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Heikema, Astrid P., Deborah Horst-Kreft, Stefan A. Boers, et al. "Comparison of Illumina versus Nanopore 16S rRNA Gene Sequencing of the Human Nasal Microbiota." Genes 11, no. 9 (2020): 1105. http://dx.doi.org/10.3390/genes11091105.
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Illumina and nanopore sequencing technologies are powerful tools that can be used to determine the bacterial composition of complex microbial communities. In this study, we compared nasal microbiota results at genus level using both Illumina and nanopore 16S rRNA gene sequencing. We also monitored the progression of nanopore sequencing in the accurate identification of species, using pure, single species cultures, and evaluated the performance of the nanopore EPI2ME 16S data analysis pipeline. Fifty-nine nasal swabs were sequenced using Illumina MiSeq and Oxford Nanopore 16S rRNA gene sequencing technologies. In addition, five pure cultures of relevant bacterial species were sequenced with the nanopore sequencing technology. The Illumina MiSeq sequence data were processed using bioinformatics modules present in the Mothur software package. Albacore and Guppy base calling, a workflow in nanopore EPI2ME (Oxford Nanopore Technologies—ONT, Oxford, UK) and an in-house developed bioinformatics script were used to analyze the nanopore data. At genus level, similar bacterial diversity profiles were found, and five main and established genera were identified by both platforms. However, probably due to mismatching of the nanopore sequence primers, the nanopore sequencing platform identified Corynebacterium in much lower abundance compared to Illumina sequencing. Further, when using default settings in the EPI2ME workflow, almost all sequence reads that seem to belong to the bacterial genus Dolosigranulum and a considerable part to the genus Haemophilus were only identified at family level. Nanopore sequencing of single species cultures demonstrated at least 88% accurate identification of the species at genus and species level for 4/5 strains tested, including improvements in accurate sequence read identification when the basecaller Guppy and Albacore, and when flowcell versions R9.4 (Oxford Nanopore Technologies—ONT, Oxford, UK) and R9.2 (Oxford Nanopore Technologies—ONT, Oxford, UK) were compared. In conclusion, the current study shows that the nanopore sequencing platform is comparable with the Illumina platform in detection bacterial genera of the nasal microbiota, but the nanopore platform does have problems in detecting bacteria within the genus Corynebacterium. Although advances are being made, thorough validation of the nanopore platform is still recommendable.
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Lin, Bo, Jianan Hui, and Hongju Mao. "Nanopore Technology and Its Applications in Gene Sequencing." Biosensors 11, no. 7 (2021): 214. http://dx.doi.org/10.3390/bios11070214.
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In recent years, nanopore technology has become increasingly important in the field of life science and biomedical research. 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. One of the most successful applications of nanopore technology, the Oxford Nanopore Technology, marks the beginning of the fourth generation of gene sequencing technology. In this review, the operational principle and the technology for signal processing of the nanopore gene sequencing are documented. Moreover, this review focuses on the applications using nanopore gene sequencing technology, including the diagnosis of cancer, detection of viruses and other microbes, and the assembly of genomes. These applications show that nanopore technology is promising in the field of biological and biomedical sensing.
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Lu, Hengyun, Francesca Giordano, and Zemin Ning. "Oxford Nanopore MinION Sequencing and Genome Assembly." Genomics, Proteomics & Bioinformatics 14, no. 5 (2016): 265–79. http://dx.doi.org/10.1016/j.gpb.2016.05.004.
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Eisenstein, Michael. "Oxford Nanopore announcement sets sequencing sector abuzz." Nature Biotechnology 30, no. 4 (2012): 295–96. http://dx.doi.org/10.1038/nbt0412-295.
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Sereika, Mantas, Rasmus Hansen Kirkegaard, Søren Michael Karst, et al. "Oxford Nanopore R10.4 long-read sequencing enables the generation of near-finished bacterial genomes from pure cultures and metagenomes without short-read or reference polishing." Nature Methods 19, no. 7 (2022): 823–26. http://dx.doi.org/10.1038/s41592-022-01539-7.
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AbstractLong-read Oxford Nanopore sequencing has democratized microbial genome sequencing and enables the recovery of highly contiguous microbial genomes from isolates or metagenomes. However, to obtain near-finished genomes it has been necessary to include short-read polishing to correct insertions and deletions derived from homopolymer regions. Here, we show that Oxford Nanopore R10.4 can be used to generate near-finished microbial genomes from isolates or metagenomes without short-read or reference polishing.
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MacKenzie, Morgan, and Christos Argyropoulos. "An Introduction to Nanopore Sequencing: Past, Present, and Future Considerations." Micromachines 14, no. 2 (2023): 459. http://dx.doi.org/10.3390/mi14020459.
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There has been significant progress made in the field of nanopore biosensor development and sequencing applications, which address previous limitations that restricted widespread nanopore use. These innovations, paired with the large-scale commercialization of biological nanopore sequencing by Oxford Nanopore Technologies, are making the platforms a mainstay in contemporary research laboratories. Equipped with the ability to provide long- and short read sequencing information, with quick turn-around times and simple sample preparation, nanopore sequencers are rapidly improving our understanding of unsolved genetic, transcriptomic, and epigenetic problems. However, there remain some key obstacles that have yet to be improved. In this review, we provide a general introduction to nanopore sequencing principles, discussing biological and solid-state nanopore developments, obstacles to single-base detection, and library preparation considerations. We present examples of important clinical applications to give perspective on the potential future of nanopore sequencing in the field of molecular diagnostics.
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Салахов, Р. Р., М. В. Голубенко, Е. Н. Павлюкова, et al. "Application of monomolecular sequencing technology to the diagnostics of hypertrophic cardiomyopathy." Nauchno-prakticheskii zhurnal «Medicinskaia genetika», no. 5(214) (May 29, 2020): 9–10. http://dx.doi.org/10.25557/2073-7998.2020.05.9-10.
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В работе представлены результаты секвенирования пяти генов, ассоциированных с гипертрофической кардиомиопатией, с использованием технологии мономолекулярного секвенирования компании Oxford Nanopore Technologies. В результате анализа данных с помощью различных алгоритмов были выявлены миссенс-варианты в исследованных генах, которые могут являться причиной заболевания у пациентов. The paper presents the results of sequencing of five genes associated with hypertrophic cardiomyopathy, using monomolecular sequencing (Oxford Nanopore Technologies). As a result of data analysis with various algorithms, missense variants were identified in the studied genes that may be the cause of the disease in the patients.
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Burns, Adam, David Robert Bruce, Pauline Robbe, et al. "Detection of Clinically Relevant Molecular Alterations in Chronic Lymphocytic Leukemia (CLL) By Nanopore Sequencing." Blood 132, Supplement 1 (2018): 1847. http://dx.doi.org/10.1182/blood-2018-99-110948.
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Abstract Introduction Chronic Lymphocytic Leukaemia (CLL) is the most prevalent leukaemia in the Western world and characterised by clinical heterogeneity. IgHV mutation status, mutations in the TP53 gene and deletions of the p-arm of chromosome 17 are currently used to predict an individual patient's response to therapy and give an indication as to their long-term prognosis. Current clinical guidelines recommend screening patients prior to initial, and any subsequent, treatment. Routine clinical laboratory practices for CLL involve three separate assays, each of which are time-consuming and require significant investment in equipment. Nanopore sequencing offers a rapid, low-cost alternative, generating a full prognostic dataset on a single platform. In addition, Nanopore sequencing also promises low failure rates on degraded material such as FFPE and excellent detection of structural variants due to long read length of sequencing. Importantly, Nanopore technology does not require expensive equipment, is low-maintenance and ideal for patient-near testing, making it an attractive DNA sequencing device for low-to-middle-income countries. Methods Eleven untreated CLL samples were selected for the analysis, harbouring both mutated (n=5) and unmutated (n=6) IgHV genes, seven TP53 mutations (five missense, one stop gain and one frameshift) and two del(17p) events. Primers were designed to amplify all exons of TP53, along with the IgHV locus, and each primer included universal tails for individual sample barcoding. The resulting PCR amplicons were prepared for sequencing using a ligation sequencing kit (SQK-LSK108, Oxford Nanopore Technologies, Oxford, UK). All IgHV libraries were pooled and sequenced on one R9.4 flowcell, with the TP53 libraries pooled and sequenced on a second R9.4 flowcell. Whole genome libraries were prepared from 400ng genomic DNA for each sample using a rapid sequencing kit (SQK-RAD004, Oxford Nanopore Technologies, Oxford, UK), and each sample sequenced on individual flowcells on a MinION mk1b instrument (Oxford Nanopore Technologies, Oxford, UK). We developed a bespoke bioinformatics pipeline to detect copy-number changes, TP53 mutations and IgHV mutation status from the Nanopore sequencing data. Results were compared to short-read sequencing data obtained earlier by targeted deep sequencing (MiSeq, Illumina Inc, San Diego, CA, USA) and whole genome sequencing (HiSeq 2500, Illumina Inc, San Diego CA, USA). Results Following basecalling and adaptor trimming, the raw data were submitted to the IMGT database. In the absence of error correction, it was possible to identify the correct VH family for each sample; however the germline homology was not sufficient to differentiate between IgHVmut and IgHVunmut CLL cases. Following bio-informatic error correction and consensus building, the percentage to germline homology was the same as that obtained from short-read sequencing and nanopore sequencing also called the same productive rearrangements in all cases. A total of 77 TP53 variants were identified, including 68 in non-coding regions, and three synonymous SNVs. The remaining 6 were predicted to be functional variants (eight missense and two stop-gains) and had all been identified in early MiSeq targeted sequencing. However, the frameshift mutation was not called by the analysis pipeline, although it is present in the aligned reads. Using the low-coverage WGS data, we were able to identify del(17p) events, of 19Mb and 20Mb length, in both patients with high confidence. Conclusions Here we demonstrate that characterization of the IgHV locus in CLL cases is possible using the MinION platform, provided sufficient downstream analysis, including error correction, is applied. Furthermore, somatic SNVs in TP53 can be identified, although similar to second generation sequencing, variant calling of small insertions and deletions is more problematic. Identification of del(17p) is possible from low-coverage WGS on the MinION and is inexpensive. Our data demonstrates that Nanopore sequencing can be a viable, patient-near, low-cost alternative to established screening methods, with the potential of diagnostic implementation in resource-poor regions of the world. Disclosures Schuh: Giles, Roche, Janssen, AbbVie: Honoraria.
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Dumschott, Kathryn, Maximilian H.-W. Schmidt, Harmeet Singh Chawla, Rod Snowdon, and Björn Usadel. "Oxford Nanopore sequencing: new opportunities for plant genomics?" Journal of Experimental Botany 71, no. 18 (2020): 5313–22. http://dx.doi.org/10.1093/jxb/eraa263.
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Abstract DNA sequencing was dominated by Sanger’s chain termination method until the mid-2000s, when it was progressively supplanted by new sequencing technologies that can generate much larger quantities of data in a shorter time. At the forefront of these developments, long-read sequencing technologies (third-generation sequencing) can produce reads that are several kilobases in length. This greatly improves the accuracy of genome assemblies by spanning the highly repetitive segments that cause difficulty for second-generation short-read technologies. Third-generation sequencing is especially appealing for plant genomes, which can be extremely large with long stretches of highly repetitive DNA. Until recently, the low basecalling accuracy of third-generation technologies meant that accurate genome assembly required expensive, high-coverage sequencing followed by computational analysis to correct for errors. However, today’s long-read technologies are more accurate and less expensive, making them the method of choice for the assembly of complex genomes. Oxford Nanopore Technologies (ONT), a third-generation platform for the sequencing of native DNA strands, is particularly suitable for the generation of high-quality assemblies of highly repetitive plant genomes. Here we discuss the benefits of ONT, especially for the plant science community, and describe the issues that remain to be addressed when using ONT for plant genome sequencing.
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Leger, Adrien, and Tommaso Leonardi. "pycoQC, interactive quality control for Oxford Nanopore Sequencing." Journal of Open Source Software 4, no. 34 (2019): 1236. http://dx.doi.org/10.21105/joss.01236.
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Chen, Weigang, Peng Zhang, Lifu Song, Jinsheng Yang, and Changcai Han. "Simulation of Nanopore Sequencing Signals Based on BiGRU." Sensors 20, no. 24 (2020): 7244. http://dx.doi.org/10.3390/s20247244.
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Oxford Nanopore sequencing is an important sequencing technology, which reads the nucleotide sequence by detecting the electrical current signal changes when DNA molecule is forced to pass through a biological nanopore. The research on signal simulation of nanopore sequencing is highly desirable for method developments of nanopore sequencing applications. To improve the simulation accuracy, we propose a novel signal simulation method based on Bi-directional Gated Recurrent Units (BiGRU). In this method, the signal processing model based on BiGRU is built to replace the traditional low-pass filter to post-process the ground-truth signal calculated by the input nucleotide sequence and nanopore sequencing pore model. Gaussian noise is then added to the filtered signal to generate the final simulated signal. This method can accurately model the relation between ground-truth signal and real-world sequencing signal through experimental sequencing data. The simulation results reveal that the proposed method utilizing the powerful learning ability of the neural network can generate the simulated signal that is closer to the real-world sequencing signal in the time and frequency domains than the existing simulation method.
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Czmil, Anna, Michal Wronski, Sylwester Czmil, et al. "NanoForms: an integrated server for processing, analysis and assembly of raw sequencing data of microbial genomes, from Oxford Nanopore technology." PeerJ 10 (March 29, 2022): e13056. http://dx.doi.org/10.7717/peerj.13056.
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Background Next Generation Sequencing (NGS) techniques dominate today’s landscape of genetics and genomics research. Though Illumina still dominates worldwide sequencing, Oxford Nanopore is one of the leading technologies currently being used by biologists, medics and geneticists across various applications. Oxford Nanopore is automated and relatively simple for conducting experiments, but generates gigabytes of raw data, to be processed by often ambiguous set of alternative bioinformatics command-line tools, and genomics frameworks which require a knowledge of bioinformatics to run. Results We established an inter-collegiate collaboration across experimentalists and bioinformaticians in order to provide a novel bioinformatics tool, free for academics. This tool allows people without extensive bioinformatics knowledge to simply process their raw genome sequencing data. Currently, due to ICT resources’ maintenance reasons, our server is only capable of handling small genomes (up to 15 Mb). In this paper, we introduce our tool, NanoForms: an intuitive and integrated web server for the processing and analysis of raw prokaryotic genome data, coming from Oxford Nanopore. NanoForms is freely available for academics at the following locations: http://nanoforms.tech (webserver) and https://github.com/czmilanna/nanoforms (GitHub source repository).
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Lamb, Harrison J., Ben J. Hayes, Loan T. Nguyen, and Elizabeth M. Ross. "The Future of Livestock Management: A Review of Real-Time Portable Sequencing Applied to Livestock." Genes 11, no. 12 (2020): 1478. http://dx.doi.org/10.3390/genes11121478.
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Oxford Nanopore Technologies’ MinION has proven to be a valuable tool within human and microbial genetics. Its capacity to produce long reads in real time has opened up unique applications for portable sequencing. Examples include tracking the recent African swine fever outbreak in China and providing a diagnostic tool for disease in the cassava plant in Eastern Africa. Here we review the current applications of Oxford Nanopore sequencing in livestock, then focus on proposed applications in livestock agriculture for rapid diagnostics, base modification detection, reference genome assembly and genomic prediction. In particular, we propose a future application: ‘crush-side genotyping’ for real-time on-farm genotyping for extensive industries such as northern Australian beef production. An initial in silico experiment to assess the feasibility of crush-side genotyping demonstrated promising results. SNPs were called from simulated Nanopore data, that included the relatively high base call error rate that is characteristic of the data, and calling parameters were varied to understand the feasibility of SNP calling at low coverages in a heterozygous population. With optimised genotype calling parameters, over 85% of the 10,000 simulated SNPs were able to be correctly called with coverages as low as 6×. These results provide preliminary evidence that Oxford Nanopore sequencing has potential to be used for real-time SNP genotyping in extensive livestock operations.
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Loman, Nick, Sarah Goodwin, Hans J. Jansen, and Matt Loose. "A disruptive sequencer meets disruptive publishing." F1000Research 4 (October 15, 2015): 1074. http://dx.doi.org/10.12688/f1000research.7229.1.
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Nanopore sequencing was recently made available to users in the form of the Oxford Nanopore MinION. Released to users through an early access programme, the MinION is made unique by its tiny form factor and ability to generate very long sequences from single DNA molecules. The platform is undergoing rapid evolution with three distinct nanopore types and five updates to library preparation chemistry in the last 18 months. To keep pace with the rapid evolution of this sequencing platform, and to provide a space where new analysis methods can be openly discussed, we present a new F1000Research channel devoted to updates to and analysis of nanopore sequence data.
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Zhao, Kaishun, Chunlin Tu, Wei Chen, et al. "Rapid Identification of Drug-Resistant Tuberculosis Genes Using Direct PCR Amplification and Oxford Nanopore Technology Sequencing." Canadian Journal of Infectious Diseases and Medical Microbiology 2022 (March 28, 2022): 1–8. http://dx.doi.org/10.1155/2022/7588033.
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Mycobacterium tuberculosis antimicrobial resistance has been continually reported and is a major public health issue worldwide. Rapid prediction of drug resistance is important for selecting appropriate antibiotic treatments, which significantly increases cure rates. Gene sequencing technology has proven to be a powerful strategy for identifying relevant drug resistance information. This study established a sequencing method and bioinformatics pipeline for resistance gene analysis using an Oxford Nanopore Technologies sequencer. The pipeline was validated by Sanger sequencing and exhibited 100% concordance with the identified variants. Turnaround time for the nanopore sequencing workflow was approximately 12 h, facilitating drug resistance prediction several weeks earlier than that of traditional phenotype drug susceptibility testing. This study produced a customized gene panel assay for rapid bacterial identification via nanopore sequencing, which improves the timeliness of tuberculosis diagnoses and provides a reliable method that may have clinical application.
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Tytgat, Olivier, Yannick Gansemans, Jana Weymaere, Kaat Rubben, Dieter Deforce, and Filip Van Nieuwerburgh. "Nanopore Sequencing of a Forensic STR Multiplex Reveals Loci Suitable for Single-Contributor STR Profiling." Genes 11, no. 4 (2020): 381. http://dx.doi.org/10.3390/genes11040381.
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Nanopore sequencing for forensic short tandem repeats (STR) genotyping comes with the advantages associated with massively parallel sequencing (MPS) without the need for a high up-front device cost, but genotyping is inaccurate, partially due to the occurrence of homopolymers in STR loci. The goal of this study was to apply the latest progress in nanopore sequencing by Oxford Nanopore Technologies in the field of STR genotyping. The experiments were performed using the state of the art R9.4 flow cell and the most recent R10 flow cell, which was specifically designed to improve consensus accuracy of homopolymers. Two single-contributor samples and one mixture sample were genotyped using Illumina sequencing, Nanopore R9.4 sequencing, and Nanopore R10 sequencing. The accuracy of genotyping was comparable for both types of flow cells, although the R10 flow cell provided improved data quality for loci characterized by the presence of homopolymers. We identify locus-dependent characteristics hindering accurate STR genotyping, providing insights for the design of a panel of STR loci suited for nanopore sequencing. Repeat number, the number of different reference alleles for the locus, repeat pattern complexity, flanking region complexity, and the presence of homopolymers are identified as unfavorable locus characteristics. For single-contributor samples and for a limited set of the commonly used STR loci, nanopore sequencing could be applied. However, the technology is not mature enough yet for implementation in routine forensic workflows.
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Kinimi, Edson, Mana Mahapatra, Tebogo Kgotlele, et al. "Complete Genome Sequencing of Field Isolates of Peste des Petits Ruminants Virus from Tanzania Revealed a High Nucleotide Identity with Lineage III PPR Viruses." Animals 11, no. 10 (2021): 2976. http://dx.doi.org/10.3390/ani11102976.
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Peste des petits ruminants virus (PPRV) causes a highly devastating disease of sheep and goats that threatens food security, small ruminant production and susceptible endangered wild ruminants. With policy directed towards achieving global PPR eradication, the establishment of cost-effective genomic surveillance tools is critical where PPR is endemic. Genomic data can provide sufficient in-depth information to identify the pockets of endemicity responsible for PPRV persistence and viral evolution, and direct an appropriate vaccination response. Yet, access to the required sequencing technology is low in resource-limited settings and is compounded by the difficulty of transporting clinical samples from wildlife across international borders due to the Convention on International Trade in Endangered Species (CITES) of Wild Fauna and Flora, and Nagoya Protocol regulations. Oxford nanopore MinION sequencing technology has recently demonstrated an extraordinary performance in the sequencing of PPRV due to its rapidity, utility in endemic countries and comparatively low cost per sample when compared to other whole-genome (WGS) sequencing platforms. In the present study, Oxford nanopore MinION sequencing was utilised to generate complete genomes of PPRV isolates collected from infected goats in Ngorongoro and Momba districts in the northern and southern highlands of Tanzania during 2016 and 2018, respectively. The tiling multiplex polymerase chain reaction (PCR) was carried out with twenty-five pairs of long-read primers. The resulting PCR amplicons were used for nanopore library preparation and sequencing. The analysis of output data was complete genomes of PPRV, produced within four hours of sequencing (accession numbers: MW960272 and MZ322753). Phylogenetic analysis of the complete genomes revealed a high nucleotide identity, between 96.19 and 99.24% with lineage III PPRV currently circulating in East Africa, indicating a common origin. The Oxford nanopore MinION sequencer can be deployed to overcome diagnostic and surveillance challenges in the PPR Global Control and Eradication program. However, the coverage depth was uneven across the genome and amplicon dropout was observed mainly in the GC-rich region between the matrix (M) and fusion (F) genes of PPRV. Thus, larger field studies are needed to allow the collection of sufficient data to assess the robustness of nanopore sequencing technology.
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Chen, Zhao, David L. Erickson, and Jianghong Meng. "Benchmarking Long-Read Assemblers for Genomic Analyses of Bacterial Pathogens Using Oxford Nanopore Sequencing." International Journal of Molecular Sciences 21, no. 23 (2020): 9161. http://dx.doi.org/10.3390/ijms21239161.
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Oxford Nanopore sequencing can be used to achieve complete bacterial genomes. However, the error rates of Oxford Nanopore long reads are greater compared to Illumina short reads. Long-read assemblers using a variety of assembly algorithms have been developed to overcome this deficiency, which have not been benchmarked for genomic analyses of bacterial pathogens using Oxford Nanopore long reads. In this study, long-read assemblers, namely Canu, Flye, Miniasm/Racon, Raven, Redbean, and Shasta, were thus benchmarked using Oxford Nanopore long reads of bacterial pathogens. Ten species were tested for mediocre- and low-quality simulated reads, and 10 species were tested for real reads. Raven was the most robust assembler, obtaining complete and accurate genomes. All Miniasm/Racon and Raven assemblies of mediocre-quality reads provided accurate antimicrobial resistance (AMR) profiles, while the Raven assembly of Klebsiella variicola with low-quality reads was the only assembly with an accurate AMR profile among all assemblers and species. All assemblers functioned well for predicting virulence genes using mediocre-quality and real reads, whereas only the Raven assemblies of low-quality reads had accurate numbers of virulence genes. Regarding multilocus sequence typing (MLST), Miniasm/Racon was the most effective assembler for mediocre-quality reads, while only the Raven assemblies of Escherichia coli O157:H7 and K. variicola with low-quality reads showed positive MLST results. Miniasm/Racon and Raven were the best performers for MLST using real reads. The Miniasm/Racon and Raven assemblies showed accurate phylogenetic inference. For the pan-genome analyses, Raven was the strongest assembler for simulated reads, whereas Miniasm/Racon and Raven performed the best for real reads. Overall, the most robust and accurate assembler was Raven, closely followed by Miniasm/Racon.
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Liem, Michael, Hans J. Jansen, Ron P. Dirks, et al. "De novo whole-genome assembly of a wild type yeast isolate using nanopore sequencing." F1000Research 6 (August 3, 2018): 618. http://dx.doi.org/10.12688/f1000research.11146.2.
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Background: The introduction of the MinION sequencing device by Oxford Nanopore Technologies may greatly accelerate whole genome sequencing. Nanopore sequence data offers great potential for de novo assembly of complex genomes without using other technologies. Furthermore, Nanopore data combined with other sequencing technologies is highly useful for accurate annotation of all genes in the genome. In this manuscript we used nanopore sequencing as a tool to classify yeast strains. Methods: We compared various technical and software developments for the nanopore sequencing protocol, showing that the R9 chemistry is, as predicted, higher in quality than R7.3 chemistry. The R9 chemistry is an essential improvement for assembly of the extremely AT-rich mitochondrial genome. We double corrected assemblies from four different assemblers with PILON and assessed sequence correctness before and after PILON correction with a set of 290 Fungi genes using BUSCO. Results: In this study, we used this new technology to sequence and de novo assemble the genome of a recently isolated ethanologenic yeast strain, and compared the results with those obtained by classical Illumina short read sequencing. This strain was originally named Candida vartiovaarae (Torulopsis vartiovaarae) based on ribosomal RNA sequencing. We show that the assembly using nanopore data is much more contiguous than the assembly using short read data. We also compared various technical and software developments for the nanopore sequencing protocol, showing that nanopore-derived assemblies provide the highest contiguity. Conclusions: The mitochondrial and chromosomal genome sequences showed that our strain is clearly distinct from other yeast taxons and most closely related to published Cyberlindnera species. In conclusion, MinION-mediated long read sequencing can be used for high quality de novo assembly of new eukaryotic microbial genomes.
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Chalapati, Sachin, Conor A. Crosbie, Dixita Limbachiya, and Nimesh Pinnamaneni. "Direct oligonucleotide sequencing with nanopores." Open Research Europe 1 (August 24, 2021): 47. http://dx.doi.org/10.12688/openreseurope.13578.2.
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Third-generation DNA sequencing has enabled sequencing of long, unamplified DNA fragments with minimal steps. Direct sequencing of ssDNA or RNA gives valuable insights like base-level modifications, phosphoramidite synthesis yield estimates and strand quality analysis, without the need to add the complimentary strand. Direct sequencing of single-stranded nucleic acid species is challenging as they are non-compatible to the double-stranded sequencing adapters used by manufacturers. The MinION platform from Oxford Nanopore Technologies performs sequencing by passing single-strands of DNA through a layer of biological nanopore sensors; although sequencing is performed on single-strands, the recommended template by the manufacturer is double-stranded. We have identified that the MinION platform can perform sequencing of short, single-strand oligonucleotides directly without amplification or second-strand synthesis by performing a single annealing step before library preparation. Short 5’ phosphorylated oligos when annealed to an adapter sequence can be directly sequenced in the 5' to 3' direction via nanopores. Adapter sequences were designed to bind to the 5’ end of the oligos and to leave a 3’ adenosine overhang after binding to their target. The 3’ adenosine overhang of the adapter and the terminal phosphate makes the 5’ end of the oligo analogous to an end-prepared dsDNA, rendering it compatible with ligation-based library preparation for sequencing. An oligo-pool containing 42,000, 120 nt orthogonal sequences was phosphorylated and sequenced using this method and ~90% of these sequences were recovered with high accuracy using BLAST. In the nanopore raw data, we have identified that empty signals can be wrongly identified as a valid read by the MinION platform and sometimes multiple signals containing several strands can be fused into a single raw sequence file due to segmentation faults in the software. This direct oligonucleotide sequencing method enables novel applications in DNA data storage systems where short oligonucleotides are the primary information carriers.
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Chalapati, Sachin, Conor A. Crosbie, Dixita Limbachiya, and Nimesh Pinnamaneni. "Direct oligonucleotide sequencing with nanopores." Open Research Europe 1 (May 12, 2021): 47. http://dx.doi.org/10.12688/openreseurope.13578.1.
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Third-generation DNA sequencing has enabled sequencing of long, unamplified DNA fragments with minimal steps. Direct sequencing of ssDNA or RNA gives valuable insights like base-level modifications, phosphoramidite synthesis yield estimates and strand quality analysis, without the need to add the complimentary strand. Direct sequencing of single-stranded nucleic acid species is challenging as they are non-compatible to the double-stranded sequencing adapters used by manufacturers. The MinION platform from Oxford Nanopore Technologies performs sequencing by passing single-strands of DNA through a layer of biological nanopore sensors; although sequencing is performed on single-strands, the recommended template by the manufacturer is double-stranded. We have identified that the MinION platform can perform sequencing of short, single-strand oligonucleotides directly without amplification or second-strand synthesis by performing a single annealing step before library preparation. Short 5’ phosphorylated oligos when annealed to an adapter sequence can be directly sequenced in the 5' to 3' direction via nanopores. Adapter sequences were designed to bind to the 5’ end of the oligos and to leave a 3’ adenosine overhang after binding to their target. The 3’ adenosine overhang of the adapter and the terminal phosphate makes the 5’ end of the oligo analogous to an end-prepared dsDNA, rendering it compatible with ligation-based library preparation for sequencing. An oligo-pool containing 42,000, 120 nt orthogonal sequences was phosphorylated and sequenced using this method and ~90% of these sequences were recovered with high accuracy using BLAST. In the nanopore raw data, we have identified that empty signals can be wrongly identified as a valid read by the MinION platform and sometimes multiple signals containing several strands can be fused into a single raw sequence file due to segmentation faults in the software. This direct oligonucleotide sequencing method enables novel applications in DNA data storage systems where short oligonucleotides are the primary information carriers.
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Diubo, Yulia V., Artur E. Akhremchuk, Leonid N. Valentovich, and Yevgeny A. Nikolaichik. "Restriction-modification systems and DNA methylation profile of Pectobacterium carotovorum 2A." Journal of the Belarusian State University. Biology, no. 3 (November 4, 2021): 71–77. http://dx.doi.org/10.33581/2521-1722-2021-3-71-77.
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The methylation profile of Pectobacterium carotovorum 2A genome was studied using the Oxford Nanopore sequencing technology. The specificity of the methylase subunits of the three restriction-modification systems of this strain was determined. Analysis of homologous systems showed the uniqueness of the type I restriction-modification system and the type IV restriction system specific to methylated DNA of this strain. The work confirms the applicability of Oxford Nanopore technology to the analysis of bacterial DNA modifications and is also the first example of such an analysis for Pectobacterium spp.
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Du, Chenghao. "The Power of Using Novel Nanopore Sequencing Technology for Diagnosis, Genomic and Pathological Studies of Covid-19." E3S Web of Conferences 271 (2021): 04024. http://dx.doi.org/10.1051/e3sconf/202127104024.
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The novel coronavirus disease 2019 (COVID‐19), originally identified in December 2019 Wuhan, China, has propagated to worldwide pandemic, causing many cases of death and morbidity. Since the development of COVID-19 vaccines is still under experimental stages without public access, different types of testing and detection ensuring rapid and accurate results are urgently required to prevent delaying isolation of infected patients. The traditional diagnostic and analytical methods of COVID-19 relied heavily on nucleic acid and antibody-antigen methods but are subject to assembly bias, restricted by reading length, showed some false positive/negative results and had a long turnaround time. Hence, three styles of nanopore sequencing techniques as complementary tools for COVID-19 diagnosis and analysis are introduced. The long-read nanopore sequencing technology has been adopted in metagenomic and pathological studies of virosphere including SARS-CoV-2 recently by either metagenomically, directly or indirectly sequencing the viral genomic RNA of SARS-CoV-2 in real-time to detect infected specimens for early isolation and treatment, to investigate the transmission and evolutionary routes of SARS-CoV-2 as well as its pathogenicity and epidemiology. In this article, the Nanopore-Based Metagenomic Sequencing, Direct RNA Nanopore Sequencing (DRS), and Nanopore Targeted Sequencing (NTS) become the main focus of the novel COVID-19 detecting analytical methods in sequencing platforms, which are discussed in comparison with other traditional and popular diagnostic methods. Finally, different types of nanopore sequencing platforms that are developed by Oxford Nanopore Technologies (ONT) due to various purposes and demands in viral genomic research are briefly discussed.
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Du, Chenghao. "The Power of Using Novel Nanopore Sequencing Technology for Diagnosis, Genomic and Pathological Studies of Covid-19." South Florida Journal of Development 2, no. 3 (2021): 4014–28. http://dx.doi.org/10.46932/sfjdv2n3-017.
Full textAbstract:
The novel coronavirus disease 2019 (COVID‐19), originally identified in December 2019 Wuhan, China, has propagated to worldwide pandemic, causing many cases of death and morbidity. Since the development of COVID-19 vaccines is still under experimental stages without public access, different types of testing and detection ensuring rapid and accurate results are urgently required to prevent delaying isolation of infected patients. The traditional diagnostic and analytical methods of COVID-19 relied heavily on nucleic acid and antibody-antigen methods but are subject to assembly bias, restricted by reading length, showed some false positive/negative results and had a long turnaround time. Hence, three styles of nanopore sequencing techniques as complementary tools for COVID-19 diagnosis and analysis are introduced. The long-read nanopore sequencing technology has been adopted in metagenomic and pathological studies of virosphere including SARS-CoV-2 recently by either metagenomically, directly or indirectly sequencing the viral genomic RNA of SARS-CoV-2 in real-time to detect infected specimens for early isolation and treatment, to investigate the transmission and evolutionary routes of SARS-CoV-2 as well as its pathogenicity and epidemiology. In this article, the Nanopore-Based Metagenomic Sequencing, Direct RNA Nanopore Sequencing (DRS), and Nanopore Targeted Sequencing (NTS) become the main focus of the novel COVID-19 detecting analytical methods in sequencing platforms, which are discussed in comparison with other traditional and popular diagnostic methods. Finally, different types of nanopore sequencing platforms that are developed by Oxford Nanopore Technologies (ONT) due to various purposes and demands in viral genomic research are briefly discussed.
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Deynichenko, K. A., K. G. Ptitsyn, S. P. Radko, et al. "Splice variants of mRNA of cytochrome P450 genes: analysis by the nanopore sequencing method in human liver tissue and HepG2 cell line." Biomeditsinskaya Khimiya 68, no. 2 (2022): 117–25. http://dx.doi.org/10.18097/pbmc20226802117.
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The analysis of cytochrome P450 transcripts was carried out by the nanopore sequencing in liver tissue samples of three donors and HepG2 line cells. It has been demonstrated that direct mRNA sequencing with a MinION nanopore sequencer (Oxford Nanopore Technologies) allows one to obtained quantitative profiles for transcripts (and their splice variants) of cytochrome P450 superfamily genes encoding isoforms involved in metabolism of the large (~80%) part of drugs. The splice variant profiles substantially differ for donors. The cytochrome P450 gene expression at the transcript level is significantly weaker in cells of the HepG2 line compared with that in the normal liver tissue. This limits the capability of the direct mRNA nanopore sequencing for studying alternative splicing of cytochrome P450 transcripts in HepG2 cells. Both quantitative and qualitative profiles of the cytochrome P450 gene expression at the transcript level are notably differ in human liver tissue and HepG2 cells.
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Tanaka, Mami, Sayaka Mino, Yoshitoshi Ogura, Tetsuya Hayashi, and Tomoo Sawabe. "Availability of Nanopore sequences in the genome taxonomy for Vibrionaceae systematics: Rumoiensis clade species as a test case." PeerJ 6 (June 18, 2018): e5018. http://dx.doi.org/10.7717/peerj.5018.
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Whole genome sequence comparisons have become essential for establishing a robust scheme in bacterial taxonomy. To generalize this genome-based taxonomy, fast, reliable, and cost-effective genome sequencing methodologies are required. MinION, the palm-sized sequencer from Oxford Nanopore Technologies, enables rapid sequencing of bacterial genomes using minimal laboratory resources. Here we tested the ability of Nanopore sequences for the genome-based taxonomy of Vibrionaceae and compared Nanopore-only assemblies to complete genomes of five Rumoiensis clade species: Vibrio aphrogenes, V. algivorus, V. casei, V. litoralis, and V. rumoiensis. Comparison of overall genome relatedness indices (OGRI) and multilocus sequence analysis (MLSA) based on Nanopore-only assembly and Illumina or hybrid assemblies revealed that errors in Nanopore-only assembly do not influence average nucleotide identity (ANI), in silico DNA-DNA hybridization (DDH), G+C content, or MLSA tree topology in Vibrionaceae. Our results show that the genome sequences from Nanopore-based approach can be used for rapid species identification based on the OGRI and MLSA.
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Radko, S. P., L. K. Kurbatov, K. G. Ptitsyn, et al. "Prospects for the use of third generation sequencers for quantitative profiling of transcriptome." Biomedical Chemistry: Research and Methods 1, no. 4 (2018): e00086. http://dx.doi.org/10.18097/bmcrm00086.
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Transcriptome profiling is widely employed to analyze transcriptome dynamics when studying various biological processes at the cell and tissue levels. Unlike the second generation sequencers, which sequence relatively short fragments of nucleic acids, the third generation DNA/RNA sequencers developed by biotechnology companies “PacBio” and “Oxford Nanopore Technologies” allow one to sequence transcripts as single molecules and may be considered as potential molecular counters capable to measure the number of copies of each transcript with high throughput, sensitivity, and specificity. In the present review, the features of single molecule sequencing technologies offered by “PacBio” and “Oxford Nanopore Technologies” are considered alongside with their utility for transcriptome analysis, including the analysis of transcript isoforms. The prospects and limitations of the single molecule sequencing technology in application to quantitative transcriptome profiling are also discussed.
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De Coster, Wouter, Endre Bakken Stovner, and Mojca Strazisar. "Methplotlib: analysis of modified nucleotides from nanopore sequencing." Bioinformatics 36, no. 10 (2020): 3236–38. http://dx.doi.org/10.1093/bioinformatics/btaa093.
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Abstract Summary Modified nucleotides play a crucial role in gene expression regulation. Here, we describe methplotlib, a tool developed for the visualization of modified nucleotides detected from Oxford Nanopore Technologies sequencing platforms, together with additional scripts for statistical analysis of allele-specific modification within-subjects and differential modification frequency across subjects. Availability and implementation The methplotlib command-line tool is written in Python3, is compatible with Linux, Mac OS and the MS Windows 10 Subsystem for Linux and released under the MIT license. The source code can be found at https://github.com/wdecoster/methplotlib and can be installed from PyPI and bioconda. Our repository includes test data, and the tool is continuously tested at travis-ci.com. Supplementary information Supplementary data are available at Bioinformatics online.
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Il Jun, Kang, Jangsup Moon, Taek Soo Kim, et al. "238. Direct identification of Bacterial Species with MinION Nanopore Sequencer In Clinical Specimens Suspected of Polybacterial Infection." Open Forum Infectious Diseases 6, Supplement_2 (2019): S136. http://dx.doi.org/10.1093/ofid/ofz360.313.
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Abstract Background Conventional culture tests usually identify only a few bacterial species, which can grow well in the culture system, in the cases of polybacterial infection. 16S rRNA gene nanopore sequencing enables semi-quantitative identification of bacterial genetic materials. We aimed to evaluate usefulness of 16s rRNA gene nanopore sequencing in the cases suspected of polybacterial infection. Methods The research was conducted in a single university hospital for one year. Conventional bacterial culture identification and nanopore sequencing of 16s rRNA gene were carried out simultaneously for cases where polybacterial infection is strongly suspected. Blood agar plate was used for conventional culture, and Microscan (Beckman Coulter, United States) and Vitek 2 (Biomerieux, FR) automated systems were used for identification. For nanopore sequencing, 16S rRNA gene PCR was performed from the clinical specimens, and sequencing libraries were generated from the PCR products using the rapid barcoding sequencing kit (Oxford nanopore technologies, UK). MinION sequencing was performed for 1–3 hours and the generated reads were analyzed using the EPI2ME 16S BLAST workflow. Results Specimens were obtained from 15 patients; 6 liver abscess, 2 psoas abscess, 2 thigh abcess, 1 paraspinal abscess, 1 mycotic aneurysm, 1 necrotizing fasciitis, 1 fingertip gangrene and 1 abscess in coccyx area. 16s rRNA gene nanopore sequencing showed monobacterial organism in 8 (53.3%) specimens and polybacterial organisms in 7 (46.6%) specimens. In three (37.5%) cases of 8 cases with monobacterial infections identified by 16s rRNA gene sequencing, no organism was grown in conventional culture, possibly due to previous antibiotic administration. Notably, among 8 cases with polybacterial infection by 16s rRNA gene nanopore sequencing test, traditional culture test showed polybacterial infection in only two (25%) cases and single bacterial organism was identified in the other 6 (75%) cases. Conclusion Nanopore sequencing of 16s rRNA gene using the MinION sequencer may be useful for identification of causing microorganism and differentiation between monobacterial and polybacterial infection when polybacterial infection is suspected. Disclosures All authors: No reported disclosures.
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Jansen, Hans J., Ron P. Dirks, Michael Liem, et al. "De novo whole-genome assembly of a wild type yeast isolate using nanopore sequencing." F1000Research 6 (May 3, 2017): 618. http://dx.doi.org/10.12688/f1000research.11146.1.
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Background: The introduction of the MinIONTM sequencing device by Oxford Nanopore Technologies may greatly accelerate whole genome sequencing. It has been shown that the nanopore sequence data, in combination with other sequencing technologies, is highly useful for accurate annotation of all genes in the genome. However, it also offers great potential for de novo assembly of complex genomes without using other technologies. In this manuscript we used nanopore sequencing as a tool to classify yeast strains. Methods: We compared various technical and software developments for the nanopore sequencing protocol, showing that the R9 chemistry is, as predicted, higher in quality than R7.3 chemistry. The R9 chemistry is an essential improvement for assembly of the extremely AT-rich mitochondrial genome. Results: In this study, we used this new technology to sequence and de novo assemble the genome of a recently isolated ethanologenic yeast strain, and compared the results with those obtained by classical Illumina short read sequencing. This strain was originally named Candida vartiovaarae (Torulopsis vartiovaarae) based on ribosomal RNA sequencing. We show that the assembly using nanopore data is much more contiguous than the assembly using short read data. Conclusions: The mitochondrial and chromosomal genome sequences showed that our strain is clearly distinct from other yeast taxons and most closely related to published Cyberlindnera species. In conclusion, MinION-mediated long read sequencing can be used for high quality de novo assembly of new eukaryotic microbial genomes.
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Morsli, Madjid, Quentin Kerharo, Jeremy Delerce, Pierre-Hugues Roche, Lucas Troude, and Michel Drancourt. "Haemophilus influenzae Meningitis Direct Diagnosis by Metagenomic Next-Generation Sequencing: A Case Report." Pathogens 10, no. 4 (2021): 461. http://dx.doi.org/10.3390/pathogens10040461.
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Current routine real-time PCR methods used for the point-of-care diagnosis of infectious meningitis do not allow for one-shot genotyping of the pathogen, as in the case of deadly Haemophilus influenzae meningitis. Real-time PCR diagnosed H. influenzae meningitis in a 22-year-old male patient, during his hospitalisation following a more than six-metre fall. Using an Oxford Nanopore Technologies real-time sequencing run in parallel to real-time PCR, we detected the H. influenzae genome directly from the cerebrospinal fluid sample in six hours. Furthermore, BLAST analysis of the sequence encoding for a partial DUF417 domain-containing protein diagnosed a non-b serotype, non-typeable H.influenzae belonging to lineage H. influenzae 22.1-21. The Oxford Nanopore metagenomic next-generation sequencing approach could be considered for the point-of-care diagnosis of infectious meningitis, by direct identification of pathogenic genomes and their genotypes/serotypes.
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David, Matei, L. J. Dursi, Delia Yao, Paul C. Boutros, and Jared T. Simpson. "Nanocall: an open source basecaller for Oxford Nanopore sequencing data." Bioinformatics 33, no. 1 (2016): 49–55. http://dx.doi.org/10.1093/bioinformatics/btw569.
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Ding, Hongxu, Andrew D. Bailey, Miten Jain, Hugh Olsen, and Benedict Paten. "Gaussian mixture model-based unsupervised nucleotide modification number detection using nanopore-sequencing readouts." Bioinformatics 36, no. 19 (2020): 4928–34. http://dx.doi.org/10.1093/bioinformatics/btaa601.
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Abstract Motivation Nucleotide modification status can be decoded from the Oxford Nanopore Technologies nanopore-sequencing ionic current signals. Although various algorithms have been developed for nanopore-sequencing-based modification analysis, more detailed characterizations, such as modification numbers, corresponding signal levels and proportions are still lacking. Results We present a framework for the unsupervised determination of the number of nucleotide modifications from nanopore-sequencing readouts. We demonstrate the approach can effectively recapitulate the number of modifications, the corresponding ionic current signal levels, as well as mixing proportions under both DNA and RNA contexts. We further show, by integrating information from multiple detected modification regions, that the modification status of DNA and RNA molecules can be inferred. This method forms a key step of de novo characterization of nucleotide modifications, shedding light on the interpretation of various biological questions. Availability and implementation Modified nanopolish: https://github.com/adbailey4/nanopolish/tree/cigar_output. All other codes used to reproduce the results: https://github.com/hd2326/ModificationNumber. Supplementary information Supplementary data are available at Bioinformatics online.
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Salakhov, R. R., M. V. Golubenko, E. N. Pavlukova, et al. "Experience in genetic testing of hypertrophic cardiomyopathy using nanopore DNA sequencing." Russian Journal of Cardiology 26, no. 10 (2021): 4673. http://dx.doi.org/10.15829/1560-4071-2021-4673.
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Aim. To investigate the application of the Oxford Nanopore Technologies’ third generation sequencing for the genetic testing of hypertrophic cardiomyopathy.Material and methods. The study involved 12 patients with hypertrophic cardiomyopathy aged 18 to 67 years (women, 9; men, 3). Using the PCR barcoding amplicons (SQK-LSK109) protocol, DNA libraries were created which contained long-range PCR fragments of the MYH7, MYBPC3, TNNT2, TNNI3 and TPM1 genes. The sequencing was performed using the MinION system by Oxford Nanopore Technologies (UK). Bioinformatic algorithms for data analysis included Guppy v.5.0.7, Nanopolish and Clairvoyante. The identified genetic variants were confirmed by Sanger sequencing.Results. Data on the complete sequence of the five major sarcomeric genes for hypertrophic cardiomyopathy were obtained. We found eight potentially disease-causing sequence variants in MYH7, MYBPC3 and TNNT2 genes by monomolecular sequencing. However, only three mutations p.Arg243Cys, p.Tyr609Asn, p.Arg870His in the MYH7 gene, and one mutation p.Lys985Asn in the MYBPC3 were confirmed by Sanger sequencing. Cascade screening of pathogenic variant p.Arg870His in the MYH7 gene was performed. We found one asymptomatic carrier.Conclusion. It appears that monomolecular sequencing technology is a feasible approach to identify mutations in patients with hypertrophic cardiomyopathy. Although improvement in accuracy of DNA sequencing, as well as optimization and simplification of bioinformatic algorithms for identification of the genetic variants are needed.
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Fukasawa, Yoshinori, Luca Ermini, Hai Wang, Karen Carty, and Min-Sin Cheung. "LongQC: A Quality Control Tool for Third Generation Sequencing Long Read Data." G3: Genes|Genomes|Genetics 10, no. 4 (2020): 1193–96. http://dx.doi.org/10.1534/g3.119.400864.
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We propose LongQC as an easy and automated quality control tool for genomic datasets generated by third generation sequencing (TGS) technologies such as Oxford Nanopore technologies (ONT) and SMRT sequencing from Pacific Bioscience (PacBio). Key statistics were optimized for long read data, and LongQC covers all major TGS platforms. LongQC processes and visualizes those statistics automatically and quickly.
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Noone, J. Christopher, Karin Helmersen, Truls Michael Leegaard, Inge Skråmm, and Hege Vangstein Aamot. "Rapid Diagnostics of Orthopaedic-Implant-Associated Infections Using Nanopore Shotgun Metagenomic Sequencing on Tissue Biopsies." Microorganisms 9, no. 1 (2021): 97. http://dx.doi.org/10.3390/microorganisms9010097.
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Conventional culture-based diagnostics of orthopaedic-implant-associated infections (OIAIs) are arduous. Hence, the aim of this study was to evaluate a culture-independent, rapid nanopore-based diagnostic protocol with regard to (a) pathogen identification, (b) time to pathogen identification, and (c) identification of antimicrobial resistance (AMR). This prospective proof-of-concept study included soft tissue biopsies from 32 patients with OIAIs undergoing first revision surgery at Akershus University Hospital, Norway. The biopsies were divided into two segments. Nanopore shotgun metagenomic sequencing and pathogen and antimicrobial resistance gene identification using the EPI2ME analysis platform (Oxford Nanopore Technologies) were performed on one segment. Conventional culture-based diagnostics were performed on the other. Microbial identification matched in 23/32 OIAI patients (72%). Sequencing detected additional microbes in 9/32 patients. Pathogens detected by culturing were identified by sequencing within a median of 1 h of sequencing start [range 1–18 h]. Phenotypic AMR was explained by the detection of resistance genes in 11/23 patients (48%). Diagnostics of OIAIs using shotgun metagenomics sequencing are possible within 24 h from biopsy using nanopore technology. Sequencing outperformed culturing with respect to speed and pathogen detection where pathogens were at sufficient concentration, whereas culture-based methods had an advantage at lower pathogen concentrations. Sequencing-based AMR detection may not yet be a suitable replacement for culture-based antibiotic susceptibility testing.
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Chen, Zhiao, and Xianghuo He. "Application of third-generation sequencing in cancer research." Medical Review 1, no. 2 (2021): 150–71. http://dx.doi.org/10.1515/mr-2021-0013.
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Abstract In the past several years, nanopore sequencing technology from Oxford Nanopore Technologies (ONT) and single-molecule real-time (SMRT) sequencing technology from Pacific BioSciences (PacBio) have become available to researchers and are currently being tested for cancer research. These methods offer many advantages over most widely used high-throughput short-read sequencing approaches and allow the comprehensive analysis of transcriptomes by identifying full-length splice isoforms and several other posttranscriptional events. In addition, these platforms enable structural variation characterization at a previously unparalleled resolution and direct detection of epigenetic marks in native DNA and RNA. Here, we present a comprehensive summary of important applications of these technologies in cancer research, including the identification of complex structure variants, alternatively spliced isoforms, fusion transcript events, and exogenous RNA. Furthermore, we discuss the impact of the newly developed nanopore direct RNA sequencing (RNA-Seq) approach in advancing epitranscriptome research in cancer. Although the unique challenges still present for these new single-molecule long-read methods, they will unravel many aspects of cancer genome complexity in unprecedented ways and present an encouraging outlook for continued application in an increasing number of different cancer research settings.
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Istace, Benjamin, Caroline Belser, Cyril Falentin, et al. "Sequencing and Chromosome-Scale Assembly of Plant Genomes, Brassica rapa as a Use Case." Biology 10, no. 8 (2021): 732. http://dx.doi.org/10.3390/biology10080732.
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With the rise of long-read sequencers and long-range technologies, delivering high-quality plant genome assemblies is no longer reserved to large consortia. Not only sequencing techniques, but also computer algorithms have reached a point where the reconstruction of assemblies at the chromosome scale is now feasible at the laboratory scale. Current technologies, in particular long-range technologies, are numerous, and selecting the most promising one for the genome of interest is crucial to obtain optimal results. In this study, we resequenced the genome of the yellow sarson, Brassica rapa cv. Z1, using the Oxford Nanopore PromethION sequencer and assembled the sequenced data using current assemblers. To reconstruct complete chromosomes, we used and compared three long-range scaffolding techniques, optical mapping, Omni-C, and Pore-C sequencing libraries, commercialized by Bionano Genomics, Dovetail Genomics, and Oxford Nanopore Technologies, respectively, or a combination of the three, in order to evaluate the capability of each technology.
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de Siqueira, Guilherme Marcelino Viana, Felipe Marcelo Pereira-dos-Santos, Rafael Silva-Rocha, and María-Eugenia Guazzaroni. "Nanopore Sequencing Provides Rapid and Reliable Insight Into Microbial Profiles of Intensive Care Units." Frontiers in Public Health 9 (August 27, 2021). http://dx.doi.org/10.3389/fpubh.2021.710985.
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Fast and accurate identification of pathogens is an essential task in healthcare settings. Second-generation sequencing platforms such as Illumina have greatly expanded the capacity with which different organisms can be detected in hospital samples, and third-generation nanopore-driven sequencing devices such as Oxford Nanopore's minION have recently emerged as ideal sequencing platforms for routine healthcare surveillance due to their long-read capacity and high portability. Despite its great potential, protocols and analysis pipelines for nanopore sequencing are still being extensively validated. In this work, we assess the ability of nanopore sequencing to provide reliable community profiles based on 16S rRNA sequencing in comparison to traditional Illumina platforms using samples collected from Intensive Care Units of a hospital in Brazil. While our results demonstrate that lower throughputs may be a shortcoming of the method in more complex samples, we show that the use of single-use Flongle flowcells in nanopore sequencing runs can provide insightful information on the community composition in healthcare settings.
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Neumann, Don, Anireddy S. N. Reddy, and Asa Ben-Hur. "RODAN: a fully convolutional architecture for basecalling nanopore RNA sequencing data." BMC Bioinformatics 23, no. 1 (2022). http://dx.doi.org/10.1186/s12859-022-04686-y.
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Abstract Background Despite recent progress in basecalling of Oxford nanopore DNA sequencing data, its wide adoption is still being hampered by its relatively low accuracy compared to short read technologies. Furthermore, very little of the recent research was focused on basecalling of RNA data, which has different characteristics than its DNA counterpart. Results We fill this gap by benchmarking a fully convolutional deep learning basecalling architecture with improved performance compared to Oxford nanopore’s RNA basecallers. Availability The source code for our basecaller is available at: https://github.com/biodlab/RODAN.
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Silvestre-Ryan, Jordi, and Ian Holmes. "Pair consensus decoding improves accuracy of neural network basecallers for nanopore sequencing." Genome Biology 22, no. 1 (2021). http://dx.doi.org/10.1186/s13059-020-02255-1.
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AbstractWe develop a general computational approach for improving the accuracy of basecalling with Oxford Nanopore’s 1D2 and related sequencing protocols. Our software PoreOver (https://github.com/jordisr/poreover) finds the consensus of two neural networks by aligning their probability profiles, and is compatible with multiple nanopore basecallers. When applied to the recently-released Bonito basecaller, our method reduces the median sequencing error by more than half.
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Qi, Weihong, Andrea Colarusso, Miriam Olombrada, et al. "New insights on Pseudoalteromonas haloplanktis TAC125 genome organization and benchmarks of genome assembly applications using next and third generation sequencing technologies." Scientific Reports 9, no. 1 (2019). http://dx.doi.org/10.1038/s41598-019-52832-z.
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Abstract Pseudoalteromonas haloplanktis TAC125 is among the most commonly studied bacteria adapted to cold environments. Aside from its ecological relevance, P. haloplanktis has a potential use for biotechnological applications. Due to its importance, we decided to take advantage of next generation sequencing (Illumina) and third generation sequencing (PacBio and Oxford Nanopore) technologies to resequence its genome. The availability of a reference genome, obtained using whole genome shotgun sequencing, allowed us to study and compare the results obtained by the different technologies and draw useful conclusions for future de novo genome assembly projects. We found that assembly polishing using Illumina reads is needed to achieve a consensus accuracy over 99.9% when using Oxford Nanopore sequencing, but not in PacBio sequencing. However, the dependency of consensus accuracy on coverage is lower in Oxford Nanopore than in PacBio, suggesting that a cost-effective solution might be the use of low coverage Oxford Nanopore sequencing together with Illumina reads. Despite the differences in consensus accuracy, all sequencing technologies revealed the presence of a large plasmid, pMEGA, which was undiscovered until now. Among the most interesting features of pMEGA is the presence of a putative error-prone polymerase regulated through the SOS response. Aside from the characterization of the newly discovered plasmid, we confirmed the sequence of the small plasmid pMtBL and uncovered the presence of a potential partitioning system. Crucially, this study shows that the combination of next and third generation sequencing technologies give us an unprecedented opportunity to characterize our bacterial model organisms at a very detailed level.
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Sun, Kai, Yi Liu, Xin Zhou, et al. "Nanopore sequencing technology and its application in plant virus diagnostics." Frontiers in Microbiology 13 (July 25, 2022). http://dx.doi.org/10.3389/fmicb.2022.939666.
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Plant viruses threaten crop yield and quality; thus, efficient and accurate pathogen diagnostics are critical for crop disease management and control. Recent advances in sequencing technology have revolutionized plant virus research. Metagenomics sequencing technology, represented by next-generation sequencing (NGS), has greatly enhanced the development of virus diagnostics research because of its high sensitivity, high throughput and non-sequence dependence. However, NGS-based virus identification protocols are limited by their high cost, labor intensiveness, and bulky equipment. In recent years, Oxford Nanopore Technologies and advances in third-generation sequencing technology have enabled direct, real-time sequencing of long DNA or RNA reads. Oxford Nanopore Technologies exhibit versatility in plant virus detection through their portable sequencers and flexible data analyses, thus are wildly used in plant virus surveillance, identification of new viruses, viral genome assembly, and evolution research. In this review, we discuss the applications of nanopore sequencing in plant virus diagnostics, as well as their limitations.
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Liu, Yang, Wojciech Rosikiewicz, Ziwei Pan, et al. "DNA methylation-calling tools for Oxford Nanopore sequencing: a survey and human epigenome-wide evaluation." Genome Biology 22, no. 1 (2021). http://dx.doi.org/10.1186/s13059-021-02510-z.
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Abstract Background Nanopore long-read sequencing technology greatly expands the capacity of long-range, single-molecule DNA-modification detection. A growing number of analytical tools have been developed to detect DNA methylation from nanopore sequencing reads. Here, we assess the performance of different methylation-calling tools to provide a systematic evaluation to guide researchers performing human epigenome-wide studies. Results We compare seven analytic tools for detecting DNA methylation from nanopore long-read sequencing data generated from human natural DNA at a whole-genome scale. We evaluate the per-read and per-site performance of CpG methylation prediction across different genomic contexts, CpG site coverage, and computational resources consumed by each tool. The seven tools exhibit different performances across the evaluation criteria. We show that the methylation prediction at regions with discordant DNA methylation patterns, intergenic regions, low CG density regions, and repetitive regions show room for improvement across all tools. Furthermore, we demonstrate that 5hmC levels at least partly contribute to the discrepancy between bisulfite and nanopore sequencing. Lastly, we provide an online DNA methylation database (https://nanome.jax.org) to display the DNA methylation levels detected by nanopore sequencing and bisulfite sequencing data across different genomic contexts. Conclusions Our study is the first systematic benchmark of computational methods for detection of mammalian whole-genome DNA modifications in nanopore sequencing. We provide a broad foundation for cross-platform standardization and an evaluation of analytical tools designed for genome-scale modified base detection using nanopore sequencing.
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Riaz, Nasir, Preston Leung, Kirston Barton, et al. "Adaptation of Oxford Nanopore technology for hepatitis C whole genome sequencing and identification of within-host viral variants." BMC Genomics 22, no. 1 (2021). http://dx.doi.org/10.1186/s12864-021-07460-1.
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Abstract Background Hepatitis C (HCV) and many other RNA viruses exist as rapidly mutating quasi-species populations in a single infected host. High throughput characterization of full genome, within-host variants is still not possible despite advances in next generation sequencing. This limitation constrains viral genomic studies that depend on accurate identification of hemi-genome or whole genome, within-host variants, especially those occurring at low frequencies. With the advent of third generation long read sequencing technologies, including Oxford Nanopore Technology (ONT) and PacBio platforms, this problem is potentially surmountable. ONT is particularly attractive in this regard due to the portable nature of the MinION sequencer, which makes real-time sequencing in remote and resource-limited locations possible. However, this technology (termed here ‘nanopore sequencing’) has a comparatively high technical error rate. The present study aimed to assess the utility, accuracy and cost-effectiveness of nanopore sequencing for HCV genomes. We also introduce a new bioinformatics tool (Nano-Q) to differentiate within-host variants from nanopore sequencing. Results The Nanopore platform, when the coverage exceeded 300 reads, generated comparable consensus sequences to Illumina sequencing. Using HCV Envelope plasmids (~ 1800 nt) mixed in known proportions, the capacity of nanopore sequencing to reliably identify variants with an abundance as low as 0.1% was demonstrated, provided the autologous reference sequence was available to identify the matching reads. Successful pooling and nanopore sequencing of 52 samples from patients with HCV infection demonstrated its cost effectiveness (AUD$ 43 per sample with nanopore sequencing versus $100 with paired-end short read technology). The Nano-Q tool successfully separated between-host sequences, including those from the same subtype, by bulk sorting and phylogenetic clustering without an autologous reference sequence (using only a subtype-specific generic reference). The pipeline also identified within-host viral variants and their abundance when the parameters were appropriately adjusted. Conclusion Cost effective HCV whole genome sequencing and within-host variant identification without haplotype reconstruction are potential advantages of nanopore sequencing.
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Kerkhof, Lee J. "Is Oxford Nanopore sequencing ready for analyzing complex microbiomes?" FEMS Microbiology Ecology 97, no. 3 (2021). http://dx.doi.org/10.1093/femsec/fiab001.
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ABSTRACT This minireview will discuss the improvements in Oxford Nanopore (Oxford; sequencing technology that make the MinION a viable platform for microbial ecology studies. Specific issues being addressed are the increase in sequence accuracy from 65 to 96.5% during the last 5 years, the ability to obtain a quantifiable/predictive signal from the MinION with respect to target molecule abundance, simple-to-use GUI-based pathways for data analysis and the modest additional equipment needs for sequencing in the field. Coupling these recent improvements with the low capital costs for equipment and the reasonable per sample cost makes MinION sequencing an attractive option for virtually any laboratory.
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Goodwin, Sara, Robert Wappel, and W. Richard McCombie. "1D Genome Sequencing on the Oxford Nanopore MinION." Current Protocols in Human Genetics 94, no. 1 (2017). http://dx.doi.org/10.1002/cphg.39.
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Jain, Miten, Hugh E. Olsen, Benedict Paten, and Mark Akeson. "The Oxford Nanopore MinION: delivery of nanopore sequencing to the genomics community." Genome Biology 17, no. 1 (2016). http://dx.doi.org/10.1186/s13059-016-1103-0.
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Sultan, Madiha, and Anastassia Kanavarioti. "Nanopore device-based fingerprinting of RNA oligos and microRNAs enhanced with an Osmium tag." Scientific Reports 9, no. 1 (2019). http://dx.doi.org/10.1038/s41598-019-50459-8.
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Abstract Protein and solid-state nanopores are used for DNA/RNA sequencing as well as for single molecule analysis. We proposed that selective labeling/tagging may improve base-to-base resolution of nucleic acids via nanopores. We have explored one specific tag, the Osmium tetroxide 2,2′-bipyridine (OsBp), which conjugates to pyrimidines and leaves purines intact. Earlier reports using OsBp-tagged oligodeoxyribonucleotides demonstrated proof-of-principle during unassisted voltage-driven translocation via either alpha-Hemolysin or a solid-state nanopore. Here we extend this work to RNA oligos and a third nanopore by employing the MinION, a commercially available device from Oxford Nanopore Technologies (ONT). Conductance measurements demonstrate that the MinION visibly discriminates oligoriboadenylates with sequence A15PyA15, where Py is an OsBp-tagged pyrimidine. Such resolution rivals traditional chromatography, suggesting that nanopore devices could be exploited for the characterization of RNA oligos and microRNAs enhanced by selective labeling. The data also reveal marked discrimination between a single pyrimidine and two consecutive pyrimidines in OsBp-tagged AnPyAn and AnPyPyAn. This observation leads to the conjecture that the MinION/OsBp platform senses a 2-nucleotide sequence, in contrast to the reported 5-nucleotide sequence with native nucleic acids. Such improvement in sensing, enabled by the presence of OsBp, may enhance base-calling accuracy in enzyme-assisted DNA/RNA sequencing.
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Prall, Trent M., Emma K. Neumann, Julie A. Karl, et al. "Consistent ultra-long DNA sequencing with automated slow pipetting." BMC Genomics 22, no. 1 (2021). http://dx.doi.org/10.1186/s12864-021-07500-w.
Full textAbstract:
Abstract Background Oxford Nanopore Technologies’ instruments can sequence reads of great length. Long reads improve sequence assemblies by unambiguously spanning repetitive elements of the genome. Sequencing reads of significant length requires the preservation of long DNA template molecules through library preparation by pipetting reagents as slowly as possible to minimize shearing. This process is time-consuming and inconsistent at preserving read length as even small changes in volumetric flow rate can result in template shearing. Results We have designed SNAILS (Slow Nucleic Acid Instrument for Long Sequences), a 3D-printable instrument that automates slow pipetting of reagents used in long read library preparation for Oxford Nanopore sequencing. Across six sequencing libraries, SNAILS preserved more reads exceeding 100 kilobases in length and increased its libraries’ average read length over manual slow pipetting. Conclusions SNAILS is a low-cost, easily deployable solution for improving sequencing projects that require reads of significant length. By automating the slow pipetting of library preparation reagents, SNAILS increases the consistency and throughput of long read Nanopore sequencing.