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Nature Biotechnology Nov 2021Rapid advances in nanopore technologies for sequencing single long DNA and RNA molecules have led to substantial improvements in accuracy, read length and throughput.... (Review)
Review
Rapid advances in nanopore technologies for sequencing single long DNA and RNA molecules have led to substantial improvements in accuracy, read length and throughput. These breakthroughs have required extensive development of experimental and bioinformatics methods to fully exploit nanopore long reads for investigations of genomes, transcriptomes, epigenomes and epitranscriptomes. Nanopore sequencing is being applied in genome assembly, full-length transcript detection and base modification detection and in more specialized areas, such as rapid clinical diagnoses and outbreak surveillance. Many opportunities remain for improving data quality and analytical approaches through the development of new nanopores, base-calling methods and experimental protocols tailored to particular applications.
Topics: Computational Biology; High-Throughput Nucleotide Sequencing; Nanopore Sequencing; Nanopores; Sequence Analysis, DNA; Technology
PubMed: 34750572
DOI: 10.1038/s41587-021-01108-x -
Biosensors Jun 2021In recent years, nanopore technology has become increasingly important in the field of life science and biomedical research. By embedding a nano-scale hole in a thin... (Review)
Review
In recent years, nanopore technology has become increasingly important in the field of life science and biomedical research. By embedding a nano-scale hole in a thin membrane and measuring the electrochemical signal, nanopore technology can be used to investigate the nucleic acids and other biomacromolecules. One of the most successful applications of nanopore technology, the Oxford Nanopore Technology, marks the beginning of the fourth generation of gene sequencing technology. In this review, the operational principle and the technology for signal processing of the nanopore gene sequencing are documented. Moreover, this review focuses on the applications using nanopore gene sequencing technology, including the diagnosis of cancer, detection of viruses and other microbes, and the assembly of genomes. These applications show that nanopore technology is promising in the field of biological and biomedical sensing.
Topics: High-Throughput Nucleotide Sequencing; Nanopores; Sequence Analysis, DNA; Technology; Viruses
PubMed: 34208844
DOI: 10.3390/bios11070214 -
Nature Biotechnology May 2016A long-held goal in sequencing has been to use a voltage-biased nanoscale pore in a membrane to measure the passage of a linear, single-stranded (ss) DNA or RNA molecule... (Review)
Review
A long-held goal in sequencing has been to use a voltage-biased nanoscale pore in a membrane to measure the passage of a linear, single-stranded (ss) DNA or RNA molecule through that pore. With the development of enzyme-based methods that ratchet polynucleotides through the nanopore, nucleobase-by-nucleobase, measurements of changes in the current through the pore can now be decoded into a DNA sequence using an algorithm. In this Historical Perspective, we describe the key steps in nanopore strand-sequencing, from its earliest conceptualization more than 25 years ago to its recent commercialization and application.
Topics: Conductometry; DNA; High-Throughput Nucleotide Sequencing; Lipid Bilayers; Nanopores; Sequence Analysis, DNA
PubMed: 27153285
DOI: 10.1038/nbt.3423 -
RATTLE: reference-free reconstruction and quantification of transcriptomes from Nanopore sequencing.Genome Biology Jul 2022Nanopore sequencing enables the efficient and unbiased measurement of transcriptomes. Current methods for transcript identification and quantification rely on mapping...
Nanopore sequencing enables the efficient and unbiased measurement of transcriptomes. Current methods for transcript identification and quantification rely on mapping reads to a reference genome, which precludes the study of species with a partial or missing reference or the identification of disease-specific transcripts not readily identifiable from a reference. We present RATTLE, a tool to perform reference-free reconstruction and quantification of transcripts using only Nanopore reads. Using simulated data and experimental data from isoform spike-ins, human tissues, and cell lines, we show that RATTLE accurately determines transcript sequences and their abundances, and shows good scalability with the number of transcripts.
Topics: High-Throughput Nucleotide Sequencing; Humans; Nanopore Sequencing; Nanopores; Protein Isoforms; Transcriptome
PubMed: 35804393
DOI: 10.1186/s13059-022-02715-w -
Philosophical Transactions of the Royal... Aug 2017Biological nanopores are a class of membrane proteins that open nanoscale water conduits in biological membranes. When they are reconstituted in artificial membranes and... (Review)
Review
Biological nanopores are a class of membrane proteins that open nanoscale water conduits in biological membranes. When they are reconstituted in artificial membranes and a bias voltage is applied across the membrane, the ionic current passing through individual nanopores can be used to monitor chemical reactions, to recognize individual molecules and, of most interest, to sequence DNA. In addition, a more recent nanopore application is the analysis of single proteins and enzymes. Monitoring enzymatic reactions with nanopores, i.e. nanopore enzymology, has the unique advantage that it allows long-timescale observations of native proteins at the single-molecule level. Here, we describe the approaches and challenges in nanopore enzymology.This article is part of the themed issue 'Membrane pores: from structure and assembly, to medicine and technology'.
Topics: Biosensing Techniques; Enzymes; Membranes, Artificial; Nanopores; Nanotechnology; Proteins; Sequence Analysis, DNA; Single Molecule Imaging
PubMed: 28630164
DOI: 10.1098/rstb.2016.0230 -
Nature Biotechnology Jan 2024Transfer RNAs (tRNAs) play a central role in protein translation. Studying them has been difficult in part because a simple method to simultaneously quantify their...
Transfer RNAs (tRNAs) play a central role in protein translation. Studying them has been difficult in part because a simple method to simultaneously quantify their abundance and chemical modifications is lacking. Here we introduce Nano-tRNAseq, a nanopore-based approach to sequence native tRNA populations that provides quantitative estimates of both tRNA abundances and modification dynamics in a single experiment. We show that default nanopore sequencing settings discard the vast majority of tRNA reads, leading to poor sequencing yields and biased representations of tRNA abundances based on their transcript length. Re-processing of raw nanopore current intensity signals leads to a 12-fold increase in the number of recovered tRNA reads and enables recapitulation of accurate tRNA abundances. We then apply Nano-tRNAseq to Saccharomyces cerevisiae tRNA populations, revealing crosstalks and interdependencies between different tRNA modification types within the same molecule and changes in tRNA populations in response to oxidative stress.
Topics: RNA; Nanopore Sequencing; Nanopores; RNA, Transfer; Sequence Analysis, RNA
PubMed: 37024678
DOI: 10.1038/s41587-023-01743-6 -
Chembiochem : a European Journal of... Sep 2021Covalently attaching ubiquitin (Ub) to cellular proteins as a post-translational modification can result in altered function of modified proteins. Enzymes regulating Ub...
Covalently attaching ubiquitin (Ub) to cellular proteins as a post-translational modification can result in altered function of modified proteins. Enzymes regulating Ub as a post-translational modification, such as ligases and deubiquitinases, are challenging to characterize in part due to the low throughput of in-vitro assays. Single-molecule nanopore based assays have the advantage of detecting proteins with high specificity and resolution, and in a label-free, real-time fashion. Here we demonstrate the use of a MspA nanopore for discriminating and quantifying Ub proteins. We further applied the MspA pore to measure the Ub-chain disassembly activity of UCH37, a proteasome associated deubiquitinase. The implementation of this MspA system into nanopore arrays could enable high throughput characterizations of unknown deubiquitinases as well as drug screening against disease related enzymes.
Topics: Nanopores
PubMed: 34060221
DOI: 10.1002/cbic.202100092 -
Proteomics Sep 2018Resistive pulse nanopore sensing enables label-free single-molecule analysis of a wide range of analytes. An increasing number of studies have demonstrated the... (Review)
Review
Resistive pulse nanopore sensing enables label-free single-molecule analysis of a wide range of analytes. An increasing number of studies have demonstrated the feasibility and usefulness of nanopore sensing for protein and peptide characterization. Nanopores offer the potential to study a variety of protein-related phenomena that includes unfolding kinetics, differences in unfolding pathways, protein structure stability, and free-energy profiles of DNA-protein and RNA-protein binding. In addition to providing a tool for fundamental protein characterization, nanopores have also been used as highly selective protein detectors in various solution mixtures and conditions. This review highlights these and other developments in the area of nanopore-based protein and peptide detection.
Topics: Animals; Biosensing Techniques; Humans; Nanopores; Nanotechnology; Peptide Fragments; Proteins
PubMed: 29952121
DOI: 10.1002/pmic.201800026 -
Human Genomics Aug 2023Long-read DNA sequencing technologies have been rapidly evolving in recent years, and their ability to assess large and complex regions of the genome makes them ideal... (Review)
Review
Long-read DNA sequencing technologies have been rapidly evolving in recent years, and their ability to assess large and complex regions of the genome makes them ideal for clinical applications in molecular diagnosis and therapy selection, thereby providing a valuable tool for precision medicine. In the third-generation sequencing duopoly, Oxford Nanopore Technologies and Pacific Biosciences work towards increasing the accuracy, throughput, and portability of long-read sequencing methods while trying to keep costs low. These trades have made long-read sequencing an attractive tool for use in research and clinical settings. This article provides an overview of current clinical applications and limitations of long-read sequencing and explores its potential for point-of-care testing and health care in remote settings.
Topics: Humans; High-Throughput Nucleotide Sequencing; Sequence Analysis, DNA; Nanopores
PubMed: 37553611
DOI: 10.1186/s40246-023-00522-3 -
Biochimica Et Biophysica Acta.... Sep 2021Protein nanopores have emerged as an important class of sensors for the understanding of biophysical processes, such as molecular transport across membranes, and for the... (Review)
Review
Protein nanopores have emerged as an important class of sensors for the understanding of biophysical processes, such as molecular transport across membranes, and for the detection and characterization of biopolymers. Here, we trace the development of these sensors from the Coulter counter and squid axon studies to the modern applications including exquisite detection of small volume changes and molecular reactions at the single molecule (or reactant) scale. This review focuses on the chemistry of biological pores, and how that influences the physical chemistry of molecular detection.
Topics: Chemistry, Physical; Models, Molecular; Nanopores
PubMed: 33989531
DOI: 10.1016/j.bbamem.2021.183644