This site needs JavaScript to work properly. Both fast5 and fastq files are output if the base-calling mode is applied during the sequencing experiment. official website and that any information you provide is encrypted The dynamics of RNA metabolism were also analyzed by labeling nascent RNAs with base analogs (for example, 5-ethynyluridine186 and 4-thiouridine187) followed by ONT direct RNA sequencing (Table 1). Careers, Unable to load your collection due to an error. Processive translocation of a DNA or RNA molecule leads to a characteristic current shift that is determined by multiple consecutive nucleotides (that is, k-mer) defined by the length of the nanopore sensing region1. The publisher's final edited version of this article is available at, GUID:551B4020-1B7F-4708-9296-3996E6D27FD1. Assemblers (Table 1) such as Canu88 and Miniasm107 are based on the overlaplayoutconsensus algorithm, which builds a graph by overlapping similar sequences and is robust to sequencing error58,67,108 (Fig. Fig. official website and that any information you provide is encrypted Similarly, multiple epigenetic features, including the endogenous 5mC methylome (at CpG sites), nucleosome occupancy and chromatin accessibility, can be simultaneously characterized on single long human DNA molecules by MeSMLR-seq (K.F.A., unpublished data, and ref. Oxford Nanopore technology update. Epub 2023 May 20. c, Direct RNA-sequencing library preparation with or without a reverse transcription step, where only the RNA strand is ligated with an adapter and thus only the RNA strand is sequenced. Before With ONT data alone, there remain drawbacks in estimating gene/isoform abundance, detecting splice sites and mapping alternative polyadenylation sites, although recent improvements in accuracy and throughput have advanced these analyses. Nanopore sequencing is one of the most exciting new technologies that undergo dynamic development. Benchmarking the MinION: Evaluating long reads for microbial - Nature 2 |. We would like to thank K. Aschheim and G. Riddihough for critical reading and editing of the manuscript. was invited by ONT to present at the conference London Calling 2020. For example, Flye improves genome assembly at long and highly repetitive regions by constructing an assembly graph from concatenated disjoint genomic segments110; Miniasm uses all-versus-all read self-mapping for ultrafast assembly107, although postassembly polishing is necessary for higher accuracy. GraphMap progressively refines candidate alignments to handle high error rates and uses fast graph transversal to align long reads with high speed and precision. Similarly, several other methods have combined various biochemical techniques with ONT sequencing (Table 1). In addition to sequencing length and accuracy, throughput is another important consideration for ONT sequencing applications. Please enable it to take advantage of the complete set of features! The .gov means its official. Given that single long reads can encompass multiple variants, including both SNVs and SVs, it is possible to perform phasing of multiploid genomes as well as other haplotype-resolved analyses112,114,115 with appropriate bioinformatics software, such as LongShot116 for SNV detection and WhatsHap117 for haplotyping/phasing. Nanopore sequencing technology (NST), also known as single molecule real-time sequencing technology, allows deciphering single DNA and RNA molecules without polymerase chain reaction (PCR). This approach avoids PCR amplification bias, but it requires a relatively large amount of input material and longer library preparation time, making it unsuitable for many clinical applications. 3 |. Nevertheless, ONT cDNA sequencing was also tested in individual B cells from mice120 and humans122,166. K.F.A. Previous versions of MinKNOW output one fast5 file for each single read (named single-fast5), but later versions output one fast5 file for multiple reads (named multi-fast5) to meet the increasing throughput. Because of its fast real-time sequencing capabilities and small size, MinION has been used for rapid pathogen detection, including diagnosis of bacterial meningitis199, bacterial lower respiratory tract infection200, infective endocarditis201, pneumonia202 and infection in prosthetic joints203. After removing the hairpin sequence, the template and complement reads, called the 1D reads, are used to generate a consensus sequence, called the 2D read, of higher accuracy. 2b and Supplementary Table 1). It is a process of transforming a raw signal obtained from a sequencer into a string of nucleotides. Nanopores: a sequencer in your backpack | Nature Methods The classifications are further categorized by specific topics, and the slice area is proportional to the number of publications (in log2 scale). ONT long reads have been applied to characterize complex genomic rearrangements in individuals with genetic disorders. Thus, ONT read lengths depend crucially on the sizes of molecules in the sequencing library. Bethesda, MD 20894, Web Policies MinION characterized pathogenic microbes, virulence genes and antimicrobial resistance markers in the polluted Little Bighorn River, Montana, United States230. In parallel, accuracy has been improved through new base-calling algorithms, including many developed through independent research32,44 (see below). 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. HHS Vulnerability Disclosure, Help Hear about the latest technology performance and innovations in the pipeline. The other key experimental barrier to be addressed is the large amount of input DNA and RNA required for ONT sequencing, which is up to a few micrograms of DNA and hundreds of nanograms of RNA. 102) and deSALT103, for ONT transcriptome data. In April 2015, MinION devices were shipped to Guinea for real-time genomic surveillance of the ongoing Ebola outbreak. 211), intellectual disability and seizures212, epilepsy213,214, Parkinsons disease215, Gaucher disease215, ataxia-pancytopenia syndrome and severe immune dysregulation114. K.F.A., Yunhao Wang, Y.Z., A.B. 2023 Jun;28(26):2300291. doi: 10.2807/1560-7917.ES.2023.28.26.2300291. Johannesen TB, Munkstrup C, Edslev SM, Baig S, Nielsen S, Funk T, Kristensen DK, Jacobsen LH, Ravn SF, Bindslev N, Gubbels S, Voldstedlund M, Jokelainen P, Hallstrm S, Rasmussen A, Kristinsson KG, Fuglsang-Damgaard D, Dessau RB, Olsn AB, Jensen CS, Skovby A, Ellermann-Eriksen S, Jensen TG, Dzajic E, stergaard C, Lomborg Andersen S, Hoffmann S, Andersen PH, Stegger M. Euro Surveill. Each current segment contains multiple measurements, and the corresponding mean, variance and duration of the current measurements together make up the event data. The .gov means its official. Reads of up to 2.273 megabases (Mb) were demonstrated in 2018 (ref. Read lengths are reported for 1D reads. 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. Picky, in addition to detecting regular SVs, also reveals enriched short-span SVs (~300 bp) in repetitive regions, as long reads cover the entire region including the variations. In parallel, ONT sequencing will benefit from the development of an end-to-end system. 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. Early versions of ONT sequencing used a 2D library preparation method to sequence each dsDNA molecule twice; the two strands of a dsDNA molecule are ligated together by a hairpin adapter, and a motor protein guides one strand (the template) through the nanopore, followed by the hairpin adapter and the second strand (the complement)4042 (Fig. A powerful application of ONT long reads is to identify large SVs (especially from humans) in biomedical contexts, such as the breast cancer cell line HCC1187 (ref. The former strategy requires less sample manipulation and is quicker and thus is good for on-site applications, whereas the latter produces a more stable library for longer sequencing courses and therefore produces higher yields. This primer set is designed . Our offering includes DNA sequencing, as well as RNA and gene expression analysis and future technology for analysing proteins. Bioinformatics of nanopore sequencing | Journal of Human Genetics - Nature It is the only sequencing technology that offers real-time analysis (for rapid insights), in fully scalable formats from pocket to population scale, that can analyse native DNA or RNA and sequence any length of fragment to achieve short to ultra-long read lengths. 4, top right). Trends Genet. NIHMS1789593-supplement-Supplementary_Table_1.xlsx, https://github.com/nanoporetech/megalodon, https://nanoporetech.com/resource-centre/tip-iceberg-sequencing-lettuce-genome, https://www.savetheredwoods.org/project/redwood-genome-project/, https://nanoporetech.com/resource-centre/beauty-and-beast, https://doi.org/10.1038/s41587-021-01108-x, Guppy, Metrichor, Nanonet, Albacore, Scrappie, Flappie, Taiyaki, Bonito, Non-reference transposable element detection, Transcriptome construction and quantification, DNA methylation and chromatin accessibility, DNA replication (replication fork detection), Epitranscriptomics (RNA secondary structure). Genome Biol. Two other experimental assays, DiMeLo-seq181 and BIND&MODIFY182, use ONT sequencing to map histone modifications (H3K9me3 and H3K27me3), a histone variant (CENP-A) and other specific proteinDNA interactions (for example, CTCF binding profile). 2b). 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). At the heart of the technology is the biological nanopore, a protein pore . 8600 Rockville Pike Similar results were achieved using another engineered nanopore, Mycobacterium smegmatis porin A (MspA)16,17, that has a similar channel diameter (~1.2 nm)18,19. HMW DNA can also be sheared to the desired size by sonication, needle extrusion or transposase cleavage (Fig. Fig. Nanopore sequencing: Review of potential applications in - PubMed 1 Europe PMCrequires Javascript to function effectively. National Library of Medicine 2018 Jul 13;19(1):90. doi: 10.1186/s13059-018-1462-9. The site is secure. Likewise, a hospital outbreak of Salmonella was monitored with MinION, with positive cases identified within 2 h (ref. MinION was also used to conduct genomic surveillance for Zika virus222, yellow fever virus223 and dengue virus224 outbreaks in Brazil. Applications of ONT sequencing. Inclusion in an NLM database does not imply endorsement of, or agreement with, 1. 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. The sequencing of single molecules has a low signal-to-noise ratio, in contrast to bulk sequencing of molecules as in Illumina sequencing. Nanoraw (integrated into the Tombo software package) was the first tool to identify the DNA modifications 5mC, 6mA and N4-methylcytosine (4mC) from ONT data74. ONT long reads have been used extensively to assemble the initial reference genomes of many non-model organisms. By contrast, direct RNA sequencing currently produces about 1,000,000 reads (13 Gb) per MinION flow cell due in part to its relatively low sequencing speed (~70 bases per s). Later, bioinformatics tools were developed to identify three kinds of DNA modifications (6mA, 5mC and 5hmC) from ONT data71,75. Because such context-independent signals minimize the complex signal interference between adjacent modified bases, they could also make it possible to detect base modifications at single-molecule and single-nucleotide resolutions. a, Special experimental techniques for ultralong genomic DNA sequencing, including HMW DNA extraction, fragmentation and size selection. Increase in invasive group A streptococcal infections and emergence of novel, rapidly expanding sub-lineage of the virulent. As described above, many limitations can be overcome by improved software fine-tuned for use with nanopore data. In particular, phi29 DNA polymerase was found to have superior performance in ratcheting DNA through the nanopore23,24. The technology identifies a nucleic acid sequence by threading a molecule through a pore a few nanometers in diameter. See this image and copyright information in PMC. 2023 May 30;14:1136727. doi: 10.3389/fimmu.2023.1136727. 4, middle center), although the resolution varies from the bulk level to the single-molecule level. Genes (Basel). 2c), primarily due to improvements in HMW DNA extraction methods and size selection strategies. 82), although few follow-up applications were published. c, Average and maximum read lengths. 239). Nanopore-based sequencers, as the fourth-generation DNA sequencing technology, have the potential to quickly and reliably sequence the entire human genome for less than $1000, and possibly for even less than $100. ONT sequencing applications are classified into three major groups (basic research, clinical usage and on-site applications) and are shown as a pie chart. When used in transcriptome analyses, ONT reads can be clustered and assembled to reconstruct full-length gene isoforms or aligned to a reference genome to characterize complex transcriptional events42,120123 (Fig. Unable to load your collection due to an error, Unable to load your delegates due to an error, A MinION flow cell contains 512 channels with 4 nanopores in each channel, for a total of 2,048 nanopores used to sequence DNA or RNA. The possibility of directly detecting N6-methyladenosine (m6A) modifications in RNA molecules was demonstrated using PacBio in 2012 (ref. PDF Wei Gu1 HHS Public Access Steve Miller , and Charles Y. Chiu - eScholarship Improved data accuracy would advance single-molecule omics studies. However, information may be lost when converting raw current measurement into event data, potentially diminishing base-calling accuracy. Ionic current passes through the nanopore because a constant voltage is applied across the membrane, where the trans side is positively charged. Fungus includes Candida auris, bacterium includes Salmonella, Neisseria meningitidis and Klebsiella pneumoniae and virus includes severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Ebola, Zika, Venezuelan equine encephalitis, yellow fever, Lassa fever and dengue; HLA, human leukocyte antigens. The DNA extraction and purification methods used in these independent studies are summarized in Supplementary Table 1. The portable palm-sized nanopore sequencer called the MinION produced by Oxford Nanopore Technologies (ONT) can perform direct selective sequencing, which rejects the genomic reads that are not of interest. 2d, dashed line). 2d, solid line) for DNA sequencing through faster chemistry (increasing from ~30 bases per s by R6 nanopore to ~450 bases per s by R9.4 nanopore) and longer run times with the introduction of the Rev D ASIC chip. Abstract. Benchmarking low-coverage nanopore long-read sequencing for the assembling of mitochondrial genomes using the vulnerable silky shark Carcharhinus falciformis. 5). When a reference genome is available, ONT data can be used to study sample-specific genomic details, including SVs and haplotypes, with much higher precision than other techniques. Applications for Nanopore sequencing | Oxford Nanopore Technologies Data points shown in b (accuracy), c (read length) and d (yield) are from independent studies. Like conventional RNA sequencing, ONT can be used to perform cDNA sequencing by utilizing existing full-length cDNA synthesis methods (for example, the SMARTer PCR cDNA Synthesis kit of Takara Bio and the TeloPrime Full-Length cDNA Amplification kit of Lexogen) followed by PCR amplification42,55 (Fig. ONT sequencing was also used to discover a new 3.8-Mb duplication in the intronic region of the F8 gene in an individual with hemophilia A208. With the increasing throughput of ONT sequencing, real-time surveillance has been applied to pathogens with larger genomes over the years, ranging from viruses of a few kilobases (for example, Ebola virus220, 1819 kb; Zika virus222, 11 kb; Venezuelan equine encephalitis virus225, 11.4 kb; Lassa fever virus226, 10.4 kb and SARS-CoV-2 coronavirus151, 29.8 kb) to bacteria of several megabases (for example, Salmonella221, 5 Mb; N. meningitidis227, 2 Mb and K. pneumoniae228, 5.4 Mb) and to human fungal pathogens with genomes of >10 Mb (for example, Candida auris229, 12 Mb). The wells are inserted into an electrically resistant polymer membrane supported by an array of microscaffolds connected to a sensor chip. The R10 and R10.3 nanopores with two sensing regions may result in different signal features compared to previous raw current data, which will likely drive another wave of method development to improve data accuracy and base modification detection. Nanopore sequencing, also known as third-generation sequencing (TGS) or single-molecule real-time DNA sequencing, enables the identification of single DNA molecules without requiring the polymerase chain reaction (PCR). BMC Genomics. The classifications are further categorized by specific topics, and the slice area is proportional to the number of publications (in log. Editorial: Bioinformatics analysis tools for nanopore sequencing and Once read lengths reach a certain range, or even cover entire chromosomes, genome assembly would become trivial, requiring little computation and having superior completeness and accuracy. Genome assembly is one of the main uses of ONT sequencing (~30% of published ONT applications; Fig. 4, top left). However, development of corresponding bioinformatics tools, especially for quantitative analyses, remains inadequate. Porechop_ABI: discovering unknown adapters in Oxford Nanopore Only one nanopore in each channel is measured at a time, allowing concurrent sequencing of up to 512 molecules. 2b). Details for these data points are summarized in Supplementary Table 1. Especially for ONT direct RNA-sequencing reads with dense base modifications, Graphmap2 has a higher alignment rate than minimap2 (ref. Many applications combine long reads and short reads in the bioinformatics analyses, termed hybrid sequencing. Nanopore sequencing technology, bioinformatics and applications Nanopore sequencing technology, bioinformatics and applications Accessibility Epub 2021 Oct 25. 2023 Mar 28;11:1189769. doi: 10.3389/fbioe.2023.1189769. The dependence of event data on neighboring nucleotides is Markov chain-like, making HMM-based methods a natural match to decode current shifts to nucleotide sequence, such as early base callers (for example, cloud-based Metrichor by ONT and Nanocall46). government site. Subsequently, many developmental efforts were focused on DNA sequencing 1 . Early MinION users reported typical yields of hundreds of megabases per flow cell, while current throughput has increased to ~1015 gigabases (Gb) (Fig. MinIT is a data analysis device that eliminates the need for a computer to run MinION. Are we there yet? SmidgION is a smartphone-compatible device under development. Similar progress has been achieved in other model organisms and closely related species (for example, Escherichia coli109, Saccharomyces cerevisiae137, Arabidopsis thaliana138 and 15 Drosophila species139) as well as in non-model organisms, including characterizing large tandem repeats in the bread wheat genome140 and improving the continuity and completeness of the genome of Trypanosoma cruzi (the parasite causing Chagas disease)141. Motivation: Nanopore sequencing may be the next disruptive technology in genomics, owing to its ability to detect single DNA molecules without prior amplification, lack of reliance on expensive optical components, and the ability to sequence long fragments. 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. In particular, Nanonet used a bidirectional method to include information from both upstream and downstream states on base calling. Unauthorized use of these marks is strictly prohibited. Furthermore, ONT direct RNA sequencing has been used to measure the poly(A) tail length of native RNA molecules in humans131, C. elegans167, A. thaliana168 and Locusta migratoria169, corroborating a negative correlation between poly(A) tail length and gene expression167,168. Poretools: a toolkit for analyzing nanopore sequence data - Oxford Academic Reducing the sample size requirement would make ONT sequencing useful for the many biomedical studies in which genetic material is limited. Nanopore-based fourth-generation DNA sequencing technology 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. However, both of these approaches were limited in that each molecule could only be measured twice. 4, bottom right). Bookshelf 169 Altmetric Metrics Abstract Rapid advances in nanopore technologies for sequencing single long DNA and RNA molecules have led to substantial improvements in accuracy, read length and. Recently, ONT direct RNA sequencing has yielded robust data of reasonable quality, and several pilot studies have detected bulk-level RNA modifications by examining either error distribution profiles (for example, EpiNano (m6A)73 and ELIGOS (m6A and 5-methoxyuridine (5moU))83) or current signals (for example, Tombo extension (m6A and m5C)74 and MINES (m6A)84). Fig. Federal government websites often end in .gov or .mil. Overcoming these challenges will require further breakthroughs in nanopore technology, molecular experiments and bioinformatics software. For example, graphene-based nanopores are capable of DNA sensing and have high durability and insulating capability in high ionic strength solutions240242, where their thickness (~0.35 nm) is ideal for capturing single nucleotides243. 135). Editorial: Bioinformatics analysis tools for nanopore sequencing and Obtaining megabase-scale or longer reads will require the development of HMW DNA extraction and size selection methods as well as protocols to maintain ultralong DNA fragments intact. An introduction to nanopore sequencing - Oxford Nanopore Technologies Nanopore technology was initially developed for the practicable stochastic sensing of ions and small molecules 2, 27, 28. For species with available reference genomes, ONT long reads are useful for closing genome gaps, especially in the human genome. strategies that use sequence-by-synthesis methods. Nanopore-based Fourth-generation DNA Sequencing Technology government site. In contrast to hybrid correction of long reads for general purposes, many hybrid sequencing-based methods integrate long reads and short reads into the algorithms and pipeline designs to harness the strengths of both types of reads to address specific biological problems. ONT developed new base callers as technology demonstrator software (for example, Nanonet, Scrappie and Flappie), which were subsequently implemented into the officially available software packages (for example, Albacore and Guppy). Next, we describe the main bioinformatics methods applied to ONT data. NanoGalaxy: Nanopore long-read sequencing data analysis in Galaxy 100), and a new mode of STAR, which was originally developed for short reads101, have been widely used in splice-aware alignment of error-prone transcriptome long reads to genomes. Nonetheless, current ONT sequencing techniques have several limitations, including relatively high error rates and the requirement for relatively high amounts of nucleic acid material. MinION is a flow cell containing 512 channels, with four nanopores per channel. Oxford Nanopore Technologies Compared to existing antibody-based approaches (which are usually followed by short-read sequencing), ONT direct RNA sequencing opens opportunities to directly identify RNA modifications (for example, m6A) and RNA editing (for example, inosine), which have critical biological functions. In the SARS-CoV-2 pandemic151, ONT sequencing was used to reconstruct full-length SARS-CoV-2 genome sequences via cDNA and direct RNA sequencing152155, providing valuable information regarding the biology, evolution and pathogenicity of the virus. Computational tools and experimental assays for ONT data analysis and applications. 3 |. eCollection 2023. Beyond optimizing the nanopore and motor protein, several strategies have been developed to improve accuracy. We developed a comprehensive multiplexed set of primers adapted for the Oxford Nanopore Rapid Barcoding library kit that allows universal SARS-CoV-2 genome sequencing. Nanopore sequencing and its application to the study of microbial Under the control of a motor protein, a double-stranded DNA (dsDNA) molecule (or an RNADNA hybrid duplex) is first unwound, then single-stranded DNA or RNA with negative charge is ratcheted through the nanopore, driven by the voltage. Alternatively, a cDNA strand can be synthesized to obtain an RNAcDNA hybrid duplex, followed by ligation of the adapter. 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. In this review, we first present an introduction to the technology development of nanopore sequencing and discuss improvements in the accuracy, read length and throughput of ONT data. Some applications span two categories, such as SV detection and rapid pathogen detection. 3b). MinION and MinIT devices were brought to farms in sub-Saharan Africa for early and rapid diagnosis (<3 h) of plant viruses and pests in cassava231.