Double-terminal molecular tag connector, application thereof and sequencing library comprising same

Abstract

Disclosed are a double-terminal molecular tag connector, application thereof and a sequencing library comprising the same. The double-terminal molecular tag connector comprises a first chain sequenceand a second chain sequence; the 3' terminal of the first chain sequence comprises a molecular tag composed of 2-4 basic groups, and at least one basic group site with basic group balancing effects; the 5' terminal of the second chain sequence comprises a molecular tag composed of 2-4 basic groups, and at least one basic group site with basic group balancing effects; the molecular tag of the firstchain sequence and that of the second chain sequence are in complementary pairing, and the basic group site with basic group balancing effects of the first chain sequence and that of the second chainsequence are in complementary pairing. When applied to sequencing, the double-terminal molecular tag connector can reduce sequencing read waste, solve the problem of basic group imbalance and improvethe quality of sequencing data.

Description

technical field [0001] The invention relates to the technical field of sequencing, in particular to a paired-terminal molecular tag joint, its use and a sequencing library with the joint. Background technique [0002] Due to its high-throughput and low-cost advantages, high-throughput sequencing technology has become an important genetic detection technology. At present, mainstream high-throughput sequencing technology providers include Illumina, Thermo Fisher, Pacbio in the United States, nanopore in the United Kingdom, and BGI in China. All of these sequencing technologies basically adopt the strategy of library construction before sequencing and sequencing while synthesizing. Since there are multiple DNA amplification steps in the process of library construction and sequencing, each amplification has a certain probability of introducing wrong bases, resulting in artificial mutations and background noise in sequencing. Different sequencing technologies have different err...

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Problems solved by technology

[0003] When detecting somatic mutations, the frequency of somatic mutations in DNA is often relatively low, even lower than 0.1% in many cases, while the background noise in high-throughput sequencing is often higher than 0.1%. Will miss true low-frequency mutations, leading to false negative results

Another situation is that in RNA sequencing, it is often necessary to accurately distinguish and quantify the types and numbers of the original RNA molecules. Duplication, errors, and biases caused by DNA amplification can cause qualitative errors or quantitative distortions in the final RNA.

The third source of sequencing noise is that when biological samples are exposed to certain chemical substances, some bases in the double strand of DNA will undergo asymmetric mutations. For example, paraffin-embedded formaldehyde-fixed tissue samples (FFPE) will appear very A high ratio of C>T mutations, this asymmetric variation caused by in vitro chemicals can also interfere with the detection of low-frequency mutations by high-throughput sequencing technologies

Another patent "a method for preparing a molecular tag" (application number 201610496676.3) adopted a two-step single-strand extension method to avoid enzyme cleavage, but it did not avoid multiple linker reactions and purification processes, and the operation was cumbersome. serious loss

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