Specific recognition sequence based T cell receptor high-throughput sequencing library construction and sequencing data analysis method

Abstract

The present invention discloses a specific recognition sequence based T cell receptor high-throughput sequencing library construction and sequencing data analysis method. According to the method, a specific reverse transcription primer is designed for an mRNA sequence of a C region of a TCR constant region, cDNA is obtained by reverse transcription, and the 3' end of the cDNA is connected with a library construction linker with a specific recognition sequence; then the linker with the specific recognition sequence is added by using a splint connection method, and a TCR gene rearrangement sequence is amplified under the action of DNA polymerase by utilizing a gene specific primer with a tag; and finally a DNA library is added with a sequencing linker by PCR amplification to prepare a high-throughput sequencing library, and the high-throughput sequencing library is used for sequencing. The TCR gene diversity is comprehensively analyzed by bioinformatics, and a rearrangement rule of TCR genes including J region, D region and V region genes can be accurately obtained. The method is high in library construction efficiency, few in library construction step, low in required RNA initial quantity and low in library construction cost.

Description

technical field [0001] The invention belongs to the technical field of gene sequencing, in particular to a high-throughput sequencing library construction and sequencing data analysis method for a T lymphocyte receptor library based on a unique recognition sequence, which is applied to the diversity of T cell receptor (TCR) detection. Background technique [0002] T cell receptor (T cell receptor, TCR) is a molecule expressed on the surface of T cells that mediates specific immune responses and can specifically recognize antigens. The heterodimer composed of two peptide chains of αβ or γδ constitutes 95-99% of T cell TCRαβ and 1-5% of T cell TCRγδ. Peripheral blood T cells are mainly TCRαβ T cells, which are the main cells that mediate the body's specific cellular immune response. The TCR sequence has the "traceability" ability similar to the two-dimensional code structure, and is closely related to the variation of TCR and cell subsets and the health status of the body. ...

AI Technical Summary

Problems solved by technology

[0007] In the present invention, there are high amplification bias and sequencing errors in the multiple PCR targeted sequencing technology for TCR gene detection in the prior art; secondly, DNA damage and errors introduced by PCR cannot be identified, so the accuracy is poor, and There may be technical problems of false positives in the detection results, and a high-throughput sequencing library construction and sequencing data analysis method for the T lymphocyte receptor library based on the unique recognition sequence is provided

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