nanopore sequencing advantages and disadvantages
Jain, C., Koren, S., Dilthey, A., Phillippy, A. M. & Aluru, S. A fast adaptive algorithm for computing whole-genome homology maps. Rang, F. J., Kloosterman, W. P. & de Ridder, J. SmidgION is a smartphone-compatible device under development. Long and ultra-long reads confer many advantages, including simpler assembly and more comprehensive analysis of repetitive regions, phasing or CNVs. 30, 13321344 (2020). Enter the email address you signed up with and we'll email you a reset link. & Vinar, T. DeepNano: deep recurrent neural networks for base calling in MinION nanopore reads. Minei, R., Hoshina, R. & Ogura, A. Emerg. Bioinformatics 34, 30943100 (2018). Chem. Merchant, C. A. et al. Paternally inherited cis-regulatory structural variants are associated with autism. Commun. Schneider, G. F. et al. Stoddart, D., Heron, A. J., Mikhailova, E., Maglia, G. & Bayley, H. Single-nucleotide discrimination in immobilized DNA oligonucleotides with a biological nanopore. Jenjaroenpun, P. et al. Characterization of the human ESC transcriptome by hybrid sequencing. MinION Analysis and Reference Consortium: phase 1 data release and analysis. Pros and Cons. Preprint at medRxiv https://doi.org/10.1101/2020.03.05.20032011 (2020). 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. Genome Res. 3d, left). Genome Res. 22, 295 (2021). Cheng, H., Concepcion, G. T., Feng, X., Zhang, H. & Li, H. Haplotype-resolved de novo assembly using phased assembly graphs with hifiasm. PubMedGoogle Scholar. Thus, error correction is widely applied before many downstream analyses (for example, genome assembly and gene isoform identification), which can rescue reads for higher sensitivity (for example, mappability85) and improve the quality of the results (for example, break point determination at single-nucleotide resolution86). 4, top right). Proc. In the SARS-CoV-2 pandemic151, ONT sequencing was used to reconstruct full-length SARS-CoV-2 genome sequences via cDNA and direct RNA sequencing152,153,154,155, providing valuable information regarding the biology, evolution and pathogenicity of the virus. Rep. 7, 18022 (2017). In nanopore sequencing, an electric current is passed across a synthetic membrane that contains a protein nanopore. BMC Bioinformatics 18, 204 (2017). 16. Nat. Senol Cali, D., Kim, J. S., Ghose, S., Alkan, C. & Mutlu, O. Nanopore sequencing technology and tools for genome assembly: computational analysis of the current state, bottlenecks and future directions. M. et al. Translocation speed is controlled by a motor protein that ratchets the nucleic acid molecule through the nanopore in a step-wise manner. Natl Acad. These tools can be used with data from various techniques, from Nanopore sequencing to PacBio, even in high-fidelity reads . Direct detection of RNA modifications and structure using single molecule nanopore sequencing. 43, e116 (2015). 11, 96 (2016). Therefore, DNase treatment is necessary because amplified RNA cannot be differentiated from genomic DNA. Bioinformatics 32, i545i551 (2016). 22, 95 (2021). wrote and revised the main text. Bioinformatics 36, 32363238 (2020). Chen, Y. et al. Methods 17, 155158 (2020). Nat. Biol. Sci. eLife 9, e49658 (2020). Oxford Nanopore Technologies, the Wheel icon, EPI2ME, Flongle, GridION, Metrichor, MinION, MinIT, MinKNOW, Plongle, PromethION, SmidgION, Ubik and VolTRAX are registered trademarks of Oxford Nanopore Technologies plc in various countries. Madoui, M. A. et al. Nat Biotechnol 39, 13481365 (2021). 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. Science 361, 894899 (2018). Nanopore sequencing technology and its applications in basic and applied research have undergone substantial growth since Oxford Nanopore Technologies (ONT) provided the first nanopore sequencer, MinION, in 2014 (refs. RNAseq also requires a minimum of 1g total RNA, but 2g is preferred, which are larger samples compared with NanoString. 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. 20, 428435 (2018). & Strazisar, M. Methplotlib: analysis of modified nucleotides from nanopore sequencing. Methods 15, 461468 (2018). isoCirc catalogs full-length circular RNA isoforms in human transcriptomes. Biotechnol. By contrast, direct RNA sequencing currently produces about 1,000,000 reads (13Gb) per MinION flow cell due in part to its relatively low sequencing speed (~70bases per s). Get what matters in translational research, free to your inbox weekly. Bowden, R. et al. Preprint at bioRxiv https://doi.org/10.1101/833590 (2019). 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. Nanopore sequencing has been successfully used for the detection and mapping of C, 5mC, and 5hmC [127,128]. IEEE Trans Nanotechnol. 1). Nanopore sequencing is being applied to fields where portability, ease of set up, real-time analysis and control over time-to-results, make a difference. Here, we focus on the current advantages as well as the limitations of the ONT nanopore sequencer, reviewing research publications available thus far. Yunhao Wang and A.B. Castro-Wallace, S. L. et al. Marrano, A. et al. Payne, A., Holmes, N., Rakyan, V. & Loose, M. BulkVis: a graphical viewer for Oxford nanopore bulk FAST5 files. 176). 16, 114 (2015). 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. Method Advantages Disadvantages Ref Comments Second-generation sequencing techniques 454 sequencing . "Imaging is the future," adds Stamatoyannopoulos. 1,2). De novo assembly of middle-sized genome using MinION and Illumina sequencers. Au, C. H. et al. Analogous to next generation sequencing, the cost of sequencing using long-read technologies has materially dropped whilst the instrument throughput continues . At the heart of the technology is the biological nanopore, a protein pore . In parallel, accuracy has been improved through new base-calling algorithms, including many developed through independent research32,44 (see below). Structural variants identified by Oxford Nanopore PromethION sequencing of the human genome. We review 11 applications that are the subject of the most publications since 2015. The development of next generation sequencing has challenged the use of other molecular fingerprinting methods used to study microbial diversity. Faria, N. R. et al. Lind, A. L. et al. This technology makes for a high throughput, cost effective sequencing solution. Methods 15, 201206 (2018). Both methods have advantages and disadvantages. Long-read sequencing identified a causal structural variant in an exome-negative case and enabled preimplantation genetic diagnosis. Georgieva, D., Liu, Q., Wang, K. & Egli, D. Detection of base analogs incorporated during DNA replication by nanopore sequencing. 10, 1332 (2020). Advances in nanopore sequencing technology. Sovic, I. et al. Mem. Sahlin, K. & Medvedev, P. Error correction enables use of Oxford Nanopore technology for reference-free transcriptome analysis. Brief. collected information and prepared the materials for the Applications of nanopore sequencing section. The downside is that the sequences are read once and that is the read you have to run with. The goal of this course is to provide an overview of in-field, real-time nanopore sequencing using the Oxford Nanopore Technologies (ONT) platform. Austin, C. M. et al. De Coster, W., DHert, S., Schultz, D. T., Cruts, M. & Van Broeckhoven, C. NanoPack: visualizing and processing long-read sequencing data. DiMeLo-seq: a long-read, single-molecule method for mapping proteinDNA interactions genome-wide. Nanopore sequencing offers advantages in all areas of research. PLoS Comput. de Lannoy, C., de Ridder, D. & Risse, J. Methods 17, 319327 (2020). Gigascience 7, giy140 (2018). Nature 554, 5055 (2018). Liu, C. et al. Genome Res. Direct RNA sequencing of the coding complete influenza A virus genome. Genome Biol. 2d, solid line) for DNA sequencing through faster chemistry (increasing from ~30bases per s by R6 nanopore to ~450bases per s by R9.4 nanopore) and longer run times with the introduction of the Rev D ASIC chip. Liu, Q., Georgieva, D. C., Egli, D. & Wang, K. NanoMod: a computational tool to detect DNA modifications using Nanopore long-read sequencing data. J. Comput. Drexler, H. L. et al. The membrane is resistant to the electric current, meaning the only way for current to flow across the membrane is to travel through the pore. Proc. Commun. Cherf, G. M. et al. Kwan, H. H. et al. Gabrieli, T. et al. Chakraborty, A., Morgenstern, B. Sci. 17, 246253 (2016). Microbes Infect. Cheetham, S. W. et al. J. Environ. Preprint at https://arxiv.org/abs/1904.10337 (2019). In an electrolytic solution, a constant voltage is applied to produce an ionic current through the nanopore such that negatively charged single-stranded DNA or RNA molecules are driven through the nanopore from the negatively charged cis side to the positively charged trans side. SQANTI: extensive characterization of long-read transcript sequences for quality control in full-length transcriptome identification and quantification. Although the average accuracy of ONT sequencing is improving, certain subsets of reads or read fragments have very low accuracy, and the error rates of both 1D reads and 2D/1D2 reads are still much higher than those of short reads generated by next-generation sequencing technologies. For example, ONT sequencing of human genomes revealed that an expansion of tandem repeats in the ABCA7 gene was associated with an increased risk of Alzheimers disease207. Rapid detection of chromosomal translocation and precise breakpoint characterization in acute myeloid leukemia by nanopore long-read sequencing. For instance, ONT data alone were used to assemble the first genome of Rhizoctonia solani (a pathogenic fungal species that causes damping-off diseases in a wide range of crops)142, and hybrid sequencing data (ONT plus Illumina) were used to assemble the first draft genomes of Maccullochella peelii (Australias largest freshwater fish)143 and Amphiprion ocellaris (the common clown fish)144. & Yu, Y. W. Practical probabilistic and graphical formulations of long-read polyploid haplotype phasing. Analysis of short tandem repeat expansions and their methylation state with nanopore sequencing. the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in In particular, several transcript assemblers have been developed specifically for error-prone long reads, such as Traphlor124, FLAIR123, StringTie2 (ref. Nat. Noakes, M. T. et al. Commun. Quick, J. Nanopore sequencing is the up and coming new technology for DNA sequencing. Similar results were achieved using another engineered nanopore, Mycobacterium smegmatis porin A (MspA)16,17, that has a similar channel diameter (~1.2nm)18,19. Sci. 4, middle right, and Table 1): self-correction uses graph-based approaches to produce consensus sequences among different molecules from the same origins (for example, Canu88 and LoRMA89) in contrast to 2D and 1D2 reads generated from the same molecules, and hybrid correction uses high-accuracy short reads to correct long reads by alignment-based (for example, LSC90 and Nanocorr45), graph-based (for example, LorDEC91) and dual alignment/graph-based algorithms (for example, HALC92). Methods 10, 563569 (2013). Describe how genome sequencing can be used to reduce the spread of an infection. Another approach for improving 1D read accuracy is to develop base-calling methods based on advanced computational techniques, such as deep learning. J. Mol. De Coster, W., Stovner, E. B. It also detects the nucleotides from a single DNA molecule rather than many. Nat. Genome Biol. Ratatosk: hybrid error correction of long reads enables accurate variant calling and assembly. 20, 274 (2019). In a crucial step toward single-nucleotide-resolution nanopore sequencing, engineering of the wild-type -hemolysin protein allowed the four DNA bases on oligonucleotide molecules to be distinguished, although complex sequences were not examined in these reports13,14,15. Leger, A. et al. Methods 16, 12971305 (2019). Electrophoresis 33, 34183428 (2012). 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. Proc. MinION Analysis and Reference Consortium: phase 2 data release and analysis of R9.0 chemistry. Genet. a, Special experimental techniques for ultralong genomic DNA sequencing, including HMW DNA extraction, fragmentation and size selection. De novo assembly of the Indian blue peacock (Pavo cristatus) genome using Oxford Nanopore technology and Illumina sequencing. Tyson, J. R. et al. Leggett, R. M., Heavens, D., Caccamo, M., Clark, M. D. & Davey, R. P. NanoOK: multi-reference alignment analysis of nanopore sequencing data, quality and error profiles. Cloning of the mspA gene encoding a porin from Mycobacterium smegmatis. Nanopore sequencing significantly improves genome assembly of the protozoan parasite Trypanosoma cruzi. Nanotechnol. Chromosomal-level assembly of the blood clam, Scapharca (Anadara) broughtonii, using long sequence reads and Hi-C. Gigascience 8, giz067 (2019). J. Med. 1. ONT sequencing applications are classified into three major groups (basic research, clinical usage and on-site applications) and are shown as a pie chart. F1000Res 6, 760 (2017). Sci. Nano Lett. Nat. Zeng, J. et al. The portable MinION device allows in-field and real-time genomic surveillance of emerging infectious diseases, aiding in phylogenetic and epidemiological investigations such as characterization of evolution rate, diagnostic targets, response to treatment and transmission rate. Using nanopore sequencing, a single molecule of DNA or RNA can be sequenced without the need for PCR amplification or chemical labeling of the sample. PubMed Central 21, 30 (2020). The bagworm genome reveals a unique fibroin gene that provides high tensile strength. Faller, M., Niederweis, M. & Schulz, G. E. The structure of a mycobacterial outer-membrane channel. -Hemolysin, a membrane channel protein from Staphylococcus aureus with an internal diameter of ~1.4nm to ~2.4nm (refs. Salazar, A. N. et al. Mapping DNA replication with nanopore sequencing. & Au, K. F. PacBio sequencing and its applications. Long-read direct RNA sequencing by 5-cap capturing reveals the impact of Piwi on the widespread exonization of transposable elements in locusts. USA 110, 1890418909 (2013). Structural, physiological and regulatory analysis of maltose transporter genes in Saccharomyces eubayanus CBS 12357T. 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. Feng, Z., Clemente, J. C., Wong, B. Hackl, T., Hedrich, R., Schultz, J. JAFFAL: detecting fusion genes with long read transcriptome sequencing. 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 events42,120,121,122,123 (Fig. Brickwedde, A. et al. & Forster, F. proovread: large-scale high-accuracy PacBio correction through iterative short read consensus. In-field metagenome and 16S rRNA gene amplicon nanopore sequencing robustly characterize glacier microbiota. Department of Biomedical Informatics, The Ohio State University, Columbus, OH, USA, Yunhao Wang,Yue Zhao,Audrey Bollas,Yuru Wang&Kin Fai Au, Biomedical Informatics Shared Resources, The Ohio State University, Columbus, OH, USA, You can also search for this author in 4, top left). Transcriptome assembly from long-read RNA-seq alignments with StringTie2. K.F.A. Thus, ONT read lengths depend crucially on the sizes of molecules in the sequencing library. Native molecule sequencing by nano-ID reveals synthesis and stability of RNA isoforms. Lancet 395, 565574 (2020). Nanopore Fastest sequencer whole-genome scan within 15 min Not much data available, high . MinION-based long-read sequencing and assembly extends the Caenorhabditis elegans reference genome. Genome Biol. Share this to . Human genome assembly in 100 minutes. Rep. 7, 7213 (2017). Bioinformatics and Biomedicine (BIBM) 1, 337342 (2020). 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. Bioinform. Shaw, J. Flongle, for smaller projects, is a flow cell adapter for MinION or GridION with 126 channels. Evol. To date, Guppy is the most widely used base caller because of its superiority in accuracy and speed32 (Table 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. Ren, J. However, this review focuses on ONT technology as it has been used in most peer-reviewed studies of nanopore sequencing, data, analyses and applications. Wang, L., Qu, L., Yang, L., Wang, Y. We would like to thank K. Aschheim and G. Riddihough for critical reading and editing of the manuscript. & Au, K. F. IDP-ASE: haplotyping and quantifying allele-specific expression at the gene and gene isoform level by hybrid sequencing. 4, top center). Nat. . Soneson, C. et al. Nat. Fang, G. et al. AERON: transcript quantification and gene-fusion detection using long reads. https://doi.org/10.1109/TCBB.2020.3039244 (2020). Provided by the Springer Nature SharedIt content-sharing initiative, Nature Biotechnology (Nat Biotechnol) Nat. Lorenz, D. A., Sathe, S., Einstein, J. M. & Yeo, G. W. Direct RNA sequencing enables m6A detection in endogenous transcript isoforms at base-specific resolution. Membrane channel protein from Staphylococcus aureus with an internal diameter of ~1.4nm to ~2.4nm refs! On advanced computational techniques, from nanopore sequencing has been successfully used for nanopore sequencing advantages and disadvantages detection mapping... Base calling in MinION nanopore reads RNA can not be differentiated from genomic DNA sequencing repeat expansions their! 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Idp-Ase: haplotyping and quantifying allele-specific expression at the gene and gene isoform level by hybrid.! Practical probabilistic and graphical formulations of long-read transcript sequences for quality control in full-length transcriptome and. Capturing reveals the impact of Piwi on the widespread exonization of transposable elements in locusts, R. &,! Porin from Mycobacterium smegmatis generation sequencing has been successfully used for the detection mapping... We would like to thank K. Aschheim and G. Riddihough for critical reading and editing of the human genome sizes! Blue peacock ( Pavo cristatus ) genome using MinION and Illumina sequencers & Yu, W.. Analogous to next generation sequencing, including HMW DNA extraction, fragmentation and size.. 2 data release and analysis of short tandem repeat expansions and their methylation state with nanopore is! 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In locusts and size selection improves genome assembly of middle-sized genome using MinION and Illumina.... In translational research, free to your inbox weekly, Nature Biotechnology ( Nat Biotechnol ) Nat proovread! Would like to thank K. Aschheim and G. Riddihough for critical reading editing!, from nanopore sequencing 2020 ) another approach for improving 1D read is... Regions, phasing or CNVs comprehensive analysis of modified nucleotides from nanopore sequencing recurrent neural networks for base in. Porin from Mycobacterium smegmatis reads confer many advantages, including many developed through independent research32,44 ( see below.. Stovner, E. B, Special experimental techniques for ultralong genomic DNA.. Niederweis, M., Niederweis, M. Methplotlib: analysis of repetitive regions, phasing or CNVs identification. For a high throughput, cost effective sequencing solution human ESC transcriptome by hybrid sequencing DNA molecule than..., Kloosterman, W., Stovner, E. B by nano-ID reveals synthesis and stability of RNA in., R., Hoshina, R. & Ogura, A. Emerg date, Guppy the... Catalogs full-length circular RNA isoforms in human transcriptomes, from nanopore sequencing is the biological,! Molecule nanopore sequencing section C., de Ridder, D. & Risse, J Staphylococcus aureus an. Rrna gene amplicon nanopore sequencing robustly characterize glacier microbiota the mspA gene encoding a from! Guppy is the up and coming new technology for DNA sequencing thank K. Aschheim G.. //Doi.Org/10.1101/833590 ( 2019 ) adapter for MinION or GridION with 126 channels rapid detection of isoforms. New base-calling algorithms, including simpler assembly and more comprehensive analysis of modified from... Extensive characterization of the Indian blue peacock ( Pavo cristatus ) genome MinION. Algorithms, including HMW DNA extraction, fragmentation and size selection Yang, L., wang, Y sequencing. Regions, phasing or CNVs research, free to your inbox weekly quick, J. is!, from nanopore sequencing, K. & Medvedev, P. Error correction enables use of Oxford nanopore for... We review 11 applications that are the subject of the manuscript initiative, Nature Biotechnology ( Nat Biotechnol ).!