78 results about "Amplicon sequencing" patented technology

The invention discloses a method for detecting copy number change based on an amplicon capture technology and application of the method. According to the method, the sequencing data difference caused by amplicon sequencing and the deviation caused by PCR reaction can be reduced, so that accurate and stable analysis of the copy number change of the target area is realized, and the method can be applied to analysis of the copy number change of a specific gene or genome area.

The invention discloses a fiveleaf virus GI.2 whole genome sequencing primer based on an amplicon sequencing technology and application thereof, and belongs to the technical field of virus gene sequencing. The primer group composed of 18 pairs of primers is preferably obtained according to an amplicon sequencing technology, the primer group can effectively improve the primer amplicon coverage degree, it is guaranteed that the primer amplicon coverage degree is 1.5-1.8 times that of a virus sequence, the amplification time is shortened, the amplification difficulty is reduced, the primer amplicon coverage degree and the whole genome capture efficiency are improved, and the primer amplification efficiency is improved. And the method has important significance on epidemic situation traceability and propagation trend of the fiveleaf virus GI.2.

Provided herein are multi-modal methods and systems of diagnosing one or more disease, as described elsewhere herein.

The invention provides a multi-modal soil microorganism classification method based on deep learning. The method comprises the following steps of: cleaning and formatting two types of modal data by using a data preprocessing technology to ensure the quality and consistency of the data, then constructing an efficient feature extraction module, respectively performing feature extraction on the image and amplicon sequencing data in combination with a Transform network and a self-normalization network (SNN) suitable for the sequencing data, and then performing feature extraction on the image and the amplicon sequencing data to obtain a feature extraction result; according to the method, a common attention fusion module is introduced to effectively integrate image features and gene sequence features and capture correlation among different modals, in addition, a multi-modal feature alignment strategy is adopted, feature representation is optimized through a contrast learning method, and therefore the recognition capacity of the model for microbial species is enhanced.

The invention discloses an application of intestinal bacteria in an ulcerative colitis prediction technology, and belongs to the technical field of biology, and the intestinal bacteria are Alistipes finegoldii; the method comprises the following steps: detecting the relative abundance of Alistipes finegii in an excrement sample of a patient, and using the low relative abundance of Alistipes finegii to predict the disease activity of ulcerative colitis; according to the application disclosed by the invention, not only is the association between the Alistipes finegili and the UC disease activity verified, but also the potential action mechanism of the Alistipes finegili is further explored. By means of 16s amplicon sequencing and transcriptomics combined analysis, it is found that the Alistipes finegili plays an anti-inflammatory role through an Hsp90 / AMPK / ACC signal channel, and the intestinal barrier function is maintained. The invention verifies that a new view angle is provided for the effect of the Alistipes finegili in the UC pathogenesis, and provides a theoretical basis for developing a treatment strategy based on the strain.

Methods are provided for amplifying microbial cell free DNA (mcfDNA) in blood samples. In contrast to amplicon sequencing, in which regions between two conserved sequences are targeted for PCR amplification, the methods herein use a single conserved sequence as the primer annealing site to initiate PCR amplification. Enrichment PCR (E- PCR) is performed using a degenerate primer complementary to a conserved region in a microbial phylogenetic marker gene and an additional primer to the opposite end of the mcfDNA. The conserved region is adjacent to a hypervariable region. Amplification PCR is performed on the enriched mcfDNA fragments using a modified degenerate primer having a 3 '-end extension relative to the degenerate primer, and the additional primer, thereby generating enriched amplified mcfDNA fragments. Sequencing the amplified mcfDNA enables identification of microbial species, which is useful for diagnosing and monitoring inflammatory diseases and cancer, including stratification of cancer by stage, type, and tumor subtype.

The invention discloses a microsatellite instability detection method and device based on a next-generation sequencing technology, and belongs to the technical field of biological detection. The method comprises the following steps: selecting MSI sites, and carrying out capture probe design and / or amplicon primer design based on any combination of the sites; selecting a plurality of samples, and calculating the missing fragment proportion and the normal fragment proportion of each sample at each MSI site; selecting a plurality of samples which are judged to be in an MSI-L or MSS state by a PCR method as baseline samples, and setting different depth levels for each baseline sample to construct a baseline; carrying out MSI state detection; selecting a sample of which the MSI state is MSI-H, and judging whether the sample is an abnormal sample or not through a Spearman rank correlation coefficient; and if yes, adjusting the base line and re-judging the MSI state. The method has high stability and high accuracy, does not need a control sample, and can be simultaneously applied to microsatellite instability detection of hybridization capture and amplicon sequencing data.

The invention discloses a method for detecting gene copy number variation based on an amplicon sequencing technology, which can accurately calculate the input molecule number of a target area based on the amplicon sequencing technology by introducing a correction template into a PCR amplification reaction system for correcting the amplification efficiency and amplification preference of a to-be-detected sample. The variation condition of the copy number of the target area of the to-be-detected sample can be accurately judged according to the comparison condition of the copy number of the to-be-detected sample and the number of the input molecules of the internal reference area.

The application provides a STR typing method applied to second-generation sequencing data, and relates to the technical field of biology.The method specifically comprises the following steps: customizing an STR locus configuration file; constructing a k-mer index library of a flanking sequence of the STR locus; extracting STR allele sequences from each READ of a sequence alignment file in combination with a CIGAR value and the k-mer index; constructing a PCR ghost peak model of the STR locus, and estimating parameters of the PCR ghost peak model by using a maximum likelihood estimation method; estimating candidate allele frequencies by using a second-generation sequencing sample data set; calculating posterior probabilities of all candidate alleles by using an allele set extracted from a given sequence alignment file, and inferring the most possible allele.The STR typing method has higher detection rate and typing accuracy, and has faster typing speed, and can be used for whole genome sequencing data, high-coverage targeted sequencing data or amplicon sequencing data.

The invention discloses a fiveleaf virus GII.3 whole genome sequencing primer based on an amplicon sequencing technology and application thereof, and belongs to the technical field of virus gene sequencing. According to the whole genome sequencing primer and the sequencing method for the fiveleaf virus GII.3, interference of other microorganisms on sequencing can be avoided, the gene sequencing depth is high, the advantages of being wider in whole genome coverage, high in detection accuracy and high in specificity are achieved, the sequencing result can reflect mutation characteristics of main epidemic strains of the fiveleaf virus, and the sequencing result can be used for detecting the mutation characteristics of the main epidemic strains of the fiveleaf virus GII.3. And a powerful tool is provided for rapid diagnosis and full prevention of the fiveleaf akebia viruses.

The invention discloses a megalurothrips megalurothrips methylamino abamectin benzoate resistance molecule detection method which comprises the following steps: (1) collecting megalurothrips megalurothrips female adults in a plurality of regions, and extracting DNA (deoxyribonucleic acid) in a single region in a mixed manner; (2) designing glutamic acid gated chloride ion channel gene specific primers of the megalurothrips megalurothrips, respectively adding specific tags at the front ends of the primers in all regions, and carrying out PCR (Polymerase Chain Reaction) amplification; mixing the PCR products of each region to obtain a library; (3) performing second-generation amplicon sequencing on the library to obtain off-machine data; and (4) analyzing offline data, merging the data in the same region, calculating the mutation frequency of each region by taking the ratio of the mutation (G232S) of the target sequence as an index, and judging the resistance of the megalurothrips usitatus to the emamectin benzoate. The method can be used for rapidly and massively monitoring the emamectin benzoate resistance condition of the field megalurothrips megalurothrips, and an important basis is provided for high-level emamectin benzoate resistance early warning and megalurothrips megalurothrips chemical treatment.

The invention discloses a specific amplification primer pair for human intestinal archaea flora 16S rRNA amplicon sequencing and application of the specific amplification primer pair, and belongs to the technical field of biology.The primer pair is designed for 16S rRNA high-throughput sequencing and can specifically amplify human intestinal archaea, the length of an amplification product is about 385bp, and the amplification product covers a V4-V5 variable region of a 16S rRNA gene; the invention also optimizes amplification conditions of the primer pair, and experimental results show that the primer pair is superior to a conventional universal primer for bacteria and archaea in the aspect of amplifying species diversity and species abundance of the intestinal archaea, and a standardized method is provided for efficient and accurate amplification of intestinal archaea florae.

The invention provides an excrement sample preserving fluid as well as a preparation method and application thereof, and belongs to the technical field of biological detection. The problems in the prior art are solved. The excrement sample preserving fluid comprises the following components: a buffer reagent, a chelating agent, sodium chloride, Tween 20, sodium citrate and glycine, and the weight volume ratio of the sodium chloride is 0.5 to 1.3 percent, the weight volume ratio of the Tween 20 is 0.2 to 1.0 percent, the weight volume ratio of the sodium citrate is 0.6 to 1.4 percent, the weight volume ratio of the glycine is 0.5 to 1.0 percent, and the balance is sterile deionized water. The preparation method of the fecal sample preserving fluid comprises the steps of proportioning, weighing, dissolving, pH adjusting, volume metering, filtering and preserving, and the fecal sample preserving fluid is used for extracting intestinal microorganism DNA from a fecal sample. The method not only has a good preservation effect, but also does not contain toxic components such as guanidine thiocyanate and the like, is safer to operate, and can be used for downstream 16S amplicon sequencing and metagenome sequencing.

This invention discloses a method for constructing a multi-target amplicon abundance standard for microorganisms and its application, belonging to the fields of molecular biology detection and microbiome analysis. This standard material screens sequences of common human gut microbiota strains, designs and adds universal primers, and obtains 14 DNA fragments of different lengths through gene synthesis, cloning, and purification, which are then mixed according to a preset abundance. It retains natural characteristics such as primer binding sites and GC content, and can systematically correct technical deviations in amplicon sequencing, solving the problem of species abundance distortion. This standard material functions as both a non-homologous internal reference and a homologous external reference, and can be used for laboratory proficiency testing, reagent kit performance evaluation, and cross-platform data calibration, promoting the leap from relative qualitative to absolute quantitative research in microbiome studies and providing metrological support for the standardization and precision of detection results.

The present invention discloses a method for identifying, quantifying and visualizing the genome of integron-resistance gene boxes in environmental samples, which is a high-throughput identification, quantification and genome visualization of mobile genetic element integrons and drug-resistant genes mediated by them based on third-generation sequencing; including: 1) identification and quantification of integrons: using the "collection-screening-verification" strategy to construct an integrase gene database and apply it to integron identification and quantification; 2) identification of drug-resistant gene boxes and genome visualization: using the preferred variable region gene box primers combined with PCR technology to obtain its DNA, using PacBio third-generation full-length amplicon sequencing to obtain the gene box diversity long sequence, identifying the integron-mediated drug-resistant gene box category based on a professional database, using a custom R script to visualize the integron-multiple drug-resistant gene box array, and quantification based on short sequence and drug-resistant gene box long fragment mapping. The present invention provides technical support for monitoring and controlling the development of bacterial drug resistance.

The invention discloses application of bitter gourd exosomes in regulating intestinal flora. The method comprises the following steps: establishing an Alzheimer's disease mouse model, injecting the extracted balsam pear exosome into the mouse body in a gavage manner to serve as an experimental group drug, taking normal saline as a model negative control group, detecting the change of the intestinal flora of the AD mouse by applying a 16S amplicon sequencing principle, and detecting the change of the intestinal flora metabolite of the AD mouse by applying an LS-MS technology. An open field experiment, a nesting experiment and a water maze experiment are adopted to verify the treatment effect of the balsam pear exosome on AD mouse intestinal flora and metabolic level changes thereof. Results prove that the bitter gourd exosome extracted by the invention can effectively improve the intestinal flora micro-ecology of AD mice; the compound can be used for preparing a microecological preparation for regulating intestinal flora of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, different types of spinal cerebellar ataxia and the like and diseases such as cerebral apoplexy, cerebral injury, epilepsy, tumor and the like.

The invention relates to a targeted sequencing primer composition for simultaneously detecting 41 infectious disease pathogens, a kit and a library building method. The primer composition comprises a forward specific primer group, a reverse specific primer group, a bridge primer, a universal primer 1 and a universal primer 2, which are specifically as shown in SEQ ID NO.1-139. The invention also provides a kit containing the primer composition, and a method for constructing an amplicon sequencing library by using three primers in a single tube with a non-diagnostic purpose. According to the invention, 68 pairs of specific primers covering viruses, bacteria and parasites are designed aiming at 41 infectious disease pathogens determined by the state, so that comprehensive and specific pathogen coverage is realized. And through bioinformatics verification and experimental verification, high specificity of the primer is ensured, a target gene conserved region can be accurately recognized, and non-specific amplification is effectively avoided.

A primer group for simultaneous detection of 15 porcine pathogens through high-throughput targeted amplicon sequencing and use thereof are provided. The primer group for simultaneous detection of 15 porcine pathogens through high-throughput targeted amplicon sequencing includes: primers having nucleotide sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 302. The nucleotide sequences of pathogenic target genes are set forth in SEQ ID NO: 303 to SEQ ID NO: 344. The primer group of the present invention is used for amplicon sequencing detection analysis method for 42 target genes of 15 porcine respiratory pathogens, has the technical advantages of rapidness, high efficiency, high targeting, strong specificity, and the like, and is suitable for rapid and accurate simultaneous diagnosis of multiple pathogens of diseased pigs in pig farms.

This invention belongs to the field of genetics and provides a technique and data analysis method for simultaneously detecting 193 Y-STRs in the Chinese population based on an NGS platform. This invention discloses 193 Y-STR loci for human Y-chromosome STR genotyping; the selected 193 Y-STRs are used for library construction using a two-step PCR method. This invention has advantages such as rapid amplification, consistent efficiency, high sensitivity, and good data visualization; due to the high depth of high-throughput sequencing, it is more conducive to the analysis of mixed and degraded materials.

Embodiments disclosed herein provide methods of using synthetic DNA spike-ins (SDSIs) to detect, prevent, and quantify contamination in amplicon sequencing. These embodiments may, but are not limited to, reveal sample swaps, intra-batch contamination, and, on a larger scale, intra-laboratory contamination. Embodiments disclosed herein also provide synthetic DNA spike-ins for use in amplicon-based sequencing methods.

The application discloses a method for in-situ collection and statistics of marine cyanobacterial symbiotic system and free cyanobacteria, which comprises the following steps: firstly, multiple gradient filtration pretreatment is performed on seawater samples, and the samples are sequentially filtered through 5 μm, 3 μm (including secondary filtration and slight ultrasonic) and 0.22 μm filter membranes, so that the samples are preliminarily separated according to particle sizes and different samples are obtained, and false symbiotic systems are removed; then, flow cytometry sorting is performed, eukaryotic algae interference is removed according to the differences of five light signals of FSC, SSC, APC, PE and PerCP-cy5.5, and symbiotic cyanobacteria, symbiotic heterotrophic bacteria and free cyanobacteria are sorted out; and the sorting results can be verified through 16S amplicon sequencing. The application does not intervene in biochemistry in the whole process, can keep the original state and natural proportion of microorganisms to the maximum extent, and realizes in-situ, accurate separation and statistics of marine cyanobacterial symbiotic system and free cyanobacteria.

The invention discloses a mutation back noise filtering algorithm based on next-generation sequencing data, relates to the technical field of high-throughput sequencing data analysis, and aims to solve the problems that the prior art depends on a baseline, and is poor in adaptability and high in false positive. The algorithm comprises the following steps: preprocessing sequencing data to obtain a pileup file, classifying sites according to a sequence context, calculating an error rate, constructing a layered back noise model to check mutation, qualitatively determining real mutation and back noise by combining a sample type, and evaluating sample quality. The method does not need to construct a base line in advance, adapt to hybrid capture and amplicon sequencing, can improve mutation detection specificity and repeated sample consistency, guarantees result reliability, and is suitable for scenes such as tumor gene detection.

The invention belongs to the technical field of fungus detection, and particularly relates to a method for rapidly screening high-frequency cross-border fungi based on high-throughput sequencing, which comprises the following steps: acquiring an imported plant sample, carrying out epiphytic microorganism extraction, DNA extraction, PCR amplification, purification, carrying out high-throughput sequencing, automatic data filtering statistics, automatic OTUs clustering analysis and the like. According to the invention, high-throughput amplicon sequencing is used for identifying common fungi, and the method is suitable for high-throughput rapid identification of common plant pathogenic fungi in imported plants and plant products.

The invention relates to a method for analyzing and evaluating the corrosion degree of power grid derrick carbon steel in different regions by utilizing 16S rRNA amplicon sequencing. The method comprises the following steps: performing genome DNA extraction and quality control on microorganisms of soil samples at contact corrosion positions of derricks and soil layers in different regions of a power grid system, performing 16S rRNA amplicon sequencing, performing OTU clustering analysis, performing species annotation and the like. Through bioinformatics analysis and combination with a corrosion degree standard, a method for evaluating the corrosion degrees of the carbon steel in different regions through a result of microorganism composition is established, and carbon steel corrosion prevention is guided.

The invention relates to a method for detecting chromosome translocation of HLA related genes based on an optimized primer design strategy. The method comprises the following steps: sequencing individual HLA related genes, and comparing the sequenced HLA related genes with HLA-A, HLA-B and HLA-C gene reference sequences to realize preliminary classification; through SNP analysis, HLA-A1 and HLA-A2, HLA-B1 and HLA-B2, and HLA-C1 and HLA-C2 are further divided from individual HLA related genes and then are compared pairwise, an obtained comparison group is subjected to editing distance calculation, a corresponding specific amplification primer is designed after a specific region is screened out, then amplicon sequencing is performed on a gene editing sample, and a specific amplification primer is designed after the specific region is screened out. And carrying out chromosome translocation detection after removing a non-specific amplification sequence. According to the method, specific primers are designed according to sequence diversification and individual differentiation of HLA genes, different HLA alleles can be effectively recognized and distinguished, and chromosome translocation results can be accurately and rapidly judged.

Provided are a method for characterizing inter-well connectivity, a device, a medium, and a product. The method includes: collecting a fluid microbial sample from an injection well and each of the production wells in a selected well field; subjecting each fluid microbial sample to 16S rRNA gene amplicon sequencing to obtain raw sequencing data of the injection fluid and raw sequencing data of each production fluid; extracting amplicon sequence variants (ASVs) from the raw sequencing data of the injection fluid and the raw sequencing data of each production fluid to obtain an ASV table of the injection fluid and an ASV table of each production fluid; and subjecting the ASV tables of each injection-production well pair to two-circle Venn diagram analysis to obtain the number of ASVs overlapped for each injection-production well pair, and determining inter-well connectivity between each injection-production well pair accordingly.

The taxonomic resolution obtained with conventional sequencing methods like Sanger (longer read lengths) takes a huge amount of time. While, NGS technologies (shorter read lengths) involves a lot of cost in sequencing. In addition to that the accuracy and depth of taxonomic classification is also less. A method and system for improving accuracy of amplicon sequencing based taxonomic profiling of microbial communities has been provided. The proposed strategy relies on obtaining taxonomic abundance profiles of a microbial community from two paired-end sequencing experiments, each of which targets different pair-wise combinations of non-contiguous (or contiguous) V-regions. The two taxonomic profiles are then combined based on (pre-estimated) accuracies of the individual V-regions (targeted in the experiments) in resolving each of the taxonomic groups under consideration.

The invention discloses a hepatitis B virus combined mutation detection method and device based on high-throughput sequencing. A novel detection and analysis method is established on the basis of targeted amplicon sequencing, the combined mutation of a basic core promoter and a front core region on a single virus DNA molecule can be accurately analyzed, and the relevance of the combined mutation with clinical phenotypes and the influence of the combined mutation on virus functions are further explored on the basis of the combined mutation. The invention provides a new technical means for detecting the combined mutation in the specific region of the hepatitis B virus, makes up for the deficiency of the recognition capability of the traditional detection method on the combined mutation, not only perfects the research on the mutation in the specific region of the hepatitis B virus, but also is beneficial to the individualized diagnosis and treatment of the hepatitis B virus and the prognosis evaluation of diseases.

The invention provides an STR typing method applied to next-generation sequencing data, and relates to the technical field of biology. The method specifically comprises the following steps: customizing an STR locus configuration file; constructing a flanking sequence k-mer index database of the STR gene locus; an STR allele sequence is extracted from each READ of the sequence comparison file in combination with the CIGAR value and the k-mer index; constructing a PCR shadow peak model of the STR gene locus, and estimating parameters of the PCR shadow peak model by using a maximum likelihood estimation method; estimating a candidate allele frequency by using the next-generation sequencing sample data set; and calculating posterior probabilities of all candidate alleles by using an allele set extracted from a given sequence comparison file, and deducing the most probable allele. The STR typing method has the advantages of higher detection rate, higher typing accuracy and higher typing speed, and can be used for whole genome sequencing data and high-coverage targeted sequencing data or amplicon sequencing data.

Methods and apparatus for assessing quality of a genetic variant for inclusion as a biomarker in a circulating tumour DNA (ctDNA) assay. The method includes receiving genetic variants associated with a sample collected from a patient, the set of genetic variants including a first genetic variant, determining values for a plurality of characteristics associated with the first genetic variant, providing the values for the plurality of characteristics as input to a trained machine learning (ML) model, the trained ML model being trained to output a quality of a genetic variant, the quality of the genetic variant representing a likelihood that the genetic variant is both somatic and will sufficiently amplify using amplicon sequencing, and including the first genetic variant in a panel of genetic variants for use in a ctDNA assay for the patient based on the quality of the first genetic variant output from the trained ML model.