Genome Biol. Nanopore sequencing has the potential to offer relatively low-cost genotyping, high mobility . Liu, H. et al. Marrano, A. et al. Antimicrobial-resistant Klebsiella pneumoniae carriage and infection in specialized geriatric care wards linked to acquisition in the referring hospital. Lv, X., Chen, Z., Lu, Y. Edwards, A. et al. Schreiber, J. et al. Nat. 67, 161170 (2018). PLoS Comput. Advantages of High-Throughput Sequencing: Process more samples to improve statistical power, and cost-effectively run emerging data-rich methods, including single-cell and spatial analyses. Genome Res. Gigascience 7, giy140 (2018). Slider with three articles shown per slide. 12, 2 (2021). Biol. De novo identification of DNA modifications enabled by genome-guided nanopore signal processing. SmidgION is a smartphone-compatible device under development. Nanopore sequencing significantly improves genome assembly of the protozoan parasite Trypanosoma cruzi. For instance, biological pores offer atomically precise structure . Rep. 7, 3935 (2017). 49, e7 (2020). BMC Bioinformatics 22, 64 (2021). DNA sequencing & Metabolic engineering 6 all sanger sequencing; took 13 years and cost more than 1bln USD now a whole human genome sequence costs c. 1000 USD and takes only a few days What is nanopore DNA sequencing physical method physical 'observation' of a base in DNA to determine its identity based on 'single molecule sequencing . 4, 1236 (2019). Rep. 6, 31602 (2016). Bioinformatics 30, 30043011 (2014). Method Advantages Disadvantages Ref Comments Second-generation sequencing techniques 454 sequencing . Evol. High-throughput targeted long-read single cell sequencing reveals the clonal and transcriptional landscape of lymphocytes. De novo yeast genome assemblies from MinION, PacBio and MiSeq platforms. Translocation speed is controlled by a motor protein that ratchets the nucleic acid molecule through the nanopore in a step-wise manner. Commun. Biotechnol. Wu, T. D. & Watanabe, C. K. GMAP: a genomic mapping and alignment program for mRNA and EST sequences. Oikonomopoulos, S., Wang, Y. C., Djambazian, H., Badescu, D. & Ragoussis, J. Benchmarking of the Oxford Nanopore MinION sequencing for quantitative and qualitative assessment of cDNA populations. Leggett, R. M. & Clark, M. D. A world of opportunities with nanopore sequencing. 46, e87 (2018). Commun. Miculini, N., Ratkovi, M. & iki, M. MinCall-MinION end2end convolutional deep learning basecaller. Nat. Nat. Sanger sequencing (Enzymatic DNA sequencing) Jyoti Pawar M.Sc IV sem - Biotechnology 2. Science 306, 666669 (2004). 7, 11307 (2016). I am interested in using high throughput DNA sequencing methods to understand microbial evolution and genomics as well as implement novel pathogen diagnostics. Mol. Yang, Y. et al. Nat. Laszlo, A. H. et al. PubMed 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. Bioinformatics 21, 18591875 (2005). Gigascience 7, giy037 (2018). IEEE Trans Nanotechnol. Nonetheless, current ONT sequencing techniques have several limitations, including relatively high error rates and the requirement for relatively high amounts of nucleic acid material. It may also be possible to increase data accuracy by recapturing DNA molecules into the same nanopore244 or by using multilayer nanopores for multiple sequencing of each molecule. Marchet, C. et al. Genes 10, 632 (2019). While for PacBio these values were slightly lower at 82 and 95%. 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). BMC Genomics 16, 327 (2015). Smith, A. M., Jain, M., Mulroney, L., Garalde, D. R. & Akeson, M. Reading canonical and modified nucleobases in 16S ribosomal RNA using nanopore native RNA sequencing. Dobin, A. et al. MinION nanopore sequencing identifies the position and structure of a bacterial antibiotic resistance island. Nucleic Acids Res. USA 115, 55105515 (2018). Oxford Nanopore has built a device for genetic sequencing that uses a technology they refer to as "nanopore sequencing" which . For example, the movement of DNA replication forks on single DNA molecules has been measured by detection of nucleotide analogs (for example, 5-bromodeoxyuridine (5-BrdU)) using ONT sequencing177,178,179, and the 3D chromatin organization in human cells has been analyzed by integrating a chromatin conformation capture technique and ONT sequencing to capture multiple loci in close spatial proximity by single reads180. A technology-agnostic long-read analysis pipeline for transcriptome discovery and quantification. 7, 146 (2018). Bioinformatics 16, btab526 (2021). 21, 11641181 (2019). 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, 1819kb; Zika virus222, 11kb; Venezuelan equine encephalitis virus225, 11.4kb; Lassa fever virus226, 10.4kb and SARS-CoV-2 coronavirus151, 29.8kb) to bacteria of several megabases (for example, Salmonella221, 5Mb; N. meningitidis227, 2Mb and K. pneumoniae228, 5.4Mb) and to human fungal pathogens with genomes of >10Mb (for example, Candida auris229, 12Mb). Preprint at bioRxiv https://doi.org/10.1101/2021.06.15.448494 (2021). Several advantages are offered by nanopore sequencing if it can be achieved. Nat. Selective nanopore sequencing of human BRCA1 by Cas9-assisted targeting of chromosome segments (CATCH). Jain, M. et al. The recently developed assembler wtdbg2 runs much faster than other tools without sacrificing contiguity and accuracy111. Early MinION users reported typical yields of hundreds of megabases per flow cell, while current throughput has increased to ~1015gigabases (Gb) (Fig. Faller, M., Niederweis, M. & Schulz, G. E. The structure of a mycobacterial outer-membrane channel. Nat. De novo clustering of long reads by gene from transcriptomics data. Bioinformatics 37, 573574 (2021). PLoS ONE 14, e0216471 (2019). Kim, D. et al. Lieberman, K. R. et al. 16, 13431375 (2021). Nat. . Nat. Long and ultra-long reads confer many advantages, including simpler assembly and more comprehensive analysis of repetitive regions, phasing or CNVs. The Illumina sequencing technique, like the other second generation techniques, is based on three main steps: Construction of a library for next generation techniques (NGS) which involves the addition of specific adapters to the DNA or cDNA fragments to be sequenced. Y.Z. In another clinical application, human leukocyte antigen genotyping benefited from the improved accuracy of the R9.5 nanopore216,217,218. 10, 31633167 (2010). Article Preprint at Preprints https://doi.org/10.20944/preprints201905.0113.v1 (2019). Biotechnol. & Quinlan, A. R. Poretools: a toolkit for analyzing nanopore sequence data. Sci. 1,2). Nat. 26, 11461153 (2008). Conclusions. Subsequent devices, such as PromethION, run more flow cells with more nanopores per flow cell. Methods 18, 165169 (2021). Tan, M. H. et al. K.F.A. 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. 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. Microorganisms play vital roles in the environment including cycling of nutrients, causing and preventing diseases in other organisms, and a host of other essential functions. As early as 2013, independent reports demonstrated that methylated cytosines (5mC and 5hmC) in DNA could be distinguished from native cytosine by the characteristic current signals measured using the MspA nanopore172,173. In 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. 20, 428435 (2018). A. et al. Microbiol. Nat. Int. Nat. 2010;9(3):281-294. doi: . Genome Biol. In more complicated cases, ONT long reads have been integrated with one or more other techniques (for example, Illumina short reads, PacBio long reads, 10x Genomics linked reads, optical mapping by Bionano Genomics and spatial distance by Hi-C) to assemble the initial reference genomes of many species, such as Maniola jurtina (the meadow brown butterfly, a model for ecological genetics)145, Varanus komodoensis (the largest extant monitor lizard)146, Pavo cristatus (the national bird of India)147, Panthera leo (the lion)148 and Eumeta variegate (a bagworm moth that produces silk with potential in biomaterial design)149. Bioinformatics 29, 1521 (2013). 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. Cloning of the mspA gene encoding a porin from Mycobacterium smegmatis. It also detects the nucleotides from a single DNA molecule rather than many. 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. These approaches take into account not only general assembly performance but also certain specific aspects, such as complex genomic regions and computational intensity. Oxford Nanopore sequencing, hybrid error correction, and de novo assembly of a eukaryotic genome. Exp. 68, 54195429 (2017). 39, 336346 (2020). Konishi, H., Yamaguchi, R., Yamaguchi, K., Furukawa, Y. 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. 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. 11, 1438 (2020). MinION was also used to conduct genomic surveillance for Zika virus222, yellow fever virus223 and dengue virus224 outbreaks in Brazil. Genome Res. Kin Fai Au. 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. Full-length transcript characterization of SF3B1 mutation in chronic lymphocytic leukemia reveals downregulation of retained introns. CAS Although each technology has advantages and disadvantages, HiFi and ONT are the most promising for future applications. Leukemia reveals downregulation of retained introns Although each technology has advantages and Disadvantages, HiFi and ONT are the promising... 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