CN110820050A - Whole genome methylation non-bisulfite sequencing library and construction - Google Patents

Whole genome methylation non-bisulfite sequencing library and construction Download PDF

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CN110820050A
CN110820050A CN201911159400.6A CN201911159400A CN110820050A CN 110820050 A CN110820050 A CN 110820050A CN 201911159400 A CN201911159400 A CN 201911159400A CN 110820050 A CN110820050 A CN 110820050A
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张燕艳
杨玲
管彦芳
姬利延
贾明玺
张珍
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Suzhou Jiyinjia Biomedical Engineering Co Ltd
Geneplus - Beijing
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Geneplus - Beijing
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Abstract

The invention relates to the technical field of bioinformatics, in particular to a sequencing library of a whole genome methylated non-bisulfite, construction and application thereof, wherein the sequencing library comprises TET enzyme reaction liquid which comprises the following independently packaged components: TET enzyme oxidation buffer; the TET enzyme oxidation buffer comprises the following micromolar components: HEPES or Tris-Cl (20-167). times.103Fraction, NaCl (100-3Aliquot, α -KG or 2-oxoglutarate 3.3X 103Portions of ascorbic acid 6.67X 103And adenosine triphosphate 4 × 103Preparing; fe (NH) is combined by adopting the kit4)2(SO4)2And TET enzyme can oxidize 5mc into 5cac, the 5cac is reduced into dihydrouracil under the action of a reducing agent, and the dihydrouracil is identified as T through PCR sequencing, so that the effect of non-heavy reaction is realizedThe conversion from DNA methylation from 'C' to 'T' under the bisulfite condition solves the defects of unbalanced bases and low utilization rate of sequencing data of the existing methylation sequencing library constructed based on bisulfite conversion.

Description

Whole genome methylation non-bisulfite sequencing library and construction
Technical Field
The invention relates to the technical field of bioinformatics, in particular to a sequencing library of whole genome methylation non-bisulfite and construction thereof.
Background
DNA methylation is one of the major epigenetic modifications of genomic DNA. In recent years, numerous studies have shown that DNA methylation modification plays a crucial role in maintaining normal cellular function, transmitting genomic genetic imprinting, embryonic development, and human tumorigenesis.
One gold standard for methylation sequencing is Whole Genome Bisulfite Sequencing (WGBS), abbreviated as BS-seq. The detection principle is that cytosine (C) which is not methylated in a genome is deaminated and converted into uracil (U) by bisulfite treatment, U base is recognized as thymine (T) by polymerase which is tolerant to U base in the subsequent PCR amplification process, so that C- > T conversion is realized, and the conversion is separated from C base which originally has methylation modification, thereby achieving the purpose of separating unmodified C base from methylation modified C base. WGBS can convert 95% of unmethylated cytosine to thymine, and can cover 70-99% of cytosine, so that methylation sequencing with single base resolution is realized.
According to the step of adding bisulfite conversion (BS) in traditional library construction, the method can be divided into a BS pre-library construction method and a BS post-library construction method. Since the current PCR process cannot completely retain the information of methylcytosine (mC), and the library before transformation cannot be amplified, the first method of library construction and BS transformation is to perform BS transformation on the adaptor-ligated dsDNA, such as the whole genome methylation sequencing library disclosed in chinese patent document CN104532360B and the construction method thereof. In the method of building a library before a BS, a large amount of template degradation (0.1-99.9%) is caused in the BS conversion process, the lower the initial amount of the linker-dsDNA is, the more the template degradation is, and the library building strategy before the BS needs to adopt a methylated linker to connect the dsDNA, so as to avoid the problem that the linker is converted in the BS conversion process to cause library building failure. Therefore, the first method of building a library and then building a BS requires that the initial amount is more than 200ng, the AT ratio in the built library is more than 80%, and the bases are unbalanced;
the second BS post-banking approach, with bisulfite conversion first, produces large amounts of fragmented single-stranded DNA, requiring synthesis of the single-stranded DNA into dsDNA, two of which are common: direct single-stranded ligation and random primer amplification. Most of the strategies used are random primer amplification of single stranded DNA. The random primer amplification method requires random primers with lengths of 4N,6N,8N and 9N, and synthesizes double-stranded DNA with the single strand as a template. Sequencing reads of the random primer method have certain preference in combination at the 3 '-end, which is shown in that the SNPs at the 3' -end are more and the comparison rate is lower (50 percent), so that a large amount of sequencing data can be lost. Some researchers find that the utilization rate of the single-chain template is higher by adopting a special single-chain ligase and adding a tail to be directly connected with a sequencing joint, and the method is more suitable for building a library of low-initial-quantity substances. The single-strand connection strategy can realize methylation detection of low initial amount of substances, however, the established library still has the defects that AT ratio is more than 80%, base imbalance exists, and the P5 and P7 truncation sequences of Illumina are connected to the single strand, so that the sequencing compatibility of other platforms is difficult to realize.
According to the method, the AT proportion of the library constructed by the library construction method based on bisulfite conversion is more than 80%, the base imbalance is defective, the operation is inconvenient because other whole genome libraries need to be mixed for sequencing in the on-computer detection, and meanwhile, the utilization rate of the constructed library sequencing data is low, and the effective data only accounts for 20-30% of the sequencing amount.
Disclosure of Invention
Therefore, the first technical problem to be solved by the present invention is to provide a DNA methylation conversion kit, which can perform conversion from "C" to "T", has low usage amount of each component, low cost and high conversion rate, can meet the requirement of sequencing, and avoids using bisulfite conversion, so that the constructed whole genome methylation sequencing library has balanced bases and high usage rate of sequencing data.
The second technical problem to be solved by the invention is that the methylation sequencing library constructed based on bisulfite conversion in the prior art has the defects of unbalanced bases and low utilization rate of sequencing data, and further provides a whole genome methylation non-bisulfite sequencing library and construction thereof.
Therefore, the invention provides the following technical scheme:
the invention provides a DNA methylation conversion kit, which comprises TET enzyme reaction liquid, wherein the TET enzyme reaction liquid comprises the following independently packaged components: TET enzyme oxidation buffer; the TET enzyme oxidation buffer comprises the following micromolar components: HEPES or Tris-Cl (20-167). times.103Fraction, NaCl (100-3Aliquot, α -KG or 2-oxoglutarate 3.3X 103Portions of ascorbic acid 6.67X 103And adenosine triphosphate 4 × 103Preparing; the TET enzyme oxidation buffer pH was 8.0.
Preferably, the TET enzyme oxidation buffer comprises the following components of HEPES or Tris-Cl (20-167) mM, NaCl (100-333) mM, α -KG or 2-oxogluterate 3.3mM, ascorbic acid 6.67mM and adenosine triphosphate 4mM, and the pH of the TET enzyme oxidation buffer is 8.0.
Further, the TET enzyme reaction solution also comprises independently packaged Fe (NH)4)2(SO4)2Solution of said Fe (NH)4)2(SO4)2Fe (NH) in solution4)2(SO4)2Is in a concentration of 0.75-1.5 mM.
Further, the TET enzyme reaction solution also comprises an independently packaged TET enzyme solution, and the concentration of the TET enzyme is 8-10.5 mu M.
Further, the TET enzyme includes TET1 enzyme or TET2 enzyme.
In the kit, a pyridine borane reducing agent packaged independently is also included.
Preferably, the pyridine borane reducing agent comprises the following components in micromole parts which are packaged independently: pyridine borane solution, the concentration of the pyridine borane is 10M; and sodium acetate solution, sodium acetate concentration 3M, pH 4.3.
The invention provides application of the kit in whole genome methylation sequencing, DNA methylation sequencing or target region DNA methylation sequencing.
The invention provides a method for constructing a sequencing library of a genome-wide methylated non-bisulfite, which comprises the step of converting 'C' into 'T' by using the kit.
In the method, in the oxidation step of the TET enzyme reaction solution, a library to be converted from 'C' to 'T' is mixed with the TET enzyme reaction solution to obtain a TET enzyme oxidation reaction system, and 50 μ L of the library is:
30-100 ng of DNA library;
Fe(NH4)2(SO4)2the final concentration is 95-105 mu M;
1 XTET enzyme oxidation buffer, 10-20 μ L;
tet enzyme, final concentration 0.048-0.06 μ M;
make up to 50. mu.L of enzyme-free water.
In the method, in the oxidation step of the TET enzyme reaction solution, the reaction conditions are as follows: reacting at 37 deg.C for 1-3h, adding proteinase K, and incubating at 37-50 deg.C for 0.5-1 h.
In the method, a product oxidized by using TET enzyme reaction liquid is subjected to a pyridine borane reduction step, the product is mixed with a pyridine borane reducing agent, and the mixture is incubated for 16h-32h at 37 ℃ and 850 rpm.
In the method, the library after the conversion from "C" to "T" is subjected to PCR amplification using PCR amplification enzymes including KAPA HIFI Urail + polymerase, NEB Q5Urail enzyme, Pfu Turbo Cx Hotstart polymerase or Taq enzyme.
Preferably, the reaction procedure for PCR amplification of the library after conversion from "C" to "T" is as follows: initial denaturation at 98 ℃ for 45 sec; denaturation at 98 ℃ for 15sec, annealing at 60 ℃ for 30sec, extension at 72 ℃ for 30sec, 8 cycles; extending for 1min at 72 ℃; 4 ℃ and infinity.
In the method, the library after amplification is subjected to second generation sequencing, and a sequencing platform comprises but is not limited to an Illumina platform, an MGI or a Gene + Seq platform; preferably, the sequencing platform is a Gene + Seq platform.
In the method, the library construction method to be converted from "C" to "T" comprises:
fragmenting the DNA;
repairing the tail end of the fragmented DNA and adding A;
the A product is added to the linker to obtain the linker dsDNA.
The invention provides a whole genome methylation sequencing library constructed by the method.
The technical scheme of the invention has the following advantages:
1. the invention provides a DNA methylation conversion kit, which comprises TET enzyme reaction liquid, wherein the TET enzyme reaction liquid comprises independently packaged components: TET enzyme oxidation buffer; the TET enzyme oxidation buffer comprises the following micromolar components: HEPES or Tris-Cl (20-167). times.103Fraction, NaCl (100-3Aliquot, α -KG or 2-oxoglutarate 3.3X 103Portions of ascorbic acid 6.67X 103And adenosine triphosphate 4 × 103Preparing; fe (NH) is combined by adopting the kit4)2(SO4)2And TET enzyme can perform DNA methylation conversion, oxidize 5mc (5-methyl cytosine) into 5cac (5-carboxyl cytosine), the 5cac obtained by the oxidation can be reduced into Dihydrouracil (DHU) under the action of a reducing agent, and the dihydrouracil can be identified as T through PCR sequencing, so that the effect of non-bisulfite conversion is realizedThe conversion from DNA methylation from 'C' to 'T' under the condition of the bisulfite solves the defects of unbalanced basic group and low utilization rate of sequencing data of a methylation sequencing library constructed based on bisulfite conversion in the prior art, and meanwhile, the TET enzyme reaction solution of the invention ensures higher conversion rate, further ensures the base balance of the constructed methylation sequencing library, satisfactory genome comparison rate and satisfactory coverage depth, and has the advantages of simple components, extremely low TET enzyme consumption when the TET enzyme oxidation buffer solution is used for oxidation, low TET enzyme consumption of 0.048-0.06 mu M when a TET enzyme oxidation reaction system is prepared, and obvious cost reduction effect.
2. The DNA methylation conversion kit provided by the invention further comprises an independently packaged pyridine borane reducing agent; the pyridine borane reducing agent comprises the following components in independent package: pyridine borane solution, the concentration of the pyridine borane is 10M; and a sodium acetate solution, the sodium acetate concentration being 3M, pH 4.3; the reducing agent can reduce 5cac obtained by oxidizing the TET enzyme reaction solution of the invention into Dihydrouracil (DHU), and the reduced product is identified as T through PCR sequencing, so that the conversion from DNA methylation C to T under the condition of no bisulfite is realized.
3. The invention provides a method for constructing a sequencing library of a full genome methylation non-bisulfite, which is characterized in that a library to be converted from 'C' to 'T' is mixed with TET enzyme reaction liquid to obtain a TET enzyme oxidation reaction system, and the total weight of the TET enzyme oxidation reaction system is 50 mu L: 30-100 ng of DNA library; fe (NH)4)2(SO4)2The final concentration is 95-105 mu M; 1 XTET enzyme oxidation buffer, 10-20 μ L; tet enzyme, final concentration 0.048-0.06 μ M; supplementing enzyme-free water to 50 μ L; the TET enzyme oxidation reaction system can oxidize 5mc (5-methyl cytosine) into 5cac (5-carboxyl cytosine), realizes the first step of conversion from DNA methylation from C to T, ensures higher conversion rate from DNA methylation from C to T, meets the sequencing requirement of a constructed library, and has the advantages of simplified components, extremely low use amount of TET enzyme and obvious cost reduction.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a photograph of agarose gel electrophoresis in example 4 of the present invention;
FIG. 2 is a library quality control map constructed in example 4 of the present invention;
FIG. 3 is a diagram showing the ratio of mononucleosides and dinucleosides of the methylation sequencing library and the bisulfite library constructed in example 3 of the experimental example of the present invention.
Detailed Description
EXAMPLE 1 DNA methylation transformation kit
The DNA methylation conversion kit provided by the embodiment comprises a TET enzyme oxidation buffer solution packaged independently;
TET enzyme oxidation buffer (pH 8) consisted of the following components in table 1:
TABLE 1 TET enzyme Oxidation buffer composition
Further, the kit also comprises Fe (NH) packaged independently4)2(SO4)2Solution of said Fe (NH)4)2(SO4)2Fe (NH) in solution4)2(SO4)2Is 1.0 mM.
Further, the kit also comprises an independently packaged TET enzyme solution, and the concentration of the TET enzyme is 8 mu M. The TET enzyme is the TET1 enzyme of wisegene.
Further, the kit further comprises a separately packaged pyridine borane reducing agent;
the pyridine borane reducing agent consists of the individually packaged components of table 2 below:
TABLE 2 pyridine borane reducing agent composition
Components Concentration of Volume of
Reagent bottle 1 Sodium acetate pH 4.3 3M(3mol/L) 10μL
Reagent bottle 2 Pyridine borane 10M 5μL
EXAMPLE 2 DNA methylation transformation kit
The DNA methylation conversion kit provided by the embodiment comprises a TET enzyme oxidation buffer solution packaged independently;
TET enzyme oxidation buffer (pH 8) consisted of the following components in table 3:
TABLE 3 TET enzyme Oxidation buffer composition
Components Concentration of
Tris–Cl 20mM
NaCl 100mM
2-oxoglutarate 3.3mM
Ascorbic acid 6.67mM
Adenosine Triphosphate (ATP) 4mM
Further, the kit also comprises Fe (NH) packaged independently4)2(SO4)2Solution of said Fe (NH)4)2(SO4)2Fe (NH) in solution4)2(SO4)2Is 0.75 mM.
Further, the kit also comprises an independently packaged TET enzyme solution, and the concentration of the TET enzyme is 10.5 mu M. The TET enzyme is NEB EM conversion module.
Further, the kit further comprises a separately packaged pyridine borane reducing agent;
the pyridine borane reducing agent consists of the individually packaged components of table 4 below:
TABLE 4 pyridine borane reducing agent composition
Components Concentration of Volume of
Reagent bottle 1 Sodium acetate pH 4.3 3M 10μL
Reagent bottle 2 Pyridine borane 10M 5μL
EXAMPLE 3 DNA methylation transformation kit
The DNA methylation conversion kit provided by the embodiment comprises a TET enzyme oxidation buffer solution packaged independently;
TET enzyme oxidation buffer (pH 8) consisted of the following components in table 5:
TABLE 5 TET enzyme Oxidation buffer composition
Components Concentration of
HEPES (4-hydroxyethyl piperazine ethanesulfonic acid) 167mM
NaCl 333mM
α -KG (α -ketoglutaric acid) 3.3mM
Ascorbic acid 6.67mM
Adenosine Triphosphate (ATP) 4mM
Further, the kit also comprises Fe (NH) packaged independently4)2(SO4)2Solution of said Fe (NH)4)2(SO4)2Fe (NH) in solution4)2(SO4)2Is 1.5 mM.
Further, the kit also comprises an independently packaged TET enzyme solution, and the concentration of the TET enzyme is 9 mu M. The TET enzyme is NEB EM conversion module.
Further, the kit further comprises a separately packaged pyridine borane reducing agent;
the pyridine borane reducing agent consists of the individually packaged components of table 6 below:
TABLE 6 pyridine borane reducing agent composition
Components Concentration of Volume of
Reagent bottle 1 Sodium acetate pH 4.3 3M 10μL
Reagent bottle 2 Pyridine borane 10M 5μL
Example 4 construction of Whole genome methylation non-bisulfite sequencing library
The embodiment provides a construction method of a genome-wide methylated non-bisulfite sequencing library, which comprises the following steps:
(1) DNA fragmentation
Breaking 100 ng-1000 ng of genome gDNA (physical breaking or enzyme breaking), wherein the genome gDNA comprises human genome gDNA and exogenous internal reference DNA (in the embodiment, the exogenous positive internal reference DNA is methylated pUC19(Zymoresearch), and the negative internal reference is unmethylated lambda DNA), and the broken gDNA is purified by using 1X magnetic beads and eluted by enzyme-free water with the elution volume of 67 mu L;
(2) end repair and addition of A
1) Taking the fragmented DNA in the step (1) for end repair and A addition, wherein the reaction system of end repair and A addition is shown in the following table 7, and the repair reaction conditions are shown in the following table 8;
TABLE 7 end-repair and A addition reaction System
Figure BDA0002285655030000111
Note: end repair & addition a-buffer provided by KAPA Hyper Prep Kit; end repair & add a-enzyme mix supplied by KAPA Hyper Prep Kit;
TABLE 8 repair reaction conditions
Step (ii) of Temperature of Time of day
Tip repair 20℃ 30min
Adding A 65℃* 30min
HOLD 4℃** ∞
Note: the hot lid temperature was 85 ℃. If the linker ligation reaction is directly performed, the holding temperature may be set to 20 ℃.
(3) Ligation reaction
1) Connection of
Ligating the end repair and A addition product in the step (2) by using ligase to obtain a ligated dsDNA, wherein the ligation reaction system is shown in the following table 9;
TABLE 9 ligation reaction System
Figure BDA0002285655030000112
Figure BDA0002285655030000121
Note: enzyme and buffer should be prepared fresh; ligation buffer, DNA ligase, and adaptor stock were supplied by KAPAbiosystems.
Incubation conditions were as follows: incubating at 20 deg.C for 15-30 min.
2) Post-ligation purification
Purifying the linker-ligated product of step 1) according to the following Table 10;
TABLE 10 purification System
Components Volume of
Joint ligation product 110μL
Magnetic bead 88μL
Total volume: 198μL
the purification steps are as follows:
1) shaking, mixing and centrifuging.
2) And incubating for 5-10min at room temperature to combine the DNA with the magnetic beads.
3) The EP tube was placed on a magnetic stand for 2min and the solution was clarified.
4) The supernatant was aspirated.
5) 200. mu.L of 80% ethanol by volume were added.
6) And standing for 30 s.
7) The ethanol was aspirated off.
8) The ethanol was evaporated at room temperature or 37 ℃ without significant brightening or cracking of the magnetic beads.
9) DNA was eluted by adding 24. mu.L of enzyme-free water.
(3) TET enzymatic oxidation
The adaptor-ligated DNA library obtained in step (2) and the TET enzyme reaction solution in the kit provided in example 3 were used to prepare a TET enzyme oxidation reaction system (pH 8) on ice, as shown in table 11 below:
TABLE 11 TET enzymatic Oxidation reaction System
Reaction mixture Dosage of
Adaptor-ligated DNA libraries 30～100ng
Fe(NH4)2(SO4)2 Final concentration 100. mu.M
1 XTET enzyme oxidation buffer (pH 8) 15μL
Tet enzyme Final concentration 0.048-0.06. mu.M
Enzyme-free water Make up to 50. mu.L
Total volume 50μL
The TET enzyme oxidation step is as follows:
1) reaction conditions are as follows: reacting for 1-3h at 37 ℃.
2) Adding 1 μ L proteinase K (0.8U) into the oxidation reaction system, and incubating at 37-50 deg.C for 0.5-1 h.
3) The product was added to 1.8X magnetic beads.
4) Shaking, mixing and centrifuging.
5) And incubating for 5-10min at room temperature to combine the DNA with the magnetic beads.
6) The EP tube was placed on a magnetic stand for 2min and the solution was clarified.
7) The supernatant was aspirated.
8) 200. mu.L of 80% ethanol by volume were added.
9) And standing for 30 s.
10) The ethanol was aspirated off.
11) The ethanol was evaporated at room temperature or 37 ℃ without significant brightening or cracking of the magnetic beads.
12) Eluting with 17-36 μ L of enzyme-free water.
(4) Pyridine borane reduction reaction
1) Reduction reaction system configuration
Taking the oxidized DNA library obtained in the step (3) and the pyridine borane reducing agent in the kit provided in example 3 to prepare a reduction reaction system, and preparing according to the following table 12:
TABLE 12 reduction reaction System preparation
Reaction mixture Volume of
3M sodium acetate pH 4.3 10μL
10M pyridine borane 5μL
DNA library 35μL
2) Reaction conditions are as follows: incubation was carried out at 37 ℃ and 850rpm for 16h-32 h.
3) The DNA was purified using a spin column (Zymo-IC spin column in this example). Elution volume 21. mu.L.
(5) Library amplification
1) PCR amplification reaction configuration:
amplifying the reduced DNA library obtained in the step (4), and configuring a PCR amplification reaction system as shown in the following table 13:
TABLE 13 PCR amplification reaction System
Figure BDA0002285655030000141
Figure BDA0002285655030000151
Note: index used is determined by the platform used and the joint used. The final concentration of the primers in the recommended system is 0.5-2. mu.M.
2) Reaction conditions are as follows: the PCR amplification reaction procedure described above is as follows in Table 14:
TABLE 14 PCR amplification reaction procedure
Figure BDA0002285655030000152
3) The PCR product was washed twice with 0.9X magnetic beads and eluted at a volume of 27. mu.L.
(6) Verification of conversion
Exogenous internal reference DNA was used for transformation rate validation:
1) exogenous internal reference Taq enzyme amplification reaction
Taking 2ng of the purified PCR product obtained in the step (5) for PCR amplification. Setting a control group: unmethylated pUC19 DNA and H2O as a control group. The PCR amplification reaction system was configured as follows:
TABLE 15 PCR amplification reaction System
Reagent Volume of Final concentration
PCR product DNA template Various 2ng
10 XTaq buffer 2.5μL 1X
dNTP[10mM each] 0.5μL 0.2mM
5mC_pUC19_Forward primer[10μM] 0.5μL 0.2μM
5mC_pUC19_Reverse primer[10μM] 0.5μL 0.2μM
Taq DNA polymerase 0.125μL 0.625U
Enzyme-free water Various NA
Total volume 25μL
Note: 5mC _ pUC19_ Forward primer, 5mC _ pUC19_ Reverse primer, Zymoresearch.
2) The PCR amplification conditions are shown in table 16 below:
TABLE 16 PCR amplification conditions
Figure BDA0002285655030000161
3) Taking 100ng of PCR amplification product obtained in the step 2), carrying out Taq α I enzyme digestion reaction, wherein the reaction system configuration is as follows:
TABLE 17 restriction reaction System
Reagent Volume of Final concentration
194bp PCR product
10 XCutSmart buffer 2μL 1X
Taq α I enzyme 1μL 10U
Enzyme-free water NA
Total volume 20μL
Reaction conditions are as follows: incubating for 0.5-1 h at 65 ℃.
4) Taking 10ul of the enzyme digestion product in the step 3) and the PCR product in the step 2), adding 6X fluorescent yellow and 1-3% agarose gel for electrophoresis. Electrophoresis results show that only one band (216bp) can be seen in the successfully transformed enzyme digestion library, and two clear bands (39bp and 176bp) can be seen in the non-successfully transformed enzyme digestion library. The PCR product was not digested, and only one band was visible. FIG. 1 is a gel diagram of the partial conversion success, and the conversion rates of the 3 samples of whole genome methylation "C" to "T" were calculated to be 70.4%, 71.5% and 64.49%, respectively, according to the calculation in FIG. 1.
(7) The constructed library is subjected to LabChip (Agilent) quality control, and a quality control qualified map is shown in figure 2.
Example 5 construction of Whole genome methylation non-bisulfite sequencing library
This example is substantially the same as example 4 except that "(3) TET enzyme oxidation reaction" TET enzyme reaction liquid in the kit provided in example 1 was used to prepare TET enzyme oxidation reaction system as shown in Table 18 below:
TABLE 18 TET enzymatic Oxidation reaction System
Figure BDA0002285655030000181
Example 6 construction of Whole genome methylation non-bisulfite sequencing library
This example is substantially the same as example 4 except that "(3) TET enzyme oxidation reaction" TET enzyme reaction liquid in the kit provided in example 2 was used to prepare TET enzyme oxidation reaction system as shown in the following Table 19:
TABLE 19 TET enzymatic Oxidation reaction System
Reaction mixture Dosage of
Adaptor-ligated DNA libraries 30～100ng
Fe(NH4)2(SO4)2 Final concentration 105. mu.M
1 XTET enzyme oxidation buffer (pH 8) 20μL
Tet enzyme Final concentration 0.06. mu.M
Enzyme-free water Make up to 50. mu.L
Total volume 50μL
Experimental example comparison of methylation sequencing library with bisulfite library constructed in example 4
The construction method of the sequencing library of the genome-wide methylated bisulfite (hereinafter referred to as bisulfite library or WGBS library) comprises the following steps: the genome-wide methylation sequencing library disclosed in Chinese patent document CN104532360B and the construction method thereof are adopted for construction, wherein the conversion treatment from 'C' to 'T' adopts bisulfite conversion.
Two, ratio of monobasic to dinucleoside bases in different pooling protocols
The methylation sequencing library constructed in example 4 was compared with the bisulfite library at A, T, C, G base ratios, and the ratio of mononucleotides to dinucleotides is shown in FIG. 3. Bisulfite methylation detection converts unmethylated C to T bases, and the protocol of example 4 achieves T conversion of methylated (mC, hmC, etc.) bases. It can be seen that the C ratio increases from 1.4% of the WGBS library to 20% of the inventive scheme; the C content is improved from 0.86% to 4.2%; the ratio of C and CG for the protocol of example 4 was comparable to the expected ratio of C and CG for the whole genome library (4.4% vs 21%). Therefore, the methylation sequencing library constructed by the scheme of the embodiment 4 of the invention has balanced bases and does not need to be mixed on a machine.
Third, the genome comparison rate and the coverage depth are improved
After sequencing the bisulfite library and the methylation sequencing library of example 4 by using Geneseq +2000, performing linker removal and low-quality filtering on the original data, comparing the quality control result of the methylation library of example 4 with the bisulfite sequencing data, it can be seen that the comparison rate, depth and coverage of the method are superior to those of the bisulfite sequencing data under the equivalent sequencing quantity, and the result of the quality control parameters is shown in Table 20 below.
TABLE 20 second generation sequencing quality control parameters for different technical schemes
Figure BDA0002285655030000191
As can be seen from the above table, the library constructed by the invention has a certain improvement in genome alignment and coverage due to heavier bisulfite conversion library in sequencing data.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

Claims (10)

1. A DNA methylation conversion kit, which is characterized by comprising a TET enzyme reaction solution, wherein the TET enzyme reaction solution comprises the following independently packaged components: TET enzyme oxidation buffer; the TET enzyme oxidation buffer comprises the following micromolar components: HEPES or Tris-Cl (20-167). times.103Fraction, NaCl (100-3Aliquot, α -KG or 2-oxoglutarate 3.3X 103Portions of ascorbic acid 6.67X 103And adenosine triphosphate 4 × 103Preparing; the TET enzyme oxidation buffer pH was 8.0.
2. The kit according to claim 1, wherein said TET enzyme reaction solution further comprises Fe (NH) in an independent package4)2(SO4)2Solution of said Fe (NH)4)2(SO4)2Fe (NH) in solution4)2(SO4)2In a concentration of 0.75-1.5 mM; preferably, the TET enzyme reaction solution further comprisesComprises independently packaged TET enzyme solution, wherein the concentration of the TET enzyme is 8-10.5 mu M.
3. The kit of claim 1 or 2, further comprising a separately packaged pyridine borane reducing agent; preferably, the pyridine borane reducing agent comprises the following components in independent packages: pyridine borane solution, the concentration of the pyridine borane is 10M; and sodium acetate solution, sodium acetate concentration 3M, pH 4.3.
4. Use of the kit of any one of claims 1-3 for whole genome methylation sequencing, DNA methylation sequencing or targeted region DNA methylation sequencing.
5. A method of whole genome methylation non-bisulfite sequencing library construction, comprising performing a "C" to "T" conversion using the kit of any one of claims 1-3.
6. The method according to claim 5, wherein in the TET enzyme reaction solution oxidation step, the library to be converted from "C" to "T" is mixed with the TET enzyme reaction solution to prepare a TET enzyme oxidation reaction system in an amount of 50. mu.L:
30-100 ng of DNA library;
Fe(NH4)2(SO4)2the final concentration is 95-105 mu M;
1 XTET enzyme oxidation buffer, 10-20 μ L;
tet enzyme, final concentration 0.048-0.06 μ M;
make up to 50. mu.L of enzyme-free water.
7. The method according to claim 5 or 6, wherein in the oxidation step of the TET enzyme reaction solution, the reaction conditions are: reacting at 37 ℃ for 1-3h, then adding proteinase K, and incubating at 37-50 ℃ for 0.5-1 h; preferably, in subjecting the product after oxidation using the TET enzyme reaction solution to a pyridine borane reduction step, the product is mixed with a pyridine borane reducing agent and incubated at 37 ℃ and 850rpm for 16h to 32 h.
8. The method according to any one of claims 5 to 7, wherein the library after transformation from "C" to "T" is subjected to PCR amplification using PCR amplification enzymes comprising KAPA HIFI Urail + polymerase, NEB Q5Urail enzyme, Pfu TurboCx hotspot polymerase or Taq enzyme; preferably, the reaction procedure for PCR amplification of the library after conversion from "C" to "T" is as follows: initial denaturation at 98 ℃ for 45 sec; denaturation at 98 ℃ for 15sec, annealing at 60 ℃ for 30sec, extension at 72 ℃ for 30sec, 8 cycles; extending for 1min at 72 ℃; 4 ℃ and infinity.
9. The method of claim 8, wherein the amplified library is subjected to next generation sequencing, and the sequencing platforms include but are not limited to Illumina platform, MGI or Gene + Seq platform; preferably, the sequencing platform is a Gene + Seq platform.
10. A whole genome methylation sequencing library constructed according to the method of any one of claims 4 to 9.
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CN114195902A (en) * 2021-10-18 2022-03-18 翌圣生物科技(上海)股份有限公司 Recombinant protein domain enhanced TET enzyme and whole genome DNA methylation detection method
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