85 results about "Immune repertoire" patented technology

The invention is directed to methods for detecting and quantifying nucleic acid contamination in a tissue sample of an individual containing T cells and/or B cells, which is used for generating a sequence-based clonotype profile. In one aspect, the invention is implemented by measuring the presence and/or level of an endogenous or exogenous nucleic acid tag by which nucleic acid from an intended individual can be distinguished from that of unintended individuals. Endogenous tags include genetic identity markers, such as short tandem repeats, rare clonotypes or the like, and exogenous tags include sequence tags employed to determine clonotype sequences from sequence reads.

The invention is directed to methods of measuring an immune response by comparing sequence-based clonotype frequency data from successively measured clonotype profiles. In particular, the invention includes immunotherapies of cancers, such as lymphomas, that include sensitive pre- and post-vaccination sequence-based measurements of changes in a patient's immune repertoire, thereby providing a sensitive measure of the likelihood of treatment success.

The invention is directed to methods for determining clonotypes and clonotype profiles in assays for analyzing immune repertoires by high throughput nucleic acid sequencing of somatically recombined immune molecules. In one aspect, the invention comprises generating a clonotype profile from an individual by generating sequence reads from a sample of recombined immune molecules; forming from the sequence reads a sequence tree representing candidate clonotypes each having a frequency; coalescing with a highest frequency candidate clonotype any lesser frequency candidate clonotypes whenever such lesser frequency is below a predetermined value and whenever a sequence difference therebetween is below a predetermined value to form a clonotype. After such coalescence, the candidate clonotypes is removed from the sequence tree and the process is repeated. This approach permits rapid and efficient differentiation of candidate clonotypes with genuine sequence differences from those with experimental or measurement errors, such as sequencing errors.

The invention is directed to a method of prognosing in an individual a transformation from follicular lymphoma to diffuse large B-cell lymphoma (DLBCL) by measuring changes and/or lack of changes in certain groups of related clonotypes, referred to herein as “clans,” in successive clonotype profiles of the individual. A clan may arise from a single lymphocyte progenitor that gives rise to many related lymphocyte progeny, each possessing and/or expressing a slightly different immunoglobulin receptor due to somatic mutation(s), such as base substitutions, inversions, related rearrangements resulting in common V(D)J gene segment usage, or the like. A higher likelihood of transformation from follicular lymphoma to DLBCL is correlated with the persistence of clans in successive clonotype profiles whose clonotype membership fails to undergo diversification over time.

The invention provides a method for detecting BCR and TCR immune repertoire in blood plasma cfDNA. The method includes: blood plasma cfDNA extraction, BCR H chain overall length multiple PCR amplification, TCR beta chain CDR3 multiple PCR amplification, PCR amplification after the products of the two previous amplifications are mixed in an equal-volume manner, high-throughput sequencing, and immune repertoire precise information analysis. The primers of the three amplifications are respectively shown in SEQ ID No. 1-28, SEQ ID No. 29-77 and SEQ ID No. 78-79. The invention further provides a kit, which contains the primers, for detecting the BCR and TCR in the blood plasma cfDNA. By the method and the kit which are promising in application prospect in the field of non-invasive detection, the BCR and TCR in the blood plasma cfDNA can be detected at the same time, precise information analysis and precise quantification of immune repertoire subcloning are achieved.

Methods are provided for predicting a subject's infection status using high-throughput T cell receptor sequencing to match the subject's TCR repertoire to a known set of disease-associated T cell receptor sequences. The methods of the present invention may be used to predict the status of several infectious agents in a single sample from a subject. Methods are also provided for predicting a subject's HLA status using high-throughput immune receptor sequencing.

Disclosed herein include systems, methods, compositions, and kits for immune repertoire profiling. There are provided, in some embodiments, primer panels enabling the determination of the nucleotide sequence of the complete variable region of nucleic acids encoding mouse B cell receptor (BCR) and T cell receptor (TCR) polypeptides. In some embodiments, the method comprises single cell transcriptomic analysis.

Disclosed herein include systems, methods, compositions, and kits for 5′-based gene expression profiling. Some embodiments provide synthetic particles (e.g., beads) associated with a first plurality of oligonucleotide barcodes and a second plurality of oligonucleotide barcodes. In some embodiments, nucleic acid targets (e.g., mRNAs) are initially barcoded on the 3′ end with the first plurality of oligonucleotide barcodes and subsequently barcoded on the 5′ end following a template switching reaction and intermolecular hybridization with the first plurality of oligonucleotide barcodes and extension. Immune repertoire profiling methods are also provided in some embodiments.

Disclosed herein include systems, methods, compositions, and kits for labeling nucleic acid targets in a sample. In some embodiments, nucleic acid targets (e.g., mRNAs) are initially barcoded on the 3′ end and are subsequently barcoded on the 5′ end following a template switching reaction and intermolecular and/or intramolecular hybridization and extension. 5′- and/or 3′-barcoded nucleic acid targets can serve as templates for amplification reactions and/or random priming and extension reactions to generate a sequencing library. The method can comprise generating a full-length sequence of the nucleic acid target by aligning a plurality of sequencing reads. Immune repertoire profiling methods are also provided in some embodiments.

The invention provides a primer group for developing a rhesus T cell immune repertoire, a high-flux sequencing method and application of method. The method comprises the following steps: setting 26 forward primer sequences in a variable region, namely a V region aiming at rhesus TCR; setting 13 reverse primer sequences in a joining region, namely a J region aiming at TCR; performing multiple PCR amplification on the TCR sequence with the magnitude order of about 10<7> in peripheral blood or other immunologic tissues, preparing a high-flux sequencing library, sequencing, and performing comprehensive analysis on the rhesus TCR gene immune repertoire by virtue of bioinformatics. According to the technical scheme, the primer group has significance for constructing a rhesus analysis model, contributes to promoting the application of the rhesus model in research on autoimmune system diseases and infectious diseases and also contributes to deeply and widely recognizing the T cell mediated diseases by the humans.

The invention discloses a method and system for processing immune repertoire sequencing data of samples. The method includes the steps that 1, data filtering processing is carried out on the immune repertoire sequencing data of the samples; 2, sequence splicing processing is carried out on the sequencing data obtained after data filtering processing; 3, local comparison is carried out on the sequencing data obtained after sequence splicing processing; 4, re-comparison is carried out on local comparison results; 5, the result with the highest score is screened out of the re-comparison results, and filtering is carried out; 6, error correction processing is carried out on the final comparison result; 7, sequence structure determination and translation are carried out based on the final comparison result obtained after error correction processing. By means of the method, analysis of a large quantity of immune repertoire sequencing data can be effectively achieved, analysis of TCR data and analysis of BCR data can be achieved at the same time, the PCR and sequencing errors can be effectively handled, the accuracy is high, the repeatability is good, and thus the whole condition of an immune repertoire can be truly and effectively reflected.

The present disclosure provides methods, compositions, kits, and systems useful in the determination and evaluation of the immune repertoire. In one aspect, target-specific primer panels provide for the effective amplification of sequences of T cell receptor and/or B cell receptor chains with improved sequencing accuracy and resolution over the repertoire. Variable regions associated with the immune cell receptor are resolved to effectively portray clonal diversity of a biological sample and/or differences associated with the immune cell repertoire of a biological sample.

The invention provides a whole blood immune repertoire detection method based on high-flux sequencing technology. The method comprises the following steps: (1) directly extracting RNA (ribonucleic acid) from a whole blood sample; (2) carrying out reverse transcription on the RNA to generate single chain cDNA (complementary deoxyribonucleic acid), connecting an oligonucleotide joint sequence to the tail end of the single chain cDNA, carrying out PCR (polymerase chain reaction) reaction on the single chain cDNA subjected to oligonucleotide joint sequence connection by using a universal primer and a special targeted primer P-VDJ (of which the sequence is GAC CTC GGG TGG GAA CAC) as a special primer pair for PCR reaction, and amplifying the immune receptor genome; and (3) carrying out high-flux sequencing. The method of directly extracting the RNA from the whole blood sample is more suitable for clinic; the single target specific primer is utilized to uniformly amplify the variable region of the subtype T cell receptor gene; and a Hi-SEQ 2500 of the Illumina corporation and a bar code are utilized for sequencing, thereby ensuring the lower cost, the sequencing time of less than 48 hours and favorable sequencing depth.

The invention provides a non-invasive technique for the detection and quantification of the immune repertoire, in a biological sample containing a plurality of distinct cell populations. Methods are conducted using sequencing technology to detect and enumerate immune repertoire within a heterogeneous biological sample.

Disclosed are methods for determining the immunological status of the adaptive immune system of a subject by identifying and quantifying rearranged DNA (and/or subsequently transcribed RNA) sequences encoding T cell receptor (TCR) and/or immunoglobulin (IG) polypeptides, in a lymphoid DNA-containing sample from the subject. TCR and/or IG sequence diversity and sequence distribution permit immunocompetence and immune repertoire assessment and reflect the degree of T cell or B cell clonality and clonal expansion in the sample. Methods for stratifying patient populations on the basis of immunocompetence including likelihood of responding to immunotherapy are also described.

The invention discloses a library construction scheme for introducing UM, namely molecular bar codes, based on a principle of 5'-RACE. The scheme specifically comprises the following steps: performingreverse transcription by designing single primers in a constant region, performing equivalent amplification to obtain all types of TCR/BCR CDR whole gene sequences, and introducing UMI, so that errors introduced in the subsequent PCR (Polymerase Chain Reaction) or sequencing process can be corrected. According to the method disclosed by the invention, the experimental requirements on library construction of trace samples as low as 100 cells can be met, and the TCR/BCR CDR whole gene sequences can be equivalently amplified.

The invention discloses an immune repertoire biological information analysis process based on molecular markers. In the process, single-chain immune globulin genes with the same sequence are selected,and primers and UMI sequences with the same structure are added to 3' ends or 5' ends of the selected single-chain immune globulin genes; after addition is completed, PCR amplification is performed on the immune genes to obtain a test sequence, and the test sequence is filtered; after filtering, the UMI sequence carried by the immune genes is corrected by establishing a directed acyclic tree, then the immune genes marked by the corrected UMI sequence are corrected, and the corrected immune genes are assembled; and finally the assembled immune genes are subjected to statistical analysis and reporting. According to the process, through correction of UMI and correction of the immune genes, the influences of amplification errors and sequencing errors on immune gene sequencing are effectivelyremoved, and the accuracy of sequencing data is improved.

The invention is directed to methods for detecting and quantifying nucleic acid contamination in a tissue sample of an individual containing T cells and/or B cells, which is used for generating a sequence-based clonotype profile. In one aspect, the invention is implemented by measuring the presence and/or level of an endogenous or exogenous nucleic acid tag by which nucleic acid from an intended individual can be distinguished from that of unintended individuals. Endogenous tags include genetic identity markers, such as short tandem repeats, rare clonotypes or the like, and exogenous tags include sequence tags employed to determine clonotype sequences from sequence reads.

The invention discloses a method for analyzing single-cell immune repertoire sequencing data. The method comprises the following steps of S1, performing data quality statistics and basic quality control on original data; S2, performing sequence alignment and noting on high-quality reads which are obtained in the step S1 and a reference V(D)J fragments sequence, thereby obtaining a Consensus sequence; S3, performing Clonotype typing on a CDR3 amino acid sequence in the Consensus which is obtained through splicing in the step S2; S4, performing CDR3, V/J gene and V-J paired characteristic analysis on the Clonotype in the sample in the step S3; and S5, combining the clonotypes in different samples, and performing multiple-sample analysis. The method has advantages of integral analysis process, abundant content, visual result displaying and comprehensive displaying of immune repertoire information, thereby making a user comprehensively understand and evaluate the immune repertoire condition and more accurately reflecting the immune function in an organism.

The invention discloses a construction method of an immune repertoire sequencing library and a test kit, belongs to the field of biological medicines, and relates to a detection method of immune repertoire diversity. One of the purposes of the invention is to provide the construction method of the immune repertoire sequencing library; the construction method comprises the following steps of (a) extracting sample RNA, (b) carrying out reverse transcription by using a reverse transcription primer to synthesize cDNA, and adding a specific linker containing a molecular tag sequence at the tail end; (c) by taking the product obtained in the step b as a template, carrying out amplification through a specific linker primer and a gene specific primer containing a molecular tag sequence; and (d) finally, adding a sequencing library linker to an amplification product, so as to complete library construction. When sequencing data is analyzed, original RNA molecules are identified through moleculartag sequences, and amplification bias is excluded. By using the method, all CDR regions of TCR and BCR can be covered, PCR amplification bias interference is effectively eliminated, the specificity is higher, and the analysis result is more accurate. The other purpose of the invention is to provide the test kit for immune repertoire sequencing.