Method for constructing human TCR alpha CDR3 region library

Abstract

The invention relates to a method for constructing a human TCR alpha CDR3 region sequencing library. The method mainly comprises the following steps: (1) extracting total RNA from human tissues or whole blood; (2) reversely transcribing the RNA into cDNA, ligating a linker sequence to the end of the long-stranded cDNA, and amplifying TCR alpha containing a CDR3 region through a universal C-terminal primer and a linker sequence primer; (3) fragmenting the TCR alpha through Tn5 transposase, and enriching a CDR3 region sequence; and (4) carrying out sequencing by an illumina high-throughput sequencing platform. The method is suitable for library construction of tissues and body fluids, containing various types of T cells, can highly-efficiently amplify the human TCR alpha through a 5'RACE technology in an agonic manner, and solves the problems of difficulty in control of the template copy number and the offset of the product, caused by multiplex PCR amplification; and the Tn5 enzyme's ability to simultaneously complete DNA fragmentation and linker ligation and a specific primer are used to fast and accurately enrich and construct a library of the hypervariable region (CDR3 region) ofthe TCR alpha, so pertinent sequencing, easy data analysis and sequencing cost saving are achieved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a primer and library preparation method required for efficient and unbiased construction of a high-throughput sequencing library in the CDR3 region of human TCR alpha. Background technique [0002] Lymphocytes exert immune function through the recognition of specific antigens by their surface antibodies. The specificity of antigen recognition is reflected at the clonal level, that is, lymphocytes of the same clone can recognize the same antigen receptor and recognize the same antigen epitope. T cell antigen receptor (TCR) is a structure that T cells specifically recognize and bind antigen peptide-MHC molecules. T cell receptor is a heterodimer composed of two different subunits. 95% of T cell receptors are composed of α subunits and β subunits, and the other 5% of receptors are composed of γ subunits and δ subunits, and the ratio will change due to individual development...

AI Technical Summary

Problems solved by technology

[0005] In view of the fact that the traditional amplification of the CDR3 region requires multiplex PCR for amplification, there are problems such as the template copy number and amplification efficiency are difficult to control during the amplification process, the product shifts, and cannot reflect the initial TCR distribution of the sample; at the same time, multiple primers amplify It will increase mismatching and mutual interference, resulting in high amplification background, poor repeatability and other problems, and eventually the sequencing results cannot truly reflect the sample conditions

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