a single chromosome, given the advances in long-read sequencing platforms such as that of Oxford Nanopore Technologies. This chapter describes the experimental as well as computational steps in the sequencing and assembly of microbial genomes.

The Current State of Nanopore Sequencing.

Nanopore sensing is a disruptive, revolutionary way in which to sequence nucleic acids, including both native DNA and RNA molecules. First commercialized with the MinIONTM sequencer from Oxford Nanopore TechnologiesTM in 2015, this review article looks at the current state of nanopore sequencing as of June 2022. Covering the unique characteristics of the technology and how it functions, we then go on to look at the ability of the platform to deliver sequencing at all scales-from personal to high-throughput devices-before looking at how the scientific community is applying the technology around the world to answer their biological questions.

How to Sequence and Assemble Plant Genomes.

Although nanopore sequencer is a great tool, many plant scientists have suffered from bad sequencing results, even though they have exactly followed the official protocol in preparing a library. This is because the protocol is not optimized for plant genomic DNA. The protocol may be good for sequencing animal or bacterial genomes, but not for plants. However, if the protocol is properly modified, one can obtain lots of long reads and achieve a telomere-to-telomere assembly. Here I present a protocol to that end.

Detection of DNA Modification Using Nanopore Sequencers.

DNA modification is a crucial factor of epigenetic modification and has vital functions for gene regulation and phenotype control. A profound understanding of DNA modification requires precise mapping of the modified bases on genomic DNA. In addition to methods such as bisulfite sequencing and single-molecule real-time (SMRT) sequencing of PacBio sequencers, nanopore sequencers can be also utilized for the detection of DNA modification. Here, I will briefly review the three methods for the detection of DNA modification with nanopore sequencers and introduce a protocol using MinION and Megalodon.

Finding Rearrangements in Nanopore DNA Reads with LAST and dnarrange.

Long-read DNA sequencing techniques such as nanopore are especially useful for characterizing complex sequence rearrangements, which occur in some genetic diseases and also during evolution. Analyzing the sequence data to understand such rearrangements is not trivial, due to sequencing error, rearrangement intricacy, and abundance of repeated similar sequences in genomes.The LAST and dnarrange software packages can resolve complex relationships between DNA sequences and characterize changes such as gene conversion, processed pseudogene insertion, and chromosome shattering. They can filter out numerous rearrangements shared by controls, e.g., healthy humans versus a patient, to focus on rearrangements unique to the patient. One useful ingredient is last-train, which learns the rates (probabilities) of deletions, insertions, and each kind of base match and mismatch. These probabilities are then used to find the most likely sequence relationships/alignments, which is especially useful for DNA with unusual rates, such as DNA from Plasmodium falciparum (malaria) with ∼80% a+t. This is also useful for less-studied species that lack reference genomes, so the DNA reads are compared to a different species' genome. We also point out that a reference genome with ancestral alleles would be ideal.

Nanopore Sequencing Data Analysis of 16S rRNA Genes Using the GenomeSync-GSTK System.

With the development of nanopore sequencing technology, long reads of DNA sequences can now be determined rapidly from various samples. This protocol introduces the GenomeSync-GSTK system for bacterial species identification in a given sample using nanopore sequencing data of 16S rRNA genes as an example. GenomeSync is a collection of genome sequences designed to provide easy access to genomic data of the species as demanded. GSTK (genome search toolkit) is a set of scripts for managing local homology searches using genomes obtained from the GenomeSync database. Based on this protocol, nanopore sequencing data analyses of metagenomes and amplicons could be efficiently performed. We also noted reanalysis in conjunction with future developments in nanopore sequencing technology and the accumulation of genome sequencing data.