Construction method of high-flux sequencing library and reagent kit for library construction

Abstract

The invention provides a construction method of a high-flux sequencing library and a reagent kit for library construction. The reagent kit comprises one or more of the following components: a high-flux sequencing Y-shaped joint, a universal primer for single-end linear PCR amplification, a biotin labeling specific primer for single-end linear multiplex PCR amplification, forward and backward library amplification primers, a UDG enzyme and the like. The invention relates to a method for constructing a targeting dimolecular identifier (UMI (unique molecular identifier) and chain unique molecularidentifier) high-flux sequencing library based on the reagent kit. According to the method, double error correcting mechanisms of a random UMI and a chain unique molecular identifier having sequencepolymorphism in a targeting sequencing system based on multiplex PCR amplification are realized, and disadvantages of most of conventional multiplex PCR amplification targeting sequencing systems areavoided, so that all false positives and false negatives in mutation detection are avoided, and high-sensitivity high-accuracy high-depth detection can be performed on low-frequency nucleic acid mutation in samples.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing a high-throughput sequencing library and a kit for constructing the library. Background technique [0002] A genetic mutation is a permanent change in the nucleic acid sequence of an organism's genome. Mutations often result from errors during gene replication or from other forms of single-strand DNA damage that escape the body's DNA error-correction repair mechanisms. It is generally believed that mutations in organisms include somatic mutations and germline mutations. Among them, somatic mutation is one of the main features of cancer, so the accuracy, sensitivity, and specificity of somatic mutation detection are crucial for early diagnosis of cancer, companion diagnosis and follow-up treatment with mutation-targeted small molecule drugs. As for mutations from the germline, taking the human diploid genome as an example, the allelic frequencies of genetic...

AI Technical Summary

Problems solved by technology

At present, the error correction function of the targeted sequencing technology based on multiplex PCR amplification is mostly to use the coupling random base UMI to the 5' end of the site-specific primer, and perform 2 to 3 rounds of amplification to obtain a complete high-throughput sequencing library structure; As the number of UMI random bases increases, the specific binding of site-specific primers will decrease, resulting in a decrease in the on-target rate; at the same time, in the conventional multiplex PCR targeted Reverse primers will produce non-specific amplification products during amplification, and these non-specific amplification products often dominate the amplification, seriously affecting the amplification efficiency of the target region; the design of conventional forward and reverse primers leads to Very low utilization of short DNA fragments

[0006] From the perspective of clinical applicability, the core of liquid-phase hybridization capture-based targeted sequencing—probe synthesis is almost entirely dependent on foreign companies such as Roche, Agilent, and IDT, and the cost remains high; at the same time, customized probes often use Mix Delivery in form, unable to realize the delivery and use of single-tube probes, lack of flexibility

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