Methods for detecting methylation and mutation status of DNA samples

Abstract

The present disclosure provides a method for detecting the methylation and mutation status of a DNA sample, specifically, a method for simultaneously detecting the methylation and mutation status of DNA molecules in a sample, the method comprising treating the sample with a methylation-sensitive restriction enzyme, performing hybrid capture on amplification products of the restriction enzyme-treated sample and a sample that has not been subjected to enzyme cleavage treatment with a probe set including a mutation capture probe and a methylation capture probe, and sequencing the captured products.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims priority to a Chinese patent application filed with the China Patent Office on June 29, 2023, bearing application number 202310782691.4, the entire contents of which are hereby incorporated by reference.

[0002] The present invention relates to a method for simultaneously detecting methylation and mutation status, which uses a capture approach to complete the methylation and / or mutation detection of a target region. [Background technology]

[0003] According to statistics, there will be approximately 4.82 million new cancer cases and 3.21 million cancer deaths in China in 2022, making cancer a globally recognized public health issue. Early cancer screening and diagnosis are key routes to reducing cancer mortality. According to a report on multi-cancer early detection published by GrandView Research, Inc., the global multi-cancer early detection market is expected to reach $2.77 billion by 2030, growing at a compound annual growth rate of 15.80% from 2022 to 2030. The emergence of multi-cancer early detection technology is a groundbreaking advancement in early cancer screening and diagnosis. Compared with single-cancer detection, multi-cancer detection allows for the detection of multiple cancers at once, setting a new trend for the industry's future development. With the development of the early cancer screening industry and the expansion of the market, more and more companies are focusing on the field of early cancer screening, making the leap from single-cancer to multi-cancer.

[0004] Clinically, early tumor screening methods include radiology, endoscopy, and tumor marker detection. However, these detection techniques have drawbacks, such as being invasive, uncomfortable, and having low detection sensitivity. Traditional methods have various limitations, and clinically, there is a pressing need for early screening techniques with high sensitivity / specificity and ease of operation. Liquid biopsy, as a new non-invasive detection technology, has great potential for early tumor screening. Liquid biopsy tumor markers mainly include genomic, epigenetic, transcriptomic, microbiome, proteomic, and metabolomic levels. Tumor marker characteristics at the ctDNA level include point mutations, fragmentation, copy number changes, and methylation. Mutation detection is the most convenient technique for gene detection companies, but early cancer mutation signals are relatively weak and difficult to detect. Mutation signals are also susceptible to interference from clonal hematopoietic signals, leading to poor tumor specificity and difficulty in tracing tumor origin. Methylation markers are characterized by their stable presence in plasma, their large number of mutation sites, and the ability to track tissues, making them the leading candidate markers for early cancer screening. Because cancer development and progression involve changes at multiple levels, and single-level evaluation has certain limitations, "multi-omics" has become a popular technology term for early cancer screening in the field of tumor detection. The combination of mutation and methylation detection technologies complements each other's strengths and can help establish early multi-cancer screening technology.

[0005] There are many different methods for methylation sequencing. First- and second-generation sequencing technologies can only distinguish between the four different bases (ATCG) and cannot distinguish between methylated and unmethylated C bases. Traditional methods use bisulfite conversion to convert unmethylated C bases to U bases, followed by PCR amplification to convert the U bases to T and compare them with the unconverted sequence to determine whether the site is methylated. However, because the converted sequence differs from the original sequence, existing systems rely on liquid-phase hybridization capture methods, which prevent the experimental goal of detecting both mutations and methylation in the same hybridization capture system, making simultaneous methylation and mutation detection impossible. Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention provides a method for simultaneously detecting the methylation and mutational status of DNA molecules in a sample. [Means for solving the problem]

[0007] The method in this article is 1) adding adapter molecules to both ends of DNA molecules in the sample to obtain an adapter ligation product sample, and dividing the adapter ligation product sample into a first adapter ligation product sample and a second adapter ligation product sample; 2) treating the first adaptor ligation product sample with one or more methylation-sensitive restriction enzymes, so that when the DNA molecule is free of methylation at the recognition site of the methylation-sensitive restriction enzyme, an enzymatic cleavage reaction can be carried out to obtain an enzymatically cleaved sample, and the second adaptor ligation product sample is not treated with the restriction enzyme; 3) amplifying the enzyme-cleaved sample and the second adapter-ligated product sample with a primer specific to the adapter molecule and having an Index sequence to obtain an enzyme-cleaved sample amplification product and a second adapter-ligated product sample amplification product, respectively, and mixing the enzyme-cleaved sample amplification product and the second adapter-ligated product sample amplification product to obtain an amplification product mixture; 4) contacting the amplification mixture with a probe set comprising one or more mutation capture probes and one or more methylation capture probes to obtain a capture product, wherein the methylation capture probe targets a target sequence whose DNA molecule includes the recognition site, and the mutation capture probe targets a target sequence whose DNA molecule includes the mutation site; 5) sequencing the capture product, determining the methylation status of the DNA molecule containing the recognition site based on the sequencing depth ratio of the recognition site from the enzyme-cleaved sample amplification product and the second adapter ligation product sample amplification product, and determining the mutation status based on the sequencing result of the mutation site.

[0008] In some embodiments, the mutation capture probe and / or the methylation 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, such that when two or more of the mutation capture probes and / or the methylation capture probes bind to their target sequences in an adjacent manner, complementary bonds can be formed between adjacent mutation capture probes and / or the methylation capture probes via the first probe binding sequence and the second probe sequence.

[0009] In some embodiments, the probe set further comprises one or more uniform probes.

[0010] In some embodiments, the length of the first probe binding sequence and the second probe binding sequence is 8 to 30 nt.

[0011] In some embodiments, the target-specific sequence is 20 to 80 nt in length.

[0012] In some embodiments, the method further comprises fragmenting the DNA molecule prior to step 1).

[0013] In some embodiments, the methylation-sensitive restriction enzyme is selected from HpaI, HpaII, HhaI, AciI, and any combination thereof.

[0014] In some embodiments, the sample comprises ctDNA.

[0015] In some embodiments, the methylation status includes the presence or absence of methylation and / or the level of methylation.

[0016] In some embodiments, the mutation status includes the presence or absence of a mutation and / or the type of mutation.

[0017] In some embodiments, the mutation type is selected from base insertions, deletions and substitutions, chromosomal copy number variations, microsatellite instability and gene fusions. [Brief explanation of the drawings]

[0018] [Figure 1] 1 shows the capture library construction flow.

[0019] [Figure 2]The experimental flow for capture and mutation co-detection and the corresponding probe design scheme are shown below. (A) The experiment is initiated according to the experimental flow described in Figure 1. After adapter ligation, enzymatic digestion is performed using a methylation-sensitive restriction enzyme. The library constructed with the enzymatic digestion products is used for methylation status detection, and the non-enzymatically digested library is used for mutation detection. (B) The experiment is initiated according to the experimental flow described in Figure 1. After adapter ligation, enzymatic digestion is performed using a methylation-sensitive restriction enzyme. The library constructed with the enzymatic digestion products is used for methylation status and mutation detection status analysis. (C) The probe design concept is that for methylation detection, the probe must cover the methylation-sensitive restriction enzyme cleavage site. For mutation site detection, the probe must cover the target gene site. When using experimental flow B, the mutation detection probe design must balance methylation detection and the designed probe cannot cover the methylation-sensitive restriction enzyme cleavage site.

[0020] [Figure 3] The experimental flow for mutation and methylation detection is compared between the traditional detection method and the method adopted in the present invention. (A) The traditional detection method is used to perform mutation and methylation detection. This method requires the construction of two sets of libraries and the implementation of two hybridization capture experiments. However, the methylation library uses methylation-modified adapters, which are relatively expensive. The adapter ligation product is processed using a BS or EM conversion module to convert unmethylated C bases to U bases. (B) The mutation and methylation co-detection method of the present invention uses standard DNA adapters and performs enzymatic cleavage using MSRE to distinguish between methylated and unmethylated C bases. A DNA library is systematically constructed, or the adapter ligation product is divided into two, one amplified after enzymatic cleavage and the other amplified directly. The two amplified products can be hybridized in the same hybridization capture system.

[0021] [Figure 4] The experimental flow and post-run quality control results are shown. (A) Experimental flow. (B) Performance results: [On-Target] Fraction of Target Reads in mapped reads, the proportion of on-target read lengths to matched read lengths; Mappability, the match rate, the proportion of data matched to the genome. (C) 0.5 × MeanCoverage, 0.5 × mean coverage percentage; 0.2 × MeanCoverage, 0.2 × mean coverage percentage.

[0022] [Figure 5] Figure 1 shows the sequencing depth and corresponding methylation levels of methylation-sensitive endonuclease-associated CpG sites within the target regions of different libraries. Methylation levels were calculated as follows: enzyme cleavage depth / control depth. (A) Average CpG methylation levels for the two enzyme cleavage experimental groups. (B) methylation levels corresponding to each CpG site.

[0023] [Figure 6] The results of an analysis of the consistency between the mutation detection analysis in the capture data and the theoretical mutation frequency are shown. DETAILED DESCRIPTION OF THE INVENTION

[0024] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art.

[0025] The term "or" refers to a single element of a list of alternative elements, unless the context clearly dictates otherwise. The term "and / or" refers to any one, any two, any three, any more, or all of the listed alternative elements.

[0026] As used herein, the terms "comprising," "containing," "having," and similar expressions are intended to be indicative of a non-exclusion of elements. These terms also include a composition consisting only of the listed elements.

[0027] The term "DNA molecule" as used herein refers to deoxyribose nucleic acid, a polymer of deoxyribose nucleotides. It may be double-stranded or single-stranded, or, for a population of DNA molecules, may include double-stranded DNA molecules or single-stranded DNA molecules. DNA molecules may be of any length, such as genomic DNA, DNA fragments, or free extracellular DNA. Free DNA refers to DNA that is free outside of cells or cell nuclei and may be extracted from biological materials such as various body fluids, extracorporeal 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. The term "DNA fragment" as used herein refers to a relatively short DNA molecule, e.g., between 50 and 700 bp in length, e.g., between 100 and 500 bp, particularly between 100 and 350 bp. The DNA fragments contained in a biological sample are usually heterogeneous, i.e., of different lengths, and accordingly, the above lengths may refer to the average lengths of these DNA fragments. These DNA fragments may have different sequences and, for example, may originate from different regions of the genome of the same organism, or even from different organisms. The DNA fragments may have single-stranded breaks and may be blunt-ended or non-blunt-ended (with a 3' or 5' protrusion).

[0028] "DNA methylation," as used herein, refers to the methylation of bases in DNA molecules or DNA fragments, specifically the modification of cytosine to 5-methylcytosine (5mC). DNA methylation in vertebrates generally occurs at CpG sites (i.e., sites in the DNA sequence where cytosine is immediately followed by guanine) and involves the conversion of cytosine to 5-methylcytosine by DNA methyltransferase. While many CpG sites are methylated in the human genome, 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 can suppress tumor suppressor genes, while demethylation can stimulate the expression of certain oncogenes. Both of these conditions can lead to cancer development. A small number of cytosine bases are also modified to 5-hydroxymethylcytosine (5hmC), 5-formylation (5fC), and 5-carboxylation (5caC), although these occur less frequently than 5mC. When referring to methylation in this text, it can refer to modifications to 5hmC, 5fC, and 5caC, unless the context dictates otherwise.

[0029] "Cytosine base methylation status" or "DNA methylation information," as used herein, 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 methods (e.g., 5mC or 5hmC). "Methylation level," also known as "methylation degree," refers to the rate (or frequency) of methylation at a specific methylation site in a sample. Multiple methods can be used to detect whether a site is methylated. Common methods include chemical or enzymatic conversion, in which one of the methylated and unmethylated cytosines is converted to uracil (U) or a base that is essentially equivalent to uracil in base pairing (e.g., dihydrogen uracil, DHU). During the subsequent amplification process, the corresponding uracil is paired with adenine (A) as thymidine (T). The final result is that the cytosine or methylated cytosine at this methylated site appears as thymidine in the detection results (e.g., sequencing results). By comparing a DNA molecule or DNA fragment with a reference sequence, it can be determined whether a cytosine is methylated. This reference sequence can be a sequence derived from the same sample but unaltered as described above, or a corresponding sequence in a healthy population. Several methods can 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, etc.), especially early screening and diagnosis. Identification of methylation status can also be used for non-diagnostic (or therapeutic) purposes, such as scientific research, to analyze factors affecting methylation, to determine the effect of methylation on gene function, or to detect methylated products.

[0030] The term "methylation-sensitive restriction enzyme" refers to a restriction enzyme that is sensitive to the presence or absence of a methylated base in its recognition site; such an enzyme typically cannot cleave a DNA molecule at its recognition site if the recognition site contains a single methylated base. As used herein, a methylation-sensitive restriction enzyme specifically refers to a restriction enzyme that cannot cleave a DNA molecule at its recognition site if the recognition site contains at least one 5mC. Methylation-sensitive restriction enzymes known in the art include, but are not limited to, HpaI, HpaII, HhaI, and AciI.

[0031] The term "capture probe" as used herein refers to a single-stranded DNA fragment for hybridization with a target DNA molecule. In this context, capture probes include methylated capture probes and mutant capture probes. The methylated capture probe preferably targets a target sequence containing a restriction enzyme recognition site in the DNA molecule. When a DNA molecule is treated with a specific restriction enzyme, if the methylation state of the recognition site allows the restriction enzyme to cleave the DNA molecule, the capture probe can only partially or insufficiently bind to the target DNA molecule, thereby preventing effective separation of the restriction enzyme-cleaved target DNA molecule from the sample. In this case, the target DNA molecules that can be separated from the restriction enzyme-treated sample using the methylated capture probe are mainly uncleaved DNA molecules. Therefore, the overall methylation status of the DNA molecules in the sample can be determined based on the amount of separable uncleaved DNA molecules relative to the amount of separable DNA molecules in the sample not treated with the restriction enzyme. For example, when treated with a methylation-sensitive restriction enzyme, a relatively high amount of cleaved DNA molecules (a relatively low amount of uncleaved DNA molecules) indicates that the DNA molecules in the sample have a relatively low methylation level at the restriction enzyme recognition site. In a preferred embodiment, the methylation capture probe is a μCaler-format probe (for the design of the μCaler probe, see Chinese Patent Publication CN116083423A, the entire contents of which are incorporated herein by reference), which 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, such that when two or more capture probes bind to the target sequence in an adjacent manner, complementary bonds can be formed between adjacent capture probes via the first probe-binding sequence and the second probe sequence.The advantages of using μCaler-format probes over conventional capture probes include at least significantly improved probe-target binding ability, improved hybridization capture efficiency of target regions, and improved overall coverage uniformity and stability. Capture probes typically have a detachable tag, such as biotin, which facilitates the separation of probe-bound target DNA molecules. Separated target DNA molecules can be sequenced to obtain their sequence information and simultaneously determine the methylation status at the restriction enzyme recognition site. A mutation capture probe targets a mutation site of interest in a DNA molecule, and sequencing the DNA molecule captured by the mutation capture probe can provide information on the mutation status of the specific mutation site. Preferably, the mutation capture probe is also a μCaler-format probe. The methylation capture probe and mutation capture probe may be located in the same probe set, enabling simultaneous capture of each target sequence. Furthermore, to achieve uniformity in detection results across different samples, the probe set used herein may further include a uniformization probe. Preferably, this uniformization probe is also a μCaler-format probe.

[0032] In some embodiments, the present disclosure provides a process for constructing a target methylation library based on methylation-sensitive restriction enzymes, including HpaI, HpaII, HhaI, AciI, etc.

[0033] In some embodiments, the target capture system described herein is designed for unconverted library capture, capturing the original DNA sequence, and detecting the methylation modification information in the target region by methylating the CpG sites in the restriction enzyme recognition sites of the captured fragment target region.

[0034] In some embodiments, the methods described herein are used to detect mutations in DNA samples, including mutation types such as base substitutions, chromosome copy number mutation analysis, insertions / deletions, microsatellite instability, or gene fusions.

[0035] In some embodiments, the capture probes used are designed against the original DNA strand to detect methylation-associated sites, and the target capture probes must cover the corresponding methylation-sensitive restriction enzyme cleavage sites.

[0036] In some implementations, two different libraries are used for mutation and methylation co-detection, respectively, and the design of the mutation capture probe does not need to be traded off with the design of the methylation capture probe; in some implementations, the same library is used for mutation and methylation co-detection, and the mutation capture probe cannot cover the methylation-sensitive restriction enzyme recognition sequence relevant to methylation detection.

[0037] In some implementations, this document provides a process for constructing a targeted methylation library based on methylation-sensitive restriction enzymes, which can be used to detect the sequencing depth of CpG sites and use controls to analyze the methylation level of sample CgG sites.

[0038] In some embodiments, the methods herein employ adapter modules containing molecular tags, which are applied to filter background noise and detect low frequency mutation signals.

[0039] The target capture library construction flow in this paper is shown in Figure 1. This flow is applied to the detection of methylation status and mutation signals of target regions in gDNA samples, plasma-free DNA, or circulating tumor ctDNA samples after physical disruption. Libraries were constructed from fragmented samples using the Nadprep Universal DNA Library Construction Kit. The library construction flow is divided into two methods: Workflow A: End repair, addition of A, and adapter ligation; the adapter ligation product was enzymatically digested using methylation-sensitive restriction enzymes, including HpaI, HpaII, HhaI, and AciI. The other group served as a control group without enzymatic digestion. PCR amplification was performed on the two sets of products, and index sequences were added. The amplification products were hybridized using the μCaler hybrid capture system. All probes were designed using the μCaler probe design method to match the original DNA sequence. The probes covered methylation-sensitive restriction enzyme cleavage sites, which were used to detect methylation status, and mutation detection sites, which were used to detect mutation signals. Workflow B: Enzyme cleavage was performed using methylation-sensitive restriction enzymes, including HpaI, HpaII, HhaI, and AciI. The cleavage products were purified and recovered, and then libraries were constructed. The library construction products were hybridized using the μCaler hybrid capture system. All probes were designed using the μCaler probe design method to match the original DNA sequence. The probes covered methylation-sensitive restriction enzyme cleavage sites, which were used to detect methylation status, and mutation detection sites, which were used to detect mutation signals. The captured libraries were then sequenced on an Illumina or MGI platform sequencer.

[0040] The experimental flow for capture and mutation co-detection and the corresponding experimental design are shown in Figure 2. Step A: Start the experiment according to the experimental flow described in Figure 1. After adapter ligation, enzymatic digestion is performed using a methylation-sensitive restriction enzyme. The library constructed with the enzymatic digestion products is used for methylation status detection, and the non-enzymatically digested library is used for mutation detection. Step B: Start the experiment according to the experimental flow described in Figure 1. After adapter ligation, enzymatic digestion is performed using a methylation-sensitive restriction enzyme. The library constructed with the enzymatic digestion products is used for methylation status and mutation detection status analysis. Step C: Design the probe for this system. For methylation detection, the probe must cover the methylation-sensitive restriction enzyme cleavage site. For mutation site detection, the probe must cover the target gene site. When using step B, the mutation detection probe design must be balanced with methylation detection; the designed probe cannot cover the methylation-sensitive restriction enzyme cleavage site.

[0041] The use of this system for co-detection of mutations and methylation has clear advantages over traditional methods. See Figure 3 for details. Figure 3 compares the experimental flow for mutation and methylation detection using traditional detection methods and the method used in this system. The traditional detection method (Figure 3A) for mutation and methylation detection requires the construction of two sets of libraries and two sets of hybridization capture experiments. The methylation library uses methylated adapters, which are relatively expensive. The adapter ligation products are processed using a BS (2-hour experiment) or EM (8-hour experiment) conversion module. The BS conversion process is relatively cumbersome and the conversion damage is relatively severe. This method results in the loss of a large amount of original sample information. Because the conversion damage of the NEB conversion module is relatively low, we recommend using the NEB Next Enzymatic Conversion module to convert unmethylated C bases to U bases and then PCR amplify them using an amplification enzyme capable of recognizing U bases (KAPA HiFi HotStart Uracil Mix). For mutation detection, we use a standard adapter module. Given the altered DNA sequence of the converted library, the probes used in hybridization capture should be designed to target the converted DNA sequence. Methylated and unmethylated libraries should be run in separate hybridization capture systems. In contrast, our system's mutation and methylation co-detection method (Figure 3B) uses a standard DNA adapter, MSRE, for enzymatic cleavage (1 h), which distinguishes between methylated and unmethylated C bases. A single DNA library can be systematically constructed, or the adapter ligation product can be split into two parts: one amplified after enzymatic cleavage and the other directly amplified. The two amplified products can then be hybridized in the same hybridization capture system. Using traditional methods to detect mutations and methylation may take 2-3 days, but using the method of this system can shorten the experiment time to 1 day. This system significantly reduces experiment time, simplifies the experiment process, and saves reagent costs, and its advantages are obvious.

[0042] Methylation level analysis method: For the enzyme-digested samples, non-enzyme-digested samples with different methylation levels were used as controls to calculate the methylation levels of different samples. Methylation level calculation method: Each site was normalized using an endogenous control gene, i.e., the normalization depth for each CpG site in the enzyme-digested sample / the normalization depth for each CpG site in the control group sample.

[0043] Mutation detection analysis method: Variant calling was performed using Vardict or GATK software. When analyzing with Vardict software, Hg19 was used as a bed file and the coordinates of hg19 were displayed. When analyzing with GATK software, a control sample was used for variant calling.

[0044] This paper aims to develop a technical solution that can complete the simultaneous detection of mutations and methylation using the same hybridization capture system. Compared with conventional detection solutions, this solution shortens the detection time, simplifies the experimental procedure, increases sample utilization, and reduces reagent costs, and has broad market application prospects.

[0045] Common experimental steps:

[0046] [Step 1: Fragmentation of the sample] DNA samples can be fragmented according to laboratory conditions. We recommend using a Covaris™ series DNA sonicator to fragment samples to an average fragment length of 200-250 bp. gDNA or FFPE DNA samples proceeded to Step 2 after a break. cfDNA samples proceeded directly to Step 2.

[0047] [Step 2: Adding end repair & A] 1. Remove the End Repair & A-Tailing Buffer, thaw it at room temperature, mix it evenly, and place it on ice. 2. The EndRepair&A-Tailing Enzyme was taken out and placed on ice to melt naturally, then mixed uniformly and immediately centrifuged. 3. Prepare reactions in 0.2 mL PCR tubes on ice according to the following table: [Table 1] NOTE: If the DNA is less than 20 µL, it can be made up to 20 µL with Nuclease Free Water. 4. Mix thoroughly and immediately centrifuge to deposit all the reaction mixture at the bottom of the PCR tube. 5. Start the following reaction program (Cycling Program I) on the PCR machine, and once the temperature stabilized at 20°C, place the PCR tube in the PCR machine. [Table 2] Note: No thermal cap is required when running a 20°C program, and the thermal cap is 70°C when running a 65°C program.

[0048] [Step 3: Adapter ligation] 1. Remove the Ligation Buffer, thaw it at room temperature, mix it evenly, and place it on ice. CAUTION: Ligation Buffer is very viscous, and pipetting and dispensing must be done slowly and gently to ensure accurate volume. 2. The DNA ligase was removed and placed on ice to allow it to melt naturally, then mixed uniformly and immediately centrifuged. 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: [Table 3]

[0049] Step 4: Purification of the ligation product Purification and recovery were performed using 0.5x beads, and the purification flow was as follows: 1. 20 μL of NadPrep® SP Beads was added to the ligation reaction product from step 3, mixed evenly, and incubated at 20-25°C for 10 minutes. 2. The PCR tube was immediately centrifuged and then placed on a magnetic holder for 5 minutes until the liquid was completely clear. The supernatant was then aspirated using a pipette and discarded. Note: It must be thoroughly clarified, and different brands of magnetic holders require adjusting the standing time. 3. Slowly add 150 μL of 80% ethanol along the side of the PCR tube, being careful not to disturb the magnetic beads, let stand for 30 seconds, and then aspirate the supernatant using a pipette and discard. 4. Step 3 was repeated once. 5. After immediate centrifugation, the PCR tube was placed on the magnetic holder and a small amount of residual ethanol was removed using a 10 μL suction head, taking care not to aspirate the magnetic beads. 6. Open the lid of the PCR tube and let it stand at 20-25°C for about 2-3 minutes until the ethanol had completely evaporated. 7. The PCR tube was removed, 44 μL of Nuclease-Free Water was added to the PCR tube, the magnetic beads were uniformly suspended using a pipette, and the mixture was incubated at 25°C for 2 minutes. 8. The PCR tube was immediately centrifuged and then placed on a magnetic holder for 2 minutes until the liquid was completely clear. The supernatant was then carefully transferred to a new 0.2 mL PCR tube using a pipette.

[0050] [Step 5: Enzymatic digestion with methylation-sensitive restriction enzymes] 1. Remove the MeEnzyme & MeBuffer, place it on ice to allow natural lysis (NEB restriction enzyme), mix evenly, and immediately centrifuge. 2. Arrange the reaction in a 0.2 mL PCR tube on ice according to the following table, using a methylation-sensitive restriction enzyme. The format is as follows: [Table 4] 3. The following reaction program was started on the PCR machine, and once the temperature had stabilized to 37°C, the PCR tube was placed in the PCR: 37°C for 1 hour, 80°C for 20 minutes.

[0051] [Step 6: Purification of enzymatic cleavage products] Purification and recovery were performed using 1x Beads, and the purification flow was as described in step 4, with elution using 20 μL of nuclease-free water.

[0052] [Step 7: PCR amplification of enzyme cleavage products] 1. Remove the 2x HiFi PCR Master Mix and NadPrep® Universal UDI-Index Primer Mix, place them on ice to thaw, mix thoroughly, and immediately centrifuge. 2. Prepare PCR Amplification Mix in a 0.2 mL PCR tube on ice according to the following table: [Table 5] 3. Place the PCR tube in the PCR machine and run the following program: [Table 6]

[0053] Step 8: Purification of the amplification product Purification and recovery were performed using 1x Beads, and the purification flow was as described in step 4, with elution using 20 μL of nuclease-free water.

[0054] Step 9: Library Hybridization 1. The hybridization reaction system was prepared according to the following table: [Table 7] 2. The hybridization reaction mixture was vortexed for at least 10 seconds to mix evenly, and then immediately centrifuged to collect the reaction mixture at the bottom of the PCR tube (avoiding the generation of air bubbles). 3. The PCR tube was placed in a PCR machine and the following reaction program was initiated: 98°C for 2 minutes, 60°C for 1 hour.

[0055] [Step 10: Library capture and elution] Washing magnetic beads 1. Streptavidin Beads were vortexed for 15 seconds to ensure thorough mixing. 2. n x 25 μL of Streptavidin Beads were collected, mixed, and washed in a 0.2 mL centrifuge tube (n is the number of captured libraries, and n<5). Note: If n>5, washing must be done in multiple tubes. 3. The StreptavidinBeads were placed on a magnetic holder and left to stand for approximately 2 minutes. After the liquid had completely clarified, the supernatant was discarded using a pipette. 4. Remove the centrifuge tube from the magnetic holder, add 100 μL of preheated Wash buffer A, and mix thoroughly by gently blowing and sucking at least 10 times. 5. The centrifuge tube was placed on a magnetic holder and left to stand for about 2 minutes. After the liquid had completely clarified, the supernatant was discarded using a pipette. 6. Steps 4 and 5 were repeated once. 7. The tube was immediately centrifuged and all of the Wash buffer A at the bottom of the tube was discarded using a 10 μL aspiration head. 8. Take n x 8 μL of μHyb#1 and resuspend the Streptavidin Beads. Wash the beads by gently blowing them over 10 times to mix evenly.

[0056] Magnetic bead capture 1. After 1 hour of hybridization, the PCR machine was left running for the capture step. 2. While keeping the PCR tube in the PCR machine, immediately add 8 μL of resuspended Streptavidin Beads to each hybridization reaction mixture and mix thoroughly by gently blowing or sucking at least 10 times. 3. Incubate at 60°C for 10 minutes. 4. After incubation, remove the PCR tube from the PCR device and place it on a magnetic holder for 2 minutes. After the liquid had completely cleared, discard the supernatant using a pipette (remove as much of the supernatant as possible).

[0057] Elution 1. Remove the PCR tube from the magnetic holder and add 150 μL of preheated Wash Buffer A. Gently blow in and out at least 10 times to mix thoroughly (avoid creating bubbles). 2. The PCR tube was placed on a magnetic holder and left to stand for 2 minutes. After the liquid had completely cleared, the supernatant was discarded using a pipette. 3. The PCR tube was removed from the magnetic holder, 100 μL of preheated Wash Buffer A was added, and the mixture was mixed evenly by gently blowing and sucking at least 10 times. The reaction mixture was then transferred to a new PCR tube. CAUTION: In this step, avoid the generation of air bubbles as much as possible, as air bubbles may come into contact with the tube cover, causing a decrease in the hit rate. 3. The PCR tube was placed in a PCR machine and incubated at 60°C for 3 minutes. 4. The PCR tube was placed in a PCR machine and incubated at 60°C for 3 minutes. 5. After incubation, the PCR tube was removed from the PCR device and placed on a magnetic holder for 2 minutes. After the liquid had completely cleared, the supernatant was discarded using a pipette. 6. Add 150 μL of Wash Buffer B, which had been left at room temperature, to the PCR tube and gently blow in and out 10 times to mix evenly. 7. The PCR tube was placed on a magnetic holder and left to stand for 2 minutes. After the liquid had completely cleared, the supernatant was discarded using a pipette. 8. After instant centrifugation, the PCR tube was placed in the magnetic holder and a 10 μL aspiration head was used to remove a small amount of residual liquid, taking care not to aspirate the magnetic beads. 9. Add 22.5 μL of Nuclease-Free Water and resuspend the magnetic beads by gently blowing and washing at least 10 times.

[0058] Step 11: PCR amplification of the hybridization capture library 1. Remove the 2x HiFi PCR Master Mix and NadPrep® Universal UDI-Index Primer Mix, place them on ice to thaw, mix thoroughly, and immediately centrifuge. 2. Prepare PCR Amplification Mix in a 0.2 mL PCR tube on ice according to the following table: [Table 8] 3. Place the PCR tube in the PCR machine and run the following program: [Table 9]

[0059] Step 12: Purification of the amplification product Purification and recovery were performed using 1x Beads, and the purification flow was as described in step 4, with elution using 20 μL of nuclease-free water.

[0060] [Example: Co-detection of mutations and methylation using standard products] Two libraries were constructed using the OGTM800 standard. The library construction process is briefly described below: After sonication, 40 ng of the standard was collected and subjected to end repair, A addition, and adapter ligation. The adapter ligation products were divided into two groups: one group was digested with the methylation-sensitive restriction enzyme HhaI, and the other group was used as a control without enzyme digestion. PCR amplification was performed on each of the two sets of products, and index sequences were added, as shown in Figure 4A. The amplified products were hybridized using the μCaler hybrid capture system and simultaneously captured using three panels: the 10K Me Panel, the Normalized Panel, and the OGTM800 Panel. Here, the 10K probe covers the methylation-sensitive restriction enzyme digestion site, the Normalized Panel is used for normalization, and the OGTM800 Panel is used for mutation detection analysis. Specific experimental steps are described in the experimental flow. The probe design scheme is based on the Chinese patent disclosure "CN116083423A Probe for Enriching Nucleic Acid Targets."

[0061] The MePanel probe sequences are as follows: [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4]

[0062] The normalized panel probe sequences are as follows: [Table 11-1] [Table 11-2] Table 11-3

[0063] Results analysis: The OGTM-CK experimental group served as a control; no enzymatic digestion was performed using a methylation-sensitive restriction enzyme. Samples from this group may be used for mutation detection analysis. The OGTM-MSRE group was the enzymatic digestion group, which performed enzymatic digestion using a methylation-sensitive restriction enzyme. The OGTM800 Panel designed in this flow does not involve methylation-sensitive restriction enzyme cleavage sites. Samples from this group may be used for methylation and mutation detection analysis. Figure 4B shows the per-target rate and mappability, with both mappability values ​​greater than 99%. Figure 4C shows the coverage depth of different experimental groups, where 0.2×Mean Coverage > 95% and 0.5×Mean Coverage > 70%. Figure 5 shows the sequencing depth and corresponding methylation levels of methylation-sensitive endonuclease-associated CpG sites within different library target regions. Methylation level calculation method: enzyme digestion depth / control depth. (A) Average CpG methylation level in the two enzyme digestion experimental groups. (B) Methylation level corresponding to each CpG site. Figure 6 shows the consistency analysis of the detected mutation frequency with the theoretical mutation frequency. The designed probes covered 15 mutation sites, including 11 mutation sites, 2 fusion sites, and 2 copy number variations (base insertion and deletion), which are common mutation detection sites in cancer. The corresponding mutation frequencies were unequal, ranging from 1% to 7%. The OGTM-CK experimental group did not undergo enzyme digestion, while the OGTM-MSRE experimental group underwent enzyme digestion using a methylation-sensitive restriction enzyme. Since none of the sites covered by the mutation panel selected in this study contained endonuclease recognition sites, the detection results of both the OGTM-CK and OGTM-MSRE groups can reflect the true mutation level of the sample. The consistency analysis of the detected mutation frequency with the theoretical mutation frequency showed that the results detected in the different experimental groups met theoretical expectations. OGTM-CK shows the mutation detection status of the control group (Figure 2A). This experimental design is recommended when the mutation detection site contains a methylation-sensitive restriction enzyme cleavage site.OGTM-MSRE shows the mutation detection results shown in Figure 2B. We recommend using this experimental design when the mutation detection site does not involve a methylation-sensitive restriction enzyme cleavage site.

Claims

1. 1. A method for simultaneously detecting the methylation status and mutation status of DNA molecules in a sample, comprising: 1) adding adapter molecules to both ends of DNA molecules in the sample to obtain an adapter ligation product sample, and dividing the adapter ligation product sample into a first adapter ligation product sample and a second adapter ligation product sample; 2) treating the first adaptor ligation product sample with one or more methylation-sensitive restriction enzymes, so that when the DNA molecule is free of methylation at the recognition site of the methylation-sensitive restriction enzyme, an enzymatic cleavage reaction can be carried out to obtain an enzymatically cleaved sample, and the second adaptor ligation product sample is not treated with the restriction enzyme; 3) amplifying the enzyme-cleaved sample and the second adaptor-ligated product sample with a primer specific to the adaptor molecule and having an index sequence to obtain an enzyme-cleaved sample amplification product and a second adaptor-ligated product sample amplification product, respectively; and mixing the enzyme-cleaved sample amplification product and the second adaptor-ligated product sample amplification product to obtain an amplification product mixture; 4) contacting the amplification mixture with a probe set comprising one or more mutation capture probes and one or more methylation capture probes to obtain a capture product, wherein the methylation capture probe targets a target sequence in which the DNA molecule includes the recognition site, and the mutation capture probe targets a target sequence in which the DNA molecule includes the mutation site; 5) A method for simultaneously detecting the methylation state and mutation state of a DNA molecule in a sample, comprising: sequencing the capture; determining the methylation state of the DNA molecule containing the recognition site based on the sequencing depth ratio of the recognition site from the enzyme-cleaved sample amplification product and the second adapter ligation product sample amplification product; and determining the mutation state based on the sequencing result of the mutation site.

2. 2. The method of claim 1, wherein the mutation capture probe and / or the methylation 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, such that when two or more of the mutation capture probes and / or the methylation capture probes bind to their target sequence in an adjacent manner, complementary bonds can be formed between adjacent mutation capture probes and / or the methylation capture probes via the first probe binding sequence and the second probe sequence.

3. The method of claim 1 or 2, wherein the probe set further comprises one or more uniformization probes.

4. The method according to any one of claims 1 to 3, wherein the length of the first probe binding sequence and the second probe binding sequence is 8 nt to 30 nt.

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

6. The method of any one of claims 1 to 5, further comprising fragmenting the DNA molecule prior to step 1).

7. The method according to any one of claims 1 to 6, wherein the methylation-sensitive restriction enzyme is selected from HpaI, HpaII, HhaI, AciI and any combination thereof.

8. The method of any one of claims 1 to 7, wherein the sample comprises ctDNA.

9. The method according to any one of claims 1 to 8, wherein the methylation status comprises the presence or absence of methylation and / or the methylation level.

10. The method of any one of claims 1 to 9, wherein the mutation status comprises the presence or absence of a mutation and / or the mutation type.

11. The method according to any one of claims 1 to 10, wherein the type of mutation is selected from base insertions, deletions and substitutions, chromosomal copy number variations, microsatellite instability and fusion genes.

Images