A method and application of constructing a hybrid capture sequencing library

Abstract

The invention belongs to the field of gene detection, and in particular relates to a strategy for direct hybridization of genomic DNA and a method for constructing a DNA library, as well as its application in the field of nucleic acid detection. The steps are as follows: fragment and phosphorylate the genomic DNA; hybridize the fragmented genomic DNA with the capture probe; elute the captured single-stranded DNA after hybridization; connect the single-stranded DNA with the adapter to obtain a ligation product; use the ligation product as The template is amplified by PCR to obtain an amplified product, and the amplified product is purified to obtain a sequencing library. The invention provides a direct hybridization method using fragmented genomic DNA, which is generally applicable to solid-phase chip capture hybridization systems and liquid-phase capture hybridization systems, and greatly improves capture efficiency and balance. At the same time, the present invention further provides a DNA library construction method, which has the characteristics of simple method, low cost, wide applicability, etc., and can be used for the construction of DNA or RNA high-throughput sequencing library.

Description

technical field [0001] The invention belongs to the field of gene detection, and in particular relates to a strategy for direct hybridization of genomic DNA and a method for constructing a DNA library, as well as its application in the field of nucleic acid detection. Background technique [0002] The target region capture method based on next-generation sequencing has been widely used in scientific research and clinical gene detection. Since the capture sequencing technology requires a small amount of sequencing data, it not only reduces the detection cost, but also reduces the calculation amount and calculation time in the data analysis process. At present, a large number of capture sequencing methods have been developed, mainly PCR-based capture technology and hybridization-based capture technology. [0003] Hybridization-based capture techniques are divided into chip hybridization and liquid phase hybridization. Chip hybridization capture technology is a method of synt...

AI Technical Summary

Problems solved by technology

In the process of hybridization and capture, since the prior art uses a DNA library containing an adapter for hybridization, this will cause the capture efficiency of the DNA library to be greatly reduced due to the annealing between the complementary sequences of the library adapter when the sample is hybridized; at the same time, Non-specific DNA library fragments may also be captured due to annealing between adapters, and there will be a large number of non-target sequence libraries in the form of cascade amplification; in addition, the process of generating DNA libraries requires a certain number of cycles of PCR amplification , needs to be amplified and enriched again after capture, which introduces errors and imbalances in the potential PCR process

These greatly affect the quality of the capture library, waste a lot of data and even affect the results of genetic testing

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