Target gene enrichment library building method

Abstract

The invention relates to a target gene enrichment library building method, which comprises: a phosphorylation step: modifying a phosphate group at the 5' end of a template molecule; an extension step: adding a first primer, modifying the 5' end of the first primer with a labeled molecule, annealing and extending in the target region of the template molecule to obtain a double-stranded target nucleotide, the double-stranded target nucleotide containing an extension chain obtained by extending the first primer of which the 5' end is modified with the labeled molecule; a first sequencing linker ligation step comprising ligation of a first sequencing linker to the double stranded target nucleotide sequence; an unlinking step: carrying out unlinking treatment on a product obtained in the previous step, and removing a first primer extension chain of which the 5' end is modified with a labeled molecule to obtain a template molecule connected with a first sequencing linker, namely a single-stranded target nucleotide molecule; a second sequencing joint connection step; and an amplification step: adding a second primer and a third primer, and carrying out PCR reaction to obtain a complete library. Library building and target gene enrichment are integrated into a whole, and the process is remarkably shortened.

Description

technical field [0001] The invention relates to the technical field of gene sequencing, in particular to a method for enriching a target gene and building a library. Background technique [0002] The preparation process of high-throughput sequencing target gene sequencing library is generally divided into two sets of processes. The mainstream capture library construction method needs to go through library construction (including single-strand library construction by a new library construction method), pre-capture amplification, hybrid capture, post-capture There are four necessary steps for amplification, and the whole process generally takes 2 to 3 days. Another common method is called amplicon library building. Generally, multiplex PCR is performed first, and then the PCR product library is built. Some commercial kits will add corresponding NGS to the outside of the 5′ end of the primer when doing multiplex PCR. The linker sequence of the platform to integrate the above t...

AI Technical Summary

Problems solved by technology

[0003] The first mainstream technical route must strictly separate library construction and hybridization capture, with many steps and a long cycle, and the connection between modules is technically difficult, and the synthesis of reagents and capture-specific probes required for hybridization capture is heavily dependent on imports

Although the second technical route is simpler than the former, it has the following problems because it is based on multiplex PCR: 1. Because it is based on PCR amplification, there are a large number of amplified copies of the same molecule in the library, resulting in the result of sequencing The redundancy rate is high, the amount of sequencing data is wasted, and the effective data rate is reduced, making it difficult or impossible to detect ultra-low frequency gene mutations; 2. Due to mutual interference between PCR primers, the number of PCR multiplexes in the same reaction system (that is, how many single PCRs are mixed in multiple PCR reactions) cannot be too many (generally no more than 1000 multiples), which makes it difficult to complete the gene detection of a large panel through a single-tube reaction, and can only be divided into multiple single-tube reactions, and then merge the products 3. PCR requires pairing of primers at both ends, which makes it impossible to detect genomic structural variations such as unknown fusion genes (novel fusion) and virus insertion sites; 4. 1. Due to the exponential amplification of PCR, the copy numbers between different genes are not linearly amplified in equal proportions, which makes it impossible to detect gene copy number variation; 5. The inevitable amplification bias of multiplex PCR leads to low uniformity, which leads to Some regions are not well covered (mutations in these regions cannot be accurately detected), while some regions are over-covered (causing a waste of local sequencing data)

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