348 results about "Nanopore sequencing" patented technology

The invention provides an amplification primer and method for a porcine epidemic diarrhea virus whole genome. The detection primer group for the porcine epidemic diarrhea virus comprises 60 pairs of amplification primers, and the specific sequences of the amplification primers are as shown in SEQ ID NO.1-120. The detection primer group provided by the invention can realize relatively uniform coverage of the porcine epidemic diarrhea virus, and 100% region coverage of a genome is realized in nanopore sequencing. The preparation method of the sequencing fragment of the porcine epidemic diarrhea virus whole genome is simple, convenient and easy to operate and good in amplification effect, nanopore sequencing has the advantages of sequencing and real-time analysis at the same time, the detection time can be greatly shortened, infection of the porcine epidemic diarrhea virus can be rapidly identified and diagnosed, meanwhile, the whole genome sequence of the porcine epidemic diarrhea virus is obtained, and the application prospect is wide. The obtained genome sequence can provide a scientific basis for virus traceability, pathogenic variation tracking, novel strain identification, early warning and the like.

The invention relates to a DNA methylation detection method and device and electronic equipment, and the method comprises the following steps: obtaining a DNA sequence to be detected; obtaining original electric signal data and a base sequence corresponding to the DNA sequence to be detected through a nanopore sequencing technology; obtaining a methylation detection result corresponding to the DNA sequence to be detected according to the original electric signal data, the base sequence and a deep learning model; wherein the deep learning model comprises a sequence feature extraction module, a signal feature extraction module, a feature fusion module and a classification module; the sequence feature extraction module comprises a Transform encoder; the signal feature extraction module comprises a Conformer encoder; the feature fusion module comprises a Transform decoder; the classification module comprises a full connection layer; according to the method provided by the invention, the identification accuracy of the methylation site can be improved, high-precision methylation detection is realized, and the problems of gradient disappearance and gradient explosion can be effectively relieved.

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 an amplification primer and method for a porcine pseudorabies virus whole genome. The detection primer group for the porcine pseudorabies virus comprises 271 pairs of amplification primers, and the specific sequences of the amplification primers are as shown in SEQ ID NO.1-542. According to the detection primer group provided by the invention, relatively uniform coverage on the porcine pseudorabies virus can be realized, and 100% region coverage of a genome is realized in nanopore sequencing. The preparation method of the sequencing fragment of the porcine pseudorabies virus whole genome is simple, convenient and easy to operate, the amplification effect is good, nanopore sequencing has the advantage of simultaneous sequencing and real-time analysis, the detection time can be greatly shortened, porcine pseudorabies virus infection can be identified and diagnosed at the soonest 15 hours, meanwhile, the whole genome sequence of the porcine pseudorabies virus is obtained, and the method is suitable for large-scale popularization and application. The obtained genome sequence can provide a scientific basis for virus traceability, pathogenic variation tracking, novel strain identification, early warning and the like.

The invention provides an amplification primer and method for an avian leukosis virus whole genome. The detection primer group for the avian leukosis virus comprises 18 pairs of amplification primers, and the specific sequences of the amplification primers are as shown in SEQ ID NO.1-36. The detection primer group provided by the invention can realize relatively uniform coverage on the avian leukosis virus, and 100% region coverage of a genome is realized in nanopore sequencing. The preparation method of the sequencing fragment of the avian leukosis virus whole genome is simple, convenient and easy to operate, the amplification effect is good, nanopore sequencing has the advantage of simultaneous sequencing and real-time analysis, the detection time can be greatly shortened, infection of the avian leukosis virus can be rapidly identified and diagnosed, meanwhile, the whole genome sequence of the avian leukosis virus is obtained, and the application prospect is wide. The obtained genome sequence can provide a scientific basis for virus traceability, pathogenic variation tracking, novel strain identification, early warning and the like.

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 relates to the field of nanopore sequencing, and particularly discloses a nanopore sequencing polymeric membrane and a membrane forming method.The polymeric membrane is a single-layer molecular membrane and is formed by a block polymer, the block polymer comprises a hydrophilic-end polymer and a hydrophobic-end polymer, the polymerization degree of the hydrophilic-end polymer is 4-6, and the hydrophobic-end polymer is 2-6. The polymerization degree of the hydrophobic end polymer is between 18 and 33; the preparation method of the polymeric membrane comprises the following steps: S1, adding a first buffer solution into a microstructure of a chip, introducing a non-polar solvent dissolved with a block polymer into a runner of the microstructure, and pushing away the redundant first buffer solution to form a lower interface of the polymeric membrane; s2, introducing gas into the microstructure to push away the redundant non-polar solvent to form an upper interface of the polymeric membrane; and S3, adding a second buffer solution, emptying upper-layer gas, and forming a polymeric membrane on the surface of the microstructure of the chip after the block polymer is stably arranged. The film forming process provided by the invention is easy to operate in film forming, low in film rupture rate, and relatively good in stability and voltage tolerance.

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 invention relates to the technical field of biomedical detection, in particular to a genetic typing primer group, a kit and a genetic typing method for accurately realizing an ultrahigh resolution level of 11 sites of human leukocyte antigen (HLA) based on a nanopore sequencing technology. According to the genetic typing primer group for the HLA ultrahigh resolution level, the primer group is used for HLA gene multiple amplification, and the primer group comprises an HLA-A site primer pair, an HLA-B site primer pair, an HLA-C site primer pair, an HLA-DRB1 site primer pair, an HLA-DRB3 / 4 / 5 site primer pair, an HLA-DQA1 site primer pair, an HLA-DQB1 site primer pair, an HLA-DPA1 site primer pair and an HLA-DPB1 site primer pair. According to the primer group for multiple amplification of 11 genes for HLA typing, disclosed by the invention, the amplification of each gene can be simultaneously carried out on the loci of HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DQB1, HLA-DPA1 and HLA-DPB1 of the HLA genes in a reaction system, so that the integrity, the amplification efficiency and the amplification uniformity of the gene amplification as well as the accuracy of subsequent HLA gene typing are improved.

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 nanopore state signal type identification method based on deep learning. The method comprises the following steps: acquiring multiple pieces of state signal data from nanopore sequencing signal data; calculating the fragment number of each piece of state signal data in the plurality of pieces of state signal data according to a preset threshold value; judging whether each piece of state signal data is abnormal state signal data or not according to the fragment number of each piece of state signal data; deleting the abnormal state signal data from the plurality of state signal data to obtain normal state signal data; and identifying the type of the normal state signal data by using the trained deep learning model.

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 DNA (deoxyribonucleic acid) storage method for quickly reading short fragments in nanopore sequencing, which comprises the following steps of: coding an information sequence by using a block code, sparsening a code word fragment, superposing the sparsened code word fragment with a known pseudo-random sequence, combining an equal-length coding bit sequence, mapping the equal-length coding bit sequence into a data DNA sequence, and constructing a label DNA sequence by using the pseudo-random sequence along with a short block error correction code; the method comprises the following steps of: aiming at nanopore sequencing, realizing quick clustering of sequencing reads by utilizing primer recognition and label recognition, constructing a hidden Markov model, deducing the maximum symbol probability by utilizing a forward-backward algorithm, performing upper-layer bit sequence merging on the reads in the clusters, correcting insertion and deletion errors, obtaining a consistent upper-layer bit sequence, and correcting a lower-layer bit sequence in the clusters; and calculating the likelihood probability of the consistency sequence, executing soft decision error correction decoding, and recovering the original data. The method can effectively utilize nanopore sequencing to quickly read short DNA fragments, and has the advantage that a probability-based multi-sequence merging method is used to correct high-insertion deletion errors of nanopore sequencing.

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.