CN103233072B - High-flux mythelation detection technology for DNA (deoxyribonucleic acid) of complete genome - Google Patents

Abstract

The purpose of the present invention is to provide a high-throughput genome-wide DNA methylation detection method, which uses the methylation modification-dependent restriction enzyme FspEI to digest the genomic DNA, and the methylation at the CG or CHG site in the genome All modifications can be recognized by FspEI, and after digestion, an equal-length tag with a core methylation site is generated. High-throughput sequencing technology for the tag library can obtain genome-wide methylation site sequence information. This method can realize accurate positioning of methylation modification sites, enrich genome methylation sites by enzymatic digestion, and directly sequence and quantify methylation tag sequences, which can effectively reduce sequencing costs. The experimental process only takes two days, and has the advantages of high throughput, simple operation, low cost, and good reliability. It is an excellent high-throughput genome-wide methylation detection method suitable for non-model organisms.

Description

A high-throughput genome-wide DNA methylation detection technology

technical field

The invention belongs to the technical field of DNA methylation detection, and in particular relates to a high-throughput genome-wide DNA methylation detection technology.

Background technique:

DNA methylation is an important epigenetic modification of eukaryotic genomic DNA, that is, under the action of DNA methyltransferase (DNA methyltransferase, DNMT), S-adenosylmethylthio The process in which the methyl group of amino acid (SAM) is covalently bound to cytosine of DNA molecule to form 5-methylcytosine (5- ^mC ). DNA methylation plays an important role in the maintenance of normal cell function in higher organisms, genetic imprinting, embryonic development, aging and the occurrence of human tumors and other biological processes. In invertebrates, genomic DNA methylation is involved in regulating the adaptation process of the organism to the environment by regulating the expression pattern of genes. Therefore, obtaining the methylation data of all cytosine sites in the whole genome is of great significance for the study of spatiotemporal specificity of epigenetics.

With the deepening of methylation research, there have been a variety of methylation analysis methods to meet the needs of different methylation research. In the research on DNA methylation in the whole gene range, the means of detection are mainly the screening of genome-wide methylation sites based on chip platform and the analysis of methylation map based on high-throughput sequencing platform. Among them, chip technology is a relatively complete and mature detection tool in the methylation research of model organisms. It has the characteristics of high coverage, convenience, and high cost performance. For example, the Human Methylation HD 450 chip launched by Illumina contains 450,000 CpG sites, able to cover all NCBI annotated genes. The operation procedure is simple, and it can accurately detect high-throughput methylation sites. However, for non-model organisms with relatively scarce genetic information, the cost of the chip is expensive, and the flexibility of methylation site selection is not high, so it is difficult to use the existing chip platform to conduct high-throughput genome-wide methylation research on non-model organisms .

With the continuous improvement of next-generation sequencing technology throughput and cost reduction, there are currently many genome-wide methylation detection methods based on next-generation sequencing platforms that have been applied, including whole genome bisulfite sequencing (whole genome bisulfite sequencing, BS-seq) and simplified expression bisulfite sequencing (reduced representation bisulfite sequencing, RRBS), methylated DNA immunoprecipitation sequencing (methylated DNA immunoprecipitation sequencing, MeDIP-seq), methylated DNA enrichment combined with Qualcomm DNA sequencing (methylated DNA binding domain sequencing, MBD-Seq) and methylation-sensitive restriction enzyme sequencing (methylation-sensitive Restriction Enzyme sequencing, MRE-seq), etc. These technologies have their advantages, disadvantages and scope of application, such as the DNA methylation detection method based on sulfite treatment, although it is the gold standard for DNA methylation detection, the operation is complicated (bisulfite treatment) , the cost required for sequencing is high, and it is not suitable for species with large genomes such as shellfish. MeDIP-Seq is a high-throughput sequencing of methylated regions on the genome enriched by specific antibodies to obtain genome-wide methylated sites. But the technique requires large amounts of DNA, and antibodies are expensive.

Contents of the invention

The purpose of the present invention is to provide a high-throughput whole-genome DNA methylation detection method, that is, a low-cost, simple and fast high-throughput whole-genome DNA methylation detection method suitable for non-model organisms, to make up for the existing There are technical deficiencies.

The whole genome DNA methylation detection method of the present invention comprises the following steps:

1) Genomic DNA is digested with endonuclease FspEI to obtain digested fragments;

2) Connect the two ends of the enzyme-digested fragments to adapters as the binding points of the amplification primers;

3) Perform the first round of PCR amplification of the enzyme-cut fragments connected with the adapters with primers, so as to enrich the enzyme-cut fragments with the correct adapter ligation;

4) The first round of PCR amplification products were gel-purified, and primers were used for the second round of PCR amplification, and Barcode was introduced to construct a sequencing library;

5) The sequencing library is sequenced; the sequencing data is analyzed to obtain the genome-wide methylation information.

Wherein, the linkers in step 2) are linkers slx1 and slx2, wherein the two nucleotide fragments constituting slx1 have the sequences respectively 5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′ (SEQ ID NO: 1) and 3′-CGAGAAGGCTAGANNNNN- 5' (SEQ ID NO: 2), wherein N is any one of the bases A, T, G, and C;

Two nucleotide fragments constituting slx2, the sequences of which are 5′-GTGACTGGAGTTCAGAC

GTGTGCTCTTCCGATCT-3' (SEQ ID NO: 3) and 3'-CGAGAAGGCTAGANNNNN-5' (SEQ ID NO: 2).

The primers in the step 3) are Slx-Primer 1 and Slx-Primer 2, and their nucleotide sequences are respectively:

Slx-Primer1: 5′-AATGATACGGCGACCACCGAGATCTACACTCTTTTCCCTA

CACGAC-3' (SEQ ID NO: 4);

Slx-Primer 2: 5'-GTGACTGGAGTTCAGACGTGT-3' (SEQ ID NO: 5);

The primers in the step 4) are Slx-Primer 1 and Slx-Index Primer, and their nucleotide sequences are respectively:

Slx-Primer1: 5′-AATGATACGGCGACCACCGAGATCTACACTCTTTTCCCTA

CACGAC-3' (SEQ ID NO: 4);

Slx-Index Primer:

5'-CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGT-3' (SEQ ID NO: 6); wherein N is any one of bases A, T, G, and C.

This method can realize accurate positioning of methylation modification sites, enrich genome methylation sites by enzymatic digestion, and directly sequence and quantify methylation tag sequences, which can effectively reduce sequencing costs. The experimental process only takes two days, and has the advantages of high throughput, simple operation, low cost, and good reliability. It is an excellent high-throughput genome-wide methylation detection method suitable for non-model organisms.

Description of drawings

Figure 1: Schematic diagram of the process and principle of the genome-wide methylation detection method of the present invention.

Detailed ways:

The present invention has developed a new genome-wide methylation detection and analysis technology MethylRAD-Seq (Methylation-dependent restriction-site associated DNA sequencing) based on a high-throughput sequencing platform, which combines methyl modification-dependent endonucleases and high-throughput sequencing It is characterized by large-scale mining of methylation sites across the genome without the need for genomic background information, and direct and accurate detection of methylated cytosine sites. The principle of this technology is to use the methylation modification-dependent restriction enzyme FspEI to digest genomic DNA. The methylation modification at the CG or CHG site in the genome can be recognized by FspEI, and the DNA with core formazan is produced after digestion. The high-throughput sequencing technology of the tag library can obtain the sequence information of the methylation site in the whole genome. The library construction process is simple and fast, and the whole genome DNA methylation analysis can be performed on multiple samples at the same time. Moreover, the adapters and primers used in the present invention can efficiently operate on genomic DNA, improving the efficiency. The method of the present invention is a low-cost, easy-to-operate high-throughput genome-wide methylation analysis method for non-model organisms with relatively few epigenetic backgrounds.

For the terms involved in the present invention, the definitions are as follows:

1. Endonuclease, also known as endonuclease (endonuclease) in nucleolytic enzymes, is an enzyme that can hydrolyze the internal phosphodiester bond of the molecular chain to generate oligonucleotides; the FspEI endonuclease used in the present invention is A methylation-dependent endonuclease that cleaves DNA molecules depending on the methylation of cytosine sites. The enzyme was purchased from NEB (New England Biolabs) Co., Ltd.

2. Adapter: adapter DNA is a short artificially synthesized DNA fragment that contains a restriction site and can be matched with a blunt end or a sticky end. Adapter DNA is often used to connect a blunt end DNA to a sticky end DNA. Sometimes the linker DNA ligated to the cohesive end is to provide a known sequence to the unknown DNA fragment, based on which primers are designed to amplify the unknown DNA fragment.

3. N is any one of the bases A, T, G, and C; wherein A, T, G, and C represent the four deoxynucleosides that make up the DNA molecule.

4. Barcode is a short characteristic sequence. When high-throughput sequencing is performed on multiple samples at the same time, sequencing a specific short sequence (ie barcode) on each reads can accurately identify the source of the sample.

The method of the present invention comprises the following steps:

1) Preparation of biological genome DNA: extract the genome DNA of the organism and store it in a refrigerator at 4°C for later use.

The extracted genomic DNA was cleaved with the methylation modification-dependent endonuclease FspEI to obtain methylation tags in the whole genome. The enzyme digestion system was 20 μl, containing 300ng of genomic DNA, and 4U of FspEI endonuclease ( NEB), 1×NE Buffer4, 1×Enzyme Activator Solution, 1×BSA, kept at 37°C for 4 hours.

2) Adapters designed with sticky ends, ligated tags

The tags produced by the enzyme digestion reaction have a 4-base overhang at the 5' end, and design adapters Slx-Adaptor 1 and Slx-Adaptor 2 with 4 merged bases at the 3' end. The ligation reaction system is 20 μl, including 10 μl of the previous step 800U T4 DNA ligase (NEB), 1×T4 Ligase Buffer, 4μM Slex-Ad1, 4μM Slx-Ad2, 20mM ATP, 4℃ for 16h.

The sequence information of the linker is shown in Table 1.

3) Carry out the first round of PCR amplification to enrich the tags. The PCR reaction system is 20 μL, including 7 μl of enzyme-cut fragments connected with adapters as reaction templates, and 4 μM Slx-Primer1 primer

(5'- AATGATACGGCGACCACCGAGATTCACACTCTTTCCCTACACGAC -3'), 4μM Slx-Primer2 primer (5'- GTGACTGGAGTTCAGACGTGT -3'), 0.3mM dNTPs, 0.4U Phusion ultra-fidelity DNA polymerase (NEB), 1×HF buffer; the reaction conditions are Denaturation at 98°C for 5 s, annealing at 60°C for 20 s, extension at 72°C for 10 s, 14-18 cycles, and finally extension at 72°C for 5 min. The PCR product was detected by 8% non-denaturing polyacrylamide agar gel electrophoresis, the size of the amplified product was about 120bp, and the PCR product was recovered by cutting the gel.

4) Second-round PCR amplification with Barcode-specific primers

In order to realize the mixed sequencing of multiple individuals for methylation detection, it can be distinguished by adding different Barcodes to each individual, and using different primers in the PCR reaction to introduce different Barcodes. The PCR reaction system is 20 μL, including 25ng round PCR amplification purification product, 4μM Slx-Primer1 primer 5'-AATGATACGGCGACCACCGAGATTCACTCTTTCCCTACACGAC-3',

4μM Slx-Index Primer primer:

5'- CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGT-3', where NNNNNN can be changed according to different Barcode sequences), 0.3mM dNTPs, 0.4UPhusion ultra-fidelity DNA polymerase (NEB), 1× HF buffer; the reaction conditions are denaturation at 98°C for 5 s, 60 Anneal at ℃ for 20 s, extend at 72°C for 10 s, perform 5-7 cycles, and finally extend at 72°C for 5 min. Three tubes were amplified in parallel, and the PCR product was detected by 8% non-denaturing polyacrylamide gel electrophoresis. The size of the amplified product was about 150 bp, and the PCR product was recovered and purified using the QIAGEN PCR Product Purification Kit. The Solexa Hiseq2000 sequencing platform was used for sequencing, and this part was completed by the sequencing company.

5) Data analysis:

The method adopted in this embodiment is as follows:

1. The result file generated by Illunima/Solexa sequencing is in fastq format. First, use the SolexaQA software package to perform quality filtering on the original sequence, and remove sequences containing N and reads with a quality value greater than 5 bases and less than 10;

2. Use CD-HIT software to cluster and analyze short sequences to obtain the type of methylation tag in the sequencing library and the abundance information of the representative tag. The number of covered reads at the methylation site can measure the methylation of the site. Basic level;

3. Using SOAP software to compare the sequence of the methylation site with the genome reference sequence, the genome source information of the site can be obtained.

Linker and primer sequence list involved in the present invention in table 1

The present invention is described in detail below by taking the scallop as an example. For the reagents used in the present invention, those skilled in the art can select from the existing reagents according to the technical scheme of the present invention, and are not limited to the specific implementation of the present invention. Example limitations.

1) Extraction of scallop genome DNA

Take 12 scallops and golden scallops from the wild population at age II, add about 0.1 g of adductor muscle to each individual, add 500 ul of STE lysis buffer (NaCl: 100 mM; EDTA: 1 mM, pH=8.0; Tris-HCl, 10nM, pH=8.0), cut into pieces, then added 50μl 10% SDS, and 5μl proteinase K (20mg/ml), digested in a water bath at 56°C until the tissue fragments were completely lysed and the lysate was clarified. Add an equal volume of saturated phenol (250 μl) and chloroform/isoamyl alcohol (24:1) (250 μl), extract 3 times, take the supernatant, add an equal volume of chloroform/isoamyl alcohol (24:1) (500ul) to extract Extract once, take the supernatant, add 1/10 volume of NaAc (3M, pH 5.2) (50μl) and 2 times the volume of absolute ethanol (1000μl) stored at -20°C, shake slowly; precipitate at -20°C for 30min.12000rpm Centrifuge for 10 minutes, and the nucleic acid will precipitate at the bottom of the tube. Wash the precipitate with 70% ethanol (1000 μl) and dry until the ethanol evaporates completely, add 100 μl sterile water and a small amount (1-2 μl) of RNase A, and store in a 4°C refrigerator for later use.

Digestion of scallop genomic DNA

Use the methylation-dependent endonuclease FspEI to digest the genome to obtain methylation tags across the genome: the digestion system is 20 μl, containing 300ng genomic DNA, 4U of FspEI endonuclease (NEB), 1×NEBuffer4 , 1×Enzyme Activator Solution, 1×BSA, keep warm at 37°C for 4 hours.

2) Connect the two ends of the enzyme-digested fragments to adapters as the binding points of the amplification primers

The tags produced by the enzyme digestion reaction have a 4-base overhang at the 5' end, and design adapters Slx-Ad1 and Slx-Ad2 with 4 merged bases at the 3' end, and the ligation reaction system is 20ul, including 10ul of the enzyme in the previous step Digested product, 800U T4 DNA ligase (NEB), 1×T4 LigaseBuffer, 4uM Adapter 1, 4uM Adapter 2, 20mM adenosine triphosphate ATP, ligated at 4°C for 16h.

3) Perform the first round of PCR amplification of the enzyme-cut fragments connected with the adapters with primers, so as to enrich the enzyme-cut fragments connected with the adapters correctly;

The PCR reaction system is 20μL, including 7ul reaction template, 4μM Slx-Primer1 primer (5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC-3'), 4μM Slx-Primer2 primer (5'- GTGACTGGAGTTCAGACGTGT -3'), 0.3mMdNTPs, 0.4U Phusion ultra-fidelity DNA polymerase (NEB), 1× HFbuffer; the reaction conditions are denaturation at 98°C for 5 s, annealing at 60°C for 20 s, extension at 72°C for 10 s, 14-18 cycles, and finally extension at 72°C for 5 min. The PCR product was detected by 8% non-denaturing polyacrylamide agar gel electrophoresis, the size of the amplified product was about 120bp, and the PCR product was recovered by cutting the gel.

4) Perform the second round of PCR amplification of the first round of PCR amplification products with primers, and introduce Barcode to construct a sequencing library;

In order to realize the mixed sequencing of multiple individuals for methylation detection, it can be distinguished by adding different Barcodes to each individual, and using different primers in the PCR reaction to introduce different Barcodes. The PCR reaction system is 20uL, including 25ng round PCR amplification purification product, 4μM Slx-Primer1 primer 5'-AATGATACGGCGACCACCGAGATTCTACACTCTTTCCCTACACGAC-3', 4μM Slx-Index Primer primer

5'- CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGT-3', where NNNNNN can be changed according to different Barcode sequences, the Slx-Index Primer primer sequence used in the 24 scallop methylation libraries is shown in Table 2, 0.3mM dNTPs, 0.4UPhusion ultra-fidelity DNA polymerase (NEB), 1× HF buffer; the reaction conditions are denaturation at 98°C for 5 s, annealing at 60°C for 20 s, extension at 72°C for 10 s, 5-7 cycles, and finally extension at 72°C for 5 min. Three tubes were amplified in parallel, and the PCR product was detected by 8% non-denaturing polyacrylamide gel electrophoresis. The size of the amplified product was about 150 bp, and the PCR product was recovered and purified using the QIAGEN PCR Product Purification Kit. The Solexa Hiseq2000 sequencing platform was used for sequencing, and this part was completed by the sequencing company.

Table 2 Slx-Index Primer primer sequence list involved in the present invention

1 CAAGCAGAAGACGGCATACGAGATAACCTGGTGACTGGAGTTCAGACGTGT 2 CAAGCAGAAGACGGCATACGAGATAACTGAGTGACTGGAGTTCAGACGTGT 3 CAAGCAGAAGACGGCATACGAGATACAGTGGTGACTGGAGTTCAGACGTGT 4 CAAGCAGAAGACGGCATACGAGATAGACTCGTGACTGGAGTTCAGACGTGT 5 CAAGCAGAAGACGGCATACGAGATATCACGGTGACTGGAGTTCAGACGTGT 6 CAAGCAGAAGACGGCATACGAGATATGTCAGTGACTGGAGTTCAGACGTGT 7 CAAGCAGAAGACGGCATACGAGATCACGATGTGACTGGAGTTCAGACGTGT 8 CAAGCAGAAGACGGCATACGAGATCATCGGGTGACTGGAGTTCAGACGTGT 9 CAAGCAGAAGACGGCATACGAGATCATGGCGTGACTGGAGTTCAGACGTGT 10 CAAGCAGAAGACGGCATACGAGATCCATCTGTGACTGGAGTTCAGACGTGT 11 CAAGCAGAAGACGGCATACGAGATCGACATGTGACTGGAGTTCAGACGTGT 12 CAAGCAGAAGACGGCATACGAGATCGTACGGTGACTGGAGTTCAGACGTGT 13 CAAGCAGAAGACGGCATACGAGATCGTGCAGTGACTGGAGTTCAGACGTGT 14 CAAGCAGAAGACGGCATACGAGATCTCAGAGTGACTGGAGTTCAGACGTGT 15 CAAGCAGAAGACGGCATACGAGATCTGTAGGTGACTGGAGTTCAGACGTGT 16 CAAGCAGAAGACGGCATACGAGATGACATCGTGACTGGAGTTCAGACGTGT 17 CAAGCAGAAGACGGCATACGAGATGCACTAGTGACTGGAGTTCAGACGTGT 18 CAAGCAGAAGACGGCATACGAGATGCTCCAGTGACTGGAGTTCAGACGTGT 19 CAAGCAGAAGACGGCATACGAGATGTAGAGGTGACTGGAGTTCAGACGTGT 20 CAAGCAGAAGACGGCATACGAGATGTCTAGGTGACTGGAGTTCAGACGTGT twenty one CAAGCAGAAGACGGCATACGAGATTAGTCAGTGACTGGAGTTCAGACGTGT twenty two CAAGCAGAAGACGGCATACGAGATTCGGCAGTGACTGGAGTTCAGACGTGT twenty three CAAGCAGAAGACGGCATACGAGATTGAGTGGTGACTGGAGTTCAGACGTGT twenty four CAAGCAGAAGACGGCATACGAGATTGTAGCGTGACTGGAGTTCAGACGTGT

Note: Bold indicates the barcode sequence

5) The sequencing library was sequenced using the Solexa Hiseq2000 sequencing platform; the sequencing data was analyzed to obtain genome-wide methylation information.

1. The result file generated by Illumina/Solexa sequencing is in the fastq format. First, use the compiled perl script to perform quality filtering on the original sequences of the 24 libraries, and remove sequences containing N and reads with a quality value greater than 5 bases and less than 10. The number of high-quality reads obtained in the 24 methylated libraries ranged from 6,749,208 to 14,661,303, and the number of high-quality reads in each library accounted for more than 99% of the original reads.

2. Use CD-HIT software to perform cluster analysis on high-quality tag sequences, and obtain the type of methylation tag in the sequencing library and the abundance of the representative tag, that is, the methylation level information of the site. 98,754 methylation sites were obtained within the genome range of the wild-type scallop and a new mutant line of scallop "Haida Jinbei", and the methylation sites were compared to the genome reference sequence and genetically Annotation analysis of . showed that genes with methylation sites were involved in a variety of biological functions and metabolic pathways, indicating that the growth, reproduction and immunity of shellfish and other related pathways may be regulated by epigenetic mechanisms.

3. Using the edgeR software to analyze the difference in methylation level between wild-type scallops and "Haida Jinbei" genome-wide loci, 2452 differentially methylated sites were obtained, among the "Haida Jinbei" The methylation level of 8 sites showed a strong correlation with the carotenoid content of adductor muscle, and the correlation coefficient was above 0.90. By comparing these sites to the genome reference sequence, several results related to carotenoids were obtained. Candidate genes associated with carotene accumulation. In order to test the reliability of the technology of the present invention, two groups of technical repeated experiments were designed, and the Poisson coefficients of the two groups of parallel controls could reach more than 0.99. The stability and reliability of the technology of the present invention are verified.

Experiments have proved that the technology of the present invention can be used not only for the screening of methylation sites in the whole genome, but also for the differential analysis of methylation site modifications among different cells, tissues or samples, and to explore the influence of DNA methylation on genes. Expression regulation mechanism.

The invention has the characteristics of high throughput, high efficiency and low cost, is suitable for analyzing and detecting methylation sites in the whole genome, and has good application potential in the whole genome methylation detection of non-model organisms.

Claims (1)

1. A whole genome DNA methylation detection method, comprising the steps of: 1) Genomic DNA is digested with endonuclease FspEI to obtain digested fragments; 2) connecting the two ends of the enzyme-cut fragments with adapters as the binding points of the amplification primers; 3) performing the first round of PCR amplification on the enzyme-cut fragments connected with the adapters with primers, so as to enrich the enzyme-cut fragments connected with the adapters correctly; 4) The first round of PCR amplification products were gel-purified, and primers were used for the second round of PCR amplification, and Barcode was introduced to construct a sequencing library; 5) The sequencing library is sequenced; the sequencing data is analyzed to obtain the methylation information of the whole genome; Wherein the joint in step 2) is joint slx1 and slx2, wherein constitutes two nucleotide fragments of slx1, and its sequence is respectively SEQ ID NO:1 and SEQ ID NO:2; Constitutes two nucleotide fragments of slx2 , whose sequences are respectively SEQ ID NO:3 and SEQ ID NO:2; The nucleotide sequences of the primers in the step 3) are respectively SEQ ID NO:4 and SEQ ID NO:5; The nucleotide sequences of the primers in the step 4) are respectively SEQ ID NO:4 and SEQ ID NO:6.

Images