Identification of four single-stranded DNA homopolymers with a solid-state nanopore in alkaline CsCl solution

Goto, Yusuke; Yanagi, Itaru; Matsui, Kazuma; Yokoi, Takeshi; Takeda, Kenta

doi:10.1039/c8nr04238a

Cited by 25 publications

(25 citation statements)

References 43 publications

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“…Nanopore-based detection methods are flexible for incorporating additional options, such as recording epigenetic modifications (11,63) and primary structure of RNA (64), proteins (65), and polysaccharides (66). Alternative nanopore-based DNA sequencing methods are also evolving (67). The potential of different nanopores to record various biomolecules indicates great promise of nanopore-based molecular diagnostics in the future.…”

Section: Discussionmentioning

confidence: 99%

Relative Performance of MinION (Oxford Nanopore Technologies) versus Sequel (Pacific Biosciences) Third-Generation Sequencing Instruments in Identification of Agricultural and Forest Fungal Pathogens

Loit

Adamson

Bahram

et al. 2019

Appl Environ Microbiol

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Culture-based molecular identification methods have revolutionized detection of pathogens, yet these methods are slow and may yield inconclusive results from environmental materials. The second-generation sequencing tools have much-improved precision and sensitivity of detection, but these analyses are costly and may take several days to months. Of the third-generation sequencing techniques, the portable MinION device (Oxford Nanopore Technologies) has received much attention because of its small size and possibility of rapid analysis at reasonable cost. Here, we compare the relative performances of two third-generation sequencing instruments, MinION and Sequel (Pacific Biosciences), in identification and diagnostics of fungal and oomycete pathogens from conifer (Pinaceae) needles and potato (Solanum tuberosum) leaves and tubers. We demonstrate that the Sequel instrument is efficient for metabarcoding of complex samples, whereas MinION is not suited for this purpose due to a high error rate and multiple biases. However, we find that MinION can be utilized for rapid and accurate identification of dominant pathogenic organisms and other associated organisms from plant tissues following both amplicon-based and PCR-free metagenomics approaches. Using the metagenomics approach with shortened DNA extraction and incubation times, we performed the entire MinION workflow, from sample preparation through DNA extraction, sequencing, bioinformatics, and interpretation, in 2.5 h. We advocate the use of MinION for rapid diagnostics of pathogens and potentially other organisms, but care needs to be taken to control or account for multiple potential technical biases. IMPORTANCE Microbial pathogens cause enormous losses to agriculture and forestry, but current combined culturing- and molecular identification-based detection methods are too slow for rapid identification and application of countermeasures. Here, we develop new and rapid protocols for Oxford Nanopore MinION-based third-generation diagnostics of plant pathogens that greatly improve the speed of diagnostics. However, due to high error rate and technical biases in MinION, the Pacific BioSciences Sequel platform is more useful for in-depth amplicon-based biodiversity monitoring (metabarcoding) from complex environmental samples.

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Section: Discussionmentioning

confidence: 99%

Relative Performance of MinION (Oxford Nanopore Technologies) versus Sequel (Pacific Biosciences) Third-Generation Sequencing Instruments in Identification of Agricultural and Forest Fungal Pathogens

Loit

Adamson

Bahram

et al. 2019

Appl Environ Microbiol

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“…20,21 Similar studies used the same approach to detect if Gqs are folded with silicon nitride nanopores. 22,23 Here, detection is based on the physical interaction of Gq with the nanopore, again affecting Gq formation. When monitoring competition of Gq DNA and Watson-Crick DNA, [24][25][26][27] fluorescent labeling might affect Gq formation and hence the ratio between Gq and duplex.…”

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confidence: 99%

Monitoring G-Quadruplex Formation with DNA Carriers and Solid-State Nanopores

et al. 2019

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G-quadruplexes (Gq) are guanine-rich DNA structures formed by single-stranded DNA. They are of paramount significance to gene expression regulation, but also drug targets for cancer and human viruses. Current ensemble and single-molecule methods require fluorescent labels, which can affect Gq folding kinetics. Here we introduce, a single-molecule Gq nanopore assay (smGNA) to detect Gqs and kinetics of Gq formation. We use ~5 nm solid-state nanopores to detect various Gq structural variants attached to designed DNA carriers. Gqs can be identified by localizing their positions along designed DNA carriers establishing smGNA as a tool for Gq mapping. In addition, smGNA allows for discrimination of (un-)folded Gq structures, provides insights into singlemolecule kinetics of G-quadruplex folding, and probes quadruplex-to-duplex structural transitions. smGNA can elucidate the formation of G-quadruplexes at the single-molecule level without labelling and has potential implications on the study of these structures both in single-stranded DNA and in genomic samples..

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“…Also, SSN have been introduced for the investigation of secondary structures of DNA owing to their remarkable thermal and mechanical stability, as well as extraordinary surface, shape and size versatility compared with their biological counterparts. For instance, Long's group has utilized silicon nitride nanopores for ready determination of i-motif structure as well as DNA dynamics; [37][38][39] the employment of tiny SSN for sieving out G4 structure was also reported; 40,41 Jiang's group have observed the G4 DNA conformational switching in a responsive nanochannel; 42,43 imotif folding dynamics 44 and G4 formation with DNA carrier in SSN was also reported. 45 However, inter-and intramolecular and consecutive G4 have not been detected with SSN since interand intramolecular DNA with distinct base permutation and stability may perform differently in organisms, and consecutive G4 units are the dominating factor for diseases.…”

Section: Introductionmentioning

confidence: 99%

Label-free single-molecule identification of telomere G-quadruplexes with a solid-state nanopore sensor

et al. 2020

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Identification of four single-stranded DNA homopolymers with a solid-state nanopore in alkaline CsCl solution

Abstract: Single-stranded DNA homopolymers of each nucleotide were statistically identified according to their blockade currents obtained with the same single 5 nm-thick SiN nanopore and an alkaline CsCl solution.

Cited by 25 publications

References 43 publications

Relative Performance of MinION (Oxford Nanopore Technologies) versus Sequel (Pacific Biosciences) Third-Generation Sequencing Instruments in Identification of Agricultural and Forest Fungal Pathogens

Relative Performance of MinION (Oxford Nanopore Technologies) versus Sequel (Pacific Biosciences) Third-Generation Sequencing Instruments in Identification of Agricultural and Forest Fungal Pathogens

Monitoring G-Quadruplex Formation with DNA Carriers and Solid-State Nanopores

Label-free single-molecule identification of telomere G-quadruplexes with a solid-state nanopore sensor

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