45 results about "Amplicon sequencing" patented technology

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 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.

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 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.

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 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 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 application discloses a sequencing data calibration system and method based on microbial amplicon sequencing, and belongs to the field of microbial detection and bioinformatics analysis. The application takes ddPCR technology as the core, first screens common human intestinal microorganisms, designs and obtains 14 reconstructed sequences containing bacterial 16S and fungal 18S / ITS, prepares high-purity DNA standard substances through chemical synthesis and molecular cloning, completes standardization and uniformity verification, then designs high-specificity primers and probes, optimizes ddPCR reaction conditions, establishes a calibration method, completes uncertainty evaluation through joint calibration of 9 laboratories, and prepares multi-target amplicon abundance standard substances. The application realizes the transformation of amplicon sequencing data from relative quantification to absolute quantification, corrects the system error of the whole sequencing process, improves the comparability and reliability of cross-platform and cross-batch data, and provides technical support for the standardization of microbiome research.

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker related to a Ningxiang pig body length character and an application of the SNP molecular marker. The molecular marker comprises an SNP1 and an SNP2; the SNP1 molecular marker takes a Sus Scrofa Build11.1 genome as a reference genome and is located at the 15776004 site of a No.1 chromosome, the polymorphism of the SNP1 molecular marker is T / C, and the serial number of the SNP1 molecular marker is rs334683505; the SNP2 molecular marker takes a Sus Scrofa Build11.1 genome as a reference genome and is located at the 15832958th site of a No.1 chromosome, the polymorphism of the SNP2 molecular marker is A / C, and the serial number of the SNP2 molecular marker is rs321288338. The molecular marker obviously related to the Ningxiang pig body length character is obtained, and an amplicon sequencing primer group capable of being used for identification analysis and large-scale detection is designed. The Ningxiang pig individuals with the BL sizes meeting the requirements can be efficiently and accurately identified and screened out.

The invention discloses a multi-dimensional comprehensive evaluation method for microbial diversity of a hot spring, which comprises the following steps: collecting a water sample, a sediment and a biological membrane along the temperature gradient of the hot spring to form a sample, and carrying out pretreatment and in-situ fixation on the sample; carrying out metagenome, metatranscriptome and amplicon sequencing treatment on the sample; calculating three-dimensional indexes of a structural stability index SSI, a functional activeness score FAS and abundance of an environmental adaptability gene set through multi-omics integration analysis; and generating a biodiversity comprehensive evaluation report through a three-dimensional index based on the environmental adaptation index EAI. According to the method, three-dimensional quantitative analysis of structure-function-environment adaptability is realized. Through multi-omics stack analysis and environment factor coupling modeling, the problem of integrity deficiency of extreme environment biological resource evaluation is solved.

This invention discloses a method for constructing a next-generation sequencing library using long PCR amplicon sequences, comprising: performing PCR amplification on a target sequence using primers to obtain long PCR amplicon sequences; digesting and fragmenting the sequences using a mixed enzyme system, followed by end-repair and A addition, wherein the mixed enzyme system includes Exo I enzyme, UDG enzyme, and Taq DNA polymerase; ligating the A-added product to sequencing adapters to obtain adapter ligation products in the form of "adapter-DNA insert-adapter"; and performing a first round of purification to obtain a next-generation sequencing library, which can be used directly for sequencing or enriched by further PCR amplification before sequencing. This invention enables rapid construction of long PCR amplicon libraries for next-generation sequencing platforms, is simple to operate, and is suitable for large-scale applications. It uses UDG enzyme and dUTP to fragment the long PCR amplicon sequences, eliminating the need for specific restriction enzyme recognition sites and improving randomness.

The invention belongs to the technical crossing field of microbiomics, and relates to a method for constructing a steady-state microbiome on the surface of a pear based on serratia rubra to inhibit postharvest diseases. The method comprises the following steps: firstly, preparing serratia rubra B11 suspension, and spraying the serratia rubra B11 suspension on the surfaces of pear fruits; the method comprises the following steps: standardly collecting epiphytic microorganisms on the surfaces of fruits by adopting a mode of combining physical elution and vacuum filtration, extracting total DNA, respectively carrying out high-throughput amplicon sequencing on a 16S rRNA gene V3-V4 region and a fungus ITS2 region, and carrying out dynamic analysis on alpha diversity, beta diversity, community composition and key class groups, so as to determine the content of the epiphytic microorganisms on the surfaces of the fruits. And finally, associating with the fruit rotting rate and the physiological quality index, establishing a set of full-process standardized system of multi-dimensional bioinformatics analysis, and revealing an ecological mechanism that beneficial bacteria realize disease inhibition by remodeling a fruit surface microbial community structure and regulating and controlling key function class groups. And reliable technical support is provided for mechanism research, efficiency evaluation and application development of the biocontrol microbial inoculum.

Described herein are methods and compositions for analyzing nucleic acid sequences. Amplicon sequencing is particularly useful for genome targeting and detection of hot-spot mutations, copy number variations, gene fusions, InDels and single-nucleotide polymorphisms (SNPs). However, for applications such as personalized cancer monitoring, wide tracking of a plurality of variants requires use of a primer sequences of extraneous length. Methods and compositions are described herein that support reduction of the length of primers with benefits including creased purity, error reduction, reduced manufacture time and savings in cost and time if a shared primer could be applied.

This invention belongs to the field of plant rhizosphere research and discloses a method for determining the regulatory effect of exogenous selenium application on the rhizosphere microecology of Amorphophallus bulbifera. The method includes: experimental design and sample collection; soil microbial DNA extraction and amplicon sequencing; and quality control and analysis of the microbiome data. This invention utilizes Illumina MiSeq high-throughput sequencing technology to analyze the changes in the composition and diversity of rhizosphere soil bacteria communities in Amorphophallus bulbifera under exogenous selenium treatment, and further analyzes the functions of related communities through PICRUSt functional prediction analysis. The conclusions are as follows: exogenous selenium treatment induces significant changes in the rhizosphere soil bacterial community of Amorphophallus bulbifera, which is beneficial for creating a stable and more diverse rhizosphere microbial community structure.

The present invention discloses primers for sequencing the whole genome of Saruvirus GI.2 based on amplicon sequencing technology and their applications, belonging to the field of viral gene sequencing technology. Based on this technology, the present invention preferably obtains a primer set consisting of 18 primer pairs. This primer set can effectively improve primer amplicon coverage, ensuring coverage of 1.5-1.8 times the viral sequence, shortening amplification time and difficulty, and improving primer amplicon coverage and whole-genome capture efficiency. This is of great significance for tracing the source and spread of Saruvirus GI.2 epidemics.

The present application belongs to the field of bioinformatics analysis, and relates to an analysis method for predicting the drug-resistant gene composition and abundance and drug resistance rate of a bacterial community in a sample based on 16S rRNA amplicon sequencing results. The method of the present application greatly expands the application range of 16S rRNA amplicon sequencing, and provides a simple and easy-to-use and low-cost data re-mining method for the study of the spread of drug-resistant genes in the environment.

The invention relates to the technical field of bioinformatics and computational biology, in particular to a multiple PCR amplicon sequencing data detection method based on deep learning. The technical problem that the accuracy of overall detection of complex sequencing data is low in the prior art is solved. The method comprises the following steps: acquiring a to-be-detected sequencing data set; obtaining a reference sequence database; constructing a feature extraction model for extracting amplicon features based on the historical amplification condition information and the multiple groups of historical sequencing data of the target tag; and processing a to-be-detected sequencing data set based on the feature extraction model, and determining a target category corresponding to each sequencing sequence in the sequencing data set. The multi-PCR amplicon sequencing method is used for a multi-PCR amplicon sequencing scene.

The application discloses a mutation background noise filtering calculation method based on second-generation sequencing data, and relates to the technical field of high-throughput sequencing data analysis, to solve the problems that the prior art depends on a baseline, has poor adaptability and high false positives. The algorithm first preprocesses sequencing data to obtain a pileup file, classifies sites according to sequence context and calculates error rates, constructs a hierarchical background noise model to test mutations, combines sample types to qualitatively determine real mutations and background noise, and simultaneously evaluates sample quality. The algorithm does not need to construct a baseline in advance, is suitable for hybrid capture and amplicon sequencing, can improve mutation detection specificity and consistency of repeated samples, guarantees reliable results, and is suitable for tumor gene detection and other scenes.

The present invention discloses a method for designing and ligating primers for DNA amplification, particularly a library construction method for increasing the effective data volume of PacBio amplicon sequencing. The method includes designing different primer pairs for DNA molecules, and using the different primer pairs to perform PCR amplification on the template DNA in different amplification systems. The method ligates shorter PCR products to generate a longer library, which is then used for sequencing on a sequencing machine. This method can significantly improve data utilization while ensuring data quality.

The invention provides a ciliate specific primer, a ciliate specific long primer suitable for an amplicon sequencing technology, a kit and application, and particularly belongs to the technical field of molecular biological detection and high-throughput gene sequencing. According to the present invention, the ciliate specific primer comprises a forward primer CS322F and a reverse primer CS819R, the ciliate specific long primer suitable for amplicon sequencing comprises a forward long primer LCS322F and a reverse long primer LCS819R. The ciliate specific primer disclosed by the invention has ciliate broad-spectrum universality, is adaptive to a short-read-length high-throughput sequencing method, and can be used for comprehensively analyzing the diversity and ecological functions of ciliate communities. Accurate detection and efficient analysis of ciliate communities can be realized, and technical support is provided for environmental monitoring, germplasm resource development and ecological assessment.

The invention relates to the technical field of tumor immunotherapy, and particularly discloses a screening method of a cancer cell mitochondrial DNA (deoxyribonucleic acid) coding new antigen, mitochondrial DNA with higher purity is obtained by means of microdissection, linear DNA splitting and the like, and nuclear metastasis mitochondrial fragment interference in mitochondrial sequencing is overcome. On the basis, mitochondrial amplicon sequencing is used for identifying the base sequence change caused by mutation from the DNA level, multiple types of tumor vaccines including polypeptide vaccines, RNA vaccines and dendritic vaccines can be prepared according to requirements, and even the tumor vaccines can be used for TCR-T therapy. According to the screening method of the mitochondrial DNA coding new antigen and the application of the mitochondrial DNA coding new antigen in preparation of the vaccine, the feasibility of the mitochondrial coding new antigen and the effectiveness of the mitochondrial coding new antigen as a tumor vaccine target are accurately and comprehensively confirmed, and a transcription translation process and a screening mechanism of DNA-RNA-polypeptide are systematically constructed; and more diversified, personalized, safe and effective treatment strategies can be selected and designed for the patient.

The application discloses an eDNA data bioinformatics automation and user-friendly analysis method, which is suitable for amplicon sequencing environmental DNA (eDNA) bioinformatics data analysis in ecological environment biological monitoring and biodiversity investigation. The method obtains original sequencing files provided by a user, automatically identifies sample information and generates a sample list; according to an analysis engine and an analysis strategy selected by the user, a preset process template is called to automatically generate executable analysis code and run; meanwhile, quality indexes are collected at key analysis nodes, abnormal samples are identified and a warning report is generated. The method realizes automatic construction, execution and quality monitoring of an eDNA amplicon sequencing data analysis process, can reduce a technical threshold of eDNA data analysis, and enables a user who lacks a bioinformatics professional background to also complete eDNA data analysis tasks of multiple analysis engines, multiple primer systems and various bioinformatics methods.

The invention belongs to the technical field of biological safety evaluation, and particularly relates to a method for evaluating non-target biological safety of transgenic insect-resistant corn in soil and application of the method. According to the method, an artificial soil method is adopted to simulate natural exposure conditions, and leaves of transgenic insect-resistant corn and non-transgenic control corn in the elongation stage are freeze-dried and then added into artificial soil for culturing eisenia fetida. After in-vivo DNA of eisenia fetida in different culture periods is extracted respectively, PCR amplification is carried out on bacterial 16S rRNA genes and fungus ITS regions, a library is constructed, and amplicon sequencing is carried out. The safety of the transgenic insect-resistant corn to a non-target organism eisenia fetida is comprehensively evaluated by analyzing the change of the diversity of Alpha and Beta of microorganisms in eisenia fetida. According to the invention, a scientific, accurate and efficient evaluation method is successfully established, and the method has important significance for perfecting an evaluation system for non-target biological safety of transgenic crops.

The application describes a method for constructing an amplicon sequencing library with reduced cross contamination, comprising the following steps: obtaining sample DNA; performing first PCR amplification on the sample DNA using a first primer, a second primer and a third primer to obtain an amplification product of the sample; performing purification; performing second PCR amplification on the purified product using a fourth primer and a fifth primer to obtain an amplicon sequencing library; wherein the first primer comprises, from 5' end to 3' end, a first sequencing tag sequence and an amplicon-specific forward primer; the second primer comprises, from 5' end to 3' end, a second sequencing tag sequence and an amplicon-specific reverse primer; the third primer comprises, from 5' end to 3' end, a second sequencing adapter sequence, a sample tag sequence and a second sequencing tag sequence; the fourth primer comprises, from 5' end to 3' end, a first sequencing adapter sequence and a first sequencing tag sequence; and the fifth primer is a second sequencing adapter sequence. According to the application, a method for constructing a sequencing library with reduced cross contamination can be provided.