Academic literature on the topic 'Oxford Nanopore sequencing'

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.

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.

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.

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.

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.

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.

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.