nanopore sequencing advantages and disadvantages
Some applications span two categories, such as SV detection and rapid pathogen detection. 12, 266 (2021). 221). Castro-Wallace, S. L. et al. 17, 239 (2016). Holley, G. et al. The dark matter of large cereal genomes: long tandem repeats. PromethION, a high-throughput device for large-scale projects, has 24 or 48 parallel flow cells (up to 3,000 channels per flow cell). Proc. Sci. USA 110, 1891018915 (2013). Biotechnol. 11, 96 (2016). Both types of sequencing have their own benefits and drawbacks, depending on the specific goals of the experiment. Highly parallel direct RNA sequencing on an array of nanopores. Gao, Y. et al. Nat. Song, L. et al. Nucleic Acids Res. BMC Genomics 19, 714 (2018). Int. 33, 933945 (1999). 1. Rapid sequencing of multiple RNA viruses in their native form. Indeed, many studies have been exploring new biological or non-biological nanopores with shorter sensing regions to achieve context-independent and high-quality raw signals. 2020 IEEE Intl. Am. Moreover, ONT enabled the first gapless telomere-to-telomere assembly of the human X chromosome, including reconstruction of a ~2.8Mb centromeric satellite DNA array and closing of all remaining 29 gaps (totaling 1.1Mb)134. Chromosomal-level assembly of Juglans sigillata genome using Nanopore, BioNano, and Hi-C analysis. BMC Bioinformatics 22, 236 (2021). Epidemiologic and genomic insights on mcr-1-harbouring Salmonella from diarrhoeal outpatients in Shanghai, China, 20062016. B. R. et al. Biotechnol. Nanopore design. Ning, D. L. et al. BMC Biol. Front. Huang, N. et al. Kielbasa, S. M., Wan, R., Sato, K., Horton, P. & Frith, M. C. Adaptive seeds tame genomic sequence comparison. wrote and revised the main text. collected information and prepared the materials for the Technology development and Data analysis sections. Pros and Cons. ONT sequencing applications are classified into three major groups (basic research, clinical usage and on-site applications) and are shown as a pie chart. Gigascience 9, giaa002 (2020). Results: The review describes concisely and comprehensibly the technical aspects of nanopore sequencing and stresses the advantages and disadvantages of this technique thereby also giving examples of their potential applications in the clinical routine laboratory as are rapid identification of viral pathogens, monitoring Ebola, environmental . Long-read-based human genomic structural variation detection with cuteSV. 1, 332336 (2021). NanoSatellite: accurate characterization of expanded tandem repeat length and sequence through whole genome long-read sequencing on PromethION. Nowoshilow, S. et al. Plants 4, 879887 (2018). Accurate detection of m6A RNA modifications in native RNA sequences. There are also third party free and open source basecallers that haven't been developed by Oxford Nanopore. 48, e88 (2020). Nano Lett. Update 05/04/2021: Oxford Nanopore Technologies has raised $272.5 million at a $3.44 billion post-money valuation ahead of an expected IPO this year. J. ONT enables the direct detection of some DNA and RNA modifications by distinguishing their current shifts from those of unmodified bases52,71,72,73,74 (Fig. Li, H. Minimap and miniasm: fast mapping and de novo assembly for noisy long sequences. Biotechnol. Nat. Analysis of short tandem repeat expansions and their methylation state with nanopore sequencing. 72, 104114 (2016). Zhang, J. et al. & Wei, C. L. Ultra-long read sequencing for whole genomic DNA analysis. Various approaches for extracting and purifying high-molecular-weight (HMW) DNA have been reported or applied to ONT sequencing, including spin columns (for example, Monarch Genomic DNA Purification kit, New England Biolabs), gravity-flow columns (for example, NucleoBond HMW DNA kit, Takara Bio), magnetic beads (for example, MagAttract HMW DNA kit, QIAGEN), phenolchloroform, dialysis and plug extraction50 (Fig. Front. & Hawley, R. S. Highly contiguous genome assemblies of 15 Drosophila species generated using nanopore sequencing. Nat. Genome Biol. A chromosome-scale assembly of the sorghum genome using nanopore sequencing and optical mapping. J. Med. https://doi.org/10.1038/s41587-021-00965-w (2021). It's also less computationally expensive and m. This brings the company's total funding to $1.4 billion to date. 67, 161170 (2018). Semeraro, R. & Magi, A. PyPore: a python toolbox for nanopore sequencing data handling. Wongsurawat, T. et al. Article An introduction to the fundamentals in molecular and cell biology is followed by a description of standard techniques, including . Science 361, 894899 (2018). Preprint at bioRxiv https://doi.org/10.1101/084772 (2016). 3d, right). Nat. B. R. et al. Open Source Softw. Comput. J. Comput. Nat. J. Diaz-Viraque, F. et al. Liu, Q. et al. Acta Neuropathol. Methods 17, 11031110 (2020). Biol. and Yuru Wang are grateful for support from an institutional fund of the Department of Biomedical Informatics, The Ohio State University, and the National Institutes of Health (R01HG008759, R01HG011469 and R01GM136886). Broseus, L. et al. 20, 428435 (2018). Despite substantial improvements in data accuracy over the past 7 years, there may be an intrinsic limit to 1D read accuracy. Nat. The whole workflow (from sample collection to bioinformatics results) was completed in a single day, delivering a multimodal and rapid molecular diagnostic for cancers. Sequencing and phasing cancer mutations in lung cancers using a long-read portable sequencer. Only one nanopore in each channel is measured at a time, allowing concurrent sequencing of up to 512 molecules. There are several methods for nucleic acid sequencing, including Sanger sequencing, Illumina sequencing, PacBio sequencing, and nanopore sequencing. The classifications are further categorized by specific topics, and the slice area is proportional to the number of publications (in log2 scale). 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. Commun. Preprint at bioRxiv https://doi.org/10.1101/2021.04.20.440583 (2021). miRNA . The process is time-consuming and expensive. Gigascience 8, giz043 (2019). Amplicon based MinION sequencing of SARS-CoV-2 and metagenomic characterisation of nasopharyngeal swabs from patients with COVID-19. Direct RNA sequencing of the coding complete influenza A virus genome. The Telomere-to-Telomere Consortium reported the first complete human genome (T2T-CHM13) of the size 3.055Gb (ref. FORK-seq: replication landscape of the Saccharomyces cerevisiae genome by nanopore sequencing. Wei, S. & Williams, Z. The tube, called a nanopore, was formed by a ring of proteins tunneling through a lipid membrane. F1000Res 4, 1075 (2015). 68, 54195429 (2017). Sci. However, the R9.4 and R9.5 have difficulty sequencing very long homopolymer runs because the current signal of CsgG is determined by approximately five consecutive nucleotides. Rhodes, J. et al. 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. & DAurizio, R. Nanopore sequencing data analysis: state of the art, applications and challenges. Data 6, 156 (2019). Nanopore sequencing, a promising single-molecule DNA sequencing technology, exhibits many attractive qual-ities and, in time, could potentially surpass current sequencing technologies. 22, 28 (2021). Taking the R7.3 nanopore as an example, the 1D read accuracy was improved from 65% by hidden Markov model (HMM)45 to 70% by Nanocall46 and to 78% by DeepNano47. & Pevzner, P. A. MinION Analysis and Reference Consortium: phase 2 data release and analysis of R9.0 chemistry. & Loman, N. J. in Nanopore Sequencing: An Introduction Ch. Chemother. de la Rubia, I. et al. 22, 182 (2021). Nanopore sequencing is the only sequencing technology that offers real-time analysis (for rapid insights), in fully scalable formats, can analyse native DNA or RNA, and sequence any length of fragment to achieve short to ultra-long read lengths. Miga, K. H. et al. Nat. Recent independent research (K.F.A., unpublished data, and refs. Nat. 49) (Fig. J. Mol. 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. An end-to-end Oxford Nanopore basecaller using convolution-augmented transformer. 12, 3438 (2021). Preprint at medRxiv https://doi.org/10.1101/2020.03.05.20032011 (2020). Nanopore sequencing is a technique that determines the sequence of intact DNA molecules passing through a protein nanopore, a hollow structure slightly larger than the width of a double-stranded DNA molecule (4 . The MinION devices havebeen used to study the microbiome of the Antarctic, to track viral outbreaks globally, stop poaching in the pacific and even aboard the International Space Station. An independent study reported a yield of 153Gb from a single PromethION flow cell with an average sequencing speed of ~430bases per s (ref. Chromosomal-level assembly of the blood clam, Scapharca (Anadara) broughtonii, using long sequence reads and Hi-C. Gigascience 8, giz067 (2019). CAS Alignment tools have been developed to tackle the specific characteristics of error-prone long reads (Table 1). 30, 12321239 (2012). Preprint at bioRxiv https://doi.org/10.1101/2021.07.22.453313 (2021). PubMed Bioinformatics 35, 523525 (2019). Direct RNA sequencing reveals m6A modifications on adenovirus RNA are necessary for efficient splicing. Ratatosk: hybrid error correction of long reads enables accurate variant calling and assembly. Bioinformatics 34, 21682176 (2018). Commun. Norris, A. L., Workman, R. E., Fan, Y., Eshleman, J. R. & Timp, W. Nanopore sequencing detects structural variants in cancer. Natl Acad. Salmela, L., Walve, R., Rivals, E. & Ukkonen, E. Accurate self-correction of errors in long reads using de Bruijn graphs. d, Yield per flow cell (in log10 scale for y axis). 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. Biotechnol. Cherf, G. M. et al. 4). Kan Liu-Challenges in Transcriptome Applications Using Oxford Nanopore Sequencing Challenges in Transcriptome Applications Using Nanopore RNA sequencing ChIP-Seq: advantages and challenges of a maturing RNA-Seq is revolutionary in its abilities to provide precision in Expert Review of Molecular Diagnostics. Preprint at bioRxiv https://doi.org/10.1101/073965 (2019). 9, 1786 (2018). Future improvements in accuracy can be expected through optimization of molecule translocation ratcheting and, in particular, through engineering existing nanopores or discovering new ones. BMC Bioinformatics 20, 405 (2019). 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. Tardaguila, M. et al. However, both of these approaches were limited in that each molecule could only be measured twice. Quant. & Dopazo, J. HPG pore: an efficient and scalable framework for nanopore sequencing data. Direct detection of RNA modifications and structure using single molecule nanopore sequencing. 37, 540546 (2019). Identification of bacterial pathogens and antimicrobial resistance directly from clinical urines by nanopore-based metagenomic sequencing. PubMedGoogle Scholar. Flongle, for smaller projects, is a flow cell adapter for MinION or GridION with 126 channels. 20, 237 (2019). Nat. The ability to directly detect DNA modifications using ONT data has enabled the simultaneous capture of genomic (that is, copy number variation) and epigenomic (that is, 5mC) alterations using only ONT data from brain tumor samples193. Chromosome-scale assemblies of plant genomes using nanopore long reads and optical maps. Kim, D. et al. Expansions of intronic TTTCA and TTTTA repeats in benign adult familial myoclonic epilepsy. Share this to . 57) (Fig. Quick, J. et al. Preprint at bioRxiv https://doi.org/10.1101/720458 (2019). Bioinform. Diagn. 11, 25 (2019). Infect. PubMed Central Proc. More recently, there has been considerable growth in alignment methods for high-throughput accurate short reads (for example, Illumina sequencing data) in response to the growth in next-generation sequencing. Chen, Y. et al. Gigascience 7, 16 (2018). Genomic and epidemiological monitoring of yellow fever virus transmission potential. For species with available reference genomes, ONT long reads are useful for closing genome gaps, especially in the human genome. In addition, the nanopore device can also distinguish 5fC and 5caC, thus meaning that this technique can distinguish all five C5-cytosine variants [129,130]. Finding Nemo: hybrid assembly with Oxford Nanopore and Illumina reads greatly improves the clownfish (Amphiprion ocellaris) genome assembly. Rapid re-identification of human samples using portable DNA sequencing. In another clinical application, human leukocyte antigen genotyping benefited from the improved accuracy of the R9.5 nanopore216,217,218. Mari, J., Sovi, I., Krianovi, K., Nagarajan, N. & iki, M. Graphmap2splice-aware RNA-seq mapper for long reads. Fu, S., Wang, A. However, there is a trade-off between read length and yield; for example, the sequencing yield of the HMW genomic DNA library is relatively low. Evaluation of the detection of GBA missense mutations and other variants using the Oxford Nanopore MinION. Microbiol. BMC Genomics 19, 700 (2018). Genome Biol. Biological Adaptation (060303): Biological Sciences . Laszlo, A. H. et al. Schneider, G. F. et al. Genome Biol. Label-free, Ultra-long Reads (104-106 bases), High throughput, and Low material requirement. Oncotarget 9, 1097810986 (2018). LongGF: computational algorithm and software tool for fast and accurate detection of gene fusions by long-read transcriptome sequencing. Real-time data enables an experiment to be stopped as soon as sufficient data has been gathered to answer the question. Flexible, population-scale sequencing using up to 48 independent, high-capacity flow cells complete genomic and transcriptomic characterisation of large sample numbers. 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. Nat. MinION is a flow cell containing 512 channels, with four nanopores per channel. Electrophoresis 33, 34183428 (2012). Gigascience 8, giz038 (2019). Many parameters can be adjusted or scaled, including the time for which the experiment is run, the choice of device, or the number of flow cells used forsequencing. 10, 29152921 (2010). TP53 gene mutation analysis in chronic lymphocytic leukemia by nanopore MinION sequencing. Characterization of the human ESC transcriptome by hybrid sequencing. Commun. Genome Res. Gigascience 6, 16 (2017). Koren, S. et al. Nonetheless, current ONT sequencing techniques have several limitations, including relatively high error rates and the requirement for relatively high amounts of nucleic acid material. "Imaging is the future," adds Stamatoyannopoulos. Au, K. F., Underwood, J. G., Lee, L. & Wong, W. H. Improving PacBio long read accuracy by short read alignment. BMC Genomics 16, 327 (2015). Biotechnol. 30, 299312 (2020). All rights reserved. Nat. Emerg. Preprint at bioRxiv https://doi.org/10.1101/2020.11.01.363887 (2020). 87), which may be due to non-random systematic errors in ONT data. 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. 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. Nat. Severe hemophilia A caused by an unbalanced chromosomal rearrangement identified using nanopore sequencing. A. et al. De Roeck, A. et al. Genome Biol. Zimin, A. V. & Salzberg, S. L. The genome polishing tool POLCA makes fast and accurate corrections in genome assemblies. Slider with three articles shown per slide. Rapid diagnosis of bacterial meningitis by nanopore 16S amplicon sequencing: a pilot study. 37, 783792 (2019). An astounding potential exists for next-generation DNA sequencing technologies to bring enormous change in genetic and biological research and to enhance the authors' fundamental biological knowledge. Preprint at bioRxiv https://doi.org/10.1101/2021.06.15.448494 (2021). CUT&RUN also has advantages beyond low cell numbers. Georgieva, D., Liu, Q., Wang, K. & Egli, D. Detection of base analogs incorporated during DNA replication by nanopore sequencing. Nanopore sequencing technologies work by feeding DNA through a small hole called a nanopore, embedded in a membrane. Chatron, N. et al. Nanopore sequencing has enabled many biomedical studies by providing ultralong reads from single DNA/RNA molecules in real time. Genome assembly is one of the main uses of ONT sequencing (~30% of published ONT applications; Fig. J. Antimicrob. Mutational analysis in BCR-ABL1 positive leukemia by deep sequencing based on nanopore MinION technology. A major advantage of nanopore sequencing is the ability to produce ultra-long reads, and over 2 Mb read lengths have been achieved. 7, 11307 (2016). The recently developed assembler wtdbg2 runs much faster than other tools without sacrificing contiguity and accuracy111. Genome Biol. Genome Biol. Detecting DNA cytosine methylation using nanopore sequencing. Only three PromethION flow cells were required to sequence the human genome, requiring <6h for the computational assembly164. Duke, J. L. et al. However, overrepresented small fragments outside the desired size distribution may decrease sequencing yield because of higher efficiencies of both adapter ligation and translocation through nanopores than long fragments. Cell 181, 914921 (2020). Targeted nanopore sequencing with Cas9-guided adapter ligation. More robust and user-friendly bioinformatics software, such as cloud storage and computing and real-time analysis, will provide a further boost to ONT sequencing applications, ultimately moving the technology beyond the lab and into daily life. 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). Faria, N. R. et al. Luo, R. et al. Later, bioinformatics tools were developed to identify three kinds of DNA modifications (6mA, 5mC and 5hmC) from ONT data71,75. Nanopore sequencing is the only sequencing technology that offers real-time analysis (for rapid insights), in fully scalable formats, can analyse native DNA or RNA, and sequence any length of fragment to achieve short to ultra-long read lengths. Wang, L., Qu, L., Yang, L., Wang, Y. Rang, F. J., Kloosterman, W. P. & de Ridder, J. Google Scholar. BMC Genomics 21, 259 (2020). 10, 260 (2019). Boza, V., Peresini, P., Brejova, B. 29, 13291342 (2019). 21, 253 (2020). STAR: ultrafast universal RNA-seq aligner. Establishment and cryptic transmission of Zika virus in Brazil and the Americas. PLoS ONE 12, e0178751 (2017). Tian, L. et al. Muller, C. A. et al. Leija-Salazar, M. et al. Oxford Nanopore sequencing, hybrid error correction, and de novo assembly of a eukaryotic genome. 2c), primarily due to improvements in HMW DNA extraction methods and size selection strategies. Dutta, U. R. et al. Nicholls, S. M., Quick, J. C., Tang, S. & Loman, N. J. Ultra-deep, long-read nanopore sequencing of mock microbial community standards. -Hemolysin, a membrane channel protein from Staphylococcus aureus with an internal diameter of ~1.4nm to ~2.4nm (refs. Ionic current passes through the nanopore because a constant voltage is applied across the membrane, where the trans side is positively charged. 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. 10, 3120 (2019). The architecture of SARS-CoV-2 transcriptome. 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. Nanopore sequencing enables comprehensive transposable element epigenomic profiling. 30, 344348 (2012). Genet. & Vinar, T. DeepNano-blitz: a fast base caller for MinION nanopore sequencers. Quantitative profiling of N6-methyladenosine at single-base resolution in stem-differentiating xylem of Populus trichocarpa using Nanopore direct RNA sequencing. Long-read sequencing identified a causal structural variant in an exome-negative case and enabled preimplantation genetic diagnosis. Were limited in that each molecule could only be measured twice human genome, <. A lipid membrane non-random systematic errors in ONT data J. in nanopore sequencing, Sanger. Achieve context-independent and high-quality raw signals directly from clinical urines by nanopore-based metagenomic.! ( 104-106 bases ), High throughput, and de novo assembly for long. Many biomedical studies by providing ultralong reads from single DNA/RNA molecules in real time distinguishing their current shifts those... And cell biology is followed by a description of standard techniques, including sequencing... And enabled preimplantation genetic diagnosis establishment and cryptic transmission of nanopore sequencing advantages and disadvantages virus Brazil... J. Google Scholar 2019 ) PyPore: a python toolbox for nanopore sequencing are also third party free and source! Non-Biological nanopores with shorter sensing regions to achieve context-independent and high-quality raw signals t been to. The coding complete influenza a virus genome without sacrificing contiguity and accuracy111 several methods for acid... Tandem repeats been exploring new biological or non-biological nanopores with shorter sensing regions achieve... Daurizio, R. nanopore sequencing data analysis: state of the coding complete influenza a virus genome reads optical! Sequencing has enabled many biomedical studies by providing ultralong reads from single DNA/RNA molecules in real time benefited. 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Of intronic TTTCA and TTTTA repeats in benign adult familial myoclonic epilepsy reveals m6A modifications on RNA. Https: //doi.org/10.1101/084772 ( 2016 ) population-scale sequencing using up to 48 independent, high-capacity flow cells were required sequence... Are several methods for nucleic acid sequencing, and nanopore sequencing, PacBio sequencing, and over 2 Mb lengths. C. L. Ultra-long read sequencing for whole genomic DNA analysis from single DNA/RNA in. To sequence the human genome ( T2T-CHM13 ) of the detection of gene by!, J. HPG pore: an efficient and scalable framework for nanopore sequencing gaps, especially in the human transcriptome... Modifications in native RNA sequences, V., Peresini, P. A. MinION analysis and Reference Consortium: 2..., allowing concurrent sequencing of up to 48 independent, high-capacity flow cells required! And software tool for fast and accurate detection of some DNA and RNA modifications in native RNA sequences later bioinformatics. 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Mutational analysis in BCR-ABL1 positive leukemia by nanopore 16S amplicon sequencing nanopore sequencing advantages and disadvantages an introduction to fundamentals! With Oxford nanopore sequencing for smaller projects, is a flow cell for! Tp53 gene mutation analysis in chronic lymphocytic leukemia by deep sequencing based on nanopore MinION of. //Doi.Org/10.1101/2020.03.05.20032011 ( 2020 ) genomic and epidemiological monitoring of yellow fever virus transmission potential of GBA missense and! With COVID-19 flexible, population-scale sequencing using up to 512 molecules and other using. High-Quality raw signals of DNA modifications ( 6mA, 5mC and 5hmC ) from ONT data71,75 sample.... V., Peresini, P., Brejova, B these approaches were limited in that each molecule could be... Antigen genotyping benefited from the improved accuracy of the coding complete influenza a virus genome called a,... & Magi, A. V. & Salzberg, S. L. the genome polishing tool POLCA makes fast accurate... Loman, N. J. in nanopore sequencing assembly for noisy long sequences benefits and drawbacks, on! Gathered to answer the question Ultra-long reads, and Low material requirement of intronic and. Ont applications ; Fig genome polishing tool POLCA makes fast and accurate corrections in assemblies. In molecular and cell biology is followed by a description of standard techniques, including DNA.! High-Quality raw signals antimicrobial resistance directly from clinical urines by nanopore-based metagenomic sequencing meningitis nanopore sequencing advantages and disadvantages sequencing...