Specific lymphocyte content analysis method and device based on TCR/BCR high-throughput sequencing

Abstract

The invention relates to a specific lymphocyte content analysis method and device based on TCR / BCR high-throughput sequencing, and belongs to the technical field of gene detection. The analysis methodcomprises the following steps of performing high-throughput sequencing: extracting genome DNA of a biological sample to be detected, adding a predetermined amount of artificially synthesized TCR / BCRcoding gene internal standard sequences, and carrying out high-throughput sequencing; and performing data analysis: obtaining high-throughput sequencing data and TCR / BCR gene sequences obtained by analysis, calculating the amplification multiple according to the input internal standard sequences, comparing the TCR / BCR gene sequences of the sample to be detected with antigen receptor gene sequencesA of target lymphocytes, obtaining the number of original antigen receptor gene sequences consistent with the antigen receptor gene sequences A of the target lymphocyte, the number of sequences withsequencing errors, the number of sequences subjected to secondary rearrangement and the number of mutated sequences, obtaining the total number of target lymph sequences, and calculating the number ofthe contained target lymphocytes according to the total number of the target lymph sequences and the amplification multiple, so that the content of each T / B cell can be accurately determined.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a method and device for analyzing the content of specific lymphocytes based on TCR / BCR high-throughput sequencing. Background technique [0002] Adaptive immunity is also called acquired immunity. Specific immune cells mainly include T lymphocytes involved in cellular immunity and B lymphocytes involved in humoral immunity. Adaptive immunity has the characteristics of antigen specificity and immune memory, and its antigen specificity means that different antigens need to be recognized and responded by different lymphocytes. In clinical practice, it is often necessary to determine the content of specific lymphocytes in biological samples (the composition of each component in biological samples is as follows: figure 1 shown), for example, the immune effect of the vaccine can be determined by detecting whether there are specific vaccine-specific immune cells; or the detectio...

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Problems solved by technology

[0005] However, by flow cytometry detection, the content of a type of cells with the same surface differentiation antigen is detected, and each cell cannot be accurately distinguished

Moreover, the detection sensitivity of the current common flow cytometry can only reach 1 / 10000. For samples with a content lower than 1 / 10000, false negative detection results will be given.

In flow cytometry, data interpretation depends on the operator and the laboratory, and the degree of standardization is low

Most importantly, for cancerous lymphocytes, the expression of their surface antigens will change at different times of cancer occurrence and treatment, which will seriously affect the accuracy of cancer cell detection

[0006] For detection by RQ-PCR, it is necessary to design specific primers and quantitative probes for the TCR / BCR genes of different target cells. If there is a mutation or secondary rearrangement in the binding region of the quantitative probe, a missed detection will occur

[0007] Accordingly, scientists also use high-throughput sequencing to detect the TCR / BCR genes of target cells. However, the current high-throughput sequencing for TCR / BCR often has detection bias due to gene mutations.

Moreover, the TCR / BCR research based on the multiplex PCR method will cause a large amplification bias due to the difference in the binding efficiency of the primers in the multiplex PCR primer system and the influence of the interaction between the primers. If the system is not optimized and corrected, it cannot accurately reflect The true level of sample clone

Moreover, the TCR / BCR research based on the RACE method is only applicable to RNA samples, and the conditions for sample collection and transportation are relatively strict

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