FOIA 5). ONT also offers a direct cDNA sequencing protocol without PCR amplification, in contrast to many existing cDNA sequencing methods. was invited by ONT to present at the conference London Calling 2020. ONT sequencing data improvement. In contrast to the previous DNA translocation tests that were poorly controlled1317, the addition of the motor protein reduced the fluctuations in translocation kinetics, thus improving data quality. Ionic current passes through the nanopore because a constant voltage is applied across the membrane, where the trans side is positively charged. The possibility of directly detecting N6-methyladenosine (m6A) modifications in RNA molecules was demonstrated using PacBio in 2012 (ref. 1. 3d, right). 184,185) has revealed that it is possible to probe RNA secondary structure using a combination of ONT direct RNA sequencing and artificial chemical modifications (Table 1). How nanopore sequencing works - Oxford Nanopore Technologies Introduction. Indeed, this motor protein provided the last piece of the puzzle; in February 2012, two groups demonstrated processive recordings of ionic currents for single-stranded DNA molecules that could be resolved into signals from individual nucleotides by combining phi29 DNA polymerase and a nanopore (-hemolysin24 and MspA25). Our offering includes DNA sequencing, as well as RNA and gene expression analysis and future technology for analysing proteins. However, both of these approaches were limited in that each molecule could only be measured twice. Abstract. A recent benchmarking study demonstrated that ONT sequencing of RNA, cDNA or PCR-cDNA for the identification and quantification of gene isoforms provides similar results56. 4, middle center). In the case of nanopore sequencing, it is a computational processing of electric signal. 1 Europe PMCrequires Javascript to function effectively. In May 2017, ONT released the 1D2 method together with the R9.5 nanopore; in this method, instead of being physically connected by a hairpin adapter, each strand is ligated separately to a special adapter (Fig. Using the R9.4 nanopore as an example, the average accuracy of 2D reads is 94% versus 86% for 1D reads33 (Fig. Nanopore5| - For example, MinION was used to identify 51 acquired resistance genes directly from clinical urine samples (without culture) of 55 that were detected from cultivated bacteria using Illumina sequencing204, and a recent survey of resistance to colistin in 12,053 Salmonella strains used a combination of ONT, PacBio and Illumina data205. Frontiers | Portable nanopore-sequencing technology: Trends in Later, ONTs open-source base caller Scrappie (implemented into both Albacore and Guppy) and the third-party software Chiron70 adopted neural networks to directly translate the raw current data into DNA sequence. J Microbiol. Applications of ONT sequencing. Some applications that use all three strengths span all three layers (for example, antimicrobial resistance profiling). However, ONT no longer offers or supports the 2D and 1D2 libraries. 2b). The nanopore technology enables direct readout of sequences from individual DNA or RNA molecules including base modifications since no synthesis or amplification is required. MinKNOW also manages data acquisition and real-time analysis and performs local base calling and outputs the binary files in fast5 format to store both metadata and read information (for example, current measurement and read sequence if base calling is performed). Genome Biol. See this image and copyright information in PMC. The concept of nanopore sequencing emerged in the 1980s and was realized through a series of technical advances in both the nanopore and the associated motor protein1,48. For small DNA/RNA viral genomes (for example, the 27-kb human coronavirus genome86), the assembly process is not required given the long read length. The Oxford Nanopore MinION: delivery of nanopore sequencing to the genomics community, van Dijk EL, Jaszczyszyn Y, Naquin D & Thermes C, The third revolution in sequencing technology, Advances in nanopore sequencing technology, A world of opportunities with nanopore sequencing, Increasing the accuracy of nanopore DNA sequencing using a time-varying cross membrane voltage, The potential and challenges of nanopore sequencing, Structure of staphylococcal -hemolysin, a heptameric transmembrane pore, Kasianowicz JJ, Brandin E, Branton D & Deamer DW, Characterization of individual polynucleotide molecules using a membrane channel, Akeson M, Branton D, Kasianowicz JJ, Brandin E & Deamer DW, Microsecond time-scale discrimination among polycytidylic acid, polyadenylic acid, and polyuridylic acid as homopolymers or as segments within single RNA molecules, Meller A, Nivon L, Brandin E, Golovchenko J & Branton D, Rapid nanopore discrimination between single polynucleotide molecules, Stoddart D, Heron AJ, Mikhailova E, Maglia G & Bayley H, Single-nucleotide discrimination in immobilized DNA oligonucleotides with a biological nanopore, Nucleobase recognition in ssDNA at the central constriction of the -hemolysin pore, Stoddart D, Maglia G, Mikhailova E, Heron AJ & Bayley H, Multiple base-recognition sites in a biological nanopore: two heads are better than one, Butler TZ, Pavlenok M, Derrington IM, Niederweis M & Gundlach JH, Single-molecule DNA detection with an engineered MspA protein nanopore, Cloning of the mspA gene encoding a porin from, The structure of a mycobacterial outer-membrane channel, Sequence-specific detection of individual DNA polymerase complexes in real time using a nanopore, Single-molecule analysis of DNAprotein complexes using nanopores, Cockroft SL, Chu J, Amorin M & Ghadiri MR, A single-molecule nanopore device detects DNA polymerase activity with single-nucleotide resolution, Processive replication of single DNA molecules in a nanopore catalyzed by phi29 DNA polymerase, Automated forward and reverse ratcheting of DNA in a nanopore at 5-A precision, Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase, Faster sequencers, larger datasets, new challenges, De novo assembly of middle-sized genome using MinION and Illumina sequencers, MinION nanopore sequencing identifies the position and structure of a bacterial antibiotic resistance island, Robust long-read native DNA sequencing using the ONT CsgG Nanopore system, Performance of neural network basecalling tools for Oxford Nanopore sequencing, Picky comprehensively detects high-resolution structural variants in nanopore long reads, Structural, physiological and regulatory analysis of maltose transporter genes in, Causalcall: nanopore basecalling using a temporal convolutional network, DNA extraction of microbial DNA directly from infected tissue: an optimized protocol for use in nanopore sequencing, Nanopore sequencing of a forensic STR multiplex reveals loci suitable for single-contributor STR profiling, W. Homopolish: a method for the removal of systematic errors in nanopore sequencing by homologous polishing, MinION Analysis and Reference Consortium: phase 1 data release and analysis, MinION Analysis and Reference Consortium: phase 2 data release and analysis of R9.0 chemistry, Comprehensive comparison of Pacific Biosciences and Oxford Nanopore Technologies and their applications to transcriptome analysis, Evaluation and application of RNA-seq by MinION, From squiggle to basepair: computational approaches for improving nanopore sequencing read accuracy, Oxford Nanopore sequencing, hybrid error correction, and de novo assembly of a eukaryotic genome, David M, Dursi LJ, Yao D, Boutros PC & Simpson JT, Nanocall: an open source basecaller for Oxford Nanopore sequencing data, DeepNano: deep recurrent neural networks for base calling in MinION nanopore reads, Gong L, Wong CH, Idol J, Ngan CY & Wei CL, Ultra-long read sequencing for whole genomic DNA analysis, BulkVis: a graphical viewer for Oxford nanopore bulk FAST5 files, A chromosome-scale assembly of the sorghum genome using nanopore sequencing and optical mapping, Highly parallel direct RNA sequencing on an array of nanopores, Direct RNA sequencing of the coding complete influenza A virus genome, Evaluating the genome and resistome of extensively drug-resistant, Cartolano M, Huettel B, Hartwig B, Reinhardt R & Schneeberger K, cDNA library enrichment of full length transcripts for SMRT long read sequencing, A systematic benchmark of Nanopore long read RNA sequencing for transcript level analysis in human cell lines, Ultra-deep, long-read nanopore sequencing of mock microbial community standards, Magi A, Semeraro R, Mingrino A, Giusti B & DAurizio R, Nanopore sequencing data analysis: state of the art, applications and challenges, Cao MD, Ganesamoorthy D, Cooper MA & Coin LJ, Realtime analysis and visualization of MinION sequencing data with npReader, poRe: an R package for the visualization and analysis of nanopore sequencing data, Leggett RM, Heavens D, Caccamo M, Clark MD & Davey RP, NanoOK: multi-reference alignment analysis of nanopore sequencing data, quality and error profiles, Tarraga J, Gallego A, Arnau V, Medina I & Dopazo J, HPG pore: an efficient and scalable framework for nanopore sequencing data, Bolognini D, Bartalucci N, Mingrino A, Vannucchi AM & Magi A, NanoR: a user-friendly R package to analyze and compare nanopore sequencing data, Poretools: a toolkit for analyzing nanopore sequence data, De Coster W, DHert S, Schultz DT, Cruts M & Van Broeckhoven C, NanoPack: visualizing and processing long-read sequencing data, PyPore: a python toolbox for nanopore sequencing data handling, Senol Cali D, Kim JS, Ghose S, Alkan C & Mutlu O, Nanopore sequencing technology and tools for genome assembly: computational analysis of the current state, bottlenecks and future directions, Opportunities and challenges in long-read sequencing data analysis, Chiron: translating nanopore raw signal directly into nucleotide sequence using deep learning, Mapping DNA methylation with high-throughput nanopore sequencing, Single-molecule long-read sequencing reveals the chromatin basis of gene expression, Accurate detection of m6A RNA modifications in native RNA sequences, De novo identification of DNA modifications enabled by genome-guided nanopore signal processing, Detecting DNA cytosine methylation using nanopore sequencing, Detection of DNA base modifications by deep recurrent neural network on Oxford Nanopore sequencing data, DeepSignal: detecting DNA methylation state from Nanopore sequencing reads using deep-learning, NanoMod: a computational tool to detect DNA modifications using Nanopore long-read sequencing data, Systematic benchmarking of tools for CpG methylation detection from nanopore sequencing, DNA methylation-calling tools for Oxford Nanopore sequencing: a survey and human epigenome-wide evaluation, Genome-wide mapping of methylated adenine residues in pathogenic, The birth of the epitranscriptome: deciphering the function of RNA modifications, Decoding the epitranscriptional landscape from native RNA sequences, A comparative evaluation of hybrid error correction methods for error-prone long reads, Direct RNA nanopore sequencing of full-length coronavirus genomes provides novel insights into structural variants and enables modification analysis, Comparative assessment of long-read error correction software applied to Nanopore RNA-sequencing data, Canu: scalable and accurate long-read assembly via adaptive, Accurate self-correction of errors in long reads using de Bruijn graphs, Improving PacBio long read accuracy by short read alignment, LoRDEC: accurate and efficient long read error correction, HALC: high throughput algorithm for long read error correction, FMLRC: hybrid long read error correction using an FM-index, Altschul SF, Gish W, Miller W, Myers EW & Lipman DJ, Fast and sensitive mapping of nanopore sequencing reads with GraphMap, Minimap2: pairwise alignment for nucleotide sequences, Kielbasa SM, Wan R, Sato K, Horton P & Frith MC, Adaptive seeds tame genomic sequence comparison, Accurate detection of complex structural variations using single-molecule sequencing, Evaluating nanopore sequencing data processing pipelines for structural variation identification, GMAP: a genomic mapping and alignment program for mRNA and EST sequences, STAR: ultrafast universal RNA-seq aligner, Mari J, Sovi I, Krianovi K, Nagarajan N & iki M, Graphmap2splice-aware RNA-seq mapper for long reads, deSALT: fast and accurate long transcriptomic read alignment with de Bruijn graph-based index, Quantitative profiling of pseudouridylation dynamics in native RNAs with nanopore sequencing, De novo yeast genome assemblies from MinION, PacBio and MiSeq platforms, Hybrid metagenomic assembly enables high-resolution analysis of resistance determinants and mobile elements in human microbiomes, Minimap and miniasm: fast mapping and de novo assembly for noisy long sequences, The long reads ahead: de novo genome assembly using the MinION, A complete bacterial genome assembled de novo using only nanopore sequencing data, Assembly of long, error-prone reads using repeat graphs, Fast and accurate long-read assembly with wtdbg2, Mapping and phasing of structural variation in patient genomes using nanopore sequencing, NanoVar: accurate characterization of patients genomic structural variants using low-depth nanopore sequencing, Sequencing of human genomes with nanopore technology, Multi-platform discovery of haplotype-resolved structural variation in human genomes, Longshot enables accurate variant calling in diploid genomes from single-molecule long read sequencing, Haplotype threading: accurate polyploid phasing from long reads, Nanopore sequencing enables comprehensive transposable element epigenomic profiling, Bolognini D, Magi A, Benes V, Korbel JO & Rausch T, TRiCoLOR: tandem repeat profiling using whole-genome long-read sequencing data, Nanopore long-read RNAseq reveals widespread transcriptional variation among the surface receptors of individual B cells, Oikonomopoulos S, Wang YC, Djambazian H, Badescu D & Ragoussis J, Benchmarking of the Oxford Nanopore MinION sequencing for quantitative and qualitative assessment of cDNA populations, Improving nanopore read accuracy with the R2C2 method enables the sequencing of highly multiplexed full-length single-cell cDNA, Full-length transcript characterization of SF3B1 mutation in chronic lymphocytic leukemia reveals downregulation of retained introns, Kuosmanen A, Sobih A, Rizzi R, Mkinen V & Tomescu AI, On using longer RNA-seq reads to improve transcript prediction accuracy, Transcriptome assembly from long-read RNA-seq alignments with StringTie2, A technology-agnostic long-read analysis pipeline for transcriptome discovery and quantification, Characterization of the human ESC transcriptome by hybrid sequencing, IDP-denovo: de novo transcriptome assembly and isoform annotation by hybrid sequencing, Reference-free reconstruction and quantification of transcriptomes from Nanopore long-read sequencing, Nanopore native RNA sequencing of a human poly(A) transcriptome, A comprehensive examination of Nanopore native RNA sequencing for characterization of complex transcriptomes, Nanopore sequencing and assembly of a human genome with ultra-long reads, Linear assembly of a human centromere on the Y chromosome, Telomere-to-telomere assembly of a complete human X chromosome, MinION-based long-read sequencing and assembly extends the Caenorhabditis elegans reference genome, Nanopore sequencing enables near-complete de novo assembly of, Miller DE, Staber C, Zeitlinger J & Hawley RS, Highly contiguous genome assemblies of 15, The dark matter of large cereal genomes: long tandem repeats, Nanopore sequencing significantly improves genome assembly of the protozoan parasite, De novo genome assembly and annotation of Australias largest freshwater fish, the Murray cod (, Finding Nemo: hybrid assembly with Oxford Nanopore and Illumina reads greatly improves the clownfish (, De novo genome assembly of the Meadow Brown Butterfly, Genome of the Komodo dragon reveals adaptations in the cardiovascular and chemosensory systems of monitor lizards, De novo assembly of the Indian blue peacock (, Long live the king: chromosome-level assembly of the lion (, The bagworm genome reveals a unique fibroin gene that provides high tensile strength, Rapid sequencing of multiple RNA viruses in their native form, Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding, The architecture of SARS-CoV-2 transcriptome, Amplicon based MinION sequencing of SARS-CoV-2 and metagenomic characterisation of nasopharyngeal swabs from patients with COVID-19, Direct RNA sequencing and early evolution of SARS-CoV-2, Nanopore targeted sequencing for the accurate and comprehensive detection of SARS-CoV-2 and other respiratory viruses, De novo assembly of the olive fruit fly (, Kadobianskyi M, Schulze L, Schuelke M & Judkewitz B. Chromosomal-level assembly of the blood clam, Chromosome-scale assemblies of plant genomes using nanopore long reads and optical maps, High-quality chromosome-scale assembly of the walnut (, The giant sequoia genome and proliferation of disease resistance genes, Nanopore sequencing and the Shasta toolkit enable efficient de novo assembly of eleven human genomes, Structural variants identified by Oxford Nanopore PromethION sequencing of the human genome, High-throughput targeted long-read single cell sequencing reveals the clonal and transcriptional landscape of lymphocytes, Nanopore direct RNA sequencing maps the complexity of Arabidopsis mRNA processing and m, Long-read direct RNA sequencing by 5-cap capturing reveals the impact of Piwi on the widespread exonization of transposable elements in locusts, Comprehensive profiling of circular RNAs with nanopore sequencing and CIRI-long, isoCirc catalogs full-length circular RNA isoforms in human transcriptomes, Detection and mapping of 5-methylcytosine and 5-hydroxymethylcytosine with nanopore MspA, Error rates for nanopore discrimination among cytosine, methylcytosine, and hydroxymethylcytosine along individual DNA strands, Long-range single-molecule mapping of chromatin accessibility in eukaryotes, Simultaneous profiling of chromatin accessibility and methylation on human cell lines with nanopore sequencing, Detection of base analogs incorporated during DNA replication by nanopore sequencing, Mapping DNA replication with nanopore sequencing, Capturing the dynamics of genome replication on individual ultra-long nanopore sequence reads, Nanopore sequencing of DNA concatemers reveals higher-order features of chromatin structure, DiMeLo-seq: a long-read, single-molecule method for mapping protein-DNA interactions genome-wide, Long-range single-molecule mapping of chromatin modification in eukaryotes, Smith AM, Jain M, Mulroney L, Garalde DR & Akeson M, Reading canonical and modified nucleobases in 16S ribosomal RNA using nanopore native RNA sequencing, Determination of isoform-specific RNA structure with nanopore long reads, Direct detection of RNA modifications and structure using single molecule nanopore sequencing, Maier KC, Gressel S, Cramer P & Schwalb B, Native molecule sequencing by nano-ID reveals synthesis and stability of RNA isoforms, Splicing kinetics and coordination revealed by direct nascent RNA sequencing through nanopores, Design and MinION testing of a nanopore targeted gene sequencing panel for chronic lymphocytic leukemia, Rapid detection of chromosomal translocation and precise breakpoint characterization in acute myeloid leukemia by nanopore long-read sequencing, Same-day genomic and epigenomic diagnosis of brain tumors using real-time nanopore sequencing, Detection of subclonal L1 transductions in colorectal cancer by long-distance inverse-PCR and Nanopore sequencing, Norris AL, Workman RE, Fan Y, Eshleman JR & Timp W, Nanopore sequencing detects structural variants in cancer, Sequencing and phasing cancer mutations in lung cancers using a long-read portable sequencer, A nanopore sequencing-based assay for rapid detection of gene fusions, Rapid diagnosis of bacterial meningitis by nanopore 16S amplicon sequencing: a pilot study, Nanopore metagenomics enables rapid clinical diagnosis of bacterial lower respiratory infection, Identification of pathogens in culture-negative infective endocarditis cases by metagenomic analysis, Real-time analysis of nanopore-based metagenomic sequencing from infected orthopaedic devices, Identification of bacterial pathogens and antimicrobial resistance directly from clinical urines by nanopore-based metagenomic sequencing, Implications of error-prone long-read whole-genome shotgun sequencing on characterizing reference microbiomes, NanoSatellite: accurate characterization of expanded tandem repeat length and sequence through whole genome long-read sequencing on PromethION, Severe hemophilia A caused by an unbalanced chromosomal rearrangement identified using nanopore sequencing, Interchromosomal template-switching as a novel molecular mechanism for imprinting perturbations associated with Temple syndrome, Long-read sequencing identified a causal structural variant in an exome-negative case and enabled preimplantation genetic diagnosis, Breakpoint mapping of a novel de novo translocation t(X;20)(q11.1;p13) by positional cloning and long read sequencing, Expansions of intronic TTTCA and TTTTA repeats in benign adult familial myoclonic epilepsy, Long-read sequencing identified intronic repeat expansions in, Full-length HLA class I genotyping with the MinION nanopore sequencer, Accurate typing of human leukocyte antigen class I genes by Oxford Nanopore sequencing, Resolving MiSeq-generated ambiguities in HLA-DPB1 typing by using the Oxford Nanopore Technology, Rapid short-read sequencing and aneuploidy detection using MinION nanopore technology, Real-time, portable genome sequencing for Ebola surveillance, Rapid draft sequencing and real-time nanopore sequencing in a hospital outbreak of, Establishment and cryptic transmission of Zika virus in Brazil and the Americas, Genomic and epidemiological monitoring of yellow fever virus transmission potential, Genomic detection of a virus lineage replacement event of dengue virus serotype 2 in Brazil, 2019, Unbiased strain-typing of arbovirus directly from mosquitoes using nanopore sequencing: a field-forward biosurveillance protocol, Metagenomic sequencing at the epicenter of the Nigeria 2018 Lassa fever outbreak, Acquisition of virulence genes by a carrier strain gave rise to the ongoing epidemics of meningococcal disease in West Africa, Dong N, Yang X, Zhang R, Chan EW & Chen S, Tracking microevolution events among ST11 carbapenemase-producing hypervirulent, Genomic epidemiology of the UK outbreak of the emerging human fungal pathogen, Metagenomic profiling of microbial pathogens in the Little Bighorn River, Montana, Tree Lab: portable genomics for early detection of plant viruses and pests in sub-Saharan Africa, Rapid re-identification of human samples using portable DNA sequencing, Runtuwene LR, Tuda JSB, Mongan AE & Suzuki Y, Radiation tolerance of nanopore sequencing technology for life detection on Mars and Europa, Nanopore DNA sequencing and genome assembly on the International Space Station, Ducluzeau A, Lekanoff RM, Khalsa NS, Smith HH & Drown DM, Introducing DNA sequencing to the next generation on a research vessel sailing the Bering Sea through a storm, In-field metagenome and 16S rRNA gene amplicon nanopore sequencing robustly characterize glacier microbiota, Next-generation technologies applied to age-old challenges in Madagascar, Single-molecule sequencing of an individual human genome, DNA translocation through graphene nanopores, Graphene as a subnanometre trans-electrode membrane, Electric field effect in atomically thin carbon films, Recapturing and trapping single molecules with a solid-state nanopore, De novo assembly and phasing of a Korean human genome, DeepNano-blitz: a fast base caller for MinION nanopore sequencers, BasecRAWller: streaming nanopore basecalling directly from raw signal, WaveNano: a signal-level nanopore base-caller via simultaneous prediction of nucleotide labels and move labels through bi-directional WaveNets, MinCall-MinION end2end convolutional deep learning basecaller, Nanopore basecalling from a perspective of instance segmentation, An end-to-end Oxford Nanopore basecaller using convolution-augmented transformer, 2020 IEEE Intl.
