Immunological group library method for discriminating self-cross-reaction of independent sample

Abstract

The invention relates to an immunological group library method for discriminating self-cross-reaction of an independent sample. The method is characterized in that with RNA of more than 10 ng as a starting amount, a principle of RACE is utilized to perform template conversion when RNA is reversely transcribed into cDNA. two pre-amplification experiments are performed before PCR amplification, the first pre-amplification uses an upstream primer with UMI, so that the 5' end of an original template takes UMI, and the second pre-amplification uses a downstream primer with UMI, so that the 3' end of the template takes UMI, the product with UMI at double ends is used as a template for PCR amplification, and a finally obtained product is purified and ligated to a sequencing linker sequence to obtain a final sequencing library. Two ends of each cDNA are labeled with different and unique UMI sequences, after the PCR amplification is completed, multiple samples can be combined to construct a library, which can solve the cross-reaction problem generated by similar sequences in the PCR amplification reaction in a sample; for samples with different cell receptor sources, PCR amplification can be performed by using only one pair of primers, thus achieving equivalent amplification and solving the primer preference problem caused by multiple primer amplification.

Description

technical field [0001] The invention belongs to the field of biomedical services, and in particular relates to PCR amplification of human immune repertoire, library construction and application in next-generation sequencing, in particular to an immune repertoire method for screening self-cross-reactivity of independent samples. 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 lambda or kappa (IGL / K) and T cell rece...

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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 from sequences in independent samples when building a library

3. Existing sequencing analysis methods and experimental techniques have not mitigated the impact of chimera sequence products produced in the late stage of PCR, and these products will exist in both amplification methods and affect the diversity of samples. Quantitative analysis of sex has caused great interference

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