CN-121992095-A - Method for detecting cleavage sites in human chromosome structural variation

Abstract

The invention provides a method for detecting a cleavage site in human chromosome structural variation, belonging to the technical field of gene detection. The method comprises the following steps of S1, cutting genome DNA to be detected into 2-10kb long fragments, cyclizing after terminal modification, capturing cyclized fragments for library establishment, S2, screening abnormal reading pairs after sequencing, clustering and locking candidate regions, S3, designing a cross-breakpoint primer for PCR amplification, and sequencing a product to determine breakpoint coordinates. The detection method provided by the invention can realize single base resolution positioning of chromosomal structural variation including translocation, inversion, insertion, repetition, deletion and the like, is suitable for low-quality samples, and successfully solves the technical problems of narrow detection range, dependence on high-quality samples, low precision and the like of the existing method.

Inventors

• CHANG LIANG
• QIAO JIE
• LI RONG

Assignees

• 北京大学第三医院（北京大学第三临床医学院）

Dates

Publication Date

20260508

Application Date

20251204

Claims (10)

1. 1. A method for detecting a human chromosomal structural variation cleavage site comprising the steps of: S1, shearing genome DNA to be detected into a large fragment with a target length, performing terminal modification, cyclizing after connecting a specific joint, capturing cyclized fragments and establishing a library, wherein the total amount of the DNA in a cyclized system is not more than 600ng; S2, screening abnormal reading pairs after sequencing, and clustering and locking candidate areas; s3, designing a PCR (polymerase chain reaction) amplification of a cross-breakpoint primer, and sequencing a product to determine breakpoint coordinates.
2. 2. The method of claim 1, wherein in S1, the target length is 2-10kb.
3. 3. The method of claim 1, wherein in S1, the terminal modification comprises at least one of terminal filling, 5 'terminal phosphorylation, and/or 3' terminal addition of a tail.
4. 4. The method of claim 1, wherein in S1, the specific linker comprises biotin.
5. 5. The method of claim 1, wherein in S2, the sequencing comprises double-ended sequencing.
6. 6. The method of claim 1, wherein in S2, the clustering method comprises a machine learning algorithm.
7. 7. The method of claim 6, wherein the machine learning algorithm comprises at least one of a K-means unsupervised clustering algorithm, a DBSCAN algorithm, a spectral clustering algorithm, a mean shift clustering algorithm, a constrained K-means algorithm, a label propagation algorithm, a random forest algorithm, a support vector machine algorithm, a convolutional neural network algorithm, or a graph neural network algorithm.
8. 8. A kit for detecting a site of disruption of structural variation in a human chromosome, comprising detection reagents for carrying out the method of any one of claims 1 to 7.
9. 9. An electronic device comprising detection means, computing means and output means, the detection means comprising a sample injector, a sample processor and a detector to implement the method of any of claims 1-7.
10. 10. A computer readable program medium, characterized in that it stores computer readable instructions, which when executed by a processor, cause a computer to perform the method of any of claims 1-7.

Description

Method for detecting cleavage sites in human chromosome structural variation Technical Field The invention relates to the field of C12Q1/6869, in particular to a method for detecting a cleavage site in human chromosome structural variation. Background The structural stability of the chromosome, which serves as a core carrier of human genetic information, directly determines the normal functioning of the gene function. In the course of life activities and disease occurrence and development, large-scale structural variations (Structural Variation, SV) frequently occur in chromosomes, including types such as translocation, inversion, insertion, repetition, deletion and the like, and complex rearrangements, and the like, and such variations usually involve DNA fragment changes of more than or equal to 50bp, and are one of key molecular mechanisms leading to genetic diseases and tumorigenesis and development. The translocation, inversion and other "equilibrium variation" do not change the copy number of the gene, but the cleavage fusion site may directly destroy the coding region or the regulatory region of the key functional gene, or promote the recombination of originally non-adjacent gene fragments to form a fusion gene, for example, the mutual translocation of chromosomes may cause the generation of an oncogene and further drive the abnormal proliferation of tumor cells, and the structural variation of embryogenesis may cause serious consequences such as abortion, congenital genetic diseases and the like, so that the precise positioning (especially single base resolution) of the SV cleavage site is realized, and the method has irreplaceable significance for disease molecular diagnosis, etiology research, genetic information inquiry and clinical treatment scheme formulation. With the development of molecular biology technology, various SV detection technologies have been developed in the scientific research and clinical fields, but the existing methods still have significant limitations, and are difficult to meet the requirements of accurate detection. Early cytogenetic techniques such as karyotyping and Fluorescence In Situ Hybridization (FISH) can primarily identify chromosome structural abnormalities, but the karyotyping can only detect large fragment variation at the level of Mb and has extremely low resolution, while the FISH technique has the advantages that the resolution is increased to the level of kb, probes are designed aiming at specific breakpoints, high-throughput detection cannot be realized, and the detection sensitivity of low-abundance variation or complex genome regions is insufficient. The popularization of Next Generation Sequencing (NGS) technology provides a new path for SV detection, and detection methods based on Whole Genome Sequencing (WGS) are currently mainstream, and the core of the detection methods depends on signal identification variations such as Read-Pair (RP), split-Read (SR), or Read coverage depth (RD). However, the conventional WGS method has three key defects of low cost, extremely high sequencing depth (generally > 30X) for covering a whole genome and capturing rare variation signals, huge data amount and high detection cost, limited positioning precision, limited dependence on sparse abnormal signal clustering to deduce break points, often only locking candidate intervals of thousands of bases, incapability of realizing single base-level accurate positioning, follow-up verification by means of Sanger sequencing and the like, complicated flow and low success rate, low signal-to-noise ratio, easy occurrence of comparison errors in short-reading long-sequencing in complex genome areas such as high GC and repeated sequences, generation of a large number of false positive or false negative results, and complicated filtering algorithm and manual examination assistance. In view of the above-mentioned shortcomings, some prior art attempts to improve SV detection performance by technical optimization, but still have significantly shorter plates. For example, chinese patent CN117059173A discloses a precise breakpoint identification method of Copy Number Variation (CNV), which optimizes breakpoint identification precision through global CNV region expansion, window merging and Cyclic Binary Segmentation (CBS) algorithm and kmeans clustering, but only aims at the copy number variation of CNV, can not detect balanced SV without copy number variation, such as translocation, inversion and the like, and relies on raw signal analysis in the whole course, has no experimental verification link, the breakpoint precision is still limited to kb level, and can not meet the clinical requirement on balanced variation single base positioning, chinese patent CN113416770A discloses a positioning method of chromosome structure variation breakpoint, which utilizes an optical map platform to perform variation primary screening with a fragment resolution of 500bp at most, but positions the structur