Multi-omics detection method for plasma cfdna and use thereof

Abstract

Provided are a multi-omics detection method for plasma cfDNA and the related use thereof. The method comprises: directly adding a methyltransferase, an enzyme buffer reagent, a protease inhibitor and EDTA to plasma of a sample to be tested, and labeling an open region of plasma cfDNA of said sample by using the methyltransferase; and extracting the cfDNA with the labeled open region, and performing end repair, A-tailing, methylated adapter ligation, and conversion on the extracted cfDNA, followed by library construction and sequencing; or, extracting the cfDNA with the labeled open region, and performing library construction and sequencing on the extracted cfDNA. By means of the provided detection method, multiple types of information of all cfDNA molecules can be obtained at once, comprising three-dimensional combination information of fragment lengths (long fragments and short fragments), information of nucleosome positions and of endogenous methylation thereon.

Description

A multi-omics detection method for plasma cfDNA and its application Technical Field

[0001] This invention relates to the field of cfDNA analysis, specifically to a multi-omics detection method for plasma cfDNA and its related applications. Background Technology

[0002] cfDNA, or circulating free DNA, refers to the partially degraded endogenous DNA circulating in blood and existing outside of cells. In plasma, cfDNA mainly exists in the form of nucleosomes (cf nucleosomes). Nucleosomes are the basic building blocks of chromosomes, formed by DNA encapsulating histone octamers. Beyond the DNA sequence information, the combination of different chemical modifications of histones endows nucleosomes with tissue-specific epigenetic information and provides insights into the state of gene expression and regulation within cells. As is generally believed, cfDNA nucleosomes retain some epigenetic information, making them highly valuable for research. In clinical applications, blood cfDNA has significant potential value in early disease diagnosis, prognosis, and monitoring. For example, non-invasive detection technologies are increasingly prevalent in early pregnancy screening and early cancer screening, and cfDNA technology is one such technology. It can accurately detect fetal chromosomes and gene mutations by analyzing cell-free DNA fragments in the blood, determining tumor staging—a non-invasive detection method. However, current technologies for cfDNA detection still have some limitations.

[0003] In the field of short-read sequencing, low-depth whole-genome sequencing based on cfDNA fragmentation detection methods can only obtain sequence and quantity information of short cfDNA fragments. Detection methods based on cfDNA mutations and methylation require extremely high sequencing depths to improve sensitivity and are prone to false positives. Currently, short-read cfDNA detection technologies only provide one-dimensional information, and the limited amount of information provided makes it difficult to provide effective standards for research and related disease diagnosis. NGS technology typically has a total sequencing length of ≤200 bp and only obtains base sequence information. Therefore, for larger cfDNA molecules, mid-segment sequence information is missing, and related epigenetic information is also lost, especially information about the nucleosome location of cfDNA (i.e., the location within cfDNA covered by nucleosomes), which has not yet been reported.

[0004] The gradual maturation of long-read high-throughput sequencing technology has compensated for the aforementioned shortcomings of NGS, and the characteristics of long cfDNA at the kb level have been revealed. In recent years, with the development of long-read sequencing technology, sequencing of cfDNA on the PacBio and ONT platforms has solved the sequence information and methylation information of long cfDNA. However, current methods, because they directly sequence cfDNA extracted from plasma without PCR amplification, require a very large amount of plasma, at least 10 ml, which is also expensive.

[0005] Furthermore, current information regarding the nucleosome location on cfDNA is all indirectly inferred through bioinformatics methods based on breakpoint information in cfDNA. There are currently no direct reports of nucleosome location information; even fewer reports on the combined information of nucleosome location and methylation, yet nucleosome location information profoundly influences gene regulation. Therefore, there is currently no technology that can obtain multi-dimensional information about a single cfDNA molecule from a single detection. Summary of the Invention

[0006] To address the aforementioned issues, this invention provides a multi-omics detection method for plasma cfDNA. By directly labeling cfDNA in plasma, multiple information about the cfDNA molecule can be obtained in one go through subsequent experiments. This includes sequence information of all fragment lengths (long and short fragments), as well as three-dimensional combined information of nucleosome position information and endogenous methylation information.

[0007] In a first aspect, the present invention provides a multi-omics detection method for plasma cfDNA, as shown in Figure 1, comprising: adding methyltransferase, enzyme buffer reagent, protease inhibitor and EDTA to the plasma sample to be tested; using methyltransferase to label the open region (i.e. the region without nucleosomes) of cfDNA in the sample to be tested; methylating specific bases in the open region of cfDNA that is not wrapped by nucleosomes; extracting the cfDNA labeled with the open region, performing end repair, adding A-methylated adapters, and transforming and then constructing a library for sequencing; or extracting the cfDNA labeled with the open region and constructing a library for sequencing.

[0008] Preferably, in the above detection method, PCR amplification and library reconstruction sequencing can be performed after transformation.

[0009] In a specific embodiment of the present invention, the specific base methylation in the above detection method specifically includes: adding methyltransferase, enzyme buffer, protease inhibitor, and EDTA to the plasma sample to be tested; and using methyltransferase to label the open regions of cfDNA in the sample to be tested under conditions of 25℃-37℃ and / or replacing the acid-base buffer in the enzyme buffer, thereby methylating specific bases in the open regions of cfDNA that are not encapsulated by nucleosomes. Further, the above temperature conditions are preferably 25℃-33℃. The replacement of the acid-base buffer in the enzyme buffer refers to replacing Tris-HCl with any one of HEPS-KOH, Tris-acetate, Glycine-KOH, Potassium Acetate, Bis-Tris-Propane-HCl, or Sodium Phosphate.

[0010] In certain specific embodiments of the present invention, the above detection method specifically includes: adding methyltransferase, enzyme buffer, protease inhibitor, and EDTA to the plasma sample to be tested; labeling the open regions (i.e., regions without nucleosomes) of cfDNA in the sample to be tested using methyltransferase at 25℃-37℃ and / or replacing the acid-base buffer in the enzyme buffer; and methylating specific bases in the open regions of cfDNA not encapsulated by nucleosomes. Extracting the cfDNA with the labeled open regions, and performing end repair by adding an A-methylated adapter. Converting the unmethylated C in the adapter-added cfDNA to U, followed by PCR amplification, sequencing library construction, and single-molecule sequencing. The methyltransferases mentioned above include, but are not limited to, CviPI methyltransferase (GC), AluI methyltransferase (AGCT), Hhal methyltransferase (GCGC), HaeIII methyltransferase (GGCC), or MspI methyltransferase (CCGG).

[0011] In certain specific embodiments of the present invention, the above detection method specifically includes: adding an m6A-labeled methyltransferase, an enzyme buffer, a protease inhibitor, and EDTA to the plasma sample to be tested; labeling the open regions (i.e., regions without nucleosomes) of cfDNA in the sample to be tested using the methyltransferase at 25℃-37℃ and / or by replacing the acid-base buffer in the enzyme buffer; and methylating specific bases in the open regions of cfDNA not encapsulated by nucleosomes. The cfDNA labeled with the open regions is then extracted, and a sequencing library is constructed and single-molecule sequencing is performed. The m6A-labeled methyltransferase includes, but is not limited to, EcoGII methyltransferase.

[0012] Secondly, similar to the first aspect above, the present invention provides a method for labeling open regions of plasma cfDNA and its application in cfDNA detection. The method includes: adding a specific methyltransferase, an enzyme buffer reagent, a protease inhibitor, and EDTA to the plasma sample to be tested; using the methyltransferase to label open regions of cfDNA in the sample to be tested, thereby methylating specific bases in the open regions of cfDNA that are not encapsulated by nucleosomes.

[0013] In certain embodiments of the present invention, the labeling method includes: adding methyltransferase, enzyme buffer, protease inhibitor, and EDTA to the plasma sample to be tested; and using methyltransferase to label the open regions of cfDNA in the sample to be tested under conditions of 25°C-37°C and / or replacing the acid-base buffer in the enzyme buffer, thereby methylating specific bases in the open regions of cfDNA that are not encapsulated by nucleosomes. Further, the above temperature conditions are preferably 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, or 37°C, and more preferably 25°C-33°C. The replacement of the acid-base buffer in the enzyme buffer refers to replacing Tris-HCl with any one of HEPS-KOH, Tris-acetate, Glycine-KOH, Potassium Acetate, Bis-Tris-Propane-HCl, or Sodium Phosphate. The aforementioned methyltransferases include, but are not limited to, CviPI methyltransferase (GC), EcoGII methyltransferase, AluI methyltransferase (AGCT), Hhal methyltransferase (GCGC), HaeIII methyltransferase (GGCC), or MspI methyltransferase (CCGG).

[0014] Thirdly, the present invention provides the application of the detection method of the first aspect and the labeling method of the second aspect in the detection and diagnosis of multiple cancers in plasma cfDNA, cancer tracing, pregnancy diseases, etc.

[0015] Fourthly, the present invention provides a kit for labeling plasma cfDNA using the labeling method of the second aspect, the kit comprising a methyltransferase, an enzyme buffer, a protease inhibitor, and EDTA. Further, the methyltransferase in the kit comprises any one of CviPI methyltransferase, EcoGII methyltransferase, AluI methyltransferase, Hhal methyltransferase, HaeIII methyltransferase, or MspI methyltransferase. The acid-base buffer in the enzyme buffer is any one of HEPS-KOH, Tris-acetate, Glycine-KOH, Potassium Acetate, Bis-Tris-Propane-HCl, or Sodium Phosphate.

[0016] Fifthly, the present invention provides a kit for detecting plasma cfDNA using the detection method of the first aspect, the kit comprising a methyltransferase, an enzyme buffer, a protease inhibitor, and EDTA. Further, the methyltransferase in the kit comprises any one of CviPI methyltransferase, EcoGII methyltransferase, AluI methyltransferase, Hhal methyltransferase, HaeIII methyltransferase, or MspI methyltransferase. The acid-base buffer in the enzyme buffer is any one of HEPS-KOH, Tris-acetate, Glycine-KOH, Potassium Acetate, Bis-Tris-Propane-HCl, or Sodium Phosphate.

[0017] The plasma cfDNA multi-omics detection method provided by this invention enables direct labeling of open regions of cfDNA in plasma, thereby preserving its nucleosome location information. This allows for subsequent transformation using a methylation method that causes less DNA damage. Through post-labeling detection, this invention can simultaneously obtain multiple molecular information for all cfDNA molecules, including all fragment lengths (long and short fragments), and a three-dimensional combination of nucleosome location information and endogenous methylation information. This provides a combination of multiple information for subsequent research, such as cancer tracing, overcoming the limitation of incomplete information dimensions in long-read cfDNA sequencing technology. Attached Figure Description

[0018] Figure 1 is a schematic diagram of the steps of the plasma cfDNA multi-omics detection method of the present invention;

[0019] Figure 2 is a distribution diagram of sequencing fragment length and abundance in an embodiment of the present invention;

[0020] Figure 3 is a diagram showing the multi-omics information of a specific sequencing fragment in an embodiment of the present invention. Detailed Implementation

[0021] Current labeling methods for methyltransferases are primarily used in cell samples, involving two labeling processes performed by preparing cell nuclei. In cell samples, nucleic acids retain information about the openness of chromatin. By fixing cells with formaldehyde or similar agents, the cells act as reaction vessels for enzymatic reactions, thus preserving the openness information of chromatin and the location information of nucleosomes. Simultaneously, fixation inactivates various intracellular enzymes, which helps prevent interference with the labeling reaction. In recent years, the development of single-molecule sequencing technology has sparked interest in longer cell-free DNA (cfDNA) in plasma. However, current research on the nucleosome location of plasma cfDNA mainly focuses on inference through data analysis, lacking a direct experimental method for visual detection by labeling the open regions of cfDNA. The main challenge lies in the fact that plasma is a fluid medium, unlike cells. Formaldehyde fixation of cells cannot act as a reaction vessel to immobilize the cfDNA, which retains nucleosome location information, for enzymatic reactions. While cell-like fixation of plasma can preserve nucleosomes, it still prevents the reaction from occurring. Furthermore, extracting the intact nucleosome structure from a single cfDNA molecule presents challenges, as conventional cfDNA extraction often disrupts the nucleosome structure. Therefore, nucleosome location labeling must be performed before cfDNA extraction, requiring direct labeling in plasma. However, plasma composition is highly complex, and current technology lacks a suitable processing method. Consequently, current information on the nucleosome location of plasma cfDNA is primarily estimated using bioinformatics analysis software based on sequencing results.

[0022] This invention, through detailed research on plasma cfDNA, proposes a method for directly detecting cfDNA nucleosome location information by directly labeling open regions of cfDNA in the plasma sample. Through extensive experiments, this invention provides a specific labeling method. The principle of this method is to use a specific methyltransferase to label open regions of cfDNA in plasma. These open regions refer to the DNA regions of cfDNA that are not wrapped around nucleosomes and are not protein-bound. The labeling involves the methylation of a specific base in this open region (methylation refers to the chemical modification process of transferring an active methyl group to a specific base in the open region based on a methyl donor, catalyzed by a methyltransferase). It should be understood that this specific base is determined according to the specific methyltransferase used.

[0023] It should be noted that, in this invention, the specific methyltransferase refers to the choice of methyltransferase tailored to the analyte. Generally, methylation sites differ between species; for example, when used for human plasma testing, a GC-labeled methyltransferase can be selected, but a CG-labeled methyltransferase cannot. Furthermore, GC labeling is a dibase-labeled enzyme, offering higher recognition accuracy; therefore, in this invention, the preferred methyltransferase is CviPI methyltransferase (GC).

[0024] Experiments conducted according to this invention have revealed that, in carrying out the enzymatic reaction of the aforementioned methyltransferase in plasma, it is necessary to add a protease inhibitor to prevent proteases in the blood from degrading the methyltransferase; simultaneously, EDTA is needed to protect DNA from degradation by DNases. However, in actual operation, plasma undergoes a coagulation reaction under the conditions of the enzymatic reaction, which directly affects the progress of the enzymatic reaction. Through verification using various methods, we found that adjusting the reaction temperature and / or replacing the acid-base buffer in the enzyme buffer reagent can effectively improve the reaction.

[0025] In one specific embodiment of the present invention, by repeatedly testing the reaction temperature, it was finally determined that the reaction temperature should be lowered to prolong the time for solidification reaction. The reaction temperature applicable to the present invention is 25℃-33℃, and the preferred reaction time is 30℃.

[0026] In addition to adjusting the reaction temperature, this invention also found that adjusting the acid-base buffer in the enzyme buffer solution in the above reaction is also possible. It should be understood that in the above enzymatic reaction, an enzyme buffer solution needs to be added. The enzyme buffer solution contains various reagents, including an acid-base buffer solution to provide a suitable pH value for the enzyme reaction—such as 50 mM Tris-HCl (pH 8.5), a salt ion solution to provide a suitable salt ion concentration for the enzyme reaction—such as 50 mM NaCl, and 10 mM DTT to prevent the enzyme from forming disulfide bonds. Under conditions where the reaction temperature is not adjusted (i.e., at the optimal reaction temperature of 37°C for the enzyme), replacing the 50 mM Tris-HCl (pH 8.5) in the conventional enzyme buffer solution with 50 mM HEPS-KOH (pH 8.5) can delay the coagulation reaction, thereby ensuring the continuous progress of the enzymatic reaction. In addition, replacing Tris-HCl with any of Tris-acetate, Glycine-KOH, Potassium Acetate, Bis-Tris-Propane-HCl, or Sodium Phosphate can achieve the same effect.

[0027] Regarding the specific implementation of the labeling, in one embodiment of the present invention, CviPI methyltransferase can be used to perform GpC methylation labeling on open DNA in plasma cfDNA that is not wrapped around nucleosomes. Converting C in GpC to methylated C can distinguish endogenous methylation (mCpG) from methylated C. Considering that there are more methods for converting and sequencing methylated C, this preferred approach also facilitates subsequent experiments. As another embodiment of the present invention, AluI methyltransferase (AGCT), Hhal methyltransferase (GCGC), HaeIII methyltransferase (GGCC), or MspI methyltransferase (CCGG) can also be used. These four methyltransferases recognize four bases and methylate C. While their accuracy in nucleosome location recognition is slightly lower than that of CviPI methyltransferase, they can still achieve nucleosome location labeling and direct detection. After directly labeling the open regions of plasma cfDNA in the test sample using the above methyltransferases, the labeled open regions of cfDNA are extracted from the plasma. It should be understood that subsequent detection steps can be performed following other steps in a standard methylation library preparation and sequencing workflow. Those skilled in the art can select and adjust relevant steps according to actual needs. Standard steps include, but are not limited to, end repair and A-methylation adapter addition to the extracted cfDNA, conversion of unmethylated C to U, PCR amplification of the transformed cfDNA, and finally, construction of a sequencing library from the amplified cfDNA and single-molecule sequencing to obtain sequencing results. Those skilled in the art can choose appropriate transformation methods, such as the TAPS method, depending on the specific circumstances.

[0028] In another specific embodiment of the present invention, a methyltransferase labeled m6A (e.g., EcoGII methyltransferase, 4-base recognition) can be used to methylate adenine in the open region. In this case, no transformation and PCR are required, and sequencing can be performed directly to achieve direct detection of nucleosome location information of cfDNA.

[0029] It should be understood that after directly labeling cfDNA in plasma using the above labeling method, the problem of low initial cfDNA levels can be overcome by PCR amplification of the labeled cfDNA during the library construction and sequencing process. This addresses the issue of the large plasma requirement (usually greater than 10 ml) in long-read cfDNA sequencing technologies. Using the detection method of this invention, the required cfDNA level can be as low as below 10 ng, and only 500 μL of plasma is needed, effectively reducing the pressure and difficulty of sample provision and facilitating the practical application of the detection method.

[0030] Regarding the specific sequencing method applicable to this invention, single-molecule sequencing, such as SMRT technology or nanopore single-molecule sequencing technology, is preferred. It should be understood that third-generation single-molecule sequencing can obtain multi-dimensional information for all fragment lengths. However, if only short fragment information is needed, second-generation short-read sequencing can be performed, which can obtain multi-omics information for short cfDNA fragments (i.e., cfDNA fragments with only one nucleosome). Those skilled in the art can choose the appropriate specific sequencing method according to actual needs.

[0031] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be noted that the manufacturers and products provided in the following embodiments are merely illustrative and do not constitute a limitation of the present invention. Those skilled in the art can select appropriate specific reagents based on the following specific examples and actual circumstances.

[0032] Example 1:

[0033] Taking multi-omics detection of cfDNA in normal human plasma as an example

[0034] 1) Take 500 μL of normal human plasma and add reagents in the order shown in Table 1 below.

[0035] Table 1. Reagents related to the labeled reaction

[0036] Place in a 30°C constant temperature mixer and mix at 300 rpm for 1 hour.

[0037] 2) Nucleic acid extraction reagent (BGI Genomics, P2-96) was used to extract cfDNA according to the standard procedure of the kit. After extraction, the concentration was measured by qubit dsDNA hi-sensitivity, and about 10 ng of cfDNA was obtained.

[0038] 3) The cfDNA was subjected to the Rapid Plus DNA Li Prep kit for Illumina (NO PCR) (Abcolonal) to add methylated adapters and then the DNA was purified.

[0039] 4) Adopt The Enzymatic Methyl-seq Kit, following the standard operating procedure of the kit, deaminates unmethylated C in DNA, converting it into U bases.

[0040] 5) The transformed DNA was prepared and sequenced using the ONT (Oxford Nanopore technology) library preparation method.

[0041] The amplification-related reagent system is shown in Table 2.

[0042] Table 2 PCR amplification system

[0043] After mixing, amplification was carried out according to the reaction procedure shown in Table 3.

[0044] Table 3 PCR reaction procedure

[0045] The PCR products were purified using 1X magnetic beads. Libraries were constructed using the ONT PCR library preparation kit and sequenced on a PromethION instrument, or libraries were constructed using the BGI Cyclone library preparation kit and sequenced on a wt-02 sequencer.

[0046] Data analysis and visualization:

[0047] 1) The length and abundance distribution of sequencing fragments are shown in Figure 2.

[0048] Most cfDNA does not exist in the circulatory system as a single DNA fragment, but rather is wrapped around one or more nucleosomes, forming cfDNA fragments of varying sizes. cfDNA fragments exhibit a main peak around 166 bp (monucleosome), with smaller peaks around 300 bp (binucleosome) and 500 bp (triucleosome). The size distribution of the sequenced fragments obtained in this example shows that the cfDNA fragment length exhibits three peaks, including 172 bp, 342 bp, and 516 bp, with a few longer fragments. This indicates that our method can obtain mononuclear, binucleate, and trinucleate cfDNA, as well as longer fragments. This demonstrates the reliability of our method in obtaining cfDNA of all lengths.

[0049] 2) Select a sequencing data point and display its multi-omics information, as shown in Figure 3: This sequence is 583 bp in length, contains three nucleosomes and two open regions, and displays the methylation sites. Figure 3 shows the nucleosome location information, containing a total of three nucleosome regions, with black representing the nucleosome regions. This region is determined by methylated GpCs. The original methylation information of this cfDNA, as well as fragment length, sequence information, and other multi-dimensional information, are also displayed. These conclusions indicate that this method can obtain multi-omics information of cfDNA in one go.

[0050] Example 2: Temperature conditions and enzyme buffer reagents for the labeling reaction

[0051] 1. Experiment on plasma labeling reaction conditions:

[0052] 1) Take 50 μL of normal human plasma, add reagents in the order shown in Table 4 below, and prepare the solution.

[0053] Table 4. Reagents related to the labeled reaction

[0054] The mixtures were placed in constant temperature mixers at 37°C and 30°C respectively, and the reaction time was observed and recorded.

[0055] Experiments showed that the reaction completely solidified after 30 minutes at 37℃, after which the reaction could not proceed; however, at 30℃, the restriction endonuclease remained in a solution state for 1 hour. To further verify the effect of temperature on the above labeling reaction, the reaction and DNA labeling were tested at 25℃, 27℃, 30℃, 33℃, and 37℃. Subsequent experiments were conducted to verify the labeling efficiency at different temperatures. The results after 30 minutes of enzymatic reaction are shown in Table 5.

[0056] Table 5 Enzyme-catalyzed reactions at different temperatures

[0057] Labeling efficiency = Labeled DNA / Total DNA

[0058] Based on the length of coagulation time and the reaction temperature (the closer the temperature is to 37℃, the higher the enzyme activity), the preferred temperature conditions are 25℃-33℃, and 30℃ is finally determined as the optimal reaction temperature.

[0059] 2. Test for replacing acid-base buffer reagents in enzyme buffer reagents

[0060] The effect of components in the 10XGC reaction buffer on plasma coagulation was tested.

[0061] It was found that 50 mM Tris-HCl had the greatest impact on the coagulation reaction. When mixed with plasma, the coagulation reaction proceeded rapidly at 37°C. However, when the 50 mM Tris-HCl in the 10X GC reaction buffer was replaced with 50 mM Heps-KOH, the coagulation time of the plasma was delayed. Subsequent experiments verified that the replacement did not affect the methyltransferase reaction.

[0062] The above examples illustrate the present invention only to aid in understanding it and are not intended to limit the scope of the invention. Those skilled in the art can make various simple deductions, modifications, or substitutions based on the principles of this invention.

Claims

1. A multi-omics detection method for plasma cfDNA, the method comprising: Methyltransferase, enzyme buffer, protease inhibitor and EDTA are added to the plasma of the test sample. The methyltransferase is used to label the open regions of cfDNA in the test sample and methylate specific bases in the open regions of cfDNA that are not wrapped by nucleosomes. cfDNA labeled with open regions was extracted, and end repair, A-methylation adapters, and transformation were performed followed by library construction and sequencing; or, cfDNA labeled with open regions was extracted, and library construction and sequencing were performed.

2. The detection method according to claim 1, characterized in that, The detection method also includes: performing PCR amplification and library reconstruction sequencing after transformation.

3. The detection method according to claim 1, characterized in that, Methyltransferase, enzyme buffer, protease inhibitor and EDTA are added to the plasma of the test sample. Under the conditions of 25℃-37℃ and / or replacing the acid-base buffer in the enzyme buffer, the open regions of cfDNA in the test sample are labeled by methyltransferase, and specific bases in the open regions of cfDNA that are not wrapped by nucleosomes are methylated.

4. The detection method according to claim 3, characterized in that, Methyltransferase, enzyme buffer, protease inhibitor and EDTA were added to the plasma of the test sample. The open regions of cfDNA in the test sample were labeled by methyltransferase at 25℃-33℃, and specific bases in the open regions of cfDNA that were not wrapped by nucleosomes were methylated.

5. The detection method according to claim 3, characterized in that, Methyltransferase, enzyme buffer, protease inhibitor and EDTA are added to the plasma of the test sample. Under the condition of replacing the acid-base buffer in the enzyme buffer, the methyltransferase is used to label the open region of cfDNA in the test sample, and specific bases in the open region of cfDNA that are not wrapped by nucleosomes are methylated.

6. The detection method according to claim 5, characterized in that, The acid-base buffer in the enzyme buffer is obtained by replacing Tris-HCl with any one of HEPS-KOH, Tris-acetate, Glycine-KOH, Potassium Acetate, Bis-Tris-Propane-HCl, or Sodium Phosphate.

7. The detection method according to claim 3, characterized in that, The detection method includes: Methyltransferase, enzyme buffer, protease inhibitor, and EDTA were added to the plasma sample to be tested. Under conditions of 25℃-37℃ and / or with the acid-base buffer in the enzyme buffer, the open regions of cfDNA in the sample to be tested were labeled using methyltransferase, and specific bases in the open regions of cfDNA not wrapped by nucleosomes were methylated. The cfDNA with labeled open regions was extracted, and end repair and A-methylated adapters were added to it. The unmethylated C of the adapter-added cfDNA was converted to U, and then PCR amplification, sequencing library construction, and single-molecule sequencing were performed. The methyltransferase is selected from any one of CviPI methyltransferase, AluI methyltransferase, Hhal methyltransferase, HaeIII methyltransferase, or MspI methyltransferase.

8. The detection method according to claim 3, characterized in that, The detection method includes: A methyltransferase labeled with m6A, an enzyme buffer, a protease inhibitor, and EDTA were added to the plasma sample to be tested. Under conditions of 25℃-37℃ and / or with the acid-base buffer in the enzyme buffer replaced, the open regions of cfDNA in the sample to be tested were labeled using the methyltransferase, and specific bases in the open regions of cfDNA not encapsulated by nucleosomes were methylated. The cfDNA labeled with the open regions was extracted, and sequencing libraries were constructed and single-molecule sequencing was performed. Preferably, the m6A-labeled methyltransferase is an EcoGII methyltransferase.

9. A method for labeling open regions of plasma cfDNA, the method comprising: Methyltransferase, enzyme buffer, protease inhibitor and EDTA are added to the plasma of the test sample. The methyltransferase is used to label the open regions of cfDNA in the test sample, and specific bases in the open regions of cfDNA that are not wrapped by nucleosomes are methylated.

10. The marking method according to claim 9, characterized in that, Methyltransferase, enzyme buffer, protease inhibitor and EDTA are added to the plasma of the test sample. Under the conditions of 25℃-37℃ and / or replacing the acid-base buffer in the enzyme buffer, the open regions of cfDNA in the test sample are labeled by methyltransferase, and specific bases in the open regions of cfDNA that are not wrapped by nucleosomes are methylated.

11. The marking method according to claim 10, characterized in that, Methyltransferase, enzyme buffer, protease inhibitor and EDTA were added to the plasma of the test sample. The open regions of cfDNA in the test sample were labeled by methyltransferase at 25℃-33℃, and specific bases in the open regions of cfDNA that were not wrapped by nucleosomes were methylated.

12. The marking method according to claim 10, characterized in that, Methyltransferase, enzyme buffer, protease inhibitor and EDTA are added to the plasma of the test sample. Under the condition of replacing the acid-base buffer in the enzyme buffer, the methyltransferase is used to label the open region of cfDNA in the test sample, and specific bases in the open region of cfDNA that are not wrapped by nucleosomes are methylated.

13. The marking method according to claim 12, characterized in that, The buffer reagent in the replacement enzyme buffer reagent is to replace the acid-base buffer reagent in the enzyme buffer reagent with Tris-HCl and any one of HEPS-KOH, Tris-acetate, Glycine-KOH, Potassium Acetate, Bis-Tris-Propane-HCl or Sodium Phosphate.

14. The marking method according to claim 9, characterized in that, The methyltransferase is selected from any one of CviPI methyltransferase, EcoGII methyltransferase, AluI methyltransferase, Hhal methyltransferase, HaeIII methyltransferase, or MspI methyltransferase.

15. The application of the labeling method according to any one of claims 9-14 in plasma cfDNA detection.

16. The application of the detection method as described in any one of claims 1-8 and / or the labeling method as described in any one of claims 9-14 in early screening of multiple cancers in plasma cfDNA, cancer tracing, detection and diagnosis of diseases during pregnancy.

17. A kit for labeling plasma cfDNA using the labeling method as described in any one of claims 9-14, characterized in that, The kit includes methyltransferase, enzyme buffer, protease inhibitor and EDTA.

18. The kit according to claim 17, characterized in that, The methyltransferase includes any one of CviPI methyltransferase, EcoGII methyltransferase, AluI methyltransferase, Hhal methyltransferase, HaeIII methyltransferase, or MspI methyltransferase.

19. The kit as described in claim 17 or 18, characterized in that, The acid-base buffer in the enzyme buffer is any one of HEPS-KOH, Tris-acetate, Glycine-KOH, Potassium Acetate, Bis-Tris-Propane-HCl, or Sodium Phosphate.

20. A kit for detecting plasma cfDNA using the detection method as described in any one of claims 1-8, characterized in that, The kit includes methyltransferase, enzyme buffer, protease inhibitor and EDTA.

21. The kit according to claim 20, characterized in that, The methyltransferase includes any one of CviPI methyltransferase, EcoGII methyltransferase, AluI methyltransferase, Hhal methyltransferase, HaeIII methyltransferase, or MspI methyltransferase.

22. The kit as described in claim 20 or 21, characterized in that, The acid-base buffer in the enzyme buffer is any one of HEPS-KOH, Tris-acetate, Glycine-KOH, Potassium Acetate, Bis-Tris-Propane-HCl, or Sodium Phosphate.

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