A nanopore-based devices are extremely sensitive analytical techniques, which uses the electrophoretic translocation of molecules in solution through a nano-scale pores. The nanopores, which mimic the functions of natural ion channels, seems to be the promissing tool for future fast and low-cost DNA sequencing. However, some difficulties in generating usable sequence data have to be solved. In this article the nanopores were reviewed. In the first part the development of nanopore technique was described and the ubiquitous presence of nanopores in living cells was highlighted. Next, the most important part of the principles of nanopore analysis was described, and the knowledges about biological and solid-state nanopores were summarized. Also the pros and cons of both kinds of nanopores and different approaches designed to circumvent the issues were mentioned.
- Keywords
- biopóry, solid-state nanopóry,
- MeSH
- Hemolysin Proteins MeSH
- Nanopores * MeSH
- Sequence Analysis, DNA * methods instrumentation MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
Nanofluidics is becoming an extensively developing technique in the field of bioanalytical chemistry. Nanoscale hole embed in an insulating membrane is employed in a vast variety of sensing platforms and applications. Although, biological nanopores have several attractive characteristics, in this paper, we focused on the solid-state nanopores due to their advantages as high stability, possibility of diameter control, and ease of surface functionalizing. A detection method, based on the translocation of analyzed molecules through nanochannels under applied voltage bias and resistive pulse sensing, is well established. Nevertheless, it seems that the new detection methods like measuring of transverse electron tunneling using nanogap electrodes or optical detection can offer significant additional advantages. The aim of this review is not to cite all related articles, but highlight the steps, which in our opinion, meant important progresses in solid-state nanopore analysis.
Recent medical applications have specific requirements on materials and Nitinol can fulfill them due to its exceptional characteristics, which can be further improved by modifications of the material surface. Various surface nanostructuring methods are utilized to enhance characteristics of oxide layer, which naturally develops on the Nitinol surface, leading to improved biocompatibility and corrosion resistance. This review is focused on studies investigating the behavior of various cell types on surface nanotubes and ordered nanopores prepared by anodic oxidation, a technique allowing fabrication of nanostructures with defined parameters. Results showed that certain dimensions of nanotubes positively affect adhesion and viability of osteoblasts and endothelial cells on the surface, contrary to negative effect on smooth muscle cells, both required by the medical applications. Furthermore, increased antibacterial effect correlated with the nanostructure topography and release rates of Ni ions.
Long-read technologies hold the promise to obtain more complete genome assemblies and to make them easier. Coupled with long-range technologies, they can reveal the architecture of complex regions, like centromeres or rDNA clusters. These technologies also make it possible to know the complete organization of chromosomes, which remained complicated before even when using genetic maps. However, generating a gapless and telomere-to-telomere assembly is still not trivial, and requires a combination of several technologies and the choice of suitable software. Here, we report a chromosome-scale assembly of a banana genome (Musa acuminata) generated using Oxford Nanopore long-reads. We generated a genome coverage of 177X from a single PromethION flowcell with near 17X with reads longer than 75 kbp. From the 11 chromosomes, 5 were entirely reconstructed in a single contig from telomere to telomere, revealing for the first time the content of complex regions like centromeres or clusters of paralogous genes.
Nanoporous surfaces are promising for label-free electrochemical biosensing. We formed nanopores directly on the electrode surface by means of assembling a dense layer of nonconductive nanoparticles. In our model affinity biosensor, covalent attachment of albumin protein on top of 40 nm polystyrene nanoparticles represented a capture of an analyte, resulting in blockage of the nanopores. Different bulk concentrations of the ferro/ferricyanide redox pair were probed by Faradaic electrochemical impedance spectroscopy and fast chronoamperometry. The character of the redox probe permeation towards the electrode surface differed in dependence on its concentration. These data were compared with the theoretical behavior of the free diffusion according to the Cottrell equation. Both the bulk concentration of the redox probe and the timescale of the experiment affected the performance of the electrochemical detection, demonstrating the importance of controlling these parameters in immunosensing applications.
Amplification of monomer sequences into long contiguous arrays is the main feature distinguishing satellite DNA from other tandem repeats, yet it is also the main obstacle in its investigation because these arrays are in principle difficult to assemble. Here we explore an alternative, assembly-free approach that utilizes ultra-long Oxford Nanopore reads to infer the length distribution of satellite repeat arrays, their association with other repeats and the prevailing sequence periodicities. Using the satellite DNA-rich legume plant Lathyrus sativus as a model, we demonstrated this approach by analyzing 11 major satellite repeats using a set of nanopore reads ranging from 30 to over 200 kb in length and representing 0.73× genome coverage. We found surprising differences between the analyzed repeats because only two of them were predominantly organized in long arrays typical for satellite DNA. The remaining nine satellites were found to be derived from short tandem arrays located within LTR-retrotransposons that occasionally expanded in length. While the corresponding LTR-retrotransposons were dispersed across the genome, this array expansion occurred mainly in the primary constrictions of the L. sativus chromosomes, which suggests that these genome regions are favourable for satellite DNA accumulation.
- MeSH
- Centromere MeSH
- Chromosomes, Plant MeSH
- DNA, Plant genetics MeSH
- Gene Frequency * MeSH
- Genome, Plant MeSH
- Heterochromatin MeSH
- Lathyrus genetics MeSH
- Evolution, Molecular MeSH
- Nanopores * MeSH
- Retroelements * MeSH
- DNA, Satellite * MeSH
- Tandem Repeat Sequences * MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
In our study we present an overview of the use of Oxford Nanopore Technologies (ONT) sequencing technology on the background of Enteric fever. Unlike traditional methods (e.g., qPCR, serological tests), the nanopore sequencing technology enables virtually real-time data generation and highly accurate pathogen identification and characterization. Blood cultures were obtained from a 48-year-old female patient suffering from a high fever, headache and diarrhea. Nevertheless, both the initial serological tests and stool culture appeared to be negative. Therefore, the bacterial isolate from blood culture was used for nanopore sequencing (ONT). This technique in combination with subsequent bioinformatic analyses allowed for prompt identification of the disease-causative agent as Salmonella enterica subsp. enterica serovar Paratyphi A. The National Reference Laboratory for Salmonella (NIPH) independently reported this isolate also as serovar Paratyphi A on the basis of results of biochemical and agglutination tests. Therefore, our results are in concordance with certified standards. Furthermore, the data enabled us to assess some basic questions concerning the comparative genomics, i.e., to describe whether the isolated strain differs from the formerly published ones or not. Quite surprisingly, these results indicate that we have detected a novel and so far, unknown variety of this bacteria.
- MeSH
- Typhoid Fever * MeSH
- Middle Aged MeSH
- Humans MeSH
- Nanopore Sequencing * MeSH
- Salmonella paratyphi A genetics MeSH
- Salmonella MeSH
- Check Tag
- Middle Aged MeSH
- Humans MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Case Reports MeSH
The recent human Monkeypox outbreak underlined the importance of studying basic biology of orthopoxviruses. However, the transcriptome of its causative agent has not been investigated before neither with short-, nor with long-read sequencing approaches. This Oxford Nanopore long-read RNA-Sequencing dataset fills this gap. It will enable the in-depth characterization of the transcriptomic architecture of the monkeypox virus, and may even make possible to annotate novel host transcripts. Moreover, our direct cDNA and native RNA sequencing reads will allow the estimation of gene expression changes of both the virus and the host cells during the infection. Overall, our study will lead to a deeper understanding of the alterations caused by the viral infection on a transcriptome level.
- MeSH
- DNA, Complementary MeSH
- Humans MeSH
- Nanopore Sequencing * MeSH
- Mpox, Monkeypox * MeSH
- Gene Expression Profiling MeSH
- Transcriptome MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Dataset MeSH
In order to characterize unauthorized genetically modified petunia, an integrated strategy has been applied here on several suspected petunia samples from the European market. More precisely, DNA fragments of interest were produced by DNA walking anchored on key targets, earlier detected by real-time PCR screening analysis, to be subsequently sequenced using the MinION platform from Oxford Nanopore Technologies. This way, the presence of genetically modified petunia was demonstrated via the characterization of their transgene flanking regions as well as unnatural associations of elements from their transgenic cassette.
- MeSH
- European Union MeSH
- Plants, Genetically Modified MeSH
- Real-Time Polymerase Chain Reaction MeSH
- Nanopores MeSH
- Petunia classification genetics MeSH
- Chromosome Walking methods MeSH
- Sequence Analysis, DNA instrumentation MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Czech Republic MeSH
- Hungary MeSH
Recently, nanopore sequencing has come to the fore as library preparation is rapid and simple, sequencing can be done almost anywhere, and longer reads are obtained than with next-generation sequencing. The main bottleneck still lies in data postprocessing which consists of basecalling, genome assembly, and localizing significant sequences, which is time consuming and computationally demanding, thus prolonging delivery of crucial results for clinical practice. Here, we present a neural network-based method capable of detecting and classifying specific genomic regions already in raw nanopore signals-squiggles. Therefore, the basecalling process can be omitted entirely as the raw signals of significant genes, or intergenic regions can be directly analyzed, or if the nucleotide sequences are required, the identified squiggles can be basecalled, preferably to others. The proposed neural network could be included directly in the sequencing run, allowing real-time squiggle processing.
- Publication type
- Journal Article MeSH
