CN115820801A - Construction method of DNA methylation site sequencing library - Google Patents

Abstract

The invention provides a method for constructing a DNA methylation site sequencing library, which adopts an unmethylated joint and a methylated joint to analyze methylation sites and construct the methylation sequencing library, wherein the methylated joint is digested by methylation-dependent restriction enzymes to generate a sticky end, the sticky end is introduced by ligase, a specific site containing methylated C bases is identified, and the side wings of the identification site are digested. The non-methylated linker uses a blunt-fill A linker for fragmented nucleic acid or a non-methylation related sticky-end producing endonuclease linker, and when a methylation sensitive restriction endonuclease is used, the present invention performs a double recognition process for a partially methylated site. The methylation sequencing library construction method provided by the invention has the advantages that the sample demand is small, the methylation sequencing library construction method can be used for methylation qualitative sequencing analysis of a single cell level and quantitative analysis of a low-abundance sample, the coverage range of methylation analysis is wide, and the methylation enrichment efficiency is high.

Description

Method for constructing DNA methylation site sequencing library

technical field

The invention relates to the field of biomedicine, in particular to a method for constructing a DNA methylation site sequencing library.

Background technique

With the advancement of epigenetics, there is an increasing demand for methylation sequencing, so a variety of methylation sequencing technologies have emerged, mainly including: methylation sequencing based on sodium bisulfite treatment, based on methylation Methylation-dependent endonuclease-based methylation analysis technology, methylation-sensitive endonuclease-based methylation sequence analysis technology, and deaminase-based methylation sequencing. Among them: the sodium bisulfite method has a large demand for sample nucleic acid and cannot be used for single-cell methylation sequencing and is difficult to use for low-abundance methylation site sequencing; the deaminase method cannot be used for methylation of single cells Sequencing and low-abundance mixed samples; Methylation-sensitive and methylation-dependent endonucleases do not provide high coverage of methylation sites and cannot be used for single-cell methylation sequencing And it is difficult to be used for sequencing low-abundance methylation sites.

The above-mentioned traditional methylation sequencing methods cannot satisfy the methylation sequencing of single cells, and are difficult to be used for sequencing analysis of low-abundance methylation sites in mixed samples.

Contents of the invention

Based on the above technical problems, one of the objectives of the present invention is to provide a new method for the construction of a methylation sequencing library. The methylation sequencing library constructed using the corresponding adapters in the new method can meet the methylation sequencing requirements of a single cell. , can also be used for sequencing of low- and medium-abundance methylation sites.

The purpose of the present invention can be achieved through the following technical solutions:

A method for constructing a DNA methylation site sequencing library, the construction method comprising the steps of:

Provide the DNA to be tested; adopt one or more of the methods shown in a) and b) to prepare the DNA fragment to be tested into an adapter product; use the adapter product as a template to perform PCR amplification to construct a methyl K-site sequencing library;

A) the method shown, comprises the steps:

Using methylation-dependent restriction endonucleases and other restriction endonucleases to digest the DNA to be tested to prepare restriction fragment a;

Ligase is used to connect the first sequencing linker a to the nicked end of the other restriction endonuclease of the enzyme-cut fragment a, and to connect to the nicked end of the methylation-dependent restriction endonuclease of the enzyme-digested fragment a The second sequencing adapter is used to prepare an adapter product a, which is used as a template for PCR amplification;

b) the shown method, comprising the steps of:

Using a mechanical method to fragment the DNA to be tested, fill in the ends, fill in A, and use ligase to connect the first sequencing adapter b to prepare an intermediate adapter product;

Digesting the intermediate linker product with a methylation-dependent restriction endonuclease to prepare restriction fragment b;

Using ligase to connect the enzyme-cut fragment b and the second sequencing adapter to prepare an adapter product b, which is used as a template for PCR amplification;

The base sequence protruding from the 5' end of the cohesive end of the second sequencing adapter is NNNN, and each N is independently selected from any one of A, T, C and G.

In some embodiments of the present invention, the base sequence protruding from the 5' end of the cohesive end of the second sequencing adapter is YNNY, RNNR, NYYN or NRRN, and each Y is independently selected from any of C and T species, each R is independently selected from any of A and G.

In some embodiments of the present invention, the methylation-dependent restriction enzyme is selected from one or more of BstNI, MspI, FspEI, LpnPI and MspJI.

In some embodiments of the invention, the other restriction enzyme is a methylation sensitive restriction enzyme.

In some embodiments of the present invention, the methylation-sensitive restriction enzyme is selected from AciI, HinP1I, HpyCH4IV, HpaII, ClaI, BsaHI, SalI, AvaI, BsiEI, Hpy99I, PvuI, MluI, EagI, Bst One or more of NI and PcilI.

In some embodiments of the present invention, the methylation-sensitive restriction endonuclease is ClaI, and the base sequence protruding from the 5' end of the sticky end of the first sequencing adapter a is CG.

In some embodiments of the invention, the ligase is T4 DNA ligase.

In some embodiments of the invention, said mechanical method comprises ultrasonic disruption.

In some embodiments of the present invention, the DNA to be tested includes artificially synthesized DNA samples.

In some embodiments of the present invention, the DNA to be tested includes genome DNA of an organism.

Compared with the conventional technology, the present invention has the following beneficial effects:

In the present invention, a non-methylated linker (ie, the first sequencing linker) and a methylated site-specific linker (ie, the second sequencing linker) are used to analyze the methylation site and construct a methylation sequencing library, wherein, A Kylated linkers are digested with methylation-dependent restriction endonucleases to generate cohesive ends and introduced with ligase. Methylation-dependent restriction enzymes recognize specific sites containing methylated C bases and cleave flanking recognition sites. The introduction of unmethylated linkers is independent of methylation-dependent restriction enzymes. Non-methylated adapters can be filled with fragmented nucleic acid and added with adapters, or non-methylation-related endonucleases that can produce sticky ends can be used to add adapters, including the use of methylation-sensitive restriction enzymes Create sticky ends plus adapters. Methylation-sensitive restriction enzymes recognize and cleave specific sites containing unmethylated C bases. For example: the CGAT sequence at the 5' end of the linker corresponding to the methylation-sensitive endonuclease in the example ensures the one-way ligation reaction between the linker and the nucleic acid fragment to be analyzed. Under the action of the enzyme ClaI, the self-ligation of the linker is prevented. When methylation-sensitive restriction enzymes are used, the present invention performs two recognition processes on partially methylated sites, that is, indirect recognition that is not digested by sensitive enzymes, and subsequent digestion by methylation-dependent enzymes direct identification. Using methylation-sensitive endonucleases and methylation-dependent endonucleases, on the one hand, methylation sites with partially overlapping sequences can be repeatedly decoded to improve accuracy, and at the same time, the coverage of the method can be expanded by non-overlapping parts scope. The method for constructing a methylation sequencing library that does not rely on sodium bisulfite provided by the present invention requires a small amount of samples, and can be used for qualitative sequencing analysis of methylation at the single cell level and quantitative analysis of low-abundance samples, methylation analysis It covers a wide range, has high methylation enrichment efficiency, and uses repeated identification of some methylation sites to ensure high accuracy, and has practical value in the field of methylation analysis of animal and plant specimens.

Description of drawings

In order to more clearly illustrate the specific implementation of the present invention or the technical solutions in the prior art, the following will briefly introduce the accompanying drawings that need to be used in the specific implementation or description of the prior art. Obviously, the accompanying drawings in the following description The drawings show some implementations of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative work.

Fig. 1 is a schematic diagram of the introduction of sequencing joints in the upstream and downstream of the methylation site in Example 1;

2 is a schematic diagram of the working principle of the disappearance of the original enzyme cleavage site after the CGAT linker is connected to the methylation-sensitive endonuclease product in Example 1;

Fig. 3 is the gel electrophoresis figure that ClaI prevents the self-connection of the CGAT linker in embodiment 1;

Fig. 4 is the result figure that prevents the self-connection of NNNN joint in embodiment 2;

Fig. 5 is the first-generation sequencing figure in embodiment 3;

Fig. 6 is the first-generation sequencing figure in embodiment 4;

Fig. 7 is the first-generation sequencing figure in embodiment 5;

Fig. 8 is the first-generation sequencing figure in embodiment 6;

Fig. 9 is the PCR result figure of embodiment 7;

FIG. 10 is a graph showing the results of next-generation sequencing of the library in Example 7.

Detailed ways

In order to facilitate the understanding of the present invention, the present invention will be described in more detail below. It should be understood, however, that the present invention can be embodied in many different forms and is not limited to the embodiments or examples described herein. On the contrary, the purpose of providing these embodiments or examples is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field of the invention. The terms used in the description of the present invention are only for the purpose of describing specific embodiments or examples, and are not intended to limit the present invention. The optional range of the term "and/or" used herein includes any one of two or more of the relevant listed items, and also includes any and all combinations of the relevant listed items, and any and all of the Combinations include combinations of any two of the related listed items, any more of the related listed items, or all of the related listed items.

In the present invention, "the first aspect", "the second aspect", and "the third aspect" are used for descriptive purposes only, and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the indicated The importance or number of technical characteristics.

In the present invention, the technical features described in open form include closed technical solutions composed of the listed features, and also include open technical solutions with the listed features.

In the present invention, when referring to a numerical interval, unless otherwise specified, both endpoints of the numerical interval are included.

The percentage content involved in the present invention, unless otherwise specified, refers to mass percentage for solid-liquid mixing and solid-solid phase mixing, and refers to volume percentage for liquid-liquid mixing.

The percentage concentration involved in the present invention refers to the final concentration unless otherwise specified. The final concentration refers to the proportion of the added component in the system after the component is added.

The temperature parameters in the present invention, unless otherwise specifically limited, allow either constant temperature treatment or treatment within a certain temperature range. The isothermal treatment allows the temperature to fluctuate within the precision of the instrument control.

The invention provides a method for constructing a DNA methylation site sequencing library, the construction method comprising the following steps:

Provide the DNA to be tested; adopt one or more of the methods shown in a) and b) to prepare the DNA fragment to be tested into an adapter product; use the adapter product as a template to perform PCR amplification to construct a methyl K-site sequencing library;

A) the method shown, comprises the steps:

Using methylation-dependent restriction endonucleases and other restriction endonucleases to digest the DNA to be tested to prepare restriction fragment a;

Ligase is used to connect the first sequencing linker a to the nicked end of the other restriction endonuclease of the enzyme-cut fragment a, and to connect to the nicked end of the methylation-dependent restriction endonuclease of the enzyme-digested fragment a The second sequencing adapter is used to prepare an adapter product a, which is used as a template for PCR amplification;

b) the shown method, comprising the steps of:

Using a mechanical method to fragment the DNA to be tested, fill in the ends, fill in A, and use ligase to connect the first sequencing adapter b to prepare an intermediate adapter product;

Digesting the intermediate linker product with a methylation-dependent restriction endonuclease to prepare restriction fragment b;

Using ligase to connect the enzyme-cut fragment b and the second sequencing adapter to prepare an adapter product b, which is used as a template for PCR amplification;

The base sequence protruding from the 5' end of the cohesive end of the second sequencing adapter is NNNN, and each N is independently selected from any one of A, T, C and G.

In conjunction with Figure 1, the present invention revolves around methyl-dependent restriction endonucleases, introduces a methylation site-specific linker (ie, the second sequencing linker in the construction method of the present invention), and passes one or more formazan In addition to methylation-dependent endonucleases, non-methylation site adapters (ie, the second sequencing adapters in the construction method of the present invention) are introduced to achieve methylation site-specificity in the downstream of the methylation site. As many unmethylated site adapters as possible are connected upstream of the methylated site on the nucleic acid molecule to be tested in the linker, so as to maximize the analysis efficiency of the methylated site. The introduction of sequencing adapters at both ends of the methylation site simplifies the process of methylation enrichment and expands the scope of methylation recognition. On the whole, the methylation analysis and the methylation sequencing library construction method provided by the present invention overcome the shortcomings of sodium bisulfite treatment in the traditional sodium bisulfite-related technologies that cause a large amount of nucleic acid damage in the sample to be analyzed, and the enrichment efficiency High, the identification accuracy of methylation sites is high, and the sample requirement is small, which can be used for quantitative analysis of low-abundance samples such as methylation qualitative sequencing analysis at the single cell level and free nucleic acid methylation sequencing.

The library construction scheme of the present invention can be used for methylation sequencing of free nucleic acid and genomic nucleic acid, including single-cell genome. The difference in its application is that free nucleic acid is fragmented nucleic acid, while genomic nucleic acid is large fragment nucleic acid. The methylation library construction step of the free nucleic acid is to introduce the first non-methylation site sequencing adapter, introduce the second methylation site-specific sequencing adapter, and use the two adapters to perform nucleic acid amplification. Among them, the first non-methylated site sequencing adapter can be used alone or in combination through the following four ways of adding adapters: 1) adding adapters by filling in the ends and complementing A and T/A; 2) adding adapters through non-methylation Methylation-associated endonuclease cleavage produces sticky ends to add adapters; 3) Methylation-sensitive endonucleases generate sticky ends to add adapters; 4) When methylation sites have palindromic sequences, form Kylation-dependent enzymes generate cohesive ends to add adapters. For the construction of the genomic nucleic acid methylation library, if the first adapter is introduced by filling in A, the genomic nucleic acid needs to be mechanically fragmented in advance, such as by ultrasonic disruption. For the construction of a single-cell methylation-sequencing library, one option for the introduction of the first sequencing adapter can be the use of methylation-sensitive restriction enzymes and unmethylated sequencing adapters with CGAT complementary ends.

In some embodiments of the present invention, the base sequence protruding from the 5' end of the cohesive end of the second sequencing adapter is YNNY, RNNR, NYYN or NRRN, and each Y is independently selected from any of C and T species, each R is independently selected from any of A and G.

In some embodiments of the present invention, the methylation-dependent restriction enzyme is selected from one or more of BstNI, MspI, FspEI, LpnPI and MspJI. For the introduction of the second sequencing adapter, a double-stranded methylation-associated adapter with a four-base overhang at the five ends is introduced by using a methylation-dependent restriction endonuclease. Methylation-dependent endonucleases can recognize methylated CG, CHG, CHH, where H is any base other than G, which can be A, C or T.

In some embodiments of the invention, the other restriction enzyme is a methylation sensitive restriction enzyme.

In some embodiments of the present invention, the methylation-sensitive restriction enzyme is selected from AciI, HinP1I, HpyCH4IV, HpaII, ClaI, BsaHI, SalI, AvaI, BsiEI, Hpy99I, PvuI, MluI, EagI, Bst One or more of NI and PcilI.

In some embodiments of the present invention, the methylation-sensitive restriction endonuclease is ClaI, and the base sequence protruding from the 5' end of the sticky end long chain of the first sequencing adapter is CG.

It can be understood that, in the construction method of the present invention, the type of methylation-dependent restriction endonuclease can be selected from one type, or multiple types can be selected. According to requirements, when it is necessary to expand the range of methylation sequencing analysis in the DNA to be tested, multiple methylation-dependent restriction endonucleases can be used in combination, for example, two kinds of MspJI and FsPEI can be combined.

Methylation-dependent restriction endonucleases MspJI and FsPEI are selected, which will generate a 4-base nick, which needs to be ligated with sequencing adapters containing 256 different combinations of 4 bases at the 5' end. In order to reduce the self-ligation of methylation-specific adapters, YNNY, RNNR, NYYN, and NRRN four kinds of sequencing adapters with four base overhangs can be chemically synthesized.

The methylation-dependent restriction endonucleases MspJI and EspEI used in the present invention have corresponding recognition sites of CmNNR and CCm, the former recognizes 75% of the potential targets of CpG methylation in the genome, and the latter recognizes 75% of the potential targets of CpG methylation in the genome Based on the identification of 21.8% (7/32), that is, 96.8% of all targets, the combined use of the two directly identifies all potential methylation targets to solve 97%.

When the methylation-specific linker is synthesized, a six-base Esp3I restriction site of GAGACG or other restriction endonucleases that can produce a 4-base protruding sticky end at the 5-terminal is implanted downstream of NNNN during the synthesis of the linker, so that the linker The product after self-ligation contains two enzyme cleavage sites, while the product ligated with the adapter and the target fragment only contains one enzyme cleavage site. By adjusting the amount of T4 ligase and the amount of endonuclease, the ligase activity is greater than one endonuclease site but lower than two endonuclease sites, thus forming the effect of dynamically preventing the self-ligation of the linker, that is, containing two The chemical reaction with two endonuclease sites points to the digestion of the self-ligation product and restoration of the methylation-specific adapter, while the chemical reaction with only one endonuclease site points to the formation of the ligation product of the adapter and the target fragment.

In some embodiments of the invention, the ligase is T4 DNA ligase.

In some embodiments of the invention, said mechanical method comprises ultrasonic disruption. It can be understood that after the DNA to be tested is fragmented by a mechanical method, the ends are blunted and A is filled, and the first sequencing linker a is introduced through A:T complementation.

In some embodiments of the present invention, the DNA to be tested includes artificially synthesized DNA samples.

In some embodiments of the present invention, the DNA to be tested includes genome DNA of an organism. The genomic DNA can be from a cell sample, a tissue sample, a blood sample, and the like.

The methylation site sequencing library constructed in the present invention can be used for non-diagnostic research.

specific embodiment

Example 1: Cla I can inhibit the self-connection of the linker of the CGAT linker

1. Materials

The sequence of the CGAT linker is shown in SEQ ID NO: 1:

CGATCTGTCTCTCTTATACACATCTCCCGAGCCCAGGACGGATGTGTATAAGAGACAGAT.

2. Method

The separate CGAT adapters were reacted under three conditions, blank control without enzyme group, single T4 ligase group, T4 ligase and ClaI restriction endonuclease group, and agarose gel electrophoresis was used to observe whether the CGat adapter was self-contained. Even and whether it can be controlled by Cla I.

The reaction system of the blank control without enzyme group is: CGat linker 400ng, 10×Ligation Buffer 2μL, make up to 20μL with water;

The reaction system of the T4 ligase group is: CGat linker 400ng, 10×Ligation Buffer 2μL, water to 20μL, T4 DNA Ligase 5μL (add at the end);

The reaction system of adding T4 ligase and ClaI restriction endonuclease group at the same time is: CGat linker 400ng, 10×Ligation Buffer 2μL, 10×Cutsmart Buffer 2μL, ClaI restriction endonuclease 1μL, make up water to 20μL, T4 DNA Ligase 5μL (add at the end).

The reaction conditions are as follows: room temperature or 37°C water bath overnight.

After the reaction is completed, take 1 μL of the reaction product, make up to 5 μL with water, and perform agarose gel electrophoresis. The electrophoresis conditions are 100V and 30min. T4 ligase group, T4 ligase and ClaI restriction endonuclease group.

3. Results

In the reaction of only adding T4 ligase group, the linker self-ligates, the band size becomes larger, and the electrophoresis speed becomes slower. In the reaction of adding T4 ligase and ClaI restriction endonuclease group at the same time, there is no obvious self-ligation product, showing that the band is the same size as the blank control.

(image 3).

4. Conclusion

Results Figure 3. According to Figure 3, it can be seen that ClaI prevents the self-ligation of the CGAT linker.

The principle of using the CGAT linker to promote the bidirectional reaction to the single direction of the ligation reaction product is shown in Figure 2. After the cohesive ends produced by the methylation-sensitive endonucleases are ligated with adapters containing CGAT bases, the original cleavage sites disappear. In this way, in the presence of ClaI, the self-ligation of the CGAT linker is inhibited, and the linking reaction between the linker and the sample to be tested is the main reaction direction.

Example 2: YNNY and RNNR joints can prevent self-ligation of joints cut by methylation-dependent enzymes

1. Linker sequence

YNNY linker: positive strand is shown in SEQ ID NO:2, reverse strand is shown in SEQ ID NO:5.

RNNR linker: the forward strand is shown in SEQ ID NO:3, and the reverse strand is shown in SEQ ID NO:5.

NNNN linker: the positive strand is shown in SEQ ID NO:4, and the reverse strand is shown in SEQ ID NO:5.

SEQ ID NO: 2: 5'-P-YNNYTTCCCACCATCCAGCACATATATCTACTCCTGAGCGCACCTGTTGTCTATGTGAT-3'

SEQ ID NO: 3: 5'-P-RNNRTTCCCACCATCCAGCACATATATCTACTCCTGAGCGCACCTGTTGTCTATGTGAT-3'

SEQ ID NO: 4: 5'-P-NNNNTTCCCACCATCCAGCACATATATCTACTCCTGAGCGCACCTGTTGTCTATGTGAT-3'

SEQ ID NO: 5': ATCACATAGACAACAGGTGCGCTCAGGAGTAGATATATGTGCTGGATGGTGGGAA-3'.

2. Experimental method

Synthesize forward and reverse strands of YNNY, RNNR, NNNN linkers.

Each 1 μL of 100 μmol forward and reverse strands was annealed under the condition of 1× annealing buffer to synthesize double-stranded YNNY linker, RNNR linker and NNNN linker.

Add T4 ligase for ligation.

Each experimental group is shown in Table 1.

Table 1

3. Results

The results are shown in Figure 4. In Figure 4, the YNNY linker and RNNR linker plus T4 ligase are not connected, but the NNNN linker plus T4 ligase can be connected, the linker is self-ligated, the band size becomes larger, and the electrophoresis speed becomes slower. The YNNY and RNNR adapters can prevent the self-ligation of the adapters cut by methylation-dependent enzymes.

The experimental materials of Examples 3-6 include: the artificial template sequence used, the CGAT linker, the TAAT linker, the NNNN linker and the primer F and primer R sequences used. in:

(1) Artificial template: the positive strand is shown in SEQ ID NO:6, and the reverse strand is shown in SEQ ID NO:7.

SEQ ID NO: 6: 5'-CAATCGCTCCGGttaaCTGCTC/i5MedC/GcATTCGTGTGCGCGGGCTGCGCCGAGCGCTGGGCA-3'

SEQ ID NO: 7: 5'-TGCCCAGCGCTCGGCGCAGCCCGCGCACACGAATGCGGAGCAGTTAACCGGAGCGATTG-3'.

(2) CGAT linker: forward chain is shown in SEQ ID NO:8, and reverse chain is shown in SEQ ID NO:9.

SEQ ID NO: 8: 5'-CGATGGTGGGGTGGGGTT-3'

SEQ ID NO: 9: 5'-GAGTTGGATGCTGGATGGNNNNNNNNNNNNNAACCCACCCCCACCAT-3'.

(3) TAAT linker: the forward strand is shown in SEQ ID NO:10, and the reverse strand is shown in SEQ ID NO:11.

SEQ ID NO: 10: 5'-TAATGGTGGGGTGGGGTT-3'

SEQ ID NO: 11: 5'-GAGTTGGATGCTGGATGGNNNNNNNNNNNNAACCCACCCCCACCAT-3'

(3) NNNN linker: the forward strand is shown in SEQ ID NO:12, and the reverse strand is shown in SEQ ID NO:13.

SEQ ID NO: 12: 5'-NNNNCTCAAGCGCGCGCGCG-3'

SEQ ID NO: 13: 5'-GGAGTGAGTACGGTGTGCNNNNNNNNNNNNNCGCGCGCGCGCTTGAG-3'

(4) Primers:

Primer F (SEQ ID NO: 14): GGAGTGAGTACGGTGTGC;

Primer R (SEQ ID NO: 15): GAGTTGGATGCTGGATGG.

Example 3

1. Digestion

The artificial template was digested with methylation-sensitive restriction enzyme HpaII and methylation-dependent restriction enzyme FspE1, wherein:

The enzyme digestion reaction system is: artificial template 200ng, 10×CutSmart ^TM Buffer 2.5μL, HpaII and FspEI each 1μL, add double distilled water to a total volume of 25μL.

The program of enzyme digestion reaction is: 37°C, 30min; 80°C, 20min; 4°C, save.

2. Connection connector

Connect the CGAT connector and the NNNN connector, where:

The ligation reaction system is: 25 μL of the digestion product obtained in the previous step, 2.5 μL of 10×CutSmart ^TM Buffer, 0.6 μL of CGAT linker (15 μM), 1 μL of NNNN linker (15 μM), 5 μL of ATP (10 μM), 1 μL of T4 DNA Ligase, supplemented with ddH ₂ O to 50 μL of the total system.

The reaction procedure of the ligation reaction was: 1h at 24°C, 20min at 65°C to inactivate T4 ligase.

3. PCR reaction

The PCR reaction system is: 25 μL of 2×taq Master Mix, 2 μL of primer F, 2 μL of primer R, 21 μL of the ligation product obtained in the previous step, and the total volume is 50 μL.

The program of PCR reaction is: 95°C 2min 1cycle; 95°C 10s, 60°C 20s, 72°C 30s, 20cycle; 72°C 1min1cycle; 4°C storage.

4. Sequencing and Results

The results are shown in Figure 5. Combined with Figure 5, it can be seen that the artificial template HpaII+FspE1 was digested, and the result of the first generation PCR product with the linker was in line with expectations.

Example 4

1. Digestion

The artificial template was digested with methylation-sensitive restriction enzyme HpaII and methylation-dependent restriction enzyme MspJ1, wherein:

The enzyme digestion reaction system is: artificial template 200ng, 10×CutSmart ^TM Buffer 2.5μL, HpaII and MspJI each 1μL, add double distilled water to a total volume of 25μL.

The reaction program of enzyme digestion is: 37°C, 30min; 80°C, 20min; 4°C, save.

2. Connection connector

Connect the CGAT connector and the NNNN connector, where:

The ligation reaction system is: 25 μL of the digestion product of the previous step, 2.5 μL of 10×CutSmart ^TM Buffer, 0.6 μL of CGAT linker (15 μM), 1 μL of NNNN linker (15 μM), 5 μL of ATP (10 μM), 1 μL of T4 DNA Ligase, supplemented with ddH ₂ O to 50 μL of the total system.

The ligation reaction procedure was: 1h at 24°C, 20min at 65°C to inactivate T4 ligase.

3. PCR reaction

The PCR reaction system was: 25 μL of 2×taq Master Mix, 2 μL of primer F, 2 μL of primer R, 21 μL of the ligation product from the previous step, and the total volume was 50 μL.

The program of PCR reaction is: 95°C 2min 1cycle; 95°C 10s, 60°C 20s, 72°C 30s, 20cycle; 72°C 1min1cycle; 4°C storage.

4. Sequencing and Results

The results are shown in Figure 6. According to Figure 6, it can be known that the artificial template HpaII+MspJ1 was digested, and the result of the first generation of PCR products with adapters was in line with expectations.

Example 5

1. Digestion

The artificial template was digested with the methylation-sensitive restriction enzyme MseI and the methylation-dependent restriction enzyme FspEI, wherein:

The enzyme digestion reaction system is: artificial template 200ng, 10×CutSmart ^TM Buffer 2.5μL, MseI and FspEI each 1μL, add double distilled water to a total volume of 25μL.

The program of enzyme digestion reaction is: 37°C, 30min; 80°C, 20min; 4°C, save.

2. Connection connector

Connect the TAAT connector and the NNNN connector, where:

The system for the ligation reaction is: 25 μL of the digestion product of the previous step, 2.5 μL of 10X CutSmart ^TM Buffer, 0.6 μL of TAAT adapter (15 μM), 1 μL of NNNN adapter (15 μM), 5 μL of ATP (10 μM), 1 μL of T4 DNA Ligase, supplemented with ddH2O to total System 50μL.

The ligation reaction procedure was: 1h at 24°C, 20min at 65°C to inactivate T4 ligase.

3. PCR reaction

The PCR reaction system is: 25 μL of 2×taq Master Mix, 2 μL of primer F, 2 μL of primer R, 21 μL of the ligation product obtained in the previous step, and the total volume is 50 μL.

The program of PCR reaction is: 95°C 2min 1cycle; 95°C 10s, 60°C 20s, 72°C 30s, 20cycle; 72°C 1min1cycle; 4°C storage.

4. Sequencing and Results

The results are shown in Figure 7. According to Fig. 7, it can be seen that: the artificial template MseI+FspE1 was digested, and the first generation result of the PCR product with the linker was in line with expectations.

Example 6

1. Digestion

The artificial template was digested with the methylation-sensitive restriction enzyme MseI and the methylation-dependent restriction enzyme MspJI, wherein:

The enzyme digestion reaction system is: artificial template 200ng, 10×CutSmart ^TM Buffer 2.5μL, MseI and MspJI each 1μL, add double distilled water to a total volume of 25μL.

The program of enzyme digestion reaction is: 37°C, 30min; 80°C, 20min; 4°C, save.

2. Connection connector

Connect the TAAT connector and the NNNN connector, where:

The system for the ligation reaction is: 25 μL of the digestion product of the previous step, 2.5 μL of 10X CutSmart ^TM Buffer, 0.6 μL of TAAT adapter (15 μM), 1 μL of NNNN adapter (15 μM), 5 μL of ATP (10 μM), 1 μL of T4 DNA Ligase, supplemented with ddH2O to total System 50μL

The ligation reaction procedure was: 1h at 24°C, 20min at 65°C to inactivate T4 ligase.

3. PCR reaction

The PCR reaction system is: 25 μL of 2×taq Master Mix, 2 μL of primer F, 2 μL of primer R, 21 μL of the ligation product obtained in the previous step, and the total volume is 50 μL.

The program of PCR reaction is: 95°C 2min 1cycle; 95°C 10s, 60°C 20s, 72°C 30s, 20cycle; 72°C 1min1cycle; 4°C storage.

4. Sequencing and Results

The results are shown in Figure 8. According to Fig. 8, it can be seen that: the artificial template MseI+MspJ1 was digested, and the first generation result of the PCR product with the linker was in line with expectations.

Example 7: Genome methylation analysis of colorectal cancer cell lines

In this embodiment, a methylation library is constructed by filling in A and adding a linker and relying on enzyme digestion and adding a linker, including the following steps:

1. Mechanical breaking, end filling, A filling, and sequencing adapters

20 ng of genomic DNA extracted from colorectal cancer cell lines was ultrasonically fragmented using an M200 ultrasonic fragmentation instrument, and the process of filling in + A+ sequencing adapters was performed using the KAPAHyper Prep Kit.

The reaction components included 50 μL (20 ng) of the genomic nucleic acid of the colorectal cancer cell line, 7 μL of end-filling buffer A, 3 μL of end-repair and tail-adding enzymes, and added double distilled water to 70 μL. React at 20°C for 30min; then 65°C for 30min; 4°C, store.

Take 40 μL of the above reaction product, add 7 μL of ligase buffer, 3 μL of T4 DNA ligase, and add 10 ml of the corresponding non-methylation-specific T:A complementary linker (as shown in SEQ ID NO:16 and SEQ ID NO:17); React at 20°C for 15 minutes. Then purification and recovery: 0.8×XP purification, 12 μL DB elution. Qubit quantification.

SEQ ID NO:16(AT-R):5'-P-CTGTCTCTTATACACATCTCCGAGCCCACGAGAC-3'

SEQ ID NO: 17 (AT-F): 5'-GCTTGACTAGTACATGACCACTTGA (public sequence 25bp) AGATGTGTATAAGAGACAG-T-3'.

Take 10 μL of the ligation product connected with the T:A adapter, 2.5 μL of 10×CutSmart ^TM Buffer, 1 μL of FspEI, and add water to 25 μL. Carry out methylation-dependent restriction endonuclease digestion, reaction program: 37°C, 60min; 65°C, 20min; 4°C, save.

Ligation of methylated adapters: 25 μL of digested products, 3 μL of T4 ligase, 50 mM ATP, 4 μL of 10X CutSmart ^TM Buffer, 5 μL of pre-annealed methylation-dependent enzyme digested adapters.

SEQ ID NO:18(MR-R):5'-P-NNNNATACTGTCTCTTATACACATCTCCGAGCCCACGAGAC-3'

SEQ ID NO: 19 (MR-F): 5'-TCGTCGGCAGCGTCAGATGTGTATAAAGAGACAGTAT-3'.

Add water to 40 μL. Reaction program: 20°C, 15 min. 1×XP purified and recovered, eluted with 23μLDB.

2. Construction of library PCR system

2×KAPA HiFi HotStart Ready Mix 25μL, forward and reverse primers 2μL each (10μM), template 21μL, add water to 50μL.

Forward primer MR-R (SEQ ID NO:20): 5'-P-NNNNNGTGATANNNNN(Barcode-5bp)CTGTCTCTTATACACATCTCCGAGCCCACGAGAC-3'

Reverse primer MR-F (SEQ ID NO: 21): 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNTATTCAC-3'

Reaction program: one cycle at 98°C for 45 seconds, then 20 cycles (98°C for 15 seconds, 60°C and 72°C for 30 seconds each). Then 72°C for one minute. 5 μL of gel was run, 1×XP purified and recovered, and 30 μL of DB was eluted.

3. Sequencing and results

The results are shown in Figure 9 and Figure 10. Figure 9 shows the results of library construction, and the final library samples in the fifth lane were selected for sequencing on the machine. The final library was sequenced using the NextSeq500/550 High Output 300cycles Sequencing Kit of the NextSeq500 model. The sequencing capacity is 24G/per library. Sequencing quality control indicators are shown in Figure 10. Combining with Figure 10, it can be seen that the number of methylated CpG sites measured in this sample is 1,298,178, which is between 1,050,000 and 1,400,000 of the theoretical estimate.

In the above examples, after sequencing, for the data analysis of the library directly using the Illumina sequencing adapter, the off-machine data was identified according to the methylation site-specific adapter sequence and the paired primers, and the quality control of the off-machine data was performed first. Remove adapters, remove low-quality and adapters; then perform sequence alignment, and determine the enzyme cleavage site according to the alignment position. If FspEI is used, for FspEI (recognition of CCG): backtrack upstream from the end position of the comparison, take the 18th, 17th, and 16th positions of 3 bases, and judge whether the sequence is CCG, if so, the position is the methylation cut position . If MspJI is used, for MspJI (recognition of CNNR): compare the end position and backtrack upstream, take the 17th, 16th, 15th, and 14th 4 bases, and judge whether the sequence is CNNR, if so, the position is methylated by the enzyme cut position.

The various technical features of the above-mentioned embodiments can be combined arbitrarily. For the sake of concise description, all possible combinations of the various technical features in the above-mentioned embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, should be considered as within the scope of this specification.

The above-mentioned embodiments only express several implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but should not be construed as limiting the patent scope of the invention. It should be pointed out that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

Claims (10)

1. A method for constructing a DNA methylation site sequencing library, which is characterized by comprising the following steps:

providing a DNA to be detected; preparing the DNA fragment to be detected into a joint product by adopting one or more methods shown as a) and b); performing PCR amplification by taking the joint product as a template to construct a methylation site sequencing library;

a) The method comprises the following steps:

carrying out enzyme digestion on the DNA to be detected by adopting methylation dependent restriction enzyme and other restriction enzymes to prepare an enzyme digestion fragment a;

adopting ligase, connecting the cut ends of other restriction enzymes of the enzyme digestion fragment a with a first sequencing adaptor a, and connecting the cut ends of methylation-dependent restriction enzymes of the enzyme digestion fragment a with a second sequencing adaptor to prepare an adaptor product a serving as a template for PCR amplification;

b) The method comprises the following steps:

fragmenting the DNA to be detected by adopting a mechanical method, filling the tail end, supplementing A, and connecting a first sequencing joint b by adopting ligase to prepare an intermediate joint product;

carrying out enzyme digestion on the intermediate linker product by using methylation dependent restriction enzyme to prepare an enzyme digestion fragment b;

adopting ligase to connect the enzyme digestion fragment b and a second sequencing linker to prepare a linker product b which is used as a template for PCR amplification;

the base sequence protruding from the 5' -end of the cohesive end of the second sequencing adaptor is NNNN, and each N is independently selected from any one of A, T, C and G.

2. The method of claim 1, wherein the overhanging nucleotide sequence at the 5' terminus of the sticky end of the second sequencing adapter is YNY, RNNR, NYN or NRRN, each Y is independently selected from any one of C and T, and each R is independently selected from any one of A and G.

3. The method for constructing a DNA methylation site sequencing library according to claim 1, wherein the methylation dependent restriction enzyme is selected from one or more of Bst NI, mspI, fspEI, lpnPI and MspJI.

4. The method for constructing a DNA methylation site sequencing library according to any one of claims 1 to 3, wherein the other restriction enzyme is a methylation sensitive restriction enzyme.

5. The method for constructing a DNA methylation site sequencing library according to claim 4, wherein the methylation sensitive restriction enzymes are selected from one or more of AciI, hinP1I, hpyCH IV, hpaII, claI, bsaHI, salI, avaI, bsiEI, hpy99I, pvuI, mluI, eagI, bst NI and PciI.

6. The method for constructing the DNA methylation site sequencing library according to claim 5, wherein the methylation sensitive restriction enzyme is ClaI, and the base sequence protruding from the 5' terminus of the cohesive end of the first sequencing adaptor a is CG.

7. The method for constructing a DNA methylation site sequencing library according to any one of claims 1 to 3, 5 and 6, wherein the ligase is T4 DNA ligase.

8. The method of constructing a DNA methylation site sequencing library according to any one of claims 1 to 3, 5 and 6, wherein the mechanical method comprises sonication.

9. The method for constructing a DNA methylation site sequencing library according to any one of claims 1 to 3, 5 and 6, wherein the DNA to be tested comprises an artificially synthesized DNA sample.

10. The method for constructing a DNA methylation site sequencing library according to any one of claims 1 to 3, 5 and 6, wherein the test DNA comprises genomic DNA of an organism.

Images