7, 11307 (2016). 104). Nucleic Acids Res. Biotechnol. 3a). Faria, N. R. et al. MinION Analysis and Reference Consortium: phase 2 data release and analysis of R9.0 chemistry. Albacore development is now discontinued in favor of Guppy, which can also run on graphics processing units in addition to central processing units to accelerate base calling. Tree Lab: portable genomics for early detection of plant viruses and pests in sub-Saharan Africa. Nat. The technology is proven with a variety of input material such as genomic DNA, amplified DNA, cDNA and native RNA. Microbiol Antimicrob. Carter, J. M. & Hussain, S. Robust long-read native DNA sequencing using the ONT CsgG Nanopore system. Haghshenas, E., Sahinalp, S. C. & Hach, F. lordFAST: sensitive and fast alignment search tool for long noisy read sequencing data. Au, K. F., Underwood, J. G., Lee, L. & Wong, W. H. Improving PacBio long read accuracy by short read alignment. Methods 17, 11031110 (2020). In addition to pathogen detection, ONT sequencing can accelerate profiling antibiotic/antimicrobial resistance in bacteria and other microbes. There are several methods for nucleic acid sequencing, including Sanger sequencing, Illumina sequencing, PacBio sequencing, and nanopore sequencing. 13, 175 (2012). 7, 43 (2018). Kolmogorov, M. et al. Genome Biol. 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. Stoddart, D., Heron, A. J., Mikhailova, E., Maglia, G. & Bayley, H. Single-nucleotide discrimination in immobilized DNA oligonucleotides with a biological nanopore. Biol. Huang, N. et al. Methods 15, 455460 (2018). Flexible, population-scale sequencing using up to 48 independent, high-capacity flow cells complete genomic and transcriptomic characterisation of large sample numbers. Nanopore sequencing of single-cell transcriptomes with scCOLOR-seq. Li, H. Minimap and miniasm: fast mapping and de novo assembly for noisy long sequences. Leggett, R. M. & Clark, M. D. A world of opportunities with nanopore sequencing. J. Med. Reference-free reconstruction and quantification of transcriptomes from Nanopore long-read sequencing. Advances in nanopore sequencing technology. Jansen, H. J. et al. Nanopore sequencing technology, bioinformatics and applications. Using our rapid kits, library prep can be completed in under 10 minutes. 17, 10971103 (2019). 4, bottom right). 22, 22 (2021). Genome Biol. The significant advantages of nanopore seq. Genome Biol. situ sequencing by synthesis that can determine the template DNA sequence by detecting the exact nucleotide extended by its tagged fluorescent moiety . 13, 45214538 (2013). 3, 12411252 (2019). M. et al. Genome Biol. Get what matters in translational research, free to your inbox weekly. 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. 3d, middle). 17, 239 (2016). Nat. Nature 467, 190193 (2010). & Shih, P. W. Homopolish: a method for the removal of systematic errors in nanopore sequencing by homologous polishing. Genome Biol. When a small (~100mV) voltage bias is imposed across a nanopore in a membrane separating Commun. 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). Sedlazeck, F. J. et al. The long reads ahead: de novo genome assembly using the MinION. 2b). Biol. Genome Biol. Bioinformatics 29, 1521 (2013). 21, 30 (2020). Genome-wide mapping of methylated adenine residues in pathogenic Escherichia coli using single-molecule real-time sequencing. DNA Res. 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. Chin, C. S. & Khalak, A. Trends Genet. 30, 349353 (2012). 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. Sequencing and phasing cancer mutations in lung cancers using a long-read portable sequencer. Metagenomic sequencing at the epicenter of the Nigeria 2018 Lassa fever outbreak. Charalampous, T. et al. Suzuki, A. et al. Cheetham, S. W. et al. Picky, in addition to detecting regular SVs, also reveals enriched short-span SVs (~300bp) in repetitive regions, as long reads cover the entire region including the variations. Nat. 22, 182 (2021). USA 93, 1377013773 (1996). Altemose, N. et al. 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. Rang, F. J., Kloosterman, W. P. & de Ridder, J. Bioinformatics 11, btab354 (2021). Edge, P. & Bansal, V. Longshot enables accurate variant calling in diploid genomes from single-molecule long read sequencing. Karst, S. M. et al. Biotechnol. BMC Bioinformatics 18, 204 (2017). 8, 1326 (2017). Detection and mapping of 5-methylcytosine and 5-hydroxymethylcytosine with nanopore MspA. A major advantage of nanopore sequencing is the ability to produce ultra-long reads, and over 2 Mb read lengths have been achieved. HMW DNA can also be sheared to the desired size by sonication, needle extrusion or transposase cleavage (Fig. Xiao, C. L. et al. Nat Biotechnol 39, 13481365 (2021). J. Mol. Whole-genome sequencing (WGS) analysis is a highly discriminatory method capable of assessing genetic relatedness among isolates and therefore constitutes an important tool for outbreak . MinION and MinIT devices were brought to farms in sub-Saharan Africa for early and rapid diagnosis (<3h) of plant viruses and pests in cassava231. Unbiased strain-typing of arbovirus directly from mosquitoes using nanopore sequencing: a field-forward biosurveillance protocol. Rep. 7, 14521 (2017). Nanopore DNA sequencing with MspA. BMC Bioinformatics 20, 405 (2019). 5). Sci. Diagn. Gong, L., Wong, C. H., Idol, J., Ngan, C. Y. Single-molecule analysis of DNAprotein complexes using nanopores. Helmersen, K. & Aamot, H. V. DNA extraction of microbial DNA directly from infected tissue: an optimized protocol for use in nanopore sequencing. Genet. Nat. Science 274, 18591866 (1996). Single-molecule sequencing detection of N6-methyladenine in microbial reference materials. Wang, Y., Yang, Q. Nowoshilow, S. et al. Nat. Biotechnol. MinION nanopore sequencing identifies the position and structure of a bacterial antibiotic resistance island. & Vinar, T. DeepNano-blitz: a fast base caller for MinION nanopore sequencers. FEMS Yeast Res. Gigascience 6, 16 (2017). Each method has its advantages and disadvantages and can be used for different applications depending on factors such as sequencing accuracy, read length, throughput . 50, 581590 (2018). Drexler, H. L., Choquet, K. & Churchman, L. S. Splicing kinetics and coordination revealed by direct nascent RNA sequencing through nanopores. Nat. Nat. 22, 28 (2021). Nanopore sequencing significantly improves genome assembly of the protozoan parasite Trypanosoma cruzi. Basic local alignment search tool. Sci. 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. Blanco, M. B. et al. & Rausch, T. TRiCoLOR: tandem repeat profiling using whole-genome long-read sequencing data. Proc. 47, e2 (2019). PubMed 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. & Li, H. Fast and accurate long-read assembly with wtdbg2. We analysed the bacterial diversity in the rumen of defaunated sheep following the introduction of different protozoal populations, using both next generation sequencing (NGS: Ion Torrent PGM) and terminal restriction fragment length polymorphism (T . The size of a staplerand USB-powered, the MinION is beingused outside the traditional lab environment, enabling users to takeanalysis to the sample. An independent study reported a yield of 153Gb from a single PromethION flow cell with an average sequencing speed of ~430bases per s (ref. 134, 691703 (2017). Sutton, M. A. et al. Cheng, H., Concepcion, G. T., Feng, X., Zhang, H. & Li, H. Haplotype-resolved de novo assembly using phased assembly graphs with hifiasm. Microbiol. Rep. 6, 31602 (2016). Boykin, L. M. et al. 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. In this regard, it should be noted that the adapters used are different . Additionally, the sequencing flow cells from Oxford Nanopore Technologies are relatively inexpensive and sequencing can be performed in any laboratory without the need for dedicated sequencing equipment. Magi, A., Semeraro, R., Mingrino, A., Giusti, B. Genome Res. 2010;9(3):281-294. doi: . Chiron: translating nanopore raw signal directly into nucleotide sequence using deep learning. The birth of the epitranscriptome: deciphering the function of RNA modifications. Similarly, several other methods have combined various biochemical techniques with ONT sequencing (Table 1). DiMeLo-seq: a long-read, single-molecule method for mapping proteinDNA interactions genome-wide. Many applications combine long reads and short reads in the bioinformatics analyses, termed hybrid sequencing. iScience 23, 101223 (2020). Nat. 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. Hornblower, B. et al. The evolution of nanopore sequencing. Preprint at bioRxiv https://doi.org/10.1101/2020.11.01.363887 (2020). Proc. Nat. & Pevzner, P. A. Chem. Only 1560min of sequencing per sample was required220. 2b). Zeng, S. et al. Nat. Zimin, A. V. & Salzberg, S. L. The genome polishing tool POLCA makes fast and accurate corrections in genome assemblies.