223 results about "Nanopore sequencing" patented technology

In one aspect, the disclosed technology relates to nanopore sequencing with a polynucleotide comprising a plurality of nucleotides, wherein each nucleotide comprises a cleavable cyclic loop between two positions of the nucleotide, wherein the cyclic loop comprises reporter moiety corresponding to the identity of the nucleotide. The reporter may be comprised of an amino acid sequence. The cyclic loop may further comprise modifications or arresting constructs for slowing or halting the polynucleotide translocation through a nanopore. Also disclosed are methods of detecting nucleotides and a kit for determining the sequence of nucleotides.

The invention provides a construction method of a bacterial pathogen detection model based on nanopore adaptive sampling and deep learning, and a method, equipment, a medium and a program product for detecting bacterial pathogens by applying the model, and relates to the technical field of nanopore sequencing. The bacterial pathogen detection method comprises the following steps: obtaining a water sample to be detected; and inputting a to-be-detected water sample into the detection model constructed in the invention to obtain a detection result of the to-be-detected water sample. The sensitivity and the accuracy of pathogen detection are obviously improved, and the improvement on prevention and control of water-borne diseases is facilitated.

The invention discloses a pathogenic microorganism detection system based on nanopore sequencing and intelligent interpretation. The system comprises six modules including a biomolecule electric signal acquisition module and a pathogen characteristic sequence extraction module. Ion current change signals are collected through a nanopore chip array, a pathogen characteristic sequence is extracted through differential coding, a hypergraph topological structure self-adaption building module is used for building a pathogen relation model, a multi-dimensional heterogeneous pathogen parameter fusion analysis module integrates morphological parameters, metabonomics parameters and other multi-dimensional parameters, and the multi-dimensional heterogeneous pathogen parameter fusion analysis module is used for analyzing the multi-dimensional parameters. The optimized hypergraph neural network reasoning module realizes pathogen category prediction, and finally, the nanopore sequencing result intelligent interpretation output module generates a detection report containing pathogen categories, subtypes and risk levels. The system is combined with nanopore sequencing and an intelligent algorithm, pathogenic microorganism detection can be efficiently and accurately completed, the accuracy, reliability and efficiency of detection are greatly improved, and the requirement for rapid and accurate detection of pathogens in practical application is met.

The present invention provides primers and methods for amplifying the entire genome of avian leukosis virus. The detection primer set for avian leukosis virus includes 18 pairs of amplification primers, the specific sequences of which are shown in SEQ ID NOs. 1 to 36. The detection primer set provided by the present invention can achieve relatively uniform coverage of avian leukosis virus, and in nanopore sequencing, 100% regional coverage of the genome is achieved. The method for preparing sequencing fragments of the entire genome of avian leukosis virus provided by the present invention is simple and easy to operate, with good amplification effect. Nanopore sequencing has the advantage of real-time analysis while sequencing, which can greatly shorten the detection time and can quickly identify and diagnose avian leukosis virus infection. At the same time, its entire genome sequence can be obtained. The obtained genome sequence can provide a scientific basis for virus tracing, pathogen mutation tracking, new strain identification and early warning, etc.

The invention is suitable for the technical field of gene sequencing, and provides a nanopore sequencing signal identification method and system based on Transform, and the method comprises the following steps: carrying out normalization processing on a nanopore sequencing signal, and segmenting the nanopore sequencing signal into signal windows with fixed lengths; signal features are extracted through a Transformer encoder comprising a convolutional layer and a multi-head self-attention module; generating a prediction probability of a current base through a cross attention mechanism by adopting an autoregressive Transform decoder and combining the signal features and context information of a decoded base sequence; iteratively generating an optimal base sequence based on a beam search algorithm, and optimizing decoding efficiency in combination with key value cache; and carrying out overlapping region fusion and quality control on a segmented decoding result, and outputting a final sequence. According to the method, the precision and reliability of nanopore sequencing in complex biological sequence analysis are remarkably improved, and the recognition accuracy on multiple gene sequencing data sets is higher than that of an existing CTC-based model.

In one aspect, the disclosed technology relates to nanopore sequencing with a polynucleotide including a plurality of ribonucleotides, wherein each ribonucleotide comprises a cyclic loop between two positions of the ribonucleotide, wherein the cyclic loop comprises a spacer or reporter moiety corresponding to the identity of the ribonucleotide, one or more linkers, and optionally one or more arresting constructs. The cyclic loop may be selectively cleaved in a ribose-mediated pathway to cause cleavage at the 5' P-O bond of the phosphate backbone.

Disclosed herein are systems and methods for nanopore sequencing basecalling. In one embodiment, the method can include: receiving raw nanopore sequencing data comprising a plurality of continuous data acquisition (DAC) values corresponding to a biomolecule; normalizing the raw nanopore sequencing data to generate normalized nanopore sequencing data comprising a plurality of normalized DAC values; generating, using a first neural network (NN) and a normalized DAC value of the plurality of normalized DAC values, a vector of transformed probability values, segmenting the plurality of normalized DAC values into a plurality of discrete events; generating, using a second neural network and the event vector, an element determination of the biomolecule.

The invention discloses a primer composition and a kit for identifying testudinate species and application of the primer composition and the kit. The primer composition comprises primers (SEQ ID NO.1-6) for targeted amplification of specific marker genes 16S, COI and Cytb of testudinate mitochondrial species and an enrichment universal primer (SEQ ID NO.7). According to the invention, full lengths of three mitochondrial genes 16S, COI and Cytb are taken as targets, a specific primer group added with an enrichment tag and an enrichment primer are designed, and a targeted nanopore sequencing method capable of realizing testudinate species identification is established and optimized, and the method can simultaneously capture testudinate mitochondrial species specific marker gene information of a family species level, and can be used for identifying testudinate species. The problem that the existing DNA bar code is difficult to accurately identify the testudinate species due to the lack of database information is effectively solved; and each marker gene can obtain a complete coding sequence, so that genetic information is richer, and the method can be used for geographical traceability analysis of testudinate species.

The invention discloses a species identification kit for animal mitochondrial bar code multiplex PCR amplification and nanopore sequencing, and relates to the technical field of species identification. The species identification kit comprises optimized primer pairs aiming at four hyper-variable genes (Cytb, COI, 16S rRNA and 12S rRNA) of mitochondria, by introducing R, Y, D and other degenerate bases at key sites, the binding capacity of the primers and template DNA of different species is remarkably improved, the problems that an existing universal primer is prone to mismatch, low in efficiency and even failed in amplification during cross-species amplification are solved, and the species identification kit has the advantages of being high in specificity, high in specificity and high in specificity. The method can be used for species identification of a mixed DNA sample containing at least two different animal species sources. The species identification primer combination is applied to preparation of an animal species identification kit, and the detection coverage range of the kit is expanded. According to the animal species identification method provided by the invention, a multiple PCR amplification system and a nanopore length reading sequencing technology are combined, congeneric sibling species with highly similar sequences can be distinguished, and the method has good accuracy.

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.

Methods of detection and identification of microbial eye infections by molecular analysis of nucleic acid in tear samples are provided. 16S rDNA is amplified by PCR directly from tear samples without extracting DNA and the amplification products are sequenced by nanopore sequencing so that the microbes can be identified.

A combination of lipid-binding molecules and / or lipid-binding proteins with lipid components (i.e., mispids) is provided for use in modifying the interaction between target molecules and lipid membranes. This includes, for example, the use of lipid-binding molecules and / or mispids to improve the sequencing efficiency and throughput of nanopore-based sequencing systems. To sequence target molecules such as nucleic acid sequences or nucleic acid substitute polymers derived therefrom, lipid-binding molecules and / or their mispids are combined with the target molecules. The mixture is then applied to a nanopore-based sequencing chip. The target molecules are then sequenced in the presence of lipid-binding molecules and / or nanodiscs, thereby improving the capture, arrival time, and effective concentration of the target molecules across the chip membrane. Such improved efficiency is particularly beneficial, for example, when the concentration of the target molecule is low.

The disclosure describes sequencing workflows and reagents used in those workflows that are suitable for sequencing a target nucleic acid using a nanopore sequencing device. The disclosure also describes detergent and air-flow based methods and compositions used to clean a device pre- and post-sequencing to enable reuse of a consumable device in a nanopore sequencer.

The invention discloses a detection system and method for identifying mycobacteria and drug resistance genes of the mycobacteria based on nanopore sequencing, and relates to the field of biological medicine, the detection system comprises a specific targeted enrichment module, a nanopore sequencing module and a biological information analysis module; the specific targeted enrichment module comprises a probe combination, and the probe combination covers a mycobacterium tuberculosis complex conservative identification gene, species-specific genes of common nontuberculous mycobacteria and mycobacterium leprosy, and full-length or partial sequences of drug resistance related genes in a targeted manner; by utilizing the characteristics of nanopore length reading length and real-time sequencing and a tuberculosis specific targeted enrichment strategy, accurate identification of a mycobacterium tuberculosis complex group, synchronous typing of 42 mycobacteria, analysis of 24 drug-resistant genes, and efficient detection of structural variation and low-abundance heterogeneity drug-resistant mutation are realized, the detection period is shortened, the detection cost is reduced, and the detection efficiency is improved. And a comprehensive and reliable technical basis is provided for accurate diagnosis and treatment of mycobacterium infection.

The invention discloses helicase ToPif 1, a preparation method of the helicase ToPif 1 and application of the helicase ToPif 1 in high-throughput sequencing. The amino acid sequence of the ToPif 1 helicase mutant is as shown in SEQ ID NO. 1. The helicase can be used for control and characterization of nucleic acid and applied to nanopore sequencing.

The invention discloses an HLA (human leukocyte antigen) gene amplification primer based on a nanopore sequencing platform and a typing method, and relates to the technical field of gene engineering, the amplification primer comprises an HLA-A gene amplification primer pair: a forward primer HLA-A-F5; a reverse primer HLA-A-R5; an HLA-B gene amplification primer pair: a forward primer HLA-B-F3; a reverse primer HLA-B-R3; the HLA-DQB1 gene amplification primer pair comprises a forward primer HLA-DQB1-F7, a reverse primer HLA-DQB1-F7 and a reverse primer HLA-DQB1-F7; a reverse primer of HLA-DQB1-R7; the HLA-DRB1 gene amplification primer pair comprises a forward primer HLA-DRB1-F1, a reverse primer HLA-DRB1-F2, a reverse primer HLA-DRB1 A reverse primer (HLA-DRB1-R1); the HLA-DRB3 gene amplification primer pair comprises a forward primer HLA-DRB3-F6, a reverse primer HLA-DRB3-F6, a reverse primer A reverse primer (HLA-DRB3-R8); by designing the specific amplification primer group and combining with the nanopore sequencing length reading technology, the problems of insufficient resolution and the like of the existing typing technology are solved, the typing accuracy and resolution are improved, and the performance is superior to that of the traditional technology; meanwhile, the process is optimized, the limitation of long detection period and the like is broken through, the whole process is rapidly completed, the technical threshold and detection expenditure are reduced, high efficiency, convenience and economy are considered, and support is provided for clinical popularization and scientific research application.

The disclosure relates to nanopore preparations with reduced concentrations of detergents including detergent-free preparations. The disclosure provides lipid nanoparticles (LNPs) comprising a lipid, saposin, and a nanopore, methods for producing such LNPs, as well as systems and methods for nanopore-based sequencing using such LNPs. The new nanopore preparation avoids the pitfalls of using detergent-based compositions, such as disrupting the interactions between a target molecule and a nanopore during sequencing, disruption of the lipid bilayer, and batch-to-batch variability in nanopore preparations.

The invention relates to a nanopore sequencing method using single-stranded specific nuclease in the field of sequencing. The nanopore sequencing method can improve the sequencing flux, the capture efficiency of a target sequencing object, the pore (channel) quantity, the average reading length of the target sequencing object and / or the pore protein gating frequency.

The present invention provides a system and method for identifying and analyzing unknown pathogenic microorganisms based on nanopore sequencing, including: microbial database construction and updating, standardized annotation of microbial information, nanopore sequencing analysis, sequencing data quality control, host sequencing read removal, microbial database alignment annotation, assembly of same-genus sequencing reads, and microbial database alignment calculation; for each representative sequencing read, the representative sequencing read is computationally aligned with any genome sequence in the microbial database; when a first computational alignment condition is met, an output is output that the representative sequencing read is consistent with the species to which the genome sequence meeting the first computational alignment condition belongs; when a second computational alignment condition is met, an output is output that the representative sequencing read is homologous to the species to which the sequence meeting the second computational alignment condition belongs; when the representative sequencing read does not meet both conditions with any sequence in the microbial database, an output is output that the representative sequencing read is not aligned in the microbial database.

Systems and methods for long-read sequencing of immune receptor are disclosed. A method in accordance with the present disclosure comprises generating a 3'-barcoded cDNA library comprising an immune receptor target sequence, wherein the immune receptor target sequence comprises a variable region and a barcoded region; amplifying the immune receptor target sequence; linking the variable region of the immune receptor target sequence to a probe to generate a labeled immune receptor target sequence; capturing the labeled immune receptor target sequence; amplifying the captured immune receptor target sequence; passing the captured immune receptor target sequence through a nanopore sequencing device; and generating, by way of the nanopore sequencing device, a full-length immune receptor target sequence.

To provide a homopolymer-encoded nucleic acid memory.SOLUTION: A nucleic acid memory chain that encodes digital data by using a sequence of homopolymer tract of a repeated nucleotide provides a more inexpensive and faster alternative method for a conventional digital DNA storage technique. Using a homopolymer tract makes it possible to read out data encoded in a memory chain by a high throughput sequencing technique of lower fidelity, that is, nanopore sequencing for example. A specialized synthesis technique makes it possible to synthesize a long memory chain capable of encoding a large volume of data despite that data density obtained by a homopolymer tract is reduced compared to a conventional single nucleotide sequence.SELECTED DRAWING: None

The invention relates to a novel HfaB nanopore protein mutant and application thereof in sequencing, and belongs to the technical field of nanopores. The polynucleotide for coding the nanopore protein mutant and the recombinant expression vector can express the nanopore protein mutant, and can be used for constructing recombinant cells or recombinant strains for expressing the nanopore protein mutant. The wild-type nanopore protein provided by the invention has relatively high pore stability and is suitable for nanopore sequencing, the sequencing current property of the nanopore protein mutant obtained by modification on the basis of the wild-type nanopore protein is further improved, the sequencing precision is obviously improved, the sequencing signal amplitude and step number are obviously increased, the pore current stability is enhanced, and the nanopore protein mutant can be applied to nanopore sequencing. The spontaneous blocking condition is reduced, and the abnormal noise on the via hole signal is reduced.

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.

The invention provides a method and a device for simulating a pulsed field gel electrophoresis band based on a sequencing technology. The method for simulating the pulsed field gel electrophoresis band based on the sequencing technology comprises the following steps: acquiring nanopore sequencing data of a biological sample to be analyzed, and assembling based on the nanopore sequencing data to obtain a genome sequence; according to the sequence characteristics of the restriction endonuclease, identifying a restriction enzyme cutting site corresponding to the restriction endonuclease from the genome sequence; when the restriction enzyme cutting site is the restriction enzyme cutting site subjected to methylation modification, removing the restriction enzyme cutting site to obtain a residual restriction enzyme cutting site; calculating the distance difference between two adjacent restriction enzyme cutting sites according to all residual restriction enzyme cutting sites, and taking the distance difference as the molecular weight of a simulated band; aiming at the molecular weight of each simulated strip, determining a strip position corresponding to the molecular weight; and drawing a simulated pulse field gel electrophoresis band result according to the position of each band and the molecular weight corresponding to the band position so as to accurately simulate the band result.

The invention provides a method for performing nanopore sequencing by using a sealer. According to the method disclosed by the invention, the sequencing library can only be combined in the capture range of the nanopores, the library is efficiently captured by the nanopores in the sequencing process, and the utilization efficiency of the library is improved.

The invention discloses a ganoderma lucidum binuclear genome assembly method based on haplotype analysis, and belongs to the technical field of bioinformatics. The purpose of the present invention is to overcome the accuracy and accuracy of high hybrid genome assembly. The invention provides a ganoderma lucidum binuclear genome assembly method based on haplotype analysis, which comprises the following steps: obtaining mononuclear ganoderma lucidum cells, respectively carrying out whole genome sequencing by utilizing Illumina NovaSeq, Nanopore, Hi-C and PacBio HiFi, and obtaining accurate genome information according to the sequencing data of the Illumina NovaSeq and the sequencing data of the Nanopore. And an assembly strategy of haplotype separation is realized.

The invention discloses a sequencing linker, a sequencing linker compound, a method for multiple amplification of a target nucleic acid sequence and a nanopore sequencing method. Wherein the sequencing linker comprises a first chain and a second chain, the first chain comprises a sequencing guide sequence, a helicase binding sequence, a limiting structure, a first complementary sequence and a first primer sequence from the 5'end to the 3 'end in sequence, the limiting structure prevents helicase from moving, and the first chain is provided with a 3' free end; the second chain comprises a second complementary sequence and a restraint sequence or a sequence complementary with the nucleic acid sequence of the restraint sequence from the 5'end to the 3 'end in sequence, and the restraint sequence comprises a nucleic acid sequence of which the tail end is connected with a hydrophobic molecule; the first complementary sequence and the second complementary sequence are reversely complementary, and the sequencing linker is formed by annealing a first chain and a second chain; the sequencing linker further comprises a structure for preventing the second strand from being replaced by the polymerase. The sequencing linker and the sequencing linker compound are simple in construction process and high in yield.

Provided are a method for representing a target biomolecule, a ligation method or a preparation method for a sequencing library, an activation linker, a method for preparing the activation linker, a kit for nanopore sequencing, and a use of the activation linker in nanopore sequencing.

The present inventive concept relates to methods of nucleic acid sequencing, and applications and uses of the same. Aspects of the inventive concept include nanopore sequencing on nucleic acids, such as ribonucleic acids (RNAs), including, but not limited to, transfer RNAs (tRNAs). Further aspects include kits for performing the methods, and applications of the methods of the inventive concept as substantively described herein.