Method for constructing B cell receptor (BCR) high-throughput sequencing library

Abstract

The invention relates to a method for constructing a B cell receptor (BCR) high-throughput sequencing library. The method is characterized in that sets of primer sets are respectively designed on thebasis of a heavy chain IgG, a light chain IgL and a light chain IgK of a BCR, and different alleles of all the IgG, the IgL and the IgK which are discovered at present can be amplified; meanwhile, a set of internal reference sequences are added in the amplification process of each of the alleles, and internal reference is used for correction in a later stage, so that the real situation of the BCRcan be truly restored, and then the method for constructing the high-throughput sequencing library can be achieved. The method can realize that all known alleles contained in IgG, IgL and IgK can be completely amplified by the three sets of primers, so that the defect of a part of alleles are missed during amplification of existing mixed primers is solved; and an internal reference sequence is added during PCR amplification, and later-stage data analysis is corrected according to the internal reference, so that the preference problem generated in the PCR process can be effectively corrected. The method can meet the requirement of establishing a library for samples with RNA total amount more than 100ng.

Description

technical field [0001] The invention belongs to the field of biomedical services, and in particular relates to the amplification of human BCR, library construction and application in next-generation sequencing. Background technique [0002] The immune system binds to pathogens or antigens derived from pathogens through the surface receptors of immune B cells, and then exerts immune function to protect the body from infringement. BCR immunoglobulin heavy chains (IGH) 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 have similar rearrangements, thus forming the diversity of the immune system. The antibody produced by B cells has a Y-shaped structure, consisting of two identical heavy chains and light chains. When V(D)J is recombined, o...

AI Technical Summary

Problems solved by technology

[0005] Although next-generation sequencing technology can quickly and high-throughput scan the panorama of BCR, there are still obvious shortcomings in the two current methods.

1. The multiplex PCR method uses a variety of primer reactions and cannot 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 effectively combine the double-ended barcode and double-ended UMI method to distinguish the cross-reaction in the sample due to the length control when building the 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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