Method for constructing a hybrid capture sequencing library and application thereof

Abstract

The invention belongs to the field of gene detection, in particular to a strategy for direct hybridization of genomic DNA and a method for constructing a DNA library and an application thereof in thefield of nucleic acid detection. The method comprises the following steps: genomic DNA is fragmented and phosphorylated; Fragmented genomic DNA hybridized with capture probes; after hybridization, thecaptured single-stranded DNA was eluted. A ligation product is obtained by linking a single strand DNA with a linker; The ligation product was used as template for PCR amplification to obtain the amplified product, and the amplified product was purified to obtain the sequencing library. The invention provides a method for directly hybridizing fragmented genomic DNA, which is universally applicable to a solid-phase chip capture hybridization system and a liquid-phase capture hybridization system, and greatly improves capture efficiency and balance. At that same time, the invention further provide a DNA library construction method, which has the characteristics of simple method, low cost, wide applicability and the like, and can be use for the construction of a high-throughput sequencing document library of DNA or RNA.

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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