8 results about "Amplification bias" patented technology

The current invention pertains to a reliable method for determining the relative frequency of a sequence variant of interest in a nucleic acid sample derived from at least one polyploid cell, wherein the method uses a UMI to correct for any amplification biases. The invention further pertains to the use of a UMI for accurately determining the relative frequency of a sequence variant of interest in a nucleic acid sample derived from at least one polyploid cell.

The invention belongs to the technical field of forensic medicine and biological medicine, discloses a method for forensic medicine identification of corpses in water based on metagenomics markers and application, and aims to solve the problems that existing drowning diagnosis is insufficient in accuracy and the deduction of post-death submerging time (PMSI) is limited. The method comprises the following steps: collecting a corpse lung tissue in water and a water sample in a corresponding water area, and obtaining a microbial DNA sequence by adopting a metagenome sequencing technology; the method comprises the following steps: processing data through bioinformatics analysis (such as fastp quality control, host sequence removal by KneadData and Kraken2 species annotation), and selecting and screening species level microbial markers in combination with a random forest algorithm and Boruta characteristics; and respectively establishing a drowning diagnosis model and a PMSI inference model based on the screened markers, and verifying the performance of the models through indexes such as AUC and MAE. Complete microbial communities of bacteria, eukaryotes (including fungi), archaea and viruses in lung tissues of corpses in water are comprehensively analyzed for the first time, 17 bacterial markers and 9 eukaryote markers are screened out for drowning diagnosis (verification experiment bacterial model AUC = 1, the accuracy rate is 89.29%, eukaryote model AUC = 0.95, the accuracy rate is 87.5%), and 17 markers are screened out for PMSI inference (integrated model MAE = 0.66 days). According to the method, the species resolution reaches the species level, the method is not influenced by amplification bias, the method is suitable for different water area environments, and an efficient and objective forensic medicine tool is provided for identifying the cause of the dead body in water and deducing the submerged time after the dead body.

The invention discloses an RNA methylation detection kit based on a CRISPR / CtCas12a system and a detection method, and belongs to the technical field of gene detection and molecular diagnosis. According to the invention, heat-resistant Cas12a protein derived from clostridium butyricum is utilized, and an ss-ds DNA activator is rationally designed, so that the difference between trans-cleavage activity of CtCas12a activated by methylated RNA and trans-cleavage activity of CtCas12a activated by unmethylated RNA is remarkably amplified; by further combining with a detection probe, high-sensitivity and single-base-resolution quantitative analysis of methylation modification of multiple RNAs such as m6A and m1A is synchronously realized under the condition that nucleic acid amplification is not needed, and an efficient tool is provided for disease marker screening and clinical precise diagnosis and treatment; besides, the method overcomes antibody dependence and amplification deviation, and is suitable for rapid detection of clinical samples and dynamic monitoring of epitranscriptome, so that the method has a better application prospect in RNA methylation detection.

The invention relates to the technical field of biological information detection, in particular to a metagenome sequencing diagnosis method and system for Aspergillus pulmonary infection, and the method comprises the following steps: acquiring lavage fluid lysis monitoring data, adjusting shear power output, analyzing fluorescence accumulation trend, adjusting temperature control frequency and substrate interval, identifying mutation points and constructing indexes, screening entropy inversion fragments, and obtaining the metagenome sequencing diagnosis result of Aspergillus pulmonary infection. According to the method, through dynamic matching of the lysate flow rate and the shearing power, the nucleic acid release balance is improved, the temperature control frequency and the substrate interval are adjusted according to the fluorescence signal accumulation trend in the amplification stage, the reaction equilibrium state is established, and the amplification deviation is corrected; and identifying a mutation boundary and reconstructing an index hierarchy according to basic group joint distribution and feature vector change, judging and screening feature sections in combination with information entropy inversion, establishing a probability convergence structure based on coverage depth and repetition frequency, and realizing stable identification of pathogen features and convergence output of classification confidence.

The invention discloses an MALBAC linear amplification method and an application thereof. DNA polymerase of the MALBAC linear amplification is Deep, a reaction buffer solution of the Deep contains Mg < 2 + >, and Mg < 2 + > is not additionally added into a reaction system of the MALBAC linear amplification. According to the method, the technical problem of amplification preference in the traditional amplification technology is effectively solved, the detection data quality is high, and the detection accuracy and the result credibility of the newborn RNA molecule in the single cell are remarkably improved. The method is suitable for the field of single cell transcriptomics research, and provides an effective technical means for realizing high-precision and high-reliability single cell gene expression analysis.

The invention relates to the technical field of biology, and particularly provides a circular RNA (circRNA) full-length sequencing method. The method comprises the following steps: carrying out rRNA removal, poly (A) tailing and RNase R treatment on an RNA sample so as to enrich circRNA; carrying out reverse transcription and double-chain cDNA synthesis through a specific primer with a unique molecular marker (UMI); determining an optimal amplification cycle number under the guidance of real-time fluorescent quantitative PCR so as to construct a library; performing long-read-long sequencing through a three-generation sequencing (TGS) platform; and in combination with UMI clustering and cyclic consistency sequence generation, realizing circRNA full-length structure recognition and quantitative analysis by adopting a ucRG algorithm. According to the method, amplification bias and sequencing errors can be reduced, the accuracy and specificity of circRNA recognition are improved, the consistency of TGS and a next-generation sequencing (NGS) platform in circRNA detection results is remarkably improved, and the method has high sensitivity and quantitative reliability and can be widely applied to fundamental research and clinical detection of circular RNA.

The invention relates to the technical field of biological information, in particular to a haplotype recognition method based on a single molecule sequencing technology. According to single molecule sequencing data of amplicons of known haplotype samples in a target area, the amplification efficiency of each primer pair in the multiple PCR primers on known haplotypes is obtained, then an amplification coefficient adjustment model is constructed according to the amplification efficiency, and the amplification coefficient adjustment model outputs amplification coefficients of each primer pair. The method is used for correcting amplification bias of different haplotypes in haplotype recognition. According to the method provided by the invention, by accurately correcting the efficiency difference of the primer pair in the amplification process, dominant and vulnerable haplotypes can be accurately identified and distinguished under the condition of extremely unbalanced coverage caused by amplification efficiency deviation, so that the overall accuracy and reliability of haplotype typing are greatly improved.

The purpose of the present invention is to provide a method for reducing amplification bias in multiplex PCR. It was found that amplification bias is reduced by amplifying nucleic acids using proliferating cell nuclear antigens. The present invention relates to a method for reducing amplification bias using a proliferating cell nuclear antigen, and a reagent, a kit, and the like used in the method.