288 results about "Nanopore sequencing" patented technology

Systems and methods for sequencing polynucleotides using nanopores are disclosed. In some embodiments, a polynucleotide including a single-stranded region and a double-stranded region, in which the single-stranded region is disposed through a nanopore. The polynucleotide can be moved relative to the nanopore by electric forces while one or more structural locks keep the polynucleotide close to the nanopore. A characteristic signal based on nanopore ionic current blockade and associated with the regions of the polynucleotide at or near the nanopore recognition zone is measured and used to infer the nucleobase sequence of the polynucleotide. In some examples, the double-stranded region is extended by a polymerase, and the polymerase is removed from the polynucleotide. In some examples, signals measured under different applied voltages provide nonredundant information regarding the polynucleotide sequence.

The invention provides a sequencing linker, a sequencing linker compound and a method for multiple nanopore sequencing of a target nucleic acid sequence. 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 the first strand and the second strand. The sequencing linker and the sequencing linker compound are simple in construction process and high in yield.

The invention discloses an unknown pathogenic microorganism identification and analysis system and method based on nanopore sequencing. Comprising the steps of microorganism database construction and updating, microorganism information standardization labeling, nanopore sequencing analysis, sequencing data quality control, host sequencing read segment removal, microorganism database comparison annotation, congeneric sequencing read segment assembly and microorganism database comparison calculation. For each representative sequencing read segment, calculating and comparing the representative sequencing read segment with any genome sequence in the microorganism database, when a first calculation and comparison condition is met, outputting that the representative sequencing read segment is consistent with the species to which the genome sequence meeting the first calculation and comparison condition belongs, and when a second calculation and comparison condition is met, outputting that the representative sequencing read segment is consistent with the species to which the genome sequence meeting the first calculation and comparison condition belongs. And outputting that the representative sequencing read and the species to which the sequence meeting the second calculation comparison condition belongs are in a homologous relationship, and when the representative sequencing read and each sequence in the microorganism database do not meet the two conditions, outputting that the representative sequencing read is not compared in the microorganism database.

The invention provides a primer group for detecting a porcine epidemic diarrhea virus based on a whole genome, a library building method and application. The primer group comprises 27 pairs of primers, and sequences of upstream primers and downstream primers of the 27 pairs of primers are shown as SEQ ID NO.1-SEQ ID NO.54. The invention further provides a kit for detecting the porcine epidemic diarrhea virus based on the whole genome. The primer group disclosed by the invention has good specificity, sensitivity and repeatability, and has a good application prospect in amplification of the whole genome of the porcine epidemic diarrhea virus and preparation of reagents or kits required by amplification. The library building method provided by the invention comprises the following steps: carrying out multiple PCR amplification by using the primer group provided by the invention; the library building method is suitable for a next-generation sequencing platform and a nanopore sequencing platform, the automation degree is high, 95% or above of porcine epidemic diarrhea virus sequences can be obtained even in a sample with the virus content as low as 1 copies / microliter, and the sequencing depth can reach 1000 X or above.

The present invention relates to a method for sequencing small non-coding RNAs on a nanopore platform. In this method, sequence-specific structured adapters are used to specifically capture small non-coding RNAs in a single ligation step reaction, thus enabling direct RNA sequencing.

The invention provides a biological nanopore sensor and a preparation method thereof. The method for preparing the biological nanopore sensor comprises the following steps: a) preparing hydrophilic protein into lipidosome; and b) mixing the lipidosome with the membrane layer to promote the lipidosome to be inserted into the membrane layer to obtain the biological nanopore sensor, and the hydrophilic protein has a nanopore channel. The method can solve the problem that the hydrophilic protein is difficult to prepare into the biological nanopore sensor in the prior art, and is suitable for the field of nanopore sequencing.

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 relates to the technical field of pathogenic microorganism detection, in particular to a pathogenic microorganism detection system based on nanopore sequencing, which comprises: a microflow sample introduction module, which uses nanopore sequencing, regulates and controls a microorganism liquid channel and adjusts the opening of a sample introduction valve at the same time, setting the flow rate of the trace liquid and comparing the ion concentration of the conductive pore channel with the pH value of the solution by using a sensor. According to the invention, liquid channel regulation and control are carried out through nanopore sequencing, and accurate control of a fluid environment is realized by combining opening adjustment of a sample introduction valve, flow velocity setting and comparison of ion concentration and pH value of a conductive pore channel, so that a microbial sample is ensured to enter a sequencing system under stable physical and chemical conditions, and the stability of sequencing data is improved. In the cell lysis process, the nucleic acid release rate is adjusted through temperature monitoring, and the particle removal operation is combined, so that the nucleic acid component in the sample is more uniform, and the reliability of subsequent detection is improved.

The invention relates to a method and a device for detecting chromosome uploidy. The method comprises the following steps: acquiring nanopore sequencing data of a detection sample; obtaining window division information of the reference genome; for each detection sample, determining a standard comparison sequence number of the detection sample in each window based on the nanopore sequencing data of the detection sample and the window division information; for each negative sample, eliminating discrete values in the standard alignment sequence number of each negative sample in each window, and correcting the standard alignment sequence number of each negative sample in each window to obtain a standard alignment sequence number baseline value of each window; and according to the standard comparison sequence number of the to-be-analyzed sample in each window and the corresponding standard comparison sequence number baseline value of each window, evaluating the chromosome uploidy condition of the to-be-analyzed sample. According to the method, the third-generation sequencing data is creatively applied to chromosome aneuploidy analysis through a new baseline data construction mode.

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.

In one aspect, the disclosed technology relates to nanopore sequencing with a polynucleotide comprising a plurality of nucleotides, wherein each nucleotide comprises a chelating moiety bound to a metal ion. In some embodiments, the chelating moieties are configured for slowing or halting the polynucleotide translocation through a nanopore until a specific stimulus is achieved. In some embodiments, the chelating moieties, as well as any bound metal ions, can serve as reporters for specific nucleotides.

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 provides a nanopore sensor and an application of the nanopore sensor in sequencing. The nanopore sensor comprises a film layer and pore protein inserted in the middle of the film layer to form a pore channel, and when an electric field force is applied across the film layer, the pore channel generates conductivity; wherein the pore protein comprises a pore protein monomer, and the pore protein monomer comprises (a) a protein with an amino acid sequence as shown in SEQ ID NO: 2; or (b) a protein mutant, the amino acid sequence of the protein mutant is subjected to substitution, deletion and / or addition of one or more amino acids at at least one of the following sites of SEQ ID NO: 2: 80, 82, 83 and the like, and the mutant has the function of forming a pore structure through polymerization; or (c) the mutant has at least 70% of identity with the protein, and the mutant has a function of forming a pore structure through polymerization. The invention provides a novel nanopore sensor which can be used for nanopore sequencing and is suitable for the field of single-molecule sequencing.

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.

The invention relates to the technical field of pathogen detection, in particular to a rapid pathogen detection system based on nanopore sequencing, which comprises an acquisition and pretreatment module, which is used for precipitating and centrifuging serum, carrying out layered monitoring, carrying out supernate separation and concentration, and measuring pH value and temperature. According to the invention, the serum is precipitated and centrifuged, and layered monitoring is combined, so that the layered state of the serum sample in the centrifugation process is visualized, and the stability of precipitated particles and the separation precision of supernate are improved. And in the supernatant concentration link, the dynamic monitoring and optimization of the sample purity are realized by measuring the pH value and the temperature and combining the recording of the protein concentration and the turbidity value. Absorbance and viscosity measurement are combined, so that the change of liquid properties can be captured in real time and compared with a pathogen directory range threshold value, and an abnormal sample can be recognized in an early stage.

The machine learning-based nanopore sequencing signal state identification method comprises the following steps: acquiring nanopore sequencing signal data X1, X2,..., XN, and carrying out feature extraction on the nanopore sequencing signal data X1, X2,..., XN to generate a feature value vector; the state of the nanopore sequencing signal data is identified using the generated eigenvalue vector and a trained machine learning model. The feature extraction comprises the following steps: dividing the nanopore sequencing signal data into G groups according to the size of values, wherein G is a natural number greater than 1 and less than N; wherein the characteristic value comprises at least one of a number ratio, a standard deviation and a variable coefficient; respectively calculating the number ratio of each group in the G groups, and generating G number ratio characteristic values; respectively calculating the standard deviation of the data of each group in the G groups, and generating G standard deviation characteristic values; calculating a variable coefficient of data of each group in the G groups, and generating G variable coefficient characteristic values; and generating a feature value vector based on the generated plurality of feature values.

The invention provides a primer group for detecting a porcine reproductive and respiratory syndrome virus based on a whole genome, a library building method and application. The primer group comprises 16 primer pairs, and sequences of an upstream primer and a downstream primer of each primer pair are shown as SEQ ID NO.1-SEQ ID NO.32. The invention further provides a kit for detecting the porcine reproductive and respiratory syndrome virus based on the whole genome. The primer group disclosed by the invention has good specificity and sensitivity, and has a good application prospect in amplification of the whole genome of the porcine reproductive and respiratory syndrome virus and preparation of reagents or kits required by amplification. The library building method provided by the invention comprises the following steps: carrying out multiple PCR amplification by using the primer group provided by the invention; the library building method is suitable for a next-generation sequencing platform and a nanopore sequencing platform, the automation degree is high, 95% or above of porcine reproductive and respiratory syndrome virus sequences can be obtained even in a sample with the virus content as low as 1 copies / microliter, and the sequencing depth can reach 1000 X or above.

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 relates to a method and device for simultaneously detecting multiple gene mutations related to thalassemia, and the method comprises the following steps: obtaining nanopore sequencing data of a to-be-detected sample, the to-be-detected sample comprising amplification products of multiple PCR primer pairs for simultaneously detecting multiple gene mutations related to thalassemia; performing quality control screening on the nanopore sequencing data according to the amplification length of the multiple PCR primer pair to obtain first target sequencing data; according to the first target sequencing data, obtaining the sequence number of the amplification product of the PCR multiple primer pair, and according to the sequence number of the amplification product of the PCR multiple primer pair, determining the gene mutation information related to thalassemia of the to-be-detected sample. According to the application, deletion mutation and non-deletion mutation related to thalassemia can be detected at the same time, and the accuracy of HBA1 and HBA2 gene detection results is improved.

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.

To provide a practical method for detecting repeat expansion diseases that can comprehensively test for repeat expansion diseases without the need for advanced specialized knowledge in repeat expansion diseases.SOLUTION: The method for detecting a repeat expansion disease according to the present invention includes a library preparation process, a sequencing process, a consensus sequence construction process, a ranking process, and a determination process. In the sequencing process, multiple disease-associated repeat regions and their neighboring genomic regions are designated as target regions, and nanopore sequencing is performed to obtain long-read sequence data for each target region in a subject's genome. In the ranking process, repeat number data for each disease-associated repeat region is compared between a subject and a group of normal healthy persons, and the disease-associated repeat regions in the subject's genome are ranked in the descending order of the possibility of pathological repeat expansion. In the determination process, evaluation is started from the disease-associated repeat region ranked first according to specific steps S1 to S7, and it is determined whether or not the subject has pathological repeat expansion.SELECTED DRAWING: Figure 1

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 relates to the technical field of sequencing, in particular to a sequencing joint for nanopore sequencing and application of the sequencing joint. According to the sequencing joint provided by the invention, different non-B-form structures are designed in the sequencing joint and are used for preventing motor protein from unwinding nucleic acid in a non-sequencing state, so that the motor protein can pass through a secondary structure region for sequencing only when a pilot chain enters a nanopore and under the action of electric field force. The synthesis cost of the sequencing linker for sequencing by using the nanopore is lower than that of the existing sequencing linker.

The invention discloses an MMLV reverse transcriptase mutant. The invention provides an MMLV reverse transcriptase mutant, and compared with a wild type MMLV reverse transcriptase amino acid sequence, the mutant mutates at a plurality of sites such as a 62 site and the like, and the mutant has the characteristics that the template conversion performance is improved, the non-template basic group addition performance is improved or the heat resistance is improved. The MMLV reverse transcriptase mutant with improved conversion performance compared with a wild type template is screened out, and the MMLV reverse transcriptase mutant also has improved non-template base addition performance or thermal stability; the screened mutant is suitable for RNA (Ribonucleic Acid) sequencing, such as Smart-Seq, nanopore sequencing, 5 'RACE (Rapid Amplification of cDNA Ends) and the like, adopting a library preparation method based on template conversion, and can also be applied to RT-qPCR (Reverse Transcription-Quantitative Polymerase Chain Reaction) and the like.