33 results about "Nanopore sequencing" patented technology

The application relates to a sarcoma fusion gene nanopore sequencing primer set and application thereof, wherein the primer set comprises five pairs of specific primers (Primer 1-5) with fixed sequences, each primer follows the design principle of 18-25 bp in length, 40%-60% in GC content and strict matching of a target sequence at a 3' end, and the length of an amplification product is 300-800 bp, which is suitable for nanopore sequencing. Through qPCR verification, the primer set has single-peak melting curves, close-to-100% amplification efficiency, strong specificity and excellent amplification performance.

Provided are a method for constructing a nucleic acid library and a nanopore sequencing method. The method for constructing a nucleic acid library comprises: processing ends of a double-stranded target nucleic acid to obtain an end-processed product, the end-processed product containing a first end and a second end, and the first end and the second end each containing a terminal overhang sequence; and directionally ligating a first adaptor and a second adaptor to the first end and the second end of the end-processed product, respectively, to obtain a nucleic acid library, the first adaptor being different from the second adaptor. The method is capable of addressing the problems of a low proportion of effective double-stranded sequencing sequences and low efficiency in double-stranded sequencing libraries constructed in the existing technology, and is applicable to the field of nucleic acid library construction.

Some embodiments of the methods and compositions provided herein relate to nanopore sequencing of a target polypeptide. In some embodiments, a sequencing substrate is prepared by Edman degradation from a target polypeptide immobilized on a substrate. De novo sequencing is performed with the sequence substrate and a nanopore embedded in a membrane.

This disclosure relates to novel nanoporous proteins and their applications in sequencing, belonging to the field of nanoporous technology. The nanoporous proteins provided in this disclosure enable the observation of resolvable DNA perforation signals, can be used for nanoporous sequencing, and enrich the variety of nanoporous proteins. The mutant proteins obtained after modification show significantly increased expression levels, a marked improvement in spontaneous blockage, and solve the problem of difficulty in assembling into bilayer membranes. They exhibit uniform electrophysiological properties, stable pore currents, relatively high sequencing accuracy of DNA perforation signals, and outstanding sequencing current properties.

This invention relates to the fields of molecular diagnostics and gene sequencing technology, specifically to a complete solution for high-precision genotyping of the human Lewis blood group system. The invention provides primer pairs for specifically amplifying long fragments of the Lewis blood group coding genes FUT2 and FUT3, and an optimized single amplification system; a library construction method adapted for nanopore sequencing; and a Lewis (FUT2, FUT3) genotyping method based on long-read data for haplotype analysis. This invention overcomes the detection limitations of conventional techniques, simultaneously acquiring the complete sequence and phase information of key regions of the FUT2 and FUT3 genes through a single amplification system, achieving accurate genotyping of common Lewis blood group phenotypes (Le(a+b−), Le(a−b+), Le(a−b−)) and effective identification of rare variants such as weakly expressed and deleted variants.

This invention relates to the fields of molecular biology and forensic identification, specifically to a method for bird species identification based on multi-gene co-amplification and nanopore sequencing. The method involves extracting genomic DNA from avian biological samples; using the extracted DNA as a template, amplification is performed using four independent PCR primer pools that specifically target the avian mitochondrial cytochrome C oxidase subunit I gene, cytochrome B gene, 12S rRNA gene, and 16S rRNA gene; after purification of the amplification products, a nanopore sequencing library is constructed and sequenced; finally, bioinformatics analysis, including read length clustering, draft consensus sequence generation, and polishing, is performed on the raw sequencing data to obtain consensus sequences for each target gene. Species identification is then completed by comparing the sequence with a avian sequence database. This method effectively solves the problem that existing technologies cannot simultaneously meet the practical needs of large-scale, rapid, and high-success-rate identification of avian biological samples.

This invention discloses a primer combination for maize genotyping suitable for nanopore sequencing platforms and its application. The primer combination includes at least one of 11 pairs of ONT primers, the sequences of which are shown in SEQ ID NO: 1-SEQ ID NO: 22. The primers of this invention can specifically amplify target regions rich in genetic variation in the maize genome. The amplified PCR products are mixed and then sequenced using nanopore sequencing. By analyzing the species attributes and genome alignment positions of the sequencing sequences, combining experimental data from the test samples to calculate the targeting rate of the amplified products, and associating them with known genomic information, the purity and identification accuracy of maize seeds can be determined. This primer combination combines maize material specificity with population compatibility, enabling economical and efficient genotyping of maize inbred lines and hybrid populations. It can also be applied to verify the authenticity of maize varieties, analyze the genetic background of breeding materials, and screen for genotyping.

The application provides a polypeptide and protein sequencing method, a kit and application. The polypeptide sequencing method comprises the following steps: S1, attaching a target polypeptide to a nanopore; S2, introducing a recognition molecule specifically recognizing a terminal amino acid of the target polypeptide into a nanopore sequencing system; under the action of an electric field, the detection of the terminal amino acid of the target polypeptide is realized by utilizing the electric signal change generated in the binding and dissociation process of the recognition molecule and the target polypeptide. By utilizing the unique electric signal change generated in the binding and dissociation process of the recognition molecule and the terminal amino acid of the target polypeptide, the detection of a single amino acid is realized.

The application discloses a nanopore base recognition method based on Libtorch and C++, which comprises the following steps: obtaining nanopore sequencing data of a target sample and preprocessing the data, wherein the preprocessing comprises normalization and overlapping segmentation; delivering the preprocessed data to a model inference module based on a convolutional neural network and a bidirectional long short-term memory network in an asynchronous mode, performing GPU inference on each time step, and outputting base sequence weights of each time step; decoding the base sequence weights of each time step output by the model inference module through a CTC decoding module based on a greedy search and a prefix beam search; reassembling the decoded result; and outputting the result. The model inference module is optimized, efficient data loading and multi-GPU inference are realized, the inference time is significantly shortened, the memory occupation is effectively reduced, the memory consumption is reduced while the high accuracy is maintained, and the running speed and throughput of base recognition are accelerated.

The application discloses a pathogen DNA detection optimization method based on deep learning, and belongs to the technical field of bioinformatics and deep learning. The first electric signal data and the first DNA sequence data of nanopore sequencing are acquired; the first electric signal feature vector is output through an electric signal feature enhancement extraction network; the first sequence feature vector is output through a sequence feature coding network; the first fusion feature vector is obtained through element-by-element weighted summation of a feature layer fusion network; the first pathogen existence probability value is output through a first pathogen classification network and compared with the first filtering threshold value 0.15 for rapid screening; the second fusion feature vector is obtained through a time sequence context coding network and a cross attention fusion network for the sample; the second pathogen existence probability value is output through a second pathogen classification network; and the detection is completed according to the application scene parameter selected 0.35, 0.50 or 0.65 determination threshold value. The application realizes the cooperative utilization of electric signal and sequence features and the dynamic adjustment of the detection strategy.

This invention belongs to the field of biological detection technology, and particularly relates to a reproductive tract HPV-mRNA-E6 / E7 detection system and method based on nanopore sequencing. The system includes a sample pretreatment module, an RNA extraction and purification module, a reverse transcription module, a nanopore sequencing module, and a bioinformatics analysis module. The method includes the following steps: (1) reproductive tract sample collection and pretreatment; (2) extraction and purification of HPV-mRNA in the sample; (3) reverse transcription of the purified HPV-mRNA into cDNA; (4) nanopore sequencing of the cDNA to obtain raw sequencing data; (5) filtering, comparing, and annotating the sequencing data through the bioinformatics analysis module to achieve HPV type identification and quantitative analysis of E6 / E7 gene expression levels. This invention utilizes the advantages of nanopore sequencing, such as long read length and real-time detection, combined with the specificity of E6 / E7 mRNA as a biomarker of active infection. It can directly detect latent and active HPV infection, improve detection sensitivity, accurately distinguish infection types, and shorten the detection cycle. It enables rapid, accurate, and high-throughput detection of active HPV infection in the genital tract, providing reliable technical support for early screening, disease monitoring, and prognostic assessment of HPV-related diseases such as cervical cancer.

This application provides a membrane structure solvent for nanopore sequencing, its preparation method, and its application. The membrane structure solvent for nanopore sequencing has the membrane structure located within a crosslinkable solvent; the crosslinkable solvent self-crosslinks to form a network structure, encapsulating the membrane structure. This application crosslinks the solvent of the membrane-forming solution to form a network structure, thereby confining the formed membrane structure within this network structure and ensuring its stability, preventing membrane rupture and other issues.

The invention provides methods of selecting polynucleotides based on their size in globular form. The polynucleotides may be treated with a condensation medium. e.g. comprising polyethylene glycol. e.g. PEG8000, and salt, in order to adopt a globular form. The method may employing filtering in order to retain the larger nucleic acids and make the larger nucleic acids available for sequencing applications, such as nanopore sequencing. The invention also relates to kits for selecting polynucleotides.

This disclosure provides a method, apparatus, and electronic device for indexing and identifying gene sequencing sequences. The method includes: acquiring multiple index signal templates; acquiring nanopore sequencing time-series electrical signal data of the gene sequencing sequence to be identified; extracting index signal data from the nanopore sequencing time-series electrical signal data; calculating the distance fraction between the index signal data and each index signal template; determining a target index signal template from the multiple index signal templates based on the distance fraction; and determining the target index sequence corresponding to the target index signal template as the indexing and identification result of the gene sequencing sequence to be identified. This method can improve the accuracy of indexing and identifying gene sequencing sequences.

Disclosed in the present disclosure are a molecular detection unit and chip, a preparation method, and a use. The molecular detection unit comprises: a substrate, a first structural layer, second structural layers, sensing electrodes, third structural layers, first buffer solution flow channels, sample flow channels, single-hole liquid storage cavities, liquid resistor flow channels, and second buffer solution flow channels; the liquid resistor flow channels and the single-hole liquid storage cavities are provided in the first structural layer; the bottom of the corresponding single-hole liquid storage cavity is communicated with one end of the corresponding liquid resistor flow channel; the sample flow channels are arranged in the second structural layers and are communicated with the single-hole liquid storage cavities; the first buffer solution flow channels are provided in the third structural layers; the bottom of the corresponding second buffer solution flow channel is communicated with the other end of the corresponding liquid resistor flow channel; the top of the corresponding second buffer solution flow channel is communicated with the corresponding first buffer solution flow channel; and the corresponding sensing electrode is arranged at the position where the corresponding single-hole liquid storage cavity is communicated with the corresponding liquid resistor flow channel. The application of the present disclosure can improve the arrangement density of nanopore devices per unit area, so that the chip manufacturing efficiency can be improved, thereby improving the throughput of nanopore sequencing devices.

This application provides a solvent carrier for nanopore sequencing, its preparation method, and its application. The solvent carrier for nanopore sequencing comprises a nanopore sequencing unit containing microwells, the upper end of which is covered with a nonpolar film-forming solution. The nonpolar film-forming solution includes a copolymer membrane and a nonpolar solvent. The end groups of the nonpolar solvent contain unsaturated bonds I, and the surface of the upper end of the microwell contains unsaturated bonds II. The nonpolar solvent is cross-linked with the surface of the upper end of the microwell to form the solvent carrier. This application cross-links the solvent of the film-forming solution to form a network structure, thereby allowing the formed membrane structure to be confined within this network structure and to exist stably, avoiding membrane rupture and other issues.

The application provides an amplification primer and method for a whole genome of chicken Marek's disease virus. The detection primer set of the chicken Marek's disease virus comprises 206 pairs of amplification primers, and specific sequences are shown as SEQ ID NO. 1-412. The detection primer set provided by the application can realize relatively uniform coverage of the chicken Marek's disease virus, and in nanopore sequencing, 100% area coverage of the genome is realized. The sequencing fragment preparation method for the whole genome of the chicken Marek's disease virus provided by the application is simple and easy to operate, has good amplification effect, and nanopore sequencing has the advantage of real-time analysis during sequencing, so that the detection time can be greatly shortened, and the infection of the chicken Marek's disease virus can be differentially diagnosed in 15h at most, meanwhile, the whole genome sequence thereof is obtained, and the obtained genome sequence can provide a scientific basis for virus tracing, pathogen variation tracking, new strain identification and early warning and the like.

The application provides a micro-well surface for nanopore sequencing and a preparation method and application thereof. The processing method comprises that the micro-well is in a concave character structure with high sides and a concave middle part, an electrode is arranged at the concave part, the micro-well is prepared by using photoresist, and the method comprises the following steps: (1) covering a material containing solidified glue to the micro-well; an electroplating layer is deposited on the electrode; (2) adding a crosslinking agent to the micro-well to perform a chemical reaction; and (3) cleaning the micro-well to remove the un-solidified glue. By processing the micro-well surface, the contact angle of the micro-well surface is changed, the top of the micro-well can be fully immersed in a film forming liquid, the film is better and faster to form, and the stability of the film is better; meanwhile, by modifying the electrode surface and selecting an initiator, the electrode can be avoided to be covered, and the possibility that sequencing cannot be performed is reduced.

This invention provides a nanopore sequencing method, terminal device, and storage medium. Utilizing the excellent mechanical and chemical stability of solid-state sub-nanopores, anionic surfactant SDS is used to denature proteins. SDS disrupts the higher-order structure of proteins while simultaneously coating their surfaces with a uniform negative charge, achieving linear translocation of full-length proteins under electric field-driven conditions. A self-supervised learning model, NanoSSL, is employed. This model is pre-trained using masked autoencoders and attention mechanisms, and fine-tuned using a small amount of labeled data. This model can specifically identify translocation events of trained target proteins from the fluctuation patterns of ion current signals in mixed samples. Combined with a K-means clustering algorithm based on dynamic time warping optimization, the translocation direction of single molecules can be further determined, constructing sequence-specific consensus current trajectories, thereby simultaneously completing the identification of target proteins and the extraction of sequence information in a mixed background.

This invention discloses an optimized method for pathogen DNA detection based on deep learning, belonging to the interdisciplinary field of bioinformatics and deep learning. The invention acquires first electrical signal data and first DNA sequence data from nanopore sequencing; outputs a first electrical signal feature vector through an electrical signal feature enhancement extraction network; outputs a first sequence feature vector through a sequence feature encoding network; obtains a first fused feature vector by element-wise weighted summation through a feature layer fusion network; outputs the probability value of the presence of a first pathogen through a first pathogen classification network and compares it with a first filtering threshold of 0.15 for rapid screening; obtains a second fused feature vector for the samples using a temporal context encoding network and a cross-attention fusion network; outputs the probability value of the presence of a second pathogen through a second pathogen classification network; and completes the detection by selecting a judgment threshold of 0.35, 0.50, or 0.65 according to the application scenario parameters. This invention achieves the synergistic utilization of electrical signals and sequence features and dynamic adjustment of the detection strategy.

This invention discloses a nanopore sequencing method for detecting full-length 16S rRNA amplicon in human semen samples. The method includes sample pretreatment and DNA extraction; nested PCR amplification using outer primers 16S-27F / 1492R and inner primers 16S-340F / 1390R to obtain target long amplicon lengths of 1.0kb to 1.1kb; construction of a nanopore library and single-molecule long-read sequencing; deep filtering of the raw data with a quality score Q≥20 and read lengths between 1000bp and 1700bp; and finally, feature clustering and taxonomic annotation. This invention addresses the characteristics of low biomass and high host DNA background in human semen samples by employing innovative specific nested primer design and customized data cleaning strategies. This effectively avoids non-specific interference and significantly improves the classification resolution and data fidelity of the semen microbiome at the genus and species levels, providing a precise detection method for analyzing the reproductive microecology of infertile men.

This invention discloses a method for detecting reverse transcriptase activity. The method includes: S1. mixing the reverse transcriptase to be tested, RNA template, reverse transcription primers, and dNTPs to perform a reverse transcription reaction to obtain a reverse transcription product; S2. preparing a nanopore sequencing library using the reverse transcription product; S3. sequencing the nanopore sequencing library using nanopores to obtain sequencing data; S4. determining the activity of the reverse transcriptase to be tested using the sequencing data. This method can accurately and comprehensively evaluate the transcriptional ability of different reverse transcriptases for RNA of various lengths, thus providing precise and detailed data support for the evaluation and optimization of reverse transcriptases.

Some embodiments of the methods and compositions provided herein relate to nanopore sequencing of a target polypeptide. In some embodiments, a nanopore is embedded in a membrane having a cis and trans surface. Some embodiments include translocating an end of the target polypeptide through the nanopore, measuring a signal generated from the target polypeptide in the nanopore, and removing a terminal residue of the target polypeptide from the target polypeptide with an Edmanase. In some embodiments, a trans solution comprises a blocker capable of attaching to the N-terminal end of the target polypeptide. In some embodiments, a cis solution comprises a leader composition attached to a lock oligonucleotide capable of forming a hairpin structure.

This invention relates to a nanopore sequencing detection method for sarcoma EWS-ETS fusion genes based on single-end primers. It designs an amplification strategy combining a 3' gene-specific single primer with a universal primer, leveraging the long read advantage of nanopore sequencing. A complete detection workflow is constructed, encompassing sample pretreatment, nucleic acid extraction, single-end primer amplification, nanopore library construction and sequencing, and fusion gene identification. The clinical application pathway is also clearly defined. This invention eliminates the need for prior knowledge of the fusion partner gene, significantly improving the detection sensitivity of rare EWS-ETS fusion genes. It offers high breakpoint localization accuracy and standardized procedures, enabling efficient screening and confirmation of sarcoma fusion genes, providing reliable technical support for accurate diagnosis, prognostic assessment, and targeted therapy of sarcoma.

This invention relates to the fields of molecular diagnostics and gene sequencing technology, specifically to a primer set, amplification system, amplification method, library construction method, and sequencing method for Kidd blood group system genotyping amplification based on long-read nanopore sequencing. This invention provides primers and an optimized amplification system for specifically amplifying long fragments of the Kidd blood group encoding gene SLC14A1; a library construction method adapted for nanopore sequencing; and a SLC14A1 genotyping method based on long-read data for haplotype analysis. This invention overcomes the limitations of conventional techniques that only detect single SNPs, enabling the simultaneous acquisition of the complete sequence and phase information of key regions of the SLC14A1 gene, achieving accurate genotyping of JKA / JKB and effective identification of rare variants such as weakly expressed and deleted variants. This invention provides a powerful molecular tool for clinical transfusion safety assurance, rare blood group bank construction, and genetic research.

The application relates to the field of animal bacteriology and provides a Streptococcus suis serotype rapid typing method based on nanopore sequencing. The detection primer group of the Streptococcus suis serotype comprises 64 amplification primers, and the specific sequences are shown as SEQ ID NO. 1-64. The detection primer group provided by the application can realize rapid typing of the Streptococcus suis serotype. The sequencing method for the rapid typing of the Streptococcus suis serotype provided by the application is simple and easy to operate, has good amplification effect, and the nanopore sequencing has the advantage of real-time analysis during sequencing, can greatly shorten the detection time, can quickly diagnose and differentiate the infection of the Streptococcus suis serotype, and can provide a scientific basis for disease tracing, variation tracking, new strain identification and early warning.

The present disclosure relates to methods of directly measuring absolute length of individual telomeres without any PCR amplification or fluorescent probe hybridization. This method can be performed in a high-throughput 96-well plate format. The disclosure provides a method of processing genomic DNA samples comprising telomeric DNA to release the telomeric DNA from the non-telomeric genomic DNA that has one end tagged with a telomere adapter and the other end ligated with a DNA barcode adapter. The telomeric DNA samples are pooled and purified prior to long-read sequencing in certain embodiments, for example via a nanopore.

Provided are a nucleic acid-polypeptide-nucleic acid ternary complex and application thereof in polypeptide nanopore sequencing. The ternary complex has the following structural formula: single-stranded nucleic acid 1-polypeptide-single-stranded nucleic acid 2, wherein the single-stranded nucleic acid 1, the polypeptide and the single-stranded nucleic acid 2 are sequentially covalently connected, the single-stranded nucleic acid 1 is used for connecting with a sequencing adaptor, and the single-stranded nucleic acid 1 and / or the single-stranded nucleic acid 2 each independently further comprises an extension chain, the extension chain is arranged at any terminal end or in the middle of the single-stranded nucleic acid 1 and / or the single-stranded nucleic acid 2, the extension chain comprises one or more molecules randomly arranged to form a chain: nucleoside, nucleotide or organic linker, and the extension chain is not a poly-mononucleotide sequence when located on the single-stranded nucleic acid 2. The length of the single-stranded nucleic acid at least one end of the extension polypeptide is conducive to reducing the jamming phenomenon generated in the process of polypeptide nanopore sequencing.

Provided herein are methods of characterising a target polypeptide by expanding the target polypeptide to form a peptide linker construct. Also provided are methods of moving one or more peptide portions of a target polypeptide with respect to a nanopore; and to methods of forming a peptide-linker construct. Also provided are related conjugates, constructs and kits.