Method for high-throughput sequencing of tcr or bcr and method for correcting multiple pcr primer bias by using tag sequence

Abstract

The invention provides a method for carrying out high-throughput sequencing on a TCR (T cell receptor) or a BCR (B cell receptor). The method is characterized by designing upstream primers according to gene features of a V region of the TCR or the BCR and designing downstream primers according to gene features of a C region or a J region of the TCR or the BCR and obtaining sequences of the of the TCR or the BCR in combination with the multiplex PCR (polymerase chain reaction) technology and high-throughput sequencing, thus analyzing the rearrangement information of the TCR or the BCR. Compared with 25-30 cycles of existing multiplex PCR, two cycles of the multiplex PCR technology provided by the invention can conduce to greatly reducing the sequencing errors caused by primer amplification preference. Besides, the invention also provides a method for correcting multiplex PCR (polymerase chain reaction) primer deviation by utilizing DNA (deoxyribonucleic acid) tag sequences, thus further reducing the sequencing errors caused by primer amplification preference and intrinsic sequencing errors of high-throughput sequencing.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a method for high-throughput sequencing of TCR or BCR and a method for correcting the bias of multiple PCR primers by using tag sequences. Background technique [0002] During the development and maturation of T or B lymphocytes, a large and diverse TCR or BCR receptor library is formed through V(D)J gene combination, junctional diversity, and somatic hypermutation, gene conversion, and class switching. , the diversity of TCRs in the immune cell population is as high as 10 16 , the diversity of BCR up to 10 14 , Traditional methods such as SSCP technology, GeneScan technology, fluorescent quantitative melting curve technology, etc., often have the disadvantage of narrow coverage range, which cannot meet the detection of a large number and variety. [0003] High-throughput sequencing can meet the huge sequence sequencing requirements. However, when high-throughput s...

AI Technical Summary

Problems solved by technology

However, when high-throughput sequencing technologies such as Roche454 sequencing platform and Illumina's solexa or GenomeAnalyzer sequencing platform are used for sequencing, library construction of samples before sequencing is generally required. Due to the diversity of TCR or BCR, the amplification preference of primers and the template The design of primers is particularly important when building a library due to factors such as differences in abundance; in addition, when building a library in the prior art, a tag sequence is often connected to the 5' end of the primer to track the specific target sequence, but the tag sequence makes the primer sequence extended. The complexity of the primers is increased. When multiple pairs of primers are used at the same time, ordinary PCR cannot meet the requirements of high-throughput sequencing library construction, and may cause amplification errors of PCR primers

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