18 results about "Amplification bias" patented technology

The invention relates to the technical field of bioinformatics, and discloses a three-generation full-length transcript sequencing data-based HLA (human leukocyte antigen) typing method, system, equipment and medium, by utilizing the technical advantage of long read length of a three-generation sequencing single molecule, a complete transcript sequence of an HLA gene is directly obtained, full-length HLA typing without PCR (polymerase chain reaction) amplification and short read length splicing is realized, and the method, the system and the equipment are simple and convenient to operate. The blank of the three-generation full-field transcript in the aspect of detecting the HLA typing is filled. The method effectively solves the problem that the cost, the efficiency and the accuracy are difficult to consider in the traditional HLA typing technology, not only avoids PCR amplification preference and short read length assembly errors and remarkably improves the typing accuracy, but also reduces the analysis complexity through an optimized bioinformatics process. According to the method, an efficient solution with economical efficiency and reliability can be provided for application scenes such as clinical transplantation matching and disease association research, and the method has important clinical application value and wide market prospects.

The invention provides a full-length 16S rRNA gene sequencing method based on UMI. The full-length 16S rRNA gene sequencing method comprises the following steps: step 1, extracting genome DNA of a sample to be detected; step 2, connecting a UMI tag to the genome DNA through a PCR reaction, constructing a DNA library, and then sequencing the DNA library by adopting a long-read-long sequencing instrument to obtain sequencing data; 3, performing data analysis on the sequencing data to determine a consistent sequence; and 4, determining classification information of the consistency sequences, and / or calculating the relative abundance of each consistency sequence. The sequencing method provided by the invention has the advantages of high sequence accuracy, low sequencing cost and elimination of PCR amplification deviation.

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 present invention belongs to the field of biotechnology, and specifically discloses a method for correcting and standardizing TCRβ high-throughput sequencing data based on template sequences and reference cells, comprising the following steps: incorporating a fixed number of exogenous reference cells and a fixed number of synthetic templates into a sample, constructing a high-throughput sequencing library for TCRβ, and performing sequencing using a high-throughput sequencing platform; analyzing the amplification bias pattern by using the added template sequences; correcting the sequencing errors caused by base mutation bias; standardizing the sample sequencing data by using the exogenous reference cells; and precisely quantifying the T cell receptors in the sample. The present invention can correct and standardize TCRβ high-throughput sequencing data in a high-throughput sequencing manner, adopting a standardized process, through amplification bias correction, sequencing error correction, and sample standardization, and finally obtaining an accurate and real TCRβ library distribution.

Provided herein are methods for preparing a sequencing library comprising nucleic acids from a plurality of single cells. In one embodiment, the method includes linear amplification of a nucleic acid. In one embodiment, a sequencing library includes whole genome nucleic acids from a plurality of single cells. In one embodiment, a nucleic acid comprises three index sequences. Also provided herein are compositions, such as compositions comprising nucleic acids having three index sequences.

This invention discloses a method for quantifying TCRβ based on high-throughput sequencing, comprising the following steps: lysing samples using Trizol and adding external reference cell lysis buffer to the lysed samples; extracting total RNA from the samples and external reference cells; performing reverse transcription using C-terminal specific primers for TCRβ; adding a template to the reverse transcription product; constructing a high-throughput sequencing library of TCRβ using a set of multiplex PCR primers with optimized sequence composition and concentration, and performing sequencing; and quantifying TCRβ in the samples using two rounds of external reference data. This invention, by adding external reference cells, can effectively estimate the total number of T cells in the sample; by using multiplex PCR primers with optimized sequence composition and concentration, it can better reduce interference and amplification bias between multiplex primers; by adding a template, it can help correct and standardize TCR high-throughput sequencing data, finally obtaining an accurate and realistic TCRβ library distribution.

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 present invention relates to the field of bioinformatics technology and discloses an HLA typing method, system, device, and medium based on third-generation full-length transcript sequencing data. By leveraging the technical advantages of third-generation sequencing's single-molecule long reads, the complete transcript sequence of the HLA gene is directly obtained, enabling full-length HLA typing without the need for PCR amplification and short-read splicing. This method effectively addresses the challenges of balancing cost, efficiency, and accuracy in traditional HLA typing techniques. It not only avoids PCR amplification bias and short-read assembly errors, significantly improving typing accuracy, but also reduces analytical complexity through optimized bioinformatics workflows. The present invention can also provide an efficient, cost-effective, and reliable solution for applications such as clinical transplant matching and disease association studies, possessing significant clinical application value and broad market prospects.

When analyzing nucleic acid expression profiles, it is desirable to be able to analyze all nucleic acids including those that are expressed at a low frequency. The purpose of the present technology is therefore to provide a technique that makes it possible to suitably analyze all nucleic acids without the analysis being affected by amplification bias based on the expression frequency of the nucleic acids. As a result of intensive studies, the present inventors found that by randomly binding a plurality of types of DNA fragments, forming a plurality of cyclic nucleic acids with a circular structure, determining the nucleotide sequences of the cyclic nucleic acids, and identifying repeating units from the nucleotide sequences, it is possible to suitably analyze all nucleic acids including those that are expressed at a low frequency.

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 invention discloses an amplicon library construction method for intestinal flora sequencing, and relates to the technical field of biological sequencing, and the amplicon library construction method specifically comprises the following steps: step (1): sample collection: collecting a fresh excrement sample, and carrying out sealed storage; (2) DNA data extraction: extracting the DNA data in the excrement sample; according to the amplicon library construction method for intestinal flora sequencing, a sample collection and preservation and DNA extraction method is optimized, and the integrity of a flora structure and the DNA quality in a sample are ensured; specific primers and optimized PCR amplification conditions are adopted, so that the amplification deviation is reduced, and the amplification efficiency is improved; through strict purification and quality control steps, the quality of the amplicon library is ensured, and the method is suitable for high-throughput sequencing; blank control and negative control are arranged, so that pollution is effectively controlled, and the accuracy and reliability of results are 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.

A method to correct amplification bias in amplicon sequencing is disclosed. Amplification efficiency is not constant among different loci in a sample, nor for the same locus in different samples. Differences in 3′-end stability, primer Tm, amplicon length, amplicon GC content, and GC content of amplicon flanking regions all may contribute to amplification bias. Such bias interferes with accurate calculation of copy number for a genomic region of interest and hinders the application of amplicon sequencing for detection of minor copy number variation. The methods of the invention allow correction of amplification bias and enable detection of minor copy number variation using amplicon sequence data.

The invention provides a multi-gene identification primer group for mycoplasma and chlamydia related to lower respiratory tract infection and application of the multi-gene identification primer group, and belongs to the technical field of gene detection. The primer group comprises specific primers of mycoplasma pneumoniae, chlamydia pneumoniae, huDNA and IC, a 5 '-end primer is labeled through FAM fluorescence, a reaction system is optimized, two target gene loci, one human internal reference gene locus and one system internal reference can be detected at the same time in a PCR system, unequal amplification deviation of traditional PCR is overcome, and target gene expression can be accurately and sensitively quantified. A reagent and a kit developed on the basis of the primer group are matched with a specific using method and have the advantages of being simple in structure, convenient to operate, low in cost, high in accuracy, good in repeatability and the like, a capillary electrophoresis technology is adopted for fragment analysis, result analysis is visual and reliable, large-scale application and popularization are facilitated, and the primer group is of great significance in clinical detection of LRTI related mycoplasma and chlamydia.