Sequencing method for accurately and quantitatively detecting DNA methylation

Abstract

The invention provides a sequencing method for quantitatively detecting DNA methylation, which comprises the following steps: carrying out oxidation and transformation pretreatment on an extracted DNA sample, and then carrying out library construction and DNA methylation sequencing; the oxidizing reagent comprises potassium perruthenate or ruthenium tetroxide; the concentration of the oxidizing reagent is 100 to 300 mM; the oxidation temperature is 30 to 50 DEG C. According to the DNA methylation sequencing method, a sequencing library is constructed after a DNA sample is oxidized and converted, a Twist targeted DNA methylation hybridization technology is adopted for detection, and the DNA methylation degree is truly quantified; the oxidation temperature is improved, so that the oxidation pretreatment time is remarkably shortened, and the detection efficiency of DNA methylation is improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a sequencing method for accurately and quantitatively detecting DNA methylation. Background Art

[0002] DNA methylation and hydroxymethylation are covalent epigenetic mechanisms acting on DNA and can be exploited as DNA biomarkers. DNA methylation is generally regarded as a repressive epigenetic mark, which is mainly catalyzed by DNA methyltransferases (DNMTs) to generate 5-methylcytosine (5-mC or 5mC). In contrast, hydroxymethylation is the conversion of 5-mC to 5-hydroxymethylcytosine (5-hmC or 5hmC) through an oxidative reaction, which is catalyzed by the TETs family of dioxygenases. Through hydroxymethylation, TETs help maintain the unmethylated state of DNA, thereby playing an activating role in cellular transcription.

[0003] Twist targeted DNA methylation hybridization technology is an emerging non-destructive DNA methylation detection method that aims to achieve accurate detection of DNA methylation sites through highly specific DNA methylation probes and hybridization capture technology. In addition, Twist technology is known for its high sensitivity and high specificity. It can significantly reduce the sample volume requirements and effectively enrich DNA methylation fragments, thereby improving data quality and sensitivity. However, Twist technology uses TET2 enzyme to oxidize 5-mC to 5-hmC, and oxidize 5-hmC to 5-fC, and finally converts 5-fC to 5-carboxycytosine (5-caC), which cannot completely distinguish between 5-mC and 5-hmC.

[0004] Therefore, it is necessary to improve the existing DNA methylation sequencing technology to obtain more accurate DNA methylation data. Summary of the invention

[0005] In order to improve the shortcomings of existing DNA methylation detection methods, the present invention provides a sequencing method for accurately and quantitatively detecting DNA methylation. The method oxidizes and converts DNA samples to construct a sequencing library, and uses Twist targeted DNA methylation hybridization technology for detection to truly quantify the degree of DNA methylation. Compared with traditional enzyme conversion methods, the method of the present invention achieves higher coverage and matching in the quantitative detection of DNA methylation.

[0006] In a first aspect, the present invention provides a sequencing method for quantitatively detecting DNA methylation, comprising: performing oxidation and conversion pretreatment on an extracted DNA sample for library construction and DNA methylation sequencing;

[0007] The oxidation reagent is potassium perruthenate or ruthenium tetroxide;

[0008] The concentration of the oxidizing agent is 100-300 mM;

[0009] The oxidation temperature is 30-50°C.

[0010] The present invention performs oxidation and conversion pretreatment on the extracted DNA sample, and the oxidation reagent used can specifically and efficiently oxidize 5-hmC to 5-formylcytosine (5-fC) without changing 5-mC, and then further convert 5-fC into thymine (T) in conjunction with the conversion pretreatment. The DNA sample that has undergone oxidation and conversion pretreatment is used to construct a sequencing library and DNA methylation sequencing, achieving quantitative sequencing of 5-mC and 5-hmC at a single-base resolution, overcoming the problem that the enzyme conversion method of Twist targeted DNA methylation hybridization technology cannot completely distinguish 5-mC from 5-hmC.

[0011] Preferably, the concentration of the oxidizing agent is 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM, 200mM, 210mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280mM, 290mM or 300mM, preferably 120-180mM.

[0012] Preferably, the oxidation temperature is 30-50°C, for example, it can be 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C or 50°C, preferably 38-42°C.

[0013] Preferably, the oxidation time is 5-30 min, for example, it can be 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min or 30 min, preferably 10-15 min.

[0014] The present invention improves the oxidation temperature, significantly shortens the oxidation pretreatment time, improves the detection efficiency of DNA methylation, and helps to achieve rapid detection of DNA methylation.

[0015] The present invention also provides a sequencing kit for quantitatively detecting DNA methylation, wherein the kit comprises a DNA sample pretreatment reagent, wherein the pretreatment reagent comprises an oxidizing reagent, and wherein the oxidizing reagent comprises potassium perruthenate.

[0016] Preferably, the concentration of the oxidizing agent is 100-300 mM, preferably 120-180 mM.

[0017] The present invention also provides the use of the sequencing kit in preparing a urothelial carcinoma diagnosis kit.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] (1) The DNA methylation sequencing method of the present invention constructs a sequencing library by oxidizing and converting the DNA sample, and uses Twist targeted DNA methylation hybridization technology for detection, which truly quantifies the degree of DNA methylation, overcomes the problem that the enzyme conversion method of Twist targeted DNA methylation hybridization technology cannot completely distinguish 5-mC from 5-hmC, and achieves higher coverage and matching degree compared with the traditional enzyme conversion method;

[0020] (2) The present invention increases the oxidation temperature to 30-50°C, significantly shortens the oxidation pretreatment time to within 20 minutes, and improves the detection efficiency of DNA methylation. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the state of the DNA sample to be oxidized.

[0022] Figure 2 The status of DNA samples treated with different oxidation temperatures and oxidation times.

[0023] Figure 3 This is a graph showing the DNA methylation conversion efficiency of chromosome 2.

[0024] Figure 4 This is a graph showing the DNA methylation conversion efficiency of chromosome 5.

[0025] Figure 5 This is a diagram showing the results of DNA library fragment size detection.

[0026] Figure 6 This is a result graph of the matching region ratio of sequencing data.

[0027] Figure 7 This is the CHH ratio result diagram of sequencing data.

[0028] Figure 8 This is the coverage result diagram of sequencing data. DETAILED DESCRIPTION

[0029] In order to more clearly understand the purpose, technical scheme and technical effect of the present invention, the present invention is further described below, but the protection scope of the present invention is not limited to the following embodiments.

[0030] Example

[0031] (1) Urine DNA extraction

[0032] DNA was extracted from urine using a DNA extraction kit: urine was centrifuged at 1500 g for 15 min, the supernatant was discarded, and 100-400 μL of the precipitate was retained; an equal volume of urine precipitate digestion buffer was added to resuspend the precipitate, followed by addition of 5% proteinase K, vortexing or pipetting to mix, instantaneous centrifugation, and incubation in a 55°C metal bath for 30 min; an equal volume of lysis buffer was added and vortexed to mix; all the liquid was transferred to a centrifuge column, centrifuged at 16000 g for 1 min, 200 μL of DNA pretreatment solution was added to the centrifuge column, and centrifuged at 16000 g for 1 min; then, 700 μL of washing solution was added, centrifuged at 16000 g for 1 min, and then 200 μL of washing solution was added, and centrifuged at 16000 g for 1 min; after drying the centrifuge column, 25 μL of elution solution preheated to an appropriate temperature was added, and centrifuged at 16000 g for 2 min to obtain DNA. The DNA concentration and OD260 / 280 value were determined using Nanodrop, and the obtained DNA was quantified and quality analyzed.

[0033] (2) DNA oxidation

[0034] Take 1 μg of DNA, make up to 21.75 μL with RNase-free water, add 1.25 μL of 1M NaOH, and incubate in a metal bath at 37°C for 30 min; then transfer the sample to ice and quickly cool for 3 min; add 2 μL of oxidizing reagent (150 mM potassium perruthenate solution), gently flick to mix, centrifuge instantly, and incubate at 40°C for 10 min; purify using a centrifuge column, shake upside down for 30 s, and centrifuge at 1000 g for 2 min; add 500 μL of pure water, centrifuge at 1000 g for 2 min, and repeat the washing process for a total of 3 times; finally, add all samples to the centrifuge column, perform central centrifugation, and centrifuge at 1000 g for 2 min.

[0035] (3) DNA transformation

[0036] use Bisulfite Handbook Kit (Qiagen) was used to perform DNA conversion according to the instructions: all samples were transferred to a 0.2 mL PCR tube, 85 μL Bidulfite Mix was added, and then 35 μL DNAprotect Buffer was added. Mix by pipetting or flicking, centrifuged briefly, and reacted on a PCR instrument according to Table 1.

[0037] Table 1

[0038] temperature time 95℃ 5min 60℃ 25min 95℃ 5min 60℃ 85min 95℃ 5min 60℃ 175min 20℃ Keep

[0039] Conversion efficiency identification: 100 ng of oxidatively transformed DNA samples and 100 ng of non-oxidatively transformed DNA samples were used as templates, and Methylight PCR primers were used to perform Methylight PCR reaction. The results were analyzed by T test.

[0040] After the conversion efficiency is determined and the conversion reaction is completed, all the products in the 0.2mL PCR tube are transferred to a 1.5mL centrifuge tube, 560μL buffer BL is added, and the mixture is mixed and then centrifuged instantly; all the samples are transferred to the centrifuge column, centrifuged at 16000g for 1min, and the waste liquid is discarded; 500μL buffer BW is added to the centrifuge column, centrifuged at 16000g for 1min, and the waste liquid is discarded; 500μL buffer BD is added to the centrifuge column, incubated at room temperature for 15min, centrifuged at 16000g for 1min, and the waste liquid is discarded, and the operation is repeated once; centrifuged at 16000g for 2min, the centrifuge column is idling, and the centrifuge column is placed in a new centrifuge tube, opened and left in a 56℃ metal bath for 5min, 20μLEB buffer is added to the center of the centrifuge column membrane, and centrifuged at 16000g for 1min, which is the product after Bisulfite conversion.

[0041] (4) Library construction

[0042] Using xGen TM Methyl-Seq DNA Library Prep Kit (IDT) was used to construct the library of the transformed DNA according to the instructions. 15 μL of the fragmented sample was added to a 0.2 mL PCR tube (if the volume is less than 15 μL, it was supplemented with LowEDTATE) and the PCR instrument was preheated in advance. The sample was placed in the PCR instrument, the program was 95°C for 2 min, and the Adaptase MasterMix was prepared as shown in Table 2. After preparation, vortexed and mixed, centrifuged, and stored on ice.

[0043] Table 2

[0044] Reagents Volume (μL) Low EDTA\TE 11.5 Buffer G1 4.0 Reagent G2 4.0 Reagent G3 2.5 Enzyme G4 1.0 Enzyme G5 1.0 Enzyme G6 1.0

[0045] After the denaturation process is completed, place on ice for 3 minutes, add 25 μL of Adaptase Master Mix to each tube, mix by pipetting / lightly vortex, and centrifuge. Place the sample in the PCR instrument and follow the program as shown in Table 3.

[0046] Table 3

[0047]

[0048]

[0049] Prepare Mix 1 as shown in Table 4. After preparation, vortex to mix, centrifuge, and store on ice.

[0050] Table 4

[0051] Reagents Volume (μL) Reagent Y1 2.0 Enzyme Y2 42.0

[0052] Place on ice, add 44 μL of Mix1 to each tube, pipette to mix / lightly vortex, and centrifuge. Place the sample in the PCR instrument and program as shown in Table 5.

[0053] Table 5

[0054] temperature time 98℃ 1min 62℃ 2min 65℃ 5min 4℃ Keep

[0055] Magnetic bead washing. Prepare a new 1.5mL tube and add 101μL of magnetic beads. After the PCR program is completed, spin the sample tube, transfer the sample to the 1.5mL tube with beads, vortex to mix, spin, and place at room temperature for 5 minutes. Place the tube on the magnetic rack until the solution is clear (about 5 minutes), and discard the supernatant (do not absorb the magnetic beads). Prepare 80% anhydrous ethanol, add 200μL of 80% anhydrous ethanol to the tube, room temperature for 30 seconds, and discard the supernatant. Repeat once, spin, and place it on the magnetic rack again. Use a 10μL pipette to absorb excess liquid. Remove from the magnetic rack, open the lid and dry for 5 minutes, add 16μL Low EDTATE, and vortex gently until resuspended. Incubate at room temperature for 5 minutes. Place the tube on the magnetic rack until the solution is clear (about 5 minutes), and transfer the supernatant to a 0.2mL PCR tube.

[0056] Prepare Mix 2 as shown in Table 6. Vortex mix thoroughly, centrifuge briefly, and store on ice.

[0057] Table 6

[0058] Reagents Volume (μL) Buffer B1 3.0 Reagent B2 10.0 Enzyme B3 2.0

[0059] Place on ice, add 15 μL of Mix2 to each tube, pipette to mix / lightly vortex, and centrifuge. Place the sample in the PCR instrument and program as shown in Table 7.

[0060] Table 7

[0061] temperature time 25℃ 15min 4℃ Keep

[0062] Magnetic bead washing. Prepare a new 1.5mL tube and add 36μL of magnetic beads. After the PCR program is completed, spin the sample tube, transfer the sample to the 1.5mL tube with beads, vortex to mix, spin, and place at room temperature for 5 minutes. Place the tube on the magnetic rack until the solution is clear (about 5 minutes), and discard the supernatant (do not absorb the magnetic beads). Prepare 80% anhydrous ethanol, add 200μL of 80% anhydrous ethanol to the tube, room temperature for 30 seconds, and discard the supernatant. Repeat once, spin, and place it on the magnetic rack again. Use a 10μl pipette to absorb excess liquid. Remove from the magnetic rack, open the lid and dry for 5 minutes, add 21μL Low EDTATE, and vortex gently until resuspended. Incubate at room temperature for 5 minutes. Place the tube on the magnetic rack until the solution is clear (about 5 minutes), and transfer the supernatant to a 0.2mL PCR tube.

[0063] Place on ice and add 5 μL xGen UDIPrimer Pairs to each tube. Prepare Mix3 as shown in Table 8. Vortex to mix, centrifuge, and store on ice.

[0064] Table 8

[0065]

[0066]

[0067] Place on ice, add 25 μL of Mix3 to each tube, pipette to mix / lightly vortex, and centrifuge. Place the sample in the PCR instrument and program as shown in Table 9. The number of cycles is selected according to Table 10.

[0068] Table 9

[0069]

[0070] Table 10

[0071] Sample size Number of cycles 100ng 4-6 10ng 7-9 1ng 11-13 100pg 14-16

[0072] Magnetic bead washing. Prepare a new 1.5mL tube and add 42.5μL magnetic beads. After the PCR program is completed, spin the sample tube, transfer the sample to the 1.5mL tube with beads, vortex to mix, spin, and place at room temperature for 5 minutes. Place the tube on the magnetic rack until the solution is clear (about 5 minutes), and discard the supernatant (do not absorb the magnetic beads). Prepare 80% anhydrous ethanol, add 200μL 80% anhydrous ethanol to the tube, room temperature for 30 seconds, and discard the supernatant. Repeat once, spin, and place it on the magnetic rack again. Use a 10μl pipette to absorb excess liquid. Remove from the magnetic rack, open the lid and dry for 5 minutes, add 21μL Low EDTATE, and vortex gently until resuspended. Incubate at room temperature for 5 minutes. Place the tube on the magnetic rack until the solution is clear (about 5 minutes), and transfer the supernatant to the 1.5mL tube.

[0073] Each enriched library was validated and quantified using the Agilent Bioanalyzer High Sensitivity DNA Kit and Thermo Fisher Scientific’s Qubit dsDNA High Sensitivity Quantitation Assay.

[0074] The libraries that passed the quality inspection were sequenced for DNA methylation using the sequencer of the BGI T7 platform.

[0075] Comparative Example

[0076] After DNA extraction, the DNA was sheared using a Covaris sonicator, and then the library was constructed using the NEBNext Enzymatic Methyl-Seq kit (Twist).

[0077] Operate on ice and transfer 200 ng of fragmented DNA into a 0.2 mL PCR tube. If the volume is less than 50 μL, make up the volume with enzyme-free water.

[0078] End repair. Add 7μL NEBNext Ultra II End PrepReaction Bffer to a 0.2mL PCR tube on ice, then add 3μL NEBNext Ultra II End Prep Enzyme Mix, pipette to mix / lightly vortex, and centrifuge. Place the sample in a PCR instrument and follow the program as shown in Table 11.

[0079] Table 11

[0080] temperature time 20℃ 30min 65℃ 30min 4℃ Keep

[0081] After the program is finished, add 2.5μl NEBNext EM-seq Adapter, 1μl NEBNext Ligation Enhancer, and 30μl NEBNext Ultra II Ligation Master Mix to a 0.2ml PCR tube, pipette to mix / lightly vortex, and spin down. Place the sample in a PCR machine and incubate at 20℃ for 15min without covering the lid.

[0082] Magnetic bead washing. Prepare a new 1.5ml tube and add 110μl (1.18×) magnetic beads. After the PCR program is completed, centrifuge the sample tube, transfer the sample to the 1.5ml tube with beads, vortex to mix, centrifuge, and place at room temperature for 5 minutes. Place the tube on the magnetic rack until the solution is clear (about 5 minutes), and discard the supernatant (do not absorb the magnetic beads). Prepare 80% anhydrous ethanol, add 200μl 80% anhydrous ethanol to the tube, incubate at room temperature for 30 seconds, and discard the supernatant. Repeat once, centrifuge, and place it on the magnetic rack again. Use a 10μl pipette to absorb excess liquid. Remove from the magnetic rack, open the lid and dry for 5 minutes, add 31μl Elution buffer, and vortex gently until resuspended. Incubate at room temperature for 5 minutes. Place the tube on the magnetic rack until the solution is clear (about 5 minutes), and transfer the supernatant to a 0.2ml PCR tube.

[0083] After the oxidation program is completed, add 10μL Reconstituted TET2 Reaction Buffer (TET2 Reaction Buffer plus reconstituted TET2 Reaction Buffer Supplement), 1μl Oxidation Supplement, 1μl DTT, 1μl Oxidation Enhancer, and 1μl TET2 to a 0.2ml PCR tube, mix by pipetting / gently vortex, and centrifuge.

[0084] Add 5 μl Diluted Fe(II)Solution to a 0.2 ml PCR tube, mix by pipetting / lightly vortex, and centrifuge; incubate at 37°C for 1 h on a PCR instrument with the heated cover set to ≥45°C; transfer to ice, and add 1 μl Stop Reagent (yellow) to each 0.2 ml PCR tube; incubate at 37°C for 30 min on a PCR instrument, then at 4°C with the heated cover set to ≥45°C or open.

[0085] Magnetic bead washing. Prepare a new 1.5ml tube and add 90μl (1.8x) magnetic beads. After the PCR program is completed, centrifuge the sample tube, transfer the sample to the 1.5ml tube with beads, vortex to mix, centrifuge, and place at room temperature for 5 minutes. Place the tube on the magnetic rack until the solution is clear (about 5 minutes), and discard the supernatant (do not absorb the magnetic beads). Prepare 80% anhydrous ethanol, add 200μl 80% anhydrous ethanol to the tube, incubate at room temperature for 30 seconds, and discard the supernatant. Repeat once, centrifuge, and place it on the magnetic rack again. Use a 10μl pipette to absorb excess liquid. Remove from the magnetic rack, open the lid and dry for 5 minutes, add 16μl Elution buffer, and vortex gently until resuspended. Incubate at room temperature for 5 minutes. Place the tube on the magnetic rack until the solution is clear (about 5 minutes), and transfer the supernatant to a 0.2ml PCR tube.

[0086] For denaturation, the formamide method is recommended. Turn on the PCR instrument and preheat to 85°C in advance. Add 4μl of deionized formamide reagent to a 0.2ml PCR tube, vortex to mix or pipette up and down to mix, and centrifuge. Incubate at 85°C for 10 minutes on the PCR instrument and cover with a hot lid. Place on ice immediately after completion.

[0087] Deamination, add 68μl Nuclease-free water, 10μl APOBEC Reaction Buffer, 1μl BSA, 1μl APOBEC to a 0.2ml PCR tube, mix by pipetting / lightly vortex, and centrifuge. Incubate at 37℃ for 3h in a PCR instrument, then at 4℃, with the heated lid set to ≥45℃ or open.

[0088] Magnetic bead washing. Prepare a new 1.5ml tube and add 100μl (1.0x) magnetic beads. After the PCR program is completed, centrifuge the sample tube, transfer the sample to the 1.5ml tube with beads, vortex to mix, centrifuge, and place at room temperature for 5 minutes. Place the tube on the magnetic rack until the solution is clear (about 5 minutes), and discard the supernatant (do not absorb the magnetic beads). Prepare 80% anhydrous ethanol, add 200μl 80% anhydrous ethanol to the tube, incubate at room temperature for 30 seconds, and discard the supernatant. Repeat once, centrifuge, and place it on the magnetic rack again. Use a 10μl pipette to absorb excess liquid. Remove from the magnetic rack, open the lid and dry for 5 minutes, add 22μl Elution buffer, and vortex gently until resuspended. Incubate at room temperature for 5 minutes. Place the tube on the magnetic rack until the solution is clear (about 5 minutes), and transfer the supernatant to a 0.2ml PCR tube.

[0089] After the amplification process is complete, add 5 μl of EM-seq Index Primer plate to a 0.2 ml PCR tube, add 25 μl of NEBNext Q5U Master Mix to a 0.2 ml PCR tube, pipette to mix / lightly vortex, and centrifuge. Place the sample in the PCR instrument and follow the process as shown in Table 12.

[0090] Table 12

[0091]

[0092]

[0093] Magnetic bead washing. Prepare a new 1.5ml tube and add 45μl (0.9x) magnetic beads. After the PCR program is completed, centrifuge the sample tube, transfer the sample to the 1.5ml tube with beads, vortex to mix, centrifuge, and place at room temperature for 5 minutes. Place the tube on the magnetic rack until the solution is clear (about 5 minutes), and discard the supernatant (do not absorb the magnetic beads). Prepare 80% anhydrous ethanol, add 200μl 80% anhydrous ethanol to the tube, incubate at room temperature for 30 seconds, and discard the supernatant. Repeat once, centrifuge, and place it on the magnetic rack again. Use a 10μl pipette to absorb excess liquid. Remove from the magnetic rack, open the lid and dry for 5 minutes, add 22μl Elution buffer, and vortex gently until resuspended. Incubate at room temperature for 5 minutes. Place the tube on the magnetic rack until the solution is clear (about 5 minutes), and transfer the supernatant to the 1.5ml tube.

[0094] For hybridization capture, the mass of the individual tag libraries in each pooled library was divided by the concentration measured during the library preparation quality control (ng / μl). Eight libraries were multiplexed for each hybridization reaction, so the mass of each library was 187.5ng, and the total mass of the pooled library was 1,500ng. Add 4μl of custom methylation probe, 8μl of universal blocker, 5μl of blocker, and 2μl of methylation promoter, and centrifuge the tube briefly to reduce bubbles. Use a vacuum concentrator to dry the methylation pre-hybridization solution (library, combination, blocker) in the tube at low temperature or without heating, and use it for hybridization reaction.

[0095] Heat the Rapid Hybridization Reaction Mix at 65°C for 10 min, or until all precipitate has dissolved. Vortex and use immediately. Do not allow the Rapid Hybridization Reaction Mix to cool to room temperature. Resuspend the dried methylated pre-hybridization material in 20 μl of the Rapid Hybridization Reaction Mix. Briefly centrifuge the tube to ensure there are no bubbles. Add 30 μl of Hybridization Promoter to the methylated pre-hybridization solution. Briefly centrifuge the tube to ensure all solution is at the bottom of the tube. Transfer the tube to the preheated thermal cycler and proceed to Steps 2 and 3 of the thermal cycler program. As shown in Table 13

[0096] Table 13

[0097] temperature time 95℃ Keep 95℃ 5min 60℃ At least 4 hours

[0098] Wash the streptavidin beads by vortexing the pre-equilibrated streptavidin-bound beads until mixed. Add 100μl of streptavidin-bound beads to a 1.5ml centrifuge tube. Prepare one tube for each hybridization reaction. Add 200μl of rapid binding buffer to the tube and mix by pipetting. Let the tube sit on the magnetic rack for 1 minute, then aspirate and discard the supernatant. Do not disrupt the bead pellet. Remove the tube from the magnetic rack. Repeat the wash twice, for a total of three washes. After the third wash, remove the supernatant and add 200μl of rapid binding buffer and resuspend the beads by vortexing until mixed.

[0099] After hybridization is complete, open the lid of the thermal cycler and quickly transfer each hybridization reaction (including the hybridization promoter) to the corresponding tube of streptavidin-conjugated magnetic beads (washed). Mix the reagents by pipetting and flicking. Mix the hybridization reaction and streptavidin-conjugated magnetic beads for 30 minutes at room temperature using an oscillator, shaker, or rotary mixer at a speed that allows the solution to mix. Remove the tube containing the hybridization reaction and streptavidin-conjugated magnetic beads from the mixer and perform a brief centrifugation to ensure that all the solution is at the bottom of the tube. Allow the tube to stand on the magnetic rack for 1 minute. Aspirate and discard the supernatant, including the hybridization promoter. Do not disrupt the magnetic bead pellet. Remove the tube from the magnetic rack and add 200μl of preheated (65℃) rapid wash buffer 1. Mix by pipetting. Incubate the tube at 65℃ for 5 minutes. Allow the tube to stand on the magnetic rack for 1 minute. Aspirate and discard the supernatant. Do not disrupt the magnetic bead pellet. Remove the tube from the magnet and add another 200 μl of pre-warmed (65°C) Rapid Wash Buffer 1. Pipette to mix. Incubate the tube at 65°C for 5 min. Centrifuge briefly to ensure that all the solution is at the bottom of the tube. Transfer all the solution (approximately 200 μl) to a new 1.5 ml microcentrifuge tube, one tube per hybridization reaction. Allow the tube to sit on the magnet for 1 min. Aspirate and discard the supernatant. Do not disrupt the bead pellet. Remove the tube from the magnet and add 200 μl of Wash Buffer 2 at 48°C. Pipette to mix, then centrifuge briefly to ensure that all the solution is at the bottom of the tube. Incubate the tube at 48°C for 5 min. Allow the tube to sit on the magnet for 1 min. Aspirate and discard the supernatant. Remove the tube from the magnet and add 200 μl of Wash Buffer 2 at 48°C. Mix by pipetting and centrifuge briefly to ensure that all the solution is at the bottom of the tube. Incubate the tube at 48°C for 5 minutes. Allow the tube to stand on the magnetic rack for 1 minute. Aspirate and discard the supernatant. Remove the tube from the magnetic rack and add 200μl of Wash Buffer 2 at 48°C. Mix by pipetting and centrifuge briefly to ensure that all the solution is at the bottom of the tube. Incubate the tube at 48°C for 5 minutes. Allow the tube to stand on the magnetic rack for 1 minute. Aspirate and discard the supernatant. After the last wash, use a 10μl pipette to aspirate all the supernatant. Proceed immediately to the next step. Do not allow the beads to dry. Remove the tube from the magnetic rack and add 45μl of water. Mix by pipetting and incubate this solution (hereafter referred to as the streptavidin-bound magnetic bead suspension) on ice.

[0100] Store the remaining 22.5 μl of water / streptavidin-bound magnetic bead suspension at -20°C for future use. Transfer 22.5 μl of streptavidin-bound magnetic bead suspension to a 0.2 ml thin-walled PCR tube, place on ice, add 2.5 μl of amplification primer (ILMN), 25 μl of KAPA HiFi HotStart ReadyMix, pipette to mix / lightly vortex, and centrifuge. Place the sample in the PCR instrument and program as shown in Table 14.

[0101] Table 14

[0102]

[0103]

[0104] Magnetic bead washing. Prepare a new 1.5ml tube and add 90μl (1.8x) purified magnetic beads. After the PCR program is completed, centrifuge the sample tube, transfer the sample to the 1.5ml tube with beads, vortex to mix, centrifuge, and place at room temperature for 5 minutes. Place the tube on the magnetic rack until the solution is clear (about 5 minutes), and discard the supernatant (do not absorb the magnetic beads). Prepare 80% anhydrous ethanol, add 200μl 80% anhydrous ethanol to the tube, incubate at room temperature for 30 seconds, and discard the supernatant. Repeat once, centrifuge, and place it on the magnetic rack again. Use a 10μl pipette to absorb excess liquid. Remove from the magnetic rack, open the lid and dry for 5 minutes, add 32μl water, and vortex gently until resuspended. Incubate at room temperature for 5 minutes. Place the tube on the magnetic rack until the solution is clear (about 5 minutes), and transfer the supernatant to the 1.5ml tube.

[0105] Each enriched library was validated and quantified using the Agilent Bioanalyzer High Sensitivity DNA Kit and Thermo Fisher Scientific’s Qubit dsDNA High Sensitivity Quantitation Assay.

[0106] Experimental Results

[0107] (1) Extracted DNA concentration

[0108] In this experiment, 8 urine samples were randomly selected and analyzed using Quick-DNA TM Urine Kit was used to extract DNA. After quantification and quality inspection, the DNA concentrations of the eight urine samples were as follows:

[0109] Table 15

[0110]

[0111]

[0112] (2) Improvement of oxidation temperature and time

[0113] Improvement and optimization of the oxidation method in the embodiment: 1 μg of DNA to be oxidized was taken, 1.25 μl of 1M NaOH was added, and the solution was clear and transparent after incubation at 37°C in a metal bath for 30 minutes. Figure 1 ), and the total DNA concentration did not change; after adding the oxidizing agent, the solution turned orange; the solution was incubated at 40°C for 10 min ( Figure 2 a tube) or 15min( Figure 2 b tube) and incubate at 50°C for 8 min ( Figure 2 c tube), it was observed that the solution incubated at 50°C for 8 min had turned black with precipitation ( Figure 2 c tube), the amount of DNA was significantly reduced after purification by Bio-Rad Micro Bio-SpinColumn. In addition, the solution incubated at 40°C for 15 min also appeared black and turbid, while the color of the solution incubated at 40°C for 10 min did not change, and no significant change was found in the concentration (Tables 16 and 17).

[0114] Table 16

[0115] name DNA concentration (ng / μl) A260 / 280 A-1 56.9 1.84 A-2 57.6 1.80 B-1 57.8 1.81 B-2 56.0 1.80 C-1 56.4 1.80 C-2 56.6 1.81

[0116] Table 17

[0117]

[0118]

[0119] (3) Transformation efficiency results

[0120] After the DNA samples were oxidized and converted, the Methylight PCR technique was used to detect the DNA methylation conversion efficiency. Chromosome 2 (Chr2: 63056830-63057032) and chromosome 5 (Chr5: 176366138-176366240) were selected as examples to detect the DNA methylation levels of these two DNA sequences. The results are as follows Figure 3 and Figure 4 As shown, the relative Ct values ​​of Chr2 and Chr5 after conversion were significantly higher than those of unconverted and oxidized ones.

[0121] (4) Library quality

[0122] The above DNA samples were constructed using the methods of the embodiment and the comparative example, and the library concentration and DNA fragment size were detected using Qubit and capillary electrophoresis. The detection results are shown in Table 18. Figure 5 All showed qualified.

[0123] Table 18

[0124]

[0125]

[0126] (5) Sequencing data quality

[0127] DNA methylation sequencing was performed on the above 8 samples, and the obtained FastQC data were subjected to data quality control by TrimGalore, Bwa-Meth, Samtools and AlignmentSieve tools, and then the methylation rate of each CpG site was calculated and counted by MethylDackel tool. Figure 6 , Figure 7 and Figure 8 It is shown that the matching area ratio and coverage obtained by the comparative example are significantly lower than those of the embodiment, while the CHH ratio is significantly higher than that of the embodiment.

[0128] The above embodiments are only specific descriptions of feasible implementation methods of the present invention, and are intended to facilitate those skilled in the art to understand and use the invention. It is obvious that those familiar with the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the above embodiments should be regarded as exemplary and non-restrictive, and any equivalent implementation or modification that does not depart from the present invention should be included in the protection scope of the present invention.

Claims

1. A sequencing method for quantitatively detecting DNA methylation, characterized in that: include: The extracted DNA samples were pre-treated with oxidation and conversion before being used for library construction and DNA methylation sequencing; The oxidation reagent includes potassium perruthenate or ruthenium tetroxide; The concentration of the oxidizing agent is 100-300 mM; The oxidation temperature is 30-50°C.

2. The sequencing method according to claim 1, characterized in that The oxidation time is 5-30 minutes.

3. The sequencing method according to claim 1, characterized in that The oxidation reagent is potassium perruthenate.

4. The sequencing method according to claim 1, characterized in that The concentration of the oxidizing agent is 120-180 mM.

5. The sequencing method according to claim 1, characterized in that The temperature of the oxidation is 38-42°C.

6. The sequencing method according to claim 1, characterized in that The oxidation time is 10-15 minutes.

7. A sequencing kit for quantitatively detecting DNA methylation, characterized in that: The kit comprises a DNA sample pretreatment reagent, wherein the pretreatment reagent comprises an oxidation reagent, and the oxidation reagent comprises potassium perruthenate or ruthenium tetroxide.

8. The sequencing kit according to claim 7, characterized in that The concentration of the oxidizing agent is 100-300 mM.

9. The sequencing kit according to claim 7, characterized in that The concentration of the oxidizing agent is 120-180 mM.

10. An application in preparing a diagnostic kit for urothelial carcinoma, characterized in that: The urothelial carcinoma diagnostic kit comprises the sequencing kit according to any one of claims 7 to 9.

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