Method for constructing high-throughput sequencing library of immune group library for screening cross-reaction between samples

Abstract

The invention relates to a method for constructing a high-throughput sequencing library of an immune group library for screening a cross-reaction between samples. The method is characterized in that: RNA of more than 10 ng is taken as a starting amount, a principle of RACE is used to make the template conversion when the RNA is reversely transcribed into cDNA, a known sequence is added to the 5' ends of all cDNAs, a product with the known sequence is used as a template, PCR amplification is performed by using a double-ended Barcode primer, the resulting product is purified and ligated to a sequencing linker sequence to obtain the final sequencing library, which can be used for samples of different receptor sources, during PCR amplification, PCR amplification can be performed using only one pair of primers to achieve equivalent amplification, and the primer preference problem caused by multiplex primer amplification is solved, and the upstream and downstream primers used in PCR amplification are designed to be labeled with Barcode composed of 4 to 12 different bases, which avoids the problem of cross-reaction between samples when multiple samples are simultaneously constructed.

Description

technical field [0001] The invention belongs to the field of biomedical services, and in particular relates to a PCR amplification of human immune repertoire, library construction and application in next-generation sequencing, especially a high-throughput sequencing of an immune repertoire for screening cross-reactions between samples Library construction method. Background technique [0002] The immune system is mediated by the surface receptors of immune B cells and T cells, and binds to pathogens or antigens derived from pathogens, and then exerts immune functions to protect the body from infringement. BCR immunoglobulin heavy chain (IGH) and TCR beta chain (TCRB) generate combinatorial diversity through rearrangement of V gene, D gene and J gene segments. At the junction between V-D and D-J, deletion of nucleotides and random addition of nucleotide sequences generated diversity in receptor junctions. The light chains produced by antibody immunization, immunoglobulin la...

AI Technical Summary

Problems solved by technology

[0005] Although next-generation sequencing technology can quickly and high-throughput scan the immune repertoire in a panoramic manner, there are still obvious shortcomings in the two current methods.

1. The multiplex PCR method uses a variety of primers, and the reaction system is extremely complicated; the design of multiplex primers can only be designed based on known reference sequences, and cannot completely capture all alleles in the human immune repertoire; multiple primer reactions cannot To achieve equivalent amplification, the different amplification efficiencies between different primers lead to the existence of PCR bias, which makes the results quite different from the real situation

2. Although the simple 5`RACE method can achieve equivalent amplification, it cannot distinguish cross-reactions between samples when building a library

3. In the two amplification methods, chimera sequence products exist and occupy a considerable proportion, but the existing technology and analysis methods cannot identify most of the cross-product sequences, which will interfere with our sample sequences , resulting in a sequence error between samples

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