72 results about "Deep sequencing" patented technology

The invention relates to the field of noninvasive prenatal diagnosis, and particularly discloses a preeclampsia noninvasive screening method based on deep sequencing 8bp oligonucleotide double-fragment characteristics, which comprises the following steps: collecting preeclampsia and healthy pregnant woman peripheral blood samples, and extracting free DNA for high-throughput sequencing; the method comprises the following steps: extracting core 8-mer sequences' GTGCGCCC 'and' GATGGGGT 'in a long fragment of 150-200bp through bioinformatics analysis; an integrated support vector machine, K-nearest neighbor, extreme gradient lifting, a random forest and a multi-layer perceptron are combined with a logistic regression element classifier to construct a stacking model, the frequency of a core sequence is normalized, machine learning analysis is carried out, and the preeclampsia risk is predicted. According to the invention, two 8bp oligonucleotide characteristic fragments are specifically screened, and a deep learning architecture of multi-model fusion is combined, so that the limitations of low specificity and invasive detection of a traditional screening method are effectively broken through.

The invention discloses a tiny residual focus detection method and system based on ultra-high depth sequencing, and relates to the technical field of tiny residual focus intelligent detection.The tiny residual focus detection method comprises the following steps that on the basis of a sequencing library, splitting is conducted according to a sample index to obtain a to-be-detected sample, and a consensus sequence is obtained according to a molecular identifier of the to-be-detected sample; based on a consensus sequence, filtering out the consensus sequence of which the mass value is less than 25 or the family size is less than 3, and combining a variation type and a distance from a fragment edge as noise introduced into an original nucleic acid molecular chain; a context sequence (context) and a chain direction are used as noise for introducing the capture level of PCR amplification; on the basis of the noise level, the circulating tumor DNA level is estimated in combination with tumor priori knowledge, and the MRD state is determined by detecting the significance of molecular signal sources. According to the invention, the sensitivity and specificity of MRD detection are improved.

The invention discloses a high-specificity minimal residual disease (MRD) monitoring method and system based on circulating tumor DNA (ctDNA). The method comprises the following steps: receiving tumor tissue sequencing data of an UTUC patient, and generating a double-Panel target list containing personalized and fixed Panel; respectively extracting plasma cfDNA and leukocyte gDNA; performing vacuum concentration, hybrid capture and sequencing on the cfDNA library by using the double Panel lists, and performing deep sequencing on the leukocyte gDNA; constructing an individualized clonal hematopoietic mutation filtering database; actively filtering and rejecting clonal hematopoietic background mutation by utilizing a filtering database; and calculating an MRD load score based on the filtered tumor-derived mutation and outputting a report. The system comprises corresponding modules which are used for automatically executing the process. According to the invention, through cooperation of four major technologies of double-Panel design, process optimization, UMI error correction and active clonal hematopoietic filtration, MRD monitoring with extremely high sensitivity and specificity on UTUC is realized, false positive is significantly reduced, and the kit has drug resistance early warning potential.

The invention provides an SNP molecular marker combination for paternity test and individual recognition of dairy cows and application, the SNP molecular marker combination comprises 300 SNP markers on 29 autosomes of the dairy cows, the marker combination can be applied to paternity test and individual recognition of the dairy cows, and an identification method comprises the step of preparing a liquid phase chip of the 300 SNP markers, constructing a high-throughput sequencing library by using the genome DNA of the dairy cow to be detected; mixing the liquid phase chip probe with a high-throughput sequencing library, capturing a DNA fragment containing a target SNP site in the dairy cow DNA high-throughput sequencing library, amplifying and purifying to obtain a genetic typing of a dairy cow individual to be detected, and performing paternity test inference according to the genetic typing. The invention provides a probe and a kit for identifying SNP (Single Nucleotide Polymorphism) site information. According to the invention, the SNP polymorphism is detected through targeted capture sequencing, through high-depth sequencing, the typing result is accurate and reliable, and the accuracy of paternity test and individual recognition is ensured.

The invention provides a target character prediction method suitable for low-depth sequencing and a whole genome selection model suitable for low-depth sequencing, and relates to the technical field of biology. According to the target character prediction method suitable for low-depth sequencing and the whole-genome selection model provided by the invention, the target character of the to-be-detected sample can be accurately predicted under a limited data volume by combining low-depth whole-genome re-sequencing data with a machine learning technology, and the genome selection efficiency can be improved by matching the two; the time and economic cost of breeding or medical research are reduced, and the method is particularly suitable for large-scale genetic analysis scenes needing rapid iteration.

Disclosed herein are methods for profiling a cancerous tumor using a high-throughput sequencing technique.

The invention belongs to the technical field of animal genetic breeding, and provides an application of an SNP molecular marker related to a pig backfat thickness character. The nucleotide sequence of the molecular marker is as shown in SEQ ID NO: 7, wherein the polymorphism of the eleventh basic group is C / T. The key gene SLC26A3 related to the backfat thickness of the large white pig is identified through whole genome deep sequencing, the genotype of the backfat thickness character of the large white pig is further detected through an SLC26A3 gene molecular marker, and the backfat thickness of the CC genotype large white pig is obviously higher than that of a CT / TT genotype pig. Therefore, the breeding of the CT / TT genotype individuals is beneficial to meeting the continuous demand of large-scale production on high-efficiency utilization of feed energy. The detection method provided by the invention is simple and easy to operate, can efficiently and quickly identify the back fat thickness character of the large white pig, improves the uniformity of the back fat thickness of the pig, and provides a scientific basis for early breeding of the pig.

The invention relates to a method for evaluating and positioning nucleic acid pollution of a nucleic acid extraction kit, which comprises the following steps: 1) taking nucleic acid-free water as a negative sample, and performing nucleic acid extraction on the nucleic acid-free water by using the nucleic acid extraction kit according to nucleic acid extraction steps in the kit, the obtained nucleic acid is presumed as the polluted nucleic acid in the nucleic acid extraction kit; 2) amplifying polluted nucleic acid, purifying a PCR product, enriching double-chain cDNA, and determining the concentration of the double-chain cDNA so as to perform quantification, sample mixing and quality control treatment before library construction; (3) carrying out high-throughput deep sequencing by adopting a platform of the Illumina company, and then carrying out bioinformatics analysis to obtain classification information and gene sequences of polluted nucleic acid; and 4) designing a specific primer based on the gene sequence of the polluted nucleic acid, and carrying out pollution evaluation and positioning verification on each component of the nucleic acid extraction kit through qPCR (Quantitative Polymerase Chain Reaction).

Next Generation DNA sequencing promises to revolutionize clinical medicine and basic research. However, while this technology has the capacity to generate hundreds of billions of nucleotides of DNA sequence in a single experiment, the error rate of approximately 1% results in hundreds of millions of sequencing mistakes. These scattered errors can be tolerated in some applications but become extremely problematic when “deep sequencing” genetically heterogeneous mixtures, such as tumors or mixed microbial populations. To overcome limitations in sequencing accuracy, a method Duplex Consensus Sequencing (DCS) is provided. This approach greatly reduces errors by independently tagging and sequencing each of the two strands of a DNA duplex. As the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors will result in errors in only one strand. This method uniquely capitalizes on the redundant information stored in double-stranded DNA, thus overcoming technical limitations of prior methods utilizing data from only one of the two strands.

This invention relates to the field of molecular marker screening technology, specifically to a SNP combination and method for paternity testing in cynomolgus monkeys. The method provided by this invention utilizes 100 selected specific SNP combinations (SNP1~SNP100) combined with low-depth sequencing for paternity testing in cynomolgus monkeys. The cost per sample is only 1 / 5 to 1 / 10 of existing high-depth sequencing and 1 / 2 of SNP chips. For large-scale experimental monkey populations (such as populations of thousands), this can save hundreds of thousands of yuan in testing costs annually, significantly reducing the economic burden of breeding and management of experimental monkeys, demonstrating extremely high economic efficiency. The method provided by this invention is applicable to cynomolgus monkey population pedigree management, kinship tracing, breeding pair optimization, and genetic quality control, and can be widely applied in experimental monkey breeding farms, primate research centers, and biomedical research institutions.

The invention discloses a construction, high-throughput sequencing and transcription intensity analysis method of a promoter random library. The invention establishes a process and an implementation method for rapidly researching the relative transcription intensity of a large number of promoters in a random promoter library, the promoter random library is rapidly constructed through a random primer method, and the transcription intensity of a large number of promoters is analyzed through deep sequencing of random tags carried by plasmid DNA and transcribed mRNA. According to the promoter library construction-library construction-sequencing-analysis process disclosed by the invention, the transcription intensity of promoters with different sequences can be quickly obtained with high throughput, and the promoter library with ideal promoter transcription intensity span is obtained. The method is rapid and efficient, and the transcription intensity of massive promoter sequences can be obtained.

The application discloses an analysis method and system for noninvasive prenatal screening, and the method comprises the following steps: obtaining target high-depth sequencing data of a target sample; performing data preprocessing on the target high-depth sequencing data to obtain depth information and SNP site information of a target region; obtaining a test result through global Z test based on copy number information, obtaining a first analysis result through local microdeletion / microduplication analysis by using a forward-backward search algorithm, and obtaining a classification result through a Bayesian classifier; obtaining a second analysis result through SNP analysis based on the SNP site information; and finally comprehensively judging to obtain a target analysis result. The application can effectively reduce the complexity of experimental operation, reduce the complexity of the diagnosis process, reduce the sample error probability caused by multiple sampling, significantly improve the efficiency of clinical testing, has high practical value and application prospect, and can be widely applied to the technical field of data processing.

The invention relates to the technical field of nucleic acid molecule detection, and discloses a pre-amplification detection method of a low-abundance fusion gene. According to the invention, a high-sensitivity SNT-MRD platform technology and the specificity of a qPCR primer probe system are combined, and a two-stage PCR amplification strategy and an optimized primer probe design are adopted, so that effective enrichment and detection of trace fusion genes in plasma ctDNA are realized. Compared with the existing qPCR detection method, the method disclosed by the invention has the advantages that the target sequence is firstly enriched, and then specific detection is performed, so that the problem of non-specific amplification caused by a large number of cycles in single PCR is avoided; compared with a method purely depending on NGS deep sequencing, the method has the advantages that the dependence on sequencing depth and complex biological information analysis is reduced, sensitive detection can be realized on a conventional qPCR instrument, the cost is lower, and the speed is higher.

The application discloses a method for simulating high-depth sequencing TSS characteristics. The method comprises the following steps: (1) obtaining reference genome captured free DNA sample sequencing data and sequence coverage of the upstream and downstream regions of the TSS of each gene; (2) obtaining features for constructing TSS values of simulated high-depth sequencing results based on the low-depth sequencing results of free DNA; and (3) constructing a TSS value model of simulated high-depth sequencing results based on the features of the low-depth sequencing results of free DNA. According to the application, the features are extracted based on the low-depth sequencing results, a model is constructed, the TSS characteristics equivalent to high-depth sequencing can be obtained, the number of analyzable gene TSS regions can be effectively increased, the stability of the sequencing fragment coverage of the analyzable gene TSS regions is increased, the sequencing cost is greatly reduced while the calculation accuracy of the TSS values is ensured.

Methods and systems provided herein address current limitations of bisulfite-based methylation sequencing by improving the quality and accuracy of nucleic acid methylation sequencing and uses thereof for detection of disease. Methods that include minimally-destructive conversion methods for methylation sequencing as well as specialized UMI adapters provide for improved quality of sequencing libraries and sequencing information. Greater accuracy and more complete methylation-state information permits higher quality feature generation for use in machine learning models and classifier generation.

Methods and systems provided herein address current limitations of bisulfite-based methylation sequencing by improving the quality and accuracy of nucleic acid methylation sequencing and uses thereof for detection of disease. Methods that include minimally-destructive conversion methods for methylation sequencing as well as specialized UMI adapters provide for improved quality of sequencing libraries and sequencing information. Greater accuracy and more complete methylation-state information permits higher quality feature generation for use in machine learning models and classifier generation.

The application provides a general avian infectious bronchitis virus whole genome deep sequencing method and application. The method realizes efficient amplification of long fragments of whole genomes of different genotype IBV epidemic strains by designing and applying a set of combinations containing 24 pairs of specific primers. The specific sequences of the primer combinations are shown in SEQ NO:1 to SEQ NO:48. Compared with a traditional sequencing method, the application significantly reduces the sequencing cost. Only two PCR reactions are needed, and more than 27 kb of IBV full-length genome sequence can be successfully obtained, which provides a more economical and efficient solution for IBV related scientific research and molecular epidemiology monitoring, and helps to promote the development of related field scientific research.

This invention provides a method for detecting genome-wide ROH based on ultra-low depth sequencing. The method first acquires the raw PGT-A sequencing data of the sample, extracts high-frequency SNP loci from the population, and screens out loci on autosomes with sequencing depths of 2-4X to construct a locus set. Then, the heterozygosity index (HetRR) and its standard score (Z) are calculated. Based on thresholds, the results are judged as follows: HetRR < 0.1 and Z ≤ -3.0 indicates genome-wide ROH; 0.1 ≤ HetRR < 0.5 and Z ≤ -3.0 indicates ROH chimerism; HetRR ≥ 1.05 and Z ≥ 3.0 suggests triploidy or DNA contamination; the rest are normal. This method can utilize sequencing data as low as 0.01X and can be simultaneously detected in the routine PGT-A process without additional operations or costs, improving the accuracy of embryo screening and possessing high clinical application value.

The invention discloses a method for separating cochlea related cells in situ based on a patch clamp system. The method comprises the following steps: contacting an electrode with a target single cell under a microscope, and carrying out in-situ grabbing on the target single cell through negative pressure adsorption; the target single cell and surrounding cells are kept in a connection state before in-situ grabbing; before the electrode approaches the target single cell, positive pressure is continuously applied, and when the electrode is in contact with the surface of the target single cell, the positive pressure is instantly stopped and switched to negative pressure. According to the method provided by the invention, high-quality cochlea related cells can be obtained in situ for deep sequencing analysis, and the method can be used for basic research and accurate diagnosis and treatment of clinical hearing impairment, and has a good application prospect.

The application discloses a methylation marker combination for detecting early esophageal cancer and application thereof. The marker combination comprises 178 methylation sites of 12 specific DNA regions and a specific internal marker region, and the methylation levels of the regions in plasma of patients with early esophageal cancer and healthy people are significantly different. The application also provides a primer combination and a kit for detecting the marker combination, and supports high-throughput deep sequencing. A detection system is used to extract plasma free DNA, perform bisulfite conversion and amplification, calculate a prediction value by using a logistic regression model, multiply the average methylation rate of each region by a corresponding weight coefficient, and compare the result with a threshold value to make a positive judgment. The application greatly improves the detection sensitivity of non-invasive screening for early esophageal cancer under the premise of ensuring high specificity, and has a wide clinical application prospect.

This invention provides a mutation site screening method based on amplicon deep sequencing and sequence counting, belonging to the field of mutant screening technology. The invention constructs a sample pool by mixing multiple individual samples in a specified ratio, extracts genomic DNA, performs tagged amplification, and uses high-throughput sequencing to obtain amplified fragment sequences. After quality control, redundancy removal, and frequency screening of the sequencing data, candidate sequences are compared with target genes to identify mutation sites. If mutations are found, the individual samples in the mixed pool are further validated. This invention has the advantages of high throughput, simple analysis, and high detection sensitivity, and is suitable for mutant screening in various species.

The invention relates to the technical field of biochips, in particular to a sturgeon SNP (Single Nucleotide Polymorphism) liquid chip and application thereof. The invention provides a sturgeon SNP (Single Nucleotide Polymorphism) liquid chip based on targeted capture sequencing aiming at the current situation that sturgeon molecular markers are limited and independent research and development of sturgeon chips are lacked at present. The sturgeon SNP liquid phase chip is designed on the basis of high-depth sequencing data of multi-species populations, the coverage of SNP sites is comprehensive, and the amount of information is rich; a targeted capture sequencing technology is adopted, so that the detection efficiency is high, the accuracy is high, and flexible expansion and upgrading can be realized. The sturgeon SNP liquid phase chip disclosed by the invention can simultaneously meet various application requirements of sturgeon molecular identification, genome selection, functional gene research and the like. The invention fills the blank of independent research and development of sturgeon liquid phase chips, and has important scientific research value and industrial application prospect.

This invention belongs to the field of gene editing technology and discloses an inosine-enriched adenine base editor off-target detection method (Ino-seq) and its applications. The method of this invention utilizes endonuclease V to specifically cleave inosine-containing DNA, combined with the stabilization of a thermostable single-stranded DNA-binding protein and the high affinity enrichment of streptavidin-biotin, to achieve direct capture of ABE enzymatic products. This method can simultaneously detect sgRNA-dependent and independent off-target effects, with systematic comparisons showing F1 scores of 0.892–0.931. Targeted deep sequencing validation showed an overall accuracy of 95.3%. Ino-seq can also detect endogenous genomic inosine, observing a variation range of >1,000-fold in 10 cell lines. This invention provides a crucial safety assessment tool for the clinical translation of ABE technology, supporting comprehensive off-target analysis in preclinical and clinical studies.

The invention relates to the field of biological medicine, and discloses an integrated genome analysis method based on low-depth sequencing, and the method comprises the following steps: obtaining whole genome low-depth sequencing data; after quality control and comparison, SNV, Indel and CNV are calculated; performing multi-dimensional function annotation by combining genome position, coding influence, splicing disturbance, conservative property, regulatory element and three-dimensional chromatin interaction; database information such as ClinVar and HGMD is integrated, and according to a phenotype-driven rule engine, a clinical interpretable report is generated according to the ACMG / AMP standard. According to the method, a complete analysis chain covering sequence variation detection, multi-dimensional function annotation, three-dimensional genome association, public database integration and phenotype driven interpretation is constructed, so that the fundamental defect that only an original variation list is output and a clinical action basis cannot be provided in traditional low-depth sequencing is overcome.

The invention discloses a corn pathogen early warning method and system based on big data, and relates to the technical field of agricultural early warning, the method comprises the following steps: comparing sequences in historical small RNA deep sequencing data and verified viral pathogen monitoring data, and establishing a mapping relationship between the two to obtain a pathogen-small RNA reference corresponding relationship library; matching the small RNA deep sequencing data of the current monitoring period with the pathogen-small RNA reference corresponding relationship library, and extracting sequence fragments consistent with the mapping relationship in the library to obtain a target viral pathogen feature sequence; coupling the target viral pathogen feature sequence with the current environmental factor data; according to the method, the core value in the multi-source data can be effectively mined, the early infection signal of the pathogen can be accurately captured, the early accurate prediction of the transmission risk and potential outbreak of the corn viral pathogen is realized, a timely and reliable decision basis is provided for field prevention and control, and the corn production safety is guaranteed.

The invention provides a mutation site screening method based on amplicon depth sequencing and sequence counting, and relates to the technical field of mutant screening. The method comprises the following steps: mixing a plurality of individual samples in proportion to construct a sample pool, extracting genome DNA, performing tagging amplification, and obtaining an amplified fragment sequence by adopting high-throughput sequencing; carrying out quality control, redundancy elimination and frequency screening on the sequencing data, then screening a candidate sequence, comparing the candidate sequence with a target gene, and identifying a mutation site; and if mutation exists, further verifying the individual sample in the mixing pool. The method has the advantages of high flux, simplicity and convenience in analysis and sensitivity in detection, and is suitable for screening mutants of various species.

To provide a method for performing methylation sequencing on nucleic acid molecules of a biological sample.SOLUTION: The method comprises: (a) preparing a methylation sequencing library from cell-free deoxyribonucleic acid (cfDNA) fragments of nucleic acid molecules; (b) enriching the methylation sequencing library for sequences corresponding to CpG or CH loci; (c) sequencing the enriched methylation sequencing library at a depth greater than 100x using single-end reads or paired-end reads; (d) correcting sequencing errors present in overlapping regions of the paired-end reads for each sequenced fragment of the paired-end reads; (e) collapsing the sequenced fragments into strand read families; and (f) collapsing the strand read families into duplex read families.SELECTED DRAWING: Figure 1

The invention relates to a method for constructing a porcine endogenous retrovirus inactivated xenotransplantation donor pig, and belongs to the technical field of xenotransplantation. And the copy number of the PERVs and the genotype of the PERV-pol are determined through ddPCR, Sanger sequencing and second-generation PCR deep sequencing. The method comprises the following steps: designing and synthesizing a porcine PERV-pol gene targeting sequence based on a CRISPR / Cas9 RNP system, co-transfecting the porcine PERV-pol gene targeting sequence and spCas9 protein into a porcine fetal fibroblast line, screening to obtain positive cells, carrying out somatic cell cloning and embryo transplantation, taking out a fetus during pregnancy, identifying the PERV-pol knockout condition, and if the PERV-pol knockout condition is completely carried out, carrying out somatic cell cloning and embryo transplantation; and if the PERV-pol gene is not completely knocked out, secondarily designing and synthesizing an sgRNA targeting sequence, transfecting the sgRNA targeting sequence into the fetal fibroblast cell line, screening to obtain a cell line in which the PERV-pol gene is completely knocked out, and carrying out somatic cell cloning and embryo transplantation to obtain the PERVs inactivated xenotransplantation donor pig. The problem that the PERVs inactivation efficiency is low is solved, and the PERVs-inactivated xenotransplantation donor pig can be obtained through one round or multiple rounds.

The invention discloses a design method of a specific methylation marker Panel, which comprises the following steps: obtaining a first candidate interval based on a methylation related gene of a selected disease, screening a first target interval in the first candidate interval, and extracting a first CpG site set in the first target interval; performing methylation sequencing data on lesion tissues of the selected disease crowd to obtain a second candidate interval, screening a second target interval in the second candidate interval, and extracting a second CpG site set in the second target interval; and combining the first CpG site set and the second CpG site set to obtain a Panel interval covering the CpG sites therein. According to the design method, researchers or medical personnel can be helped to obtain the methylation Panel of the required disease, the methylation level resolution based on high-depth sequencing data is improved, and the efficiency of screening methylation markers related to the specific disease is improved.

The present application relates to the fields of molecular biology technology and livestock breeding technology, and particularly to a large white pig low back fat selection method based on SNP marker rs340365542, the SNP molecular marker is located at the 10107037th nucleotide of the 3rd chromosome of pig genome version Ensembl Sscrofa11.1, and the polymorphism is T or G; application of the SNP molecular marker rs340365542 related to pig back fat thickness in the large white pig low back fat selection method, and particularly includes application in identifying or screening low back fat pig breeds and application in genetic improvement of pig breeds for reducing pig back fat thickness; the present application adopts a specific strain-large white pig core group, a specific low-depth sequencing strategy, a customized quality control process and a population structure control model to perform whole genome correlation analysis on the back fat thickness trait, and carries out targeted technical optimization for screening out sites with dominant and universal characteristics in large white pigs.