Method for quantifying TCR beta based on high-throughput sequencing

Abstract

The invention discloses a method for quantifying TCR beta based on high-throughput sequencing, which comprises the following steps: cracking a sample by using Trizol, and adding an external reference cell lysis solution into the cracked sample; extracting total RNA (Ribonucleic Acid) of the sample and the external reference cells; carrying out reverse transcription by utilizing a C-terminal specific primer of TCR beta; adding a template into a reverse transcription product; the method comprises the following steps: constructing a high-throughput sequencing library of TCR beta by using a group of multiple PCR primers with optimized sequence composition and use concentration, and sequencing; and quantifying the TCR beta in the sample by using two rounds of external parameter data. By adding the external reference cells, the total number of the T cells of the sample can be effectively estimated; by using multiple PCR primers with optimized sequence composition and use concentration, mutual interference and amplification bias among multiple primers can be better reduced; by adding a template, correction and standardization of TCR high-throughput sequencing data can be helped, and finally accurate and real TCR beta library distribution is obtained.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for quantifying TCR β based on high-throughput sequencing. Background technique [0002] T cell receptor (TCR) is a specific receptor on the surface of T cells, which is responsible for recognizing antigens presented by the major histocompatibility complex (MHC) and mediating immune responses. The vast majority of T cells are α, β-T cells (about 90% to 95% of the total number of T cells), which are composed of highly diverse α subunits and β subunits, all of which are regenerated in the thymus by germline genes. lined up. Mature T cells migrate to the periphery and form an extremely diverse T cell receptor repertoire, which determines the form and ability of the body to adapt to environmental changes. The diversity of TCR comes from the recombination of V(D)J gene segments. At the same time, during the recombination process, there are often random insertions or deletions o...

AI Technical Summary

Problems solved by technology

However, due to the problems of amplification bias and sequencing errors in the sequencing data of the T cell receptor library, the estimation of the population characteristics of T cell receptors in previous studies is generally biased.

Moreover, we found that during the deep sequencing of T cell receptor repertoire, sequencing errors will have a great impact on the estimation of T cell repertoire diversity

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