A transposase-based 5mCpG high-throughput detection method TnM-seq, transposase TnM and single-cell multi-omics application

Abstract

The application provides a 5mCpG high-throughput detection method TnM-seq based on TnM transposase, which adopts transposase TnM including an MBD domain to detect 5mCpG in situ, and comprises the following steps: after pretreatment of cells to be detected, the cells are incubated with transposase TnM; DNA in the target sample after transposition is recovered and purified; the purified DNA is subjected to PCR library construction; and the library construction product is subjected to high-throughput sequencing. The new transposase TnM newly designed and synthesized is used, combined with high-throughput sequencing, to realize convenient, fast, low-cost and high-sensitivity detection of 5mCpG in a sample. Based on the transposase TnM, a multi-omics technology HAM-seq for simultaneously detecting histone modification H3K9me3, chromatin accessibility and DNA methylation modification 5mCpG in the same sample is further developed, and after being combined with a commercial single-cell microfluidic platform, an epigenome and gene expression four-omics technology EpiX-seq for single-cell analysis in a complex tissue is obtained.

Description

Technical Field

[0001] This invention relates to high-throughput sequencing technology and fusion enzymes, and more particularly to a 5mCpG high-throughput sequencing method based on transposases, a novel transposase designed for this method, and its application in single-cell multi-omics. Background Technology

[0002] DNA methylation, as an important epigenetic modification, has a profound impact on life processes such as chromosome structure maintenance, gene expression regulation, cell differentiation, development, aging, and disease occurrence, and has therefore always been a research hotspot in the life sciences. In recent years, with the rapid development of high-throughput sequencing technology and bioinformatics analysis, significant breakthroughs have been made in several aspects of DNA methylation research. 5mC (5-methylcytosine) refers to the methylation modification of the fifth carbon of cytosine in DNA under the action of DNA methyltransferases, and is the earliest type of DNA methylation identified in eukaryotes. In mammals, 5mC is often located upstream of guanine (G) in DNA, the so-called CpG site; therefore, mammalian DNA methylation is also called 5mCpG. Besides 5mC, other methylation types include: 4mC (4-methylcytosine), 7mG (7-methylguanine), and 6mA (6-methyladenine). 5mC has attracted widespread research attention due to its extensive presence in the genomes of higher organisms and its central role in key biological processes such as gene expression silencing, genomic imprinting, X chromosome inactivation, and transposon repression. Abnormal 5mC patterns (such as promoter region hypermethylation or genome-wide hypomethylation) are closely related to the development and progression of various diseases, particularly cancer. Therefore, accurate detection of the distribution and dynamic changes of 5mC in the genome is of great significance for understanding life processes, disease mechanisms, and developing novel diagnostic and therapeutic approaches.

[0003] Currently, methods for detecting 5mC across the entire genome can be broadly categorized into two types: WGBS (whole-genome bisulfite sequencing) based on base conversion and MeDIP-seq (immunoprecipitation-based sequencing). WGBS is currently the gold standard for DNA methylation research, accurately detecting the methylation level of all individual cytosine bases across the entire genome. First, genomic DNA is extracted and fragmented, then subjected to bisulfite treatment to convert unmethylated cytosine (C) to uracil (U), while 5mC remains unchanged. The base-converted DNA fragments are then used for library construction (e.g., end repair, adapter ligation, PCR amplification) and high-throughput sequencing. Finally, the sequencing data is aligned to a reference genome to calculate the methylation level of each cytosine (C). MeDIP seq (methylated DNA immunoprecipitation sequencing) is an immunoprecipitation and high-throughput sequencing analysis technology based on 5mC-specific antibodies. It uses anti-5mC antibodies to bind to methylated DNA fragments, enriches them using magnetic beads (such as Protein A / G), then elutes the enriched methylated DNA, performs end repair, adapter ligation, and PCR amplification to construct a sequencing library. Differential methylation analysis is then performed by identifying high-methylation regions (peak calling).

[0004] While WGBS can achieve full genome coverage and single-base resolution, it requires a large amount of sample DNA due to significant DNA double-strand breaks caused by the chemical base conversion step. Furthermore, WGBS cannot distinguish between 5mC and 5hmC (5-hydroxymethylcytosine), resulting in a mixed signal from both. MeDIP-seq, while unable to achieve single-base resolution detection of 5mC, utilizes a 5mC-specific antibody to avoid interference from 5hmC and can be used for analyzing low-volume samples. In summary, existing 5mC detection methods have several drawbacks, primarily: 1. High initial sample volume requirements; 2. Inability to achieve in situ detection in intact cells; 3. Complex and time-consuming detection procedures; 4. High cost, requiring antibodies or multiple chemical reagents; 5. Difficulty in achieving single-cell level detection.

[0005] Therefore, it is urgent to establish a novel DNA methylation detection method and integrate single-cell multi-omics technologies, which is of great significance for studying the location, methylation level, dynamic changes, and biological functions of 5mC in the genome. Summary of the Invention

[0006] The technical objective of this invention is to address the problems existing in the background art by providing a high-throughput detection method for 5mCpG based on the TnM transposase, TnM-seq. This method utilizes a newly designed and synthesized novel transposase, TnM, combined with high-throughput sequencing to achieve convenient, rapid, low-cost, and highly sensitive whole-genome detection of 5mCpG in samples. Furthermore, by combining different types of transposases, a multi-omics technology, HAM-seq, is developed that can simultaneously detect histone modification H3K9me3, chromatin accessibility, and DNA methylation modification 5mCpG in the same sample. Finally, combined with a commercial microfluidic single-cell platform, a single-cell four-omics sequencing technology, EpiX-seq, is invented.

[0007] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a high-throughput detection method for 5mCpG based on TnM transposase, wherein the high-throughput detection method uses the TnM transposase, which includes an MBD domain, for in situ detection of 5mCpG, and includes the following steps:

[0009] (1) After pretreatment of the target sample, transposition reaction was carried out using transposase TnM;

[0010] (2) Recover and purify the DNA in the target sample after transposition;

[0011] (3) Perform PCR library construction on the purified genome;

[0012] (4) The library-built products were subjected to high-throughput sequencing.

[0013] Furthermore, the amino acid sequence of the transposase is as shown in SEQ NO.1, or is an amino acid sequence with the same function formed by substitution, deletion or addition of one or more amino acids based on the sequence shown in SEQ ID NO.1.

[0014] Furthermore, the transposase TnM is constructed as follows: the human MBD1 protein-MBD domain is linked to the N-terminus of the Tn5 transposase via a flexible linker, and it functions as a dimer.

[0015] Furthermore, the human MBD1 protein-MBD domain is a 5mCpG specific recognition protein domain, and its amino acid sequence is shown in SEQ NO.2, or an amino acid sequence with the same function formed by substitution, deletion or addition of one or more amino acids based on the sequence shown in SEQ NO.2.

[0016] Furthermore, the amino acid sequence of the flexible linker is shown in SEQ NO.3.

[0017] Furthermore, the Tn5 transposase is a wild-type Tn5 transposase mutant, and its amino acid sequence is shown in SEQ NO.4, or an amino acid sequence with the same function formed by substitution, deletion or addition of one or more amino acids based on the sequence shown in SEQ ID NO.4.

[0018] Furthermore, the transposase TnM is attached with a protein purification tag, which is a His tag, a Strep tag, a GST tag, or a FLAG tag.

[0019] Furthermore, the method for constructing the TnM transposase is as follows:

[0020] (1) Design of TnM transposase structure: Select the 1-75aa of the MBD domain of human MBD1 protein and link it to the N-terminus of Tn5 transposase using a flexible linker. Use AlphaFold3 to predict the three-dimensional structure of the fusion protein. Construct TnM expression vector using gene synthesis or molecular cloning methods and verify the correctness of the expression vector sequence by sequencing.

[0021] (2) TnM transposase expression:

[0022] (2.1) Transform the correctly sequenced TnM expression plasmid into the expression strain and culture it under constant temperature and humidity;

[0023] (2.2) Inoculate the culture medium with a single-inoculation and a culture expressing TnM in two separate inoculations.

[0024] (2.3) Extract bacterial cells from the culture product, lyse and break them (any known breaking method can be used, as long as it can fully break the protein while maintaining its stability) to obtain a protein solution.

[0025] (3) Purification of TnM transposase;

[0026] (4) TnM transposase encapsulation:

[0027] (4.1) Synthesize the sequencing adapter sequence and anneal it into a double linker; mix the embedding solution, double linker and purified protein solution in a certain proportion and incubate to obtain TnM with high transposition activity, and store it below -20℃.

[0028] Secondly, the present invention provides a transposase for high-throughput detection of 5mCpG, wherein the transposase is a dimer composed of an N-terminal MBD domain, a C-terminal Tn5 transposase, and a linker region, and the amino acid sequence of the transposase is shown in SEQ ID NO.1, or is an amino acid sequence with the same function formed by substitution, deletion, or addition of one or more amino acids based on the sequence shown in SEQ ID NO.1.

[0029] Thirdly, the present invention provides the application of the novel transposase described above in constructing DNA-5mCpG high-throughput sequencing libraries of biological samples, and / or in the detection of 5mCpG in naked DNA or intact cells.

[0030] The beneficial effects of this invention are as follows: This invention designs a novel transposase TnM, which can be used for the detection of 5mCpG modification on DNA and the construction of high-throughput libraries. It can improve the accuracy and sensitivity of 5mCpG detection, reduce reaction costs and cell input, improve library construction efficiency, and realize in situ detection of 5mCpG in intact cells, providing a foundation for methylation research at the single-cell and multi-omics levels.

[0031] Specifically

[0032] (1) This invention creatively links the Tn5 transposase with the MBD domain of the human 5mCpG binding protein MBD1 via a flexible linker to form a novel transposase fusion protein, TnM. This transposase can significantly reduce the number of cells required for high-throughput 5mCpG detection, and can even achieve single-cell level studies. Compared to traditional methods such as MeDIP seq, which require millions of cells for library construction, this invention requires a maximum of only 50,000 cells or 100 nanograms of genomic DNA, and can complete the construction of a high-quality 5mCpG library with a minimum of 1,000 cells or 10 nanograms of genomic DNA.

[0033] Moreover, the cell processing in this invention is gentle, eliminating the need for extensive cell lysis and DNA extraction as in traditional methods, or for treating DNA with sulfites, which damages the DNA double strand and leads to sample loss. Instead, it preserves the integrity of the cells, allowing the transposase TnM to enter the cell nucleus and exert its function, resulting in a higher signal-to-noise ratio.

[0034] (2) The transposase TnM in this invention has a completely different function from other transposases in traditional library construction methods (regardless of the type of library, including DNA methylated libraries). If transposases are used in traditional library construction methods, their function is only to break the complete genome into small fragments that can be used for library construction, and at the same time add adapters to both ends of the fragments, which plays an auxiliary role in library construction. However, the TnM transposase in this invention can directly target and cut methylated DNA sites and add adapters required for high-throughput sequencing at both ends of the cleavage sites while targeting and cutting 5mCpG sites enriched on the genome. It completes the enrichment, fragmentation and adapter addition of DNA methylated fragments in one step, without the need for conventional end repair and A-linking adapters. The library can be directly prepared by PCR, which significantly simplifies the experimental process and improves the efficiency of library construction.

[0035] Therefore, the sequencing method TnM-seq of this invention is an improvement on ATAC-seq technology. The constructed transposase TnM can significantly reduce experimental costs and improve library construction efficiency in the 5mCpG high-throughput detection process. The library construction process of this invention does not require expensive antibodies or commercial DNA methylation library construction kits. It uses self-expressed and purified TnM for library construction, and the library construction can be completed in a maximum of three hours, with the operation time for experimental personnel within one hour.

[0036] (3) The transposase TnM involved in this invention can achieve in situ detection within cells during 5mCpG high-throughput detection, thereby effectively preserving the spatial in situ information of the sample. The detection method of this invention does not require cross-linking with chemical reagents such as formaldehyde and methanol, nor does it require lysing cells to extract genomic DNA. After permeation through the cell membrane and nuclear membrane, the TnM designed in this invention is directly reacted with the cell, cutting the 5mCpG site in situ and releasing the genome. This process does not affect the natural structure within the active cells, while traditional WGBS or MeDIP-seq require lysing cells to extract genomic DNA before subsequent experiments. Therefore, this invention expands the application of transposase TnM by combining different types of transposases to develop a multi-omics technology, HAM-seq, that can simultaneously detect histone modification H3K9me3, chromatin accessibility, and DNA methylation modification 5mCpG in the same sample. Furthermore, by integrating with a commercial microfluidic single-cell platform, EpiX-seq, a four-omics technology, was developed to simultaneously detect histone modification H3K9me3, chromatin accessibility, DNA methylation modification 5mCpG, and gene expression in a single cell. This technology can capture nearly ten thousand high-quality single-cell data points in a single experiment. This invention provides a powerful and versatile tool for single-cell multi-omics epigenetic research, with broad application prospects in developmental biology and cancer research. Attached Figure Description

[0037] Figure 1 This is a predicted structure diagram of the TnM transposase from Example 1 of the present invention (presented by PyMOL).

[0038] Figure 2 This is the construction map of the TnM transposase expression vector of Example 1 of the present invention (Presented by SnapGene8.0).

[0039] Figure 3 This is a Coq stain gel image (10% SDS-PAGE) of the TnM transposase expression and purification in Example 2 of this invention.

[0040] Figure 4This is a graph showing the TnM transposase activity detection results of Example 4 of the present invention (Generated by Agilent2100).

[0041] Figure 5 This is a graph showing the verification results of the TnM transposase specific binding ability in Example 5 of the present invention.

[0042] Figure 6 This is a flowchart of the detection process in Embodiment 6 of the present invention.

[0043] Figure 7 This is a flowchart of the detection process in Embodiment 7 of the present invention.

[0044] Figure 8 This is a comparison chart of the results of embodiments 6 and 7 of the present invention.

[0045] Figure 9 This is a diagram showing the method-specific results of Embodiment 7 of the present invention.

[0046] Figure 10 This is a graph showing the sensitivity results of the method in Embodiment 7 of the present invention.

[0047] Figure 11 This is a diagram showing the universality results of the method in Embodiment 7 of the present invention.

[0048] Figure 12 This is a result example diagram of Embodiment 7 of the present invention.

[0049] Figure 13 This is a flowchart of the detection process in Embodiment 8 of the present invention.

[0050] Figure 14 This is a schematic diagram of Embodiment 8 of the present invention.

[0051] Figure 15 This is a comparison chart of the results of Embodiment 8 of the present invention.

[0052] Figure 16 This is a diagram showing the universality results of the method in Embodiment 8 of the present invention.

[0053] Figure 17 This is a flowchart and schematic diagram of the detection process in Embodiment 9 of the present invention.

[0054] Figure 18 This is a result example diagram of Embodiment 9 of the present invention.

[0055] Figure 19 This is a diagram showing the single-cell clustering and annotation results of Embodiment 9 of the present invention.

[0056] Figure 20 This is a result example diagram of Embodiment 9 of the present invention. Detailed Implementation

[0057] To enable those skilled in the art to better implement the technical solutions of this invention, the invention will be described in detail, clearly, and completely below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Furthermore, based on the embodiments of this invention, any modifications, equivalent substitutions, improvements, etc., made by those skilled in the art to all other embodiments obtained without creative effort should be included within the protection scope of this invention.

[0058] To enable those skilled in the art to better understand the technical solutions of this invention, some technical terms of this invention are first explained. In the text of this invention, these explanations are primarily intended to help those skilled in the art understand and implement the technical solutions of this invention, rather than to limit the invention. Furthermore, when these technical terms may be interpreted differently in other disclosures outside of this invention, to avoid technical ambiguity, the interpretation of this invention should be used as the basis for understanding this invention.

[0059] 5mCpG: 5-methylcytosine in CpG dinucleotide context refers to 5-methylcytosine (5mC) formed by methylation of the 5th carbon atom of cytosine (C) at the CpG site in DNA.

[0060] WGBS: Whole-Genome Bisulfite Sequencing converts unmethylated cytosine (C) to uracil (U) by treating DNA with sulfite, and then detects the level of single-base methylation across the entire genome through sequencing.

[0061] MeDIP-seq: Methylated DNA Immunoprecipitation Sequencing utilizes 5mC antibodies to enrich methylated DNA fragments, combined with high-throughput sequencing to detect methylated regions of the genome.

[0062] Tn5: Transposase 5, a bacterial transposase, can randomly cut DNA and insert sequencing adapters at the breakpoints. It is commonly used for high-throughput sequencing library construction (such as ATAC-seq).

[0063] TnH: Tn5-HP1α, a fusion transposase reported by Martina Tedesco et al. in 2021, can be used to construct high-throughput sequencing libraries of heterochromatin histone-modified H3K9me3.

[0064] ATAC-seq:Assay for Transposase-Accessible Chromatin using sequencing: High-throughput sequencing technology for detecting open regions of chromatin using Tn5 transposase.

[0065] HAM-seq: Transposase-based H eterochromatin, chromatin A Ccessibility, and DNA M ethylation seq uencing, the present invention is a high-throughput sequencing technology based on transposases for heterochromatin, chromatin accessibility and DNA methylation.

[0066] EpiX-seq: Epigenome and eXpression sequencing, a single-cell four-omics high-throughput sequencing technology for epigenome and gene expression developed in this invention.

[0067] MBD1: Methyl-CpG Binding Domain Protein 1 is a protein that specifically recognizes and binds to methylated CpG sites, with its MBD domain responsible for binding 5mCpG.

[0068] EMSA: Electrophoretic Mobility Shift Assay (EMS) is used to detect the interaction between proteins and DNA / RNA binding.

[0069] SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis is used to separate proteins and analyze their molecular weight.

[0070] IPTG: Isopropyl β-D-1-Thiogalactopyranoside is used to induce protein expression controlled by the Lac operon in prokaryotic expression systems.

[0071] PCR: Polymerase chain reaction (PCR) amplifies specific DNA fragments using primers (such as DNA amplification in library construction).

[0072] Ni-NTA: Nickel-Nitrilotriacetic Acid (Ni-Nitrilotriacetic Acid) affinity chromatography medium is used to purify His-tagged recombinant proteins.

[0073] DMF: Dimethylformamide is an organic solvent used to improve reaction efficiency in transposition reactions.

[0074] DTT: Dithiothreitol is a reducing agent used to stabilize disulfide bonds in proteins.

[0075] EDTA: Ethylenediaminetetraacetic acid (EDTA) is a chelating agent used to inhibit metal-dependent nuclease activity.

[0076] BL21 (DE3): E. coli BL21(DE3) strain is a commonly used E. coli expression strain that carries the T7 RNA polymerase gene for recombinant protein expression.

[0077] IGV: Integrative Genomics Viewer is a genomic data visualization tool used to compare and analyze sequencing results.

[0078] LB medium: Luria-Bertani Medium is a bacterial culture medium containing tryptone, yeast extract, and sodium chloride.

[0079] OD: Optical density is a measure of the concentration of a microbial culture medium by its absorbance (typically 600 nm).

[0080] 5hmC: 5-Hydroxymethylcytosine is a type of DNA modification that is often indistinguishable from 5mC due to their similar chemical properties.

[0081] Con A beads: Concanavalin A-coated beads, magnetic beads coated with concanavalin A, are used for cell capture.

[0082] BGI: Beijing Genomics Institute (BGI) provides high-throughput sequencing platforms.

[0083] AlphaFold3:A deep learning-driven protein structure prediction tool.

[0084] PyMOL: Python Molecular Viewer is a molecular visualization software used to display the three-dimensional structure of proteins.

[0085] SnapGene: Molecular biology tools for plasmid mapping and analysis.

[0086] Agilent 2100: The Agilent 2100 Bioanalyzer is a bioanalyzer used to detect the size distribution of DNA / RNA fragments.

[0087] Example 1: Design of TnM transposase

[0088] The MBD domain (1-75 aa) of the MBD1 protein was selected and linked to the N-terminus of the Tn5 transposase using a flexible linker. The three-dimensional structure of the fusion protein was predicted using AlphaFold3, as shown below. Figure 1 As shown, the dimer structure of the original Tn5 and the TnM of the present invention after binding to the adapter DNA of high-throughput library construction is displayed.

[0089] The designed TnM sequence was inserted into the pET-28α(+) vector, as follows: Figure 2 As shown, TnM expression vectors were constructed using gene synthesis or molecular cloning methods, and the correctness of the expression vector sequence was verified by sequencing.

[0090] The amino acid sequence of the MBD domain of the MBD1 protein is shown in SEQ NO.2, the amino acid sequence of the flexible linker is shown in SEQ NO.3, and the Tn5 transposase is a wild-type Tn5 transposase mutant, the amino acid sequence of which is shown in SEQ NO.4.

[0091] The transposase TnM is attached with a protein purification tag, which is selected from at least one of His tag, Strep tag, GST tag, or FLAG tag. In this embodiment, the His tag is selected, and its sequence is shown in SEQ NO.5.

[0092] SEQ ID NO.2

[0093] MBD1-MBD structural domain:

[0094] MAEDWLDCPALGPGWKRREVFRKSGATCGRSDTYYQSPTGDRIRSKVELTRYLGPACDLTLFDFKQGILCYPAPK

[0095] SEQ ID NO.3

[0096] Linker:

[0097] GGGGSGGGGSGGGGS

[0098] SEQ ID NO.4

[0099] Tn5:

[0100] MITSALHRAADWAKSVFSSAALGDPRRTARLVNVAAQLAKYSGKSITISSEGSKAMQEGAYRFIRNPNVSAEAIRKAGAMQTVKLAQEFPELLAIEDTTSLSYRHQVAEELGKLGSIQD KSRGWWVHSVLLLEATTFRTVGLLHQEWWMRPDDPADADEKESGKWLAAAATSRLRMGSMMSNVIAVCDREADIHAYLQDKLAHNERFVVRSKHPRKDVESGLYLYDHLKNQPELGGYQ ISIPQKGVVDKRGKRKNRPARKASLSLRSGRITLKQGNITLNAVLAEEINPKGETPLKWLLLTSEPVESLAQALRVIDIYTHRWRIEEFHKAWKTGAGAERQRMEEPDNLERMVSILS FVAVRLLQLRESFTPPQALRAQGLLKEAEHVESQSAETVLTPDECQLLGYLDKGKRKRKEKAGSLQWAYMAIARLGGFMDSKRTGIASWGALWEGWEALQSKLDGFLAAKDLMAQGIKI

[0101] SEQ ID NO.5

[0102] 6×His Tag:

[0103] HHHHHH

[0104] Example 2 Expression and purification of TnM transposase

[0105] 1. Transform the correctly sequenced TnM expression plasmid into the BL21(DE3) expression strain, spread the bacterial culture on kanamycin resistance plates, and incubate upside down in a 37°C constant temperature and humidity incubator for about 14 hours.

[0106] 2. Inoculate a single TnM-expressing strain into LB medium containing 50 μg / mL kanamycin and incubate at 37°C and 220 rpm for approximately 12 h.

[0107] 3. The culture from step 2 was inoculated into LB medium containing 50 μg / mL kanamycin at a volume ratio of 1:20 and cultured at 37℃ and 220 rpm for 3 h until OD = 0.6~0.8.

[0108] 4. Place the culture medium from step 3 on ice to cool for 20 minutes, then add IPTG to bring the final concentration to 0.5 mM.

[0109] 5. The product from step 4 was cultured at 16℃ and 220rpm for 20h. The cells were collected by centrifugation at 4℃ and weighed. 7mL of bacterial lysis buffer was added to each g of cells, and the mixture was mixed by pipetting and placed on ice for lysis for 30min.

[0110] 6. The bacterial lysate from step 5 is sonicated to obtain a protein solution. The obtained protein solution is centrifuged at 10000g for 30 minutes at 4℃.

[0111] In some other implementations, in addition to ultrasonic disruption, high-pressure disruption can also be used, as long as the cells can be sufficiently disrupted while maintaining protein stability.

[0112] 7. Ni-column affinity purification: Take an appropriate amount of Ni-NTA agarose beads (agarose gel column), centrifuge at 1000g for 10s to remove the supernatant, resuspend the beads in pre-cooled equilibration buffer containing 20mM imidazole, centrifuge to remove the supernatant, and repeat the above operation, washing three times in total to equilibrate the Ni-NTA agarose beads. Then, take the supernatant protein solution from step 6 into a 15mL centrifuge tube, add the equilibrated Ni-NTA agarose beads, and incubate at 4℃ for 1h by rotation.

[0113] 8. Fill the column with the Ni-NTA agarose beads containing TnM from step 7. Wash the column multiple times with 100 mL of pre-chilled wash buffer containing 20 mM imidazole. Then elute the protein with 5 mL of elution buffer containing 250 mM imidazole. Replace the eluent with 2×dialysis buffer (100 mM HEPES, pH 7.3, 200 mM NaCl, 0.2 mM EDTA, 0.2% Triton X-100, 2 mM DTT, 10% glycerol) and concentrate using a 15 mL 30 kDa ultrafiltration tube. After concentration, determine the protein concentration using the Bradford method. Add an equal volume of glycerol and store at -80°C.

[0114] In some other embodiments, when replacing the buffer solution after protein elution, if ultrafiltration tubes or a benchtop high-capacity refrigerated centrifuge required for protein ultrafiltration are unavailable, overnight dialysis at 4°C can be used. However, dialysis takes a long time and results in significant enzyme activity loss, so ultrafiltration is recommended as the preferred method.

[0115] 9. The purified protein was subjected to SDS-PAGE and Coomassie Brilliant Blue staining. The results are as follows: Figure 3 As shown. M represents the protein marker, Tn5 represents the unmodified transposase Tn5, TnM#1 is a failed product that did not achieve the expected results (using a different flexible linker), and TnM#2 is the TnM described in this invention.

[0116] The amino acid sequence of the transposase TnM is shown in SEQ ID NO.1. Those skilled in the art can also, based on their common knowledge, substitute, delete, or add one or more amino acids to the sequence shown in SEQ ID NO.1 to achieve the function of TnM.

[0117] SEQ ID NO.1

[0118] TnM:

[0119] .

[0120] Example 3: Encapsulation of TnM transposase

[0121] 1. Synthesize the required sequencing adapter sequence and perform gradient-cooling annealing to obtain double strands. The sequencing adapter can be selected by those skilled in the art; the sequencing adapter used in this embodiment is as follows:

[0122] SEQ ID NO.6

[0123] ME: (5'P) AGATGTGTATAAGAGACAG;

[0124] SEQ ID NO.7

[0125] ME-V5:TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG;

[0126] SEQ ID NO.8

[0127] ME-V7:

[0128] GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG.

[0129] 2. Mix 4× embedding solution, annealed adapter and TnM protein solution in a volume ratio of 1:1:2 to make the final concentration of TnM and adapter both 37.5 μM. Incubate at 25℃ for 1 h to obtain TnM with high transposition activity. Store at -20℃.

[0130] Example 4: Detection of TnM transposase activity

[0131] 3 μl and 7 μl of embedded TnM were mixed with 7 μg of genomic DNA, respectively, and Tn5 buffer and H2O were added to prepare a suitable transposon system. The mixture was reacted at 55 °C for 2 h. After purification, the fragment size was determined using an Agilent 2100 bioanalyzer.

[0132] Activity detection results as follows Figure 4 As shown, compared with the K562 cell line genome without TnM treatment, the addition of TnM can effectively fragment DNA. By increasing the amount of TnM enzyme or appropriately extending the reaction time, TnM can cut genomic DNA to between 100bp and 300bp, meeting the requirements of various omics experiments for DNA fragmentation.

[0133] In vitro genome digestion experiments have confirmed that the transposase TnM of this invention has high enzyme activity and can be used for high-throughput library construction.

[0134] Example 5: Specificity verification of TnM transposase

[0135] Design and synthesize single-stranded DNA probes with or without 5mCpG modification and their complementary strands. After annealing to form double strands, perform EMSA (gel migration assay) using a kit. Results are as follows: Figure 5 As shown.

[0136] Depend on Figure 5 As observed in lanes 5 and 6, TnM exhibits a stronger binding affinity to DNA modified with 5mCpG compared to DNA without 5mCpG modification; simultaneously, [the text abruptly ends here]. Figure 5 As seen in lanes 7 and 8, there was no significant difference in the binding ability of Tn5 with either of them.

[0137] Gel migration assays have verified that the transposase TnM of this invention has a specific binding ability to DNA containing 5mCpG modification and can be used for the construction of 5mCpG libraries.

[0138] Example 6: Detection of 5mCpG in naked DNA using TnM transposase

[0139] The transposase TnM obtained in this invention was applied to genomic DNA extracted from K562 cells for whole-genome 5mCpG detection. The TnM-seq flowchart is shown below. Figure 6 As shown, the detection method includes the following steps:

[0140] 1. Mix 50 ng gDNA with 0.5 μl 37.5 μM TnM in 20 μl binding buffer (20 mM HEPES, pH 7.5; 300 mM NaCl; 0.5 mM spermidine; 1× EDTA-free protease inhibitor cocktails) and incubate at room temperature for 2 h.

[0141] 2. Add MgCl2 to the system to a final concentration of 10 mM and react at 55°C for 10 min.

[0142] 3. Use the DNA purification kit (Zymo DNA Clean and Concentrator-5 Kit) to recover the transposed DNA.

[0143] In some other implementations, EDTA or SDS may be added to the system to terminate the reaction or to inactivate the enzyme at high temperature, and then DNA may be extracted using phenol:chloroform:isoamyl alcohol.

[0144] 4. Perform PCR library construction on the purified DNA (Vazyme TruePrep Index Kit V2 for Illumina). PCR system: 15 μl DNA, 5 μl N5XX, 5 μl N7XX, 25 μl 2×Mix buffer (NEB Cat#M0541); Program: 72℃, 3 min; 98℃, 30 s; 98℃, 10 s; 63℃, 30 s; 72℃, 15 s; 72℃, 30 s, 4℃, store, for a total of 10 cycles.

[0145] 5. Purify the PCR-obtained library using DNA purification magnetic beads (Vazyme VAHTS DNA Clean Beads).

[0146] 6. The company was commissioned to perform BGI high-throughput sequencing on the library-built products.

[0147] Example 7: In situ detection of 5mCpG in the cell nucleus using TnM transposase

[0148] The transposase TnM obtained in this invention was applied to fresh, non-crosslinked cells under natural conditions for in-situ detection at 5 mCpG. The TnM-seq flowchart is shown below. Figure 7 As shown, the detection method includes the following steps:

[0149] 1. Collect K562 cells cultured in vitro by centrifugation at 200g for 3 min, resuspend in DPBS and count, take 50,000 cells into 1.5mL centrifuge tube, centrifuge at 500g for 5 min at 4℃, remove supernatant, resuspend cells in mild lysis buffer (containing 0.1% NP-40, 0.1% Tween-20) and lyse on ice for 5 min.

[0150] 2. Centrifuge at 500g for 5 min at 4℃, remove the supernatant, resuspend in 95 μl of binding buffer containing 0.01% Digitonin (20 mM HEPES, pH 7.5; 300 mM NaCl; 0.5 mM spermidine; 1× EDTA-free protease inhibitor cocktails), add 5 μl TnM, mix well by pipetting, and incubate at room temperature for 1 h.

[0151] In some other implementations, to reduce cell loss, cells can be bound to Con A beads, then incubated with TnM to bind to the 5mCpG site of the genome, unbound TnM is washed away, and then a reaction buffer is added to activate transposition activity.

[0152] 3. Centrifuge at 300g for 5 min to remove the supernatant, and wash the sample twice with 200 μl of binding buffer containing 0.1% Tween-20.

[0153] 4. Resuspend the sample in 50 μl of binding buffer containing 0.01% Digitonin and 10 mM MgCl2, and react at 37°C for 30 min.

[0154] 5. Use the DNA purification kit (Zymo DNA Clean and Concentrator-5 Kit) to recover DNA from the transposable cells.

[0155] In some other implementations, EDTA can be added to the system to terminate the reaction or the enzyme can be inactivated by high temperature, and then DNA can be extracted using phenol:chloroform:isoamyl alcohol.

[0156] 6. Perform PCR library construction on the purified DNA (Vazyme TruePrep Index Kit V2 for Illumina). PCR system: 15 μl DNA, 5 μl N5XX, 5 μl N7XX, 25 μl 2×Mix buffer (NEB Cat#M0541); Program: 72℃, 3 min; 98℃, 30 s; 98℃, 10 s; 63℃, 30 s; 72℃, 15 s; 72℃, 30 s, 4℃, store, for a total of 10 cycles.

[0157] 7. Purify the PCR-obtained library using DNA purification magnetic beads (Vazyme VAHTS DNA Clean Beads).

[0158] 8. Perform BGI sequencing on the library-constructed products.

[0159] In some other implementations, other adapters may be used during TnM embedding, and other high-throughput sequencing platforms, such as the Illumina sequencing platform, may be used.

[0160] The biological samples for which TnM is used in this application may include, but are not limited to, uncrosslinked, fixed, or frozen / crosslinked, fixed, or frozen cultured cell samples, tissue samples, or other biological samples and their extracted genomic DNA.

[0161] For the PCR library preparation step, commercially available kits or methods can be used. After obtaining DNA, other polymerases, including isothermal polymerases and other polymerases with strand displacement properties, RNA or DNA-dependent polymerases, can be used for PCR amplification and library preparation.

[0162] like Figure 8 As shown, the results obtained by applying the transposase TnM of this invention to the in situ detection of 5mCpG in naked DNA or cell nucleus are compared with the results of traditional detection methods WGBS (An integrative ENCODE resource for cancer genomics. Zhang et al., Nat Commun. 2012 11, 3696) and MeDIP-seq.

[0163] This invention, based on TnM-based 5mCpG, can capture the same positive signal as WGBS and MeDIP seq, and the peak signal of this invention is more concentrated. Simultaneously, the signal value of the 5mCpG detection method of this invention is also higher, indicating that this invention (5mCpG based on transposase TnM) effectively improves the signal-to-noise ratio compared to traditional methods. Furthermore, the signal specifically detected by this invention is significantly reduced after treatment with GSK-3484862 (a DNA methyltransferase 1 inhibitor). Figure 9 This indicates that the 5mCpG site is a real 5mCpG site on the genome, confirming that the 5mCpG detection method of this invention can effectively and accurately capture 5mCpG compared to traditional methods. Compared with WGBS and MeDIP-seq, this invention greatly reduces the sample input; a high-quality 5mCpG library can be prepared with as little as 10 ng of DNA or 1000 cells. Figure 10 Furthermore, it achieved in-situ localization of 5 mCpG within intact cells. The TnM-based one-step method completed genome fragmentation and adapter addition, reducing the entire library preparation time to within three hours.

[0164] The TnM-seq developed in this invention has broad species applicability. In addition to the human cell line K562, it can generate high-quality 5mCpG maps in genomic DNA extracted from Arabidopsis leaves, zebrafish 24hpf embryonic cells, and mouse NIH / 3T3 cell lines. Figure 11 In the Drosophila S2 cell line, which has almost no epigenetic modification system such as DNA methylation, TnM-seq struggles to generate libraries, and the weak signals it produces are consistent with ATAC-seq, indicating that TnM can serve a dual purpose: characterizing DNA methylation while retaining the ability to detect chromatin openness.

[0165] In addition, from Figure 12 The peak sites generated by TnM are clearly enriched in the promoter regions of genes, which is consistent with the known distribution characteristics of 5mCpG islands on the genome. Moreover, in the promoter regions, the TnM-seq signals of highly expressed genes are lower than those of low-expressed genes, further verifying that this method can be used to characterize the distribution and level of DNA methylation in cells and to study the regulatory role of DNA methylation on gene expression.

[0166] Example 8: Application of TnM transposase in combination with other transposases ( HAM-seq ）

[0167] The transposase TnM obtained in this invention was applied to fresh, non-crosslinked cells under natural conditions to simultaneously detect histone modification H3K9me3, chromatin accessibility, and DNA methylation 5mCpG in the same sample. The HAM-seq flowchart and schematic diagram are shown below. Figure 13 , 14 As shown, the detection method includes the following steps:

[0168] 1. Collect K562 cells cultured in vitro by centrifugation at 200g for 3 min, resuspend in DPBS and count, take 50,000 cells into 1.5mL centrifuge tube, centrifuge at 500g for 5 min at 4℃, remove supernatant, resuspend cells in mild lysis buffer (containing 0.1% NP-40, 0.1% Tween-20) and lyse on ice for 5 min.

[0169] 2. Centrifuge at 500g for 5 min at 4℃, discard the supernatant, add 15 μl DPBS, 10 μl 5× TAPS–DMF, 15 μl lddH2O and 5 μl 37.5 μM Tn5, and incubate at 37 °C for 30 min. Centrifuge at 500g for 5 min at 4℃, discard the supernatant, and wash the sample precipitate with 200 μl 1× BSA / DPBS.

[0170] 3. Centrifuge at 500g for 5 min at 4℃, discard the supernatant, add 15 μl DPBS, 10 μl 5× TAPS–DMF, 15 μl lddH2O and 5 μl 37.5 μM TnH, and incubate at 37 °C for 30 min. Add 300 μl 1×BSA / DPBS, centrifuge at 500g for 5 min at 4℃, and discard the supernatant.

[0171] 4. Resuspend the sample precipitate in 95 μl of binding buffer containing 0.01% Digitonin (20 mM HEPES, pH 7.5; 300 mM NaCl; 0.5 mM spermidine; 1× EDTA-free protease inhibitor cocktails), add 5 μl of TnM, mix well by pipetting, and incubate at room temperature for 1 h.

[0172] 5. Centrifuge at 300g for 5 min to remove the supernatant, and wash the sample twice with 200 μl of binding buffer containing 0.1% Tween-20.

[0173] 6. Resuspend the sample in 50 μl of binding buffer containing 0.01% Digitonin and 10 mM MgCl2, and react at 37°C for 30 min.

[0174] 7. Use the DNA purification kit (Zymo DNA Clean and Concentrator-5 Kit) to recover DNA from transposable cells.

[0175] 8. Perform PCR library construction on the purified DNA (Vazyme TruePrep Index Kit V2 for Illumina). PCR system: 15 μl DNA, 5 μl N5XX, 5 μl N7XX, 25 μl 2×Mix buffer (NEB Cat#M0541); Program: 72℃, 3 min; 98℃, 30 s; 98℃, 10 s; 63℃, 30 s; 72℃, 15 s; 72℃, 30 s, 4℃, store, for a total of 12 cycles.

[0176] 9. Purify the PCR-obtained library using DNA purification magnetic beads (Vazyme VAHTS DNA Clean Beads).

[0177] 10. Perform BGI sequencing on the library-constructed products.

[0178] The results are as follows Figure 15 As shown, the results of using the three transposases in combination were highly consistent with those obtained by using them individually. Furthermore, HAM-seq can detect the coexistence of H3K9me3 and 5mCpG at the single-molecule level. This technology was also validated in the HEK293T and NIH / 3T3 cell lines. Figure 16 ).

[0179] Example 9: Application of TnM transposase in single-cell multi-omics technology ( EpiX-seq ）

[0180] The transposase TnM obtained in this invention was applied to extracted mouse whole brain cell nuclei for histone modification H3K9me3, chromatin accessibility, DNA methylation 5mCpG, and simultaneous detection of gene expression in single cells. The EpiX-seq schematic is shown below. Figure 17 As shown, the detection method is as follows:

[0181] The procedures for generating single-cell droplets in EpiX-seq are the same as those in HAM-seq, except that an additional RNase inhibitor at a final concentration of 1 U / µl is added to all systems. Following the procedure described in Example 8, three rounds of transposition reactions are performed, followed by the addition of 300 μl of 1×BSA / DPBS and centrifugation at 300g for 5 min. The sample is resuspended in 20 μl of 1× Nuclei Buffer (diluted from 20× Nuclei Buffer (10x Genomics, PN-2000207)) and counted using a disposable hemocytometer. The required number of cell nuclei are collected to generate single-cell droplets according to the GEM-X Epi Multiome (10x Genomics) user guide, and libraries are constructed for sequencing.

[0182] The obtained single-cell data, after integration, are highly consistent with the population data. Figure 18 Based on the RNA data, dimensionality reduction clustering and annotation of cells can effectively distinguish multiple cell types. Figure 19 By simply dividing cells into two categories, nerve cells and non-nerve cells, different signal distribution characteristics can be observed. Figure 20 This verifies the feasibility of EpiX-seq analysis for complex organizations.

[0183] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0184] Although preferred embodiments of the present invention have been described in the specification, those skilled in the art, upon learning the basic inventive concept, may make other changes and modifications to these embodiments or combinations of embodiments without inventive effort. Therefore, the scope of protection of the present invention should be understood as the scope intended to be interpreted and covered by the claims, and should not be limited to the parts detailed in the specification and embodiments of the present invention. Furthermore, the content described in the present invention, including the preferred embodiments, includes all changes and modifications falling within the scope of the present invention.

Claims

1. A high-throughput detection method for 5mCpG based on TnM transposase, TnM-seq, characterized in that, The high-throughput detection method uses the transposase TnM, which includes the MBD domain, for 5mCpG detection and includes the following steps: (1) After pretreatment of the target sample, transposition reaction was carried out using transposase TnM; (2) Recover and purify the DNA in the target sample after transposition; (3) Perform PCR library construction on the purified genome; (4) The library-built products were subjected to high-throughput sequencing.

2. The high-throughput detection method for 5mCpG based on TnM transposase, TnM-seq, as described in claim 1, is characterized in that... The amino acid sequence of the TnM transposase is shown in SEQ NO.1, or an amino acid sequence with the same function formed by substitution, deletion or addition of one or more amino acids based on the sequence shown in SEQ ID NO.

1. SEQ ID NO.1 TnM: .

3. The high-throughput detection method for 5mCpG based on TnM transposase, TnM-seq, as described in claim 1, is characterized in that... The transposase TnM is constructed as follows: the human MBD1 protein-MBD domain is linked to the N-terminus of the Tn5 transposase via a flexible linker, and it functions as a dimer.

4. The high-throughput detection method for 5mCpG based on TnM transposase, TnM-seq, as described in claim 3, is characterized in that... The human MBD1 protein-MBD domain is a 5mCpG specific recognition protein domain, and its amino acid sequence is shown in SEQ NO.2, or an amino acid sequence with the same function formed by substitution, deletion or addition of one or more amino acids based on the sequence shown in SEQ NO.

2. SEQ ID NO.2 Human MBD1-MBD domains: MAEDWLDCPALGPGWKRREVFRKSGATCGRSDTYYQSPTGDRIRSKVELTRYLGPACDLTLFDFKQGILCYPAPK.

5. The high-throughput detection method for 5mCpG based on TnM transposase, TnM-seq, as described in claim 3, is characterized in that... The amino acid sequence of the flexible linker is shown in SEQ NO.3; SEQ ID NO.3 Linker: GGGGSGGGGSGGGGS.

6. The high-throughput detection method for 5mCpG based on TnM transposase, TnM-seq, as described in claim 3, is characterized in that... The Tn5 transposase is a wild-type Tn5 transposase mutant, and its amino acid sequence is shown in SEQ NO.4, or an amino acid sequence with the same function formed by substitution, deletion or addition of one or more amino acids based on the sequence shown in SEQ ID NO.

4. SEQ ID NO.4 Tn5: MITSALHRAADWAKSVFSSAALGDPRRTARLVNVAAQLAKYSGKSITISSEGSKAMQEGAYRFIRNPNVSAEAIRKAGAMQTVKLAQEFPELLAIEDTTSLSYRHQVAEELGKLGSIQD KSRGWWVHSVLLLEATTFRTVGLLHQEWWMRPDDPADADEKESGKWLAAAATSRLRMGSMMSNVIAVCDREADIHAYLQDKLAHNERFVVRSKHPRKDVESGLYLYDHLKNQPELGGYQ ISIPQKGVVDKRGKRKNRPARKASLSLRSGRITLKQGNITLNAVLAEEINPKGETPLKWLLLTSEPVESLAQALRVIDIYTHRWRIEEFHKAWKTGAGAERQRMEEPDNLERMVSILS FVAVRLLQLRESFTPPQALRAQGLLKEAEHVESQSAETVLTPDECQLLGYLDKGKRKRKEKAGSLQWAYMAIARLGGFMDSKRTGIASWGALWEGWEALQSKLDGFLAAKDLMAQGIKI.

7. The TnM-seq method for high-throughput detection of 5mCpG based on TnM transposase according to claim 2 or 3, characterized in that, The transposase TnM is attached with a protein purification tag, which may be a His tag, a Strep tag, a GST tag, or a FLAG tag.

8. The TnM-seq method for high-throughput detection of 5mCpG based on TnM transposase according to claim 1, 2 or 3, characterized in that, The method for constructing the TnM transposase is as follows: (1) Design of TnM transposase structure: Select MBD domain 1-75aa of MBD1 protein and link it to the N-terminus of Tn5 transposase using a flexible linker. Use AlphaFold3 to predict the three-dimensional structure of the fusion protein. Use gene synthesis or molecular cloning methods to construct TnM expression vector and verify the correctness of the expression vector sequence by sequencing. (2) TnM transposase expression: (2.1) Transform the correctly sequenced TnM expression plasmid into the expression strain and culture it under constant temperature and humidity; (2.2) Inoculate the culture medium with a single-inoculation and a culture expressing TnM in two separate inoculations. (2.3) Extract bacterial cells from the culture product, lyse and break them to obtain a protein solution; (3) Purification of TnM transposase; (4) TnM transposase encapsulation: (4.1) Synthesize the sequencing adapter sequence and anneal it into a double linker; mix the embedding solution, double linker and purified protein solution in a certain proportion and incubate to obtain TnM with high transposition activity, and store it below -20℃.

9. A novel transposase TnM, characterized in that, The transposase is a dimer composed of an N-terminal MBD domain, a C-terminal Tn5 transposase, and a linker region. The amino acid sequence of the transposase is shown in SEQ NO.1, or is an amino acid sequence with the same function formed by substitution, deletion, or addition of one or more amino acids based on the sequence shown in SEQ ID NO.

1.

10. The application of the novel transposase TnM according to claim 9 in constructing DNA-5mCpG high-throughput sequencing libraries of biological samples, and / or in the detection of 5mCpG in naked DNA or intact cells.

11. A multi-omics technique, HAM-seq, for simultaneously detecting histone modification H3K9me3, chromatin accessibility, and DNA methylation modification 5mCpG in the same sample, characterized in that... By combining multiple transposases with different functions, such as TnH and Tn5, as well as a novel transposase TnM as described in claim 10, data can be split after library sequencing using the adapter barcode sequence carried by the transposase, thereby obtaining information at different omics levels.

12. EpiX-seq, a four-omics technology for simultaneously detecting histone modification H3K9me3, chromatin accessibility, DNA methylation modification 5mCpG, and gene expression in a single cell, characterized in that, The HAM-seq technology described in claim 11 can be combined with a commercial single-cell microfluidic platform, or with other types of single-cell sequencing technologies.

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