Library construction primer group and application thereof in high-throughput detection

Abstract

The invention relates to a rapid and accurate library construction primer group and application thereof in high-throughput detection, and belongs to the technical field of gene detection. The library construction primer group comprises a plurality of amplification primers for target area amplification and a sequencing linker for library construction, each amplification primer sequentially comprises a target sequence segment matched with a target area sequence, a single-molecule tag segment playing a unique recognition role and a library construction segment matched with the sequencing linker, and the multiple single-molecule tag segments comprise random basic groups with different lengths. The rapid and accurate library construction primer group can be used for quickly and accurately constructing a base balanced library, the library construction time is shortened, pollution and sequencing cost are reduced, data are more accurately corrected, and the accuracy of the technology is improved. The rapid and accurate library construction primer group can be more conveniently and efficiently used in scientific research and clinical research. In a scientific research project, the immune group can be sequenced without a sequencer with high productivity, and the base balance library is not needed, so that the rapid and accurate library construction primer group is convenient and efficient, and the application and development of the immune group are promoted and expanded.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a rapid and accurate library construction primer set and its application in high-throughput detection. Background technique [0002] The immune repertoire (Immune Repertoire, IR) is defined as the T cell receptor (T cell receptor, TCR) and B cell receptor (B cell receptor, BCR) of the body's adaptive immune system at any given time. Sum. During the maturation process of T / B cells, rearrangement of the VDJ gene will occur. During the rearrangement process, a V, D and J gene is randomly selected and connected in series to form the variable region of TCR / BCR. Random insertion and deletion of bases occurs at the site, so the TCR / BCR genes of almost every newly generated T / B cell are different, forming a large number of TCR / BCR immune repertoires, endowing the body with the ability to recognize various antigens Ability. Analysis of TCR / BCR coding genes by high-throughput seq...

AI Technical Summary

Problems solved by technology

[0005] Specifically, the two-step PCR method cannot add barcodes to distinguish different samples at the beginning, so the data cannot be split into contaminated sequences, the contamination rate is high, and the library construction time is longer (at least 1 day), and the cost is higher

The existing one-step PCR method cannot accurately perform PCR and sequencing error correction

Regardless of the one-step or two-step PCR method, it cannot solve the problem of high sequence similarity in the V region of the immune group, which is likely to cause base imbalance, that is, when the proportion of bases at the same position is relatively single during sequencing, the base reading will be affected , resulting in the inability to read the base signal, so it is often necessary to run the same lane with a 20%-30% base-balanced library (such as the whole genome, exon, etc.) on the machine, resulting in an increase in sequencing costs

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