CN113943779A

CN113943779A - Enrichment method of DNA sequence with high CG content and application thereof

Info

Publication number: CN113943779A
Application number: CN202111201399.6A
Authority: CN
Inventors: 郭萍; 陈洪亮; 郑进
Original assignee: Xiamen Vangenes Biotechnology Co ltd
Current assignee: Xiamen Vangenes Biotechnology Co ltd
Priority date: 2021-10-15
Filing date: 2021-10-15
Publication date: 2022-01-18

Abstract

The invention provides an enrichment method of a DNA sequence with high CG content, which comprises the steps of preparing a DNA fragment with a joint and converted by CT, treating DNA endonuclease and carrying out enrichment amplification to obtain a product, namely an enriched product of the DNA sequence with high CG content. The invention adopts the method of firstly performing joint connection and CT transformation and then performing enzyme digestion enrichment, thereby not only reducing the loss of original DNA materials, but also improving the effect of enzyme digestion enrichment of DNA sequences with high CG content. The enriched product can be used for high-throughput sequencing, greatly reduces the data volume of sequencing, improves the sequencing efficiency, and is suitable for methylation analysis of a high-CG-content DNA sequence of a clinical sample with a small amount of original DNA material.

Description

Enrichment method of DNA sequence with high CG content and application thereof

Technical Field

The invention relates to the technical field of genomics and molecular biology, in particular to an enrichment method of a DNA sequence with high CG content and application thereof.

Background

DNA methylation refers to covalent modification of cytosine cyclomethyl naturally occurring in the genome of a species such as prokaryotes, eukaryotes, and the like. DNA methylation regulates gene expression without altering the DNA sequence, further affecting the phenotype of the organism. In eukaryotes, DNA methylation is an epigenetic modification that is ubiquitous and is being studied in large numbers. DNA methylation plays a key role in regulating gene expression, maintaining genome stability, regulating DNA spatial conformation, interacting with proteins and even influencing chromatin high-level structures. Furthermore, the change of biological characters and the occurrence and development of diseases have the function of DNA methylation.

One of the commonly used methylation analysis methods is to treat the DNA sample to be tested with bisulfite. Bisulfite treatment converts unmethylated modified C bases in a DNA sequence to U bases (which are subsequently converted to T bases during amplification), while methylated modified C bases remain, a process referred to herein as CT conversion. The obtained DNA sample can obtain methylation modification information of a specific site through high-throughput sequencing analysis. In addition to bisulfite, the academia and industry have developed biological enzyme-mediated CT conversion products. CT transformation in combination with high throughput sequencing is currently the most commonly used methylation analysis method.

Methylation modifications typically occur at the 5-carbon of CpG or CpHpG (H ═ a, T, C) cytosines. CpG dinucleotides are heterogeneously distributed in the human genome, and a fraction of CpG dinucleotides are in a highly aggregated state and are called CpG islands. Although there is some controversy regarding the exact definition of a CpG island, it is generally believed that a DNA region with a CG content of greater than 50% and a length of more than 200bp may be considered as a CpG island. A large number of research results show that methylation modification of the CpG island influences physiological processes such as cell division proliferation, differentiation and pathological changes to a certain extent and is closely related to various diseases such as cancer, aging and senile dementia. The analysis of CpG island methylation modification can not only promote the research of life process and disease occurrence process, but also be applied to the diagnosis and monitoring of diseases, especially tumor diseases.

Currently, genome-wide methylation analysis techniques can be broadly divided into the following four types:

first, whole genome sulfite sequencing (WGBS) can analyze the human genome for methylation modifications at approximately 2800 million CpG sites. However, the total length of the human genome is about 30 hundred million bases, the sequencing flux required by whole genome sequencing is huge, and the analysis cost of a single sample is higher. The ratio of CpG island sequences enriched in CpG sites in the whole genome sequence is less than 1%. The detection flux and cost can be greatly reduced by enriching CpG islands or DNA sequences with high CG content.

Secondly, an effective CpG island enrichment method is to simplify whole genome methylation Sequencing (RRBS), digest the genome by using restriction enzyme MspI, collect fragmented CG rich sequence DNA for CT transformation and Sequencing, and can realize enrichment and methylation analysis of CpG island regions to a certain extent. The RRBS has the advantages that the MspI enzyme digestion is used for enriching the high CG sequences for analysis, so that the sequencing flux can be obviously reduced, and the analysis cost is reduced. However, this method is only suitable for whole genome DNA which is complete or not strongly fragmented, and is not suitable for clinical DNA samples which are fragmented, such as plasma free DNA (cfDNA) or Formalin-Fixed paraffin Embedded (FFPE) DNA samples. In addition, the library construction process needs sample consumption steps such as enzyme digestion and fragment screening, the required sample size is large, and the library construction method is not suitable for detecting clinical samples with low sample size.

And thirdly, another effective target site enrichment method is to design a probe and enrich target sequences through sequence hybridization. The advantage of probe hybridization is that the enrichment site can be selected by designing the probe, reducing sequencing throughput. However, the method has limited enrichment sites, a new probe needs to be designed for converting the enrichment range, and the operation cost is higher.

Fourthly, CN109295188A discloses a simplified methylation sequencing method for cfDNA, which is characterized in that cfDNA is cut by restriction enzyme MseI capable of identifying TTAA sites, fragments rich in T/A are cut into fragments with smaller length, and enrichment of DNA with high CG content in the cfDNA is realized to a certain extent through fragment screening.

However, in the human genome sequence, the number of the MseI recognition sites TTAA is small, the enrichment effect by direct enzyme digestion is limited, and a large sequencing flux and a high sequencing cost are still required. Secondly, the method is not suitable for clinical sample analysis with relatively low DNA content due to the loss of DNA caused by direct enzyme digestion treatment and fragment screening of the original DNA material.

In order to solve the problems, a new method for enriching a DNA sequence with high CG content is needed, and the method is popularized in methylation sequencing of a high CG region of a genome or methylation site analysis of the high CG region of the genome, so that the sequencing cost is reduced.

Disclosure of Invention

In view of the above-mentioned shortcomings of the prior art, the main object of the present invention is to provide a method for enriching a DNA sequence with high CG content and the application thereof, which can improve the utilization efficiency of DNA sample, enhance the enrichment effect of CG-rich sequence, and reduce the sequencing cost of methylation analysis technology.

In order to achieve the above object, the present invention provides a method for enriching a high CG content DNA sequence, comprising the steps of:

s1, connecting the DNA fragment with a connector, and carrying out CT transformation to obtain a processed fragment;

s2, amplifying the treated fragment obtained in the step S1 by using sequence specific primers of the joint to obtain a DNA fragment which is transformed by CT and is provided with the joint;

s3, carrying out enzyme digestion treatment on the DNA fragment which is converted from the CT and is provided with the joint and obtained in the step S2 by using DNA endonuclease or enzyme combination for specifically recognizing AT rich sequences to obtain an enzyme digestion product;

s4, amplifying the enzyme digestion product obtained in S3 by using a sequence specific primer of the primer in S2;

s5, obtaining the DNA sequence enriched in the high CG content area.

Further, in step S1, the DNA fragment is ligated to the linker and then transformed by CT, which comprises the following steps: carrying out end repair on the DNA fragment to obtain an end repair product; connecting the obtained end repairing product with a joint to obtain a joint connecting product; and (3) converting the obtained adaptor connection product by CT to obtain a treated fragment.

Further, in step S1, the DNA fragment is transformed by CT and then ligated with a linker, which comprises the following steps: carrying out CT conversion treatment on the DNA fragment to obtain a CT conversion product; the obtained CT transformation product is connected with a joint in a single-stranded DNA state to obtain a treated fragment.

Further, the DNA fragment in step S1 is a DNA fragment having a length of 50-1000 bp.

Further, the DNA fragment in step S1 is a genomic DNA subjected to a fragmentation process or a DNA sample having a length of more than 1000 bp.

Further, the fragmentation treatment is ultrasonic fragmentation or enzyme digestion.

Further, in step S1, the CT conversion refers to the conversion of the unmethylated modified C base into U base or T base by bisulfite or bio-enzyme treatment.

Further, the amplification in steps S2 and S4 includes methods of polymerase chain reaction amplification, strand displacement amplification, recombinase polymerase amplification, rolling circle amplification, and helicase-dependent amplification.

Further, steps S3 and S4 are repeated at least once.

Preferably, steps S3 and S4 are repeated at least once, and step S3 is repeated once more.

More preferably, steps S3 and S4 are repeated once and step S3 is repeated once more.

Further, the DNA endonuclease specifically recognizing the AT rich sequence in step S3 includes restriction endonuclease, CRISPR gene editing enzyme, ZFN, TALEN, and meganuclease.

Further, restriction endonucleases include MlucI, MseI, SspI, PsiI, AseI, DraI, PacI, Ana I, AcsI, AgI, apoI, Afl II, Bfr I, BspT I, BstAF I, EcoRI, EcoRV, Fai I, FauND I, Hpa I, KspA I, Mfe I, MspC I, MsI, MunI, NdeI, pmeI, PshB I, SaqAI, Smii, Sse9I, Tas I, Tru1I, Tru9I, TspDT I, Tsp509I, VspI, Xap I, Hind, Nsi I, Nsp, Pag I, Pci, SfuI, SnaFriB I, BsfII, Sw I, and Sca I.

Preferably, the restriction enzymes include MlucI, Mse I, Ssp I, Psi I, Ana I, Dra I, Ase I, PshB I, SaqA I, Smi I, Sse9I, Tas I, Tru1I, Tru9I, Vsp I, Swa I and Pac I.

Further, the reaction conditions required for the cleavage treatment in step S3 include specific active cleavage conditions of the restriction enzyme and asterisk active cleavage conditions.

Further, the asterisk active enzyme cutting condition refers to the condition of adding or/and replacing one or more conditions relative to the optimal enzyme cutting condition indicated in the used restriction enzyme specificationThe star activity of the restriction enzyme is excited, the site recognition specificity of the restriction enzyme is changed, so that the restriction enzyme can recognize and cut a plurality of specific sequences, and the conditions of addition or/and replacement are 5% -43% of glycerol concentration and MnCl₂One or more of the combination of 0.1mM-100mM concentration, 2% -15% alcohol concentration, 5% -35% DMSO concentration, 0.1U/μ l-10U/μ l enzyme concentration and reaction time of 10 minutes to 48 hours.

Preferably, the restriction endonuclease, the CRISPR gene editing enzyme, the ZFN, the TALEN and the meganuclease are used independently or combined with each other to form different screening pressures, a sequencing library with different CG content screening degrees is constructed, and the diversified sequencing requirements are met.

In a second aspect of the invention, the application of the enrichment method of the high-CG-content DNA sequence in methylation sequencing of a high-CG-region genome or methylation site analysis of the high-CG-region genome is provided.

Further, methylation sequencing of the genome high CG region comprises the following steps:

preparing a high CG content DNA library by adopting a joint adapted by a high-throughput sequencing platform, a sequence specific primer of the joint and a universal primer; performing high-throughput sequencing; data alignment reference genomic analysis to obtain methylation information.

Compared with the prior art, the invention has the following advantages:

compared with a classical methylation analysis method WGBS, the method provided by the invention has the advantages that the enrichment effect of the DNA sequence with high CG content is improved by adding the steps of DNA enzyme digestion and PCR amplification, the operation is simple, and the result is stable;

compared with the CN109295188A in the prior art, the method adopts the sequence of enzyme cutting after adding the adaptor and amplifying, avoids the loss of original DNA materials caused by the steps of enzyme cutting, fragment recovery and the like to the maximum extent, and is suitable for clinical samples with less original DNA.

Compared with the prior art CN109295188A, the method adopts the sequence of carrying out the enzyme digestion enrichment treatment after carrying out the CT transformation, and utilizes the characteristic that the CG content of DNA is obviously reduced after the CT transformation, thereby obviously improving the CpG island enrichment effect, reducing the sequencing flux and reducing the sequencing cost;

and fourthly, performing enzyme digestion treatment on the PCR amplification library which is connected with the joint and subjected to CT transformation by using the method, wherein DNA samples with different lengths can be analyzed, and the DNA samples comprise but are not limited to genome DNA, free plasma DNA, FFPE DNA and the like.

Fifthly, the method can realize enrichment effects of different degrees. The invention uses a plurality of DNA endonucleases which can specifically recognize AT rich sequences, including but not limited to restriction endonucleases, CRISPR gene editing enzymes, ZFNs, TALENs, meganucleases and the like. The DNA endonucleases can be combined with each other, repeatedly processed or processed under special conditions (such as restriction endonuclease asterisk activity conditions) to form different screening pressures, so that sequencing libraries with different screening degrees are obtained, and diversified sequencing requirements are met.

Drawings

FIG. 1 is a schematic diagram of a technical route of the present invention;

FIG. 2 is an analysis of the abundance of the recognition sites for the restriction enzymes Mse I, Mluc I, Psi I and Ssp I used in example 1, example 2, example 3 and example 4 of the present invention on the CT-transformed and non-CT-transformed human genomes;

FIG. 3 is a comparison of the enrichment effect of the sequenced sequences of example 1 and comparative example of the present invention in CpG islands and other regions;

FIG. 4 is a comparison of the number of sequencing layers and the number of CpG sites for example 1 of the present invention and comparative example.

FIG. 5 shows the star activities of the restriction enzymes MlucI, MseI, SspI, and PsiI mix. The final product of the DNA with high CG content in the embodiment 1 of the invention is further subjected to enzyme digestion by MluCI, MseI, SspI and PsiI, and a shearing product cannot be generated under the optimal enzyme digestion condition; in 20% glycerol and 1mM MnCl₂Can be further sheared to generate a sheared product under the star activity conditions.

Detailed Description

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The technical route of the enrichment method of the DNA sequence with high CG content provided by the invention is shown in figure 1. The abundance analysis of the recognition sites for the restriction enzymes Mse I, Mluc I, Psi I and Ssp I used in example 1, example 2, example 3 and example 4 of the present invention on the CT-transformed and non-CT-transformed human genomes is shown in FIG. 2.

Example 1

1.1 end repair and linker attachment

1) The plasma free DNA and the linker in NEBNext Multiplex oligonucleotides for Enzymatic Methyl-seq (Unique Dual IndexPrimer Pairs) (NEB, cat # E7140) were ligated using VAHTS Universal DNA library prep kit for Illumina V3 (Novozam, cat # ND 607).

Composition (I)	Volume (μ L)
		cfDNA	50
Methylation control	1
		End Prep Mix4	15
Total volume	61

Centrifuging the reaction system after vortex, incubating for 15min at 20 ℃ and incubating for 15min at 65 ℃;

2) adding 5 mu.L of Rapid Ligation buffer2, 5 mu.L of NEB adapter and 5 mu.L of Rapid DNaligase into the DNA tube in the step 1), centrifuging after vortexing, and carrying out 15min at 20 ℃;

3) the ligated DNA sample was recovered using Magpure magnetic beads. Add 100. mu.L (1X) magpure magnetic beads to each DNA tube, vortex, centrifuge, and stand for 10min according to the protocol; placing the DNA tube on a magnetic frame until the liquid is clear; using a pipette to suck the clarified liquid and leaving magnetic beads; adding 500 μ L of new 80% ethanol, standing for 30s, and removing the supernatant; repeatedly rinsing with alcohol once; centrifuging to remove residual solution, and air drying for 5 min; add 29. mu.L of Low TE vortex for 30s to each air-dried EP tube, centrifuge, place on magnetic rack, transfer 28. mu.L of eluate to a new EP tube;

1.2 Bio-enzyme CT transformation (NEB reagent, cat # E7125)

1) Unfreezing an NEB enzymology CT conversion module reagent in advance, and carrying out vortex centrifugation; preparing TET2reaction buffer and Fe (II) diluted solution according to reagents and instructions;

2) the reagents were added as per the following table

Composition (I)	Volume (μ L)
		DNA for linker ligation	28
TET2 Reaction Buffer	10
		Oxidation Supplement	1
Oxidation Enhancer	1
		DTT	1
TET2	4

Vortex mixing and centrifuging; adding 5 mu L of diluted Fe (II) solution into a DNA reaction tube, uniformly mixing by vortex, centrifuging, and incubating for 1h at 37 ℃; adding 1 mu L of Stop Reagent into the DNA reaction tube, and continuously incubating for 30min at 37 ℃;

3) the DNA sample was collected using Magpure magnetic beads. Add 90. mu.L (1.8X) magpure magnetic beads to each DNA tube, vortex, centrifuge, and stand for 10min according to the protocol; placing the DNA tube on a magnetic frame until the liquid is clear; using a pipette to suck the clarified liquid and leaving magnetic beads; adding 500 μ L of new 80% ethanol, standing for 30s, and removing the supernatant; repeatedly rinsing with alcohol once; centrifuging to remove residual solution, and air drying for 5 min; add 17. mu.L of Low TE vortex for 30s to each air-dried EP tube, centrifuge, place on magnetic rack, transfer 16. mu.L of eluent to a new EP tube;

4) adding 4 mu L of formamide into the DNA sample tube, uniformly mixing, centrifuging, incubating in a PCR instrument preheated to 85 ℃ for 10min, and immediately placing on ice;

5) add 68. mu. L H to the DNA reaction tube₂O, 10. mu.L of APOBEC Reaction buffer, 1. mu.L of BSA, 1. mu.L of LAPOBEC, incubated at 37 ℃ for 3 h;

6) the DNA sample was collected using Magpure magnetic beads. Add 100. mu.L (1X) magpure magnetic beads to each DNA tube, vortex, centrifuge, and stand for 10min according to the protocol; placing the DNA tube on a magnetic frame until the liquid is clear; using a pipette to suck the clarified liquid and leaving magnetic beads; adding 500 μ L of new 80% ethanol, standing for 30s, and removing the supernatant; repeatedly rinsing with alcohol once; centrifuging to remove residual solution, and air drying for 5 min; add 21. mu.L of Low TE vortex for 30s to each air-dried EP tube, centrifuge, place on magnetic rack, transfer 20. mu.L of eluent to a new EP tube;

2. DNA sample amplification after CT transformation (KAPA reagent, cat # KR0413, NEB reagent, cat # E7140)

1) The following system was configured on ice:

composition (I)	Volume (μ L)
		DNA after CT transformation	20
KAPA HiFi HotStart Uracil+ReadyMix	25
		NEB INDEX primer	5
Total volume	50

2) After vortex centrifugation, the PCR tube was placed in a PCR instrument and run according to the following procedure:

3) the DNA sample was collected using Magpure magnetic beads. Add 45. mu.L (0.9X) magpure magnetic beads to each DNA tube, vortex, centrifuge, and stand for 10min according to the protocol; placing the DNA tube on a magnetic frame until the liquid is clear; using a pipette to suck the clarified liquid and leaving magnetic beads; adding 500 μ L of new 80% ethanol, standing for 30s, and removing the supernatant; repeatedly rinsing with alcohol once; centrifuging to remove residual solution, and air drying for 5 min; add 41. mu.L of Low TE vortex for 30s to each air-dried EP tube, centrifuge, place on magnetic rack, transfer 40. mu.L of eluent to a new EP tube;

3. restriction enzyme treatment (NEB # R0538, NEB # R0525, NEB # R0744S and NEB # R0132S)

1) The following system was configured on ice:

composition (I)	Volume (μ L)
		DNA transformed and amplified by CT	20
CutSmart Buffer	5
		MseI	0.5
MluCI	0.5
		PsiI	0.5
SspI	0.5
		H₂O	23
Total volume	50

After vortex centrifugation, the reaction was incubated at 37 ℃ for 30 minutes.

2) The DNA sample was collected using Magpure magnetic beads. Add 45. mu.L (0.9X) magpure magnetic beads to each DNA tube, vortex, centrifuge, and stand for 10min according to the protocol; placing the DNA tube on a magnetic frame until the liquid is clear; using a pipette to suck the clarified liquid and leaving magnetic beads; adding 500 μ L of new 80% ethanol, standing for 30s, and removing the supernatant; repeatedly rinsing with alcohol once; centrifuging to remove residual solution, and air drying for 5 min; add 21. mu.L of Low TE vortex for 30s to each air-dried EP tube, centrifuge, place on magnetic rack, 20. mu.L of eluent is transferred to a new EP tube;

4. DNA sample amplification after restriction enzyme treatment (Nodezan, cat # ND607)

1) The following system was configured on ice:

reagent	Volume (μ L)
		Restriction enzyme treated DNA	20
VAHTS HiFi Amplificationmix	25
		PCR primer mix 3	5
H₂O	10
		Total volume	50

3) the DNA sample was collected using Magpure magnetic beads. Add 45. mu.L (0.9X) magpure magnetic beads to each DNA tube, vortex, centrifuge, and stand for 10min according to the protocol; placing the DNA tube on a magnetic frame until the liquid is clear; using a pipette to suck the clarified liquid and leaving magnetic beads; adding 500 μ L of new 80% ethanol, standing for 30s, and removing the supernatant; repeatedly rinsing with alcohol once; centrifuging to remove residual solution, and air drying for 5 min; to each air-dried EP tube was added 21. mu.L of Low TE vortex for 30s, centrifuged, placed on a magnetic stand, and 20. mu.L of the eluate was transferred to a new EP tube.

5. Repeating the step 3 and the step 4 once.

6. Repeating the step 3 once to obtain the final product of the DNA with high CG content.

7. The final product was sequenced using the Illumina platform.

The final product of the DNA with high CG content in the embodiment 1 of the invention is further subjected to enzyme digestion by restriction enzymes MlucI, MseI, SspI and PsiI, and a shearing product cannot be generated under the optimal enzyme digestion condition; in 20% glycerol and 1mM MnCl₂Can be further sheared to generate a sheared product under the star activity conditions. The star activities of the restriction enzymes MluCI, MseI, SspI, and PsiI mix are shown in FIG. 5.

Example 2

1.1 end repair and linker attachment

1) The plasma free DNA was ligated to the linker in NEBNext Multiplex oligonucleotides for enzymic Methyl-seq (Unique Dual Index primers pairs) (NEB, cat # E7140) using VAHTS Universal DNA library prep kit for Illumina V3 (Novozam, cat # ND 607).

2) adding 5 mu.L of Rapid Ligation buffer2, 5 mu.L of NEB adapter and 5 mu.L of Rapid DNA ligase into the DNA tube in the step 1), centrifuging after vortexing, and carrying out centrifugation for 15min at 20 ℃;

1.2 Bio-enzyme CT transformation (NEB reagent, cat # E7125)

2) the reagents were added as per the following table

1) The following system was configured on ice:

1) The following system was configured on ice:

5. Repeating the process of the step 3 and the step 4 once to obtain the final product of the DNA with high CG content.

6. The final product was sequenced using the Illumina platform.

Example 3

1.1 end repair and linker attachment

1) The plasma free DNA and the linker in NEBNext multiple oligonucleotides for Enzymatic Methyl-seq (Unique DualIndexPrimer Pairs) (NEB, cat # E7140) were ligated using VAHTS Universal DNA library prep kit for Illumina V3 (Novozam, cat # ND 607).

1.2 Bio-enzyme CT transformation (NEB reagent, cat # E7125)

2) the reagents were added as per the following table

Composition (I)	Volume (μL)
		DNA for linker ligation	28
TET2 Reaction Buffer	10
		Oxidation Supplement	1
Oxidation Enhancer	1
		DTT	1
TET2	4

1) The following system was configured on ice:

1) The following system was configured on ice:

composition (I)	Volume (μ L)
		DNA transformed and amplified by CT	20
CutSmart Buffer	5
		10mM MnCl₂	5
Glycerol	10
		MseI	0.5
MluCI	0.5
		PsiI	0.5
SspI	0.5
		H₂O	8
Total volume	50

After vortex centrifugation, the reaction was incubated at 37 ℃ for 2 minutes.

1) The following system was configured on ice:

5. Repeating the process of step 3 and step 4 once.

6. Repeating the step 3 once, and recovering the product, namely the DNA enrichment final product with high CG content.

7. The enriched end product was sequenced using the Illumina platform.

Example 4

1.1 Bio-enzyme CT transformation (NEB reagent, cat # E7125)

2) the reagents were added as per the following table

Composition (I)	Volume (μ L)
		cfDNA	28
TET2 Reaction Buffer	10
		Oxidation Supplement	1
Oxidation Enhancer	1
		DTT	1
TET2	4

6) the DNA sample was collected using Magpure magnetic beads. Add 100. mu.L (1X) magpure magnetic beads to each DNA tube, vortex, centrifuge, and stand for 10min according to the protocol; placing the DNA tube on a magnetic frame until the liquid is clear; using a pipette to suck the clarified liquid and leaving magnetic beads; adding 500 μ L of new 80% ethanol, standing for 30s, and removing the supernatant; repeatedly rinsing with alcohol once; centrifuging to remove residual solution, and air drying for 5 min; add 21. mu.L of Low TE vortex for 30s to each air-dried EP tube, centrifuge, place on magnetic rack, transfer 20. mu.L of eluate to a new PCR tube;

1.2 Single chain construction of library (Nuo Wei Zan ND620)

1) The following system was configured on ice:

composition (I)	Volume (μ L)
		DNA after CT transformation	20
3’Ligation Buffer	10
		3’Ligation Enzyme Mix	5
3’Adapter	5

temperature of	Time
		37℃	15min
95℃	2min
		4℃	Hold

3) The following system was configured on ice:

composition (I)	Volume (μ L)
		Reaction solution of the last step	40
Extension Primer	5
		Extension Enzyme Mix	35

4) After vortex centrifugation, the PCR tube was placed in a PCR instrument and run according to the following procedure:

composition (I)	Time
		98℃	30sec
63℃	15sec
		68℃	5min
4℃	Hold

5) The DNA sample was collected using Magpure magnetic beads. Add 80. mu.L (1.2X) magpure magnetic beads to each DNA tube, vortex, centrifuge, and stand for 10min according to the protocol; placing the DNA tube on a magnetic frame until the liquid is clear; using a pipette to suck the clarified liquid and leaving magnetic beads; adding 500 μ L of new 80% ethanol, standing for 30s, and removing the supernatant; repeatedly rinsing with alcohol once; centrifuging to remove residual solution, and air drying for 5 min; add 21. mu.L of Low TE vortex for 30s to each air-dried EP tube, centrifuge, place on magnetic rack, transfer 20. mu.L of eluate to a new PCR tube;

6) the following system was configured on ice:

composition (I)	Volume (μ L)
		Purifying the product in the last step	20
5’Ligation Mix	10
		5’Adapter	10

7) After vortex centrifugation, the PCR tube was placed in a PCR instrument and run according to the following procedure:

temperature of	Time
		25℃	15min
4℃	Hold

8) The DNA sample was collected using Magpure magnetic beads. Add 40. mu.L (1X) magpure magnetic beads to each DNA tube, vortex, centrifuge, and stand for 10min according to the protocol; placing the DNA tube on a magnetic frame until the liquid is clear; using a pipette to suck the clarified liquid and leaving magnetic beads; adding 500 μ L of new 80% ethanol, standing for 30s, and removing the supernatant; repeatedly rinsing with alcohol once; centrifuging to remove residual solution, and air drying for 5 min; add 21. mu.L of TE vortex for 30s to each air-dried EP tube, centrifuge, place on magnetic rack, transfer 20. mu.L of eluate to a new PCR tube;

2. amplification of DNA samples after Single Strand Bank construction (KAPA reagent, cat # KR0413, NEB reagent, cat # E7140)

9) The following system was configured on ice:

10) After vortex centrifugation, the PCR tube was placed in a PCR instrument and run according to the following procedure:

11) the DNA sample was collected using Magpure magnetic beads. Add 45. mu.L (0.9X) magpure magnetic beads to each DNA tube, vortex, centrifuge, and stand for 10min according to the protocol; placing the DNA tube on a magnetic frame until the liquid is clear; using a pipette to suck the clarified liquid and leaving magnetic beads; adding 500 μ L of new 80% ethanol, standing for 30s, and removing the supernatant; repeatedly rinsing with alcohol once; centrifuging to remove residual solution, and air drying for 5 min; add 41. mu.L of Low TE vortex for 30s to each air-dried EP tube, centrifuge, place on magnetic rack, transfer 40. mu.L of eluent to a new EP tube;

3) The following system was configured on ice:

4) The DNA sample was collected using Magpure magnetic beads. Add 45. mu.L (0.9X) magpure magnetic beads to each DNA tube, vortex, centrifuge, and stand for 10min according to the protocol; placing the DNA tube on a magnetic frame until the liquid is clear; using a pipette to suck the clarified liquid and leaving magnetic beads; adding 500 μ L of new 80% ethanol, standing for 30s, and removing the supernatant; repeatedly rinsing with alcohol once; centrifuging to remove residual solution, and air drying for 5 min; add 21. mu.L of Low TE vortex for 30s to each air-dried EP tube, centrifuge, place on magnetic rack, 20. mu.L of eluent is transferred to a new EP tube;

4) The following system was configured on ice:

5) After vortex centrifugation, the PCR tube was placed in a PCR instrument and run according to the following procedure:

6) the DNA sample was collected using Magpure magnetic beads. Add 45. mu.L (0.9X) magpure magnetic beads to each DNA tube, vortex, centrifuge, and stand for 10min according to the protocol; placing the DNA tube on a magnetic frame until the liquid is clear; using a pipette to suck the clarified liquid and leaving magnetic beads; adding 500 μ L of new 80% ethanol, standing for 30s, and removing the supernatant; repeatedly rinsing with alcohol once; centrifuging to remove residual solution, and air drying for 5 min; to each air-dried EP tube was added 21. mu.L of Low TE vortex for 30s, centrifuged, placed on a magnetic stand, and 20. mu.L of the eluate was transferred to a new EP tube.

5. Repeating the process of step 3 and step 4 once.

7. The enriched end product was sequenced using the Illumina platform.

Comparative example

After the DNA is digested by MseI restriction endonuclease, library construction, CT transformation and amplification are carried out by the same treatment as steps 1 to 4 in the example, and after fragment screening, the generated library is sequenced by an Illumina platform. The enrichment effect of the sequenced sequences of example 1 and comparative example of the present invention in CpG islands and other regions is shown in fig. 3. The sequencing layer number and CpG site number pairs for example 1 and comparative example of the present invention are shown in FIG. 4.

Sequencing results

The content of CG in a DNA sequence can be obviously reduced by CT conversion, and the enzyme digestion recognition site frequency of AT rich in enzyme is improved. We select all CpG islands in human genome to compose CpG island sequence file, analyze sequence characteristic difference of CpG island and other sequences. As shown in fig. 2, by CT transformation, the average number of enzyme-digestion sites of the four AT rich sequence recognition enzymes is significantly increased in CpG island sequences and other sequences, and the average number increase value in other sequences is significantly higher than that in CpG island sequences, which indicates that more effective enrichment of CpG island sequences can be theoretically achieved by performing AT rich enzyme digestion after CT transformation on human genome sequences.

The sequencing data of the samples prepared in example 1 of the present invention were analyzed and compared with the method disclosed in CN 109295188A. As shown in FIG. 3, the number of reads of the CpG islands compared to the sequencing data of example 1 is about 10 times that of the comparative example, when the sequencing data is normalized and the amount of sequencing data is the same. As shown in fig. 4, under the same sequencing throughput conditions, the number of CpG island sites with a sequencing depth of more than 10 in example 1 of the method is also significantly greater than that of the comparative example method. The enrichment effect of the high CG content DNA library prepared by adopting different processing sequences, different enzyme cutting repeated combinations and asterisk condition enzyme cutting conditions in the four embodiments of the invention is detailed in Table 1.

In conclusion, the method provided by the invention utilizes the characteristic that the content of CG is remarkably reduced after DNA is converted by CT, adopts the endonuclease for identifying the AT rich sequence to treat, cuts the AT rich sequence and enriches the CG rich sequence, and remarkably improves the enrichment effect of the CpG island sequence compared with the method disclosed by CN 109295188A. Meanwhile, the invention carries out enzyme digestion treatment aiming at the amplification library, does not carry out enzyme digestion and purification on the original DNA, reduces the loss of the original DNA and has low input demand on the original DNA; the built library can be enriched by the method of the invention, and also can be enriched by other methods, such as a probe hybridization method, so that the utilization rate of precious DNA samples is improved.

TABLE 1

Claims

1. A method for enriching a DNA sequence with high CG content is characterized by comprising the following steps:

s1, connecting the DNA fragments with a joint, and carrying out CT transformation to obtain processed fragments;

s5, obtaining the DNA fragment with rich CG content sequence.

2. The method for enriching a DNA sequence with high CG content according to claim 1, wherein in step S1, the DNA fragment is connected with the linker and then transformed by CT, comprising the following steps: carrying out end repair on the DNA fragment to obtain an end repair product; connecting the obtained end repairing product with a joint to obtain a joint connecting product; and (3) converting the obtained adaptor connection product by CT to obtain a treated fragment.

3. The method for enriching a DNA sequence with high CG content according to claim 1, wherein in step S1, the DNA fragment is connected with the linker after CT transformation, the specific steps are as follows: carrying out CT conversion treatment on the DNA fragment to obtain a CT conversion product; the obtained CT transformation product is connected with a joint in a single-stranded DNA state to obtain a treated fragment.

4. The method for enriching a DNA sequence with high CG content according to claim 1, wherein the DNA fragment in step S1 is a DNA fragment with a length of 50-1000 bp.

5. The method for enriching a DNA sequence with high CG content according to claim 1, wherein the DNA fragment in step S1 is a fragmented genomic DNA or a DNA sample with a length greater than 1000 bp.

6. The method for enriching a DNA sequence with high CG content according to claim 5, wherein the fragmenting treatment is ultrasonication or enzyme digestion.

7. The method for enriching a DNA sequence with high CG content according to claim 1, wherein in the step S1, the CT conversion is to convert the unmethylated modified C base into U base or T base by bisulfite or bio-enzyme treatment.

8. The method for enriching a high CG content DNA sequence according to claim 1, wherein in the steps S2 and S4, the amplification includes PCR amplification, strand displacement amplification, recombinase PCR amplification, rolling circle amplification and helicase dependent amplification.

9. The method for enriching a DNA sequence with high CG content according to claim 1, wherein the steps S3 and S4 are repeated at least once.

10. The method for enriching a DNA sequence with high CG content according to claim 1, wherein the steps S3 and S4 are repeated at least once and the step S3 is repeated once more.

11. The method for enriching a DNA sequence with high CG content according to claim 1, wherein in the step S3, the DNA endonuclease specifically recognizing the AT rich sequence includes restriction endonuclease, CRISPR gene editing enzyme, ZFN, TALEN and meganuclease.

12. The method of claim 11, wherein the restriction enzymes that specifically recognize AT rich sequences include MlucI, Mse I, Ssp I, Psi I, Ase I, DraI, Pac I, AnaI, AcsI, AgsI, ApoI, AflII, BfrI, BspTI, BstAFI, EcoRI, FaiI, FauNDI, HpaI, KspAI, MfeI, MspCI, MssI, MunI, NdeI, PmeI, PshBI, SaqAI, SmiI, Sse9I, TasI, tau 1I, Tru9I, TspTI, Tsp509I, VspI, Xapi, HindIII, NsiI, NspV, PagI, PciI, SfuI, BfuBI, BfuI, and Clafa.

13. The method for enriching a DNA sequence with high CG content according to claim 12, wherein the restriction enzymes that specifically recognize AT rich sequences include MlucI, MseI, SspI, Psi I, AnaI, DraI, Ase I, PshBI, SaqAI, SmiI, Sse9I, TasI, Tru1I, Tru9I, VspI, SwaI and Pac I.

14. The method for enriching a DNA sequence with high CG content according to claim 1, wherein the reaction conditions required for the cleavage treatment in step S3 include restriction enzyme cleavage conditions and asterisk active cleavage conditions.

15. The method for enriching a DNA sequence with high CG content according to claim 14, wherein the asterisk active digestion conditions are conditions that are added or/and replaced to excite the asterisk activity of the restriction enzyme and change the site recognition specificity of the restriction enzyme to recognize and cut a plurality of specific sequences, relative to the optimal digestion conditions indicated in the restriction enzyme specification used, and the conditions that can be added or/and replaced are 2% -15% alcohol concentration, 5% -35% DMSO concentration, 1% -43% glycerol concentration, MnCl₂The concentration is 0.1mM-100mM, the enzyme concentration is 0.1U/mul-10U/mul, and the reaction time is 10 minutes to 48 hours.

16. The use of the method for enriching the high CG content DNA sequence according to any one of claims 1-15, wherein the use is in methylation sequencing of high CG region of genome; or the application is the application in the methylation site analysis of the high CG region of the genome.

17. The use of the method according to claim 16 for enriching a high CG content DNA sequence, wherein the methylation sequencing of the genomic high CG region comprises the following steps: preparing a high CG content DNA library according to the enrichment method of the high CG content DNA sequence; performing high-throughput sequencing; data alignment reference genomic analysis to obtain methylation information.