Library building method based on high-throughput sequencing

Abstract

The invention discloses a library building method based on high-throughput sequencing. The library building method based on the high-throughput sequencing is applicable to fragmented nucleotide samples, such as fragmented genomic DNA / FFPE RNA / cfDNActDNA and the like. Furthermore, the fragmented nucleotide samples are subjected to modification, denaturation and joint connection, followed by enrichment of target regions by two specific PCR reactions, so as to obtain a sequencing library; and the sequencing library is subjected to quality control and quantitative analysis so as to carry out high-throughput sequencing. The invention further provides a simulated double-chain connection joint. The library building method based on the high-throughput sequencing is simple and convenient, and retains advantages of multiplex PCR library building methods based on amplicons; and moreover, the library building method based on the high-throughput sequencing can also be used for detecting unknownfusion, and is especially applicable for detecting nucleotide samples (e.g. cfDNA / ctDNA sample) with relatively high degree of fragmentation. The library building method based on the high-throughput sequencing is also feasible for building libraries of DNA molecules with damages. The library building method based on the high-throughput sequencing disclosed by the invention is capable of retaining all original nucleotide information to the maximum extent; and moreover, no two-chain synthesis is required when the method is adopted for carrying out RNA library building.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for building a library based on high-throughput sequencing. Background technique [0002] Today's human whole genome sequencing has made great progress, but there are still problems such as incomplete and accurate interpretation databases related to this technology, and high sequencing costs, so it is difficult for this technology to analyze some challenging genomic regions. At the same time, the high cost and complexity of this technology limit its application in specific genomic regions such as diseases and transformations. For this reason, a variety of methods for high-throughput sequencing library construction for enrichment of target regions for specific genomic regions have been widely developed and applied, thereby improving coverage, and achieving the purpose of simplifying the process and reducing costs. The library construction method of the present in...

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Problems solved by technology

[0005] This library construction method has the following disadvantages: (1) The library is dried and dehydrated with a vacuum pump of a special instrument, and then dissolved in a hybridization buffer, which easily leads to cross-contamination during vacuum treatment; (2) It is necessary to use a variety of elution buffers to capture the product Perform cleaning to remove non-specific binding. The operation of this step is relatively complicated, and the reaction system is sensitive to temperature. The proficiency of the experimenter has a great influence on the capture efficiency of the target product; (3) if there are damaged double-stranded DNA molecules ( figure 1 B), it cannot be successfully amplified, resulting in the loss of the original DNA information; (4) If the nucleotide sample is RNA, it is necessary to reverse transcribe the RNA sample into cDNA first, and then obtain double-stranded DNA molecules through further PCR amplification. For subsequent connection and database building operations, the process is complicated and the cost is high

[0008] This library construction method has the following disadvantages: (1) It relies on the PCR amplification of the target region with clear forward and reverse primers, so it cannot be used for the detection of unknown types of fusion mutations; (2) if there is a damaged double-strand DNA molecules ( figure 2 B), it cannot be amplified smoothly, resulting in the loss of original DNA information

[0011] The above-mentioned method of building a library has the following disadvantages: (1) The use of biotin-labeled magnetic beads to enrich the ligation product leads to complex procedures, high cost, and the reaction system is sensitive to temperature. The operator's proficiency directly affects the concentration of the target product Capture efficiency; (2) If there are damaged double-stranded DNA molecules ( image 3 B), it cannot be successfully amplified, resulting in the loss of the original DNA information; (3) If the nucleotide sample is RNA, it is necessary to reverse transcribe the RNA sample into cDNA first, and then obtain double-stranded DNA molecules through further PCR amplification. For subsequent connection and database building operations, the process is complicated and the cost is high

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