WO2023082240A1 - Method for detecting genome-wide dna methylation modification - Google Patents

Abstract

Disclosed in the present invention is a method for detecting genome-wide DNA methylation modification. Provided in the present invention is a method for preparing a library for detecting the methylation modification of a nucleic acid, which method comprises: (1) transforming a methylation site in a nucleic acid sample by means of an enzyme reaction; (2) transforming an unmodified C into methylated C by means of an enzyme reaction; and (3) treating same with a bisulfite to obtain a transformed nucleic acid library. According to the present invention, unmethylated cytosine C can be prevented from being transformed, and only methylated cytosine 5mC, hydroxymethylated cytosine 5hmC, hydroformylated cytosine 5fC and carboxylated cytosine 5caC are transformed into uracil U, thereby solving the problem of low complexity of the library established using WGBS and EM-seq, improving the quality of genome-wide methylation sequencing, improving the alignment rate and alignment accuracy of sequencing data, and improving the sensitivity of methylation detection.

Description

A method for detecting DNA methylation modifications on a genome-wide scale technical field

The invention relates to a method for detecting DNA methylation modifications in the whole genome.

Background technique

There are more than a dozen known DNA modifications, among which methylation is the most common and the most studied DNA modification. DNA methylation is an important manifestation of epigenetics. DNA methylation can not only show changes in the state due to environmental differences, but also show genetic characteristics that can be passed on to offspring along with the DNA replication process. DNA methylation can regulate gene expression and play an important role in maintaining normal cell function and genetic imprinting, as well as in embryonic development and human tumorigenesis. DNA can be passed on to newborn offspring along with the DNA replication process. DNA methylation in a broad sense refers to the bases on the DNA chain under the catalysis of DNA methyltransferase, with S-adenosyl methionine (S-adenosyl methionine, SAM) as the methyl donor, A chemical modification process in which a methyl group is obtained by means of covalent bonding. Methylation can occur at the C5 position of cytosine, the N6 position of adenine and the N7 position of guanine. At present, the research on DNA methylation is mainly focused on the methylation of cytosine C5. At present, about 1% of the methylated cytosine in the human genome is studied, which is the most abundant DNA modification found so far.

DNA methylation modification is not completely stable, it can be changed, and it is dynamic within a certain period of time. For example, during embryonic development, DNA will undergo multiple demethylation and re-methylation processes. DNA methylation depends on DNA methyltransferase, and DNA demethylation can be done in two ways, one is passive demethylation of DNA, due to the lack of methylation maintenance enzymes, methylated cytosine in the genome was diluted. The second is active DNA demethylation, which is cyclical, starting from 5-methylcytosine (5mC) and ending with unmodified C. 5mC can be oxidized to 5-hydroxymethylcytosine (5hmC), and this process is accomplished by a family protein called TET (ten-eleven translocation). 5hmC can be further oxidized to 5-formylcytosine (5fC) and 5-carboxycytosine (5caC). 5fC and 5caC will be recognized and excised by thymine DNA glycosylase (TDG), remove 5fC and 5caC from DNA, and produce an unmodified C after repair.

Since DNA methylation can regulate gene expression and play an important role in a series of life activities of organisms, many methods have been applied to detect methylation on DNA, such as HPLC-Mass, qPCR/ddPCR, pyrosequencing , Microarry chips and next-generation sequencing methods, etc. With the continuous development of next-generation sequencing in recent years, the method based on next-generation sequencing has become more and more widely used, such as the detection of methylation targets in liquid biopsy and early tumor screening. Next-generation sequencing-based DNA methylation detection methods also include reduced representative bisulfite sequencing (RRBS), whole-genome bisulfite sequencing (WGBS), methylation co-immunoprecipitation sequencing (MeDIP-seq), Targeted DNA methylation sequencing, etc.

Among the many existing methods for detecting DNA methylation modification, the DNA methylation detection method based on next-generation sequencing is the most widely used. Among the DNA methylation detection methods based on next-generation sequencing, genome-wide methylation sequencing is currently one of the most commonly used methods. The following are two common genome-wide methylation sequencing methods. The schematic diagrams of the two methods are shown in Figure 1.

First, whole genome sequencing (WGBS) based on bisulfite (BS) treatment. Using bisulfite to treat DNA (commonly known as BS conversion) can "convert" unmethylated C, 5fC and 5caC into uracil (U), and finally "convert" into thymine (T) through PCR amplification, while The methylated 5mC and 5hmC are not converted, and the DNA methylation level can be obtained by analyzing the CT ratio of the base in the original position in the sequencing data. Limitations: Since 5mC and 5hmC account for only 5% of the total C, nearly 95% of the C in the whole genome is converted into T, which reduces the complexity of the library and will be due to C during sequencing. The proportion of bases is very low (close to 1%), which affects the quality of sequencing, resulting in a decrease in the amount of sequencing data and sequencing accuracy. At the same time, when performing comparisons, due to the conversion of most C into T and the degradation of sequencing quality, The comparison rate and comparison accuracy of the data are low.

Second, genome-wide methylation sequencing based on enzymatic conversion (EM-seq, from NEB), which converts unmethylated cytosine C to uracil U through a series of enzymatic reactions to identify 5mC, 5hmC, 5fC and 5caC sites. Specifically, 5mC and 5hmC were converted to 5caC by Tet-methylating cytosine dioxygenase 2 (TET2) and glycosylated to 5ghmC by T4 bacteriophage β-glucosyltransferase, thereby protecting 5mC and 5hmC Protected from downstream deamination reactions; cytosine C is then deaminated to uracil U with deaminase (APOBEC); subsequent PCR amplification can convert modified 5mC, 5hmC, 5fC, and 5caC to ordinary C, Convert uracil U to thymine T; the methylation level of DNA can be obtained by analyzing the CT ratio of the base in the original position in the sequencing data. Limitations: Enzymatic transformation-based genome-wide methylation sequencing (EM-seq) can generate more complete libraries and provide more sensitive methylation detection, but the final transformation form of genomic DNA is the same and will The same problem is caused, leaving only three bases A, T, and G in the sequence that constitutes DNA, and its complexity is greatly reduced. Will have the same problem with WGBS.

invention disclosure

The purpose of the present invention is to provide a method for detecting genomic DNA methylation modification.

The invention provides a method for preparing a library for detecting nucleic acid methylation modification, comprising the following steps:

(1) converting the methylation site in the nucleic acid sample by enzymatic reaction;

(2) converting unmodified C into methylated C by enzymatic reaction;

(3) Treatment with bisulfite (BS conversion treatment).

The "converting the methylation sites in the nucleic acid sample by enzymatic reaction" is specifically "converting 5mC, 5hmC and 5fC in the nucleic acid sample to 5caC by enzymatic reaction".

The "convert unmodified C into methylated C by enzymatic reaction" is specifically "convert unmodified C into 5mC by enzymatic reaction".

The "treatment with bisulfite" specifically means "treatment with bisulfite to convert 5caC into U while keeping 5mC unchanged".

The method also includes the following steps: using the product of step (3) as a sample for library construction to prepare a nucleic acid library.

The method further includes the following steps: after step (3), PCR amplification or extension reaction is performed, U is converted into T, and 5mC is converted into C, which is the sample for library construction.

The method also includes at least one of the following two steps: ① nucleic acid fragmentation; ② nucleic acid end repair and linker.

The step ① and/or the step ② are located between the step (2) and the step (3).

The cytosine in the linker is a methylated cytosine.

The step ① and/or step ② is located before the step (1).

The linker is a sequencing linker.

In "converting the methylation site in the nucleic acid sample by enzymatic reaction", the enzyme required for the enzymatic reaction is oxidase.

In "converting unmodified C into methylated C by enzymatic reaction", the enzymes required for the enzymatic reaction are methyltransferase and S-adenosylmethionine.

The inclusions may be sequential inclusions.

Exemplarily, the nucleic acid is DNA or RNA.

The DNA sample is genomic DNA or fragmented DNA (eg cf DNA).

The present invention also provides a method for detecting nucleic acid methylation modification, comprising the following steps:

Use any of the methods described above to prepare samples for library construction;

Perform library construction to obtain a nucleic acid library;

Perform nucleic acid sequence determination.

The inclusions may be sequential inclusions.

The library construction may be library construction by PCR.

The library construction may be library construction by single-strand circularization.

The library construction can be library construction by PCR and single strand circularization.

Exemplarily, the nucleic acid is DNA or RNA.

The present invention also protects the application of any of the above methods in detecting nucleic acid methylation modification.

The present invention also provides a kit, including the following components: oxidase, methyltransferase and S-adenosylmethionine.

Oxidases convert methylated sites in nucleic acids through enzymatic reactions. The "transforming the methylation site in the nucleic acid through an enzymatic reaction" is specifically "converting 5mC, 5hmC and 5fC in the nucleic acid into 5caC through an enzymatic reaction".

Methyltransferase and S-adenosylmethionine convert unmodified C in nucleic acid into methylated C through enzymatic reaction. The "convert unmodified C in nucleic acid to methylated C by enzymatic reaction" is specifically "convert unmodified C in nucleic acid to 5mC by enzymatic reaction".

The kit also includes the following components: a reagent or combination of reagents for bisulfite-treating nucleic acids. The "treating the nucleic acid with bisulfite" specifically means "treating the nucleic acid with bisulfite to convert 5caC into U while keeping 5mC unchanged".

The kit also includes at least one of the following components: ① a reagent or a reagent combination for nucleic acid fragmentation; ② a reagent or a reagent combination for nucleic acid end repair plus adapters.

The linker is a sequencing linker.

The cytosine in the linker is a methylated cytosine.

The kit may also include a protease.

The kit may also include nucleic acid fragmentation enzymes, methylating nucleic acid adapters, and ligases.

The kit may also include nucleic acid repair enzymes.

The kit may also include primers, reagents or kits required for nucleic acid library construction.

The kit may also include primers, reagents or kits required for single strand circularization.

The kit may also include reagents or kits required for making nucleic acid nanospheres.

Exemplarily, the nucleic acid is DNA or RNA.

The function of any of the above kits is to prepare a nucleic acid library for detecting nucleic acid methylation modification.

The present invention also protects the application of any of the kits described above in the detection of nucleic acid methylation modification.

Exemplarily, any of the oxidases described above is methylcytosine dioxygenase.

Exemplarily, the methylcytosine dioxygenase is Tet1 protein.

Exemplarily, the methylcytosine dioxygenase is Tet2 protein or Tet3 protein.

Exemplarily, any one of the above methyltransferases is a CpG methyltransferase.

The requirement for fragmentation of the nucleic acid is a nucleic acid fragmentation enzyme (eg, DNA fragmentation enzyme).

The nucleic acid repair enzymes (such as DNA repair enzymes) are required for the repair of nucleic acid ends.

The DNA repair enzyme is ERAT Enzyme.

Specifically, the DNA Methylation kit can be used for treatment with bisulfite.

Specifically, MGIEasy-whole genome methylation library preparation kit can be used for library construction by PCR.

The single-strand circularization MGIEasy circularization kit can be used to construct the library by single-strand circularization.

The production of DNA nanospheres can specifically use the MGISEQ-2000 high-throughput sequencing reagent kit.

The fragmented DNA may be obtained by fragmenting genomic DNA.

The fragmented DNA can be extracellular episomal DNA (eg cfDNA).

The sequencing linker sequence may be a sequencing linker adapted to the DNBSEQ platform or the illumina sequencing platform.

The sequencing can adopt DNBSEQ sequencing platform or illumina sequencing platform.

Specifically, the nucleic acid methylation modification is a genome-wide nucleic acid methylation modification.

Specifically, the methylation modification of the nucleic acid is the methylation modification at the C5 position of cytosine.

Specifically, the DNA methylation modification is a genome-wide DNA methylation modification.

Specifically, the DNA methylation modification is methylation modification at the C5 position of cytosine.

The principle schematic diagram of the method provided by the present invention is shown in FIG. 2 .

The flow diagram of the method provided by the present invention is shown in FIG. 3 .

The main steps of the method provided by the invention: the test DNA sample is first treated with methylcytosine dioxygenase, so that 5mC, 5hmC and 5fC are converted into 5caC; then, DNA methyltransferase is used for treatment , so that the unmodified C is converted into 5mC; then, bisulfite is used for treatment (BS conversion treatment), so that 5caC is converted into U, while 5mC remains unchanged; then, PCR amplification or extension reaction is performed, and U conversion If it is T, 5mC is converted to C, which is the library construction sample; then, the library construction sample is used for library construction to obtain a DNA library, and then the nucleic acid sequence is determined. Therefore, the core of the present invention is that, through a series of steps, from the test DNA sample to the library construction, the unmodified C remains unchanged, and the 5mC, 5hmC and 5fC in the test sample are converted into T.

For subsequent high-throughput sequencing, pre-processing is required. The pretreatment refers to: DNA fragmentation, end repair and A addition, and sequencing linker connection. The pretreatment can be carried out before Tet protein (methylcytosine dioxygenase) treatment, or between DNA methyltransferase treatment and bisulfite treatment.

Beneficial effects of the present invention: solve the problem of low library complexity existing in the existing WGBS and EM-seq, thereby improving the quality of whole-genome methylation sequencing, increasing the amount and accuracy of sequencing data, and reducing the number of whole-genome methylation sequencing. At the same time, it can also improve the alignment rate and alignment accuracy of genome-wide methylation sequencing data, thereby improving the sensitivity of genome-wide methylation detection.

The invention can be used in the fields of tumor early screening and liquid biopsy.

The present invention can prevent unmethylated cytosine C from being converted, and only convert methylated cytosine 5mC, hydroxymethylated cytosine 5hmC, formylated cytosine 5fC and carboxylated cytosine 5caC into urine Pyrimidine U, so as to solve the problem of low library complexity in WGBS and EM-seq, thereby improving the quality of genome-wide methylation sequencing, and at the same time improving the alignment rate and alignment accuracy of sequencing data, and also improving methylation Detection sensitivity.

Description of drawings

FIG. 1 is a schematic diagram of the principles of two genome-wide methylation sequencing methods in the prior art.

Fig. 2 is a schematic diagram of the principle of the method provided by the present invention.

Fig. 3 is a schematic flow chart of the method provided by the present invention.

Fig. 4 is the result graph of embodiment 1.

Fig. 5 is the result graph of embodiment 2.

The best way to practice the invention

The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention. The examples provided below can be used as a guide for those skilled in the art to make further improvements, and are not intended to limit the present invention in any way. The experimental methods in the following examples, unless otherwise specified, are conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified. Unless otherwise specified, the quantitative tests in the following examples all set three repeated tests, and the results were averaged. Unless otherwise specified, the water in the examples is molecular grade water.

Example 1. Construction and sequencing of human whole genome DNA methylation library

DNA sample: NA12878 human genomic DNA standard (commercially available from Coriell institute, catalog number NA12878).

1. Methylcytosine dioxygenase and methyltransferase pretreatment

1. Add 500ng of DNA sample to a 0.2mL PCR tube and place it on an ice box.

2. Prepare 2xTET reaction buffer according to Table 1. Table 1 shows the volume of a single reaction system (24 μl). If multiple reaction systems are needed, just double it.

Table 1

components	add volume
1M HEPES buffer(pH 8.0)	2.5μL
50mM ammonium iron(II)sulfate aqueous solution	0.1μL

100mM α-ketoglutarate aqueous solution	0.5μL
100mM ascorbic acid aqueous solution	1μL
100mM DTT aqueous solution	0.5μL
5M NaCl aqueous solution	1μL
0.1M ATP water solution	0.6μL
water	17.8μL

Ammonium iron (II) sulfate: Sigma Company, catalog number 215406-100G.

3. Take the PCR tube that completed step 1, add 24 μl 2xTET reaction buffer prepared in step 2 and 1 μl methylcytosine dioxygenase (protein content is 1 ng), then add water to a total volume of 50 μl, and then place the PCR tube in Incubate at 37°C for 3 hours on a PCR machine.

Methylcytosine dioxygenase (Recombinant Tet1protein): Active motif company, product catalog number is 31363.

4. Take the PCR tube that has completed step 3, add 0.8U Proteinase K, mix well, centrifuge briefly, then place the PCR tube on a PCR instrument, and incubate at 50°C for 50 minutes.

Proteinase K: NEB company, P8170S.

5. Take the PCR tube that completed step 4, centrifuge briefly, and then transfer the entire contents of the 0.2mL PCR tube to a 1.5ml centrifuge tube.

6. DNA purification

① After completing the previous step, take the 1.5ml centrifuge tube, add 1.8x Ampure XP beads (about 90 μl), mix with a pipette, and incubate at room temperature for 10 minutes; then, centrifuge briefly, and place the centrifuge tube on a magnetic stand Let stand until the liquid is clear (5min), suck up with a pipette and discard the supernatant.

② After completing step ①, remove the centrifuge tube from the magnetic stand, add 200 μl 75% ethanol aqueous solution, and let it stand for 30 seconds; Aspirate and discard the supernatant.

③ After completing step ②, keep the centrifuge tube on the magnetic stand, open the cap of the centrifuge tube, and dry at room temperature until the surface of the magnetic beads has no reflection and no cracks.

④ After completing step ③, remove the centrifuge tube from the magnetic stand, add 23 μL molecular grade water, gently blow and mix with a pipette, and incubate at room temperature for 5 minutes; then, centrifuge briefly, place the centrifuge tube on the magnetic stand, Let stand until the liquid is clear (5min), and transfer the supernatant to a new 1.5mL centrifuge tube.

The supernatant obtained in step ④ is the purified DNA, about 21 μL.

7. Prepare 10x M.SssI Mg ²⁺ -free reaction buffer according to Table 2. Table 2 shows the volume of a single reaction system (2.5 μl). If multiple reaction systems are needed, just double it.

Table 2

components	add volume
1M Tris-HCl aqueous solution (pH8.0)	0.25 μL
5M NaCl aqueous solution	0.05μL
0.5M EDTA aqueous solution (pH8.0)	0.1μL
water	2.1μL

8. Take a 0.2mL PCR tube, add 20.75μl of the purified DNA obtained in step 6, then add 1.25μl of CpG methyltransferase and 0.5μl of 32mM S-adenosylmethionine, and then add 2.5μl of 10x DNA prepared in step 7 M.SssI Mg ²⁺ -free reaction buffer, incubated at 37°C for 4h.

CpG methyltransferase (M.SssI), the specification is 20unit/μl; CpG methyltransferase is a commercially available product, NEB Company, the catalog number is M0226S. S-adenosylmethionine (SAM) is a supporting product of CpG methyltransferase: NEB Company, the product catalog number is M0226S.

9. Take the PCR tube that completed step 8, centrifuge briefly, and then transfer to a 1.5ml centrifuge tube.

10. DNA purification

The method is the same as step 6.

The supernatant obtained in step ④ is the purified DNA, about 21 μL.

11. Take a 0.2mL PCR tube, add 20.75μl of the purified DNA obtained in step 10, add 1.25μl of CpG methyltransferase and 0.5μl of 32mM S-adenosylmethionine, and then add 2.5μl of 10x NEB buffer 2,37 Incubate at ℃ for 8h.

10x NEB buffer 2: NEB company, catalog number is B7002S.

12. Take the PCR tube that completed step 11, centrifuge briefly, and then transfer the DNA to a 1.5ml centrifuge tube.

13. DNA purification

The method is the same as step 6.

The supernatant obtained in step ④ is the product solution, about 21 μL.

2. Build a library of processed samples

MGIEasy-Whole Genome Methylation Library Preparation Kit (MGI Company, Cat. No. 1000005251) was used for library construction. Its components include methylated DNA Adapter, DNA Ligase and its supporting Ligation Buffer, WGBS PCR Enzyme Mix, and PCR Primer Mix. Frag Enzyme and Frag Buffer are supporting products: YEASEN Company, the product catalog number is 12907ES96. ERAT Buffer: MGI Company, catalog number 1000019872. ERAT Enzyme Mix: MGI Corporation, catalog number 1000019873.

1. Take a 0.2mL PCR tube, add all the product solution obtained in step 1, make up to a total volume of 45 μL with water, and place the PCR tube on an ice box.

2. Take Frag Enzyme, gently invert and mix 10 times, centrifuge briefly and place on ice for later use.

3. Prepare the interruption reaction solution on ice according to Table 3. Table 3 shows the volume of a single reaction system (15 μl). If multiple reaction systems are needed, just double it.

table 3

components	add volume
Frag Buffer	10μL
Frag Enzyme	5μL

4. Take the PCR tube that completed step 1, add 15 μL of the interruption reaction solution prepared in step 3, vortex 3 times (3 seconds each time), and then centrifuge briefly to collect the reaction solution to the bottom of the tube.

5. Take the PCR tube that completed step 4, put it on the PCR machine, perform the reaction according to Table 4, then centrifuge briefly to collect the reaction solution to the bottom of the tube, and then transfer all the liquid to a new 1.5mL centrifuge tube.

Table 4

temperature		time
105°C hot lid	On
4°C		1min
30℃	15min
72°C	10min
4°C	hold

6. DNA purification

① After completing step 5, take the centrifuge tube, add 60 μL DNA Clean Beads, gently pipette until completely mixed, incubate at room temperature for 10 minutes, then centrifuge briefly, then place the centrifuge tube on a magnetic stand and let it stand When the liquid is clear (2-5min), the supernatant is carefully aspirated with a pipette and discarded.

② After completing step ①, keep the centrifuge tube on the magnetic stand, add 200 μL 80% ethanol aqueous solution, rinse the magnetic beads and the tube wall, let stand for 30 seconds, carefully pipette and discard the supernatant.

③ After completing step ②, keep the centrifuge tube on the magnetic stand, add 200 μL 80% ethanol aqueous solution, rinse the magnetic beads and the tube wall, let stand for 30 seconds, carefully aspirate and discard the supernatant.

④ After completing step ③, keep the centrifuge tube fixed on the magnetic stand, open the cap of the centrifuge tube, and dry at room temperature until the surface of the magnetic beads has no reflection.

⑤ After completing step ④, remove the centrifuge tube from the magnetic stand, add 42 μL TE Buffer, gently pipette until completely mixed, incubate at room temperature for 5 minutes, then centrifuge briefly, and then place the centrifuge tube on the magnetic stand , let it stand until the liquid is clear (2-5min), and transfer the supernatant (about 40 μL) to a new 0.2mL PCR tube.

7. Prepare the end repair reaction solution on ice according to Table 5. Table 5 shows the volume (10 μl) of a single reaction system. If multiple reaction systems are needed, just double it.

table 5

components	add volume
ERAT Buffer	7.1μL
ERAT Enzyme Mix	2.9μL

8. Take the PCR tube that completed step 6, add 10 μL of the end repair reaction solution prepared in step 7, vortex 3 times (3 s each time), and then centrifuge briefly to collect the reaction solution to the bottom of the tube.

9. Take the PCR tube that has completed step 8, put it on the PCR machine, perform the reaction according to Table 6, and then centrifuge briefly to collect the reaction solution to the bottom of the tube, then add 5 μL of methylated DNA Adapter, and vortex 3 times (each time 3s), and then centrifuged briefly to collect the reaction solution to the bottom of the tube.

Table 6

temperature		time
65°C hot lid	On
37°C	30min
65°C	15min
4°C	hold

10. Prepare the adapter ligation reaction solution on ice according to Table 7. Table 7 shows the volume (25 μl) of a single reaction system. If multiple reaction systems are needed, just double it.

Table 7

components	add volume
Ligation Buffer	23.4μL
DNA Ligase	1.6μL

11. Take the PCR tube that completed step 9, add 25 μL of the adapter connection reaction solution prepared in step 10, vortex 6 times (3 s each time), and then centrifuge briefly to collect the reaction solution to the bottom of the tube.

12. Take the PCR tube that completed step 11, put it on the PCR instrument, perform the reaction according to Table 8, and then centrifuge briefly to collect the reaction solution to the bottom of the tube.

Table 8

temperature		time
25°C hot lid	On
23°C	30min
4°C	hold

13. Take the PCR tube that completed step 12, add TE Buffer to 100 μL of the total system, and then transfer all to a new 1.5 mL centrifuge tube.

14. DNA purification

① After completing step 13, take the centrifuge tube, add 50 μL DNA Clean Beads, gently pipette until completely mixed, incubate at room temperature for 5 minutes, then centrifuge briefly, then place the centrifuge tube on the magnetic stand and let it stand When the liquid is clear (2-5min), the supernatant is carefully aspirated with a pipette and discarded.

② After completing step ①, keep the centrifuge tube on the magnetic stand, add 200 μL 80% ethanol aqueous solution, rinse the magnetic beads and the tube wall, let stand for 30 seconds, carefully pipette and discard the supernatant.

③ After completing step ②, keep the centrifuge tube on the magnetic stand, add 200 μL 80% ethanol aqueous solution, rinse the magnetic beads and the tube wall, let stand for 30 seconds, carefully aspirate and discard the supernatant.

④ After completing step ③, keep the centrifuge tube fixed on the magnetic stand, open the cap of the centrifuge tube, and dry at room temperature until the surface of the magnetic beads has no reflection.

⑤ After completing step ④, remove the centrifuge tube from the magnetic stand, add 21 μL TE Buffer, gently pipette until completely mixed, incubate at room temperature for 5 minutes, then centrifuge briefly, and then place the centrifuge tube on the magnetic stand Let stand until the liquid is clear (2-5min), and transfer the supernatant (that is, the purified DNA, about 19 μL) to a new 0.2mL PCR tube.

15. Use EZ DNA Methylation-Gold Kit (Zymo Research, Cat.No.D5005/D5006) to Bisulfite treat and purify the adapter ligation product.

The components in EZ DNA Methylation-Gold Kit include: M-Dilution Buffer, M-Dissolving Buffer, CT Conversion Reagent, M-Wash Buffer, Lambda DNA, M-Binding Buffer, Collection Tube, Zymo-Spin IC Column, M-Desulphonation Buffer and M-Elution Buffer. Pipette 900 μL of water, 300 μL of M-Dilution Buffer and 50 μL of M-Dissolving Buffer into a tube of CT Conversion Reagent powder (short-time centrifugation before opening the cap), and vortex frequently at room temperature for 10 minutes to obtain CT Conversion Reagent. Take the M-Wash Buffer bottle, add the correct volume of absolute ethanol according to the label to get the M-Wash Buffer.

①Take a new 0.2mL PCR tube, and add the components in Table 9 at room temperature (the total volume of the system is 150 μL); then, place the PCR tube on the PCR machine, perform the reaction according to Table 10, and then transfer the reaction product into a new 1.5mL centrifuge tube. Lambda DNA does not need to be interrupted, and the concentration is 200ng/μL.

Table 9

components	add volume
CT Conversion Reagent	130μL
Purified DNA obtained in step 14	19μL
Lambda DNA	1μL

Table 10

temperature	time
hot cover	On
98°C	10min
64°C	2.5h
4°C	hold

② Take the centrifuge tube that completed step ①, add 600 μL M-Binding Buffer, vortex 6 times (3s each time), and then centrifuge briefly to collect the reaction solution to the bottom of the tube.

③Take 2mL Collection Tube and put it into Zymo-Spin IC Column, then add the reaction solution obtained in step ② to Zymo-Spin IC Column, centrifuge at 13000rpm for 30s, discard the waste liquid; then add 100μL M-Wash Buffer to Zymo-Spin IC Column, centrifuge at 13000rpm for 30s; then add 200μL M-Desulphonation Buffer to Zymo-Spin IC Column, quickly close the cap tightly, incubate at room temperature for 15-20min, centrifuge at 13000rpm for 30s, discard the waste liquid; then add 200μL M-Wash Buffer Centrifuge at 13000rpm for 30s, discard the waste liquid; then add 200μL M-Wash Buffer to the Zymo-Spin IC Column, centrifuge at 13000rpm for 30s, discard the waste liquid; then, centrifuge at 13000rpm for 30s, discard the Collection Tube, and use a pipette Aspirate the liquid on the outer wall of the Zymo-Spin IC Column as much as possible, and place it in a new 1.5mL centrifuge tube.

④ After completing step ③, take the centrifuge tube, open the Zymo-Spin IC Column tube cap, dry at room temperature for 2 minutes, and then put the Zymo-Spin IC Column in a new 1.5mL centrifuge tube.

⑤ After completing step ④, take the centrifuge tube, add 10 μL M-Elution Buffer to the center of the Zymo-Spin IC Column filter membrane, let it stand for 1 min, and centrifuge at 13,000 rpm for 30 s. The liquid in the centrifuge tube is the product solution.

16. Transfer all the product solution obtained in step 15 to a new 0.2mL PCR tube, and add water to 20μL.

17. Prepare the PCR reaction solution on ice according to Table 11. Table 11 shows the volume of a single reaction system (30 μl). If multiple reaction systems are needed, just double it.

Table 11

components	add volume
WGBS PCR Enzyme Mix	25 μL
PCR Primer Mix	5μL

18. Take the PCR tube that completed step 16, add 30 μL of the PCR reaction solution prepared in step 17, vortex 3 times (3 seconds each time), and then centrifuge briefly to collect the reaction solution to the bottom of the tube.

19. Take the PCR tube that has completed step 18, put it on the PCR machine, perform the reaction according to Table 12, and then centrifuge briefly to collect the reaction solution to the bottom of the tube, then add TE Buffer to a total volume of 100 μL, and then transfer all to a new 1.5 μL tube. mL centrifuge tube.

Table 12

20. DNA purification

① After completing step 19, take the centrifuge tube, add 55 μL DNA Clean Beads, pipette gently until all the magnetic beads are suspended, incubate at room temperature for 5 minutes, then centrifuge briefly, then place the centrifuge tube on the magnetic stand, statically Place until the liquid is clarified (2-5min), carefully draw the supernatant with a pipette, and transfer to a new 1.5mL centrifuge tube.

② Take the centrifuge tube that has completed step ①, add 25 μL DNA Clean Beads, gently blow and mix with a pipette, incubate at room temperature for 5 minutes, then centrifuge briefly, then place the centrifuge tube on a magnetic stand, and let it stand until the liquid is clear (2 -5min), the supernatant was carefully aspirated with a pipette and discarded.

③ After completing step ②, keep the centrifuge tube on the magnetic stand, add 200 μL of 80% ethanol aqueous solution, rinse the magnetic beads and the tube wall, let stand for 30 seconds, then carefully aspirate the supernatant and discard it.

④ After completing step ③, keep the centrifuge tube on the magnetic stand, add 200 μL of 80% ethanol aqueous solution, rinse the magnetic beads and the tube wall, let stand for 30 seconds, then carefully aspirate the supernatant and discard it.

⑤ After completing step ④, keep the centrifuge tube on the magnetic stand, open the cap of the centrifuge tube, and dry at room temperature until the surface of the magnetic beads is no longer reflective.

⑥ After completing step ⑤, remove the centrifuge tube from the magnetic stand, add 32 μL TE Buffer, gently blow with a pipette until completely mixed, incubate at room temperature for 5 minutes, then centrifuge briefly, and then place the centrifuge tube on the magnetic stand , let it stand until the liquid is clear (2-5min), and transfer the supernatant to a new 1.5mL centrifuge tube with a pipette.

The supernatant obtained in step ⑥ is the purified DNA, about 30 μL.

The supernatant obtained in step ⑥ can be used as a library solution and directly sequenced on the illumina sequencing platform.

21. Take the purified DNA obtained in step 20 (DNA content is 360ng) into a new 0.2mL PCR tube, add TE Buffer to a total volume of 48μL.

22. After completing step 21, place the PCR tube on the PCR instrument, and perform the reaction according to Table 13.

Table 13

temperature		time
105°C hot lid	On
95°C	3min

23. Prepare the single-chain cyclization reaction solution on ice according to Table 14. Table 14 shows the volume of a single reaction system (12.1 μl). If multiple reaction systems are needed, just double it.

Table 14

components	add volume
Splint Buffer	11.6μL
DNA Rapid Ligase	0.5μL

DNA Rapid Ligase and Splint Buffer: Both are reagents in the MGIEasy Circularization Kit (MGI, 1000005259).

24. Immediately after the reaction in step 22, place the PCR tube on ice and let it stand for 2 minutes, then add 12.1 μL of the single-strand circularization reaction solution prepared in step 23, vortex 3 times (3s each time), and then Centrifuge briefly to collect the reaction solution to the bottom of the tube, then place the PCR tube on the PCR instrument, and perform the reaction according to Table 15.

Table 15

temperature	time
75°C hot lid	On
37°C	30min
4°C	hold

25. Prepare enzyme digestion reaction solution on ice according to Table 16. Table 16 shows the volume (4 μl) of a single reaction system. If multiple reaction systems are needed, just double it.

Table 16

components	single reaction volume
Digestion Buffer	1.4μL
Digestion Enzyme	2.6 μL

Digestion Enzyme and Digestion Buffer: Both are reagents in the MGIEasy cyclization kit (MGI, 1000005259).

26. After the reaction in step 24 is completed, centrifuge the PCR tube briefly and put it on ice, immediately add 4 μL of the enzyme digestion reaction solution prepared in step 25, vortex 3 times (3 seconds each time), and then centrifuge briefly The reaction solution was collected at the bottom of the tube, and then the PCR tube was placed on the PCR machine, and the reaction was carried out according to Table 17, and then the reaction solution was collected at the bottom of the tube by instantaneous centrifugation.

Table 17

temperature	time
75°C hot lid	On
37°C	30min
4°C	hold

27. After completing step 26, immediately add 7.5 μL Digestion Stop Buffer to the PCR tube, vortex 3 times (3 seconds each time), then centrifuge briefly to collect the reaction solution to the bottom of the tube, and transfer all the reaction solution to a new PCR tube. in a 1.5mL centrifuge tube.

Digestion Stop Buffer: Reagent in MGIEasy Circularization Kit (MGI, 1000005259).

28. DNA purification

① After completing step 27, take the centrifuge tube, add 170 μL DNA Clean Beads, gently blow with a pipette until all magnetic beads are suspended, incubate at room temperature for 10 minutes, then centrifuge briefly, and then place the centrifuge tube on a magnetic stand , let stand until the liquid is clear (2-5min), carefully draw the supernatant with a pipette and discard it.

② After completing step ①, keep the centrifuge tube on the magnetic stand, add 500 μL of 80% ethanol aqueous solution, rinse the magnetic beads and the tube wall, let it stand for 30 seconds, and then carefully aspirate the supernatant and discard it.

③ After completing step ②, keep the centrifuge tube on the magnetic stand, add 200 μL of 80% ethanol aqueous solution, rinse the magnetic beads and the tube wall, let stand for 30 seconds, then carefully aspirate the supernatant and discard it.

④ After completing step ③, keep the centrifuge tube fixed on the magnetic stand, open the cap of the centrifuge tube, and dry at room temperature until the surface of the magnetic beads has no reflection.

⑤ After completing step ④, remove the centrifuge tube from the magnetic stand, add 22 μL TE Buffer, gently pipette until completely mixed, incubate at room temperature for 10 minutes, then centrifuge briefly, and then place the centrifuge tube on the magnetic stand Leave it to stand until the liquid is clear (2-5min), and transfer the supernatant (that is, the product solution, about 20 μL) to a new 1.5mL PCR tube.

The supernatant obtained in step ⑤ can be used as a library solution.

29. Use

ssDNA Assay Kit Fluorescence Quantitative Kit, Quantify the product solution obtained in step 28 according to the operating instructions of the quantitative kit.

30. Take the product solution obtained in step 28, use the MGISEQ-2000 high-throughput sequencing reagent set (PE100) (MGI, catalog number 1000012536) to make DNB (DNA nanoballs) according to the instructions, and then use the MGISEQ-2000 sequencer to perform sequencing.

The database construction results are shown in Table 18. The PCR concentration refers to the DNA concentration of the library solution obtained in step 20. The single-stranded circular library concentration refers to the DNA concentration of the library solution obtained in step 28. The DNB concentration refers to the DNA concentration of the solution after DNB production. NA-1 and NA-2 represent different replicate treatments.

Table 18

sample name	PCR concentration (ng/μL)	Single-stranded library concentration (ng/μL)	DNB concentration (ng/μL)
NA-1	13.5	0.95	23.2
NA-2	15.7	1.02	25.8

The sequencing results are shown in Figure 4. The statistical results of the data are shown in Table 19. Sequencing data analysis shows that the proportion of C bases in the bases at each position on the reads is higher than that of WGBS.

Table 19

sample name	NA-1	NA-2
Sequencing length (bp)	150	150
Sequence ratio after filtering (%)	85.03	95.73
number of sequences after filtering	732780862	733333344
Base number after filtering (bp)	1.09917E+11	1.11044E+11
Number of aligned sequences	659612809	666856392
Aligned bases (bp)	98941921350	1.00028E+11
Contrast rate (%)	90.02	90.93
Repeat rate (%)	24.63	11.22
Number of Unique Aligned Sequences	639145206	645354142
Number of uniquely aligned bases (bp)	95871780900	96803121300
Unique comparison rate (%)	87.22	88
Number of paired-end aligned sequences	611973404	620914692
Double-ended alignment ratio (%)	83.51	84.67

Number of single-end aligned sequences	47639405	45941700
Single-ended alignment rate (%)	6.5	6.26
Number of mismatched bases (bp)	1522754409	753366881
Mismatch rate (%)	1.54	0.75
Sulphite conversion rate (%)	99.41	99.4

Example 2. Construction and sequencing of human peripheral blood cfDNA methylation library

1. Extraction of peripheral blood cfDNA

1. Take 10mL peripheral blood whole blood from a healthy person and centrifuge at 1600g for 10min at 4°C. After centrifugation, it is divided into three layers, the upper layer is the plasma layer, the middle layer is the white blood cell layer, and the lower layer is the red blood cell layer. Use a pipette to draw the plasma layer into a 1.5mL centrifuge tube, then centrifuge at 16000g at 4°C for 10min, draw the plasma, and discard the precipitate.

2. Take the plasma obtained in step 1, and use the MGIEasy Free DNA Extraction Kit (MGI Company, Cat. No. 1000017017) to extract cfDNA.

Proteinase K solution, MGIPure Particle G, Lysis Buffer, Wash Buffer 1, Wash Buffer 2 and Elution Buffer are all components in the MGIEasy cell-free DNA extraction kit.

①Take three new 2mL centrifuge tubes, add 40μL Proteinase K solution and 50μL MGIPure Particle G to each centrifuge tube.

② After completing step ①, add 600 μL of the plasma obtained in step 1 to each centrifuge tube.

③ After completing step ②, add 1100 μL Lysis Buffer to each centrifuge tube, vortex and mix for 10 seconds, place at room temperature (15-25°C) for 10-15 minutes (invert and mix once every 3-5 minutes), and then place the centrifuge tube Place it on a magnetic stand, let it stand for 3-5min to absorb the magnetic beads, and carefully discard all the supernatant.

④ After completing step ③, remove the centrifuge tube from the magnetic stand, add 700 μL Wash Buffer 1, vortex and mix for 15 seconds, and then place it on the magnetic stand for 1 min. After the magnetic beads are completely adsorbed, carefully discard all the above Serum.

⑤ After completing step ④, remove the centrifuge tube from the magnetic stand, add 700 μL Wash Buffer 2, vortex and mix for 15 seconds, and then place it on the magnetic stand for 1 min. After the magnetic beads are completely adsorbed, carefully discard all the above Serum.

⑥ After completing step ⑤, remove the centrifuge tube from the magnetic stand, add 700 μL Wash Buffer 2, vortex and mix for 15 seconds, and then place it on the magnetic stand for 1 min. After the magnetic beads are completely adsorbed, carefully discard all the above Serum.

⑦ After completing step ⑥, centrifuge briefly to centrifuge the liquid droplets remaining on the tube wall to the bottom of the tube, then place it on the magnetic stand for 1 min, and carefully discard all the supernatant.

⑧ After completing step ⑦, store the centrifuge tube on a magnetic stand and dry it at room temperature (15-25°C) for 10-1min.

⑨ After completing step ⑧, remove the centrifuge tube from the magnetic stand, add 10 μL Elution Buffer, vortex to disperse the magnetic beads, and place at room temperature (15-25°C) for 5-10 minutes (during this period, shake gently 1-2 times to accelerate DNA dissolve).

⑩After completing step ⑨, place the centrifuge tube on the magnetic stand, let it stand for 3 minutes, and collect the supernatant (the supernatant is the cfDNA sample) into a new 1.5mL centrifuge tube.

2. Methylcytosine dioxygenase and methyltransferase pretreatment

1. Add 30 μl of the cfDNA sample prepared in step 1 (containing 30ng DNA) to a 0.2mL PCR tube and place it on an ice box.

2. Prepare 2xTET reaction buffer according to Table 20. Table 1 shows the volume of a single reaction system (19 μl). If multiple reaction systems are needed, just double it.

Table 20

components	add volume
1M HEPES buffer(pH 8.0)	2.5μL
50mM ammonium iron(II)sulfate aqueous solution	0.1μL
100mM α-ketoglutarate aqueous solution	0.5μL
100mM ascorbic acid aqueous solution	1μL
100mM DTT aqueous solution	0.5μL
5M NaCl aqueous solution	1μL
0.1M ATP water solution	0.6μL
water	12.8μL

Ammonium iron (II) sulfate: Sigma Company, catalog number 215406-100G.

3. Take the PCR tube that completed step 1, add 19 μl 2xTET reaction buffer prepared in step 2 and 1 μl methylcytosine dioxygenase (protein content is 1 ng), and then place it on a PCR instrument and incubate at 37°C for 3 hours.

4. Take the PCR tube that has completed step 3, add 0.8U Proteinase K, mix well, centrifuge briefly, then place the PCR tube on a PCR instrument, and incubate at 50°C for 50 minutes.

5. Take the PCR tube that completed step 4, centrifuge briefly, and then transfer the entire contents of the 0.2mL PCR tube to a 1.5ml centrifuge tube.

6. DNA purification

Same as Step 6 of Example 1. The supernatant obtained in step ④ is the purified DNA, about 21 μL.

7. Same as Step 7 of Example 1.

8. Same as step 8 in Example 1.

9. Same as step 9 in Example 1.

10. DNA purification

Same as Step 6 of Example 1. The supernatant obtained in step ④ is the purified DNA, about 21 μL.

11. Same as step 11 in Embodiment 1.

12. Same as step 12 in Embodiment 1.

13. DNA purification

Same as Step 6 of Example 1. The supernatant obtained in step ④ is the product solution, about 21 μL.

3. Build a library of processed samples

MGIEasy-Whole Genome Methylation Library Preparation Kit (MGI Company, Cat. No. 1000005251) was used for library construction. Its components include methylated DNA Adapter, DNA Ligase and its supporting Ligation Buffer, WGBS PCR Enzyme Mix, and PCR Primer Mix. ERAT Buffer: MGI Company, catalog number 1000019872. ERAT Enzyme Mix: MGI Corporation, catalog number 1000019873.

1. Prepare the end repair reaction solution on ice according to Table 21. Table 21 shows the volume of a single reaction system (10 μl). If multiple reaction systems are needed, just double it.

Table 21

components	add volume
ERAT Buffer	7.1μL
ERAT Enzyme Mix	2.9μL

2. Take a 0.2 mL PCR tube, add about 21 μL of the product solution prepared in step 2, then add water to a total volume of 40 μL, then add 10 μL of the end repair reaction solution prepared in step 1, and vortex 3 times (3 seconds each time) , and then centrifuged briefly to collect the reaction solution to the bottom of the tube.

3. Take the PCR tube that has completed step 2, place it on the PCR machine, perform the reaction according to Table 6, and then centrifuge briefly to collect the reaction solution to the bottom of the tube, then add 5 μL methylated DNA Adapter, and vortex 3 times (each time 3s), and then centrifuged briefly to collect the reaction solution to the bottom of the tube.

4. Prepare the adapter ligation reaction solution on ice according to Table 7. Table 7 shows the volume (25 μl) of a single reaction system. If multiple reaction systems are needed, just double it.

5. Take the PCR tube that completed step 3, add 25 μL of the adapter connection reaction solution prepared in step 4, vortex 6 times (3 s each time), and then centrifuge briefly to collect the reaction solution to the bottom of the tube.

6. Take the PCR tube that completed step 5, put it on the PCR machine, perform the reaction according to Table 8, and then centrifuge briefly to collect the reaction solution to the bottom of the tube.

7. Take the PCR tube that completed step 6, add TE Buffer to 100 μL of the total system, and then transfer all to a new 1.5 mL centrifuge tube.

8. DNA purification

① After completing step 7, take the centrifuge tube, add 50 μL DNA Clean Beads, gently blow with a pipette until completely mixed, incubate at room temperature for 5 minutes, then centrifuge briefly, then place the centrifuge tube on a magnetic stand and let it stand When the liquid is clear (2-5min), the supernatant is carefully aspirated with a pipette and discarded.

② After completing step ①, keep the centrifuge tube on the magnetic stand, add 200 μL 80% ethanol aqueous solution, rinse the magnetic beads and the tube wall, let stand for 30 seconds, carefully pipette and discard the supernatant.

③ After completing step ②, keep the centrifuge tube on the magnetic stand, add 200 μL 80% ethanol aqueous solution, rinse the magnetic beads and the tube wall, let stand for 30 seconds, carefully aspirate and discard the supernatant.

④ After completing step ③, keep the centrifuge tube fixed on the magnetic stand, open the cap of the centrifuge tube, and dry at room temperature until the surface of the magnetic beads has no reflection.

⑤ After completing step ④, remove the centrifuge tube from the magnetic stand, add 21 μL TE Buffer, gently pipette until completely mixed, incubate at room temperature for 5 minutes, then centrifuge briefly, and then place the centrifuge tube on the magnetic stand Let stand until the liquid is clear (2-5min), and transfer the supernatant (that is, the purified DNA, about 19 μL) to a new 0.2mL PCR tube.

9. Use EZ DNA Methylation-Gold Kit (Zymo Research, Cat.No.D5005/D5006) to Bisulfite treat and purify the adapter ligation product.

The method is the same as step 15 in step 2 of Example 1, the only difference is that the purified DNA obtained in step 9 is used to replace the purified DNA obtained in step 14 in the original step.

10. Transfer all the product solution obtained in step 9 to a new 0.2mL PCR tube, and add water to 20μL.

11. Same as 17 in Step 2 of Example 1.

12. Take the PCR tube that completed step 10, add 30 μL of the PCR reaction solution prepared in step 11, vortex 3 times (3 seconds each time), and then centrifuge briefly to collect the reaction solution to the bottom of the tube.

13. Take the PCR tube that has completed step 12, put it on the PCR instrument, perform the reaction according to Table 12, and then centrifuge briefly to collect the reaction solution to the bottom of the tube, then add TE Buffer to a total volume of 100 μL, and then transfer all to a new 1.5 μL tube. mL centrifuge tube.

14. DNA purification

① After step 13 is completed, take the centrifuge tube, add 55 μL DNA Clean Beads, gently blow with a pipette until all the magnetic beads are suspended, incubate at room temperature for 5 minutes, then centrifuge briefly, then place the centrifuge tube on the magnetic stand, statically Place until the liquid is clarified (2-5min), carefully draw the supernatant with a pipette, and transfer to a new 1.5mL centrifuge tube.

② Take the centrifuge tube that has completed step ①, add 25 μL DNA Clean Beads, gently blow and mix with a pipette, incubate at room temperature for 5 minutes, then centrifuge briefly, then place the centrifuge tube on a magnetic stand, and let it stand until the liquid is clear (2 -5min), the supernatant was carefully aspirated with a pipette and discarded.

③ After completing step ②, keep the centrifuge tube on the magnetic stand, add 200 μL of 80% ethanol aqueous solution, rinse the magnetic beads and the tube wall, let stand for 30 seconds, then carefully aspirate the supernatant and discard it.

④ After completing step ③, keep the centrifuge tube on the magnetic stand, add 200 μL of 80% ethanol aqueous solution, rinse the magnetic beads and the tube wall, let stand for 30 seconds, then carefully aspirate the supernatant and discard it.

⑤ After completing step ④, keep the centrifuge tube on the magnetic stand, open the cap of the centrifuge tube, and dry at room temperature until the surface of the magnetic beads is no longer reflective.

⑥ After completing step ⑤, remove the centrifuge tube from the magnetic stand, add 32 μL TE Buffer, gently blow with a pipette until completely mixed, incubate at room temperature for 5 minutes, then centrifuge briefly, and then place the centrifuge tube on the magnetic stand , let it stand until the liquid is clear (2-5min), and transfer the supernatant to a new 1.5mL centrifuge tube with a pipette.

The supernatant obtained in step ⑥ is the purified DNA, about 30 μL.

The supernatant obtained in step ⑥ can be used as a library solution and directly sequenced on the illumina sequencing platform.

15. Take the purified DNA obtained in step 14 (DNA content is 360ng) into a new 0.2mL PCR tube, add TE Buffer to a total volume of 48μL.

16. After completing step 15, place the PCR tube on the PCR instrument, and perform the reaction according to Table 13.

17. According to Table 14, prepare the single-chain cyclization reaction solution on ice. Table 14 shows the volume of a single reaction system (12.1 μl). If multiple reaction systems are needed, just double it.

18. Immediately after the reaction in step 16, put the PCR tube on ice and let it stand for 2 minutes, then add 12.1 μL of the single-strand circularization reaction solution prepared in step 17, vortex 3 times (3 seconds each time), and then Centrifuge briefly to collect the reaction solution to the bottom of the tube, then place the PCR tube on the PCR instrument, and perform the reaction according to Table 15.

19. Prepare enzyme digestion reaction solution on ice according to Table 16. Table 16 shows the volume (4 μl) of a single reaction system. If multiple reaction systems are needed, just double it.

20. After the reaction in step 18 is completed, centrifuge the PCR tube briefly and place it on ice, immediately add 4 μL of the enzyme digestion reaction solution prepared in step 19, vortex 3 times (3 seconds each time), and then centrifuge briefly The reaction solution was collected at the bottom of the tube, and then the PCR tube was placed on the PCR machine, and the reaction was carried out according to Table 17, and then the reaction solution was collected at the bottom of the tube by instantaneous centrifugation.

21. After completing step 20, immediately add 7.5 μL Digestion Stop Buffer to the PCR tube, vortex 3 times (3 seconds each time), then centrifuge briefly to collect the reaction solution to the bottom of the tube, absorb all the reaction solution and transfer it to a new PCR tube. in a 1.5mL centrifuge tube.

22. DNA purification

①After completing step 21, take the centrifuge tube, add 170 μL DNA Clean Beads, gently pipette until all the magnetic beads are suspended, incubate at room temperature for 10 minutes, then centrifuge briefly, and then place the centrifuge tube on the magnetic stand , let stand until the liquid is clear (2-5min), carefully draw the supernatant with a pipette and discard it.

② After completing step ①, keep the centrifuge tube on the magnetic stand, add 500 μL of 80% ethanol aqueous solution, rinse the magnetic beads and the tube wall, let it stand for 30 seconds, and then carefully aspirate the supernatant and discard it.

③ After completing step ②, keep the centrifuge tube on the magnetic stand, add 200 μL of 80% ethanol aqueous solution, rinse the magnetic beads and the tube wall, let stand for 30 seconds, then carefully aspirate the supernatant and discard it.

④ After completing step ③, keep the centrifuge tube fixed on the magnetic stand, open the cap of the centrifuge tube, and dry at room temperature until the surface of the magnetic beads has no reflection.

⑤ After completing step ④, remove the centrifuge tube from the magnetic stand, add 22 μL TE Buffer, gently pipette until completely mixed, incubate at room temperature for 10 minutes, then centrifuge briefly, and then place the centrifuge tube on the magnetic stand Leave it to stand until the liquid is clear (2-5min), and transfer the supernatant (that is, the product solution, about 20 μL) to a new 1.5mL PCR tube.

The supernatant obtained in step ⑤ can be used as a library solution.

23. Use

ssDNA Assay Kit Fluorescence Quantitative Kit, Quantify the product solution obtained in step 22 according to the operating instructions of the quantitative kit.

24. Take the product solution obtained in step 22, use the MGISEQ-2000 high-throughput sequencing reagent set (PE100) (MGI, product number 1000012536) and follow the instructions to make DNB (DNA nanoballs), and then use the MGISEQ-2000 sequencer to perform sequencing.

The database construction results are shown in Table 22. The PCR concentration refers to the DNA concentration of the library solution obtained in step 14. The single-stranded circular library concentration refers to the DNA concentration of the library solution obtained in step 22. The DNB concentration refers to the DNA concentration of the solution after DNB production.

Table 22

sample name	PCR concentration (ng/μL)	Single-stranded library concentration (ng/μL)	DNB concentration (ng/μL)
cfDNA-1	6.84	0.82	20.2

The sequencing results are shown in Figure 5. The statistical results of the data are shown in Table 23. Sequencing data analysis shows that the proportion of C bases in the bases at each position on the reads is higher than that of WGBS.

Table 23

sample name	cfDNA-1
Sequencing length (bp)	100
Sequence ratio after filtering (%)	98.4
number of sequences after filtering	1164518152
Base number after filtering (bp)	1.16452E+11
Number of aligned sequences	1025578414
Aligned bases (bp)	1.02558E+11

Contrast rate (%)	93.23
Repeat rate (%)	14.79
Number of Unique Aligned Sequences	979187833
Number of uniquely aligned bases (bp)	97918783300
Unique comparison rate (%)	89.02
Number of paired-end aligned sequences	941240780
Double-ended alignment ratio (%)	85.57
Number of single-end aligned sequences	84337634
Single-ended alignment rate (%)	7.67
Number of mismatched bases (bp)	1300744759
Mismatch rate (%)	1.27
Sulphite conversion rate (%)	99.60

The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experiments, the present invention can be practiced in a wider range under equivalent parameters, concentrations and conditions. While specific embodiments of the invention have been shown, it should be understood that the invention can be further modified. In a word, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by using conventional techniques known in the art and departing from the disclosed scope of this application. Applications of some of the essential features are possible within the scope of the appended claims below.

industrial application

The invention disclosed has the following effects: the invention can prevent unmethylated cytosine C from being converted, and only convert methylated modified cytosine 5mC, hydroxymethylated cytosine 5hmC, and formylated cytosine 5fC and carboxylated cytosine 5caC are uracil U, so as to solve the problem of low library complexity in WGBS and EM-seq, thereby improving the quality of genome-wide methylation sequencing, and also improving the comparison rate and comparison of sequencing data Accuracy, but also improve the sensitivity of methylation detection. The invention can be used in the fields of tumor early screening and liquid biopsy.

Claims (14)

1. A method for preparing a library for detecting nucleic acid methylation modifications, comprising the steps of:

   (1) converting the methylation site in the nucleic acid sample by enzymatic reaction;

   (2) converting unmodified C into methylated C by enzymatic reaction;

   (3) Treating with bisulfite to obtain the converted nucleic acid library.
2. The method of claim 1, characterized in that:

   The "converting the methylation site in the nucleic acid sample by enzymatic reaction" is "converting 5mC, 5hmC and 5fC in the nucleic acid sample to 5caC by enzymatic reaction";

   The "convert unmodified C into methylated C by enzymatic reaction" is "convert unmodified C to 5mC by enzymatic reaction";

   The "treatment with bisulfite" means "treatment with bisulfite to convert 5caC into U while keeping 5mC unchanged".
3. The method according to claim 1 or 2, characterized in that: the method further comprises at least one of the following two steps: ① breaking; ② end repair and joint; the step ① and/or step ② Located between the step (2) and the step (3).
4. The method according to claim 3, characterized in that: the cytosine in the linker is methylated modified cytosine.
5. The method according to claim 1 or 2, characterized in that: the method further comprises at least one of the following two steps: ① nucleic acid fragmentation; ② nucleic acid end repair and adapter; the step ① and/or Step ② is located before step (1).
6. The method according to any one of claims 1 to 5, characterized in that:

   In "converting the methylation site in the nucleic acid sample by an enzymatic reaction", the enzyme required for the enzymatic reaction is an oxidase;

   In "converting unmodified C into methylated C by enzymatic reaction", the enzymes required for the enzymatic reaction are methyltransferase and S-adenosylmethionine.
7. The method according to claim 6, characterized in that: the oxidase is methylcytosine dioxygenase; the methyltransferase is CpG methyltransferase.
8. A method for detecting nucleic acid methylation modification, comprising the steps of:

   Prepare a library sample by any one of the methods described in claims 1 to 7;

   Perform library construction to obtain a nucleic acid sequencing library;

   Perform nucleic acid sequence determination.
9. A kit for preparing a library for detecting nucleic acid methylation modification, comprising the following components: oxidase, methyltransferase and S-adenosylmethionine.
10. The kit according to claim 9, characterized in that: the oxidase is methylcytosine dioxygenase; the methyltransferase is CpG methyltransferase.
11. The kit according to claim 9 or 10, characterized in that: the kit further comprises the following components: a reagent or a combination of reagents for treating nucleic acid with bisulfite.
12. The kit according to claim 9 or 10, characterized in that: the kit also includes at least one of the following components: ① reagents or reagent combinations for nucleic acid fragmentation; ② reagents for nucleic acid end repair and adapters or reagent combinations.
13. Application of the method according to any one of claims 1 to 8 or the kit according to any one of claims 9 to 12 in detecting nucleic acid methylation modification.
14. The application according to claim 13, characterized in that: the nucleic acid methylation modification is the methylation modification at the C5 position of cytosine.

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