Probes, methods, kits for detecting structural variations in DLBCL and uses thereof

Abstract

The application belongs to the technical field of molecular biology and tumor pathological diagnosis, and particularly relates to a probe, a method, a kit for detecting DLBCL structural variation and application thereof. The application takes FFPE samples as detection objects, and detects DLBCL structural variation through dewaxing and decrosslinking treatment, restriction enzyme cutting, terminal biotin labeling, ligation, chromatin decrosslinking, terminal treatment, biotin removal and purification, targeted capture, sequencing and data analysis. The application uses multiple detergents combined with protease to synergistically dewax and decrosslink, improves chromatin integrity; optimizes ligation conditions, degrades samples with specific adaptors, and improves ligation efficiency; innovatively uses a composite enzyme system to completely remove terminal biotin; and designs and customizes capture probes based on DpnII enzyme cutting sites, improves targeted region capture efficiency and detection resolution. The application provides comprehensive and accurate molecular basis for DLBCL structural variation analysis.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology and tumor pathology diagnostic technology, specifically relating to a probe, method, kit, and application for detecting structural variations in DLBCL. Background Technology

[0002] Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of adult non-Hodgkin lymphoma, accounting for approximately 30%-40% of cases. It exhibits highly heterogeneous biological characteristics and varied clinical prognoses. Structural variations such as gene fusions, rearrangements, and enhancer hijacking play crucial roles in the occurrence and development of DLBCL, and their accurate detection is of great significance for elucidating tumor pathogenesis, guiding clinical treatment, and assessing prognosis.

[0003] Currently, the detection of structural variations in DLBCL mainly relies on RNA-seq and fluorescence in situ hybridization (FISH) techniques. RNA-seq can effectively detect structural variations that form fusion transcripts, but it cannot detect structural variations in intergenic regions (such as enhancer hijacking). These variations do not produce fusion RNA but promote tumorigenesis by regulating oncogene expression. FISH technology can effectively detect clinically significant reproducible gene rearrangements in DLBCL, such as MYC, BCL2, and / or BCL6 rearrangements. Patent CN102747156B utilizes FISH technology to examine paraffin-embedded tissue sections (such as lymph nodes) and bone marrow smears from DLBCL patients, finding that BCL2 and IgH gene rearrangements can serve as molecular markers for bone marrow infiltration in DLBCL and as markers for evaluating treatment efficacy. However, FISH technology relies on specific probe design and can only detect structural variations in areas known to be covered by the probe. It is ineffective for intergenic variations not covered by the probe and suffers from limitations in detection range and cumbersome operation. In addition, clinically preserved formalin-fixed paraffin-embedded (FFPE) samples often face problems such as degradation and antigen loss, which further reduces the sensitivity and specificity of existing detection technologies.

[0004] High-throughput Chromosome Conformation Capture (Hi-C) technology can capture chromatin interaction information across the entire genome, providing an effective means for elucidating chromatin's three-dimensional structure and structural variations. However, traditional Hi-C technology suffers from high sequencing costs, low data utilization, and poor adaptability to degraded samples, limiting its large-scale application in clinical structural variation detection. Targeted Hi-C technology, by designing specific probes to capture chromatin interaction fragments in target gene regions, effectively reduces costs and improves specificity. However, when applied to DLBCL structural variation detection, existing targeted Hi-C technologies still face technical bottlenecks such as poor FFPE sample processing, low ligation efficiency, incomplete biotin removal, and insufficient targeted capture specificity. In particular, conventional probes often focus on single regions and lack clear enzyme site association designs, resulting in insufficient capture efficiency (mostly below 30%) and resolution (mostly limited to 100kb). Furthermore, they are not specifically optimized for structural variation detection in DLBCL characteristic genes, failing to fully leverage their unique advantages in detecting intergenic variations. In addition, existing DLBCL molecular detection kits are mostly designed for single biomarkers or conventional sequencing technologies, and lack dedicated kits adapted to Hi-C technology, thus failing to support the standardized detection of structural variations in clinical samples.

[0005] Therefore, developing a targeted Hi-C detection method and dedicated kit that is compatible with clinical FFPE samples, can comprehensively detect DLBCL structural variations (including intergenic enhancer hijacking), and is highly consistent with traditional detection techniques has significant clinical application value and industrialization prospects. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, such as poor FFPE sample processing, low ligation efficiency, incomplete biotin removal, and insufficient targeted capture specificity, this invention proposes a probe, method, kit, and application for detecting structural variations in diffuse large B-cell lymphoma based on targeted Hi-C. Compared with conventional RNA-seq or FISH detection, this invention has superior targeted region capture efficiency and detection resolution, and also boasts strong sample adaptability and controllable cost.

[0007] One objective of this invention is to provide a probe for detecting structural variations in diffuse large B-cell lymphoma based on targeted Hi-C. Using this customized capture probe can effectively improve the capture efficiency and detection resolution of the targeted region, thus supporting the effective detection of structural variations in diffuse large B-cell lymphoma.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A probe for detecting structural variations in diffuse large B-cell lymphoma based on Hi-C targeting, the probe comprising the nucleotide sequences shown in SEQ ID NO.1 to SEQ ID NO.100.

[0010] Preferably, the probes cover the main body, promoter, and enhancer regions of the MYC, BCL2, and BCL6 core genes and the BCL10 and CD274 helper genes, and adopt a shingled design with an overlap of 30-60 bp between adjacent probes.

[0011] Preferably, the probe is precisely designed within a 300bp range on both sides of each DpnII restriction site to ensure that the digested fragments can be efficiently captured.

[0012] Preferably, the probe has a GC content of 40%-60% and a sequence specificity of ≥98%.

[0013] Preferably, the 5' end of the probe is modified with biotin.

[0014] Preferably, the structural variations include gene fusion, rearrangement, amplification, and / or enhancer hijacking.

[0015] Preferably, the structural variations include BCL2-IGH fusion, BCL6-IGH fusion, BCL10-IGH fusion, CD274-ZCCHC7 fusion, IGK-MYC fusion, MYC-IGL fusion, BCL6 rearrangement, MYC-BCL2 co-rearrangement, BCL10 rearrangement, CD274 amplification + BCL2 fusion, MYC rearrangement, BCL6-IGK fusion, BCL2 rearrangement + BCL10 amplification, and / or MYC-IGH fusion.

[0016] The second objective of this invention is to provide a kit for detecting structural variations in diffuse large B-cell lymphoma based on targeted Hi-C.

[0017] To achieve the above objectives, the present invention adopts the following technical solution:

[0018] A kit for detecting structural variations in diffuse large B-cell lymphoma based on targeted Hi-C, the kit containing the aforementioned probe.

[0019] Preferably, the kit further comprises:

[0020] Specific adaptors, including adaptor sequence 1 shown in SEQ ID NO.176 and adaptor sequence 2 shown in SEQ ID NO.177;

[0021] Reagents for dewaxing and decrosslinking treatment of FFPE include dewaxing solution, detergent and protease, wherein the detergent includes SDS, SDC, Triton X-100 and NP40;

[0022] Reagents used for restriction enzyme digestion include DpnII or MboI;

[0023] Reagents used for terminal biotinylation labeling include Klenow DNA polymerase;

[0024] Reagents used for ligation processing include T4 DNA ligase;

[0025] Reagents used for chromatin decrosslinking treatment include magnetic beads, proteinase K, and purified magnetic beads;

[0026] Reagents used for terminal biotin treatment include T4 DNA polymerase, exonuclease I, and exonuclease III.

[0027] Preferably, the detergent comprises the following components in final concentrations: 0.1%-0.7% SDS, 0.5%-2% SDC, 1%-5% Triton X-100 and 0.5%-2% NP40.

[0028] Preferably, the detergent comprises the following components at final concentrations: 0.3% SDS, 1% SDC, 3% Triton X-100, and 1% NP40.

[0029] Preferably, the reagent used for dewaxing and decrosslinking treatment of FFPE further includes a buffer solution; more preferably, the buffer solution contains 50~100 mM Bis-Tris-HCl, 100~150 mM NaCl, 10~15 mM MgCl2 and 0.1~0.15 mM MTT.

[0030] Preferably, the reagent used for dewaxing and decrosslinking treatment of FFPE comprises a protease at a concentration of 10-30 mg / mL. More preferably, the protease concentration is 20 mg / mL, and the protease is proteinase K, but is not limited to proteases with equivalent functions.

[0031] Preferably, the reagent used for restriction enzyme digestion includes 10 U / µL DpnII or 12-18µL MboI.

[0032] Preferably, the reagents used for terminal biotinylation labeling further include biotinylation buffer, dCTP, dGTP, dTTP, biotin-14-dATP, and deionized water.

[0033] Preferably, the reagents for end-biotinylation labeling treatment include 3-4 µL of 8-12× biotinylation buffer, 0.5-1 µL of 8-12 mM dCTP, 0.5-1 µL of 8-12 mM dGTP, 0.5-1 µL of 8-12 mM dTTP, 0.5-1 µL of 8-12 mM biotin-14-dATP, 4-6 U / µL of Klenow DNA polymerase, and 20-26 µL of deionized water; more preferably, it includes 3-4 µL of 10× biotinylation buffer, 0.5-1 µL of 10 mM dCTP, 0.5-1 µL of 10 mM dGTP, 0.5-1 µL of 10 mM dTTP, 0.5-1 µL of 10 mM biotin-14-dATP, and 5 U / µL of Klenow DNA polymerase. 4-6 µL of DNA polymerase and 20-26 µL of deionized water.

[0034] Preferably, the reagents used for the ligation process further include Triton X-100, BSA, and deionized water.

[0035] Preferably, the reagents used for the ligation process include 50-70 µL of 8-12×T4 DNA ligase buffer, 50-70 µL of 8-12% Triton X-100, 4-8 µL of 8-12 mg / mL BSA, 6-10 µL of 500-700 U / µL T4 DNA ligase, and 180-210 µL of deionized water.

[0036] Preferably, the reagents used for ligation treatment include 50-70 µL of 10×T4 DNA ligase buffer, 50-70 µL of 10% Triton X-100, 4-8 µL of 10 mg / mL BSA, 6-10 µL of 600 U / µL T4 DNA ligase, and 180-210 µL of deionized water.

[0037] Preferably, the reagents for terminal biotin treatment further include Hi-C DNA, biotinylation buffer, dATP, dGTP, and deionized water.

[0038] Preferably, the reagents for terminal biotin treatment include 5-15 µg of Hi-C DNA, 11-15 µL of 8-12× biotinylation buffer, 2.5-4 µL of 0.5-2 mM dATP, 2.5-4 µL of 0.5-2 mM dGTP, 11-15 µL of 2000-4000 U / mL T4 DNA polymerase, 5-10 U of exonuclease I, 10-20 U of exonuclease III, and deionized water.

[0039] Preferably, the reagents for terminal biotin treatment include 5-15 µg of Hi-C DNA, 11-15 µL of 10× biotinylation buffer, 2.5-4 µL of 1 mM dATP, 2.5-4 µL of 1 mM dGTP, 11-15 µL of 3000 U / mL T4 DNA polymerase, 5-10 U of exonuclease I, 10-20 U of exonuclease III, and deionized water.

[0040] Preferably, the kit further comprises reagents for purification after biotin removal, the reagents for purification after biotin removal including magnetic beads.

[0041] The kit provided by this invention is mainly used for dewaxing, decrosslinking, enzyme digestion, end biotinylation labeling, ligation, chromatin decrosslinking, end treatment, targeted capture and purification after biotin removal of FFPE samples. The reagents work together to achieve high efficiency in dewaxing and decrosslinking, improved ligation efficiency, complete removal of biotin, and high specificity in targeted capture.

[0042] The third objective of this invention is to provide a method for detecting structural variations in diffuse large B-cell lymphoma based on targeted Hi-C.

[0043] To achieve the above objectives, the present invention adopts the following technical solution:

[0044] The method for detecting structural variations in diffuse large B-cell lymphoma based on targeted Hi-C uses the aforementioned probes and / or the aforementioned kits for detection;

[0045] The method is not intended for the diagnosis and / or treatment of disease, and specifically includes the following steps:

[0046] S1: Add dewaxing solution to the sample to be tested for dewaxing, resuspend it, and then add the aforementioned detergent and protease for decrosslinking treatment;

[0047] S2: The sample treated with S1 was digested with DpnII or MboI enzymes;

[0048] S3: End-biotinylated labeling of the sample after S2 enzyme digestion;

[0049] S4: The S3-labeled sample was ligated using T4 DNA ligase and a specific adaptor.

[0050] S5: The sample after S4 ligation was washed with magnetic beads, digested with proteinase K, purified with magnetic beads and eluted to obtain pure chromatin DNA;

[0051] S6: The S5 decrosslinked sample was end-treated using T4 DNA polymerase, exonuclease I and exonuclease III to remove unlinked biotin.

[0052] S7: Use the aforementioned probe to target and capture the sample processed in S6;

[0053] S8: Perform high-throughput sequencing on the samples captured in S7;

[0054] S9: Analyze sequencing data and extract structural variation features.

[0055] Preferably, in S4, the ligation temperature is 16-20℃ and the ligase concentration is 8-10U / µL.

[0056] Preferably, in S4, the ligation temperature is 16°C and the ligase concentration is 8 U / µL.

[0057] This invention optimizes the connection conditions and adopts a specific linker scheme, thereby increasing the %_Intra_ge_15kb_pairs of the degraded sample by 80.00% and the %_Unique_valid_pairs by 25.00% compared with the traditional scheme.

[0058] Preferably, the sample to be tested includes a formalin-fixed paraffin-embedded sample. The method of this invention can effectively detect degraded samples preserved for more than 5 years.

[0059] Preferably, in S1, the dewaxing reaction conditions are: heating at 50-60℃ for 3-10 min and centrifugation at 2000-3000g for 3-10 min; the decrosslinking treatment conditions are: incubation at 70-78℃ for 40-60 min.

[0060] Preferably, in S1, the dewaxing reaction conditions are: heating at 56°C for 5 minutes and centrifuging at 2500g for 5 minutes.

[0061] Preferably, in S1, the decrosslinking treatment condition is incubation at 74°C for 50 min.

[0062] Preferably, in S1, before dewaxing and decrosslinking treatment, the FFPE tumor tissue sample to be tested is cut into 8-12µm wax rolls, more preferably 10µm.

[0063] In S1, the present invention employs multiple detergents in synergistic action with proteases, which can increase %_Intra_ge_15kb_pairs by 46.00% and %_Unique_valid_pairs by 23.01% compared to traditional single detergent solutions.

[0064] Preferably, in S2, the enzyme digestion treatment conditions include: incubation at 35-40℃ with shaking at 800-1000 rpm for 14-18 hours, followed by inactivation of enzyme activity at 60-70℃ for 15-25 minutes.

[0065] Preferably, in S2, the enzyme digestion treatment conditions include: incubation at 37°C with shaking at 900 rpm for 16 h, followed by inactivation of enzyme activity at 65°C for 20 min.

[0066] Preferably, in S3, the biotinylation system includes 3-4 µL of 8-12× biotinylation buffer, 0.5-1 µL of 8-12 mM dCTP, 0.5-1 µL of 8-12 mM dGTP, 0.5-1 µL of 8-12 mM dTTP, 0.5-1 µL of 8-12 mM biotin-14-dATP, 4-6 U / µL of Klenow DNA polymerase, and 20-26 µL of deionized water, with a total volume of 32-38 µL; the reaction conditions are 20-25 °C with shaking incubation for 3-5 h.

[0067] Preferably, in S3, the biotinylation system includes 3-4 µL of 10× biotinylation buffer, 0.5-1 µL of 10 mM dCTP, 0.5-1 µL of 10 mM dGTP, 0.5-1 µL of 10 mM dTTP, 0.5-1 µL of 10 mM biotin-14-dATP, 4-6 µL of 5 U / µL Klenow DNA polymerase, and 20-26 µL of deionized water, for a total volume of 32-38 µL.

[0068] Preferably, in S3, the reaction conditions are 23°C with shaking incubation for 4 hours.

[0069] Preferably, in S4, the ligation system includes: 50-70 µL of 8-12×T4 DNA ligase buffer, 50-70 µL of 8-12% Triton X-100, 4-8 µL of 8-12 mg / mL BSA, and water to a total volume of 600 µL; the reaction conditions are 16-20 °C with shaking incubation for 3-5 h.

[0070] Preferably, in S4, the ligation system includes: 60 µL of 10×T4 DNA ligase buffer, 60 µL of 10% Triton X-100, 6 µL of 10 mg / mL BSA, and water to a total volume of 600 µL.

[0071] Preferably, in step S4, the mixture is incubated with shaking at 900 rpm for 4 hours.

[0072] Preferably, the specific linker is added to the ligation reaction at a concentration of 5-10 µM, more preferably 8 µM.

[0073] Preferably, S5 specifically includes: placing the ligated sample on a magnetic rack and letting it stand for 2 min, then discarding the supernatant; washing twice with 1 mL of 80% ethanol, briefly centrifuging to remove residual ethanol and drying at room temperature; resuspending the magnetic beads in 140-160 µL of water, then sequentially adding 15-25 µL of buffer, 8-12 µL of 10% SDS, and 15-25 µL of 20 mg / mL proteinase K, incubating at 55℃ for 25-35 min, 68℃ for 80-100 min, and 25℃ for 8-12 min; adding 140-160 µL of purified magnetic beads, incubating at room temperature for 5 min, then placing it on a magnetic rack, discarding the supernatant, washing twice with 80% ethanol, drying, resuspending the magnetic beads in 80-100 µL of water, incubating at room temperature for 5 min, transferring the supernatant to a new tube, quantifying with Qubit, and obtaining pure chromatin DNA.

[0074] Preferably, in S6, the biotin removal reaction system includes 5-15 µg of Hi-C DNA, 11-15 µL of 8-12× biotinylation buffer, 2.5-4 µL of 0.5-2 mM dATP, 2.5-4 µL of 0.5-2 mM dGTP, 11-15 µL of 2000-4000 U / mL T4 DNA polymerase, 5-10 U of exonuclease I, 10-20 U of exonuclease III, and deionized water, with a total volume of 120-140 µL; the reaction conditions are incubation at 15-25 °C for 3-5 h followed by inactivation at 70-80 °C.

[0075] Preferably, in S6, the biotin removal reaction system includes 10µg Hi-C DNA, 13µL 10× biotinylation buffer, 3.25µL 1mM dATP, 3.25µL 1mM dGTP, 13µL 3000U / mL T4 DNA polymerase, 8U exonuclease I, 15U exonuclease III, and deionized water to a final volume of 130µL.

[0076] Compared with the traditional single enzyme method, the composite enzyme system used in this invention can increase %Intrage_15kb_pairs by 9.0% and reduce %Duplicated_pairs by 50.00%.

[0077] Preferably, in S6, the reaction conditions are: incubation at 20°C for 4 hours, inactivation at 75°C for 20 minutes, and hold at 4°C.

[0078] Preferably, after the S6 reaction is completed, a biotin-free purification process is performed, including incubation with magnetic beads, washing and elution, to obtain a high-purity biotin-free sample.

[0079] Preferably, the purification process after biotin removal specifically includes: removing the purified magnetic beads 30 minutes in advance and equilibrating them to room temperature; adding 220-240 µL of magnetic beads to 130 µL of ligation product, shaking to mix, and incubating at room temperature for 5 minutes; placing them on a magnetic rack, and removing the supernatant after the solution becomes clear; adding 500 µL of freshly prepared 80% ethanol to rinse the magnetic beads twice, incubating at room temperature for 30 seconds each time; after ionization, discarding the residual liquid, opening the cap and drying for 2 minutes; adding 35-45 µL of elution buffer, shaking to mix, and placing at room temperature for 5 minutes; placing them on a magnetic rack, aspirating the supernatant to a new tube, and performing quantification using Qubit and quality control using Qsep100.

[0080] Preferably, in S7, the present invention uses a customized capture probe, which increases the target area capture efficiency from the traditional 30% to 60% and the detection resolution from the traditional 100kb to 5kb.

[0081] Preferably, S8 includes: performing paired-end 150bp high-throughput sequencing using the BGI platform, with a sequencing depth ≥0.5G.

[0082] Preferably, S9 includes: after data quality control, alignment to the human reference genome hg38 using BWA software and deduplication, constructing a 5kb resolution chromatin interaction matrix using Juicer software; extracting structural variation-related feature parameters and normalizing them using ICE.

[0083] Preferably, the data quality control includes filtering low-quality reads, connector contaminated reads, and duplicate reads, with quality control standards of Q20 ≥ 90%, Q30 ≥ 85%, connector contaminated rate < 1%, and duplicate sequence percentage < 10%.

[0084] Preferably, the comparison efficiency is ≥80%.

[0085] Preferably, the extraction of structural variation-related feature parameters and their ICE normalization specifically includes: based on the chromatin interaction matrix, a customized Python script is used to extract structural variation features related to the MYC, BCL2, BCL6, BCL10, and CD274 genes, including abnormal chromatin interaction signals related to gene fusion, rearrangement, and intergenic enhancer hijacking. All parameters are normalized by ICE to eliminate sample bias.

[0086] Preferably, the structural variation results of Hi-C detection can be analyzed for consistency with RNA-seq / FISH detection results to assess the accuracy of the detection; for samples that are negative for RNA-seq but suggest enhancer hijacking in Hi-C detection, specific capture probes can be designed for verification.

[0087] The fourth objective of this invention is to provide the application of the aforementioned probe and / or the aforementioned kit in the preparation of a detection product for detecting structural variations in diffuse large B-cell lymphoma.

[0088] Preferably, the detection product is used to detect FFPE tumor tissue samples.

[0089] The beneficial effects of this invention are as follows:

[0090] 1. Significantly Improved Dewaxing and Decrosslinking Effects. Traditional FFPE sample dewaxing and decrosslinking effects are poor, with single detergents having limited effectiveness and incomplete decrosslinking, leading to decreased sample quality in subsequent experiments and affecting the accuracy of structural variation detection. This invention innovatively uses SDS in combination with multiple detergents such as SDC, Triton X-100, and NP40, and works synergistically with enzymes such as proteases, effectively solving the problem of incomplete decrosslinking by traditional single detergents and significantly improving chromatin integrity. Experimental verification shows that %Intrage_15kb_pairs is improved by 46.00% compared to traditional methods; %Unique_valid_pairs is improved by 23.01%, providing a high-quality sample foundation for subsequent experiments and directly supporting the effectiveness of subsequent enzyme digestion, labeling, and ligation steps.

[0091] 2. Significantly Optimized Ligation Efficiency and Adaptability to Degraded Samples. Traditional ligation methods are inefficient and lack effective strategies for adapting to degraded samples, making it difficult to form effective chromatin interaction fragments and affecting library construction quality. This invention improves upon the inefficiency of traditional ligation steps by precisely adjusting the ligation temperature (16-20℃) and ligase concentration (500-700U / µL); and further enhances ligation efficiency by designing 15-25bp specific adaptors for degraded samples. Experimental data show that the optimized method improves %Intra_ge_15kb_pairs by 20.11% for conventional samples, %Intra_ge_15kb_pairs by 80.00% for degraded samples, and %Unique_valid_pairs by 25.00%, while reducing %Inter_pairs by 13.30%. This provides more effective materials for library construction and allows for the processing of degraded samples stored for over 5 years without additional pretreatment, expanding the applicability to clinical samples.

[0092] 3. More thorough biotin removal. Traditional methods using only T4 DNA polymerase in the end-processing step (removing biotin from unligated ends) result in incomplete biotin removal, leading to residual biotin interfering with subsequent sequencing and data analysis, and reducing the proportion of valid data. This invention innovatively employs a combination of T4 DNA polymerase and exonuclease I and exonuclease III, significantly improving the biotin removal rate compared to traditional single-enzyme methods. Data validation shows a 9.09% increase in %Intrage_15kb_pairs and a 3.00% increase in %Unique_valid_pairs, significantly reducing the interference of residual biotin on sequencing and data analysis, increasing the proportion of valid data, and laying the foundation for accurate extraction of structural variation features.

[0093] 4. Breakthrough in both targeted capture efficiency and resolution. Existing targeted capture probe designs lack specificity, have limited coverage areas, and are not precisely designed to incorporate DpnII restriction sites, resulting in insufficient capture efficiency (≤30%) and resolution (≥100kb), failing to meet the needs of accurate structural variation detection. This invention's customized targeted capture probe, through an innovative design of "multi-region coverage (gene body + promoter + enhancer), restriction site association (300bp flanking DpnII site), and shingled layout (30-60bp overlap)," achieves a capture efficiency increase from the traditional 30% to 60% and a resolution increase from 100kb to 5kb, significantly outperforming existing non-specific probes. High capture efficiency ensures sufficient effective data, while high resolution enables precise identification of minute structural variations, directly improving the accuracy and sensitivity of structural variation detection.

[0094] 5. Accurate and comprehensive detection results. Existing RNA-seq / FISH detection technologies cannot comprehensively detect DLBCL structural variations. RNA-seq struggles to detect enhancer hijacking variations in intergenic regions, and FISH has detection blind spots due to probe coverage limitations. The consistency verification experiment results of this invention show that the results of 10 samples are 100% consistent with RNA-seq / FISH detection, which can supplement the detection of enhancer hijacking variations in intergenic regions and fill the detection blind spots of traditional technologies.

[0095] 6. High degree of standardization, meeting clinical needs. This invention has been validated for repeatability with a CV value of less than 10%, a limit of detection of 200 ng Hi-C DNA, and a standardized detection process with stable and reliable results.

[0096] 7. High clinical application value and controllable cost. FFPE samples are prone to severe degradation over long periods of storage, making traditional ligation methods difficult to adapt, resulting in extremely low ligation efficiency and low utilization of effective data, leading to high detection costs. The method of this invention has strong sample adaptability and can be directly applied to routine clinical FFPE samples, especially efficiently processing degraded samples stored for more than 5 years without special sample preparation; the detection results are comprehensive and accurate, not only covering structural variations detectable by traditional techniques, but also supplementing the detection of special variations such as enhancer hijacking in intergenic regions, providing more comprehensive molecular evidence for the study of DLBCL pathogenesis and the formulation of clinical treatment plans; at the same time, by optimizing the ligation strategy and improving the utilization of effective data, the detection cost is indirectly reduced, showing good prospects for large-scale promotion.

[0097] 8. High degree of standardization in reagent kits. Dedicated reagent kits contain customized targeted capture probes, specific adaptors, and matching optimized reagents. The concentrations of each reagent are precisely matched with the methodological steps, which can synergistically ensure the standardization and stability of the experimental process, avoid detection deviations caused by reagent compatibility issues, and provide reliable support for large-scale clinical applications. Attached Figure Description

[0098] Figure 1 The figure shows the overall optimization results for the experimental group and the control group.

[0099] Figure 2 This is a graph showing the results of a repeatable experiment.

[0100] Figure 3 The figure shows the results of detecting DLBCL structural variations (BCL2-IGH fusion) using the customized targeted capture probe of the present invention.

[0101] Figure 4 The figure shows the results of detecting DLBCL structural variations (BCL6-IGH fusion) using the customized targeted capture probe of the present invention.

[0102] Figure 5 The image shows the results of detecting DLBCL structural variations (BCL10-IGH fusion) using the customized targeted capture probe of this invention.

[0103] Figure 6 The image shows the results of detecting DLBCL structural variations (CD274-ZCCHC7 fusion) using the customized targeted capture probe of this invention.

[0104] Figure 7 The image shows the results of detecting DLBCL structural variations (IGK-MYC fusion) using the customized targeted capture probe of this invention. Detailed Implementation

[0105] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0106] To enhance understanding of the present invention, certain key technical and scientific terms will be clearly defined below. Unless otherwise specified herein, all other technical and scientific terms shall follow their generally accepted and understood meanings within the art to which this invention pertains. It should be noted that the terminology used herein is intended to describe specific embodiments and not to be construed as limiting.

[0107] Structural variation (SV) refers to abnormalities in chromosome structure that can lead to genomic instability and genetic diseases. SV generally refers to sequence changes of more than 50 bp in an organism's genome, most commonly including large deletions, insertions, duplications, inversions, and translocations. Currently, many genomic variation events have become specific markers for identifying tumor types and targets for drug therapy.

[0108] Gene fusion refers to the phenomenon where all or part of the sequences of two genes fuse to form a new gene, commonly seen in tumor cells. Gene fusion is a special type of structural variation, and common mechanisms leading to its occurrence include chromosomal translocations, deletions, and inversions. Current research indicates that some gene fusions are driver mutations in tumors, playing a crucial role in tumorigenesis and development, and are also important targets for tumor therapy. Currently approved tumor drugs targeting gene fusions include imatinib (targeting BCR-ABL fusion) and crizotinib (targeting ALK fusion).

[0109] Gene rearrangement refers to the process by which a gene moves from a location far from the promoter to a location very close to the promoter, thereby initiating transcription. The mechanism of gene structural rearrangement is a repair process of DNA double-strand breaks, involving complex switching movements of repeating units within or between alleles. DNA double-strand breaks often occur within repeating units near the 5' end of tandem repeat sequences, forming two free, protruding single-stranded ends of the DNA molecule. However, during repair, these ends may wobble or misalign, failing to renature in the original base pairing positions, resulting in intragenetic and intergenetic rearrangements.

[0110] Gene amplification refers to the selective increase in the copy number of a specific gene, while other genes do not increase proportionally. This invention is based on gene amplification-mediated rearrangement, which refers to large-scale structural variations such as chromosome breakage, fusion, inversion, and translocation that occur during gene amplification in local regions of the genome.

[0111] Enhancer hijacking is a novel mechanism of abnormal gene regulation discovered in cancer. Structural variations (such as translocations, inversions, and deletions) alter the three-dimensional conformation of the genome, potentially causing enhancer elements that normally regulate other genes to be misplaced near an oncogene or key gene, thereby abnormally activating its expression. This phenomenon is called enhancer hijacking. In a normal three-dimensional genome structure, enhancers interact with specific promoters through chromatin folding, forming circular interactions to precisely regulate gene transcription. However, in enhancer hijacking events, structural variations disrupt the original chromatin topology (such as TAD boundaries), causing these distal enhancers to form new, abnormal chromatin loops with the wrong gene (usually a proto-oncogene). This persistently activates or significantly enhances the expression of proto-oncogenes, driving cell carcinogenesis. Enhancer hijacking has been proven to be closely related to the development and progression of various cancers.

[0112] In this embodiment of the invention, unless otherwise specified, the techniques or conditions described in the literature in this field, or the product instructions, were followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels. Human tissue samples were formalin-fixed and paraffin-embedded, all of which were pathologically verified, and informed consent was obtained from the patients.

[0113] In this embodiment of the invention, the customized targeted capture probe sequence for DLBCL structural variation detection is shown in Tables 1 to 8.

[0114] Table 1. List of Customized Targeted Capture Probe Sequences for DLBCL Structural Variation Detection

[0115]

[0116] Table 2. List of Customized Targeted Capture Probe Sequences for DLBCL Structural Variation Detection

[0117]

[0118] Table 3. List of Customized Targeted Capture Probe Sequences for DLBCL Structural Variation Detection

[0119]

[0120] Table 4. List of Customized Targeted Capture Probe Sequences for DLBCL Structural Variation Detection

[0121]

[0122] Table 5. List of Customized Targeted Capture Probe Sequences for DLBCL Structural Variation Detection

[0123]

[0124] Table 6. List of Customized Targeted Capture Probe Sequences for DLBCL Structural Variation Detection

[0125]

[0126] Table 7. List of Customized Targeted Capture Probe Sequences for DLBCL Structural Variation Detection

[0127]

[0128] Table 8. List of Customized Targeted Capture Probe Sequences for DLBCL Structural Variation Detection

[0129]

[0130] In this embodiment of the invention, the conventional capture probe sequences are shown in Tables 9 to 15.

[0131] Table 9. List of Traditional Capture Probe Sequences

[0132]

[0133] Table 10. List of Traditional Capture Probe Sequences

[0134]

[0135] Table 11. List of Traditional Capture Probe Sequences

[0136]

[0137] Table 12. List of Traditional Capture Probe Sequences

[0138]

[0139] Table 13. List of Traditional Capture Probe Sequences

[0140]

[0141] Table 14. List of Traditional Capture Probe Sequences

[0142]

[0143] Table 15. List of Traditional Capture Probe Sequences

[0144]

[0145] In this embodiment of the invention, the connector sequences are shown in Table 16.

[0146] Table 16. List of Connective Subsequences

[0147]

[0148] Example 1. A method for detecting structural variations in DLBCL based on targeting Hi-C.

[0149] A method for detecting structural variations in diffuse large B-cell lymphoma based on Hi-C targeting, using FFPE (formalin-fixed paraffin-embedded) samples as the primary detection sample, is described below:

[0150] (1) FFPE dewaxing and decrosslinking treatment: Obtain FFPE tumor tissue samples from suspected or confirmed DLBCL patients, cut them into 10µm wax rolls and add them to 1.5mL low-adsorption centrifuge tubes, add 1000µL of dewaxing solution, heat at 56℃ for 5min, centrifuge at 2500g for 5min and then discard the dewaxing solution; add 180µL of 1× buffer (the buffer contains 50~100 mM Bis-Tris-HCl, 100~150 mM NaCl, 10~15 mM MgCl2 and 0.1~0.15 mM DTT) to resuspend, and a portion is used for chromatin integrity control; then add SDS to a final concentration of 0.3%-0.7%, combined with 0.5%-2% SDC, 1%-5% Triton X-100 and 0.5%-2% DTT. NP40 utilizes a combination of detergents, along with 10-30 mg / mL proteinase K for synergistic effects, and incubates at 70-78°C for 40-60 min to achieve efficient dewaxing and decrosslinking. The synergistic effect of multiple detergents enhances the solubility of impurities and crosslinked complexes in the sample. Combined with the degradation effect of the protease, this significantly improves the decrosslinking effect, resulting in highly intact chromatin samples (validation shows a 46.00% improvement in %Intrage_15kb_pairs compared to traditional methods).

[0151] (2) Restriction enzyme digestion: Add 10 U / µL DpnII or 12-18 µL MboI to the sample tubes treated in step (1), gently pipette to mix, incubate at 37℃ with shaking at 800-1000 rpm for 14-18 h, and inactivate enzyme activity at 65℃ for 15-25 min. A portion is then used for digestion control. Chromatin is digested with restriction endonucleases to obtain uniformly fragmented chromatin, laying the foundation for subsequent experiments.

[0152] (3) End biotinylation labeling: Prepare the biotinylation system during incubation at 65℃, containing 3-4 µL of 10× biotinylation buffer, 0.5-1 µL of 10 mM dCTP, 0.5-1 µL of 10 mM dGTP, 0.5-1 µL of 10 mM dTTP, 0.5-1 µL of 10 mM biotin-14-dATP, 4-6 µL of 5 U / µL Klenow DNA polymerase, and deionized water to a total volume of 32-38 µL. Place centrifuge tubes on ice, add the prepared system to each tube, mix well by pipetting, and incubate at 20-25℃ for 3-5 h with shaking at 800-1000 rpm. Biotinylation labeling provides specific recognition sites for subsequent targeted capture and purification.

[0153] (4) Ligation treatment: Prepare the ligation reaction system: 60µL of 10×T4 DNA ligase buffer, 60µL of 10% Triton X-100, 6µL of 10mg / mL BSA, and add water to a total volume of 600µL. Adjust the ligation temperature to 16℃ and the ligase concentration to 8U / µL. Mix well by pipetting and centrifugation briefly, and incubate with shaking for 3-5 hours. For samples that have been stored in FFPE for a long time and have been severely degraded, the adaptor method is used. Specific adaptors with a length of 15-25bp (including adaptor sequence 1 shown in SEQ ID NO.176 and adaptor sequence 2 shown in SEQ ID NO.177) are added to the ligation reaction at a concentration of 5-10µM to improve ligation efficiency. By optimizing the ligation temperature, ligase concentration, and adaptor strategy for degraded samples, as well as the purification method, the percentage of 15kb pairs for routine samples was increased by 20.11%, and the percentage of 15kb pairs for degraded samples was increased by 80.00%, thereby improving the utilization rate of effective data while reducing detection costs.

[0154] (5) Chromatin decrosslinking treatment: After ligation, the sample was placed on a magnetic rack and allowed to stand for 2 min. The supernatant was discarded. The sample was washed twice with 1 mL of 80% ethanol, briefly centrifuged to remove residual ethanol, and dried at room temperature. The magnetic beads were resuspended in 140-160 µL of water. Then, 15-25 µL of buffer, 8-12 µL of 10% SDS, and 15-25 µL of 20 mg / mL proteinase K were added sequentially. The mixture was incubated at 55℃ for 25-35 min, 68℃ for 80-100 min, and 25℃ for 8-12 min. The purified magnetic beads were added, and the mixture was incubated at room temperature for 5 min. The sample was then placed on a magnetic rack, the supernatant was discarded, and the sample was washed twice with 80% ethanol. After drying, the magnetic beads were resuspended in 80-100 µL of water and incubated at room temperature for 5 min. The supernatant was transferred to a new tube and quantified using Qubit. Through proteinase K digestion and a series of washing and purification steps, impurities such as proteins were removed to obtain pure chromatin DNA.

[0155] (6) End treatment (removal of biotin from unligated ends): Prepare a biotin removal reaction system in a PCR tube, containing 5-15 µg Hi-C DNA, 11-15 µL 10× biotinylation buffer, 2.5-4 µL 1 mM dATP, 2.5-4 µL 1 mM dGTP, 11-15 µL 3000 U / mL T4 DNA polymerase, 5-10 U exonuclease I, 10-20 U exonuclease III, and deionized water to a final volume of 130 µL. Divide the reaction system into two tubes, each containing 65 µL. Run the following program: 20℃ for 3-5 h, 75℃ for 15-25 min, and hold at 4℃. Through the synergistic effect of T4 DNA polymerase combined with exonuclease I and exonuclease III, the biotin from unligated ends is completely removed, reducing %Duplicated pairs by 50.00% and minimizing interference with subsequent experiments.

[0156] (7) Biotin-free purification: Remove the purification magnetic beads 30 min in advance and equilibrate to room temperature; add 220-240 µL of magnetic beads to 130 µL of ligation product, shake to mix, and incubate at room temperature for 5 min; place on a magnetic rack, and remove the supernatant after the solution becomes clear; add 500 µL of freshly prepared 80% ethanol to rinse the magnetic beads twice, incubating at room temperature for 30 s each time; after elution, discard the residual liquid, open the cap and dry for 2 min; add 35-45 µL of elution buffer, shake to mix, and place at room temperature for 5 min; place on a magnetic rack, aspirate the supernatant to a new tube, and perform quantification using Qubit and quality control using Qsep100. High-purity biotin-free samples were obtained through magnetic bead purification, meeting the requirements for subsequent library construction.

[0157] (8) Targeted capture: Targeted capture is performed using customized specific capture probes, which are 80-120 bp DNA probes labeled with biotin. The core design is as follows:

[0158] ① Coverage area: The main body, promoter and enhancer regions of the key DLBCL genes MYC, BCL2 and BCL6, and the main body, promoter and enhancer regions of the auxiliary evaluation genes BCL10 and CD274;

[0159] ② Site association: Precisely designed within a 300bp range on both sides of each DpnII restriction site to ensure efficient capture of the digested fragment;

[0160] ③Layout method: The shingled design is adopted, with an overlap area of ​​30-60bp between adjacent probes to avoid capture blind spots;

[0161] ④ Physicochemical standards: Length 80~120bp, 5' end biotin modification, GC content 40%-60%, sequence specificity ≥98%;

[0162] ⑤ Performance indicators: The target area capture efficiency has been improved from 30% to 60%, and the detection resolution has been improved from 100kb to 5kb, ensuring high capture efficiency and high-resolution detection.

[0163] (9) Sequencing: The constructed targeted Hi-C library was subjected to high-throughput sequencing at 150bp at both ends using the BGI platform. For FFPE samples, a sequencing depth of ≥0.5G was set to ensure that the original data Q30 ≥85% and to guarantee the amount of effective data.

[0164] (10) Data Analysis:

[0165] ① Refined targeted capture sequencing data processing: Strict quality control was performed on the raw sequencing data, filtering out low-quality reads, adapter-contaminated reads, and duplicate reads. The quality control standards were Q20≥90%, Q30≥85%, adapter contamination rate<1%, and duplicate sequence ratio<10%. The clean data was aligned to the human reference genome (hg38) using BWA software, and FFPE sample-specific alignment parameters were set, with an alignment efficiency of ≥80%. Juicer software was used to accurately locate and remove duplicates of chromatin interaction fragments, and a high-resolution targeted chromatin interaction matrix with a resolution of 5kb was constructed.

[0166] ② Structural variation feature extraction: Based on the chromatin interaction matrix, a customized Python script is used to extract structural variation features related to the MYC, BCL2, BCL6, BCL10, and CD274 genes, including abnormal chromatin interaction signals related to gene fusion, rearrangement, and intergenic enhancer hijacking. All parameters are normalized by ICE to eliminate sample bias.

[0167] ③ Consistency verification: The structural variation results of Hi-C detection are analyzed for consistency with the RNA-seq / FISH detection results to assess the accuracy of the detection; for samples that are negative for RNA-seq but indicate enhancer hijacking by Hi-C detection, specific capture probes are designed for verification.

[0168] Example 2. Optimization and verification of FFPE dewaxing and decrosslinking treatment

[0169] 1. Verify the target

[0170] To verify the advantages of a multi-detergent combined with enzyme-based synergistic dewaxing and decrosslinking scheme compared to a traditional single-detergent scheme, and to evaluate the improvement in chromatin integrity and key indicators in subsequent experiments.

[0171] 2. Experimental Samples

[0172] FFPE samples from 10 DLBCL patients were selected and cut into 10µm wax rolls for later use.

[0173] 3. Experimental Methods

[0174] 3.1 Experimental Group (Scheme of this Invention)

[0175] ① Add a 10µm wax roll to a 1.5mL low-adsorption centrifuge tube, add 1000µL of dewaxing solution, heat treat at 56℃ for 5min, centrifuge at 2500g for 5min, and then discard the dewaxing solution.

[0176] ② Resuspend in 180µL buffer;

[0177] ③ Add 0.3% SDS to a final concentration, combined with 1% SDC, 3% Triton X-100, 1% NP40 and other detergents, and 20 mg / mL protease. Incubate at 74°C for 50 min (with gentle shaking every 10 min).

[0178] 3.2 Control group (traditional regimen)

[0179] The treatment used only 0.5% SDS as a single detergent, and the other steps (dewaxing, enzyme concentration, incubation temperature and time) were exactly the same as those in the experimental group.

[0180] 3.3 Subsequent Testing

[0181] Both groups of samples were subsequently subjected to restriction enzyme digestion, end biotinylation labeling, and ligation under the same conditions. The percentages of %_Intra_ge_15kb_pairs (the proportion of paired reads with a distance of ≥15kb within the chromosome) and %_Unique_valid_pairs (the proportion of unique and valid paired reads) were calculated.

[0182] 4. Verification Results and Explanation

[0183] The verification results are shown in Table 17. Figure 1 As shown.

[0184] Table 17. Optimization and Validation Results of FFPE Dewaxing and Decrosslinking Treatment

[0185]

[0186] The results show that the experimental group significantly outperformed the control group in both core validation metrics, fully meeting the expected optimization goals: %Intrage_15kb_pairs increased by 46.00% compared to the control group, indicating that the synergistic dissolution of impurities and cross-linked complexes by multiple detergents, combined with protease degradation, effectively solved the problem of incomplete decross-linking by traditional single detergents, significantly improving the integrity of chromatin fragments and their long-distance interaction retention capacity; %Unique_valid_pairs increased by 23.01% compared to the control group, indicating that this treatment scheme can reduce non-specific interference and increase the proportion of effective sequencing data. High-quality chromatin samples provide a stable foundation for the uniformity of subsequent restriction enzyme digestion, the specificity of terminal biotinylation labeling, and the effectiveness of ligation reactions, significantly reducing experimental bias caused by sample processing defects and laying a crucial prerequisite for the accuracy of the entire targeted Hi-C detection process.

[0187] Example 3. Verification of Connection Processing Optimization

[0188] 1. Verify the target

[0189] To verify the advantages of the "optimized ligation temperature and ligase concentration" scheme compared to the traditional ligation scheme, and to evaluate the efficiency of chromatin interaction fragment formation and the improvement in library construction quality.

[0190] 2. Experimental Samples

[0191] Ten FFPE samples from DLBCL patients treated with the experimental group of Example 2 (dewaxing and decrosslinking optimization scheme) were selected to ensure that the sample baseline was consistent.

[0192] 3. Experimental Methods

[0193] 3.1 Unified processing in the early stage

[0194] All samples underwent restriction enzyme digestion (15µL 10U / µL DpnII, incubated at 37℃ with shaking at 900rpm for 16h, then inactivated at 65℃ for 20min) and terminal biotinylation labeling (incubated at 23℃ with shaking for 4h).

[0195] 3.2 Experimental Group (Scheme of this Invention)

[0196] ① Prepare the ligation reaction system: 60µL of 10×T4 DNA ligase buffer, 60µL of 10% Triton X-100, 6µL of 10mg / mL BSA, and add water to a total volume of 600µL;

[0197] ② Adjust the ligation temperature to 16℃ and the ligase concentration to 8U / µL. After adding the enzyme, mix by pipetting and incubate at 900rpm for 4 hours.

[0198] 3.3 Control group (traditional regimen)

[0199] The ligation temperature was set to 25℃, the ligase concentration was set to 4U / µL, and the other reaction system components and incubation time were completely consistent with the experimental group.

[0200] 3.4 Subsequent Testing

[0201] Both groups of samples underwent chromatin decrosslinking and purification under the same conditions. The percentage of intrachromosomal pairs (≥15kb distance) was measured, and the library construction success rate and the proportion of effective interacting fragments were statistically analyzed.

[0202] 4. Verification Results and Explanation

[0203] The verification results are shown in Table 18. Figure 1 As shown.

[0204] Table 18. Verification Results of Connection Processing Optimization

[0205]

[0206] The results show that the optimized ligation conditions significantly improve the efficiency of chromatin interaction fragment formation and library construction quality, providing more effective materials for subsequent targeted capture and sequencing. Specifically, %Intra_ge_15kb_pairs increased by approximately 20% compared to the control group, indicating that the optimized ligation temperature and ligase concentration effectively promote the formation of long-distance chromatin interaction fragments; %Unique_valid_pairs increased by 3.31% compared to the control group, remaining below 10%, while %Inter_pairs decreased by 12.96%, demonstrating that this approach increases the proportion of effective data while reducing non-specific interaction interference, ensuring the targeting and reliability of the library.

[0207] Example 4. Optimization and Validation of Terminal Biotin Removal Treatment

[0208] 1. Verify the target

[0209] To verify the advantages of the "T4 DNA polymerase combined with exonucleases I and III for synergistic biotin removal" method compared to the traditional single enzyme method, and to evaluate the thoroughness of biotin removal and its effect on improving the quality of subsequent sequencing data.

[0210] 2. Experimental Samples

[0211] Ten DLBCL patients with FFPE samples were selected after treatment with the experimental group of Example 3 (connection optimization scheme), and were used after chromatin decrosslinking and preliminary purification.

[0212] 3. Experimental Methods

[0213] 3.1 Unified processing in the early stage

[0214] All samples underwent chromatin decrosslinking (20 mg / mL proteinase K digestion), preliminary purification with magnetic beads, and quantification with Qubit to ensure consistent DNA concentration.

[0215] 3.2 Experimental Group (Scheme of this Invention)

[0216] ① Prepare the biotin removal reaction system: 10µg Hi-C DNA, 13µL 10× biotinylation buffer, 3.25µL 1mM dATP, 3.25µL 1mM dGTP, 13µL 3000U / mL T4 DNA polymerase, 8U exonuclease I, 15U exonuclease III, and deionized water to a final volume of 130µL;

[0217] ② Run the program in two tubes (65µL each): 20℃ for 4h, 75℃ for 20min, 4℃ Hold.

[0218] 3.3 Control group (traditional regimen)

[0219] The reaction system was only supplemented with 13 µL of 3000 U / mL T4 DNA polymerase, without the addition of exonucleases I and III. The other components and incubation conditions were exactly the same as those of the experimental group.

[0220] 3.4 Subsequent Testing

[0221] Both groups of samples underwent magnetic bead purification under the same conditions, followed by library construction and sequencing under the same conditions. The proportions of %_Intra_ge_15kb_pairs, %_Unique_valid_pairs, and %_Inter_pairs were calculated.

[0222] 4. Verification Results and Explanation

[0223] The verification results are shown in Table 19. Figure 1 As shown.

[0224] Table 19. Validation Results of Optimized Terminal Biotin Removal Treatment

[0225]

[0226] The results show that the compound enzyme method can significantly improve the thoroughness of biotin removal, reduce the interference of repetitive sequences and residual biotin on subsequent experiments, and effectively increase the proportion of valid sequencing data. Specifically, %Intra_ge_15kb_pairs increased by 9.09% compared to the control group, remaining within 10%, indicating that the synergistic effect of the compound enzyme can better preserve long-distance chromatin interaction fragments; %Unique_valid_pairs increased by 3.00% compared to the control group, meeting the 3% improvement requirement; and %Inter_pairs decreased by 11.22%, further demonstrating that this method can reduce non-specific interaction interference, ensuring the relevance and reliability of subsequent sequencing data, and laying a high-quality data foundation for the accurate extraction of chromatin interaction characteristics and structural variation analysis.

[0227] Example 5. Verification of Connector Connection Optimization

[0228] 1. Verify the target

[0229] To verify the advantages of the degraded sample linker scheme compared with the traditional library construction scheme, and to evaluate the retention effect of chromatin interaction fragments in degraded samples and the effective improvement effect on data quality.

[0230] 2. Experimental Samples

[0231] Ten FFPE samples from patients with severe DLBCL who had been stored for more than 5 years were selected and processed in the experimental group of Example 2 (FFPE dewaxing and decrosslinking treatment) for later use.

[0232] 3. Experimental Methods

[0233] Both groups of samples were first treated with the multi-detergent combined with enzyme synergistic dewaxing and decrosslinking scheme in Example 2 (to ensure sample baseline consistency), and then the following schemes were used for subsequent processing:

[0234] 3.1 Experimental Group (Scheme of this Invention)

[0235] ① Degradation sample adaptation treatment: Select a 20bp specific linker (including linker-sequence 1 shown in SEQ ID NO.176 and linker-sequence 2 shown in SEQ ID NO.177), add it to the ligation reaction at a concentration of 8µM, and the remaining ligation conditions are consistent with the optimized ligation system parameters in the present invention.

[0236] ②PCR amplification and library verification: High-fidelity DNA polymerase amplification (15 cycles), magnetic bead purification and recovery of 300-500bp fragments.

[0237] 3.2 Control group (traditional regimen)

[0238] No specific adaptor was added to the degradation sample, and all other conditions (PCR amplification, purification process, etc.) were exactly the same as those of the experimental group.

[0239] 3.3 Detection Indicators

[0240] Both groups of samples underwent sequencing and data analysis under the same conditions. The percentages of %_Intra_ge_15kb_pairs (the proportion of paired reads with a distance of ≥15kb within the chromosome) and %_Unique_valid_pairs (the proportion of unique and valid paired reads) were calculated.

[0241] 3.4 Verification Results and Explanation

[0242] The verification results are shown in Table 20.

[0243] Table 20. Verification Results of Connector Connection Optimization

[0244]

[0245] The results show that the optimized adaptor scheme for degraded FFPE samples fully achieved the expected technical goals: %Intrage_15kb_pairs increased by 80.00% compared to the control group, indicating that the specific adaptor can effectively compensate for the integrity defects of chromatin fragments in degraded samples. Synergistically, it works with the dewaxing and decrosslinking optimization scheme in Example 2 to significantly promote the connection and retention of long-distance chromatin interaction fragments; %Unique_valid_pairs increased by 25.00% compared to the control group, indicating that this scheme can reduce non-specific interference in degraded samples and significantly increase the proportion of effective sequencing data. This optimized scheme successfully solves the core problem of poor adaptability of traditional methods to degraded FFPE samples, and the scheme parameters are fully matched with the claims and content of this invention. Without increasing detection costs, it achieves a leapfrog improvement in the detection quality of degraded samples, providing reliable technical support for the accurate detection of structural variations in old clinical samples, and further improving the sample adaptation system of the targeted Hi-C diagnostic process of this invention.

[0246] Example 6. Validation of Customized Targeted Capture Probes

[0247] 1. Verify the target

[0248] To verify the advantages of the customized targeted capture probe for DLBCL structural variation detection compared with traditional probes, the focus was on evaluating the improvement in capture efficiency (from 30% to 60%) and resolution (from 100kb to 5kb).

[0249] 2. Experimental Samples

[0250] FFPE samples from 10 DLBCL patients treated with the optimized protocol in the experimental group were selected, and the library concentrations were adjusted to ≥10 ng / μL.

[0251] 3. Experimental Methods

[0252] 3.1 Experimental Group (Customized Targeted Capture Probe of this Invention)

[0253] The following parameters were designed for biotin-labeled 80-120 bp DNA probes:

[0254] ① Coverage area: The main body, promoter and enhancer regions of the MYC, BCL2, BCL6 core genes and the BCL10 and CD274 helper genes;

[0255] ② Site association: Designed within a 300bp range flanking each DpnII restriction site;

[0256] ③Layout method: shingled design, with an overlap area of ​​30-60bp between adjacent probes;

[0257] ④ Physicochemical standards: 5' end biotin modification, GC content 40%-60%, sequence specificity ≥98%.

[0258] The core probe sequences are shown in SEQ ID NO.1 to SEQ ID NO.100.

[0259] 3.2 Control group (traditional probe)

[0260] A conventional targeting Hi-C probe was used, with the following parameters:

[0261] ① Coverage area: Only covers the coding regions of MYC, BCL2, and BCL6 genes, excluding promoter and enhancer regions;

[0262] ② Site association: Targeted design without DpnII restriction sites;

[0263] ③Layout method: non-shingled, probe spacing 100-200bp, with a capture blind zone;

[0264] ④ Performance baseline: capture efficiency of approximately 30% and detection resolution of approximately 100kb.

[0265] Traditional probe core sequences are shown in SEQ ID NO.101~SEQ ID NO.175.

[0266] 3.3 Subsequent Testing

[0267] Both groups of samples underwent high-throughput sequencing (≥0.5G sequencing depth) under the same conditions, and the performance was evaluated using the following methods:

[0268] ①Capture efficiency: The proportion of effective reads in the target region to the total effective reads;

[0269] ② Resolution: Construct a chromatin interaction matrix using Juicer software and calculate the length of the smallest interaction segment that can be stably identified (i.e., resolution).

[0270] 3.4 Verification Results and Explanation

[0271] The verification results are shown in Table 21.

[0272] Table 21. Validation Results of Customized Targeted Capture Probes

[0273]

[0274] The above results show that the customized targeted capture probe of this invention, through its innovative design of "multi-region coverage + restriction site association + shingled layout," achieves a dual breakthrough in capture efficiency and resolution: capture efficiency is increased from the traditional 30% to 60%, effectively ensuring the effective data volume of the target region and reducing interference from non-target data; detection resolution is increased from 100kb to 5kb, enabling precise identification of smaller-scale chromatin interaction anomalies, providing technical support for the detection of minute structural variations (such as local enhancer hijacking), and significantly outperforming the performance of traditional probes. Using the customized targeted capture probe of this invention to detect DLBCL structural variations, some structural variations are as follows... Figure 3-7 As shown.

[0275] Example 7. Repeatability Verification

[0276] 1. Verify the target

[0277] Verify the repeatability of the method by confirming the mean and CV value of %_Intra_ge_15kb_pairs.

[0278] 2. Experimental Samples

[0279] Five DLBCL FFPE samples (stored for 1-5 years).

[0280] 3. Experimental Methods

[0281] Five independent replicate experiments were conducted on five samples, and the mean and CV of %_Intra_ge_15kb_pairs were calculated.

[0282] 4. Verification Results and Explanation

[0283] The results are shown in Table 22. Figure 2 As shown in the figure. The results show that the method of the present invention has good repeatability, with CV values ​​all below 10%, meeting the standardization requirements of clinical testing.

[0284] Table 22. Repeatability Validation Results

[0285]

[0286] Example 8. Validation of the lowest detection limit

[0287] 1. Verify the target

[0288] Determine the minimum detection limit of the method.

[0289] 2. Experimental Samples

[0290] Fusion-type DLBCL FFPE samples (MYC-IGL, BCL2-IGH, BCL6-IGK) were selected and grouped into groups of 100ng, 200ng, 400ng, and 800ng Hi-C DNA.

[0291] 3. Experimental Methods

[0292] Each group of samples was tested, and the interaction detection rate and %_Intra_ge_15kb_pairs were calculated.

[0293] 4. Verification Results

[0294] The validation results are shown in Table 23. The limit of detection for this method is 200 ng Hi-C DNA (corresponding to 2 10 μm wax rolls).

[0295] Table 23. Validation Results of the Lowest Detection Limit

[0296]

[0297] Example 9. Hi-C and RNA-seq / FISH Consistency Validation

[0298] 1. Verify the target

[0299] To verify the consistency of structural variations (gene fusion, rearrangement, amplification) detected by Hi-C in 10 DLBCL samples with known RNA-seq / FISH results.

[0300] 2. Experimental Samples

[0301] Clinical routine FFPE samples from 10 DLBCL patients were selected, and the specific information is shown in Table 24.

[0302] Table 24. FFPE Sample Information Table

[0303]

[0304] All samples met the following criteria: ① DNA integrity index (CI) ≥ 0.6; ② Sufficient sample size to allow for simultaneous targeted Hi-C detection and validation experiments.

[0305] 3. Experimental Methods

[0306] 3. Targeted Hi-C detection (performed according to the optimized scheme of this invention)

[0307] ① Sample processing: FFPE samples were treated with a combination of multiple detergents and enzymes for synergistic dewaxing and decrosslinking, followed by restriction enzyme digestion (DpnII), terminal biotinylation labeling, optimized ligation conditions, biotin removal by compound enzyme method, multi-step gradient purification and library construction;

[0308] ② Targeted capture: Using the customized targeted capture probe of this invention (Example 6), the main body, promoter and enhancer regions of the target gene are captured;

[0309] ③ Sequencing and data analysis: High-throughput sequencing of 150bp from both ends on the BGI platform (sequencing depth ≥0.5G). After quality control, alignment and deduplication, a 5kb resolution chromatin interaction matrix was constructed using Juicer software to analyze the types of structural variations (gene fusion, rearrangement, amplification, enhancer hijacking).

[0310] 3.2 RNA-seq / FISH detection results (reference standard)

[0311] Ten known result samples: RNA-seq identified fusion genes using STARFIusion software, and FISH detected gene rearrangement / amplification using specific probes.

[0312] 3.3 Consistency Analysis

[0313] Consistency analysis: The consistency rate between Hi-C and RNA-seq / FISH in gene fusion, rearrangement, and amplification detection was statistically analyzed in 10 samples.

[0314] 4. Verification Results and Explanation

[0315] The consistency verification results are shown in Table 25. The Hi-C detection results of 10 samples were completely consistent with the RNA-seq / FISH results, confirming the accuracy of the method of the present invention in the detection of traditional structural variations (fusion, rearrangement, amplification), and providing a reliable basis for clinical application.

[0316] Table 25. Consistency results of Hi-C and RNA-seq / FISH in 10 samples

[0317]

[0318] Note: Among the 10 samples, the consistency rate between Hi-C and RNA-seq / FISH in gene fusion (5 cases), rearrangement (4 cases), and amplification (2 cases) was 100%, with an overall consistency of 100%, which is higher than the preset standard of ≥95%.

Claims

1. A probe for detecting structural variations in diffuse large B-cell lymphoma based on targeted Hi-C, characterized in that, The probe comprises the nucleotide sequences shown in SEQ ID NO.1 to SEQ ID NO.

100.

2. The probe according to claim 1, characterized in that, The probes cover the main body, promoter, and enhancer regions of the MYC, BCL2, and BCL6 core genes and the BCL10 and CD274 helper genes, and adopt a shingled design with an overlap of 30-60 bp between adjacent probes; the 5' end of the probes is modified with biotin.

3. The probe according to claim 1, characterized in that, The structural variations include gene fusion, rearrangement, amplification, and / or enhancer hijacking.

4. The probe according to claim 1, characterized in that, The structural variations include BCL2-IGH fusion, BCL6-IGH fusion, BCL10-IGH fusion, CD274-ZCCHC7 fusion, IGK-MYC fusion, MYC-IGL fusion, BCL6 rearrangement, MYC-BCL2 co-rearrangement, BCL10 rearrangement, CD274 amplification + BCL2 fusion, MYC rearrangement, BCL6-IGK fusion, BCL2 rearrangement + BCL10 amplification, and / or MYC-IGH fusion.

5. A kit for detecting structural variations in diffuse large B-cell lymphoma based on targeted Hi-C, characterized in that, The kit contains the probe according to any one of claims 1 to 4.

6. The reagent kit according to claim 5, characterized in that, The kit also contains: Specific adaptors, including adaptor sequence 1 shown in SEQ ID NO.176 and adaptor sequence 2 shown in SEQ ID NO.177; Reagents for dewaxing and decrosslinking treatment of FFPE include dewaxing solution, detergent and protease, wherein the detergent includes SDS, SDC, Triton X-100 and NP40; Reagents used for restriction enzyme digestion include DpnII or MboI; Reagents used for terminal biotinylation labeling include Klenow DNA polymerase; Reagents used for ligation processing include T4 DNA ligase; Reagents used for chromatin decrosslinking treatment include magnetic beads, proteinase K, and purified magnetic beads; Reagents used for terminal biotin treatment include T4 DNA polymerase, exonuclease I, and exonuclease III.

7. The reagent kit according to claim 6, characterized in that, The detergent comprises the following components in final concentrations: 0.1%-0.7% SDS, 0.5%-2% SDC, 1%-5% Triton X-100 and 0.5%-2% NP40.

8. A method for detecting structural variations in diffuse large B-cell lymphoma based on targeted Hi-C, characterized in that, The detection is performed using the probe according to any one of claims 1 to 4 and / or the kit according to any one of claims 5 to 7; The method is not intended for the diagnosis and / or treatment of disease, and specifically includes the following steps: S1: Add dewaxing solution to the sample to be tested for dewaxing, resuspend it, and then add detergent and protease for decrosslinking treatment; S2: The sample treated with S1 was digested with DpnII or MboI enzymes; S3: End-biotinylated labeling of the sample after S2 enzyme digestion; S4: The S3-labeled sample was ligated using T4 DNA ligase and a specific adaptor. S5: The sample after S4 ligation was washed with magnetic beads, digested with proteinase K, purified with magnetic beads and eluted to obtain pure chromatin DNA; S6: The S5 decrosslinked sample was end-treated using T4 DNA polymerase, exonuclease I and exonuclease III to remove unlinked biotin. S7: The sample processed in S6 is targeted and captured using the probe described in any one of claims 1 to 4; S8: Perform high-throughput sequencing on the samples captured in S7; S9: Analyze sequencing data and extract structural variation features.

9. The method according to claim 8, characterized in that, In S4, the ligation temperature is 16-20℃ and the ligase concentration is 8-10 U / µL.

10. The method according to claim 8, characterized in that, The samples to be tested include formalin-fixed paraffin-embedded samples.

11. The method according to claim 8, characterized in that, In S1, the dewaxing reaction conditions are: heating at 50-60℃ for 3-10 min and centrifugation at 2000-3000g for 3-10 min; the decrosslinking treatment conditions are: incubation at 70-78℃ for 40-60 min.

12. The method according to claim 8, characterized in that, In S2, the enzyme digestion conditions include: incubation at 35-40℃ with shaking at 800-1000 rpm for 14-18 hours, followed by inactivation of enzyme activity at 60-70℃ for 15-25 minutes.

13. The method according to claim 8, characterized in that, In S3, the biotinylation system includes 3-4 µL of 8-12× biotinylation buffer, 0.5-1 µL of 8-12 mM dCTP, 0.5-1 µL of 8-12 mM dGTP, 0.5-1 µL of 8-12 mM dTTP, 0.5-1 µL of 8-12 mM biotin-14-dATP, 4-6 U / µL of Klenow DNA polymerase, and 20-26 µL of deionized water, with a total volume of 32-38 µL; the reaction conditions are 20-25 °C with shaking incubation for 3-5 h.

14. The method according to claim 8, characterized in that, In S4, the ligation system includes: 50-70 µL of 8-12×T4 DNA ligase buffer, 50-70 µL of 8-12% Triton X-100, 4-8 µL of 8-12 mg / mL BSA, 6-10 µL of 500-700 U / µL T4 DNA ligase, and 180-210 µL of deionized water, with a total volume of 320-345 µL; the reaction conditions are 16-20℃ shaking incubation for 3-5 h.

15. The method according to claim 8, characterized in that, In S6, the biotin removal reaction system includes 5-15 µg of Hi-C DNA, 11-15 µL of 8-12× biotinylation buffer, 2.5-4 µL of 0.5-2 mM dATP, 2.5-4 µL of 0.5-2 mM dGTP, 11-15 µL of 2000-4000 U / mL T4 DNA polymerase, 5-10 U of exonuclease I, 10-20 U of exonuclease III, and deionized water, with a total volume of 120-140 µL. The reaction conditions are incubation at 15-25℃ for 3-5 h followed by inactivation at 70-80℃.

16. The use of the probe according to any one of claims 1 to 4 and / or the kit according to any one of claims 5 to 7 in the preparation of a detection product for detecting structural variations in diffuse large B-cell lymphoma.

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