Methods for detecting the methylation status of DNA samples

Abstract

The present specification provides a method for detecting the methylation status of a DNA sample, specifically, a method for detecting the methylation status of DNA molecules in a sample. The method involves treating the sample with a methylation-sensitive or methylation-dependent restriction endonuclease, comparing it with a sample not treated with the restriction endonuclease, and reflecting the methylation status of the DNA molecules through the degree of enzymatic cleavage. The present specification also provides a kit for detecting the methylation status of DNA molecules in a sample. The method for detecting the methylation status of DNA molecules in a sample provided herein is simple in process and produces accurate results, and can be used for early screening of multiple types of cancer using ctDNA samples.

Description

[Technical Field]

[0001] This application claims priority to a Chinese patent application with application number 2023107826859, filed with the China Patent Office on June 29, 2023, the entire text of which is incorporated herein by reference.

[0002] The present invention relates to a method for detecting methylation, and more particularly to a method for detecting methylation using a methylation-sensitive restriction endonuclease. The present invention also relates to a kit for detecting methylation. [Background technology]

[0003] DNA methylation is one of the ways cells regulate gene expression, regulating protein expression without altering the gene sequence. DNA methylation is associated with many physiological and pathological factors and carries a wealth of biological information. In cancer cells, DNA methylation changes, resulting in either hypomethylation of the promoter regions of oncogenes (activation of oncogenic factors) or hypermethylation of the promoter regions of tumor suppressor genes (downregulation of tumor suppressor gene expression). It has been reported that the methylation status of ctDNA in plasma corresponds to the methylation level in cancer tissue. At the same time, the methylation status of ctDNA can be traced back to the tissue. Therefore, ctDNA methylation offers significant advantages for implementing early cancer screening projects.

[0004] There are many different methylation sequencing methods. First- and second-generation sequencing technologies can only distinguish between four distinct bases (ATCG) and cannot distinguish between methylated and unmethylated C bases. Traditional processing methods use bisulfite conversion to convert unmethylated C bases to U bases. After PCR amplification, U bases are converted to T and compared with the unconverted sequence to determine whether methylation modification has occurred at that site. Therefore, bisulfite conversion was once the gold standard for methylation sequencing. However, bisulfite conversion uses high salt and strong alkaline conditions, which cause significant sample damage, resulting in a sample loss rate of approximately 70% after conversion. For difficult-to-obtain sample types such as ctDNA, a large amount of methylation information is lost. NEB has developed an enzyme conversion kit based on the operating principle of methylation modification enzymes in living organisms. This conversion method ultimately obtains sequence information consistent with the bisulfite conversion state. While enzymatic conversion results in relatively low conversion damage, the enzymatic reaction is highly dependent on enzyme activity, leading to incomplete conversion in complex regions. At the same time, the multi-step conversion procedure is relatively laborious, and repeated purification procedures result in a certain amount of sample loss. Reduced representation bisulfite sequencing (RRBS) is considered a cost-effective solution for DNA methylation detection. It uses MspI endonuclease to cleave the methylated CCGG enzyme cleavage site. The coverage depth and ratio of RRBS are related to the efficiency of MspI enzyme cleavage. While suitable for detecting methylation in the whole mammalian genome, it is not suitable for methylation detection and analysis of samples related to cfDNA early screening tests.

[0005] Currently, most commercially available methylation early screening products are PCR-based, covering a limited number of sites and primarily analyzing the methylation status of a single gene. As the cancer early screening market develops, it is shifting from single-cancer early screening to multi-cancer early screening. This increases the demand for early cancer screening technologies. Methylation target capture sequencing methods detect the methylation status of various sites by enriching fragments in target regions. Depending on the targeted sequencing method, they can be divided into liquid-phase hybrid capture sequencing technology and multiplex PCR amplification technology. Traditional liquid-phase hybrid capture technology has difficulty capturing small methylation panels and has a relatively complex operation process. Multiplex PCR amplification technology places high requirements on primer design, and the GC bias of the converted library is large, further increasing the difficulty of primer design. The development of methylation detection technologies with simple and convenient operation procedures, particularly suitable for cfDNA samples, can facilitate the implementation of early methylation screening projects. Summary of the Invention

[0006] In one aspect, the present invention provides a method for detecting the methylation state of DNA molecules in a sample. (1) dividing the sample into two parts, a first sample and a second sample; treating the first sample with one or more methylation-sensitive restriction endonucleases, such that if the DNA molecules are not methylated at the recognition sites of the methylation-sensitive restriction endonucleases, an enzymatic cleavage reaction can be carried out to obtain an enzymatically cleaved sample; or treating the sample with one or more methylation-dependent restriction endonucleases, such that if the DNA molecules are methylated at the recognition sites of the methylation-dependent restriction endonucleases, an enzymatic cleavage reaction can be carried out to obtain an enzymatically cleaved sample; and not treating the second sample with the restriction endonucleases; (2) contacting the enzyme-cleaved sample with one or more capture probes, wherein the capture probes target target sequences in the DNA molecules that include the recognition site; separating the DNA molecules that include the uncleaved recognition site from the enzyme-cleaved sample; and contacting the capture probes with the second sample to separate the DNA molecules that include the recognition site from the second sample; (3) determining the overall methylation status of DNA molecules in the sample depending on the amount of DNA molecules containing the recognition site that have not been enzymatically cleaved compared to the amount of DNA molecules containing the recognition site in the second sample, or sequencing the DNA molecules containing the recognition site that have not been enzymatically cleaved and the DNA molecules containing the recognition site in the second sample, and determining the methylation status of different DNA molecules in the sample by a sequencing depth ratio.

[0007] In some embodiments, the capture probe comprises a target-specific sequence, a first probe-binding sequence located at the 5' end of the target-specific sequence, and a second probe-binding sequence located at the 3' end of the target-specific sequence, wherein the first probe-binding sequence is at least partially complementary to the second probe-binding sequence, so that when two or more capture probes bind to the target sequence adjacent to each other, the adjacent capture probes can form complementary bonds with the first probe-binding sequence via the second probe sequence.

[0008] In some embodiments, the first probe binding sequence and the second probe binding sequence are 8 to 30 nt in length.

[0009] In some embodiments, the target-specific sequence is 20 to 80 nt in length.

[0010] In some embodiments, prior to step (1), the method further comprises constructing a sequencing library using DNA molecules of the sample, and optionally fragmenting the DNA molecules while constructing the sequencing library.

[0011] In some embodiments, the methylation-sensitive restriction endonuclease is selected from Hpa I, Hpa II, Hha I, Aci I, and any combination thereof.

[0012] In some embodiments, the methylation-dependent restriction endonuclease is selected from FspE I, LpnP I, MspJ I, and any combination thereof.

[0013] In some embodiments, the sample comprises ctDNA and / or gDNA, or the sample is an FFPE sample.

[0014] In one embodiment, the present invention provides a kit for detecting the methylation status of DNA molecules in a sample. (1) one or more methylation-sensitive restriction endonucleases that are capable of performing an enzymatic cleavage reaction on a DNA molecule when the DNA molecule is unmethylated at a recognition site for the methylation-sensitive restriction endonuclease, or one or more methylation-dependent restriction endonucleases that are capable of performing an enzymatic cleavage reaction on a DNA molecule when the DNA molecule is methylated at a recognition site for the methylation-dependent restriction endonuclease; (2) one or more capture probes, the capture probes comprising a target-specific sequence, a first probe-binding sequence located at the 5' end of the target-specific sequence, and a second probe-binding sequence located at the 3' end of the target-specific sequence, the first probe-binding sequence being at least partially complementary to the second probe-binding sequence, such that when two or more capture probes bind adjacently to a target sequence containing the recognition site in the DNA molecule, the adjacent capture probes are one or more capture probes that can form a complementary bond with the first probe-binding sequence via the second probe sequence.

[0015] In some embodiments, the kit further comprises enzymes and adapter molecules used to construct a sequencing library of the DNA molecules.

[0016] In some embodiments, the first probe binding sequence and the second probe binding sequence are 8 to 30 nt in length.

[0017] In some embodiments, the target-specific sequence is 20-80 nt in length.

[0018] In some embodiments, the methylation-sensitive restriction endonuclease is selected from Hpa I, Hpa II, Hha I, Aci I, and any combination thereof.

[0019] In some embodiments, the methylation-dependent restriction endonuclease is selected from FspE I, LpnP I, MspJ I, and any combination thereof.

[0020] In some embodiments, the sample comprises ctDNA. [Effects of the Invention]

[0021] The method for detecting the methylation status of DNA molecules in a sample provided herein reflects the methylation level through the enzymatic cleavage level of restriction endonucleases. Combined with the use of capture probes, the process is simple and the detection results are accurate. The method can be used for early screening of multiple types of cancer using ctDNA samples. [Brief explanation of the drawings]

[0022] [Figure 1] This paper presents the recognition sites of four methylation-sensitive restriction endonucleases and the process for constructing a target methylation capture library based on the methylation-sensitive restriction endonucleases. This process is suitable for detecting the methylation status of target regions in gDNA samples after physical shearing, plasma-free DNA, or circulating tumor ctDNA samples. [Figure 2] Target regions with different methylation status are enzymatically digested with methylation-sensitive restriction endonucleases, resulting in corresponding library capture results. [Figure 3] This is a situation where the constructed library does not contain the complete restriction endonuclease recognition site. The constructed library must contain the complete restriction endonuclease recognition site; if the library contains only three or fewer bases of site information at the enzyme cleavage site, the methylation status of the enzyme cleavage site cannot be determined. Here, we will use the Hha I restriction endonuclease cleavage site as an example. For this restriction endonuclease cleavage site, the data for this portion must be excluded during analysis. A. The first 3, 2, or 1 base of the restriction endonuclease cleavage site is ligated to the adapter. B. The last 3, 2, or 1 base of the restriction endonuclease cleavage site is ligated to the adapter. [Figure 4]One site has two sensitive endonuclease recognition sites, making it impossible to determine the methylation status of the enzyme cleavage site. (A) The Hha I and Hpa II enzyme cleavage sites are not methylated and can be cleaved. (B) The Hha I enzyme cleavage site is not methylated, but the Hpa II enzyme cleavage site is methylated and can be cleaved. (C) The Hha I enzyme cleavage site is methylated, but the Hpa II enzyme cleavage site is not methylated and can be cleaved. (D) The Hha I and Hpa II enzyme cleavage sites are methylated and cannot be cleaved. [Figure 5] The process of constructing different libraries using two standards, 100% methylated DNA and 0% methylated DNA (ZYMO) (A), and the output of four libraries (B, pre-capture library yield and C, hybrid capture library yield). [Figure 6] The target rate, coverage, and sequencing depth of libraries constructed from samples with different methylation states. In samples with a 0% methylation state, the number of targets after enzymatic digestion was reduced, resulting in lower target rates, coverage, and sequencing depth for the libraries than in the other experimental groups. (A) [On-Target] Fraction of Target Reads in mapped reads, the ratio of on-target read length to mapped read length. (B) 0.5 × Mean Coverage, 0.5 × average coverage percentage; 0.2 × Mean Coverage, 0.2 × average coverage percentage. (C) Average depth (rmdup), the depth after deduplication of the target region. [Figure 7]Sequencing depth and corresponding methylation levels of methylation-sensitive endonuclease-associated CpG sites in the target regions of different libraries. (A) 100% methylation level sample enzyme-cleaved and non-cleaved libraries, sequencing depth corresponding to the CpG sites, 100% Me-EN 100% methylation level enzyme-cleaved library, and 100% Me-C 100% methylation level non-cleaved control library. (B) 0% methylation level sample enzyme-cleaved and non-cleaved libraries, sequencing depth corresponding to the CpG sites, 0% Me-EN 0% methylation level enzyme-cleaved library, and 0% Me-C 0% methylation level non-cleaved control library. (C) Methylation level calculation: enzyme cleavage depth / control depth. The methylation level detected in the 0% methyl sample was close to 0, while the methylation level detected in the 100% methyl sample was close to 100%. The detection results were as expected. [Figure 8] Sequencing depth and corresponding methylation levels of methylation-sensitive endonuclease Aci I-associated CpG sites in the target regions of different libraries. Methylation levels detected in the 0% methyl sample and the 100% methyl sample are shown. [Figure 9] Methylation levels of CpG sites associated with methylation-sensitive restriction endonucleases in different samples. Methylation level calculation method: enzyme cleavage depth / control depth. The methylation level detected in the 0% methyl sample is close to 0, while the methylation level detected in the 10% methyl sample is close to 10%. The methylation level detected in the 50% methyl sample is close to 50%. The methylation level detected in the 100% methyl sample is close to 100%. The detection results were as expected. [Figure 10] The methylation levels of the different samples after enzymatic cleavage were calculated using non-enzymatic cleavage samples with different methylation levels as controls. DETAILED DESCRIPTION OF THE INVENTION

[0023] Unless otherwise explained, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art.

[0024] The term "or" refers to one element of a listed set of alternative elements, unless the context clearly indicates otherwise. The term "and / or" means any one, two, three, more, or all of the listed alternative elements.

[0025] As used herein, the terms "comprise," "contain," "have," and similar expressions do not exclude unlisted elements. These terms also include a composition consisting only of the listed elements.

[0026] As used herein, "DNA molecule" refers to deoxyribonucleic acid, a polymer of deoxyribonucleotides. It may be double-stranded or single-stranded, and the group of DNA molecules may include both double-stranded and single-stranded DNA molecules. DNA molecules can be of any length, including genomic DNA, DNA fragments, and free extracellular DNA. Free DNA refers to DNA that is free outside of cells or the cell nucleus. It can be extracted from various biological materials, including body fluids, in vitro cell culture media, and natural environments, including, but not limited to, peripheral blood, plasma, serum, urine, feces, saliva, cerebrospinal fluid, and lymphatic fluid. Free DNA can be obtained by extraction and purification. As used herein, "DNA fragment" refers to shorter DNA molecules, e.g., 50 bp to 700 bp, e.g., 100 bp to 500 bp, particularly 100 bp to 350 bp in length. DNA fragments contained in biological samples are typically heterogeneous, i.e., vary in length. Therefore, the above lengths may refer to the average lengths of these DNA fragments. These DNA fragments may have different sequences, for example, from different regions of the genome of the same organism, or from different organisms. The DNA fragments may contain single-strand breaks and may be blunt or non-blunt ended (with 3' or 5' overhangs).

[0027] As used herein, "DNA methylation" refers to the modification of bases in DNA molecules or DNA fragments by methylation, specifically the modification of cytosine to 5-methylcytosine (5mC). In vertebrates, DNA methylation typically occurs at CpG sites (i.e., sites in the DNA sequence where cytosine is immediately followed by guanine), where DNA methyltransferase converts cytosine to 5-methylcytosine. While most CpG sites in the human genome are methylated, certain regions, such as CpG islands rich in cytosine (C) and guanine (G), are typically unmethylated. CpG methylation can affect the transcriptional activity of associated genes. For example, methylation inhibits tumor suppressor genes, while demethylation stimulates the expression of certain oncogenes. These conditions can lead to cancer. Additionally, a small number of cytosine bases are modified to 5-hydroxymethylcytosine (5hmC), 5-formylation (5fC), or 5-carboxylation (5caC), although at lower rates than 5mC. When referring to methylation herein, it may also refer to modification to 5hmC, 5fC, or 5caC, unless otherwise indicated by the context.

[0028] As used herein, the term "methylation status of a cytosine base" or "DNA methylation information" refers to information about the methylation status of a DNA molecule or DNA fragment, including, but not limited to, methylation sites, methylation levels, and methylation patterns (such as 5mC or 5hmC). The "methylation level," also known as the "methylation degree," refers to the rate (or frequency) at which a specific methylation site in a sample is modified by methylation. There are several methods for detecting whether a site is methylated. Common methods include chemical or enzymatic conversion, during which either methylated or unmethylated cytosine is converted to uracil (U) or a base substantially identical to uracil in terms of base pairing (e.g., dihydrouracil, DHU). In the subsequent amplification process, the corresponding uracil pairs with adenine (A) as thymine (T), and as a final result, the cytosine or methylated cytosine at the methylated site is reflected as thymine in the detection result (e.g., sequencing result). Comparison with a reference sequence can determine whether a cytosine in a DNA molecule or DNA fragment is methylated. The reference sequence can be a sequence from the same sample that has not undergone these modifications, or a corresponding sequence from a healthy population. Alternatively, several methods can be used to distinguish between 5mC and 5hmC. Currently, DNA methylation information is widely used for cancer screening and diagnosis (e.g., lung cancer, breast cancer, liver cancer, colorectal cancer), especially for early screening and diagnosis. Identifying methylation status can also be used for non-diagnostic (or therapeutic) purposes, such as analyzing factors affecting methylation, scientific research into the impact of methylation on gene function, or detecting methylation standards.

[0029] "Methylation-sensitive restriction endonucleases" refer to restriction endonucleases that are sensitive to the presence or absence of methylated bases in their recognition sites. If the recognition site of such an enzyme contains a single methylated base, it is generally unable to cleave a DNA molecule at the recognition site. As used herein, methylation-sensitive restriction endonucleases specifically refer to restriction endonucleases that are unable to cleave a DNA molecule at the recognition site if the recognition site contains at least one 5mC. Methylation-sensitive restriction endonucleases known in the art include, but are not limited to, Hpa I, Hpa II, Hha I, and Aci I.

[0030] In contrast to "methylation-sensitive restriction endonucleases," "methylation-dependent restriction endonucleases" refer to restriction endonucleases that depend on the presence of a methylated base at their recognition site. If such enzymes do not contain a methylated base at their recognition site, they typically cannot cleave a DNA molecule at the recognition site. As used herein, methylation-dependent restriction endonucleases specifically refer to restriction endonucleases that can cleave a DNA molecule at their recognition site only if the recognition site contains at least one 5mC. Methylation-dependent restriction endonucleases known in the art include, but are not limited to, FspE I, LpnP I, and MspJ I.

[0031] The term "capture probe" as used herein refers to a single-stranded DNA fragment used to hybridize with a target DNA molecule. The capture probe used herein preferably targets a target sequence in a DNA molecule containing a restriction endonuclease recognition site. If the methylation state of the recognition site of a DNA molecule treated with a specific restriction endonuclease allows the restriction endonuclease to cleave the DNA molecule, the capture probe can only partially or incompletely bind to the target DNA molecule, and therefore cannot effectively separate the target DNA molecule cleaved by the restriction endonuclease from the sample. In this case, the target DNA molecules separated from the sample treated with the restriction endonuclease using the capture probe will mainly be uncleaved DNA molecules. Therefore, the overall methylation state of the DNA molecules in the sample can be determined based on the amount of uncleaved DNA molecules that can be separated relative to the amount of DNA molecules that can be separated from the sample not treated with the restriction endonuclease. For example, treatment with a methylation-sensitive restriction endonuclease results in a relatively high amount of cleaved DNA molecules (relatively low amount of uncleaved DNA molecules), indicating that the DNA molecules in the sample have a low level of methylation at the restriction endonuclease recognition site. In a preferred embodiment, the capture probe is a μCaler probe (the design of the μCaler probe is described in Chinese Patent Publication CN116083423A, the entire contents of which are incorporated herein by reference). Such a probe comprises a target-specific sequence, a first probe-binding sequence located at the 5' end of the target-specific sequence, and a second probe-binding sequence located at the 3' end of the target-specific sequence, wherein the first probe-binding sequence is at least partially complementary to the second probe-binding sequence, so that when two or more capture probes are adjacently bound to the target sequence, adjacent capture probes can form complementary bonds with the first probe-binding sequence and the second probe sequence.Compared with conventional capture probes, the advantages of using μCaler probes are significantly improved probe-target binding capacity, improved hybrid capture efficiency of target regions, and improved overall coverage uniformity and stability. Capture probes are typically tagged with a separable tag, such as biotin, to facilitate separation of probe-bound target DNA molecules. The separated target DNA molecules can then be sequenced to obtain their sequence information, as well as the methylation status of restriction endonuclease recognition sites.

[0032] The process of the method for detecting the methylation status of a DNA sample provided herein is shown in Figure 1. This process can be applied to construct methylation libraries of genomic DNA and plasma-free DNA. Briefly, genomic DNA is sheared into 200-250 bp DNA fragments by ultrasonic shearing, and the fragmented sample is subjected to library construction, using the Nadprep Universal DNA Library Construction Kit. The library construction process is as follows: A is added to the end repair and adapters are ligated. The adapter-ligated products are divided into two groups. One group is enzymatically digested with a methylation-sensitive restriction endonuclease (i.e., methylation can resist the action of the endonuclease), such as HpaI, HpaII, HhaI, or AciI. The other group serves as a control and is not enzymatically digested. PCR amplification is then performed on each of the two groups of products, and an index sequence is added. The amplified products can be subjected to hybrid capture using the μCaler hybrid capture system. All probes are designed using the μCaler probe design scheme, which covers methylation-sensitive restriction endonuclease cleavage sites for each DNA strand. The captured library can then be sent to an Illumina or MGI platform sequencer for sequencing.

[0033] Target regions with different methylation status were enzymatically cleaved with a methylation-sensitive restriction endonuclease, and the corresponding library capture results are shown in Figure 2. In this schematic diagram, Hha I restriction endonuclease is used as an example. Figure 2A shows that the C base of the GCGC restriction endonuclease recognition site in the target region is not methylated, yet Hha I recognizes and cleaves the adaptor ligation product at this site. The target cannot be detected by PCR amplification or target capture. Figure 2B shows that the C base of the GCGC restriction endonuclease recognition site in the target region is methylated, yet Hha I cannot cleave the adaptor ligation product containing this site. The target can be detected by both PCR amplification and target capture. The detection principle using other methylation-sensitive restriction endonucleases is consistent with this.

[0034] The constructed library must contain the complete restriction endonuclease recognition site. The cleavage sites of gDNA or cfDNA after sonication are random, located within the restriction site, and ligated to the upper adaptor. If the library contains only three or fewer bases of site information at the cleavage site, the methylation status of the cleavage site cannot be determined. This information must be excluded during data analysis. See Figure 3 for an example of the Hha I restriction endonuclease cleavage site. For this restriction endonuclease cleavage site, this portion of the data must be excluded during analysis. Figure 3A shows the first three, two, or one base of the restriction endonuclease cleavage site ligated to the adaptor; Figure 3B shows the last three, two, or one base of the restriction endonuclease cleavage site ligated to the adaptor.

[0035] A single site contains two sensitive endonuclease recognition sites, making it impossible to determine the methylation status of the enzyme cleavage site. See Figure 4. In this process, two or more restriction endonucleases can be used simultaneously for enzymatic cleavage. When using multiple restriction endonucleases in combination, it is necessary to consider whether the same library fragment contains two enzyme cleavage sites simultaneously. When the same DNA fragment contains two restriction endonuclease recognition sites simultaneously, it is not possible to accurately analyze the methylation status of all sites. Here, we will use the Hha I and Hpa II restriction endonuclease cleavage sites as an example. In Figure A, the Hha I and Hpa II enzyme cleavage sites are unmethylated and can be cleaved. In Figure B, the Hha I enzyme cleavage site is unmethylated, but the Hpa II enzyme cleavage site is methylated and can be cleaved. In Figure C, the Hha I enzyme cleavage site is methylated, while the Hpa II enzyme cleavage site is unmethylated and can be cleaved. In Figure D, the Hha I and Hpa II enzyme cleavage sites are methylated and cannot be cleaved. Of the four states, only state D can be detected, and states A, B, and C cannot be distinguished. Therefore, the probe used in the method of the present invention covers as many restriction endonuclease enzyme cleavage sites as possible to ensure the accuracy of the detection results.

[0036] Methylation level analysis plan: The enzyme-cleaved samples were used as controls with non-enzyme-cleaved samples of different methylation levels to calculate the methylation levels of different samples. Methylation level calculation method: Each site was normalized using an internal reference gene, and the normalized depth of each CpG site in the enzyme-cleaved sample was divided by the normalized depth of each CpG site in the control sample.

[0037] Thus, the present specification provides a process for constructing a target methylation library based on a methylation-sensitive restriction endonuclease, including HpaI, HpaII, HhaI, AciI, etc. In some embodiments, a target capture system is used to capture the original DNA sequence from an unconverted library, methylate C bases in CpG sites in the restriction endonuclease recognition sites of the captured fragment target region, and detect methylation modification information in the target region. In some embodiments, a capture probe is designed for the original DNA strand, and the target capture probe must cover the corresponding methylation-sensitive restriction endonuclease cleavage site. The methylation-sensitive restriction endonuclease-based target methylation library construction process provided herein can detect the sequencing depth of CpG sites. The system introduces an internal reference gene for normalization and uses the control to analyze the methylation degree of CpG sites in the sample.

[0038] General experimental steps: Step 1: Sample fragmentation DNA sample shearing can be performed according to laboratory conditions. To shear samples into products with an average fragment length of 200-250 bp, we recommend using a Covaris® Series DNA Sonicator. After shearing gDNA or FFPE DNA, proceed to Step 2. For cfDNA samples, proceed directly to Step 2. Step 2: Repair the ends & 1. Take out the End Repair & A-Tailing Buffer, dissolve it at room temperature, mix it evenly, and store it on ice for later use. 2. Remove the End Repair & A-Tailing Enzyme, place it on ice to dissolve naturally, mix evenly, and centrifuge immediately for later use. 3. Prepare the reaction mixture in a 0.2 mL PCR tube on ice according to the following table. TIFF2026500756000002.tif56170Note: If the amount of DNA is less than 20 μL, you can add nuclease-free water to increase the volume to 20 μL. 4. Mix thoroughly and centrifuge immediately to deposit the entire reaction mixture at the bottom of the PCR tube. 5. Start the next reaction program (Cycling Program I) in the PCR machine, and once the temperature has stabilized at 20°C, place the PCR tube into the PCR machine. TIFF2026500756000003.tif31170Note: When running a 20°C program, there is no need to heat the cap, and when running a 65°C program, the cap will be heated to 70°C.

[0039] Step 3: Connect the adapter 1. Remove the Ligation Buffer, dissolve it at room temperature, mix it evenly, and place it on ice for later use. NOTE: Ligation Buffer is very viscous and requires slow and steady pipetting to ensure accurate volume. 2. Remove the DNA Ligase, place it on ice to allow it to dissolve naturally, mix it evenly, and centrifuge it immediately for later use. 3. Remove the PCR tubes from Step 2 from the PCR machine, place them on an ice plate, and prepare the reaction mixture according to the following table. TIFF2026500756000004.tif55170

[0040] Step 4: Purification of the ligation product Purification and recovery were carried out using 0.5x beads, and the purification process was as follows. 1. Add 20 μL of NadPrep® SP beads to the ligation product of step 3, mix thoroughly, and incubate at 20-25°C for 10 minutes. 2. Centrifuge the PCR tubes immediately, then place them on a magnetic rack for 5 minutes until the liquid is completely clear. Use a pipette to aspirate and discard the supernatant. Note: It needs to be completely transparent. Placement time may need to be adjusted depending on the brand of magnetic rack. 3. Being careful not to disturb the magnetic beads, slowly add 150 µL of 80% ethanol along the side wall of the PCR tube, let stand for 30 seconds, and then aspirate and discard the supernatant using a pipette. 4. Repeat step 3 once. 5. After briefly centrifuging the PCR tube, place it on the magnetic rack and, using a 10 μL pipette tip, aspirate and remove the small amount of remaining ethanol, being careful not to aspirate the magnetic beads. 6. Open the cap of the PCR tube and leave it at 20-25°C for approximately 2-3 minutes until the ethanol has completely evaporated. 7. Remove the PCR tube, add 44 μL of Nuclease-Free Water to the PCR tube, resuspend the magnetic beads evenly using a pipette, and incubate at 25°C for 2 minutes. 8. Centrifuge the PCR tubes briefly, then place them on a magnetic rack for 2 minutes until the liquid is completely clear. Carefully transfer the supernatant to a new 0.2 mL PCR tube using a pipette.

[0041] Step 5: Perform enzymatic cleavage with a methylation-sensitive restriction endonuclease 1. Remove MeEnzyme and MeBuffer, place on ice to allow for natural dissolution (NEB restriction endonuclease), mix thoroughly, and centrifuge immediately for later use. 2. Prepare the reaction mixture in a 0.2 mL PCR tube on ice using a methylation-sensitive restriction endonuclease according to the table below: TIFF2026500756000005.tif501703.Start the following reaction program in the PCR machine, and once the temperature has stabilized at 37°C, place the PCR tube in the PCR: 37°C for 1 hour, 80°C for 20 minutes. Step 6: Purification of enzymatic cleavage products Purify and recover using 1x beads, see step 4 for the purification process, and elute using 20uL of nuclease-free water. Step 7: PCR amplification of enzyme cleavage products 1. Remove the 2x HiFi PCR Master Mix and NadPrep® Universal UDI-Index Primer Mix, place on ice to allow them to thaw, mix thoroughly, and immediately centrifuge. 2. Prepare the PCR amplification mix (Mix) in a 0.2 mL PCR tube on ice according to the following table: TIFF2026500756000006.tif501703. Place the PCR tube in the PCR machine and start the following program. TIFF2026500756000007.tif57170

[0042] Step 8: Purify the amplification product Purify and recover using 1x beads, see step 4 for the purification process, and elute using 20uL of nuclease-free water. Step 9: Library Hybridization 1. Prepare the hybrid reaction system according to the following table. TIFF2026500756000008.tif521702. Vortex the hybridization reaction mixture for at least 10 seconds to mix evenly, then immediately centrifuge to collect the reaction mixture at the bottom of the PCR tube (avoid generating bubbles). 3. Place the PCR tube in the PCR machine and start the next reaction program. 98°C for 2 minutes. 60°C for 1 hour.

[0043] Step 10: Library capture and elution Washing magnetic beads 1. Vortex the streptavidin beads for 15 seconds to mix thoroughly and homogenously. 2. Mix n x 25 µL of streptavidin beads in a 0.2 mL centrifuge tube and wash (n is the number of captured libraries, n < 5). Note: If n>5, the washes must be divided into multiple tubes and mixed. 3. Place the streptavidin beads on the magnetic rack and leave for approximately 2 minutes. When the liquid becomes completely clear, discard the supernatant using a pipette. 4. Remove the centrifuge tube from the magnetic rack, add 100 μL of preheated wash buffer A, and gently purge at least 10 times to mix evenly. 5. Place the centrifuge tube on the magnetic rack and let it sit for about 2 minutes. When the liquid becomes completely clear, use a pipette to discard the supernatant. 6. Repeat steps 4 and 5 once. 7. Immediately centrifuge the tube and discard all of the Wash buffer A at the bottom of the tube using a 10 μL pipette tip. 8. Take 8 μL of uHyb #1, resuspend the streptavidin beads, and gently purge at least 10 times to mix evenly. Magnetic bead capture 1. After 1 hour of hybridization, start the capture stage and keep the PCR machine running. 2. With the PCR tubes still in the PCR machine, immediately add 8 μL of resuspended streptavidin beads to each hybridization reaction mixture and mix thoroughly by gently purging at least 10 times. 3. Incubate at 60°C for 10 minutes. 4. After the incubation is complete, remove the PCR tube from the PCR machine and place it on the magnetic rack for 2 minutes. When the liquid becomes completely clear, use a pipette to discard the supernatant and remove it as completely as possible. Elution 1. Remove the PCR tube from the previous step from the magnetic rack and add 150 μL of preheated Wash Buffer A. Gently purge the tube at least 10 times to mix thoroughly (avoid introducing air bubbles). 2. Place the PCR tube on the magnetic rack and let it sit for 2 minutes. When the liquid becomes completely clear, discard the supernatant using a pipette, remove the PCR tube from the magnetic rack, add 100 μL of preheated Wash Buffer A, and gently purge at least 10 times to mix evenly. Transfer the reaction mixture to a new PCR tube. Note: Avoid creating air bubbles during this step as air bubbles may come into contact with the tube cap and reduce target accuracy. 3. Place the PCR tube in the PCR machine and incubate at 60°C for 3 minutes. 4. Place the PCR tube in the PCR machine and incubate at 60°C for 3 minutes. 5. After the incubation is finished, remove the PCR tube from the PCR machine and place it on the magnetic rack for 2 minutes. When the liquid becomes completely clear, use a pipette to discard the supernatant. 6. Add 150 μL of Wash Buffer B, which has been left at room temperature, to the PCR tube and gently purge at least 10 times to mix evenly. 7. Place the PCR tube on the magnetic rack and let it sit for 2 minutes. Once the liquid is completely clear, use a pipette to discard the supernatant. 8. After briefly centrifuging the PCR tube, place it on the magnetic rack and, using a 10 μL pipette tip, aspirate and remove the small amount of remaining liquid, being careful not to aspirate the magnetic beads. 9.2 Add 2.5 μL of Nuclease-Free Water and gently purge at least 10 times to resuspend the magnetic beads.

[0044] Step 11: Hybrid Capture Library PCR Amplification Remove the 1.2x HiFi PCR Master Mix and NadPrep® Universal UDI-Index Primer Mix, place on ice to allow them to thaw, mix thoroughly, and immediately centrifuge. 2. Prepare the PCR amplification mix (Mix) in a 0.2 mL PCR tube on ice according to the following table: TIFF2026500756000009.tif451703. Place the PCR tube in the PCR machine and start the following program. TIFF2026500756000010.tif55170Step 12: Purification of amplification products Purify and recover using 1x beads, see step 4 for the purification process, and elute using 20uL of nuclease-free water.

[0045] Example 1: Testing with fully methylated and fully unmethylated samples Two standards, 100% methylated DNA and 0% methylated DNA (ZYMO), were used to construct different libraries. The library construction process was briefly described as follows: 50 ng of each standard was collected, end-repaired, and adapters were ligated. The adapter-ligated products were divided into two groups: one group was digested with the methylation-sensitive restriction endonuclease HhaI, and the other group was left undigested as a control. PCR amplification was then performed on each of the two groups to add index sequences (Figure 5A). The amplification products were subjected to hybrid capture using the μCaler Hybrid Capture system. All probes used contained 10KB probes to cover the methylation-sensitive restriction endonuclease cleavage sites. The capture probe sequences used are listed in the table below. For detailed experimental procedures, see "Experimental Steps." TIFF2026500756000011.tif158170TIFF2026500756000012.tif203170TIFF2026500756000013.tif203170TIFF2026500756000014.tif82170

[0046] The library construction output is shown in Figures 5B and 5C. The library construction output indicates that the library constructed using 0% methylation standard endonuclease digestion had the lowest library construction yield and enzyme digestion yield. Because targets containing restriction endonuclease sites are not methylated, they are cleaved by methylation-sensitive restriction endonucleases, resulting in lower library construction yields than the non-enzyme digestion and 100% methylation groups. The target hit rate for the 0% methylation standard endonuclease digestion group was approximately 10%, while the target hit rates for the other groups were approximately 50%. The 0.2xMean group was approximately 35%, the 0.5xMean group was approximately 25%, the other groups were approximately 95%, and the 0.5xMean group was approximately 80%. The depth after deduplication for the 0% methylation standard endonuclease digestion group was approximately 160, while the depth for the other groups was approximately 1200 (Figure 6). Basic quality control at the final stage of the machine showed that the use of methylation-sensitive restriction endonucleases can cleave samples with a methylation level of 0, but cleave unmethylated modification sites, and the use of methylation-sensitive restriction endonucleases can cleave samples with a methylation level of 100%, but do not cleave methylated modification sites.

[0047] Figure 7 shows the sequencing depth and corresponding methylation levels of methylation-sensitive endonuclease-associated CpG sites in the target regions of different libraries. Figure A shows the sequencing depth corresponding to the CpG sites of the 100% methylation level sample enzyme-cleaved and non-enzyme-cleaved libraries. The sequencing depth ranges from hundreds to thousands. Figure B shows the sequencing depth corresponding to the CpG sites of the 0% methylation level sample enzyme-cleaved and non-enzyme-cleaved libraries. The sequencing depth of the 0% methylation level non-enzyme-cleaved library ranged from hundreds to thousands, which is consistent with the results of the 100% methylation level sample experimental group. The sequencing depth corresponding to the CpG sites of the 0% methylation level enzyme-cleaved library was essentially zero. Methylation-sensitive restriction endonucleases can cleave unmethylated modification sites, most of which are cleavable, but they cannot cleave methylated modification sites.

[0048] The results in Figure 7C show the methylation levels of CpG sites associated with methylation-sensitive restriction endonucleases in different samples. Calculation method: enzyme cleavage depth / control depth. The methylation level detected in the 0% methyl sample was close to 0, while the methylation level detected in the 100% methyl sample was close to 100%. The detection results were as expected.

[0049] Example 2: Other methylation-sensitive restriction endonucleases were tested using fully methylated and fully unmethylated samples Two standards, 100% methylated DNA and 0% methylated DNA (ZYMO), were used to construct different libraries. The database construction process was the same as in Example 1. In this example, the methylation-sensitive endonuclease Aci I was selected to test the cleavage efficiency of this endonuclease against standards with different methylation levels. The sequencing depth and corresponding methylation level of the Aci I-associated CpG sites in the target regions of the libraries were calculated, as shown in Figure 8. The methylation level detected in the 0% methylated sample was close to 0, while the methylation level detected in the 100% methylated sample was close to 100%. The detection results were as expected. This hybrid capture system can be applied to different types of methylation-sensitive restriction endonucleases, and the choice of endonuclease is primarily related to the type of restriction endonuclease contained in the target gene.

[0050] Example 3: Test using samples with different methylation levels 100% methylated DNA and 0% methylated DNA (ZYMO) were mixed at a certain ratio to produce methylation standards with methylation levels of 0%, 10%, 50%, and 100%, respectively. 50ng of each standard was taken and end-repair & A was performed, followed by adapter ligation. The adapter ligation products were divided into two groups: one group was digested with the methylation-sensitive restriction endonuclease HhaI, and the other group was not digested as a control. The grouping is shown in the table below, and PCR amplification was performed on the products of the two groups, and index sequences were added. TIFF2026500756000015.tif60170

[0051] The amplification products were subjected to hybrid capture using the μCaler hybrid capture system (the capture probes were the same as in Example 1). All probes were normalized using an internal reference gene (by designing probes in regions that did not contain enzyme cleavage sites) using a 10KB probe. The normalization probe sequences are shown in the table below. TIFF2026500756000016.tif50170TIFF2026500756000017.tif228170

[0052] The results in Figure 9 show the methylation levels of CpG sites associated with methylation-sensitive restriction endonucleases in different samples. Calculation method: enzyme cleavage depth / control depth. The methylation level detected in the 0% methyl sample is close to 0, while the methylation level detected in the 100% methyl sample is close to 100%. The methylation level detected in the 10% methyl sample is close to 10, while the methylation level detected in the 50% methyl sample is close to 50. The methylation level detected in the 100% methyl sample is close to 100%. The detection results were as expected.

[0053] For the enzymatically digested samples, non-enzymatically digested samples with different methylation levels were used as controls to calculate the methylation levels of the different samples. See Figure 10. Methylation level calculation method: Each site was normalized using an internal reference gene (by designing a probe in an area without an enzymatically digested site). The normalized depth of each CpG site in the enzymatically digested sample was calculated by dividing the normalized depth of each CpG site in the control sample. The methylation level detected in the 0% methyl sample was close to 0, while the methylation level detected in the 100% methyl sample was close to 100%. The methylation level detected in the 50% methyl sample was close to 50%. The methylation level detected in the 100% methyl sample was close to 100%. The detection results, normalized by adding an internal reference gene and using non-enzymatically digested samples with different methylation levels as controls, showed that the methylation levels corresponding to the different CpG sites calculated for the enzymatically digested samples were all as expected.

Claims

1. 1. A method for detecting the methylation status of DNA molecules in a sample, comprising: (1) dividing the sample into two parts, a first sample and a second sample; treating the first sample with one or more methylation-sensitive restriction endonucleases, such that if the DNA molecules are not methylated at the recognition sites of the methylation-sensitive restriction endonucleases, an enzymatic cleavage reaction can be carried out to obtain an enzymatically cleaved sample; or treating the sample with one or more methylation-dependent restriction endonucleases, such that if the DNA molecules are methylated at the recognition sites of the methylation-dependent restriction endonucleases, an enzymatic cleavage reaction can be carried out to obtain an enzymatically cleaved sample; and not treating the second sample with the restriction endonucleases; (2) contacting the enzyme-cleaved sample with one or more capture probes, wherein the capture probes target target sequences in the DNA molecules that include the recognition site; separating the DNA molecules that include the recognition site that have not been enzyme-cleaved from the enzyme-cleaved sample; and contacting the capture probes with the second sample to separate the DNA molecules that include the recognition site from the second sample; (3) determining the overall methylation status of DNA molecules in the sample depending on the amount of DNA molecules containing the recognition site that have not been enzymatically cleaved compared to the amount of DNA molecules containing the recognition site in the second sample, or alternatively sequencing the DNA molecules containing the recognition site that have not been enzymatically cleaved and the DNA molecules containing the recognition site in the second sample and determining the methylation status of different DNA molecules in the sample by sequencing depth ratio.

2. 2. The method of claim 1, wherein the capture probe comprises a target-specific sequence, a first probe-binding sequence located at the 5' end of the target-specific sequence, and a second probe-binding sequence located at the 3' end of the target-specific sequence, and the first probe-binding sequence is at least partially complementary to the second probe-binding sequence, so that when two or more capture probes bind to the target sequence adjacent to each other, adjacent capture probes can form complementary bonds with the first probe-binding sequence via the second probe sequence.

3. The method according to claim 1 or 2, wherein the length of the first probe binding sequence and the second probe binding sequence is 8 to 30 nt.

4. The method according to any one of claims 1 to 3, wherein the target-specific sequence is 20 to 80 nt in length.

5. 5. The method of claim 1, further comprising, prior to step (1), constructing a sequencing library using DNA molecules in the sample; and fragmenting the DNA molecules before constructing the sequencing library.

6. 6. The method of any one of claims 1 to 5, wherein the methylation-sensitive restriction endonuclease is selected from Hpa I, Hpa II, Hha I, Aci I, and any combination thereof.

7. The method of any one of claims 1 to 6, wherein the methylation-dependent restriction endonuclease is selected from FspE I, LpnP I, MspJ I, and any combination thereof.

8. The method of any one of claims 1 to 7, wherein the sample comprises ctDNA and / or gDNA or the sample is an FFPE sample.

9. 1. A kit for detecting the methylation status of DNA molecules in a sample, comprising: (1) one or more methylation-sensitive restriction endonucleases that are capable of performing an enzymatic cleavage reaction on a DNA molecule when the DNA molecule is unmethylated at the recognition site for the methylation-sensitive restriction endonuclease, or one or more methylation-dependent restriction endonucleases that are capable of performing an enzymatic cleavage reaction on a DNA molecule when the DNA molecule is methylated at the recognition site for the methylation-dependent restriction endonuclease; a target sequence containing the recognition site in the DNA molecule, wherein the first probe binding sequence is at least partially complementary to the second probe binding sequence, so that when two or more capture probes bind to the target sequence containing the recognition site in the DNA molecule adjacent to each other, the adjacent capture probes can form a complementary bond with the first probe binding sequence via the second probe sequence;

10. 10. The kit of claim 9, further comprising enzymes and adapter molecules used to construct a sequencing library of the DNA molecules.

11. The kit according to claim 9 or 10, wherein the first probe binding sequence and the second probe binding sequence have a length of 8 to 30 nt.

12. The kit according to any one of claims 9 to 11, wherein the target-specific sequence is 20 to 80 nt in length.

13. 13. The method of any one of claims 9 to 12, wherein the methylation-sensitive restriction endonuclease is selected from Hpa I, Hpa II, Hha I, Aci I, and any combination thereof.

14. The method of any one of claims 9 to 13, wherein the methylation-dependent restriction endonuclease is selected from FspE I, LpnP I, MspJ I, and any combination thereof.

15. The method according to any one of claims 9 to 14, wherein the sample comprises ctDNA.

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