Method for performing bisulfite conversion and DNA fragmentation on DNA and methylation library preparation method adopting same

Abstract

The invention relates to the technical field of methylation sequencing, in particular to a low-cost methylation library preparation method for simultaneously performing bisulfite conversion and DNA fragmentation. Compared with a method for fragmenting the genome DNA by using an ultrasonic breaking method, the method disclosed by the invention has the advantage that the cost for fragmenting the genome DNA in the methylation library preparation process is greatly reduced.

Description

Technical Field

[0001] This application relates to the field of methylation sequencing technology, and in particular to a method for preparing methylated libraries that simultaneously perform bisulfite conversion and DNA fragmentation. Background Technology

[0002] Second-generation high-throughput sequencing, also known as next-generation sequencing technology, can sequence hundreds of thousands to millions of nucleic acid molecules in a single run. Currently, in scientific research, second-generation sequencing is mainly used for genome sequencing, transcriptome sequencing, population sequencing, amplicon sequencing, metagenomic sequencing, and resequencing. Clinically, it plays an increasingly important role in assisted reproduction (e.g., non-invasive prenatal testing, NIPT), genetic diseases (e.g., screening for pathogenic mutations), and cancer research (e.g., early diagnosis, medication guidance, and prognosis).

[0003] DNA methylation is a major form of epigenetic modification of genomic DNA. Numerous studies in recent years have shown a close link between abnormal DNA methylation and the occurrence, development, and carcinogenesis of tumors. A gold standard for methylation sequencing is whole genome bisulfite sequencing (WGBS), or BS-seq for short. Its detection principle involves treating the genome with bisulfite to convert unmethylated C bases to U, which are then amplified by PCR to become T, distinguishing them from the originally methylated C bases. This is then combined with high-throughput sequencing technology and compared with a reference sequence to determine whether methylation has occurred at CpG / CHG / CHH sites (where H represents any one of A, T, or C). To achieve this process, a genome library must first be constructed, with suitable adapters added, and then sequencing performed on a sequencing platform. Depending on the order in which bisulfite is added during library construction, library construction can be divided into pre-BS (pre-transformation library construction) and post-BS (post-transformation library construction). Pre-BS library preparation requires a relatively large investment, and the constructed libraries are often AT-biased, easily losing regions with high CpG content and important epigenetic information. Post-BS library preparation, on the other hand, involves first treating the DNA with bisulfite, followed by ligation or PCR for library construction. It requires a smaller initial sample size and can meet the sequencing needs of various sample types, such as cfDNA (cell-free DNA) and FFPE DNA.

[0004] Before library construction, DNA, especially genomic DNA, needs to be fragmented. Current technologies mostly use methods such as ultrasound to fragment DNA. However, ultrasound methods require specialized imported equipment and consumables, resulting in high costs and relatively long procedures. Therefore, a low-cost and simplified method for DNA fragmentation is needed. Summary of the Invention

[0005] The purpose of this application is to provide a low-cost method for bisulfite conversion and fragmentation of DNA, and a method for preparing methylated libraries by simultaneously performing bisulfite conversion and DNA fragmentation. By performing bisulfite conversion and DNA fragmentation simultaneously during library construction, the cost of fragmenting genomic DNA during methylated library preparation is significantly reduced, and the total volume of the constructed library is significantly greater than that of the library constructed by sonication fragmentation (see the examples and accompanying drawings).

[0006] The specific technical solution of this application is as follows:

[0007] 1. A method for bisulfite conversion and fragmentation of DNA, comprising the following steps: after bisulfite conversion of DNA, the reaction product is shaken with the aid of glass beads to fragment the DNA; wherein the glass beads have a diameter of 0.1-0.2 mm.

[0008] 2. The method according to item 1, wherein the oscillation treatment is a constant temperature oscillation treatment; the constant temperature can be selected between 50-85℃; preferably between 64-80℃.

[0009] 3. The method according to item 1 or 2, wherein the oscillation time can be selected as 0.5-5 hours.

[0010] 4. The amount of glass beads added according to any one of items 1-3 is 50-200 mg.

[0011] 5. The method according to any one of items 1-4, wherein the DNA is whole genome DNA.

[0012] 6. A method for constructing a whole-genome methylation library, comprising the following steps:

[0013] (1) Extraction and isolation of target genomic DNA from biological samples;

[0014] (2) Perform bisulfite conversion and fragmentation on the extracted genomic DNA according to any one of items 1-5;

[0015] (3) Repair the ends of genomic DNA fragments and add 3' tails;

[0016] (4) Connect adapter 1 to one end of the genomic DNA fragment using ligase, and after extension reaction, purify the above product;

[0017] (5) Use ligase to connect adapter 2 to the other end of the genomic DNA fragment again, and purify the genomic DNA fragment with adapters 1 and 2 connected;

[0018] (6) The whole genome methylation library was obtained by PCR amplification and purification of the product;

[0019] The biological sample can be fresh tissue, FFPE sample (formalin-fixed paraffin-embedded sample), cells, or whole blood.

[0020] 7. The method for constructing a whole genome methylation library according to item 6, wherein the purification steps in steps (4) and (5) are performed using magnetic beads or purification columns; when using a purification column, the purification column may be ZYMO's C1003-50 purification column.

[0021] 8. The method for constructing a whole genome methylated library according to item 6 or 7, wherein in the fragmentation step, the genomic DNA is broken into fragments of 100-500 bp in length, preferably fragments of 150-400 bp in length, and most preferably fragments of 150-300 bp in length; the purification step in step (6) is purified using magnetic beads.

[0022] 9. The method for constructing a whole-genome methylated library according to any one of items 6-8, wherein step (3) includes a DNA denaturation step.

[0023] 10. The method for constructing a whole genome methylation library according to any one of items 6 to 9, wherein, in step (6), after amplifying the library by PCR and purifying the product, a quality detection step is further included: detecting the length of the amplified product fragment; when the product fragment length is 150bp-400bp, the obtained product is a whole genome methylation library; preferably, the product fragment length is 150-300bp.

[0024] Technical effect

[0025] This application provides a fragmentation method and library construction method for methylation sequencing, enabling whole-genome methylation sequencing. This method eliminates the need for expensive, dedicated imported fragmentation equipment and tubes, simultaneously performing sulfite conversion and genome mechanical fragmentation, allowing for direct use in whole-genome library construction or the construction of methylation target regions. The DNA fragmentation method described in this application is low-cost and streamlined.

[0026] This method is not only simple, fast, and cost-effective, but it can also: 1) be used for methylated whole genome library construction, and the total amount of the constructed library is significantly better than that of the library constructed by sonication fragmentation (see the attached figures in the examples and instructions); 2) be applicable to all types of DNA samples, such as fresh tissue genomes, FFPE (Formalin-Fixed and Parrffin-Embedded) genomes, cell genomes, and whole blood genomes; 3) be subsequently used for the construction of methylation target regions. Attached Figure Description

[0027] Figure 1 This is a diagram showing the effect of bisulfite on DNA disruption. Figure 1 The lane descriptions for the electrophoresis images are as follows: M1: DL10000 marker; M2: DL1000 marker; M3: DL10000 marker; 1-8 correspond to the treatment conditions in Table 1, respectively; 9 and 10: S1 and S2 were sonicated, respectively; 11 and 12 are the untreated genomes of S1 and S2, respectively.

[0028] Figures 2A-2D This is a quality control peak diagram of the whole genome library prepared in Example 2 of this application. Figure 2A Corresponding to sample number 1 in Table 17; Figure 2B Corresponding to sample number 2 in Table 17; Figure 2C Corresponding to sample number 3 in Table 17; Figure 2D This corresponds to sample number 4 in Table 17. Detailed Implementation

[0029] The present invention will now be described in detail. While specific embodiments of the invention have been shown, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the invention and to fully convey the scope of the invention to those skilled in the art.

[0030] It should be noted that certain terms are used in the specification and claims to refer to specific components. Those skilled in the art will understand that different terms may be used to refer to the same component. This specification and claims do not distinguish components based on differences in terminology, but rather on differences in function. The terms "comprising" or "including" used throughout the specification and claims are open-ended and should be interpreted as "comprising but not limited to." The following descriptions are preferred embodiments for carrying out the invention; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of the invention. The scope of protection of this invention is determined by the appended claims.

[0031] Explanation of some definitions in this application:

[0032] Library construction: also known as library building, refers to the process of randomly fragmenting DNA extracted from samples such as blood and tissue, repairing and ligating it to a known DNA fragment, namely the adapter sequence (also known as a linker), so that it can be used for high-throughput DNA sequencing on next-generation sequencing platforms (in this application, it is used for the Illumina sequencing platform).

[0033] Bisulfite is a widely used chemical reagent in DNA methylation research. Its core principle is to convert unmethylated cytosine (C) to uracil (U) through a deamination reaction, while methylated cytosine (5-methylcytosine, 5mC) remains unaffected. This difference allows subsequent PCR amplification and sequencing to distinguish between methylated and unmethylated DNA, thus enabling accurate detection of DNA methylation status.

[0034] Adapter: A universal, known sequence that can be ligated to a DNA fragment by a ligase. Adapter 1 and Adapter 2 used in this application are both derived from commercially available IDT xGen Methyl-Seq Lib KIT (catalog number 10009824).

[0035] Annealing is the process by which single-stranded DNA is denatured and then slowly cooled to form double-stranded DNA.

[0036] DNA ligases: The DNA ligases available in this application are selected from one or more of the following: NAD-dependent ligases, such as Taq DNA ligase, *Thermus filiformis* DNA ligase, *E. coli* DNA ligase, Tth DNA ligase, *Thermus scotoductus* DNA ligase (I and II), thermostable ligases, Ampligase thermostable DNA ligase, VanC-type ligase, and 9°N DNA ligase; and ATP-dependent ligases, including T4 RNA ligase, T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, Pfu DNA ligase, DNA ligase 1, DNA ligase III, and DNA ligase IV. The preferred DNA ligase is T4 DNA ligase.

[0037] NF water: refers to water without nucleases.

[0038] Specifically, this application provides the following technical solutions:

[0039] In a first aspect, this application provides a method for bisulfite conversion and fragmentation of DNA, comprising the following steps: after bisulfite conversion of DNA, the reaction product is subjected to oscillation treatment with the assistance of glass beads to fragment the DNA; the diameter of the glass beads is 0.1-0.2 mm; the oscillation treatment is a constant temperature oscillation treatment; the constant temperature is between 50-85°C.

[0040] In some implementations, the constant temperature is between 64-80°C.

[0041] In some implementations, the oscillation time is 0.5-5 hours; optionally 2-2.5 hours; further optionally 2.5 hours.

[0042] In some implementations, the amount of glass beads added is 50-200 mg; optionally 100-150 mg, and further optionally 100 mg.

[0043] In some implementations, the DNA is whole-genome DNA.

[0044] In a second aspect, this application provides a method for constructing a whole-genome methylation library, comprising the following steps: (1) extracting and separating target genomic DNA from a biological sample; (2) performing bisulfite conversion and fragmentation on the extracted genomic DNA according to the method described in the first aspect of this application; (3) repairing the ends of the genomic DNA fragments and adding 3' tails; (4) using a ligase to connect adapter 1 to one end of the genomic DNA fragment, and purifying the product after an extension reaction; (5) again using a ligase to connect adapter 2 to the other end of the genomic DNA fragment, and purifying the genomic DNA fragment connected with adapters 1 and 2; (6) amplifying the library by PCR and purifying the product to obtain a whole-genome methylation library; wherein the biological sample may be fresh tissue, FFPE sample (i.e., formalin-fixed paraffin-embedded sample), cells, or whole blood.

[0045] In some implementations, the purification steps in steps (4) and (5) are performed using magnetic beads or purification columns; when purification is performed using a purification column, the purification column may be ZYMO's C1003-50 purification column.

[0046] In some embodiments, in the fragmentation step, the genomic DNA is broken into fragments of 100-500 bp in length, preferably 150-400 bp in length, and most preferably 150-300 bp in length; the purification step in step (6) is performed using magnetic beads.

[0047] In some implementations, step (3) includes a DNA denaturation step.

[0048] In some implementations, in step (6), after amplifying the library by PCR and purifying the product, a quality detection step is also included: detecting the length of the amplified product fragment; when the product fragment length is 150bp-400bp, the product obtained is a whole genome methylation library; the product fragment length can be 150-300bp.

[0049] Example

[0050] This application provides a general and / or specific description of the materials and test methods used in the experiments. In the following examples, unless otherwise specified, % represents wt%, i.e., weight percentage. Reagents or instruments used, unless otherwise specified, are all commercially available conventional reagents or instruments.

[0051] Example 1: The effect of genomic DNA fragmentation during bisulfite conversion

[0052] To verify the effectiveness of DNA fragmentation during bisulfite conversion, we randomly selected two whole blood samples, named S1 and S2, and designed different conversion conditions to evaluate the DNA fragmentation effect. Specific conditions are shown in Table 1. Genomic DNA was extracted using a blood / cell / tissue genomic DNA extraction kit (centrifuge column type), catalog number DP304. The bisulfite conversion method used was ZYMO's EZ DNA Methylation-Gold. TM Kit, item number D5005. The specific operating procedure is as follows:

[0053] 1.1 Whole blood genomic extraction

[0054] 1.1.1. Take 200 μl of whole blood; if insufficient, supplement with GA solution.

[0055] 1.1.2. Add 20 μl of Proteinase K solution and mix well.

[0056] 1.1.3. Add 200 μl of buffer GB, mix thoroughly by inverting, incubate at 70°C for 10 min, the solution should become clear, and briefly centrifuge to remove water droplets from the inner wall of the tube cap.

[0057] 1.1.4. Add 200 μl of anhydrous ethanol and shake thoroughly for 15 seconds. At this time, flocculent precipitate may appear. Briefly centrifuge to remove water droplets from the inner wall of the tube cap.

[0058] 1.1.5. Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3 (place the adsorption column in the collection tube), centrifuge at 12,000 rpm (~13,400×g) for 30 s, discard the waste liquid, and put the adsorption column CB3 back into the collection tube.

[0059] 1.1.6. Add 500 μl of buffer GD to the adsorption column CB3 (please check whether anhydrous ethanol has been added before use), centrifuge at 12,000 rpm (~13,400×g) for 30 s, discard the waste liquid, and put the adsorption column CB3 into the collection tube.

[0060] 1.1.7. Add 600 μl of washing buffer PW to the adsorption column CB3 (please check whether anhydrous ethanol has been added before use), centrifuge at 12,000 rpm (~13,400×g) for 30 s, discard the waste liquid, and place the adsorption column CB3 into the collection tube.

[0061] 1.1.8. Repeat step 1.1.7.

[0062] 1.1.9. Place the adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm (~13,400 × g) for 2 min, and discard the waste liquid. Place the adsorption column CB3 at room temperature for several minutes to thoroughly dry any residual washing liquid in the adsorption material.

[0063] 1.1.10. Transfer the adsorption column CB3 into a clean centrifuge tube, add 50-200 μl of elution buffer TE dropwise to the middle of the adsorption membrane, incubate at room temperature for 2-5 min, centrifuge at 12,000 rpm (~13,400×g) for 2 min, and collect the solution into the centrifuge tube.

[0064] Note: The GA, GB, GD, and PW solutions used in section "1.1" and the CB3 adsorption column are all derived from the Blood / Cell / Tissue Genomic DNA Extraction Kit (centrifuge column type) with catalog number DP304.

[0065] 1.2 Bisulfite transformation and fragmentation (i.e., fragmentation) of genomic DNA

[0066] 1.2.1. Preparation of CT conversion reagent: Add 700 μl NF water, 300 μl M-Dilution Buffer and 50 μl M-Dissolving Buffer to CT conversion reagent powder (from the aforementioned kit with catalog number D5005), and mix at room temperature;

[0067] 1.2.2. Bisulfite conversion of DNA: Take PCR tubes and prepare the reaction system according to Table 1:

[0068] Table 1

[0069]

[0070]

[0071] 1.2.3. Run the PCR program:

[0072] Hot cap temperature: 105℃; 98℃, 10min; cool down to 64℃, proceed directly to the next step.

[0073] 1.2.4. In the previous step, take a new 2.0ml centrifuge tube, add 100mg of glass beads (from Bocheng Company's reagent kit, Beijing Medical Device Registration No. 20170103, kit number C-025), and place it on a preheated 64℃ or 80℃ constant temperature shaker. Set the shaker program to 64℃ or 80℃ for 2.5h.

[0074] 1.2.5. Transfer the PCR product to the 2.0 ml centrifuge tube and incubate with shaking for 2.5 h;

[0075] 1.2.6. Add 600 μl of M-Binding Buffer to the 2.0 ml centrifuge tube containing the converted material from step 1.2.5, and vortex to mix.

[0076] 1.2.7. Add the above-mixed sample to Zymo-Spin TM In the IC column, mix by inversion, centrifuge at 10,000 x g for 30 s; load the sample into a Zymo-Spindle. TM When using IC Column, try to avoid picking up glass beads;

[0077] 1.2.8. Add 100 μl of M-Wash Buffer to the column and centrifuge at 10,000 x g for 30 s;

[0078] 1.2.9. Add 200 μl of M-Desulphonation Buffer to the column, let stand at room temperature for 15-20 min, and centrifuge at 10,000 x g for 30 s;

[0079] 1.2.10. Add 200 μl of M-Wash Buffer to the column and centrifuge at 10,000 x g for 30 s;

[0080] 1.2.11. Repeat step 1.2.10 once;

[0081] 1.2.12. Place the column into a new collection tube and centrifuge again at 10,000 x g for 30 s to better remove the liquid from the column;

[0082] 1.2.13. Place the recovery column into a new 1.5 ml EP tube, add 13 μl of LOW EDTA buffer to the center of the column membrane, and centrifuge at 10,000 x g for 30 s;

[0083] Note: The NF water, M-Dilution Buffer, M-Dissolving Buffer, CT conversion reagent, M-Binding Buffer, and Zymo-Spin used in section "1.2" are... TM The IC column, M-Wash Buffer, M-Desulphonation Buffer, LOW EDTA buffer, and column are all derived from ZYMO EZ DNA Methylation-Gold. TM Kit, item number D5005.

[0084] The conditions under which the effect of simultaneous bisulfite conversion and DNA fragmentation was tested are shown in Table 2.

[0085] Table 2. Test conditions for the effect of DNA fragmentation during bisulfite conversion

[0086]

[0087]

[0088] The test results of Example 1 verified that bisulfite combined with glass beads can achieve DNA fragmentation. Based on the fragmentation effects shown in Table 2, we can see that doubling the reagent dosage has little effect. Using the normal dosage of reagent, DNA fragmentation can be achieved at both temperatures. Considering that the transformation kit's transformation temperature is 64℃, 64℃ was chosen as the final screening transformation condition.

[0089] Example 2: Preparation of whole-genome methylation library

[0090] 2.1-2.2 Genomic DNA extraction, bisulfite conversion and DNA fragmentation are performed as described in Example 1; except that the temperature of the isothermal oscillator is set to 64°C.

[0091] 2.3 Transformation:

[0092] 2.3.1. Preheat the PCR instrument to 95℃.

[0093] 2.3.2. Set the program shown in Table 3 to perform the reaction on the PCR instrument: hot lid temperature 105℃.

[0094] Table 3

[0095] temperature time 95℃ ∞ 95℃ 2min 95℃ ∞

[0096] 2.3.3. After incubation, immediately place the test tube on ice for 2 minutes.

[0097] 2.4. Connector connection and purification:

[0098] 2.4.1. Configure the reaction system according to Table 4:

[0099] Table 4

[0100]

[0101]

[0102] 2.4.2. Set the following program to perform the reaction on the PCR instrument: hot lid temperature 105℃.

[0103] Table 5

[0104] temperature time 37℃ ∞ 37℃ 15min 95℃ 2min 4℃ ∞

[0105] 2.5. Sample extension and purification:

[0106] 2.5.1. Configure the reaction system according to Table 6:

[0107] Table 6

[0108] Components volume Reagent Y1 2ul Enzyme Y2 42ul totalVolume 44ul

[0109] 2.5.2. Set the program shown in Table 7 to perform the reaction on the PCR instrument: hot lid temperature 105℃.

[0110] Table 7

[0111] temperature time 98℃ ∞ 98℃ 1min 62℃ 2min 65℃ 5min 4℃ ∞

[0112] 2.5.3. The DNA protection buffer turns the liquid blue upon addition. Gently pipette and mix well, then divide into two tubes and place them on the PCR instrument.

[0113] 2.5.4. Set the program as shown in Table 8 and run it: hot cover 105℃.

[0114] Table 8

[0115]

[0116]

[0117] 2.5.5. Prepare the purification system according to Table 9:

[0118] Table 9

[0119]

[0120] 2.5.6. Add 1.2 times the volume of the reaction system of magnetic beads (as shown in Table 9) to each sample for recovery, shake to mix and then briefly separate.

[0121] 2.5.7. Incubate at room temperature for 5 minutes.

[0122] 2.5.8. Shake to mix, then centrifuge briefly and place on a magnetic rack to adsorb until the solution is clear (~2 min). After the solution is clear, remove the supernatant.

[0123] 2.5.9. Add 200 μl of 80% ethanol to wash the magnetic beads for 30 seconds, discard the supernatant, and carefully remove all remaining ethanol from the inner wall of the dropper.

[0124] 2.5.10. Repeat the above steps.

[0125] 2.5.11. Add the volume of low EDTA TE buffer described in Table 9 for elution, then vortex to mix.

[0126] 2.5.12. Incubate at room temperature for 2 minutes.

[0127] 2.5.13. Place the solution on a magnetic rack for adsorption until the solution becomes clear (~2 min). After the solution becomes clear, remove the supernatant.

[0128] 2.5.14. Transfer the entire eluent to a new 0.2 mL PCR tube, ensuring that the eluent does not contain magnetic beads.

[0129] 2.6. Connector connection and purification:

[0130] 2.6.1. Prepare the library reaction system according to Table 10:

[0131] Table 10

[0132] Components volume Buffer B1 3ul Reagent B2 10μl Enzyme B3 2ul TotalVolume 15ul

[0133] 2.6.2. Set the program as shown in Table 11 and run it: Hot lid 50℃:

[0134] Table 11

[0135]

[0136] 2.6.3. Prepare the purification system according to Table 12:

[0137] Table 12

[0138]

[0139] 2.6.4. Add 1.2 times the volume of the reaction system of magnetic beads (as shown in Table 12) to each sample for recovery, shake to mix and then briefly separate.

[0140] 2.6.5. Incubate at room temperature for 5 minutes.

[0141] 2.6.6. Shake to mix, then centrifuge briefly and place on a magnetic rack to adsorb until the solution is clear (~2 min). After the solution is clear, remove the supernatant.

[0142] 2.6.7. Add 200 μl of 80% ethanol to wash the magnetic beads for 30 seconds, discard the supernatant, and carefully remove all remaining ethanol from the inner wall of the dropper.

[0143] 2.6.8. Repeat the above steps.

[0144] 2.6.9. Add the recommended volume of low EDTA TE buffer from Table 12 for elution, then vortex to mix.

[0145] 2.6.10. Incubate at room temperature for 2 minutes.

[0146] 2.6.11. Place on a magnetic rack for adsorption until the solution becomes clear (~2 min). After the solution becomes clear, remove the supernatant.

[0147] 2.6.12. Transfer the entire eluent to a new 0.2 mL PCR tube, ensuring that the eluent does not contain magnetic beads.

[0148] 2.7. Library amplification and purification:

[0149] 2.7.1. Prepare the library reaction system according to Table 13:

[0150] Table 13

[0151] Components volume The above reaction DNA 20ul KAPA HiFi HotStart Uracil+ReadyMix(2x) 25ul index(U001-U024) 5μl Total volume 50ul

[0152] 2.7.2. Set the program as shown in Table 14 and run it: Hot lid 105℃:

[0153] Table 14

[0154]

[0155]

[0156] 2.7.3. Recommended number of loops is shown in Table 15:

[0157] Table 15

[0158] Input Recommended number of loops 20ng cfDNA 10-11 100ng gDNA 9-10 20ng gDNA 11-12

[0159] 2.7.4. Prepare the purification system according to Table 16:

[0160] Table 16

[0161]

[0162] 2.7.5. Transfer the PCR product into a 1.5 ml centrifuge tube.

[0163] 2.7.6. Add magnetic beads of the volume ratio shown in Table 16 to each sample for recycling, shake to mix and then briefly separate.

[0164] 2.7.7. Incubate at room temperature for 5 minutes.

[0165] 2.7.8. Shake to mix, then centrifuge briefly and place on a magnetic rack to adsorb until the solution is clear (~2 min). After the solution is clear, aspirate the supernatant.

[0166] 2.7.9. Add 500 μl of 80% ethanol to wash the magnetic beads for 30 seconds, discard the supernatant, and carefully remove all remaining ethanol from the inner wall of the dropper.

[0167] 2.7.10. Repeat step 2.7.9 above.

[0168] 2.7.11. Place on the magnetic rack for 5-10 minutes until the beads are dry (avoid over-drying, as this may reduce DNA recovery rate).

[0169] 2.7.12. Add the recommended volume of low EDTA TE buffer from Table 16 for elution, then vortex to mix.

[0170] 2.7.13. Incubate at room temperature for 2 minutes.

[0171] 2.7.14. Place on a magnetic rack for adsorption until the solution becomes clear (~2 min). After the solution becomes clear, remove the supernatant.

[0172] 2.7.15. Transfer the entire eluent to a new 0.2 mL PCR tube, ensuring that the eluent does not contain magnetic beads.

[0173] 2.7.16. Extract 1 μL for qubit calibration and perform 2100 quality inspection.

[0174] Note: The Low EDTA TE buffer, Buffer G1, Reagent G2, Reagent G3, Enzyme G4, Enzyme G5, Enzyme G6, Reagent Y1, Enzyme Y2, Buffer B1, Reagent B2, Enzyme B3, KAPA HiFi HotStart Uracil+ReadyMix (2x), index (U001-U024), and magnetic beads used in steps “2.3”-“2.7” above are all derived from xGen Methyl-Seq Lib KIT (IDT, catalog number 10009824).

[0175] 2.8 Library quality control and sequencing

[0176] After library construction, fragment size and qPCR concentration were determined using an Agilent 2100 bioanalyzer. Once the samples passed the tests, sequencing was performed on a sequencer.

[0177] Table 17. Results of whole-genome library preparation

[0178] Serial Number Sample number Interruption method Initial sample size Cycle number Total number of documents 1 S1 This application 100ng 9 552ng 2 S2 This application 100ng 9 618ng 3 S1 Ultrasonic interruption 100ng 9 525ng 4 S2 Ultrasonic interruption 100ng 9 597ng

[0179] Table 18. Sequencing results of whole-genome library preparation are shown below (some key sequencing quality control indicators were selected).

[0180] As shown in Table 18, the sequencing metrics of the method described in this application are comparable to those of the sonication fragmentation method (as shown in columns 3-9 of Table 18). The library preparation and sequencing results obtained by the method described in this application are not significantly different from those obtained by the sonication fragmentation method, and can replace the traditional fragmentation method.

[0181] The method described in this application can be applied to the construction of various types of fully methylated libraries or libraries targeting specific regions. Using the fragmentation method described in this application, high-quality whole-genome libraries can be constructed, and the sequencing metrics are superior to those of commercially available post-conversion library preparation kits (such as important metrics like effective sequencing depth after deduplication, see the last column of Table 18).

Claims

1. A method of bisulfite conversion and fragmentation of DNA comprising the steps of: After the bisulfite conversion of DNA, the reaction product is treated with oscillation assisted by glass beads to fragment the DNA; The diameter of the glass beads is 0.1-0.2mm.

2. The method of claim 1, wherein, The oscillation treatment is constant temperature oscillation treatment; the constant temperature can be selected between 50-85℃; preferably between 64-80℃.

3. The method according to claim 1 or 2, wherein the oscillation time is selected between 0.5-5 hours.

4. The method according to any one of claims 1-3, wherein the amount of glass beads added is 50-200mg.

5. The method according to any one of claims 1-4, wherein the DNA is whole genome DNA.

6. A method for constructing a whole genome methylation library, comprising the following steps: (1) extracting and isolating the genomic DNA of interest from a biological sample; (2) bisulfite converting and fragmenting the extracted genomic DNA according to the method of any one of claims 1-5; (3) repairing the ends of the genomic DNA fragments and adding a tail to the 3’ end; (4) connecting adaptor 1 to one end of the genomic DNA fragments using ligase, purifying the product after extension reaction; (5) connecting adaptor 2 to the other end of the genomic DNA fragments using ligase again, purifying the genomic DNA fragments with adaptor 1 and 2 connected; (6) amplifying the library by PCR and purifying the product to obtain the whole genome methylation library; The biological sample can be fresh tissue, FFPE sample (formalin-fixed paraffin-embedded sample), cells or whole blood.

7. The method for constructing a whole genome methylation library according to claim 6, wherein the purification steps in steps (4) and (5) are completed using magnetic beads or purification columns; when using purification columns, the purification columns can be ZYMO’s purification columns with catalog number C1003-50.

8. The method for constructing a whole genome methylation library according to claim 6 or 7, wherein in the fragmentation step, the genomic DNA is broken into fragments with a length of 100-500bp, preferably 150-400bp, most preferably 150-300bp; the purification step in step (6) is completed using magnetic beads.

9. The method for constructing a whole genome methylation library according to any one of claims 6-8, wherein step (3) contains a DNA denaturation step.

10. The method for constructing a whole genome methylation library according to any one of claims 6 to 9, wherein, In step (6), after amplifying the library by PCR and purifying the product, a quality detection step is further included: detecting the length of the amplified product fragments, when the length of the product fragments is 150bp-400bp, the obtained product is a whole genome methylation library; preferably the length of the product fragments is 150-300bp.

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