High-throughput single cell nucleic acid methylation sequencing method

Abstract

The invention provides a construction method of a high-throughput sequencing library. The method comprises the following steps: carrying out nucleic acid interruption in a single-tube reaction chamber by adopting transposase with a universal joint, adding the universal joint, and then carrying out at least two rounds of ligation reactions in a branched-combination combination manner to introduce different tag combinations into genome fragments of each cell, thereby obtaining a sequencing library. The homogeneity of cell transposase breaking treatment is higher, the nucleic acid tag connection process is easier to operate, the cell flux covered by single sequencing is improved, the cost is lower, and the method is compatible with a single-cell multi-omics sequencing method.

Description

Technical Field

[0001] The present invention relates to the field of methylation sequencing, and more particularly, to a high-throughput single-cell nucleic acid methylation sequencing method. Background Art

[0002] DNA methylation is an important epigenetic modification that regulates gene expression and plays an important role in embryonic development, cell growth and differentiation, aging, and the occurrence and development of tumors. Therefore, revealing the methylation heterogeneity of cells at the single-cell level to study cell identity, fate, and function under normal biological and pathological backgrounds has become one of the hot research fields in modern biology and medicine.

[0003] Currently, the commonly used single-cell methylation sequencing technologies are mainly single-cell reduced representation bisulfite sequencing (scRRBS) and single-cell whole-genome methylation sequencing (scWGBS). For scRRBS, after single cells are isolated, the genomic DNA after cell lysis is treated with restriction enzymes Msp I and Bgl II through a single-tube enzyme reaction to generate fragments with CpG at the ends. Subsequently, the 3' sticky ends are repaired and additional adenosines are added, and then the DNA fragments are treated with bisulfite. After deaminating unmethylated cytosines to uracils, sequencing is performed to obtain the whole-genome DNA methylation map. scWGBS is a technology capable of drawing a whole-genome DNA methylation map at single-base resolution. The main steps are DNA fragmentation, methylation adapter ligation, bisulfite conversion. After DNA library amplification, sequencing is performed through the NGS platform, which can achieve absolute quantification of almost all cytosines in the genome under different biological backgrounds, with the advantages of high precision and high coverage. Currently, multiple platforms have been developed and applied, such as sci-MET, sci-METv2, sci-EM, Drop-BS, etc. These technical methods are generally complex to operate, such as requiring transposases embedding different adapters, with poor detection uniformity, limited cell throughput, and high costs.

[0004] Currently, combinatorial indexing techniques are widely used in single-cell sequencing. The prior art has disclosed scalable and efficient single-cell DNA methylation sequencing via combinatorial indexing. The common feature is that the first round of indexing is introduced by treating cell nuclei with transposases embedded with different adapters, followed by bisulfite treatment and linear amplification, and then the second round of indexing (which is also the sequencing library indexing) is introduced through library PCR amplification, thereby achieving methylation detection of nucleic acids from multiple single cells. However, such a process has two main problems: First, the cost of Tn5 transposases embedded with different adapters increases significantly, and at the same time, it limits the cell throughput and cannot be too high (published literature and patent data generally use 96 transposases, and at most 22 cells can be used in each library); Second, each reaction chamber for introducing the first round of indexing reacts with Tn5 transposases containing different adapters, and there will be deviations in the enzyme activity and fragmentation efficiency, resulting in poor uniformity of cell cleavage in different reaction chambers.

[0005] Therefore, there is an urgent need in the field of single-cell DNA methylation sequencing for a high-throughput single-cell DNA methylation sequencing method that is low-cost, has good uniformity, has flexible cell throughput and is easy to achieve high throughput, is easy to operate, does not require specific equipment, and is suitable for widespread use in laboratories. Summary of the Invention

[0006] To solve at least one problem in the prior art, the present disclosure provides a single-cell sequencing method.

[0007] According to a first aspect of the present disclosure, there is provided a method for constructing a high-throughput sequencing library, the method comprising the following steps:

[0008] 1) Prepare a suspension containing multiple single cell nuclei;

[0009] 2) Fragment the chromatin DNA of the multiple cell nuclei using Tn5 transposase embedded with a universal adapter, and add the universal adapter to the obtained chromatin DNA fragments;

[0010] 3) Divide the suspension of the multiple cell nuclei into n reaction chambers, and add the first round of indexing to the chromatin DNA fragments in each reaction chamber to obtain cell nuclei with n types of the first round of indexing;

[0011] 4) Mix the cell nuclei with n types of the first round of indexing, and then divide them into m reaction chambers, and add the second round of indexing sequences to the cell nuclei in each reaction chamber to obtain cell nuclei with m types of the second round of indexing;

[0012] 5) Use the cell nuclei with m types of the second round of indexing to construct a single-cell sequencing library;

[0013] 6) Optionally, repeat steps 3) and 4).

[0014] In some embodiments, the suspension containing multiple single cell nuclei in step 1) contains a suspension of at least 100×n or at least 100×m single cell nuclei.

[0015] In some embodiments, the suspension containing multiple single cell nuclei in step 1) contains at least 100×n, at least 200×n, at least 300×n, at least 400×n, at least 500×n, at least 600×n, at least 700×n, at least 800×n, at least 900×n, at least 1000×n, 2000×n, at least 3000×n, at least 4000×n, at least 5000×n, at least 6000×n, at least 7000×n, at least 8000×n, at least 9000×n, at least 10000×n single cell nuclei.

[0016] In some embodiments, n and m can be the same or different.

[0017] Preferably, n and m are the same.

[0018] In some embodiments, n is from 10 to 1000, and m is from 10 to 1000.

[0019] In some embodiments, in step 1), the suspension containing multiple cell nuclei is prepared from a sample.

[0020] In some embodiments, the sample contains single cells, consists essentially of single cells, or consists of single cell nuclei.

[0021] In some embodiments, the sample contains the following, consists essentially of the following, or consists of the following: at least 100,000, 200,000, 300,000, 500,000, 800,000, or 1,000,000 single cells or single cell nuclei.

[0022] In some embodiments, nucleosome disruption is performed after step 1).

[0023] In some embodiments, after step 5) or step 6), bisulfite treatment, linear amplification, and / or PCR amplification are performed.

[0024] In some embodiments, after step 5) or step 6), the cell nuclei with m types of second-round tags are mixed, and then bisulfite treatment, linear amplification, and / or PCR amplification are performed.

[0025] In some embodiments, the linear amplification includes 1 to 10 cycles.

[0026] In some embodiments, before bisulfite treatment, linear amplification, and / or PCR amplification, the cell nuclei with m types of second-round tags are sorted into q digestion reaction chambers according to the number of target cells, and the digestion of the cell nuclei is performed.

[0027] The bisulfite treatment converts the unmethylated cytosine residues of CpG dinucleotides into uracil residues and keeps the 5-methylcytosine residues unchanged.

[0028] In some embodiments, 100 to 1000 single cell nuclei are sorted into one of the digestion reaction chambers.

[0029] The specific number of the sorted digestion reaction chambers depends on the number of target cells.

[0030] In some embodiments, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 single cell nuclei are sorted into one of the digestion reaction chambers.

[0031] In some embodiments, 10 - 1000 types of first-round tags and 10 - 1000 types of second-round tags are selected, so that 100 - 1000000 types of tag combinations can be achieved, thereby realizing a throughput of 100 - 1000000 cell nuclei. If base conversion, amplification, and sequencing of 100 cell nuclei in a single reaction chamber are set up with 1 - 10000 single reaction chambers, a throughput of 100 - 1000000 cell nuclei can be achieved.

[0032] In some embodiments, in step 3), the suspension of the multiple cell nuclei is evenly distributed into the n reaction chambers.

[0033] In some embodiments, the reaction chamber can be a vial, tube, well, droplet, pellet, bead, or container.

[0034] In some embodiments, the reaction chamber is a 96-well plate or a 384-well plate.

[0035] In some embodiments, the single cell sequencing library is a single cell nucleic acid methylation sequencing library.

[0036] In some embodiments, the sample contains single cells, consists essentially of single cells, or consists of single cell nuclei. In some examples, the sample contains, consists essentially of, or consists of: at least 100,000, 200,000, 300,000, 500,000, 800,000, or 1,000,000 single cells or single cell nuclei.

[0037] In some embodiments, the nuclei distributed into n reaction chambers in step 3) and the nuclei distributed into m reaction chambers in step 4) are substantially equal.

[0038] In a second aspect, the present invention provides a method for single-cell sequencing, the method comprising the following steps:

[0039] 1) Prepare a suspension containing multiple single nuclei;

[0040] 2) Fragment the chromatin DNA of the multiple nuclei using Tn5 transposase embedded with universal adapters, and add universal adapters to the obtained chromatin DNA fragments;

[0041] 3) Divide the suspension of the multiple nuclei into n reaction chambers, and add a first round of tags to the chromatin DNA fragments in each reaction chamber to obtain nuclei with n types of first-round tags;

[0042] 4) Mix the nuclei with n types of first-round tags, then divide them into m reaction chambers, and add a second-round tag sequence to the nuclei in each reaction chamber to obtain nuclei with m types of second-round tags;

[0043] 5) Use the nuclei with m types of second-round tags to construct a single-cell sequencing library;

[0044] 6) Optionally, repeat step 3) and step 4);

[0045] 7) Perform bisulfite treatment.

[0046] In some embodiments, the suspension containing multiple single nuclei in step 1) contains a suspension of at least 100×n or at least 100×m single nuclei.

[0047] In some embodiments, n and m may be the same or different.

[0048] Preferably, n and m are the same.

[0049] In some embodiments, n is 10 to 1000, and m is 10 to 1000.

[0050] In some embodiments, in step 1), the suspension containing multiple nuclei is prepared from a sample.

[0051] In some embodiments, the sample contains single cells, consists essentially of single cells, or consists of single nuclei.

[0052] In some embodiments, the sample comprises, consists essentially of, or consists of: at least 100,000, 200,000, 300,000, 500,000, 800,000, or 1,000,000 single cells or single cell nuclei.

[0053] In some embodiments, nucleosome disruption is performed after step 1).

[0054] In some embodiments, after step 7), linear amplification and / or PCR amplification is performed.

[0055] In some embodiments, after step 5) or step 6), the cell nuclei with m types of second-round tags are mixed, followed by bisulfite treatment, linear amplification, and / or PCR amplification.

[0056] In some embodiments, the linear amplification includes 1 to 10 cycles.

[0057] In some embodiments, before performing the bisulfite treatment, linear amplification, and / or PCR amplification, the cell nuclei with m types of second-round tags are sorted into q digestion reaction chambers according to the target cell number, and the cell nuclei are digested.

[0058] The bisulfite treatment converts unmethylated cytosine residues of CpG dinucleotides into uracil residues and leaves 5-methylcytosine residues unchanged.

[0059] In some embodiments, 100 to 1000 single cell nuclei are sorted into one of the digestion reaction chambers.

[0060] The specific number of sorted digestion reaction chambers depends on the target cell number.

[0061] In some embodiments, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 single cell nuclei are sorted into one of the digestion reaction chambers.

[0062] In some embodiments, 10 - 1000 types of the first-round tags and 10 - 1000 types of the second-round tags are selected, so that 100 - 1,000,000 types of tag combinations can be achieved, thereby realizing a throughput of 100 - 1,000,000 cell nuclei. If base conversion and amplification sequencing of 100 cell nuclei in a single reaction chamber are set up with 1 - 10,000 single reaction chambers, a throughput of 100 - 1,000,000 cell nuclei can be achieved.

[0063] In some embodiments, in step 3), the suspension of the multiple cell nuclei is evenly distributed into the n reaction chambers.

[0064] In some embodiments, the reaction chamber can be a vial, tube, well, droplet, pellet, bead, or container.

[0065] In some embodiments, the reaction chamber is a 96-well plate or a 384-well plate.

[0066] In some embodiments, the single-cell sequencing library is a single-cell nucleic acid methylation sequencing library.

[0067] In some embodiments, the sample contains single cells, consists essentially of single cells, or consists of single cell nuclei.

[0068] In some embodiments, the sample contains, consists essentially of, or consists of: at least 100,000, 200,000, 300,000, 500,000, 800,000, or 1,000,000 single cells or single cell nuclei.

[0069] In some embodiments, the cell nuclei distributed into n reaction chambers in step 3) and the cell nuclei distributed into m reaction chambers in step 4) are substantially equal.

[0070] The present invention also provides the use of the method of the first aspect or the method of the second aspect in one or more of the following:

[0071] (a) Determining the methylation level of single cells or cell populations;

[0072] (c) Quantifying gene transcription;

[0073] (d) Identifying cell types and states of complex heterogeneous tissues;

[0074] (e) Identifying endogenous and exogenous dsDNA and ssDNA;

[0075] (f) Determining epigenetic modifications of single cells or cell populations, where the epigenetic modifications include DNA methylation. Description of the Drawings

[0076] Figure 1 Showing the high-throughput single-cell nucleic acid methylation library construction flow chart.

[0077] Figure 2 Showing that the proportion of correctly structured cell tags in 6 libraries averages 80%.

[0078] Figure 3 Showing that a total of 117 cells were sequenced, including 53 human cells, 52 mouse cells, and 12 unknown cells.

[0079] Figure 4The average number of reads measured for human cells was 153,488, and the average number of methylated CpG sites covered was 60,845; the average number of reads measured for mouse cells was 164,827, and the average number of methylated CpG sites covered was 53,442.

[0080] Figure 5 The average methylation level of human cells was shown to be 36.6%, and the average methylation level of mouse cells was 51.4%.

[0081] Figure 6 It was shown that the proportion of cells with correct tag structures in the two libraries was greater than 90%.

[0082] Figure 7 It was shown that a total of 204 cells were obtained by sequencing, including 174 human cells, 25 mouse cells, and 5 unknown cells.

[0083] Figure 8 The average number of reads measured for human cells was 451,556, and the average number of methylated CpG sites covered was 346,064. The average number of reads measured for mouse cells was 289,451, and the average number of methylated CpG sites covered was 189,558.

[0084] Figure 9 The average methylation level of human cells was shown to be 36.4%, and the average methylation level of mouse cells was 47.5%.

[0085] Figure 10 It was shown that the number of uniquely mapped reads was linearly correlated with the number of methylated CpG sites covered, indicating that as the sequencing depth increased, the number of methylated CpG sites increased until saturation. Detailed implementation mode

[0086] In the present invention, a transposase with a universal adaptor is used to fragment nucleic acids and add a universal adaptor in a single-tube reaction chamber. Subsequently, at least two rounds of ligation reactions are carried out in a combined manner of separating tubes and merging them to introduce different tag combinations into the genomic fragments of each cell. Then, cells labeled with different tags are merged for bisulfite conversion treatment, linear amplification, and library PCR amplification to add library tags, and a library that can be directly sequenced is obtained. See Figure 1 。

[0087] Therefore, the present invention has stronger uniformity in the transposase fragmentation treatment of cells, and the nucleic acid tag ligation process is easier to operate. The cell throughput can be expanded through two rounds or multiple rounds of combined tags, and the cost is low (for a 40×40 tag combination, 100 cells can be divided into one tube for subsequent library preparation. To achieve a throughput of 1000 cells, only 10 tubes need to be paralleled for conversion and library PCR amplification, and the average cost per cell is only about 1.1 yuan. Increasing the cell throughput can further reduce the cost).

[0088] The Tn5 transposase consists of a protein and an adapter sequence (e.g., ME sequence), which bind together to form a transposase complex. In the present application, the transpososome complex includes a transposase having two universal adapter sequences, which may be the same or different.

[0089] In some embodiments, the two universal adapter sequences are the same.

[0090] Universal adapter primer names and sequences:

[0091] PrimerM (SEQ ID NO:1):

[0092] 5’-phos- / i5MedC / TGT / i5MedC / T / i5MedC / TTATA / i5MedC / A / i5MedC / AT / i5MedC / T-NH2-3’

[0093] PrimerE (SEQ ID NO:2):

[0094] 5’-Phos-GT / i5MedC / T / i5MedC / GTGGG / i5MedC / T / i5MedC / GGAGATGTGTATAAGAGA / i5MedC / AG-3’

[0095] Wherein, "i5Med" represents a methylation modification on the 5th carbon atom of cytosine; "5’-phos-" represents a phosphorylation modification on the first nucleotide at the 5’ end; "-NH2-3’" represents an amino modification on the first nucleotide at the 3’ end.

[0096] Bisulfite treatment of nucleic acids is known to those skilled in the art and is routine. In one embodiment, bisulfite treatment converts unmethylated cytosine residues of CpG dinucleotides into uracil residues and leaves 5-methylcytosine residues unchanged. Bisulfite treatment produces bisulfite-treated nucleic acid fragments.

[0097] In the present invention, nucleosome disruption includes chemically treating isolated nuclei to produce nuclei depleted of nucleosomes while maintaining the integrity of these isolated nuclei. The isolated nuclei or cells may include nucleosomes, may be nucleosome-free, or may be subjected to conditions that deplete the nucleosomes of the nuclei to produce nucleosome-depleted nuclei. Nucleosome-depleted nuclei can be used in methods for determining the DNA sequence of the entire genome or a portion thereof of a cell.

[0098] Methods for nucleosome depletion are known and conventional and include, but are not limited to, enzymatic and chemical treatments. In one embodiment, the conditions for nucleosome depletion include chemical treatment with a chaotropic agent capable of disrupting nucleic acid-protein interactions.

[0099] The cells described in the present application can be a variety of different cells obtained from a single organism (or from organisms of the same species). The cells and cell nuclei can be from any sample, such as any organism, and any cell type or any tissue from an organism.

[0100] In some embodiments, the cells can be germ cells, e.g., sperm cells or egg cells.

[0101] In some embodiments, the tissue can be reproductive tissue, such as the epididymis. In some embodiments, the cells or cell nuclei can be from cancerous or diseased tissue. The method can further include dissociating the cells and / or isolating the cell nuclei. Methods for isolating cell nuclei from cells are known to those skilled in the art and are conventional. The number of cell nuclei or cells can be at least two. The number of cell nuclei or cells that can be used is not intended to be limiting and can reach billions. For example, in one embodiment, the number of cell nuclei or cells can be no greater than 100,000,000, no greater than 10,000,000, no greater than 1,000,000,000, no greater than 100,000,000, no greater than 10,000,000, no greater than 1,000,000, no greater than 100,000, greater than 10,000, no greater than 1,000, no greater than 500 or no greater than 50. One or more samples can be provided. For example, the sample can be one cell type or tissue from one organism.

[0102] The beneficial effects of the present invention are reflected in the following aspects:

[0103] (1) Instead of encapsulating Tn5 transposase complexes with different nucleic acid tags, which results in different activities of Tn5 transposase and nucleic acid fragmentation efficiencies in each reaction chamber, a homogenized adapter is used to encapsulate Tn5 transposase, and the Tn5 transposase nucleic acid fragmentation reaction is carried out in a single-tube reaction chamber;

[0104] (2) Different tag combinations are introduced into single cell nuclei through the combination of branch tube - merging based on the ligation method. The operation is simple, and the cell throughput only depends on the number of nucleotide tags introduced in each round. For example, 10×10 = 100 tag combinations can achieve the conversion, amplification and sequencing of 20 cell nuclei in a single tube. By dividing the experiment into 10 tubes, a throughput of 200 cell nuclei can be achieved. 40×40 = 1600 tag combinations can achieve the conversion, amplification and sequencing of 100 cell nuclei in a single tube. By dividing the experiment into 10 tubes, a throughput of 1000 cell nuclei can be achieved. Then, 96*96 = 9216 tag combinations can achieve a throughput of 10,000 cell nuclei;

[0105] (3) The cost basically comes from reagent consumables and there is no need to purchase specific instruments and equipment. For example, for 40×40 = 1600 tag combinations, with 100 in a single tube to achieve a throughput of 1000 cell nuclei, the average cost per cell nucleus is only 1.1 yuan. With 300 in a single tube to achieve a throughput of 3000 cell nuclei, the average cost per cell nucleus is only 0.4 yuan. The larger the single cell nucleus throughput, the lower the average cost per cell;

[0106] (4) The present invention is a high - throughput single - cell DNA methylation sequencing method based on the ligation method and is also compatible with single - cell multi - omics sequencing methods.

[0107] Example:

[0108] Experimental steps:

[0109] (1) Preparation of reaction buffer: NIB - Tris buffer (10 mM Tris - HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% 0.1% Tween - 20, 1× protease inhibitor); NIB - HEPES buffer (10 mM HEPES - KOH pH 7.2, 10 mM NaCl, 3 mM MgCl2, 0.1% 0.1% Tween - 20, 1× protease inhibitor); 10× NEB buffer 2.1 (100 mM Tris - HCl pH 7.9, 500 mM NaCl, 100 mM MgCl2, 1 ng / μl bovine serum albumin).

[0110] (2) Preparation of single nuclear suspensions (suspension cell lines and adherent cell lines): First, digest the cultured cell line into a single cell suspension. Resuspend 1 million cells in 1 ml of cold NIB-HEPES buffer, incubate on ice for 10 minutes, and then centrifuge at 500×g for 5 minutes in a 4°C centrifuge. Resuspend the cells again in 1 ml of cold NIB-HEPES buffer, incubate on ice for 10 minutes, and centrifuge at 500×g for 5 minutes in a 4°C centrifuge. The precipitate at the bottom of the tube is the single nucleus. Then resuspend the nucleus in 4 ml of NIB-HEPES buffer, add 925 μl of 4% paraformaldehyde, gently shake and incubate at room temperature for 10 minutes, centrifuge at 500×g for 5 minutes in a 4°C centrifuge, and wash the nucleus with 5 ml of cold PBS. Resuspend the nucleus in 1 ml of cold NIB-Tris buffer.

[0111] (3) Nucleosome disruption: Centrifuge the nucleus at 500×g for 5 minutes in a 4°C centrifuge, resuspend the nucleus in 1 ml of 1× NEB buffer 2.1, centrifuge at 500×g for 5 minutes in a 4°C centrifuge, resuspend the nucleus in 760 μl of 1× NEB buffer 2.1, add 40 μl of 1% SDS, mix well and incubate in a 37°C shaker at 300 rpm for 30 minutes. Slowly pass the nucleus through a 30-μm cell filter, centrifuge at 500×g for 5 minutes in a 4°C centrifuge. Wash with 1 ml of cold NIB-Tris buffer, centrifuge at 500×g for 5 minutes in a 4°C centrifuge, resuspend the nucleus in 500 μl of cold NIB-Tris buffer, and count and adjust the nucleus density to 1×10 6 cells per milliliter.

[0112] (4) Single adapter Tn5 transposase fragmentation: Prepare a single-tube Tn5 transposase reaction system (10,000 nuclei, 1× TD buffer, 400 ng of Tn5 transposase complex, supplemented with deionized water to a volume of 50 μl), mix well and incubate in a 55°C shaker at 500 rpm for 60 minutes. After the reaction is completed, add 1 ml of 0.25× NIB-Tris buffer, centrifuge at 1000×g for 3 minutes in a 4°C centrifuge, and resuspend the nucleus in 1 ml of 0.25× NIB-Tris buffer.

[0113] (5) Ligation of the first-round cell tags: Centrifuge the cell nuclei at 1000×g for 3 minutes in a 4°C centrifuge to precipitate the nuclei, and then resuspend the nuclei in the ligation reaction system (for 40×40 tag combinations: 372 μl of 0.25× NIB-Tris, 80 μl of 10× ligase buffer, 8 μl of 10% TritonTM X-100, 20 μl of T4 ligase). Divide the average volume of 12 μl into 40 reaction chambers, add 8 μl of the first-round annealed nucleotide primers (5 μM) to each reaction chamber, mix well, and incubate with gentle shaking at room temperature for 30 minutes. Then add 8 μl of the first-round blocked nucleotide primers (10 μM) to each reaction chamber, mix well, and incubate with gentle shaking at room temperature for 30 minutes. Combine all the reaction chambers, centrifuge at 1000×g for 3 minutes in a 4°C centrifuge, wash with 1 ml of 0.25× NIB-Tris buffer, centrifuge at 1000×g for 3 minutes in a 4°C centrifuge, and resuspend the nuclei in 1 ml of 0.25× NIB-Tris buffer.

[0114] (6) Ligation of the second-round cell tags: Centrifuge the cell nuclei at 1000×g for 3 minutes in a 4°C centrifuge to precipitate the nuclei, and resuspend the nuclei in the ligation reaction system (for 40×40 tag combinations: 372 μl of 0.25× NIB-Tris, 80 μl of 10× ligase buffer, 8 μl of 10% TritonTM X-100, 20 μl of T4 ligase). Divide the average volume of 12 μl into 40 reaction chambers, add 8 μl of the first-round annealed nucleotide primers (5 μM) to each reaction chamber, mix well, and incubate with gentle shaking at room temperature for 30 minutes. Then add 8 μl of the second-round blocked nucleotide primers (10 μM) to each reaction chamber, mix well, and incubate with gentle shaking at room temperature for 30 minutes. Combine all the reaction chambers, centrifuge at 1000×g for 3 minutes in a 4°C centrifuge, wash with 1 ml of cold NIB-Tris buffer, centrifuge at 1000×g for 3 minutes in a 4°C centrifuge, and resuspend the nuclei in 1 ml of cold NIB-Tris buffer.

[0115] (7) Nucleus sorting and digestion: Add 1 μl of 500× DAPI dye to the cell nuclei, mix well, incubate in the dark at room temperature for 3 minutes, centrifuge at 1000×g for 3 minutes in a 4°C centrifuge, wash with 1 ml of cold NIB-Tris buffer, centrifuge at 1000×g for 3 minutes in a 4°C centrifuge, and resuspend the nuclei in 100 μl of cold NIB-Tris buffer. Sort 300 single cell nuclei into a digestion reaction chamber (including 3 μl of M-digestion buffer, 1.5 μl of deionized water, 1.5 μl of proteinase K). The specific number of digestion reaction chambers depends on the number of target cells. Then incubate at 50°C for 30 minutes to digest the cell nuclei.

[0116] (8) Bisulfite conversion: Add 39 μl of Zymo CT conversion reagent to each reaction chamber, mix well and place on a thermal cycler for the following steps: react at 98 °C for 8 minutes, react at 64 °C for 3.5 hours, and then hold at 4 °C for less than 20 hours. After the reaction, prepare the nucleic acid purification bead reaction solution (each reaction chamber includes 180 μl of M-binding buffer and 3 μl of ZymoMagBinding beads), add 183 μl of the nucleic acid purification bead reaction solution to each reaction chamber, mix well by shaking and incubate at room temperature for 5 minutes, then place on a magnetic stand compatible with the reaction chamber. After the supernatant becomes clear, remove the supernatant. Resuspend the beads with 120 μl of M-wash buffer, place on the magnetic stand, and immediately remove the supernatant after the supernatant becomes clear. Add 60 μl of M-desulfonation buffer, mix well by shaking and incubate at room temperature for 15 minutes, shaking and mixing every 3 minutes. Place on the magnetic stand, and after the supernatant becomes clear, remove the supernatant. Resuspend the beads with 120 μl of M-wash buffer again, place on the magnetic stand, and immediately remove the supernatant after the supernatant becomes clear. Dry the beads at 55 °C for 10 minutes, add 21 μl of pre-warmed M-elution buffer at 55 °C to each reaction chamber, and then incubate at 55 °C for 4 minutes.

[0117] (9) Linear amplification: Transfer 20 μl of the eluate to a new reaction chamber, add 4.5 μl of the linear amplification reaction mixture (2.5 μl of reaction buffer, 1 μl of 10 mM dNTPs, 1 μl of 10 mM N5H6 nucleotide random primers), incubate at 95 °C for 45 seconds to make the nucleic acid single-stranded, immediately cool rapidly on ice and keep it on ice. After complete cooling, add 0.5 μl of Klenow-exo- polymerase, mix well and place on a thermal cycler for the following steps: react at 4 °C for 5 minutes, then increase the temperature to 37 °C at a rate of +1 °C / 15 seconds, and react at 37 °C for 1.5 hours. Repeat this step 3 more times, for a total of 4 rounds of linear amplification. For each amplification, first incubate at 95 °C for 45 seconds to make the nucleic acid single-stranded, immediately cool rapidly on ice and keep it on ice. After complete cooling, add 5 μl of the reaction mixture (0.5 μl of reaction buffer, 1.5 μl of 2.5 nM dNTPs, 1.5 μl of 25 pM N5H6 nucleotide random primers, 0.5 μl of Klenow-exo- polymerase, 1 μl of deionized water).

[0118] (10) PCR amplification: Add 1× (an amount equal to 1× the volume of the reaction chamber) of XP beads to each reaction chamber. After shaking and mixing evenly, incubate at room temperature for 5 minutes, then place on a magnetic stand compatible with the reaction chamber. After the supernatant becomes clear, remove the supernatant. Wash the beads twice with 200 μl of 80% ethanol (during the washing process, place on the magnetic stand). After completely removing the ethanol, dry the beads at room temperature for 2 - 3 minutes. Add 20 μl of 10 mM Tris-HCl pH 8.5 to elute the nucleic acid, and incubate at room temperature for 5 minutes. Transfer 19 μl of the eluate to a new reaction chamber. Add 31 μl of the PCR amplification reaction mixture (2 μl of 10 μM QP2 universal primer, 4 μl of 5 μM i5 index PCR primer, 25 μl of 2× KAPA high-fidelity polymerase). After mixing evenly, place on a thermal cycler and perform the following steps: react at 98°C for 3 minutes, 14 cycles (react at 98°C for 20 seconds, 63°C for 30 seconds, 72°C for 30 seconds), react at 72°C for 5 minutes, and then hold at 4°C. Add 0.8× (an amount equal to 0.8× the volume of the reaction chamber) of XP beads to each reaction chamber. After shaking and mixing evenly, incubate at room temperature for 5 minutes, then place on the magnetic stand. After the supernatant becomes clear, remove the supernatant. Wash the beads twice with 200 μl of 80% ethanol (during the washing process, place on the magnetic stand). After completely removing the ethanol, dry the beads at room temperature for 2 - 3 minutes, and elute the nucleic acid in 20 μl of 10 mM Tris-HCl pH 8.5.

[0119] Example 1:

[0120] Mix the human cell line GM12878 and the mouse cell line 3T3 in equal proportions; there are 10 * 10 = 100 combinations of cell labels; after introducing 2 rounds of cell label combinations according to the above experimental steps, count the cell nuclei and manually divide them into 20 single-nuclei per tube for bisulfite conversion and amplification library construction, and perform methylation sequencing on the Sailu Medical platform and conduct data processing. Among them, use the TrimGalore software to remove sequencing adapters and low-quality reads; use the Bismark software to align the reads to the multiple reference genomes of humans and mice. First, use the paired-end sequencing mode for alignment, and then align the unaligned reads using the single-end sequencing mode; use the MarkDuplicates option of the picard software to remove duplicates of the reads according to the cell barcodes; use samtools to statistically analyze the alignment results. The statistically analyzed data is then plotted using the GraphPad Prism 8 software.

[0121] The nucleotide sequences of the universal adapter primers are shown in Table 1, the nucleotide sequences of the first-round tag primers are shown in Table 2, and the nucleotide sequences of the second-round tag primers are shown in Table 3.

[0122] The sequencing analysis results are as Figures 2 to 5As shown below:

[0123] Figure 2 It shows that the average proportion of the correct structure of 6 library cell tags is 80%;

[0124] Figure 3 It shows that a total of 117 cells were obtained by sequencing, including 53 human cells, 52 mouse cells, and 12 unknown cells;

[0125] Figure 4 It shows that the average number of reads measured for human cells is 153,488, and the average number of methylated CpG sites covered is 60,845; the average number of reads measured for mouse cells is 164,827, and the average number of methylated CpG sites covered is 53,442;

[0126] Figure 5 It shows that the average methylation level of human cells is 36.6%, and the average methylation level of mouse cells is 51.4%.

[0127] Table 1. Names and sequences of universal adapter primers:

[0128]

[0129] Among them, "i5Med" represents the methylation modification on the 5th carbon atom of cytosine; "phos-" represents the phosphorylation modification on the first nucleotide at the 5' end; "-NH2" represents the amino modification on the first nucleotide at the 3' end.

[0130] Table 2. Names and sequences of the first-round tag primers

[0131]

[0132] Table 3. Names and sequences of the second-round tag primers:

[0133]

[0134] Table 3 (continued)

[0135]

[0136] Example 2:

[0137] Mix the human cell line GM12878 and the mouse cell line 3T3; there are 40 * 40 = 1600 combinations of cell labels; after introducing 2 rounds of cell label combinations according to the experimental steps described in Example 1, perform bisulfite conversion and amplification library construction with 100 single nuclei in each tube by flow sorting, perform methylation sequencing on the Sailu Medical platform, and perform data processing. Among them, use the TrimGalore software to remove sequencing adapters and low-quality reads; use the Bismark software to align the reads to the multiple reference genomes of humans and mice. First, use the paired-end sequencing mode for alignment, and then use the single-end sequencing mode for the reads that are not aligned; use the MarkDuplicates option of the picard software to remove duplicates of the reads according to cell barcodes; use samtools to perform statistics on the alignment results. The statistically processed data is then plotted using the GraphPad Prism 8 software.

[0138] Among them, the nucleotide sequences of the universal adapter primers are shown in Table 4, the nucleotide sequences of the first-round label primers are shown in Table 5, and the nucleotide sequences of the second-round label primers are shown in Table 6.

[0139] The results are as Figures 6 to 10 shown:

[0140] Figure 6 It shows that the proportion of correctly structured cell labels in the two libraries is greater than 90%;

[0141] Figure 7 It shows that a total of 204 cells were sequenced, including 174 human cells, 25 mouse cells, and 5 unknown cells;

[0142] Figure 8 It shows that the average number of reads measured for human cells is 451556, and the average number of methylated CpG sites covered is 346064; the average number of reads measured for mouse cells is 289451, and the average number of methylated CpG sites covered is 189558;

[0143] Figure 9 It shows that the average methylation level of human cells is 36.4%, and the average methylation level of mouse cells is 47.5%;

[0144] Figure 10 It shows that the number of uniquely aligned reads and the number of methylated CpG sites covered are linearly correlated, indicating that as the sequencing depth increases, the number of methylated CpG sites increases until saturation.

[0145] Table 4, names and sequences of universal adapter primers:

[0146]

[0147] Table 5. Names and Sequences of the First Round of Tag Primers:

[0148]

[0149] Table 5 (continued)

[0150]

[0151] Table 5 (continued)

[0152]

[0153] Table 6. Names and Sequences of the Second Round of Tag Primers:

[0154]

[0155] Table 6 (continued)

[0156]

[0157] Table 6 (continued)

[0158]

[0159] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A high-throughput method for constructing a sequencing library, the method comprising the following steps: 1) Prepare a suspension containing multiple single cell nuclei; 2) Fragment the chromatin DNA of the multiple cell nuclei using Tn5 transposase embedded with a universal adaptor, and add the universal adaptor to the obtained chromatin DNA fragments; 3) Divide the suspension of the multiple cell nuclei into n reaction chambers, and add a first round of tags to the chromatin DNA fragments in each reaction chamber to obtain cell nuclei with n types of first round tags; 4) Mix the cell nuclei with n types of first round tags, and then divide them into m reaction chambers, and add a second round of tag sequences to the cell nuclei in each reaction chamber to obtain cell nuclei with m types of second round tags; 5) Use the cell nuclei with m types of second round tags to construct a single cell sequencing library; 6) Optionally, repeat step 3) and step 4).

2. The method according to claim 1, wherein The suspension containing multiple single cell nuclei in step 1) contains a suspension of at least 100×n or at least 100×m single cell nuclei; Preferably, n and m are the same or different; Preferably, n and m are the same; Preferably, n is 10 to 1000, and m is 10 to 1000.

3. The method according to claim 1, wherein In step 1), prepare the suspension containing multiple cell nuclei from a sample; Preferably, the sample contains single cells, consists essentially of single cells or consists of single cell nuclei; Preferably, the sample contains: at least 100,000, 200,000, 300,000, 500,000, 800,000 or 1,000,000 single cells or single cell nuclei; Preferably, nucleosome disruption is performed after step 1); Preferably, after step 5) or step 6), bisulfite treatment, linear amplification and / or PCR amplification are performed; Preferably, after step 5) or step 6), the cell nuclei with m types of second round tags are mixed, and then the bisulfite treatment, linear amplification and / or PCR amplification are performed; Preferably, the linear amplification includes 1 to 10 cycles; Preferably, before performing the bisulfite treatment, linear amplification and / or PCR amplification, the cell nuclei with m types of second round tags are sorted into q digestion reaction chambers according to the target cell number, and the cell nuclei are digested; Preferably, 10 to 10,000 single cell nuclei are sorted into one of the digestion reaction chambers.

4. The method according to claim 1, wherein In step 3), the suspension of the multiple cell nuclei is evenly divided into the n reaction chambers.

5. The method according to claim 1, characterized in that The single cell sequencing library is a single cell nucleic acid methylation sequencing library; Preferably, the cell nuclei divided into the n reaction chambers in step 3) and the cell nuclei divided into the m reaction chambers in step 4) are substantially equal.

6. A method for single cell sequencing, the method comprising the following steps: 1) Prepare a suspension containing multiple single cell nuclei; 2) Fragment the chromatin DNA of the multiple cell nuclei using Tn5 transposase embedded with a universal adaptor, and add the universal adaptor to the obtained chromatin DNA fragments; 3) Dispense the suspension of the multiple cell nuclei into n reaction chambers, and add a first round of tags to the chromatin DNA fragments in each reaction chamber to obtain cell nuclei with n types of first-round tags; 4) Mix the cell nuclei with n types of first-round tags, and then dispense them into m reaction chambers, and add a second-round tag sequence to the cell nuclei in each reaction chamber to obtain cell nuclei with m types of second-round tags; 5) Use the cell nuclei with m types of second-round tags to construct a single-cell sequencing library; 6) Optionally, repeat step 3) and step 4); 7) Perform bisulfite treatment.

7. The method according to claim 6, wherein The suspension containing multiple single cell nuclei in step 1) contains a suspension of at least 100×n or at least 100×m single cell nuclei; Preferably, the n and the m are the same or different; Preferably, the n and the m are the same; Preferably, the n is 10 to 1000, and the m is 10 to 1000.

8. The method according to claim 6, characterized in that Prepare the suspension containing multiple cell nuclei from the sample in step 1); Preferably, the sample contains single cells, consists essentially of single cells or consists of single cell nuclei; Preferably, the sample contains: at least 100,000, 200,000, 300,000, 500,000, 800,000 or 1,000,000 single cells or single cell nuclei; Preferably, nucleosome disruption is performed after step 1); Preferably, after step 7), linear amplification and / or PCR amplification are performed; Preferably, after step 5) or step 6), the cell nuclei with m types of second-round tags are mixed, and then bisulfite treatment, linear amplification and / or PCR amplification are performed; Preferably, the linear amplification includes 1 to 10 cycles; Preferably, before performing the bisulfite treatment, linear amplification and / or PCR amplification, the cell nuclei with m types of second-round tags are sorted into q digestion reaction chambers according to the target cell number, and the cell nuclei are digested; Sort 100 to 10,000 single cell nuclei into one of the digestion reaction chambers.

9. The method according to claim 6, wherein In step 3), the suspension of the multiple cell nuclei is evenly dispensed into the n reaction chambers; Preferably, the single-cell sequencing library is a single-cell nucleic acid methylation sequencing library; Preferably, the cell nuclei dispensed into the n reaction chambers in step 3) and the cell nuclei dispensed into the m reaction chambers in step 4) are substantially equal.

10. Use of the method according to any one of claims 1 to 5 or the method according to any one of claims 6 to 9 in one or more of the following: (a) Determining the methylation level of single cells or cell populations; (c) Quantifying the transcription of genes; (d) Identifying the cell types and states of complex heterogeneous tissues; (e) Identifying endogenous and exogenous dsDNA and ssDNA; (f) Determining the epigenetic modifications of single cells or cell populations, where the epigenetic modifications include DNA methylation.

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