Method for detecting methylation status of DNA sample

Abstract

The present application provides a method for detecting the methylation status of a DNA sample, and specifically provides a method for detecting the methylation status of a DNA molecule in a sample, comprising: treating a sample with a methylation-sensitive restriction endonuclease or a methylation-dependent restriction endonuclease, comparing said sample with a sample not treated with a restriction endonuclease, and reflecting the methylation status of the DNA molecule by the degree of enzyme cutting. The present application also provides a kit for detecting the methylation status of a DNA molecule in a sample. The method for detecting the methylation status of a DNA molecule in a sample provided herein is simple in process and accurate in results, and can be used for early screening of multiple cancer types using ctDNA samples.

Description

A method for detecting the methylation status of a DNA sample

[0001] This application claims priority to Chinese patent application No. 2023107826859 filed with the Patent Office of China on June 29, 2023, the entire text of which is incorporated herein by reference. Technical Field

[0002] The present invention relates to a method for methylation detection, in particular to a method for methylation detection using a methylation-sensitive restriction endonuclease. The present invention also relates to a kit for methylation detection. Background Art

[0003] DNA methylation is one of the ways cells regulate gene expression, regulating protein expression without changing the gene sequence. DNA methylation is associated with many physiological and pathological factors and carries a large amount of biological information. In cancer cells, the methylation state of DNA is changed, with the promoter region of proto-oncogenes being hypomethylated (oncogenic factors activated) or the promoter region of tumor suppressor genes being hypermethylated (tumor suppressor gene expression downregulated). It has been reported that the methylation state of ctDNA in plasma is consistent with the methylation level in cancer tissues, and that the methylation state of ctDNA can be traced to tissues, so ctDNA methylation has great advantages in carrying out early cancer screening projects.

[0004] There are numerous methylation sequencing methods. First- and second-generation sequencing technologies can only distinguish between four different bases in ATCG and cannot distinguish between methylated and unmethylated C bases. Traditionally, bisulfite conversion is used to convert unmethylated C bases to U bases. After PCR amplification, the U bases are converted to T bases, and comparison with the unconverted sequence confirms whether methylation has occurred at that site. Therefore, bisulfite conversion was once the gold standard for methylation sequencing. However, BS conversion uses high-salt and strong-base conditions, which can severely damage the sample, resulting in a loss rate of approximately 70% after conversion. For sample types such as ctDNA, which are difficult to obtain, a significant amount of methylation information is lost. NEB has developed an enzymatic conversion kit based on the working principles of methylation-modifying enzymes in vivo. This conversion method ultimately yields sequence information consistent with the BS conversion state. Enzymatic conversion reduces conversion damage relatively little, but the enzymatic reaction is highly dependent on enzyme activity, leading to incomplete conversion in complex regions. Furthermore, the multi-step conversion process is cumbersome, and repeated purification steps also result in sample loss. Reduced Representation Bisulfite Sequencing (RRBS) is considered a cost-effective solution for DNA methylation detection. It uses the MspI endonuclease to cleave methylated CCGG sites. The coverage depth and ratio of RRBS are related to the efficiency of MspI cleavage. While it is suitable for genome-wide methylation detection in mammals, it is less suitable for methylation analysis of cfDNA samples for early detection.

[0005] Currently, most of the methylation early screening products approved for marketing are PCR-based, with a limited number of sites covered and focusing on single-gene methylation status analysis. With the development of the cancer early screening market, single-cancer early screening will transition to multi-cancer early screening. This places higher demands on cancer early screening technology. The methylation targeted capture sequencing method detects the methylation status of different sites by enriching fragments in the target region. Depending on the targeted sequencing method, it is divided into liquid phase hybridization capture sequencing technology and multiplex PCR amplification technology. Traditional liquid phase hybridization capture technology has difficulty capturing small methylation panels and the operation process is relatively cumbersome. Multiplex PCR amplification technology has high requirements for primer design, and the GC bias of the converted library is large, which further increases the difficulty of primer design. The development of a methylation detection technology with a simple operation process, high convenience, and especially suitable for cfDNA samples can promote the implementation of methylation early screening projects.

[0006] Summary of the Invention

[0007] In one aspect, the present invention provides a method for detecting the methylation status of a DNA molecule in a sample, comprising:

[0008] 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 so that the DNA molecule can undergo an enzymatic digestion reaction when methylation is not present at the recognition site of the methylation-sensitive restriction endonuclease, thereby obtaining an enzyme-digested sample; or treating the sample with one or more methylation-dependent restriction endonucleases so that the DNA molecule can undergo an enzymatic digestion reaction when methylation is present at the recognition site of the methylation-dependent restriction endonuclease, thereby obtaining an enzyme-digested sample; and the second sample is not treated with the restriction endonuclease;

[0009] 2) contacting the digested sample with one or more capture probes, wherein the capture probes target a target sequence including the recognition site in the DNA molecule, so as to separate the DNA molecule including the recognition site that has not been digested from the digested sample; contacting the second sample with the capture probes, so as to separate the DNA molecule including the recognition site from the second sample; and

[0010] 3) determining the overall methylation status of the DNA molecules in the sample based on the amount of the DNA molecules including the recognition site that have not been digested, compared with the amount of the DNA molecules including the recognition site in the second sample; alternatively, sequencing the DNA molecules including the recognition site that have not been digested and the DNA molecules including the recognition site in the second sample, and determining the methylation status of different DNA molecules in the sample by sequencing depth ratio.

[0011] In some embodiments, the capture probe includes 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 in an adjacent manner, the adjacent capture probes can form complementary binding through the first probe binding sequence and the second probe sequence.

[0012] In some embodiments, the first probe binding sequence and the second probe binding sequence are 8-30 nt in length.

[0013] In some embodiments, the target-specific sequence is 20-80 nt in length.

[0014] In some embodiments, before step 1), the method further comprises constructing a sequencing library using the DNA molecules in the sample and, optionally, fragmenting the DNA molecules before constructing the sequencing library.

[0015] In some embodiments, the methylation-sensitive restriction endonuclease is selected from Hpa I, Hpa II, Hha I, Aci I, and any combination thereof.

[0016] In some embodiments, the methylation-dependent restriction endonuclease is selected from FspE I, LpnP I, MspJ I, and any combination thereof.

[0017] In some embodiments, the sample comprises ctDNA and / or gDNA, or the sample is a FFPE sample.

[0018] In one aspect, the present invention provides a kit for detecting the methylation status of a DNA molecule in a sample, comprising:

[0019] 1) one or more methylation-sensitive restriction endonucleases, wherein the methylation-sensitive restriction endonucleases are capable of enzymatically cleaving the DNA molecule when the DNA molecule is not methylated at the recognition site of the methylation-sensitive restriction endonuclease; or one or more methylation-dependent restriction endonucleases, wherein the DNA molecule is capable of enzymatically cleaving when the recognition site of the methylation-dependent restriction endonuclease is methylated; and

[0020] 2) One or more capture probes, wherein the capture probe includes 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 capture probes or more capture probes bind to the target sequence including the recognition site in the DNA molecule in an adjacent manner, adjacent capture probes can form complementary binding through the first probe binding sequence and the second probe sequence.

[0021] In some embodiments, the kit further comprises enzymes and adapter molecules for constructing a sequencing library for the DNA molecule.

[0022] In some embodiments, the first probe binding sequence and the second probe binding sequence are 8-30 nt in length.

[0023] In some embodiments, the target-specific sequence is 20-80 nt in length.

[0024] In some embodiments, the methylation-sensitive restriction endonuclease is selected from Hpa I, Hpa II, Hha I, Aci I, and any combination thereof.

[0025] In some embodiments, the methylation-dependent restriction endonuclease is selected from FspE I, LpnP I, MspJ I, and any combination thereof.

[0026] In some embodiments, the sample comprises ctDNA.

[0027] The method provided in this article for detecting the methylation status of DNA molecules in a sample reflects the methylation level through the level of restriction endonuclease cleavage. Combined with the use of capture probes, the process is simple and the detection results are accurate. It can be used for early screening of multiple cancer types using ctDNA samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1: Recognition sites for four methylation-sensitive restriction endonucleases and the workflow for constructing a targeted methylation capture library based on methylation-sensitive restriction endonucleases. This workflow is suitable for detecting the methylation status of target regions in physically fragmented gDNA samples, plasma cell-free DNA, or circulating tumor ctDNA samples.

[0029] Figure 2: Target regions with different methylation status were digested with methylation-sensitive restriction enzymes, and the corresponding library capture results.

[0030] Figure 3: The constructed library does not contain a complete restriction endonuclease recognition site. The constructed library should contain a complete restriction endonuclease recognition site. If the library only contains information for three or fewer bases within a restriction endonuclease site, the methylation status of that site cannot be determined. This example uses the Hha I restriction endonuclease site as an example. For this restriction endonuclease site, this portion of the data needs to be filtered out during analysis. A: The first three, first two, or first bases of the restriction endonuclease site are ligated to an adapter; B: The last three, last two, or last bases of the restriction endonuclease site are ligated to an adapter.

[0031] Figure 4: A locus has two sensitive endonuclease recognition sites, making it impossible to determine the methylation status of the restriction sites. (A) Both the Hha I and Hpa II restriction sites are unmethylated, allowing cleavage; (B) The Hha I restriction site is unmethylated, while the Hpa II restriction site is methylated, allowing cleavage; (C) The Hha I restriction site is methylated, while the Hpa II restriction site is unmethylated, allowing cleavage; (D) Both the Hha I and Hpa II restriction sites are methylated, preventing cleavage.

[0032] Figure 5: The process of constructing different libraries using two standards, 100% methylated DNA and 0% methylated DNA (ZYMO) (A) and the library construction outputs of four libraries (B, pre-capture library yield and C, hybridization capture library yield).

[0033] Figure 6: On-target rate, coverage, and sequencing depth of libraries constructed from samples with different methylation statuses. For samples with 0% methylation, the number of targets is reduced after enzyme digestion, resulting in lower on-target rate, coverage, and sequencing depth than in other experimental groups. (A) [On-Target] Fraction of Target Reads in Mapped Reads, on-target read length ratio; (B) 0.5× Mean Coverage, 0.5× average coverage percentage; 0.2× Mean Coverage, 0.2× average coverage percentage; (C) Average depth (rmdup), target region depth after deduplication.

[0034] Figure 7: Sequencing depth and corresponding methylation levels for methylation-sensitive endonucleases at CpG sites within the target regions of different libraries. (A) Sequencing depth for CpG sites in a 100% methylation-level digested and undigested sample library, 100% Me-EN digested library, 100% Me-C, and a 100% methylation-level undigested control library; (B) Sequencing depth for a 0% methylation-level digested and undigested sample library, 0% Me-EN digested library, 0% Me-C, and a 0% methylation-level undigested control library; (C) Methylation level calculation: digestion depth / control depth; methylation levels detected in 0% methylated samples are close to 0, while methylation levels detected in 100% methylated samples are close to 100%. The results are consistent with expectations.

[0035] Figure 8: Sequencing depth and corresponding methylation levels of CpG sites associated with the methylation-sensitive endonuclease Aci I within the target region of different libraries. Methylation levels detected in samples with 0% methylation and samples with 100% methylation are shown.

[0036] Figure 9: Methylation levels at CpG sites in different samples using methylation-sensitive restriction enzymes. Methylation levels are calculated as: enzyme digestion depth / control depth. Methylation levels detected in samples with 0% methylation are close to 0, and those detected in samples with 10% methylation are close to 10%. Methylation levels detected in samples with 50% methylation are close to 50%, and those detected in samples with 100% methylation are close to 100%. All test results are consistent with expectations.

[0037] Figure 10: The methylation levels of the enzyme-digested samples were calculated using non-enzyme-digested samples with different methylation levels as controls. DETAILED DESCRIPTION

[0038] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0039] The term "or" refers to a single element of the listed alternative elements, unless the context clearly indicates otherwise. The term "and / or" refers to any one, any two, any three, any more or all of the listed alternative elements.

[0040] The terms "include", "comprising", "having" and similar expressions used herein do not exclude unrecited elements. These terms also include the case where the composition consists of only the listed elements.

[0041] "DNA molecule" herein refers to deoxyribonucleic acid, which is a polymer of deoxyribonucleotides. It can be in double-stranded form or single-stranded form, or for a population of DNA molecules, it can include double-stranded DNA molecules and single-stranded DNA molecules. DNA molecules can be of any length, for example, genomic DNA, DNA fragments, or extracellular free DNA. Free DNA refers to DNA that is free outside the cell or outside the cell nucleus and can be extracted from various body fluids, in vitro cell culture fluid, natural environment and other biological materials, including but not limited to: peripheral blood, plasma, serum, urine, feces, saliva, cerebrospinal fluid, lymph fluid, etc. Free DNA can be obtained through extraction and purification. "DNA fragment" herein refers to a shorter DNA molecule, for example, with a length between 50bp and 700bp, for example, 100bp to 500bp, and especially 100bp to 350bp. The DNA fragments contained in a biological sample are usually heterogeneous, that is, the lengths are different. Accordingly, the above-mentioned length can refer to the mean length of these DNA fragments. These DNA fragments may have different sequences, for example, from different regions of the genome of the same organism, or even from different organisms. The DNA fragments may have single-stranded nicks and may have blunt or non-blunt ends (with 3' or 5' overhangs).

[0042] "DNA methylation" in this context refers to the methylation of bases within a DNA molecule or DNA fragment, specifically the modification of cytosine to 5-methylcytosine (5mC). In vertebrates, DNA methylation typically occurs at CpG sites (i.e., sites in a DNA sequence where cytosine is immediately followed by a guanine), where DNA methyltransferases catalyze the conversion of cytosine to 5-methylcytosine. While most CpG sites in the human genome are methylated, certain specific regions, such as CpG islands rich in cytosine (C) and guanine (G), are typically unmethylated. CpG methylation can affect the transcriptional activity of related genes. For example, methylation can inhibit tumor suppressor genes, while demethylation can stimulate the expression of certain oncogenes, potentially leading to the development of cancer. Furthermore, although the incidence is lower than that of 5mC, a small number of cytosine bases are modified to 5-hydroxymethylcytosine (5hmC), 5-formylation (5fC), and 5-carboxylation (5caC). When methylation is mentioned herein, it may also refer to modifications such as 5hmC, 5fC, and 5caC, unless the context indicates otherwise.

[0043] "Cytosine base methylation status" or "DNA methylation information" in this article refers to information about the methylation status in a DNA molecule or DNA fragment, including but not limited to methylation sites, methylation levels, methylation patterns (such as 5mC or 5hmC), etc. "Methylation level", also known as "methylation degree", refers to the proportion (or frequency) of a specific methylation site in a sample that is modified by methylation. There are many ways to detect whether a site is methylated. Common methods include chemical or enzymatic conversion, during which one of the methylated cytosine and unmethylated cytosine is converted into uracil (U) or a base that is substantially equivalent to uracil in base pairing (such as dihydrouracil, DHU). In the subsequent amplification process, the corresponding uracil pairs with adenine (A) as thymine (T), and the final result is that the cytosine or methylated cytosine at the methylation site is reflected as thymine in the test results (such as sequencing results). By comparing with the reference sequence, it is possible to determine whether the cytosine in the DNA molecule or DNA fragment is methylated. The reference sequence can be a sequence from the same sample but without the above-mentioned conversion, or a corresponding sequence in a healthy population. 5mC and 5hmC can also be distinguished by some methods. Currently, DNA methylation information has been widely used in cancer (e.g., lung cancer, breast cancer, liver cancer, colorectal cancer, etc.) screening and diagnosis, especially early screening and diagnosis. In addition, the identification of methylation status can also be used for non-diagnostic (or therapeutic) purposes, such as for scientific research, such as analyzing factors that affect methylation, the effect of methylation on gene function, or for the detection of methylation standards, etc.

[0044] "Methylation-sensitive restriction endonucleases" refer to restriction endonucleases that are sensitive to the presence of methylated bases in their recognition sites. Such enzymes generally cannot cleave DNA molecules at their recognition sites if they contain a methylated base. Herein, methylation-sensitive restriction endonucleases specifically refer to restriction endonucleases that cannot cleave DNA molecules at their recognition sites if they contain 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.

[0045] In contrast to "methylation-sensitive restriction endonucleases," "methylation-dependent restriction endonucleases" refer to restriction endonucleases that rely on the presence of methylated bases in their recognition sites. Such enzymes typically cannot cleave DNA molecules at their recognition sites if they do not contain methylated bases. Herein, methylation-dependent restriction endonucleases specifically refer to restriction endonucleases that can only cleave DNA molecules at their recognition sites if they contain 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.

[0046] "Capture probe" refers to a single-stranded DNA fragment for hybridizing with a target DNA molecule in this article. In this article, the capture probe preferably targets a target sequence containing a restriction endonuclease recognition site in a DNA molecule. If the methylation state of the recognition site of a DNA molecule treated with a specific restriction endonuclease enables the restriction endonuclease to cut the DNA molecule, the capture probe may only partially or insufficiently bind to the target DNA molecule, thereby effectively preventing the target DNA molecule cut by the restriction endonuclease from being isolated from the sample. In this case, the target DNA molecule that can be isolated from the sample treated with the restriction endonuclease utilizing the capture probe is primarily an uncut DNA molecule. Therefore, the overall methylation state of the DNA molecule in the sample can be judged based on the amount of the uncut DNA molecule that can be isolated relative to the amount of the uncut DNA molecule that can be isolated in the sample treated with the restriction endonuclease. For example, when treated with a methylation-sensitive restriction endonuclease, the amount of the cut DNA molecule is relatively high (the amount of the uncut DNA molecule is relatively low), indicating that the DNA molecule in the sample has a lower methylation level at the restriction endonuclease recognition site. In a preferred embodiment, the capture probe is a μCaler-type probe (for the design of μCaler probes, please refer to Chinese patent publication CN116083423A, which is hereby incorporated by reference in its entirety). This probe includes 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 is at least partially complementary to the second probe binding sequence, so that when two or more capture probes bind to the target sequence in an adjacent manner, adjacent capture probes can form complementary binding through the first probe binding sequence and the second probe sequence. Compared with ordinary capture probes, the advantage of using μCaler-type probes is that it can significantly improve the ability of the probe to bind to the target, improve the hybridization capture efficiency of the target area, and improve the overall coverage uniformity and stability. The capture probe can usually carry a detachable label, such as biotin, so as to separate the target DNA molecules bound to the probe. The sequence information of the separated different target DNA molecules can be obtained by sequencing, and the methylation status at the restriction endonuclease recognition site can also be obtained.

[0047] The process of the method for detecting the methylation status of DNA samples provided in this article can be seen in Figure 1. This process is applicable to the construction of methylation libraries of genomic DNA and plasma free DNA. The process is briefly described as follows: genomic DNA is sheared into 200-250bp DNA fragments using ultrasonic shearing; the fragmented samples are subjected to library construction, and the library construction kit can use the Nadprep universal DNA library construction kit. The library construction process is as follows: end repair plus A, adapter ligation; the adapter ligation product is divided into two, one group is digested with a methylation-sensitive restriction endonuclease (i.e., methylation can resist the action of the endonuclease), and the restriction endonuclease type is selected from Hpa I, Hpa II, Hha I and Aci I, etc., and the other group is not digested as a control group. PCR amplification is performed on the two groups of products respectively, and an index sequence is added. The amplified products can be hybridized and captured using the μCaler hybrid Capture system. All probes use the μCaler probe design scheme, and the probes are designed for the DNA chain. The probes cover the methylation-sensitive restriction endonuclease cutting sites. The captured library is sent to Illumina or MGI platform sequencer for sequencing.

[0048] Target regions with different methylation states were digested using methylation-sensitive restriction endonucleases, and the corresponding library capture results are shown in Figure 2. This schematic diagram takes Hha I restriction endonuclease as an example. In Figure 2A, the C base of the GCGC restriction endonuclease recognition site in the target region has not been methylated. Hha I recognizes and cuts the adapter-ligated product with this site, and neither PCR amplification nor targeted capture can detect the target. In Figure 2B, the C base of the GCGC restriction endonuclease recognition site in the target region has been methylated. Hha I cannot cut the adapter-ligated product with this site, and both PCR amplification and targeted capture can detect the target. The detection principle using other methylation-sensitive restriction endonucleases is consistent with this.

[0049] The constructed library should contain a complete set of restriction endonuclease recognition sites. The breakpoints of gDNA or cfDNA after ultrasonic shearing are random and located within the restriction endonuclease site. Adapters are attached to the breakpoints. If only three or fewer bases of the restriction endonuclease site are retained in the library, the methylation status of the restriction endonuclease site cannot be determined. This information needs to be filtered out during data analysis. See Figure 3, which uses the Hha I restriction endonuclease site as an example for explanation. For this restriction endonuclease site, this data needs to be filtered out during analysis. Figure 3A shows that the first three, first two, or first one bases of the restriction endonuclease site are ligated to the adapter; Figure 3B shows that the last three, last two, or last one bases of the restriction endonuclease site are ligated to the adapter.

[0050] A site has two sensitive endonuclease recognition sites, making it impossible to determine the methylation status of the restriction endonucleases. See Figure 4. This process can be used with two or more restriction endonucleases simultaneously. When using multiple restriction endonucleases in combination, it is necessary to consider whether the same library fragment contains both restriction endonuclease sites. If the same DNA fragment contains both restriction endonuclease recognition sites, it is impossible to accurately analyze the methylation status of all sites. This is explained using the Hha I and Hpa II restriction endonuclease sites as an example. Figure A: Both the Hha I and Hpa II restriction endonuclease sites are unmethylated and can be cut; B: The Hha I restriction endonuclease site is unmethylated, while the Hpa II restriction endonuclease site is methylated and can be cut; C: The Hha I restriction endonuclease site is methylated, while the Hpa II restriction endonuclease site is unmethylated and can be cut; D: Both the Hha I and Hpa II restriction endonucleases are methylated and cannot be cut. Of the four states described above, only state D can be detected; states A, B, and C cannot be distinguished. Therefore, the probe used in the method of the present invention covers a single restriction endonuclease cleavage site as much as possible to ensure the accuracy of the detection result.

[0051] Methylation level analysis protocol: After digestion, use non-digested samples with varying methylation levels as controls to calculate methylation levels. Methylation level calculation: Each site is normalized using an internal reference gene, with the normalized depth per CpG site for the digested samples divided by the normalized depth per CpG site for the control samples.

[0052] Therefore, this article provides a process for constructing a targeted methylation library based on a methylation-sensitive restriction endonuclease, wherein the methylation-sensitive restriction endonuclease includes Hpa I, Hpa II, Hha I, Aci I, etc. In some embodiments, the targeted capture system is captured for non-transformed libraries, the original DNA sequence is captured, the C base in the CpG site in the restriction endonuclease recognition site of the captured fragment target region is methylated, and the methylation modification information is detected in the target region. In some embodiments, the capture probe is designed for the original DNA chain, and the targeted capture probe needs to cover the corresponding methylation-sensitive restriction endonuclease cutting site. The process for constructing a targeted methylation library based on a methylation-sensitive restriction endonuclease provided herein can detect the sequencing depth of the CpG site, the system introduces an internal reference gene for normalization, and uses a control to analyze the methylation degree of the sample CgG site.

[0053] General experimental steps:

[0054] Step 1: Sample fragmentation

[0055] DNA sample fragmentation can be performed according to laboratory conditions. Covaris is recommended. TM Use a DNA ultrasonic disruptor to fragment the sample into products with an average fragment length of 200-250 bp. gDNA or FFPE DNA fragments proceed to step 2 after fragmentation. cfDNA samples proceed directly to step 2.

[0056] Step 2: End Repair & A Addition

[0057] 1. Take out End Repair & A-Tailing Buffer and dissolve it at room temperature, mix well, and place on ice until ready to use.

[0058] 2. Take out End Repair & A-Tailing Enzyme and place it on ice to thaw naturally. Mix well and centrifuge briefly for later use.

[0059] 3. Prepare the reaction system in a 0.2 mL PCR tube on ice according to the table below:

[0060] Note: If the amount of DNA is less than 20 μL, add Nuclease Free Water to make up to 20 μL.

[0061] 4. Mix well and centrifuge briefly to allow all the reaction solution to settle at the bottom of the PCR tube.

[0062] 5. Start the following reaction program (Cycling Program I) on the PCR instrument. When the temperature stabilizes at 20°C, place the PCR tube into the PCR instrument:

[0063] Note: No heated cover is required when the 20°C program is running, and the heated cover is 70°C when the 65°C program is running.

[0064] Step 3: Connect the connector

[0065] 1. Take out the Ligation Buffer and dissolve it at room temperature, mix well, and place on ice for later use.

[0066] Note: Ligation Buffer is very viscous. Pipette aspiration and release should be slow and steady to ensure accurate volume.

[0067] 2. Take out DNA Ligase and place it on ice to thaw naturally, mix well, and centrifuge briefly for later use.

[0068] 3. Remove the PCR tube from step 2 from the PCR instrument, place it on ice, and prepare the reaction system according to the following table:

[0069] Step 4: Purification of ligation product

[0070] Use 0.5×Beads for purification and recovery. The purification process is as follows:

[0071] 1. Add 20 μL of the ligation reaction product from step 3. SP Beads, mix well, and incubate at 20-25°C for 10 min.

[0072] 2. Centrifuge the PCR tube briefly and place it on a magnetic rack for 5 minutes until the liquid is completely clear. Use a pipette to remove and discard the supernatant.

[0073] Note: It must be fully clarified that the placement time must be adjusted for different brands of magnetic racks.

[0074] 3. Slowly add 150 μL of 80% ethanol along the side of the PCR tube, being careful not to disturb the magnetic beads. Let it stand for 30 seconds and use a pipette to remove and discard the supernatant.

[0075] 4. Repeat step 3 once.

[0076] 5. Centrifuge the PCR tube briefly and place it on a magnetic rack. Use a 10 μL pipette tip to remove a small amount of residual ethanol, being careful not to absorb the magnetic beads.

[0077] 6. Open the PCR tube cap and let it stand at 20-25℃ for about 2-3 minutes until the ethanol evaporates completely.

[0078] 7. Remove the PCR tube and add 44 μL of Nuclease Free Water to the PCR tube. Use a pipette to evenly resuspend the magnetic beads and incubate at 25°C for 2 minutes.

[0079] 8. Centrifuge the PCR tube briefly and place it on a magnetic rack for 2 minutes until the liquid is completely clear. Use a pipette to carefully transfer the supernatant to a new 0.2 mL PCR tube.

[0080] Step 5: Digestion with methylation-sensitive restriction enzymes

[0081] 1. Take out MeEnzyme & MeBuffer and place them on ice to dissolve naturally (NEB restriction endonuclease), mix well, and centrifuge instantly for later use.

[0082] 2. Set up the reaction system according to the table below in a 0.2 mL PCR tube placed on ice, using a methylation-sensitive restriction endonuclease. The system is as follows:

[0083] 3. Start the following reaction program on the PCR instrument and place the PCR tube into the PCR when the temperature stabilizes at 37°C: 37°C for 1 hour; 80°C for 20 minutes.

[0084] Step 6: Purification of enzyme digestion products

[0085] Use 1×Beads for purification and recovery. Refer to step 4 for the purification process and elute with 20uL Nuclease-Free water.

[0086] Step 7: PCR amplification of enzyme digestion products

[0087] 1. Take out 2×HiFi PCR Master Mix and Thaw the Universal UDI-Index Primer Mix naturally on ice, mix well, and centrifuge briefly.

[0088] 2. Prepare PCR amplification mix in a 0.2 mL PCR tube placed on ice according to the following table:

[0089] 3. Place the PCR tube into the PCR instrument and start the following program:

[0090] Step 8: Purification of amplified products

[0091] Use 1×Beads for purification and recovery. The purification process is shown in step 4. Use 20μL Nuclease-Free water for elution.

[0092] Step 9: Library hybridization

[0093] 1. Prepare the hybridization reaction system according to the following table:

[0094] 2. Vortex the hybridization reaction mixture for more than 10 seconds, centrifuge it, and collect the reaction mixture at the bottom of the PCR tube (avoid generating bubbles).

[0095] 3. Place the PCR tube into the PCR instrument and start the following reaction program: 98℃ for 2 minutes; 60℃ for 1 hour.

[0096] Step 10: Library capture and elution

[0097] Magnetic bead washing

[0098] 1. Vortex the Streptavidin Beads for 15 seconds to ensure complete mixing.

[0099] 2. Take n × 25 μL Streptavidin Beads and mix and wash in a 0.2 mL centrifuge tube (n is the number of captured libraries, and n < 5)

[0100] Note: When n>5, it must be divided into multiple tubes for mixed cleaning.

[0101] 3. Place the Streptavidin Beads on a magnetic rack and let it stand for about 2 minutes. When the liquid is completely clear, discard the supernatant using a pipette.

[0102] 4. Remove the centrifuge tube from the magnetic stand, add 100 μL preheated Wash buffer A, and mix gently by pipetting for more than 10 times.

[0103] 5. Place the centrifuge tube on the magnetic stand and let it stand for about 2 minutes. When the liquid is completely clear, use a pipette to discard the supernatant.

[0104] 6. Repeat steps 4 and 5 once.

[0105] 7. Centrifuge the tube briefly and remove all Wash buffer A at the bottom of the tube with a 10 μL pipette tip. 8. Take n×8 μL uHyb#l to resuspend the Streptavidin Beads and gently pipette and mix for more than 10 times.

[0106] Magnetic bead capture

[0107] After 1.1 hr of hybridization reaction, enter the capture phase and maintain the PCR instrument running state.

[0108] 2. While keeping the PCR tube in the PCR instrument, take 8 μL of resuspended Streptavidin Beads and immediately add it to each hybridization reaction solution. Gently pipette and mix for more than 10 times.

[0109] Incubate at 60°C for 10 min.

[0110] 4. After the incubation is completed, remove the PCR tube from the PCR instrument and place it on a magnetic stand for 2 minutes. When the liquid is completely clear, use a pipette to discard the supernatant and try to remove as much of the supernatant as possible.

[0111] Elution

[0112] 1. Remove the PCR tube from the magnetic stand, add 150 μL preheated Wash Buffer A, and mix gently by pipetting for more than 10 times (avoid creating bubbles).

[0113] 2. Place the PCR tube on a magnetic rack and let it stand for 2 minutes. When the liquid is completely clear, use a pipette to discard the supernatant. Remove the PCR tube from the magnetic rack, add 100 μL of preheated Wash Buffer A, gently pipette and mix for more than 10 times, and transfer the reaction solution to a new PCR tube.

[0114] Note: Try to avoid generating bubbles in this step. Bubbles contacting the tube cap may reduce the target hit rate.

[0115] 3. Place the PCR tube into the PCR instrument and incubate at 60°C for 3 minutes.

[0116] 4. Place the PCR tube into the PCR instrument and incubate at 60°C for 3 minutes.

[0117] 5. After the incubation is completed, remove the PCR tube from the PCR instrument and place it on a magnetic rack for 2 minutes. Wait until the liquid is completely clear and discard the supernatant using a pipette.

[0118] 6. Add 150 μL of room temperature Wash Buffer B to the PCR tube and mix gently by pipetting up and down 10 times.

[0119] 7. Place the PCR tube on a magnetic stand and let it stand for 2 minutes. When the liquid is completely clear, discard the supernatant using a pipette.

[0120] 8. Centrifuge the PCR tube briefly and place it on a magnetic rack. Use a 10 μL pipette tip to remove a small amount of residual liquid, being careful not to absorb the magnetic beads.

[0121] 9. Add 22.5 μL Nuclease Free Water and gently blow for more than 10 times to resuspend the magnetic beads.

[0122] Step 11: PCR amplification of hybrid capture library

[0123] 1. Take out 2×HiFi PCR Master Mix and Thaw the Universal UDI-Index Primer Mix naturally on ice, mix well, and centrifuge briefly.

[0124] 2. Prepare PCR amplification mix in a 0.2 mL PCR tube placed on ice according to the following table:

[0125] 3. Place the PCR tube into the PCR instrument and start the following program:

[0126] Step 12: Purification of amplified products

[0127] Use 1×Beads for purification and recovery. The purification process is shown in step 4. Use 20μL Nuclease-Free water for elution.

[0128] Example 1: Testing using fully methylated and fully unmethylated samples

[0129] Different libraries were constructed using two standards, 100% methylated DNA and 0% methylated DNA (ZYMO). The library construction process is briefly described as follows: 50 ng of the standard was taken for end-repair and A addition, and adapter ligation was performed; the adapter ligation products were divided into two, one group was digested with the methylation-sensitive restriction endonuclease Hha I, and the other group was not digested as a control group. PCR amplification was performed on the two groups of products, and the index sequence was added (Figure 5A). The amplified products were hybridized and captured using the μCaler hybrid capture system. All probes used 10KB probes to cover the methylation-sensitive restriction endonuclease cleavage site. The capture probe sequences used are shown in the table below. For detailed experimental procedures, please refer to the experimental steps.

[0130] The library construction output is shown in Figures 5B and 5C. As can be seen from the library construction output, the library construction yield and enzyme digestion yield were the lowest when the 0% methylation standard endonuclease digestion was used to construct the library. Targets containing restriction endonuclease cleavage sites were not methylated and would be digested by the methylation-sensitive restriction endonuclease, resulting in a lower library construction yield than the non-digestion experimental group and the experimental group with a 100% methylation level. The on-target rate of the 0% methylation standard endonuclease digestion group was approximately 10%, while that of the other experimental groups was approximately 50%. The 0.2×Mean was approximately 35%, and the 0.5×Mean was approximately 25%. The 0.2×Mean and 0.5×Mean were approximately 95% and 80% of the other experimental groups. The depth of the 0% methylation standard endonuclease digestion group after deduplication was approximately 160, while that of the other experimental groups was approximately 1200 (Figure 6). Basic quality control before the machine showed that the use of methylation-sensitive restriction endonucleases can cut samples with a methylation level of 0, and non-methylated modification sites will be cut; the use of methylation-sensitive restriction endonucleases can cut samples with a methylation level of 100%, and methylated modification sites will not be cut.

[0131] Figure 7 shows the sequencing depth of methylation-sensitive restriction endonucleases and their corresponding methylation levels in the target regions of different libraries. Figure A refers to the sequencing depth corresponding to the CpG sites of the sample digested with 100% methylation level and the non-enzyme digested library, and the sequencing depth ranges from hundreds to thousands. Figure B refers to the sequencing depth corresponding to the CpG sites of the sample digested with 0% methylation level and the non-enzyme digested library, where the sequencing depth of the 0% methylation level non-enzyme digested library ranges from hundreds to thousands, which is consistent with the results of the sample experimental group with 100% methylation level. The sequencing depth corresponding to the CpG sites of the 0% methylation level enzyme digested library is basically 0. Methylation-sensitive restriction endonucleases can cut non-methylated modified sites, and the vast majority of sites can be cut, while methylated modified sites will not be cut.

[0132] Figure 7C shows the methylation levels of CpG sites in different samples using methylation-sensitive restriction enzymes. Calculation method: enzyme digestion depth / control depth. Methylation levels detected in samples with 0% methylation are close to 0, while methylation levels detected in samples with 100% methylation are close to 100%. The results are consistent with expectations.

[0133] Example 2: Other methylation-sensitive restriction enzymes were tested using fully methylated and fully unmethylated samples

[0134] Different libraries were constructed using two standards, 100% Methylated DNA and 0% Methylated DNA (ZYMO). The library construction process was the same as Example 1. In this embodiment, the methylation-sensitive endonuclease Aci I was selected to test the cutting efficiency of the endonuclease for standards with different methylation levels. The sequencing depth and corresponding methylation level of the methylation-sensitive endonuclease Aci I-related CpG sites in the target region of the library were calculated, as shown in Figure 8. The methylation level detected by the 0% Methyl sample was close to 0, and the methylation level detected by the 100% Methyl sample was close to 100%. The test results were in line with expectations. This hybrid capture system is applicable to different types of methylation-sensitive restriction endonucleases, and the selection of the endonuclease is mainly related to the type of restriction endonuclease contained in the target gene.

[0135] Example 3: Testing using samples with different methylation levels

[0136] 100% methylated DNA and 0% methylated DNA (ZYMO) were mixed in specific proportions to prepare methylation standards with methylation levels of 0%, 10%, 50%, and 100%. 50 ng of each standard was then subjected to end-repair and A addition, followed by adapter ligation. The adapter-ligated products were split into two groups. One group was digested with the methylation-sensitive restriction endonuclease Hha I, while the other group was not digested and served as a control group. The groupings are shown in the table below. PCR amplification was performed on each of the two groups of products, and index sequences were added.

[0137] Amplified products were captured by hybridization using the μCaler hybrid capture system (capture probes were the same as in Example 1). All probes used 10KB probes to cover the methylation-sensitive restriction endonuclease cleavage sites. Normalization was performed using an internal reference gene (probes were designed in regions without restriction endonuclease cleavage sites). The normalization probe sequences are shown in the table below.

[0138] Figure 9 shows the methylation levels of CpG sites in different samples using methylation-sensitive restriction enzymes. Calculation method: enzyme digestion depth / control depth. The methylation level detected in samples with 0% methylation is close to 0, and the methylation level detected in samples with 100% methylation is close to 100%. The methylation level detected in samples with 10% methylation is close to 10, and the methylation level detected in samples with 50% methylation is close to 50. The methylation level detected in samples with 100% methylation is close to 100%. The test results are consistent with expectations.

[0139] After enzyme digestion, samples with different methylation levels were used as controls for non-enzyme digestion, and the methylation levels of different samples were calculated, as shown in Figure 10. Methylation level calculation method: each site was normalized using an internal reference gene (by designing probes in regions without enzyme digestion sites), and the normalized depth of each CpG site in the enzyme digestion sample was / the normalized depth of each CpG site in the control group sample; the methylation level detected in the 0% Methyl sample was close to 0, and the methylation level detected in the 10% Methyl sample was close to 10%. 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 test results showed that when the internal reference gene was added for normalization and non-enzyme digestion samples with different methylation levels were used as controls, the methylation levels corresponding to different CpG sites calculated for the enzyme digestion group samples were all in line with expectations.

Claims

1. A method for detecting the methylation status of a DNA molecule 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 so that when the DNA molecule is not methylated at the recognition site of the methylation-sensitive restriction endonuclease, an enzyme-cutting reaction can be performed to obtain an enzyme-cutting sample; or treating the sample with one or more methylation-dependent restriction endonucleases so that when the DNA molecule is methylated at the recognition site of the methylation-dependent restriction endonuclease, an enzyme-cutting reaction can be performed to obtain an enzyme-cutting sample; the second sample is not treated with the restriction endonuclease; 2) contacting the digested sample with one or more capture probes, wherein the capture probes target the target sequence including the recognition site in the DNA molecule, so as to separate the DNA molecule including the recognition site that has not been digested from the digested sample; contacting the second sample with the capture probe to separate the DNA molecule including the recognition site from the second sample; as well as 3) determining the overall methylation status of the DNA molecules in the sample based on the amount of the DNA molecules including the recognition site that have not been digested, compared with the amount of the DNA molecules including the recognition site in the second sample; or, sequencing the DNA molecules including the recognition site that have not been digested and the DNA molecules including the recognition site in the second sample, and determining the methylation status of different DNA molecules in the sample by the sequencing depth ratio.

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 in an adjacent manner, adjacent capture probes can form complementary binding through the first probe binding sequence and the second probe sequence.

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

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

5. The method according to any one of claims 1 to 4, further comprising constructing a sequencing library with the DNA molecules in the sample before step 1) and, optionally, fragmenting the DNA molecules before constructing the sequencing library.

6. The method of any one of claims 1 to 5, wherein the methylation-sensitive restriction endonuclease is selected from the group consisting of 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 restriction endonuclease on which the methylation is dependent is selected from the group consisting of FspE I, LpnP I, MspJ I, and any combination thereof.

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

9. A kit for detecting the methylation status of DNA molecules in a sample, comprising: 1) one or more methylation-sensitive restriction endonucleases, wherein the methylation-sensitive restriction endonucleases can perform an enzymatic cleavage reaction on the DNA molecule when the DNA molecule is not methylated at the recognition site of the methylation-sensitive restriction endonuclease; or one or more methylation-dependent restriction endonucleases, wherein the DNA molecule can perform an enzymatic cleavage reaction when the DNA molecule is methylated at the recognition site of the methylation-dependent restriction endonuclease; and 2) One or more capture probes, 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 including the recognition site in the DNA molecule in an adjacent manner, adjacent capture probes can form complementary binding through the first probe binding sequence and the second probe sequence.

10. The kit according to claim 9, further comprising an enzyme and a linker molecule for constructing a sequencing library for the DNA molecule.

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

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

13. The method of any one of claims 9 to 12, wherein the methylation-sensitive restriction endonuclease is selected from the group consisting of 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 the group consisting of FspE I, LpnP I, MspJ I, and any combination thereof.

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

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