A method for whole genome methylation detection based on meDIP-msre

Abstract

The application discloses a MeDIP-MSRE-based whole genome methylation detection method, and belongs to the technical field of epigenetic detection. The method innovatively combines methylation immunoprecipitation sequencing and methylation-sensitive restriction enzyme technology, firstly uses methylation-specific antibodies to immunoprecipitate sample DNA, and enriches whole genome methylation fragments; then uses methylation-sensitive restriction enzymes to cut the enrichment products, specifically removes unmethylated DNA regions, and retains complete methylation sequences; finally, a methylation map is constructed through high-throughput sequencing. The method does not need traditional bisulfite chemical conversion, avoids DNA damage and base conversion bias caused thereby, and simultaneously combines the high sensitivity of MeDIP and the high specificity of methylation-sensitive restriction enzymes, so that the detection fidelity, sensitivity and specificity are significantly improved.

Description

Technical Field

[0001] This invention belongs to the field of epigenetic detection technology, and more specifically, relates to a whole-genome methylation detection method based on MeDIP-MSRE. Background Technology

[0002] DNA methylation, as one of the core mechanisms of epigenetic modification, plays a decisive role in maintaining normal cellular function, regulating gene expression, mediating cell differentiation, and in the development and progression of diseases. In tumorigenesis, abnormal changes in genome-wide methylation patterns—primarily manifested as global hypomethylation and hypermethylation of CpG islands in specific gene promoter regions—have become important biomarkers for early cancer diagnosis, prognostic assessment, and treatment monitoring. Therefore, genome-wide methylation detection technology has become a core tool for discovering and validating these key methylation biomarkers.

[0003] Currently, the main methods for whole-genome methylation detection include bisulfite sequencing (BS-seq), methylation immunoprecipitation sequencing (MeDIP-seq), and methylation-sensitive restriction endonuclease technology. The principle of BS-seq is to treat sample DNA with bisulfite, converting unmethylated C bases to U bases. After PCR amplification, the U bases are converted to T, and the result is compared with the unconverted sequence to determine whether methylation modification has occurred at that site. However, this conversion efficiency is unstable, easily leading to DNA degradation and base conversion errors, thus affecting detection accuracy. MeDIP-seq technology is based on the antigen-antibody specific reaction principle, using a 5-methylcytosine-specific antibody to enrich methylated DNA fragments in the genome. This method avoids DNA damage caused by chemical transformation, maintains DNA integrity, is more suitable for small sample volumes, and has high detection sensitivity. However, this technology has significant limitations: antibodies exhibit a binding preference for high CpG density regions, leading to insufficient detection sensitivity in low-density CpG regions; antibodies may bind nonspecifically, generating false positive signals; while methylation-sensitive restriction endonuclease technology can specifically identify and cleave unmethylated sites, inferring methylation status by observing changes in DNA fragments before and after cleavage. Although it offers advantages such as high specificity and low background signal, its coverage is limited when used alone.

[0004] In terms of current technological development, MeDIP-seq-based methods have been widely used for whole-genome methylation analysis. However, they are highly dependent on the quality of 5-methylcytosine antibodies. Performance differences between different batches of antibodies and non-specific binding issues lead to high background signals, affecting the specificity of the results. On the other hand, while using methylation-sensitive restriction endonucleases alone can accurately identify the methylation status of specific sequences, the limited distribution of restriction sites in the genome makes it difficult to achieve high-coverage whole-genome analysis, especially when processing low-concentration clinical samples where sensitivity is significantly insufficient. In summary, existing single-technology approaches all have insurmountable technical bottlenecks. There is an urgent need in this field for a high-precision, high-coverage whole-genome methylation detection method that can integrate the advantages of different technologies while overcoming their inherent defects. Summary of the Invention

[0005] To address the shortcomings of existing technologies in whole-genome methylation detection, such as low sensitivity and low specificity, this invention provides a MeDIP-MSRE-based whole-genome methylation detection method. This method eliminates the need for traditional bisulfite chemical conversion, avoiding the DNA damage and base conversion bias caused by it. At the same time, it combines the high sensitivity of Medip with the high specificity of methylation-sensitive restriction endonucleases, significantly improving the fidelity, sensitivity, and specificity of the detection. Its sensitivity and specificity are superior to those of existing technologies.

[0006] To solve the above problems, the present invention adopts the following technical solution:

[0007] This invention provides a method for whole-genome methylation detection based on MeDIP-MSRE, comprising the following steps:

[0008] (1) Extract DNA from the sample to be tested, including cell-free DNA from plasma, tissue gDNA or cell line gDNA;

[0009] (2) The gDNA is subjected to ultrasonic fragmentation to obtain DNA fragments of 200-500bp, and the free DNA directly enters the downstream steps;

[0010] (3) End repair, A addition and adapter ligation are performed on fragmented DNA or cell-free DNA mixed with Lambda DNA;

[0011] (4) Immunoprecipitate the ligation product with a 5-methylcytosine-specific antibody to enrich methylated DNA fragments;

[0012] (5) The enriched product was digested with a combination of methylation-sensitive restriction endonucleases to specifically remove unmethylated regions;

[0013] (6) The enzyme digestion products were enriched by PCR, purified and sequenced by high throughput;

[0014] (7) Construct a genome-wide methylation map through bioinformatics analysis.

[0015] Preferably, the DNA includes genomic DNA extracted from cell lines, tumor tissues, exfoliated cells, and free DNA extracted from plasma, urine, pleural effusion, and peritoneal fluid.

[0016] Preferably, the sample sequencing library includes the complete sequence of the sample DNA and the added exogenous DNA.

[0017] Preferably, the exogenous DNA is Lambda DNA, used to monitor the digestion efficiency of methylation-sensitive restriction endonucleases.

[0018] Preferably, the methylation-sensitive restriction endonuclease combination comprises at least two enzymes selected from HpaII, AciI, and Hin6I, and more preferably a mixture of HpaII, AciI, and Hin6I. This combination can cover a wider range of genomic regions, and in particular, can effectively detect a variety of key sequence patterns, including CGCG.

[0019] Preferably, the 5-methylcytosine-specific antibody is a monoclonal antibody derived from mice or rabbits, used at a concentration of 1-5 μg / reaction, and incubated for 12-16 hours.

[0020] Preferably, the washing step includes: first washing 2-3 times with a low-salt washing buffer, and then washing 2-3 times with a high-salt washing buffer containing 400-500 mM NaCl.

[0021] Preferably, the concentration of each enzyme in the methylation-sensitive restriction endonuclease combination is 5-20 U / μg DNA, the enzyme digestion reaction is carried out in 1×NEBuffer 4, the reaction temperature is 37℃, and the reaction time is 2-4 hours.

[0022] Preferably, the adapter comprises P5 and P7 sequences and is modified by 5' phosphorylation and 3' C3 spacer or dideoxy C blocking, and does not contain the recognition site of the methylation-sensitive restriction endonuclease combination.

[0023] This invention also provides the application of the construction method described above in DNA methylation detection.

[0024] This invention also provides the application of the above-mentioned technical solutions in cell lines, clinical tissues, and clinical plasma samples.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] (1) The present invention has higher detection accuracy: it completely avoids DNA damage and base conversion deviation caused by bisulfite conversion. Validated by methylation standards, the average absolute deviation of the methylation ratio detected by this method from the theoretical value is less than 1%, which is significantly better than the traditional BS-seq method.

[0027] (2) The present invention has better technical repeatability: the method of the present invention shows high consistency among technical repeat experiments, with a Pearson correlation coefficient of 0.995, and the repeatability is significantly higher than that of the traditional BS-seq method, ensuring the stability and reliability of the detection results;

[0028] (3) This invention has higher sensitivity and specificity: By organically combining the high sensitivity of MeDIP with the high specificity of methylation-sensitive restriction endonuclease (MSRE), this invention effectively overcomes the inherent defects of single technologies. In standard validation, the sensitivity reached 99.2% and the specificity reached 98.7%.

[0029] (4) The present invention has better cost-effectiveness: Under the same amount of sequencing data, the present invention can detect an average of more than 15% more effective methylation sites in the whole genome comparable region than the traditional BS-seq; this means that when achieving the same detection performance, the present invention requires less sequencing data, thereby significantly reducing the cost of single sample detection and providing an economical and feasible solution for large-scale clinical screening and application.

[0030] (5) The present invention has a wider range of sample applicability: it can be applied to various sample types such as blood, tissue, and cell lines, and is especially suitable for the detection of trace amounts of cfDNA;

[0031] (6) This invention provides a reliable technical platform for early cancer screening and screening of methylation markers, diagnosis of genetic diseases and epigenetic research. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0033] Figure 1 This is a flowchart illustrating the method of the present invention;

[0034] Figure 2 This is a schematic diagram showing the comparison of the number of effective methylation sites detected in the genome-wide comparable region in samples with different methylation levels using the method of the present invention and the BS-seq method in Example 1;

[0035] Figure 3 This is a schematic diagram showing the comparison of the detection accuracy of the method of the present invention and the BS-seq method in Example 1;

[0036] Figure 4 This is a schematic diagram showing the technical repeatability comparison results between the method of the present invention and the BS-seq method in Example 1;

[0037] Figure 5 This is a schematic diagram comparing the detection results of methylation sites in a specific region using the method of the present invention and the Medip method in Example 2;

[0038] Figure 6 This is a schematic diagram comparing the detection performance of the method of the present invention and the Medip method in a specific region in Example 2;

[0039] Figure 7 This is a schematic diagram comparing the false positive detection results of the present invention method and the Medip method in a specific area in Example 2;

[0040] Figure 8 This is a heatmap diagram illustrating the screening of methylation markers in plasma cfDNA of colorectal cancer, lung cancer, and healthy individuals using the method of the present invention in Example 3. Detailed Implementation

[0041] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. However, the following embodiments are only for explaining the present invention, and the scope of protection of the present invention should include all the contents of the claims. Moreover, through the description of the following embodiments, those skilled in the art can fully implement all the contents of the claims of the present invention.

[0042] This embodiment discloses a whole-genome methylation detection method based on MeDIP-MSRE, the procedure of which is as follows: Figure 1 As shown, the steps are as follows:

[0043] 1. Sample DNA extraction: Extract sample DNA using a plasma cell-free DNA or tissue extraction kit or a related body fluid extraction kit, following the steps outlined in the extraction kit.

[0044] 2. Genomic DNA samples need to be fragmented into DNA fragments of 200-500bp. Fragmentation is performed using a Covaris S220 sonicator with the following parameters: peak power of 175W, 10% duty factor, 200 cycles, temperature of 4-7 degrees Celsius, and fragmentation time of 120s. Free DNA is directly mixed with a certain proportion of Lambda DNA.

[0045] 3. End-of-pipe repair and dA tailing

[0046] Table 1: Reaction System 1

[0047] Table 2: Reaction Procedure 1

[0048] Note: The temperature of the PCR instrument's heating cap is set to 80℃;

[0049] 4. Connector Connection

[0050] Table 3: Reaction System 2

[0051] Table 4: Reaction Procedure 2

[0052] Note: The temperature of the PCR instrument's heating cap is set to 80℃;

[0053] 5. Purification after adapter ligation

[0054] After the linker connection reaction is complete, use AMPure beads for purification. Note that the AMPure beads should be equilibrated at room temperature for at least 30 minutes before use.

[0055] 5.1 Purify the AMPure beads by equilibrating them at room temperature for at least 30 minutes and vortexing them thoroughly before use.

[0056] 5.2 Add 80 µL of AMPure beads to a 100 µL sample and vortex mix.

[0057] 5.3 Let the mixture stand at room temperature for 5-10 minutes, then place it on a magnetic rack and let it stand for about 3-5 minutes until the magnetic beads are completely separated from the solution. Carefully remove the supernatant.

[0058] 5.4 Clean the magnetic beads with 200 µL of 80% ethanol, repeat once, for a total of 2 washes;

[0059] 5.5 Air dry on the magnetic rack until the magnetic beads are dry;

[0060] 5.6 Resuspend the dried magnetic beads in 25 µL of TE buffer and let stand at room temperature for about 5 minutes. Place the centrifuge tube on a magnetic rack and wait for the supernatant to become clear. Transfer the supernatant to a new PCR tube and place it on ice for later use.

[0061] 6. MeDIP-enriched methylated regions

[0062] 6.1 Take 25 µL of the ligation product, denature at 95 °C for 10 min, and immediately place in an ice bath for 5 min;

[0063] 6.2 Immunoprecipitation

[0064] Table 5: Reaction System 3

[0065] The reaction procedure is as follows: incubate overnight (approximately 12-16 hours) on a rotary mixer at 4°C.

[0066] 6.3 Magnetic Bead Capture

[0067] The Protein A / G (Thermo Fisher Scientific) magnetic beads were washed three times with IP buffer beforehand, as follows:

[0068] a. Remove the Protein A / G magnetic bead suspension from the 4°C freezer and vortex it vigorously for at least 30 seconds to ensure it is fully and evenly resuspended.

[0069] b. Aspirate 25 μL of a uniform magnetic bead suspension into a new 1.5 mL sterile centrifuge tube;

[0070] c. Place the centrifuge tube containing 25 μL of magnetic beads on a magnetic rack and let it stand for 1-2 minutes until the solution becomes clear and all the magnetic beads are adsorbed onto the tube wall near the magnet. Carefully remove the supernatant completely with a pipette, avoiding touching or disturbing the magnetic bead precipitate that has accumulated on the tube wall.

[0071] d. Remove the centrifuge tube from the magnetic rack and immediately add 500 μL of pre-cooled IP buffer (self-prepared);

[0072] e. Gently vortex or invert the centrifuge tube 10-15 times to fully resuspend the magnetic beads in the buffer solution. Do not use vigorous vortexing to avoid damaging the magnetic beads or antibodies;

[0073] f. Place the centrifuge tube back on the magnetic rack and let it stand for 1-2 minutes. Once the solution has clarified, carefully aspirate the supernatant.

[0074] g. Remove the centrifuge tube from the magnetic rack again, add 500 μL of pre-cooled IP buffer, and repeat the washing process 3 times.

[0075] h. Finally, add 25 μL of IP buffer to the washed magnetic bead precipitate, and gently pipette or briefly vortex to resuspend the magnetic beads evenly, forming a magnetic bead working solution with a concentration of 1:1 (v / v), which can be used immediately for magnetic bead capture.

[0076] Table 6: Reaction System 4

[0077] The magnetic bead capture reaction procedure is as follows: incubate for 1-2 hours on a rotary mixer at room temperature.

[0078] 6.4 Cleaning after magnetic bead capture

[0079] Wash three times with low-salt (homemade) solution, following these steps:

[0080] a. Place the centrifuge tube on a magnetic rack and let it stand for 2 minutes. Once the solution becomes clear, carefully aspirate the supernatant.

[0081] b. Add 500 μL of low-salt wash buffer, remove the centrifuge tube from the magnetic rack, and gently vortex or invert the tube 10 times to mix.

[0082] c. Place back on the magnetic rack and let stand for 2 minutes, then discard the supernatant;

[0083] d. Repeat steps b and c twice, for a total of 3 washes.

[0084] Wash three times with a high-salt (home-prepared) solution, following these steps:

[0085] a. Add 500 μL of high-salt washing buffer to the low-salt washed magnetic beads;

[0086] b. Remove the centrifuge tube from the magnetic rack and gently vortex or invert the mixture 10 times.

[0087] c. Place back on the magnetic rack and let stand for 2 minutes, then discard the supernatant;

[0088] d. Repeat step ac twice, for a total of 3 washes.

[0089] e. Finally, add 100 μL of elution buffer (containing 1% SDS) to the magnetic beads after washing with low and high salt, incubate at 65°C for 15 minutes, shaking to mix every 5 minutes, separate on a magnetic rack, and carefully transfer the supernatant to a new centrifuge tube.

[0090] 6.5 DNA purification column purification

[0091] a. Add 500 μL of binding buffer (containing 5M GuHCl and 30% isopropanol) to 100 μL of elution product and vortex to mix.

[0092] b. Transfer the entire mixture to a purification column and centrifuge at 12,000 × g for 1 minute;

[0093] c. Discard the filtrate, add 500 μL of washing buffer (containing 80% ethanol), and centrifuge at 12,000×g for 1 minute;

[0094] d. Repeat washing once;

[0095] e. Centrifuge the empty column at 12,000×g for 2 minutes to completely remove residual ethanol;

[0096] f. Transfer the purification column to a new centrifuge tube and add 30 μL of preheated sterile water (65°C).

[0097] g. After standing at room temperature for 2 minutes, centrifuge at 12,000×g for 2 minutes to elute the DNA.

[0098] 7. Purification by methylation-sensitive endonuclease digestion

[0099] Table 7: Reaction System 5

[0100] Table 8: Reaction Procedure 3

[0101] 8. Purification after digestion with methylation-sensitive endonuclease

[0102] After digestion with methylation-sensitive endonuclease, the enzyme was purified using AMPure beads. Note that the AMPure beads should be equilibrated at room temperature for at least 30 minutes before use.

[0103] 8.1 Purify AMPure beads by equilibrating at room temperature for at least 30 minutes and vortexing thoroughly before use;

[0104] 8.2 Add 40 µL of AMPure beads to a 50 µL sample and vortex mix.

[0105] 8.3 Let the mixture stand at room temperature for 5-10 minutes, then place it on a magnetic rack and let it stand for about 3-5 minutes until the magnetic beads are completely separated from the solution. Carefully remove the supernatant.

[0106] 8.4 Clean the magnetic beads with 200 µL of 80% ethanol, repeat once, for a total of 2 washes;

[0107] 8.5 Air dry on the magnetic rack until the magnetic beads are dry;

[0108] 8.6 Resuspend the dried magnetic beads in 25 µL of enzyme-free sterile water and let stand at room temperature for about 5 minutes. Place the centrifuge tube on a magnetic rack and wait for the supernatant to become clear. Transfer the supernatant to a new PCR tube and place it on ice for later use.

[0109] 9. Library amplification

[0110] Table 9: Reaction System 6

[0111] Table 10: Reaction Procedure 4

[0112] Note: The heating cap is set to 105℃;

[0113] 10. Purification after library amplification

[0114] After library amplification, purification is performed using AMPure beads. Note that the AMPure beads should be equilibrated at room temperature for at least 30 minutes before use.

[0115] 10.1 Purify AMPure beads by equilibrating at room temperature for at least 30 minutes and vortexing thoroughly before use;

[0116] 10.2 Add 44 µL of AMPure beads to a 55 µL sample and vortex mix.

[0117] 10.3 Let the mixture stand at room temperature for 5-10 minutes, then place it on a magnetic rack and let it stand for about 3-5 minutes until the magnetic beads are completely separated from the solution. Carefully remove the supernatant.

[0118] 10.4 Clean the magnetic beads with 200 µL of 80% ethanol, repeat once, for a total of 2 washes;

[0119] 10.5 Air dry on the magnetic rack until the magnetic beads are dry;

[0120] 10.6 Resuspend the dried magnetic beads in 30 µL of enzyme-free sterile water and let stand at room temperature for about 5 minutes. Place the centrifuge tube on a magnetic rack and wait for the supernatant to become clear. Transfer the supernatant to a new PCR tube and place it on ice for later use.

[0121] 11. Document Quality Inspection

[0122] 11.1 Concentration Quality Control

[0123] The library was subjected to concentration quality control using HS dsDNA reagent and Qubit 4.0 instrument. To meet the requirements of downstream sequencing, the library concentration was ≥1.5ng / µL.

[0124] 11.2 Segment Quality Control

[0125] Library fragment sizes were determined using a Qsep-1 / Labchip GX Touch (Caliper), with average fragment lengths ranging from 200 to 1000 bp.

[0126] 12. Sequencing

[0127] This invention uses a second-generation sequencing platform well-known to those skilled in the art for high-throughput sequencing, and performs sequencing of the library on the Illumina sequencing platform using a sequencing-by-synthesis method.

[0128] 13. Data Processing and Analysis

[0129] 13.1 Raw sequencing data quality control: The raw sequencing data (FASTQ format) was evaluated using FastQC (v0.11.9) software, including base quality distribution, GC content, sequence length distribution, adapter contamination and repetitive sequence levels, etc. Trimmomatic (v0.39) or cutadapt software was used for quality control filtering.

[0130] 13.2 Sequence alignment and matching with the reference genome: Using high-efficiency alignment software BWA-MEM (v0.7.17) or Bowtie2 (v2.4.5), the high-quality reads after quality control were aligned with the human reference genome (e.g., GRCh38 / hg38); Picard Tools (v2.27) or sambamba was used to mark and remove duplicate reads generated by PCR amplification to avoid the impact of amplification bias on subsequent quantitative analysis;

[0131] 13.3 Identification of Methylation-Enriched Regions: Using the callpeak command in MACS2 (v2.2.7.1), regions enriched with methylated DNA were identified across the entire genome;

[0132] 13.4 Differential Methylation Analysis: Calculations were performed in the R environment using the MethylKit (v1.20) or DSS (v2.44) software packages. The sequencing read coverage depth of each sample within a predefined genomic region was statistically analyzed; statistical tests were performed between the experimental and control groups. For data based on enrichment levels, negative binomial distribution tests or linear models were used to correct for differences in sequencing depth.

[0133] The present invention will be further described below with reference to specific embodiments.

[0134] Example 1

[0135] The samples in this example are methylated standards from Zymo Research, with product catalog numbers D5014-1 and D5014-2. D5014-1 is a non-methylated standard (methylation level of 0%), and D5014-2 is a methylated standard (methylation level of 100%).

[0136] First, mixed samples with different methylation ratios were prepared using the two standards described above: 100%, 75%, 50%, 25%, and 0%, for a total of five methylation levels. Three replicates were set up for each methylation level. The samples were fragmented into 200-500 bp fragments using a Covaris S220 sonicator according to the parameters described above. One set of samples was then prepared using the method of this invention, while the other set was prepared using the BS-seq method. Both sets of samples were simultaneously sequenced in the same batch on an Illumina NovaSeq 6000 instrument using the PE150 sequencing mode to eliminate batch effects. After quality control of the raw data, the sequencing data of each sample was randomly sampled using seqtk software to unify the effective sequencing data volume of all samples to the same level (40 million paired-end reads). The human reference genome (GRCh38) was divided into 500 bp non-overlapping windows using bedtools software. Windows with stable sequencing coverage (average sequencing depth ≥5X within the window) in all replicate samples from both methods are retained as a set of "alignable genomic regions" for subsequent comparison of detection counts and accuracy. Within the set of aligned regions, for BS-seq data, the average methylation level of all CpG sites within each window is calculated, and windows with an average methylation level ≥25% are identified as "methylated regions" of this method. For the method of this invention, MACS2 (v2.2.7.1) software is used to call methylation enrichment peaks across the entire genome (parameter: --broad -q 0.05), and aligned windows falling within these significantly enriched peaks (FDR<0.05) are identified as "methylated regions" of the method of this invention. Furthermore, the detection sensitivity and specificity of the two methods are calculated based on a "gold standard" composed of 0% and 100% methylation standards. All CpG sites in the 100% methylated standard (D5014-2) were used as the positive set, and all CpG sites in the 0% methylated standard (D5014-1) were used as the negative set. For BS-seq, a single CpG site with a methylation level ≥50% was considered positive; for the method of this invention, CpG sites located within the methylation enrichment peaks recognized by MACS2 were considered positive.

[0137] Based on the above methods, under the same sequencing data volume and comparable genomic regions, the method of this invention detected a significantly higher number of methylated regions than the BS-seq method (see Appendix). Figure 2Quantitative analysis showed that the method of this invention detected an average of 15% more methylated regions than the BS-seq method. This is mainly due to the fact that the method of this invention avoids DNA degradation caused by bisulfite conversion and achieves efficient detection in both high and low CpG density regions through the synergistic effect of MeDIP and MSRE. The detection accuracy of the two methods for methylation levels is analyzed, and the results are attached. Figure 3 The detection values ​​of the method of this invention are highly consistent with the theoretical values, with a mean absolute deviation (MAD) of less than 1%, and the regression curve is closer to the ideal Y=X line. In contrast, BS-seq exhibits systematic bias at both low and high methylation levels due to incomplete and non-specific transformations. The detection sensitivity and specificity of the two methods are analyzed, and the results are shown in Table 11. The sensitivity and specificity of the method of this invention are superior to those of the BS-seq method. The technical reproducibility of the two methods is analyzed, and the results are shown in the appendix. Figure 4 The Pearson correlation coefficients of whole-genome methylation levels between technical replicates of the two methods were calculated separately. The correlation coefficient of the invented method was as high as 0.995, which was significantly higher than that of the BS-seq method, indicating that it has excellent operational stability and reproducibility.

[0138] Therefore, the method of this invention, through the combination of MeDIP and MSRE technologies, completely avoids DNA degradation and sequence bias caused by bisulfite conversion, demonstrating significant advantages in detection accuracy, sensitivity, specificity, and technical repeatability. Most importantly, the method of this invention can obtain stable and reliable results with relatively low sequencing data volumes, resulting in a significantly lower single-sample detection cost compared to traditional BS-seq methods.

[0139] Table 11

[0140] Example 2

[0141] This embodiment uses methylated standards from Zymo Research, catalog numbers D5014-1 and D5014-2. D5014-1 is a non-methylated standard, and D5014-2 is a methylated standard. Each standard is divided into two equal parts. One part is processed and library constructed according to the method of this invention, and the other part is constructed according to the traditional MeDIP-seq method. Both sets of samples are sequenced simultaneously on an Illumina NovaSeq 6000 instrument in the same batch to eliminate batch effects. The sequencing mode is PE150.

[0142] Finally, data quality control and analysis are performed.

[0143] Based on sequencing and analysis results, 500 high CpG density regions (mainly CpG islands in tumor suppressor gene promoters) and 500 low CpG density regions (mainly intergenic or intronic regions) were selected from the methylated standards. Conversely, 500 typical unmethylated regions (mainly promoter regions of actively transcribed genes) and 500 high-risk false-positive regions (mainly low-complexity and repetitive sequence regions) were selected from the non-methylated standards for comparative analysis of the two detection methods. Results are shown below. Figure 5 , Figure 6 , Figure 7 The results show that the method of the present invention has higher detection specificity than the MeDIP-seq method while maintaining high detection sensitivity, especially in the high-risk false positive region of the MeDIP method, where it demonstrates superior detection capability. It can reduce the false positive rate of the MeDIP method from 25% to 5% of MeDIP-seq, and generate more realistic methylation profiles, providing a higher quality and more reliable data foundation for downstream biomarker discovery and mechanism research.

[0144] Example 3

[0145] This embodiment used plasma samples from 30 healthy volunteers, 30 patients with pathologically confirmed colorectal cancer, and 30 patients with pathologically confirmed lung cancer. Informed consent was obtained for all samples, and the samples were approved by the ethics committee. First, cell-free DNA (cfDNA) was extracted using a plasma cell-free DNA extraction kit, yielding an average of 15 ng. Then, 10 ng of cfDNA was precisely extracted from each sample for library construction, sequencing, and finally, data quality control and analysis were performed. A t-test was used to compare differences in methylation levels between groups, and FDR (Frequency and Determination of methylation) was used for multiple correction. The comparison results are shown in the appendix. Figure 8 .

[0146] The results showed that the method of this invention successfully screened 50 candidate methylation biomarkers. Heatmap analysis clearly demonstrated methylation patterns specific to different cancer types. In colorectal cancer patients, SEPT9 and VIM genes showed specific hypermethylation and could serve as specific methylation genes for colorectal cancer detection. In lung cancer patients, SHOX2 and RASSF1A genes showed characteristic hypermethylation and could serve as specific methylation genes for lung cancer detection. Genes showing hypermethylation in both colorectal and lung cancer patients may serve as broad-spectrum cancer screening biomarkers. Furthermore, these biomarkers remained hypomethylated in healthy controls. This indicates that the method of this invention can efficiently screen diagnostically valuable methylation biomarkers from plasma cfDNA, providing important targets for the development of multi-cancer early screening kits and showing significant clinical application potential in the screening and discovery of cancer biomarkers based on liquid biopsy.

[0147] Example 4

[0148] The samples in this embodiment were obtained from the plasma of 20 patients with early-stage (stage I-II) lung cancer and 20 healthy controls. The plasma of the lung cancer patients was separated from their peripheral blood before surgery. Informed consent was obtained for all samples and the samples were approved by the ethics committee.

[0149] First, cell-free DNA was extracted from plasma samples, with an average yield of 15 ng. Then, 10 ng of cfDNA was precisely extracted from each sample for library construction, sequencing, and finally, data quality control and analysis were performed. Based on the sequencing results, the detection results of SHOX2 and RASSF1A methylation were analyzed. The detection results are shown in Table 12.

[0150] The results showed that even with a cfDNA input as low as 10 ng, the method of the present invention achieved a high sensitivity of 95.0% and a specificity of 95.0% for detecting methylation of SHOX2 or RASSF1A in the plasma of early-stage lung cancer patients. This indicates that the methylation markers screened based on the method of the present invention can effectively distinguish early-stage lung cancer patients from healthy individuals, demonstrating the great application potential and commercial value of the present invention in the field of non-invasive early cancer screening based on liquid biopsy.

[0151] Table 12

[0152] The SHOX2 gene, a dwarf homeobox gene, is a member of the homeobox gene family, and its gene expression regulation is closely related to organ development. Studies have found that the SHOX2 gene is abnormally expressed in various solid tumors, such as lung cancer, breast cancer, and kidney cancer, and that abnormal SHOX2 gene expression levels can induce tumor development. The RASSF1A gene, a member of the Ras-associated domain family 1, is a protein-coding gene. It is abnormally expressed in various solid tumors, including lung cancer and kidney cancer, and participates in the occurrence and development of tumors. Aberrant methylation of the RASSF1A gene may occur in the early stages of tumor development. Studies have found a characteristic increase in the levels of methylated SHOX2 and RASSF1A genes in plasma samples from lung cancer patients.

[0153] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A method for detecting whole-genome methylation based on MeDIP-MSRE, wherein the method is for non-diagnostic purposes, characterized in that, Includes the following steps: (1) Extract DNA from the sample to be tested, including cell-free DNA from plasma, tissue gDNA or cell line gDNA; (2) The gDNA is subjected to ultrasonic fragmentation to obtain DNA fragments of 200-500bp, and the free DNA directly enters the downstream steps; (3) End repair, A addition and adapter ligation are performed on fragmented DNA or cell-free DNA mixed with Lambda DNA; (4) Immunoprecipitate the ligation product with a 5-methylcytosine-specific antibody to enrich methylated DNA fragments; (5) The enriched product was digested with a combination of methylation-sensitive restriction endonucleases to specifically remove unmethylated regions; (6) The enzyme digestion products were enriched by PCR, purified and sequenced by high throughput; (7) Construct a genome-wide methylation map through bioinformatics analysis; The adapter described in step (3) is modified with 5' phosphorylation and 3' end blocking, and does not contain recognition sites for HpaII, AciI, and Hin6I; The immunoprecipitation described in step (4) includes: denaturing DNA into single-stranded DNA, incubating it with 5-methylcytosine antibody at 4°C for 12-16 hours, capturing the complex with Protein A / G magnetic beads, and eluting it after washing three times each with low-salt and high-salt buffers. The methylation-sensitive restriction endonuclease combination described in step (5) includes HpaII, AciI, and Hin6I. The reaction system consists of 30 µL of Medip enriched product, 45 µL of 10×NEBuffer, and the enzyme combination is 5 U each for HpaII, AciI, and Hin6I, with the remainder being enzyme-free sterile water. The total volume is 50 µL. The reaction program is as follows: Step 1: 37 °C for 3 h, Step 2: 80 °C, Step 3: 4 °C.

2. The method as described in claim 1, characterized in that, The fragmentation process described in step (2) uses ultrasonic fragmentation, and the fragment size is 300±50 bp.

3. The method as described in claim 1, characterized in that: The Lambda DNA mixed in during step (3) is used to monitor enzyme digestion efficiency.

4. The application of the method described in any one of claims 1-3 in epigenetic studies for non-diagnostic purposes or in the construction of DNA methylation maps.

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