Three-dimensional spatial omics method for tissues based on three-coordinate spatial positioning

Abstract

The present invention discloses a method for three-dimensional spatial omics of tissues based on three-dimensional coordinate spatial positioning. The method includes performing XYZ three-dimensional spatial encoding on analytes such as DNA, RNA, proteins, chromatin, and sugars in tissue cells through multiple rounds of DNA ligation reactions, and then analyzing the corresponding spatial positions and biological information through sequencing technology, thereby realizing three-dimensional spatial omics analysis of tissues. The method for three-dimensional spatial omics of tissues based on three-dimensional coordinate spatial positioning of the present invention has the advantages of convenient operation, low technical threshold, low cost, etc., can obtain the three-dimensional spatial distribution of heterogeneous cells in intact tissues and the expression of corresponding biological information, and is widely applied to research in the directions of neuroscience, pathology, tissue development, etc.

Description

Technical Field

[0001] The present invention relates to a three-dimensional spatial omics method for tissues based on three-dimensional coordinate space positioning. Specifically, it relates to a method for indirectly or directly performing three-dimensional spatial encoding on analytes such as DNA, RNA, proteins, chromatin, and sugars in tissue cells through multiple rounds of DNA ligation reactions, and then obtaining the spatial positions and biological information in the corresponding tissue cells through sequencing, thereby realizing three-dimensional spatial omics analysis of tissues. Background Art

[0002] In multicellular organisms, different cells have complex spatial distributions and interactions in tissues, forming a functional, structured, and dynamically balanced ecological system. When the living body is healthy, tissues maintain homeostasis through the combined actions of multiple cells and undergo dynamic division of labor among different parenchymal cells and accessory cells. When a disease occurs, along with changes in the composition and structure of tissue cells, the occurrence of tissue dysfunction usually spans multiple types of cells. The histological morphology of cells and molecules at different scales usually reflects their combined functions, and analyzing the relationship between tissue structure and function is the cornerstone of research in tissue biology and pathology.

[0003] Traditional cell analysis methods generally measure the gene expression information of multiple cells in large batches, thereby masking the differences in gene expression between cells and losing key information. In recent years, single-cell sequencing technology has developed greatly. It can study the heterogeneity of single-cell gene expression but lacks the positional information of cell spatial distribution. To solve this problem, spatial omics has emerged. Researchers' combined analysis of the spatial positions and gene expression information of tissue cells can provide important information for studying cell functions, interactions between cells, and the relationship between tissue microenvironment and cell positions.

[0004] Since the development of spatial omics technology, it has been preliminarily applied in the fields of neuroscience, pathology, tissue development, etc. Currently, spatial omics is roughly divided into four types based on laser capture microdissection (LCM), fluorescence in situ hybridization (FISH), fluorescence in situ sequencing (FISSEQ), and in situ capture. Specifically, the laser microdissection method uses a physical laser cutting method to obtain cells at different sites of tissues. Although it can conduct three-dimensional spatial omics research, the experimental operation is complex, the instrument requirements are high, and the analysis throughput is low. The fluorescence in situ hybridization method is based on multiple rounds of hybridization imaging to determine the gene information expression in cells. However, it targets specific gene sequences, so it is necessary to pre-synthesize targeted probes, which has the disadvantages of complex experimental procedures, long time consumption, limited analysis throughput, and high costs. Compared with fluorescence in situ hybridization, fluorescence in situ sequencing can use non-targeted probes to bind to gene sequences for in situ sequencing, but there are still problems such as complex operation, high cost, limited analysis throughput, and short sequencing read sequences. Since 2016, spatial transcriptomics methods based on in situ capture have emerged one after another. Its basic principle is to use a dot matrix with polyT sequences to capture tissue mRNA, combined with next-generation sequencing technology to achieve high-throughput spatial transcriptomics research. Compared with the first three types of methods, the spatial omics sequencing technology based on in situ capture is more straightforward to operate, but it is mostly used for two-dimensional spatial omics analysis of tissues. Different organs and tissues have unique three-dimensional cell distributions and gene expression maps. Conducting convenient, high-coverage, and low-cost three-dimensional spatial omics research on tissues poses great technical challenges and important scientific significance. The key point of three-dimensional spatial omics lies in how to break through the two-dimensional cell research and comprehensively analyze the spatial distribution, interaction, and gene and protein expression of different cells at the three-dimensional level of tissues. Therefore, the present invention proposes a three-dimensional spatial omics method for tissues based on three-coordinate spatial positioning, aiming to achieve the analysis of three-dimensional spatial coordinates of cells, gene recognition of spatial expression patterns, and cell-cell interaction analysis, thereby promoting the application of three-dimensional spatial omics technology in the biomedical field. Summary of the Invention

[0005] The present invention designs a three-dimensional spatial omics method for tissues based on three-coordinate spatial positioning. Through multiple rounds of DNA ligation reactions, three-dimensional spatial encoding of analytes such as DNA, RNA, proteins, chromatin, and sugars in tissue cells is carried out indirectly or directly, and then the spatial positions and biological information of the corresponding tissue cells are obtained through sequencing, realizing three-dimensional spatial omics analysis of tissues.

[0006] The present invention is realized through the following solutions:

[0007] A three-dimensional spatial omics method based on three-dimensional coordinate space positioning. This method indirectly or directly performs three-dimensional spatial encoding on the analytes of cells in tissues through multiple rounds of DNA ligation reactions, and then obtains the spatial positions and biological information of cells in the corresponding tissues through sequencing, realizing three-dimensional spatial omics analysis of tissues.

[0008] The specific steps of indirectly performing three-dimensional spatial encoding on the analytes in tissue cells through multiple rounds of DNA ligation reactions are as follows:

[0009] (1) Provide L substrates, and perform physical or chemical modification on the surface of the substrates to generate groups that can be connected to the 5' end of oligonucleotides.

[0010] (2) Provide L first-group positioning oligonucleotides, named Z-DNA, where the 5' end of Z-DNA is modified with specific groups and is connected to the modified groups on the substrate surface.

[0011] (3) Connect the L types of Z-DNA to the L substrates respectively to fabricate L substrates with Z-DNA encoding.

[0012] (4) Provide splint oligonucleotide 1 that hybridizes with the 3' end of Z-DNA.

[0013] (5) Provide M second-group positioning oligonucleotides, named X-DNA, where X-DNA contains a sequence that is substantially complementary to a part of splint oligonucleotide 1, and the 5' end is modified with a phosphate group and can be connected to the 3' end of Z-DNA.

[0014] (6) Anneal and hybridize the M types of X-DNA with splint oligonucleotide 1, and connect them to the 3' end of Z-DNA along the X-axis direction to form M rows of X-DNA barcodes.

[0015] (7) Provide splint oligonucleotide 2 that hybridizes with the 3' end of X-DNA.

[0016] (8) Provide N third-group positioning oligonucleotides, named Y-DNA, where Y-DNA contains a sequence that is substantially complementary to a part of splint oligonucleotide 2, and the 5' end is modified with a phosphate group and can be connected to the 3' end of X-DNA.

[0017] (9) Anneal and hybridize the N types of Y-DNA with splint oligonucleotide 2, and connect them to the 3' end of X-DNA along the Y-axis direction to form N columns of Y-DNA barcodes, thereby generating an L-type three-dimensional spatial encoding array of M*N.

[0018] (10) Take L continuously cut tissue samples and contact them with the L-type three-dimensional spatial encoding arrays of M*N generated by the above method respectively.

[0019] (11) Release the analyte in the tissue sample and specifically bind it to the capture domain on the Y-DNA;

[0020] (12) Construct a sequencing library and perform next-generation sequencing on the machine to identify the analyte specifically bound to the capture domain and its spatial location information;

[0021] (13) Use the corresponding bioinformatics algorithm to obtain the two-dimensional coordinates (x i , y j ) based on the X-DNA coding and Y-DNA coding positions on each substrate for the obtained sequencing data, and obtain the coordinate zk based on the Z-DNA coding on different substrates, thereby obtaining the three-dimensional coordinates (x i , y j , z k ) of the analyte, so as to determine the spatial position of the analyte in the tissue sample and reconstruct the three-dimensional spatial analysis map of the tissue.

[0022] The substrate described in step (1) is one of a silicon wafer, a glass slide, a coverslip, indium tin oxide conductive glass, and a polymethyl methacrylate sheet.

[0023] In step (1), the surface of the substrate is physically modified or chemically modified to generate a group that can be linked to the 5'-end of the oligonucleotide. The physical modification includes one of etching, evaporation coating, and spin coating, and the chemical modification includes one of in-situ synthesis, covalent coupling, molecular assembly, and affinity interaction.

[0024] In step (1), a group that can be linked to the 5'-end of the oligonucleotide is generated, and the group includes one of succinimide, maleimide, thiol, amino group, aldehyde group, epoxy group, macrocyclic alkyne, and azide.

[0025] In step (2), the 5'-end of the first group of positioning oligonucleotide Z-DNA is modified with a specific group and linked to the group modified on the substrate surface. The group includes one of NHS, maleimide, thiol, amino group, aldehyde group, epoxy group, macrocyclic alkyne, and azide.

[0026] In step (2), the L types of first group of positioning oligonucleotide Z-DNA contain the spatial barcode Z and two constant sequences. The two constant sequences include the first constant sequence and the second constant sequence. The spatial barcodes Z of the Z-DNA on different substrates are different, and there are L types of spatial barcodes Z in total. The first constant sequence is a PCR amplification adapter, and the second constant sequence is complementary to a partial sequence of the splint oligonucleotide 1 described in step (4).

[0027] In step (3), the L types of Z-DNA are respectively linked to the L types of substrates, and the linking is one of covalent coupling, molecular assembly, and affinity interaction.

[0028] The second set of positioning oligonucleotide X-DNA described in step (5) contains a spatial barcode X, and the spatial barcode X of each row of X-DNA is the same, forming M rows of X-DNA barcodes.

[0029] The M types of second set of positioning oligonucleotide X-DNA described in step (5) also contain two constant sequences, a third constant sequence and a fourth sequence. The third constant sequence is complementary to a partial sequence of the splint oligonucleotide 1 described in step (4), and the fourth constant sequence is complementary to a partial sequence of the splint oligonucleotide 2 described in step (7).

[0030] In step (6), annealing and hybridizing the M types of X-DNA with the splint oligonucleotide 1, and connecting them to the 3'-end of Z-DNA along the X-axis direction. The connection method is a DNA ligation reaction, connecting the 5'-end phosphate group of X-DNA with the 3'-end hydroxyl group of Z-DNA respectively, and X-DNA needs to be added to the corresponding position by means of a microfluidic chip, a bioarrayer or a micro-well array.

[0031] The third set of positioning oligonucleotide Y-DNA described in step (8) contains a spatial barcode Y, and the spatial barcode Y of each column of Y-DNA is the same, forming N columns of Y-DNA barcodes.

[0032] The N types of third set of positioning oligonucleotide Y-DNA described in step (8) also contain a unique molecular identifier, a constant sequence and a capture domain. The constant sequence is complementary to a partial sequence of the splint oligonucleotide 2 described in step (7).

[0033] The capture domain of the Y-DNA described in step (8) is a specific sequence, preferably polyT.

[0034] In step (9), annealing and hybridizing the N types of Y-DNA with the splint oligonucleotide 2, and connecting them to the 3'-end of X-DNA along the Y-axis direction. The connection method is a DNA ligation reaction, connecting the 5'-end phosphate group of Y-DNA with the 3'-end hydroxyl group of X-DNA respectively, and Y-DNA needs to be added to the corresponding position by means of a microfluidic chip, a bioarrayer or a micro-well array.

[0035] The encoding of the M types of X-DNA and the encoding of the N types of Y-DNA described in step (9) achieve M*N two-dimensional spatial encodings. Combining with L types of Z-DNA, finally form L types of M*N three-dimensional spatial encoding arrays.

[0036] The encoding of the M types of X-DNA and the encoding of the N types of Y-DNA described in step (9) achieve M*N two-dimensional spatial encodings, and the resolution of the spatial encoding array is 100μm - 0.5μm.

[0037] In step (9), L is any integer between 1 and 100, and M and N are any integers between 10 and 1000.

[0038] The tissue sample described in step (10) is a paraffin or fresh frozen tissue section.

[0039] The L-block tissue sections described in step (10) have the same thickness, which is 5 μm - 50 μm, and the tissue sections are sequentially adhered to the corresponding spatially encoded array substrates in the up-and-down order.

[0040] The analytes in the tissue sample described in step (11) include one or several of DNA, RNA, protein, chromatin, and carbohydrates.

[0041] Releasing the analytes in the tissue sample described in step (11) includes permeabilizing the tissue using a protease or a surfactant, preferably pepsin.

[0042] When spatially localizing DNA or chromatin open regions for releasing the analytes in the tissue sample described in step (11), it is also necessary to use a transposase to cut the DNA double strand, insert a linker, and make it bind to the capture domain of the spatially encoded array.

[0043] When spatially localizing RNA for releasing the analytes in the tissue sample described in step (11), after permeabilizing the tissue, the polyT on the spatially encoded array Y-DNA can be directly hybridized with the 3'-terminal polyA tail of mRNA.

[0044] When spatially localizing protein for releasing the analytes in the tissue sample described in step (11), before permeabilizing the tissue, multiple DNA-conjugated antibodies can be pre-incubated with the tissue to make them bind to different proteins; the DNA-conjugated antibody consists of an antibody, a PCR amplification linker, an antibody code, and a 3'-terminal constant sequence, where the constant sequence is complementary to the capture domain of the Y-DNA described in step (9), and the constant sequence is a specific DNA sequence.

[0045] When spatially localizing protein for releasing the analytes in the tissue sample described in step (11), after co-incubating the tissue with the DNA-conjugated antibody, the excess DNA-conjugated antibody is washed away, and the tissue is continuously permeabilized to release the DNA-conjugated antibody, and the capture domain on the spatially encoded array Y-DNA is used to hybridize the constant sequence at the 3'-end of the DNA-conjugated antibody.

[0046] When spatially localizing carbohydrates for releasing the analytes in the tissue sample described in step (11), before permeabilizing the tissue, multiple DNA-conjugated lectins can be pre-incubated with the tissue to make them bind to different carbohydrates; the DNA-conjugated lectin consists of a lectin, a PCR amplification linker, a lectin code, and a 3'-terminal constant sequence, where the constant sequence is complementary to the capture domain of the Y-DNA described in (9), and the constant sequence is a specific DNA sequence.

[0047] For the release of the analyte in the tissue sample in step (11), if spatial localization of saccharides is required, after co-incubating the tissue with DNA-conjugated lectin, the excess DNA-conjugated lectin is washed away, and the tissue is further permeabilized to release the DNA-conjugated lectin. The capture domain on the spatially encoded array Y-DNA hybridizes with the constant sequence at the 3' end of the DNA-conjugated lectin.

[0048] For the construction of the sequencing library in step (12) and performing next-generation sequencing on the machine to identify the analyte that specifically binds to the capture domain and its spatial position information, spatial genomics, transcriptomics, proteomics, epigenomics, and glycomics sequencing can be performed separately, or spatial multi-omics sequencing can be achieved by combining genomics and transcriptomics, or transcriptomics and proteomics.

[0049] For the construction of the sequencing library in step (12) and performing next-generation sequencing on the machine to identify the analyte that specifically binds to the capture domain and its spatial position information, the specific steps include tissue section fixation, permeabilization to release tissue analytes, analyte capture, reverse transcription, collection and amplification of cDNA, and library construction using a DNA library construction kit followed by sequencing on the machine.

[0050] For the development of the corresponding tissue informatics algorithm in step (13) to determine the spatial position of the analyte in the tissue sample and reconstruct the three-dimensional spatial analysis map of the tissue, including analyzing the sequencing library, comparing the measured ZXY spatial barcodes with the pre-designed spatial barcode library, obtaining the two-dimensional coordinates (xi, yj) based on the X-DNA and Y-DNA spatial barcodes, and obtaining the spatial coordinates zk of different tissue layers based on the Z-DNA spatial barcode, thereby obtaining the three-dimensional coordinates (xi, yj, zk) of the tissue analyte; combining the DNA library, cDNA library, antibody coding, and lectin coding to achieve spatial genomics, transcriptomics, proteomics, epigenomics, glycomics, and spatial multi-omics sequencing analysis.

[0051] The three-dimensional spatial encoding of the analyte in cells in the tissue is directly performed through multiple rounds of DNA ligation reactions, specifically including the following steps:

[0052] (1) Tissue clearing, in-situ retention and fixation of the analyte in the tissue, and removal of lipids;

[0053] (2) Developing a cube "Rubik's Cube" spatial tissue sequencing system for delivering DNA-encoded probes; the front, side, and top of the cube "Rubik's Cube" are respectively integrated with parallel loading microchannels and corresponding sample interfaces. The inside of the channels is filled with gel for pre-enrichment of the encoded probes in the channels, and microelectrodes are embedded inside the corresponding two sides to apply an electric field to drive the directional movement of the encoded probes in the cleared tissue.

[0054] (3) Provide M first-group positioning oligonucleotides, named X-DNA, where the 3'-end of X-DNA is a capture domain for binding tissue analytes, and the 5'-end is modified with a phosphate group for DNA ligation reaction;

[0055] (4) Mix the M X-DNAs with the gel and pre-enrich them into different loading microchannels in front of the cube "magic cube" respectively, with one X-DNA corresponding to one loading microchannel;

[0056] (5) Provide splint oligonucleotide 1 that hybridizes with the 5'-end of X-DNA, and the 5'-end contains a sequence that is substantially complementary to a part of X-DNA;

[0057] (6) Provide N second-group positioning oligonucleotides, named Y-DNA, where Y-DNA contains a sequence that is substantially complementary to a part of splint oligonucleotide 1, and the 5'-end is modified with a phosphate group;

[0058] (7) Pre-anneal and hybridize the N Y-DNAs with splint oligonucleotide 1, mix the annealed and hybridized products with the gel, and pre-enrich them into different loading microchannels on the side of the cube "magic cube" respectively, with one Y-DNA corresponding to one loading microchannel;

[0059] (8) Provide splint oligonucleotide 2 that hybridizes with the 5'-end of Y-DNA, and the 5'-end contains a sequence that is substantially complementary to a part of Y-DNA;

[0060] (9) Provide L third-group positioning oligonucleotides, named Z-DNA, where Z-DNA contains a sequence that is substantially complementary to a part of splint oligonucleotide 2;

[0061] (10) Pre-anneal and hybridize the L Z-DNAs with splint oligonucleotide 2, mix the annealed and hybridized products with the gel, and pre-enrich them into different loading microchannels on the top of the cube "magic cube" respectively, with one Z-DNA corresponding to one loading microchannel;

[0062] (11) Use three-axis microchannel electrophoresis to accurately deliver the X-DNA, the hybridization product of Y-DNA and splint oligonucleotide 1, and the hybridization product of Z-DNA and splint oligonucleotide 2 into the three-dimensional tissue with gel transparency treatment along the channel direction. Among them, X-DNA binds to tissue analytes through the capture domain, Y-DNA is sequentially ligated to the 5'-end of X-DNA along the inherent direction under the action of DNA ligase, and Z-DNA is sequentially ligated to the 5'-end of Y-DNA along the inherent direction under the action of DNA ligase, combining to form a unique XYZ three-coordinate space code;

[0063] (12) Construct a sequencing library and perform next-generation sequencing on the machine to identify the analytes specifically binding to the capture domain and their spatial position information;

[0064] (13) Develop corresponding bioinformatics algorithms. For the obtained sequencing data, obtain the three-dimensional coordinates (xi, yj, zk) of the analyte according to the coding positions of X-DNA, Y-DNA, and Z-DNA, so as to determine the spatial position of the analyte in the tissue sample and reconstruct the three-dimensional spatial analysis map of the tissue.

[0065] The tissue clearing described in step (1) includes three tissue clearing methods: oily, hydrogel-based, and aqueous.

[0066] The tissue clearing described in step (1) in-situ retains and fixes the analyte in the tissue and removes lipids. It includes soaking the sample with a reactant solution for forming a hydrogel, so that the biomolecules in the tissue are connected to the gel monomer mediated by formaldehyde to achieve the retention and fixation of the tissue analyte; at the same time, thermally initiate the gel polymerization of the tissue, and then clear the lipid components in the tissue through electrophoresis to achieve tissue clearing; the reactants for forming the hydrogel include acrylamide, bisacrylamide, formaldehyde, and a thermal trigger.

[0067] The development of the cubic "magic cube" spatial tissue sequencing system described in step (2) for delivering DNA-encoded probes includes mold casting, metal stamping, plastic molding, and 3D printing.

[0068] The front, side, and top of the cubic "magic cube" described in step (2) are respectively integrated with parallel sample loading microchannels and corresponding sample interfaces. The front, side, and top are respectively set as the X-axis, Y-axis, and Z-axis of the corresponding tissue, and are respectively used for the sample loading of X-DNA, Y-DNA, and Z-DNA.

[0069] The front, side, and top described in step (2) are integrated with parallel sample loading microchannels, which respectively have M, N, and L channels, and M, N, and L are any integers between 5 and 1000.

[0070] The front, side, and top described in step (2) are integrated with parallel sample loading microchannels, and the channel width is 100 μm - 0.5 μm.

[0071] The inside of the channels described in step (2) is filled with a gel for pre-enrichment of the encoded probes in the channels. The gel includes agarose hydrogel, gelatin hydrogel, saccharide hydrogel, hyaluronic acid hydrogel, dextran hydrogel, and copolymer hydrogel.

[0072] The M types of first-group positioning oligonucleotide X-DNAs described in step (3) contain a spatial barcode X, a constant sequence, and a capture domain. The spatial barcodes X of X-DNAs in different channels are different, with a total of M types of spatial barcodes X. The constant sequence is complementary to a partial sequence of the splint oligonucleotide 1 described in (5). The capture domain is a specific sequence or a polyT sequence, preferably polyT.

[0073] The first-group positioning oligonucleotide X-DNAs described in step (4) contain a spatial barcode X. The spatial barcode X of each row of X-DNAs is the same, forming M rows of X-DNA barcodes.

[0074] The N types of second-group positioning oligonucleotide Y-DNAs described in step (6) contain a spatial barcode Y and two constant sequences. One constant sequence is complementary to a partial sequence of the splint oligonucleotide 1 described in step (5), and the other constant sequence is complementary to a partial sequence of the splint oligonucleotide 2 described in step (8).

[0075] The second-group positioning oligonucleotide Y-DNAs described in step (7) contain a spatial barcode Y. The spatial barcode Y of each column of Y-DNAs is the same, forming N columns of Y-DNA barcodes.

[0076] The L types of third-group positioning oligonucleotide Z-DNAs described in step (9) contain a spatial barcode Z, a constant sequence, and a PCR amplification linker. The constant sequence is complementary to a partial sequence of the splint oligonucleotide 2 described in (8).

[0077] The third-group positioning oligonucleotide Z-DNAs described in step (10) contain a spatial barcode Z. The spatial barcode Z of each vertical column of Z-DNAs is the same, forming L vertical columns of Z-DNA barcodes.

[0078] In step (11), the three-axis microchannel electrophoresis is used to accurately deliver the hybridization products of X-DNA, Y-DNA and splint oligonucleotide 1, and the hybridization products of Z-DNA and splint oligonucleotide 2 into the three-dimensional tissue subjected to gel clearing treatment along the channel direction; the M types of X-DNAs enter the tissue along the X-axis under the drive of voltage, and their 3'-end capture domains bind to the analyte. The hybridization products of the N types of Y-DNAs and splint oligonucleotide 1 enter the tissue along the Y-axis under the drive of voltage, and the 3'-end hydroxyl group is ligated to the 5'-end phosphate group of X-DNA through T4 DNA ligase. The L types of Z-DNAs and splint oligonucleotide 2 enter the tissue along the Z-axis under the drive of voltage, and the 3'-end hydroxyl group is ligated to the 5'-end phosphate group of Y-DNA through T4 DNA ligase.

[0079] The orthogonal ligation of the M types of X-DNAs encoding, the N types of Y-DNAs encoding, and the L types of Z-DNAs in step (11) realizes M*N*L three-dimensional spatial encodings.

[0080] The tissue described in step (11) is fresh or fresh frozen tissue.

[0081] The thickness of the tissue described in step (11) is 50 - 5000 μm.

[0082] The analyte in the tissue sample described in step (11) includes one or several of DNA, RNA, protein, chromatin, and carbohydrates.

[0083] To release the analyte in the tissue sample in step (11), if spatial localization of DNA or chromatin open regions is required, a transposase is also needed to cut the double-stranded DNA and insert adapters, so that it binds to the capture domain of X-DNA.

[0084] To release the analyte in the tissue sample in step (11), if spatial localization of RNA is required, the polyT on X-DNA can be directly used to hybridize with the 3'-end polyA tail of mRNA.

[0085] To release the analyte in the tissue sample in step (11), if spatial localization of protein is required, multiple DNA-conjugated antibodies can be pre-incubated with the tissue to bind to different proteins; the DNA-conjugated antibody consists of an antibody, a PCR amplification adapter, an antibody coding sequence, and a 3'-end constant sequence, where the constant sequence is complementary to the capture domain of X-DNA described in step (3), and the constant sequence is a specific DNA sequence or polyA.

[0086] To release the analyte in the tissue sample in step (11), if spatial localization of protein is required, after co-incubating the tissue with the DNA-conjugated antibody, the excess DNA-conjugated antibody is washed away, and voltage is used to drive X-DNA into the tissue, and the capture domain on X-DNA is used to hybridize with the constant sequence at the 3'-end of the DNA-conjugated antibody.

[0087] To release the analyte in the tissue sample in step (11), if spatial localization of carbohydrates is required, multiple DNA-conjugated lectins can be pre-incubated with the tissue to bind to different carbohydrates; the DNA-conjugated lectin consists of a lectin, a PCR amplification adapter, a lectin coding sequence, and a 3'-end constant sequence, where the constant sequence is complementary to the capture domain of X-DNA described in step (3), and the constant sequence is a specific DNA sequence or polyA.

[0088] To release the analyte in the tissue sample in step (11), if spatial localization of carbohydrates is required, after co-incubating the tissue with the DNA-conjugated lectin, the excess DNA-conjugated lectin is washed away, and voltage is used to drive X-DNA into the tissue, and the capture domain on X-DNA is used to hybridize with the constant sequence at the 3'-end of the DNA-conjugated lectin.

[0089] In step (12), the sequencing library is constructed and subjected to next-generation sequencing to identify the analytes specifically binding to the capture domain and their spatial position information. It can not only perform spatial genomics, transcriptomics, proteomics, epigenomics, and glycomics sequencing alone, but also achieve spatial multi-omics sequencing by combining genomics and transcriptomics, transcriptomics and proteomics, etc.

[0090] In step (12), the sequencing library is constructed and subjected to next-generation sequencing to identify the analytes specifically binding to the capture domain and their spatial position information. The specific steps include analyte capture, reverse transcription, collection and amplification of cDNA, library construction using a DNA library construction kit, and sequencing on the machine.

[0091] In step (13), the corresponding bioinformatics algorithm is developed to determine the spatial position of the analytes in the tissue sample and reconstruct the three-dimensional spatial analysis map of the tissue, including analyzing the sequencing library, comparing the measured XYZ spatial barcodes with the pre-designed spatial barcode library, and obtaining the three-dimensional coordinates (xi, yj, zk) according to the X-DNA, Y-DNA, and Z-DNA spatial barcodes; combining the DNA library, cDNA library, antibody coding, and lectin coding to achieve spatial genomics, transcriptomics, proteomics, epigenomics, glycomics, and spatial multi-omics sequencing analysis.

[0092] The beneficial effects of the present invention are as follows:

[0093] Existing in-situ capture-based spatial omics is mostly used for two-dimensional spatial omics analysis of tissues. Different organs and tissues have unique three-dimensional cell distributions and gene expression maps. Conducting convenient, high-coverage, and low-cost three-dimensional spatial omics research on tissues poses great technical challenges and important scientific significance. The three-dimensional spatial omics method for tissues based on three-coordinate spatial positioning in the present invention indirectly or directly performs three-dimensional spatial encoding on analytes such as DNA, RNA, proteins, chromatin, and carbohydrates in tissue cells through multiple rounds of DNA ligation reactions, and then obtains the spatial positions and biological information of cells in the corresponding tissues through sequencing, and can simply and conveniently achieve three-dimensional spatial omics analysis of tissues.

[0094] The present invention is easy to operate, has a low technical threshold, and is low in cost. It can obtain the three-dimensional spatial distribution and biological information of tissues, and can be widely applied to the applications and research in neuroscience, pathology, and tissue development. The method of the present application retains the original position information of cells in tissues and constructs an expression library of the complete three-dimensional spatial biology information of tissues, realizing high-throughput and high-coverage spatial omics sequencing. This method can be used for the analysis of three-dimensional spatial coordinates of cells, gene recognition of spatial expression patterns, and analysis of cell-cell interactions, and has significant significance for biomedical fields such as neuroscience, tumor microenvironment research, and tissue and organ development.

[0095] The present invention also has the following advantages:

[0096] 1. By preparing three-dimensional spatial coding sequences through multiple rounds of DNA ligation reactions, the types of DNA coding sequences are significantly reduced, effectively reducing the experimental cost.

[0097] 2. There are various types of DNA barcodes with wide applicability, which can be used for three-dimensional coding of analytes such as tissue cell mRNA, DNA, protein, chromatin, and carbohydrates, thereby realizing spatial genomics, transcriptomics, proteomics, epigenomics, glycomics, and spatial multi-omics sequencing analysis.

[0098] 3. The spatial barcodes of the XYZ spatial coding sequences are known. Compared with spatial transcriptomics sequencing methods such as slide-seq, slide-seqV2, and stereo-seq that require pre-decoding to obtain spatial positioning, etc., the operation is simpler and it is easier to trace back the spatial positions of tissue analytes.

[0099] 4. By constructing a ZXY three-dimensional spatial coding glass slide to indirectly capture tissue analytes for three-dimensional spatial multi-omics research, the method is simple and convenient to operate, reducing the technical threshold and facilitating the realization of a process-based and automated spatial multi-omics research.

[0100] 5. Based on the 3D integrated technology, a cube "magic cube" sequencing platform is printed, which is compatible with tissue gel clearing and the construction of sequencing libraries such as mRNA, protein, and genome. It also enables the automation and integration of spatial tissue sequencing, improving the operation efficiency.

[0101] 6. Based on the three-axis microchannel electrophoresis technology, three types of coding probes, namely X-DNA, Y-DNA, and Z-DNA, are accurately delivered into the tissue, which can effectively capture tissue analytes, improve the capture efficiency, and achieve high-coverage spatial multi-omics research. BRIEF DESCRIPTION OF THE DRAWINGS

[0102] Figure 1 It is a schematic flow diagram of modifying the 3D amino group on the glass slide surface;

[0103] Figure 2 It is a schematic diagram of the preparation of a tissue three-dimensional spatial positioning coding glass slide;

[0104] Figure 3 It is a diagram of a microfluidic chip and fixture for spatial coding;

[0105] Figure 4 It is a dot matrix result diagram of realizing ZXY three-dimensional coding based on two rounds of DNA ligation reactions;

[0106] Figure 5 It is a schematic flow diagram of positioning tissue mRNA for three-dimensional spatial transcriptomics sequencing;

[0107] Figure 6 It is a diagram of the results of three-dimensional spatial transcriptomics sequencing of mouse olfactory bulb tissue;

[0108] Figure 7 It is a schematic diagram of the process for modifying the surface of the glass slide with streptavidin;

[0109] Figure 8 It is a schematic diagram of the process for localizing tissue proteins and mRNAs and simultaneously performing three-dimensional spatial proteomics and transcriptomics;

[0110] Figure 9 It is a schematic diagram of the process for localizing tissue surface carbohydrates and mRNAs and simultaneously performing three-dimensional spatial glycomics and transcriptomics;

[0111] Figure 10 It is a schematic diagram of the process for localizing tissue DNA or open chromatin regions and performing three-dimensional genomics sequencing;

[0112] Figure 11 It is a flow chart of three-dimensional in situ encoding of tissue mRNAs for spatial transcriptomics;

[0113] Figure 12 It is a diagram of the clearing results of mouse olfactory bulb tissue;

[0114] Figure 13 It is a schematic diagram of the "magic cube" three-dimensional encoding delivery system based on microfluidic DNA-encoded chip electrophoresis;

[0115] Figure 14 It is a schematic diagram of three-dimensional reconstruction and spatio-temporal information analysis of the transcriptome of intact tissue at single-cell resolution;

[0116] Figure 15 It is a schematic diagram of three-dimensional in situ encoding of tissue proteins;

[0117] Figure 16 It is a schematic diagram of three-dimensional in situ encoding of tissue carbohydrates;

[0118] Figure 17 It is a schematic diagram of three-dimensional in situ encoding of tissue DNA or chromatin accessibility. Detailed implementation manners

[0119] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0120] Example 1

[0121] In this example, RNA in tissue cells is indirectly encoded in three-dimensional space through multiple rounds of DNA ligation reactions, and then the spatial positions and gene expression information of the corresponding tissue cells are obtained through sequencing, realizing three-dimensional spatial transcriptomics analysis of tissues.

[0122] (1) Covalently modify 3D amino groups on the glass slide surface: The schematic diagram of the glass slide surface modification is as Figure 1 shown. First, activate the glass slide with piranha solution (H2O2:H2SO4 = 3:7) to expose the hydroxyl groups on the glass slide. After washing the glass slide with ultrapure water and drying it with nitrogen, add 5% 3-glycidoxypropyltrimethoxysilane (GOPTS), shake and react for 7 h to cause silanization hydrolysis reaction on the glass slide surface and connect epoxy groups. Thoroughly wash away the residual unreacted GOPTS on the surface with a large amount of absolute ethanol, dry it with nitrogen, and heat and dry it at 110 °C for 1 h to stabilize the silanization reaction. Finally, add 0.035% G4PAMAM amino dendrimer, shake and react overnight, and the epoxy and amino reactions finally form a 3D amino interface.

[0123] (2) Connect the first group of positioning oligonucleotide Z-DNA: First, custom synthesize 5 kinds of spatial coding sequences Z-DNA with amino modifications at the 5' end. The spatial barcodes Z of the 5 kinds of Z-DNA are different. Subsequently, prepare a mixed solution of 2.5 mM disuccinimidyl suberate (DSS) and 5 μM Z-DNA in a sodium phosphate buffer at pH = 7.2, quickly drop different Z-DNA onto the surfaces of different 3D amino glass slides, and react at room temperature for 30 min to allow DSS to fully crosslink the amino-modified Z-DNA and the amino groups on the glass slide surface. Finally, wash the glass slide surface with 2×SSC solution (containing 0.1% SDS) to remove unreacted and non-specifically adsorbed DNA. The final modified area of Z-DNA is about 5×5 mm 2 .

[0124] (3) Use a microfluidic chip to orthogonally ligate the second set of positioning oligonucleotides X-DNA and the third set of positioning oligonucleotides Y-DNA: To avoid non-specific electrostatic adsorption of the remaining unreacted amino groups, the glass slide needs to be blocked before the ligation reaction of X-DNA, converting the remaining amino groups into carboxyl groups. The specific experimental operation is to prepare a 0.1 g / ml succinic anhydride solution using N,N-dimethylformamide (DMF) solvent, add it to the glass slide modified with Z-DNA, and shake the reaction for 40 min. Subsequently, 50 types of X-DNA, 50 types of Y-DNA, splint oligonucleotide 1, and splint oligonucleotide 2 are custom-synthesized. Both the 5'-ends of X-DNA and Y-DNA carry phosphate groups for DNA ligation reactions, but the spatial barcodes of the 50 types of DNA are all different. Use soft lithography technology to prepare two types of polydimethylsiloxane (PDMS) microfluidic chips with 50 channels each (microfluidic chip X and microfluidic chip Y) in horizontal and vertical directions, and the channel diameters are 50 μm and 15 μm respectively. Use an acrylic fixture to assist in assembling the PDMS chip on the glass slide. Pre-anneal and hybridize 50 types of 5 μM X-DNA and 5 μM splint oligonucleotide 1. Next, uniformly mix DNA T4 ligase with the hybridization product of 50 types of X-DNA and splint oligonucleotide 1, and respectively take 1 μl and add it to different loading wells of microfluidic chip X. Use negative pressure sampling to make the sample enter different channels and ligate with Z-DNA on the bottom glass slide. After reacting at 37 °C for 30 min, add 2 ml of PBS solution to the loading area, and continuously use negative pressure sampling to wash the channels to remove the unreacted X-DNA. Remove the fixture, and use 2×SSC solution (containing 0.1% SDS) to wash the surface of the glass slide to completely remove the non-specifically adsorbed X-DNA. Finally, react with 80 mM NaOH for 1 min to remove splint oligonucleotide 1. The ligation of Y-DNA is similar to the operation of X-DNA. Similarly, pre-anneal and hybridize 50 types of 5 μM Y-DNA and 5 μM splint oligonucleotide 2. Uniformly mix DNA T4 ligase with the hybridization product of 50 types of Y-DNA and splint oligonucleotide 2, and respectively take 1 μl and add it to different loading wells of microfluidic chip Y. Use negative pressure sampling to make the sample enter different channels and ligate with X-DNA on the bottom glass slide. After reacting at 37 °C for 30 min, add 2 ml of PBS solution to the loading area, and continuously use negative pressure sampling to wash the channels to remove the unreacted Y-DNA. Remove the fixture, and use 2×SSC solution (containing 0.1% SDS) to wash the surface of the glass slide to completely remove the non-specifically adsorbed Y-DNA. Finally, react with 80 mM NaOH for 1 min to remove splint oligonucleotide 2. The schematic diagram of the three-dimensional spatial positioning encoding glass slide is as shown in Figure 2 shown, and the structures of microfluidic chip X, microfluidic chip Y, and the fixture are as shown in Figure 3 shown, and the results of the ZXY three-dimensional encoding lattices with two spatial resolutions (50 μm and 15 μm) finally obtained are as shown in Figure 4 shown.

[0125] (4) Three-dimensional spatial transcriptomics study of mouse olfactory bulb tissue: Capture the mRNA of mouse olfactory bulb tissue, and the experimental procedure for three-dimensional spatial transcriptomics is as Figure 5 shown. Take fresh mouse olfactory bulb tissue, embed it with OTC embedding agent, and quickly freeze it on dry ice. After freezing, store it in a -80°C refrigerator for long-term preservation. Take out the tissue at -80°C, transport the tissue to a cryostat (-20°C) with dry ice, and pre-equilibrate the frozen tissue for 30 minutes. Use the cryostat to cut continuous sections, each section being 10 μm thick, and sequentially adhere them to the above-mentioned three-dimensional spatially encoded glass slides. Subsequently, preheat the tissue sections at 37°C for 1 minute and fix them in ice methanol solution at -20°C for 30 minutes. After fixation, take out the glass slides, add ~200 μl of isopropanol solution to each glass slide to evenly cover the tissue surface, and perform dehydration treatment for 1 minute. Air-dry the isopropanol naturally for 5 minutes.

[0126] After that, perform hematoxylin-eosin (HE) staining on the tissue. First, add hematoxylin, evenly cover the tissue on all glass slides, and incubate at room temperature for 7 minutes. After 7 minutes, discard the hematoxylin, and repeatedly immerse the glass slides in 50 ml centrifuge tubes filled with ultrapure water 5 times in sequence, repeatedly immerse them in 1 L of ultrapure water 1 15 times, and repeatedly immerse them in 1 L of ultrapure water 2 15 times. Then wipe off the excess liquid from the back of the glass slides, add the bluing solution, evenly cover all tissue sections, and incubate at room temperature for 2 minutes. After 2 minutes, discard the bluing solution, and repeatedly immerse the glass slides in 1 L of ultrapure water 2 5 times. Finally, wipe off the excess liquid from the back of the glass slides, add eosin dilution solution (eosin: acetic acid = 1:9), evenly cover all tissue sections, and incubate at room temperature for 1 minute. After 1 minute, discard the eosin dilution solution, and repeatedly immerse the glass slides in 1 L of ultrapure water 3 15 times. Wipe off the excess liquid from the back of the glass slides, air-dry until the tissue becomes opaque, then incubate at 37°C for 5 minutes, and scan and image with an HE scanner to save the HE staining pictures.

[0127] After HE staining, the tissue sections were placed in an incubation box, and 70 μl of pepsin solution was added to each tissue section. The olfactory bulb tissue was permeabilized at 37 °C for 10 min. After permeabilization, the pepsin solution was aspirated, and the tissue was gently washed 3 times with 100 μl of 0.1× SSC solution. For each tissue section, 100 μl of reverse transcription reagent was prepared: 20 μl of 5× reverse transcription buffer, 48 μl of enzyme-free water, 20 μl of 2.5 mM dNTP, 5 μl of reverse transcriptase, 2 μl of RNase inhibitor, and 5 μl of template switching primer TSO. The reverse transcription reagent was added, and the reaction was carried out at 42 °C for 8 h to reverse transcribe mRNA into cDNA. After reverse transcription, the reverse transcription reagent was aspirated, and the tissue was gently washed 3 times with 100 μl of 0.1× SSC solution. Subsequently, 75 μl of 80 mM NaOH solution was added and reacted for 5 min, and then washed 3 times with 100 μl of EB buffer to remove the mRNA strand. Next, second-strand synthesis was performed. For each tissue section, 75 μl of second-strand synthesis mixture was prepared: 22.5 μl of enzyme-free water, 30 μl of 2.5 mM dNTP, 7.5 μl of DNA polymerase, 7.5 μl of second-strand synthesis primer, and 7.5 μl of 10× DNA polymerase buffer. The second-strand synthesis mixture was added, and the reaction was carried out at 37 °C for 10 h. After second-strand synthesis, the second-strand synthesis mixture was aspirated, and the tissue was washed 3 times with 100 μl of EB buffer solution. For each tissue section, 75 μl of exonuclease mixture was prepared: 63.73 μl of enzyme-free water, 3.75 μl of exonuclease, and 7.5 μl of exonuclease buffer. The exonuclease mixture was added, and the reaction was carried out at 37 °C for 45 min to shear the remaining unreacted single strands. After the reaction, the exonuclease mixture was aspirated, and the tissue was washed 3 times with 100 μl of 1× TE buffer. Finally, 35 μl of 80 mM NaOH was added and reacted for 10 min to collect the cDNA single strand, and 5 μL of 1 M Tris-HCl was added to neutralize the NaOH.

[0128] The collected cDNA single strands were subjected to PCR amplification using 2× Kapa Biosystems HiFi Hotstart Readymix. The reaction components included 10 μl of PCR primer, 50 μl of 2× Kapa Biosystems HiFi Hotstart Readymix, and 40 μl of cDNA. The PCR amplification program was: 98 °C for 3 min; 98 °C for 20 s, 67 °C for 20 s, 72 °C for 5 min, for X cycles (X is determined by the ct value of qPCR); 72 °C for 5 min; 4 °C ∞. After amplification, the cDNA libraries of different tissue sections were mixed, and the cDNA library was purified using 0.6X VAHTS DNA purification magnetic beads, and the sample recovery volume was 30 μl. Finally, according to Instructions for DNA Library PrepKit V2 for Illumina to construct a sequencing library.

[0129] Send the sequencing library to the company for next-generation sequencing to obtain sequencing data. Develop corresponding bioinformatics algorithms for analyzing the sequencing library, compare the barcodes in the measured spatially encoded library with the barcode library, and obtain two-dimensional coordinates (x i , y j ) according to the positions of X-DNA coding and Y-DNA coding, and obtain the coordinate z k according to the Z-DNA coding, thereby obtaining the three-dimensional coordinates (x i , y j , z k ) of the cells in the tissue. Finally, integrate and align the HE staining images, the spatially encoded library and the cDNA library in the sequencing library to obtain the spatial expression matrix of genes and perform spatial transcriptomics analysis. Figure 6 Shows the spatial gene distribution map of the mouse olfactory bulb obtained by capturing the mRNA of the mouse olfactory bulb on one 3D spatial positioning encoded glass slide and tracing the spatial position.

[0130] Example 2

[0131] In this example, the RNA in tissue cells is indirectly three-dimensionally spatially encoded through multiple rounds of DNA ligation reactions, and then the spatial positions and biological information of the cells in the corresponding tissue are obtained through sequencing, realizing three-dimensional spatial transcriptomics analysis of the tissue. In Example 2, streptavidin (SA) glass slides are used to replace the 3D amino glass slides in Example 1. Except for the SA modification method and the Z-DNA ligation method being different, other operation steps, sequences, and conditions are the same as those in Example 1.

[0132] (1) Modify the surface of the glass slide with SA: Figure 7Disclosed is a method for modifying SA on the surface of a glass slide. First, the glass slide is activated with piranha solution (H2O2:H2SO4 = 3:7) to expose the hydroxyl groups on the glass slide. After washing the glass slide with ultrapure water and drying it with nitrogen, 10% 3-mercaptopropyltrimethoxysilane (MPTS) is added, and the reaction is shaken for 5 h to cause a silanization hydrolysis reaction on the glass slide surface and connect mercapto groups. A large amount of absolute ethanol is used to thoroughly wash away the residual unreacted MPTS on the surface, dried with nitrogen, and heated and dried at 110 °C for 30 min to stabilize the silanization reaction. Then, 0.01 μM / mL 4-maleimidobutyric acid N-hydroxysuccinimide ester (GMBS) is added as a cross-linking agent, and the reaction is shaken for 30 min to connect the maleimide at one end of GMBS to the mercapto group and expose the NHS group at the other end. The residual unreacted GMBS on the surface is washed away with PBS solution. Finally, 50 μg / mL SA solution is added, and the reaction is carried out at 4 °C for 12 h. SA reacts with the NHS group to finally form an SA interface.

[0133] (2) Connecting the first group of positioning oligonucleotide Z-DNA: First, 5 kinds of spatially encoded sequences Z-DNA with biotin modification at the 5' end are custom-synthesized, and the spatial barcodes Z of the 5 kinds of Z-DNA are different. Subsequently, a mixed solution of 2 μM Z-DNA is configured in 1×PBS buffer, and different Z-DNA are quickly dropped onto the surfaces of different SA glass slides, and the reaction is carried out at room temperature for 30 min to allow the biotin-modified Z-DNA and SA on the glass slide surface to fully react. Finally, the glass slide surface is washed with 2×SSC solution (containing 0.1% SDS) to remove the unreacted and non-specifically adsorbed DNA. The final modified area of Z-DNA is about 5×5 mm 2 .

[0134] The subsequent steps are the same as those in Example 1.

[0135] Example 3

[0136] In this example, three-dimensional spatial encoding of proteins and RNAs in tissue cells is indirectly carried out through multiple rounds of DNA ligation reactions, and then the spatial positions and biological information of cells in the corresponding tissue are obtained through sequencing, realizing three-dimensional spatial proteomics and transcriptomics analysis of the tissue. In Example 3, except that additional labeling of DNA-coupled antibodies and separate purification and preparation of the proteomics sequencing library are required, other operation steps, sequences, and conditions are the same as those in Example 1.

[0137] (1) Preparation of three-dimensional spatial positioning encoding glass slides: The same as in Example 1

[0138] (2) Three-dimensional spatial proteomics and transcriptomics research on mouse olfactory bulb tissue: Schematic flow chart of localizing tissue proteins and mRNAs and simultaneously performing three-dimensional spatial proteomics and transcriptomics is as Figure 8 shown.

[0139] The HE staining and previous steps were the same as in Example 1.

[0140] After HE staining, the tissue sections were placed in an incubation box, and 70 μl of DNA-conjugated antibody solution was prepared for each tissue section: 7 μl of DNA-conjugated antibody and 63 μl of PBS buffer solution. The DNA-conjugated antibody solution was added to the incubation box and incubated at 37 °C for 1 h. After incubation, the DNA-conjugated antibody was aspirated, and the tissue sections were washed slowly 3 times with 100 μl of EB buffer solution. 70 μl of pepsin solution was added to each tissue section, and the olfactory bulb tissue was permeabilized at 37 °C for 10 min. After permeabilization, the pepsin solution was aspirated, and the tissue sections were washed slowly 3 times with 100 μl of 0.1× SSC solution. Then, second-strand synthesis was performed. 75 μl of second-strand synthesis mixture was prepared for each tissue section: 30 μl of enzyme-free water, 30 μl of 2.5 mM dNTP, 7.5 μl of DNA polymerase, and 7.5 μl of 10× DNA polymerase buffer. The second-strand synthesis mixture was added and reacted at 37 °C for 10 h. After second-strand synthesis, the second-strand synthesis mixture was aspirated, and the tissue sections were washed 3 times with 100 μl of EB buffer solution. 75 μl of exonuclease mixture was prepared for each tissue section: 63.73 μl of enzyme-free water, 3.75 μl of exonuclease, and 7.5 μl of exonuclease buffer. The exonuclease mixture was added and reacted at 37 °C for 45 min to shear the remaining unreacted single strands. After the reaction, the exonuclease mixture was aspirated, and the tissue sections were washed 3 times with 100 μl of 1× TE buffer. Finally, 35 μl of 80 mM NaOH was added and reacted for 10 min to collect the DNA single strands, and 5 μL of 1 M Tris-HCl was added to neutralize the NaOH.

[0141] The collected DNA single strands were subjected to PCR amplification using 2× Kapa Biosystems HiFi Hotstart Readymix. The reaction components included 10 μl of PCR primer, 50 μl of 2× Kapa Biosystems HiFi Hotstart Readymix, and 40 μl of DNA. The amplification program was: 98 °C for 3 min; 98 °C for 20 s, 67 °C for 20 s, 72 °C for 5 min, for X cycles (X is determined by the ct value of qPCR); 72 °C for 5 min; 4 °C ∞. After amplification, the DNA libraries of different tissue sections were mixed, and 0.6× VAHTS DNA purification magnetic beads were used to sort the cDNA sequencing library, and the sample recovery volume was 30 μl. The supernatant was collected, and 1.4× VAHTS DNA purification magnetic beads were used to sort the protein sequencing library, and the sample recovery volume was also 30 μl. Finally, according to the instructions of the Small RNA LibraryPrep Kit for Illumina, the protein sequencing library was constructed. According to Instructions for the DNA Library Prep Kit V2 for Illumina to construct a gene sequencing library. The sequencing library was sent to the company for next-generation sequencing to obtain sequencing data.

[0142] Develop corresponding bioinformatics algorithms for analyzing the sequencing library. Compare the barcodes in the measured spatially encoded library with the barcode library, and obtain two-dimensional coordinates (x i , y j ) according to the positions of X-DNA coding and Y-DNA coding, and obtain the coordinate z k according to the Z-DNA coding. Thus, the three-dimensional coordinates (x i , y j , z k ) of the cells in the tissue are obtained. Finally, compare the HE staining pictures, the spatially encoded library and the antibody-encoded library in the sequencing library to obtain the spatial expression matrix of proteins for spatial proteomics analysis. Compare and integrate the HE staining pictures, the spatially encoded library and the cDNA library in the sequencing library to obtain the spatial expression matrix of genes for spatial transcriptomics analysis.

[0143] Example 4

[0144] In this example, sugars and RNAs in tissue cells were indirectly three-dimensionally spatially encoded through multiple rounds of DNA ligation reactions, and then the spatial positions and biological information of the cells in the corresponding tissue were obtained by sequencing, realizing three-dimensional spatial glycomics and transcriptomics analysis of the tissue. In Example 4, except for replacing the DNA-conjugated antibody with DNA-conjugated lectin, other operation steps, sequences and conditions were the same as in Example 3.

[0145] (1) Preparation of three-dimensional spatial positioning and encoding slides: the same as in Example 1

[0146] (2) Three-dimensional spatial glycomics and transcriptomics study of mouse olfactory bulb tissue: Locate sugars and mRNAs on the tissue surface. The schematic flow chart of simultaneous three-dimensional spatial glycomics and transcriptomics is as Figure 9 shown.

[0147] Replace the DNA-conjugated antibody in Example 3 with DNA-conjugated lectin, and the remaining experimental steps are the same as in Example 3.

[0148] Send the sequencing library to the company for next-generation sequencing to obtain sequencing data. Develop corresponding bioinformatics algorithms for analyzing the sequencing library. Compare the barcodes in the measured spatially encoded library with the barcode library, and obtain two-dimensional coordinates (x i , y j ) according to the positions of X-DNA coding and Y-DNA coding, and obtain the coordinate z according to the Z-DNA codingk , from which the three-dimensional coordinates (x i , y j , z k ) of the cells in the tissue are obtained. Finally, the HE-stained images are compared with the spatially encoded library and the lectin-encoded library in the sequencing library to obtain the spatial expression matrix of carbohydrates and perform spatial glycomics analysis. The HE-stained images are compared and integrated with the spatially encoded library and the cDNA library in the sequencing library to obtain the spatial expression matrix of genes and perform spatial transcriptomics analysis

[0149] Example 5

[0150] In this example, the three-dimensional spatial encoding of DNA or chromatin open regions in tissue cells is indirectly performed through multiple rounds of DNA ligation reactions, and then the spatial positions and biological information of the cells in the corresponding tissue are obtained by sequencing, realizing the three-dimensional spatial genomics analysis of the tissue. In Example 5, except for the capture of DNA or chromatin open regions being different, other operation steps, sequences, and conditions are the same as those in Example 1

[0151] (1) Preparation of three-dimensional spatial positioning and encoding slides: same as Example 1

[0152] (2) Spatial genomics or epigenetics research on mouse olfactory bulb tissue: The experimental procedures for capturing DNA or chromatin open regions in mouse olfactory bulb tissue, performing three-dimensional positioning, and sequencing are as Figure 10 shown

[0153] The HE staining and previous steps are the same as those in Example 1

[0154] After HE staining, the tissue sections were placed in an incubation box, 70 μl of pepsin solution was added to each tissue section, and the olfactory bulb tissue was permeabilized at 37 °C for 10 min. After permeabilization, the pepsin solution was aspirated, and the tissue was slowly washed 3 times with 100 μl of 0.1×SSC solution. Prepare 125 μl of Tn5 transposase-adaptor solution: 1.25 μl of adaptor 1, 1.25 μl of adaptor 2, and 122.5 μl of Tn5 transposase. Prepare 100 μl of Tn5 transposase solution for each tissue section: 25 μl of Tn5 transposase, 25 μl of Tn5 transposase-adaptor solution, and 50 μl of Tn5 transposase buffer solution. Add the Tn5 transposase solution to the incubation box and react at 37 °C for 1 h. After the reaction, the Tn5 transposase solution was aspirated, and the tissue was slowly washed 3 times with 100 μl of 0.1×SSC solution. Prepare 70 μl of splint oligonucleotide sequence solution for each tissue section: 7 μl of splint oligonucleotide sequence solution and 63 μl of PBS buffer solution. Add 70 μl of splint oligonucleotide sequence to the incubation box and react at 37 °C for 2 h. After the reaction, the splint oligonucleotide sequence solution was aspirated, and the tissue was washed 3 times with 100 μl of 0.1×SSC solution. Prepare 70 μl of DNA T4 ligase solution for each tissue section: 7 μl of DNA T4 ligase and 63 μl of DNA T4 ligase buffer solution. Add 70 μl of DNA T4 ligase solution to the incubation box and react at 37 °C for 1 h. After the reaction, the tissue was washed 3 times with 100 μl of 0.1×SSC solution. Then, second-strand synthesis was performed. Prepare 75 μl of second-strand synthesis mixture for each tissue section: 30 μl of enzyme-free water, 30 μl of 2.5 mM dNTP, 7.5 μl of DNA polymerase, and 7.5 μl of 10×DNA polymerase buffer. Add the second-strand synthesis mixture and react at 37 °C for 10 h.

[0155] The subsequent steps are the same as those in Example 1.

[0156] The sequencing library was sent to a company for second-generation sequencing to obtain sequencing data. A corresponding bioinformatics algorithm was developed to analyze the sequencing library. The barcode in the measured spatial encoding library was compared with the barcode library. According to the X-DNA encoding and Y-DNA encoding positions, the two-dimensional coordinates (x i , y j ) were obtained. According to the Z-DNA encoding, the coordinate z k was obtained. Thus, the three-dimensional coordinates (x i , y j , z k ) of the cells in the tissue were obtained. The spatial encoding library in the sequencing library was compared with the DNA information to obtain the spatial expression matrix of the DNA information for spatial genomics analysis.

[0157] Example 6

[0158] In this embodiment, three-dimensional spatial encoding of RNA in tissue cells is directly performed through multiple rounds of DNA ligation reactions, and then the spatial positions and biological information of cells in the corresponding tissue are obtained through sequencing, realizing three-dimensional spatial transcriptomics analysis of the tissue.

[0159] (1) Tissue clearing, in-situ retention and fixation of analytes in the tissue, and removal of obstacles such as lipids: The experimental procedure is as Figure 11 shown. First, take a 1×1×1 mm 3 fresh mouse olfactory bulb tissue and immerse it in a cold 10% methanol (1×PBS) solution for 30 min for fixation. Then wash the tissue 3 times with a cold 1×PBS solution for 5 min each time. Next, immerse it in a cold 4% SDS (1×PBS) solution for 10 minutes and wash it 3 times with cold 1×PBS. Prepare a hydrogel solution (1×PBS, 2M NaCl, 8.625% (w / v) sodium acrylate, 2.5% (w / v) acrylamide, 0.15% (w / v) N,N'-methylenebisacrylamide, 0.01% 4-hydroxy-TEMPO, 0.2% (w / v) ammonium persulfate, and 0.2% (w / v) tetramethylethylenediamine, formaldehyde), immerse the tissue sample in the hydrogel solution at 4°C and incubate for 20 minutes to in-situ retain and fix tissue RNA. Then take out the tissue sample and incubate it at 37°C for another 2 hours. Finally, wash the tissue sample 3 times with 1×PBS, put it into an electrophoresis tank, apply a voltage of 40V, and perform electrophoresis for 20 min to remove obstacles such as lipids. Figure 12 Figure of the gel clearing result of mouse olfactory bulb tissue

[0160] (2) Develop a cube "magic cube" spatial tissue sequencing system to deliver DNA-encoded probes: The schematic diagram of the "magic cube" three-dimensional encoding delivery system based on microfluidic DNA-encoded chip electrophoresis is as Figure 13 shown. First, fabricate a cube "magic cube" spatial tissue sequencing system through 3D integrated printing technology. Among them, 100 parallel loading microchannels are integrated on the front, side, and top respectively. The channel width is 10 μm, and the inside of the channel is filled with agarose hydrogel for pre-enriching encoded probes. Microelectrodes are embedded inside the two corresponding sides of the channel for driving DNA into the tissue by voltage.

[0161] (3) Three-dimensional spatial encoding of tissue mRNA: Custom synthesize 100 kinds of X-DNA, 100 kinds of Y-DNA, 100 kinds of Z-DNA, splint oligonucleotide 1, and splint oligonucleotide 2. The spatial codes of the 100 DNA sequences are different from each other. In addition, the 5'-ends of X-DNA and Y-DNA both carry phosphate groups for DNA ligation reactions. The 3'-end of X-DNA is a polyT sequence for capturing the 3'-end polyA sequence of hybridized mRNA. The 5'-end of Z-DNA is a PCR amplification sequence for cDNA amplification. Anneal and hybridize 100 kinds of Y-DNA with splint oligonucleotide 1 and 100 kinds of Z-DNA with splint oligonucleotide 2. Mix the annealing and hybridization products of 100 kinds of X-DNA, Y-DNA, and splint oligonucleotide 1 and the annealing and hybridization products of Z-DNA and splint oligonucleotide 2 with melted agarose hydrogel respectively, and inject them into 100 parallel loading microchannels corresponding to the front, side, and top of the cube "magic cube" system respectively. Place the cube "magic cube" system at 4°C and quickly cool the agarose hydrogel to achieve pre-enrichment of DNA-encoded probes. Subsequently, place the tissue sample into the cube "magic cube" system, and place the cube "magic cube" system into an electrophoresis tank. Add electrophoresis buffer, and the electrophoresis buffer is 1×TBE and contains 10 mM DTT and 1 M urea. Then connect the wires to an RND 320-KA3005D laboratory power supply, apply a voltage of 1.5 V (10 V / cm) for 20 min to drive the DNA in the agarose hydrogel into the tissue. To ensure the success of each round of sequential hybridization, subsequently incubate the tissue sample with 6×SSC high-salt concentration buffer for 5 minutes. After the incubation, wash the sample three times with 2×SSC (containing 0.1% SDS) and three times with 0.1×SSC. Then add DNA T4 ligase and react at 37°C for 1 h to achieve the ligation of X-DNA and Y-DNA, and Y-DNA and Z-DNA. After the reaction, wash three times with 0.1×SSC solution to remove the reaction reagents.

[0162] (4) Three-dimensional spatial transcriptomics study of mouse olfactory bulb tissue: Subsequent steps such as reverse transcription, second-strand synthesis, cDNA amplification, and library construction are the same as in Example 1.

[0163] Perform second-generation sequencing on the sequencing library to obtain the spatial position information and biological information in the library. Develop corresponding bioinformatics algorithms to analyze the obtained sequencing library, compare the barcode in the measured spatial encoding library with the barcode library, and obtain the three-dimensional coordinates (x i , y j , z k) Align the spatially encoded library and the cDNA library in the sequencing library to obtain the spatial expression matrix of genes and conduct spatial transcriptomics analysis. The schematic diagram of three-dimensional reconstruction and spatio-temporal information analysis is as Figure 14 shown.

[0164] Example 7

[0165] In this example, the proteins in tissue cells are directly encoded in three-dimensional space through multiple rounds of DNA ligation reactions, and then the spatial positions and biological information of cells in the corresponding tissue are obtained through sequencing, realizing three-dimensional spatial proteomics analysis of tissues. In Example 7, except that additional labeling of DNA-coupled antibodies, purification and library construction of protein sequencing libraries are different, other operation steps, sequences and conditions are the same as those in Example 6.

[0166] (1) Tissue clearing, in-situ retention and fixation of analytes in tissues, and removal of obstacles such as lipids: the same as in Example 6.

[0167] (2) Develop a cube "Rubik's Cube" spatial tissue sequencing system to deliver DNA-encoded probes: the same as in Example 6.

[0168] (3) Three-dimensional spatial encoding of tissue proteins: The schematic diagram of capturing proteins in mouse olfactory bulb tissue and performing three-dimensional spatial encoding is as Figure 15 shown. First, prepare 210 μl of DNA-coupled antibody solution: 21 μl of DNA-coupled antibody and 189 μl of PBS buffer solution. Add the DNA-coupled antibody solution to the incubation box and incubate with the tissue sample at 37 °C for 1 h. After incubation, aspirate the DNA-coupled antibody and slowly wash 3 times with EB buffer solution to remove excess reagents. The remaining steps are the same as in Example 6.

[0169] (4) Three-dimensional spatial proteomics research on mouse olfactory bulb tissue: The subsequent steps such as reverse transcription, second-strand synthesis, cDNA amplification, and construction of protein sequencing libraries are the same as those in the three-dimensional spatial proteomics research part of Example 3.

[0170] Perform second-generation sequencing on the sequencing library to obtain the spatial position information and biological information in the library. Develop corresponding bioinformatics algorithms to analyze the obtained sequencing library, compare the barcodes in the measured spatially encoded library with the barcode library, and obtain the three-dimensional coordinates (x i , y j , z k ) of cells in the tissue according to the X-DNA encoding, Y-DNA encoding, and Z-DNA encoding positions. Align the spatially encoded library and the antibody-encoded library in the sequencing library to obtain the spatial expression matrix of proteins and conduct spatial proteomics analysis.

[0171] Example 8

[0172] In this example, three-dimensional spatial encoding of sugars in tissue cells is directly performed through multiple rounds of DNA ligation reactions, and then the spatial positions and biological information of cells in the corresponding tissue are obtained through sequencing, realizing three-dimensional spatial glycomics analysis of the tissue. In Example 8, except for replacing the DNA-conjugated antibody with a DNA-conjugated lectin, other operation steps, sequences, and conditions are the same as those in Example 7.

[0173] (1) Tissue clearing, in-situ retention and fixation of analytes in the tissue, and removal of obstacles such as lipids: the same as in Example 6.

[0174] (2) Developing a cube "magic cube" spatial tissue sequencing system to deliver DNA-encoded probes: the same as in Example 6.

[0175] (3) Three-dimensional spatial encoding of tissue sugars: Capturing the sugars on the surface of the mouse olfactory bulb tissue, and the schematic diagram of three-dimensional spatial encoding is as Figure 16 shown.

[0176] Replace the DNA-conjugated antibody in Example 7 with a DNA-conjugated lectin, and the remaining experimental steps are the same as those in Example 7.

[0177] (4) Three-dimensional spatial glycomics research on mouse olfactory bulb tissue: Subsequent steps such as reverse transcription, second-strand synthesis, cDNA amplification, and construction of a sugar sequencing library are the same as those in Example 7.

[0178] Perform second-generation sequencing on the sequencing library to obtain the spatial position information and biological information in the library. Develop corresponding bioinformatics algorithms to analyze the obtained sequencing library, compare the barcode in the measured spatial encoding library with the barcode library, and obtain the three-dimensional coordinates (x i , y j , z k ) of cells in the tissue according to the X-DNA encoding, Y-DNA encoding, and Z-DNA encoding positions. Compare the spatial encoding library and the lectin encoding library in the sequencing library to obtain the spatial expression matrix of proteins and perform spatial glycomics analysis.

[0179] Example 9

[0180] In this example, three-dimensional spatial encoding of DNA or chromatin open regions in tissue cells is directly performed through multiple rounds of DNA ligation reactions, and then the spatial positions and biological information of cells in the corresponding tissue are obtained through sequencing, realizing three-dimensional spatial genomics or epigenomics analysis of the tissue. In Example 9, except for the capture of DNA or chromatin open regions being different, other operation steps, sequences, and conditions are the same as those in Example 6.

[0181] (1) Tissue transparency, in-situ retention and fixation of analytes in tissues, removal of obstacles such as lipids: same as Example 6.

[0182] (2) Development of a cube "Rubik's Cube" spatial tissue sequencing system for delivering DNA-encoded probes: same as Example 6.

[0183] (3) Three-dimensional spatial encoding of tissue DNA or epigenetics: Capture DNA or chromatin open regions of mouse olfactory bulb tissue. The schematic diagram of three-dimensional spatial encoding is as Figure 17 shown. Prepare 125 μl of Tn5 transposase-adaptor solution: 1.25 μl of adaptor 1, 1.25 μl of adaptor 2, and 122.5 μl of Tn5 transposase. Subsequently, prepare 200 μl of Tn5 transposase solution: 50 μl of Tn5 transposase, 50 μl of Tn5 transposase-adaptor solution, and 100 μl of Tn5 transposase buffer solution. Add the Tn5 transposase solution to the incubation box and incubate with the tissue sample at 37 °C for 1 h. After the reaction, aspirate the Tn5 transposase solution and wash it slowly 3 times with 0.1×SSC solution to remove excess reagents. Prepare 210 μl of splint oligonucleotide sequence solution for each tissue slice: 21 μl of splint oligonucleotide sequence solution and 189 μl of PBS buffer solution. Add the splint oligonucleotide sequence to the incubation box and react with the tissue sample at 37 °C for 2 h. After the reaction, aspirate the splint oligonucleotide sequence solution and wash it 3

[0184] (4) Three-dimensional spatial genomics or epigenetics research on mouse olfactory bulb tissue: Subsequent steps such as reverse transcription, second-strand synthesis, cDNA amplification, and library construction are the same as in Example 1.

[0185] Perform second-generation sequencing on the sequencing library to obtain the spatial location information and biological information in the library. Develop corresponding bioinformatics algorithms to analyze the obtained sequencing library, compare the barcode in the measured spatial encoding library with the barcode library, and obtain the three-dimensional coordinates (x i , y j , z k ) of cells in the tissue according to the X-DNA encoding, Y-DNA encoding, and Z-DNA encoding positions. Compare the spatial encoding library and the genomic or chromatin library in the sequencing library to obtain the spatial expression matrix of genes or chromatin, and perform spatial genomics or epigenomic analysis.

[0186] Although the technical solutions of the present invention have been described in detail and listed, it should be understood that for those skilled in the art, making modifications to the above embodiments or adopting equivalent alternative solutions are obvious to those skilled in the art. These modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. A three-dimensional spatial omics method for tissues based on three-coordinate spatial positioning, characterized in that: This method indirectly or directly performs three-dimensional spatial encoding on the analytes of cells in a tissue through multiple rounds of DNA ligation reactions, and then obtains the spatial positions and biological information of the cells in the corresponding tissue through sequencing, realizing three-dimensional spatial omics analysis of the tissue; The multiple rounds of DNA ligation reactions include the following steps: (a) Provide L substrates, and perform physical or chemical modification on the surface of the substrates; (b) Provide L types of Z-DNA, with specific groups modified at their 5' ends, which are connected to the modification groups on the substrate surface; connect the L types of Z-DNA to L substrates respectively; (c) Anneal and hybridize M types of X-DNA with splint oligonucleotide 1, and connect them to the 3' end of Z-DNA along the X-axis direction to form M rows of X-DNA barcodes; anneal and hybridize N types of Y-DNA with splint nucleotide 2, and connect them to the 3' end of X-DNA along the Y-axis direction to form N columns of Y-DNA barcodes, thereby generating an L-type three-dimensional spatial encoding array of M*N; both the 5' ends of X-DNA and Y-DNA carry phosphate groups; Among them, X-DNA and Y-DNA are added to the corresponding positions by means of a microfluidic chip; and Z-DNA contains a spatial barcode Z and two constant sequences, the two constant sequences include a first constant sequence and a second constant sequence, the spatial barcodes Z of Z-DNA on different substrates are different, and the first constant sequence is a PCR amplification linker, and the second constant sequence is complementary to a partial sequence of splint oligonucleotide 1; X-DNA contains a spatial barcode X, and the spatial barcodes X of each row of X-DNA are the same; X-DNA also contains two constant sequences, a third constant sequence and a fourth constant sequence, the third constant sequence is complementary to a partial sequence of splint oligonucleotide 1, and the fourth constant sequence is complementary to a partial sequence of splint oligonucleotide 2; Y-DNA contains a spatial barcode Y, and the spatial barcodes Y of each column of Y-DNA are the same; The N types of Y-DNA also contain unique molecular identifiers, a constant sequence, and a capture domain, and the constant sequence is complementary to a partial sequence of splint oligonucleotide 2.

2. The three-dimensional spatial omics method for tissues based on three-coordinate spatial positioning according to claim 1, wherein The specific steps for indirectly performing three-dimensional spatial encoding on the analytes in tissue cells through multiple rounds of DNA ligation reactions are as follows: (1) Provide L substrates, and perform physical or chemical modification on the surface of the substrates to generate groups that can be connected to the 5' ends of oligonucleotides; (2) Provide L types of first-group positioning oligonucleotides, named Z-DNA, where the 5' ends of Z-DNA are modified with specific groups and are connected to the modification groups on the substrate surface; (3) Connect the L types of Z-DNA to L substrates respectively to fabricate L substrates with Z-DNA encoding; (4) Provide splint oligonucleotide 1 that hybridizes with the 3' end of Z-DNA; (5) Provide M types of second-group positioning oligonucleotides, named X-DNA, where X-DNA contains a sequence that is substantially complementary to a part of splint oligonucleotide 1, and the 5' end is modified with a phosphate group and can be connected to the 3' end of Z-DNA; (6) Anneal and hybridize the M types of X-DNA with splint oligonucleotide 1, and connect them to the 3' end of Z-DNA along the X-axis direction to form M rows of X-DNA barcodes; (7) Provide a splint oligonucleotide 2 that hybridizes to the 3'-end of X-DNA; (8) Provide N third-group positioning oligonucleotides named Y-DNA, where Y-DNA contains a sequence substantially complementary to a part of splint oligonucleotide 2, and the 5'-end is modified with a phosphate group and can be ligated to the 3'-end of X-DNA; (9) Anneal and hybridize the N Y-DNAs with splint nucleotide 2, and ligate them to the 3'-end of X-DNA along the Y-axis direction to form N columns of Y-DNA barcodes, thereby generating an L×M×N three-dimensional spatial coding array; (10) Take L continuously cut tissue samples and contact them with the L×M×N spatial coding arrays generated by the above method respectively; (11) Release the analytes in the tissue samples and specifically bind them to the capture domains on Y-DNA; (12) Construct a sequencing library and perform next-generation sequencing on the machine to identify the analytes specifically bound to the capture domains and their spatial position information; (13) Using the corresponding bioinformatics algorithm, for the obtained sequencing data, two-dimensional coordinates (x i , y j ) are obtained according to the positions of X-DNA coding and Y-DNA coding on each substrate, and the coordinate z k is obtained according to the Z-DNA coding on different substrates. Thus, the three-dimensional coordinates (x i , y j , z k ) of the analyte are obtained, thereby determining the spatial position of the analyte in the tissue sample and reconstructing the three-dimensional spatial analysis map of the tissue.

3. The method according to claim 2, wherein: The substrate described in step (1) is one of a silicon wafer, a glass slide, an indium tin oxide conductive glass, and a polymethyl methacrylate sheet.

4. The method according to claim 2, characterized in that: In step (1), the surface of the substrate is physically modified or chemically modified to generate a group that can be ligated to the 5'-end of the oligonucleotide. The physical modification includes one of etching, evaporation coating, and spin coating, and the chemical modification includes one of in-situ synthesis, covalent coupling, molecular assembly, and affinity interaction.

5. The method according to claim 2, wherein: In step (1), a group that can be ligated to the 5'-end of the oligonucleotide is generated, and the group includes one of succinimide, maleimide, mercapto, amino, aldehyde, epoxy, macrocyclic alkyne, and azide.

6. The method according to claim 2, characterized in that: In step (2), the 5'-end of the first-group positioning oligonucleotide Z-DNA is modified with a specific group and ligated to the surface modification group of the substrate. The group includes one of NHS, maleimide, mercapto, amino, aldehyde, epoxy, macrocyclic alkyne, and azide.

7. The method according to claim 2, characterized in that: The L first-group positioning oligonucleotides Z-DNA described in step (2) include a spatial barcode Z and two constant sequences. The two constant sequences include a first constant sequence and a second constant sequence. The spatial barcodes Z of Z-DNA on different substrates are different, and there are L spatial barcodes Z in total. The first constant sequence is a PCR amplification adapter, and the second constant sequence is complementary to a part of the splint oligonucleotide 1 described in step (4).

8. The method according to claim 2, wherein: In step (3), the L Z-DNAs are ligated to the L substrates respectively, and the ligation is one of covalent coupling, molecular assembly, and affinity interaction.

9. The method according to claim 2, wherein: The second-group positioning oligonucleotide X-DNA described in step (5) includes a spatial barcode X. The spatial barcodes X of each row of X-DNA are the same, forming M rows of X-DNA barcodes.

10. The method according to claim 2, wherein: The M second-group positioning oligonucleotides X-DNA described in step (5) also include two constant sequences, a third constant sequence and a fourth sequence. The third constant sequence is complementary to a part of the splint oligonucleotide 1 described in step (4), and the fourth constant sequence is complementary to a part of the splint oligonucleotide 2 described in step (7).

11. The method according to claim 2, wherein: In step (6), the M types of X-DNA are annealed and hybridized with splint oligonucleotide 1 and ligated to the 3'-end of Z-DNA along the X-axis direction. The ligation method is DNA ligation reaction, ligating the 5'-end phosphate group of X-DNA and the 3'-end hydroxyl group of Z-DNA respectively.

12. The method according to claim 2, wherein: In step (8), the third set of positioning oligonucleotides Y-DNA contains the spatial barcode Y. The spatial barcode Y of each column of Y-DNA is the same, forming N columns of Y-DNA barcodes.

13. The method according to claim 2, wherein: The N types of the third set of positioning oligonucleotides Y-DNA in step (8) also contain unique molecular identifiers, a constant sequence, and a capture domain. The constant sequence is complementary to a partial sequence of the splint oligonucleotide 2 in step (7).

14. The method according to claim 2, wherein: The capture domain of Y-DNA in step (8) is a specific sequence.

15. The method according to claim 14, wherein The capture domain of Y-DNA in step (8) is a poly-thymine oligonucleotide (poly-T) sequence.

16. The method according to claim 2, wherein: In step (9), the N types of Y-DNA are annealed and hybridized with splint oligonucleotide 2 and ligated to the 3'-end of X-DNA along the Y-axis direction. The ligation method is DNA ligation reaction, ligating the 5'-end phosphate group of Y-DNA and the 3'-end hydroxyl group of X-DNA respectively.

17. The method according to claim 2, wherein: In step (9), the encoding of M types of X-DNA and the encoding of N types of Y-DNA achieve M×N two-dimensional spatial encodings. Combined with L types of Z-DNA, finally L types of M×N three-dimensional spatial encoding arrays are formed.

18. The method according to claim 2, wherein: The resolution of the spatial encoding array is 100 μm - 0.5 μm.

19. The method according to claim 2, wherein: In step (9), L is any integer from 1 to 100, and M and N are any integers between 10 and 1000.

20. The method according to claim 2, characterized in that: The tissue sample in step (10) is a paraffin or fresh frozen tissue section.

21. The method according to claim 2, characterized in that: The thickness of the L tissue samples after continuous cutting in step (10) is the same, with a thickness of 5 μm - 50 μm. The continuously cut tissue samples are adhered to the corresponding spatial encoding array substrates in order from top to bottom.

22. The method according to claim 2, wherein: The analytes in the tissue sample in step (11) include one or several of DNA, RNA, protein, chromatin, and carbohydrates.

23. The method according to claim 2, wherein: Releasing the analytes in the tissue sample in step (11) includes permeabilizing the tissue using a protease or a surfactant.

24. The method according to claim 23, wherein: The protease is pepsin.

25. The method according to claim 2, characterized in that: Releasing the analytes in the tissue sample in step (11), if spatial positioning of DNA or chromatin open regions is required, a transposase is also needed to cut the DNA double strand, insert a ligation linker, and make it bind to the capture domain of the spatial encoding array.

26. The method according to claim 15, characterized in that: Releasing the analytes in the tissue sample in step (11), if spatial positioning of RNA is required, after permeabilizing the tissue, directly hybridize the polyT on the spatial encoding array Y-DNA with the 3'-end polyA tail of mRNA.

27. The method according to claim 2, wherein: For the release of analytes in the tissue sample in step (11), if spatial localization of proteins is required, before permeabilizing the tissue, multiple DNA-conjugated antibodies are pre-incubated with the tissue to bind to different proteins; the DNA-conjugated antibody consists of an antibody, a PCR amplification linker, an antibody code, and a 3'-terminal constant sequence, where the constant sequence is complementary to the capture domain of the Y-DNA described in step (9), and the constant sequence is a specific DNA sequence.

28. The method according to claim 2, wherein: For the release of analytes in the tissue sample in step (11), if spatial localization of proteins is required, after co-incubating the tissue with the DNA-conjugated antibody, the excess DNA-conjugated antibody is washed away, and the tissue is further permeabilized to release the DNA-conjugated antibody, and the capture domain on the spatially encoded array Y-DNA is used to hybridize with the constant sequence at the 3'-end of the DNA-conjugated antibody.

29. The method according to claim 2, wherein: For the release of analytes in the tissue sample in step (11), if spatial localization of carbohydrates is required, before permeabilizing the tissue, multiple DNA-conjugated lectins are pre-incubated with the tissue to bind to different carbohydrates; the DNA-conjugated lectin consists of a lectin, a PCR amplification linker, a lectin code, and a 3'-terminal constant sequence, where the constant sequence is complementary to the capture domain of the Y-DNA described in (9), and the constant sequence is a specific DNA sequence.

30. The method according to claim 2, characterized in that: For the release of analytes in the tissue sample in step (11), if spatial localization of carbohydrates is required, after co-incubating the tissue with the DNA-conjugated lectin, the excess DNA-conjugated lectin is washed away, and the tissue is further permeabilized to release the DNA-conjugated lectin, and the capture domain on the spatially encoded array Y-DNA is used to hybridize with the constant sequence at the 3'-end of the DNA-conjugated lectin.

31. The method according to claim 2, characterized in that: For the construction of the sequencing library in step (12), next-generation sequencing is performed on the machine to identify the analytes specifically binding to the capture domain and their spatial position information, which is used for individual spatial genomics, transcriptomics, proteomics, epigenomics, and glycomics sequencing, or for combined genomics and transcriptomics, transcriptomics and proteomics to achieve spatial multi-omics sequencing.

32. The method according to claim 2, wherein: For the construction of the sequencing library in step (12), next-generation sequencing is performed on the machine to identify the analytes specifically binding to the capture domain and their spatial position information. The specific steps include tissue section fixation, permeabilization to release tissue analytes, analyte capture, reverse transcription, collection and amplification of cDNA, and library construction using a DNA library construction kit followed by sequencing on the machine.

33. The method according to claim 2, wherein: In step (13), the corresponding bioinformatics algorithm is used to obtain two-dimensional coordinates (xi, yj) from the obtained sequencing data according to the positions of X-DNA coding and Y-DNA coding on each substrate, and the coordinate zk is obtained according to the Z-DNA coding on different substrates, thereby obtaining the three-dimensional coordinates (xi, yj, zk) of the analyte, so as to determine the spatial position of the analyte in the tissue sample and reconstruct the three-dimensional spatial analysis map of the tissue, including analyzing the sequencing library, comparing the measured ZXY spatial barcodes with the pre-designed spatial barcode library, and obtaining two-dimensional coordinates (x i , y j ) according to the X-DNA and Y-DNA spatial barcodes, and obtaining the spatial coordinates z of different layers of the tissue according to the Z-DNA spatial barcode k , thereby obtaining the three-dimensional coordinates (x i , y j , z k ) of the tissue analyte; combining the DNA library, cDNA library, antibody coding and lectin coding to realize spatial genomics, transcriptomics, proteomics, epigenomics, glycomics and spatial multi-omics sequencing analysis.

34. A three-dimensional spatial omics method for tissues based on three-coordinate spatial positioning, characterized in that: This method indirectly or directly performs three-dimensional spatial encoding on the analytes of cells in the tissue through multiple rounds of DNA ligation reactions, and then obtains the spatial positions and biological information of the cells in the corresponding tissue through sequencing, realizing three-dimensional spatial omics analysis of the tissue. The direct three-dimensional spatial encoding of the analytes of cells in the tissue through multiple rounds of DNA ligation reactions specifically includes the following steps: (1) Tissue clearing, in-situ retention and fixation of analytes in the tissue, and removal of lipids; (2) Develop a spatial organization sequencing system for the cube "Rubik's Cube" for delivering DNA-encoded probes; on the front, side, and top of the cube "Rubik's Cube", parallel loading microchannels and corresponding sample interfaces are integrated respectively. The inside of the channels is filled with gel for pre-enrichment of the encoded probes in the channels, and microelectrodes are embedded inside the corresponding two sides to apply an electric field to drive the directional movement of the encoded probes in the cleared tissue; (3) Provide M first-group positioning oligonucleotides named X-DNA, where the 3'-end of X-DNA is a capture domain for binding tissue analytes, and the 5'-end is modified with a phosphate group for DNA ligation reaction; (4) Mix M kinds of X-DNA with the gel and pre-enrich them into different loading microchannels on the front of the cube "Rubik's Cube" respectively, with one kind of X-DNA corresponding to one loading microchannel; (5) Provide splint oligonucleotide 1 that hybridizes with the 5'-end of X-DNA, and the 5'-end contains a sequence substantially complementary to a part of X-DNA; (6) Provide N second-group positioning oligonucleotides named Y-DNA, where Y-DNA contains a sequence substantially complementary to a part of splint oligonucleotide 1, and the 5'-end is modified with a phosphate group; (7) Anneal and hybridize N kinds of Y-DNA with splint oligonucleotide 1 in advance, mix the annealed and hybridized products with the gel, and pre-enrich them into different loading microchannels on the side of the cube "Rubik's Cube" respectively, with one kind of Y-DNA corresponding to one loading microchannel; (8) Provide splint oligonucleotide 2 that hybridizes with the 5'-end of Y-DNA, and the 5'-end contains a sequence substantially complementary to a part of Y-DNA; (9) Provide L third-group positioning oligonucleotides named Z-DNA, where Z-DNA contains a sequence substantially complementary to a part of splint oligonucleotide 2; (10) Anneal and hybridize L kinds of Z-DNA with splint oligonucleotide 2 in advance, mix the annealed and hybridized products with the gel, and pre-enrich them into different loading microchannels on the top of the cube "Rubik's Cube" respectively, with one kind of Z-DNA corresponding to one loading microchannel; (11) Use three-axis microchannel electrophoresis to accurately deliver the X-DNA, the hybridization product of Y-DNA and splint oligonucleotide 1, and the hybridization product of Z-DNA and splint oligonucleotide 2 into the tissue treated by gel clearing along the channel direction. Among them, X-DNA binds to tissue analytes through the capture domain. Under the action of DNA ligase, Y-DNA is sequentially connected to the 5'-end of X-DNA along the inherent direction, and Z-DNA is sequentially connected to the 5'-end of Y-DNA along the inherent direction under the action of DNA ligase, combining to form a unique XYZ three-coordinate spatial code; (12) Construct a sequencing library and perform next-generation sequencing on the machine to identify the analytes specifically binding to the capture domain and their spatial position information; (13) Develop corresponding bioinformatics algorithms. For the obtained sequencing data, obtain the three-dimensional coordinates (x i , y j , z k ) of the analyte according to the X-DNA, Y-DNA, and Z-DNA coding positions, so as to determine the spatial position of the analyte in the tissue sample and reconstruct the three-dimensional spatial analysis map of the tissue.

35. The method according to claim 34, wherein: The tissue clearing described in step (1) includes 3 tissue clearing methods: oil-based, hydrogel-based, and aqueous-based.

36. The method according to claim 34, wherein: The tissue clearing described in step (1) in-situ preserves and fixes the analytes in the tissue and removes lipids, including soaking the sample with a reactant solution for forming a hydrogel, such that the biomolecules in the tissue are linked to the gel monomers mediated by formaldehyde to achieve the retention and fixation of tissue analytes; meanwhile, heat initiation is performed to initiate gel polymerization on the tissue, and then lipid components in the tissue are removed by electrophoresis to achieve tissue clearing; the reactants for forming the hydrogel include acrylamide, bisacrylamide, formaldehyde, and a heat trigger agent.

37. The method according to claim 34, characterized in that: The development of the cubic "magic cube" spatial tissue sequencing system described in step (2) for delivering DNA-encoded probes includes mold casting, metal stamping, plastic molding, and 3D printing.

38. The method according to claim 34, wherein: The front, side, and top of the cubic "magic cube" described in step (2) are respectively integrated with parallel loading microchannels and corresponding sample interfaces, and the front, side, and top are respectively set as the X-axis, Y-axis, and Z-axis of the corresponding tissue, which are respectively used for the loading of X-DNA, Y-DNA, and Z-DNA.

39. The method according to claim 32, wherein: The front, side, and top described in step (2) are integrated with parallel loading microchannels, which respectively have M, N, and L channels, and M, N, and L are respectively any integer between 5 and 1000.

40. The method according to claim 34, wherein: The front, side, and top described in step (2) are integrated with parallel loading microchannels, and the channel width is 100 μm - 0.5 μm.

41. The method according to claim 34, characterized in that, The inside of the channels described in step (2) is filled with a gel for pre-enrichment of the encoded probes in the channels, and the gel includes agarose hydrogel, gelatin hydrogel, hyaluronic acid hydrogel, dextran hydrogel, copolymer hydrogel.

42. The method according to claim 34, characterized in that: The M first-group positioning oligonucleotides X-DNA described in step (3) include a spatial barcode X, a constant sequence, and a capture domain. The spatial barcodes X of X-DNA in different channels are different, and there are M spatial barcodes X in total. The constant sequence is complementary to a partial sequence of the splint oligonucleotide 1 described in (5), and the capture domain is a specific sequence or a polyT sequence.

43. The method according to claim 42, characterized in that: The capture domain is polyT.

44. The method according to claim 34, wherein: The first-group positioning oligonucleotides X-DNA described in step (4) include a spatial barcode X, and the spatial barcodes X of each row of X-DNA are the same, forming M rows of X-DNA barcodes.

45. The method according to claim 34, wherein: The N second-group positioning oligonucleotides Y-DNA described in step (6) include a spatial barcode Y and two constant sequences. One constant sequence is complementary to a partial sequence of the splint oligonucleotide 1 described in step (5), and the other constant sequence is complementary to a partial sequence of the splint oligonucleotide 2 described in step (8).

46. The method according to claim 34, wherein: The second-group positioning oligonucleotides Y-DNA described in step (7) include a spatial barcode Y, and the spatial barcodes Y of each column of Y-DNA are the same, forming N columns of Y-DNA barcodes.

47. The method according to claim 34, wherein: The L third-group positioning oligonucleotides Z-DNA described in step (9) include a spatial barcode Z, a constant sequence, and a PCR amplification linker. The constant sequence is complementary to a partial sequence of the splint oligonucleotide 2 described in (8).

48. The method according to claim 34, characterized in that: The third-group positioning oligonucleotides Z-DNA described in step (10) include a spatial barcode Z, and the spatial barcodes Z of each vertical column of Z-DNA are the same, forming L vertical columns of Z-DNA barcodes.

49. The method according to claim 34, characterized in that: In step (11), by using three-axis microchannel electrophoresis, the hybridization products of X-DNA, Y-DNA and splint oligonucleotide 1, and the hybridization products of Z-DNA and splint oligonucleotide 2 are accurately delivered into the tissue subjected to gel clearing along the channel direction; M types of X-DNA enter the tissue along the X-axis under the drive of voltage, and the capture domain at its 3'-end binds to the analyte. N types of hybridization products of Y-DNA and splint oligonucleotide 1 enter the tissue along the Y-axis under the drive of voltage, and the 3'-end hydroxyl group is ligated to the 5'-end phosphate group of X-DNA through T4 DNA ligase. L types of Z-DNA and splint oligonucleotide 2 enter the tissue along the Z-axis under the drive of voltage, and the 3'-end hydroxyl group is ligated to the 5'-end phosphate group of Y-DNA through T4 DNA ligase.

50. The method according to claim 34, wherein: In step (11), the orthogonal ligation of M types of X-DNA encoding, N types of Y-DNA encoding and L types of Z-DNA realizes M*N*L three-dimensional spatial encoding.

51. The method according to claim 34, characterized in that: The tissue described in step (11) is fresh or fresh frozen tissue.

52. The method according to claim 34, characterized in that: The tissue thickness described in step (11) is 50 - 5000 μm.

53. The method according to claim 34, characterized in that: The tissue analyte described in step (11) includes one or several of DNA, RNA, protein, chromatin and carbohydrates.

54. The method according to claim 34, characterized in that: If the tissue analyte in step (11) needs to spatially locate DNA or chromatin open regions, a transposase is also required to cut the DNA double strand and insert an adaptor to bind to the capture domain of X-DNA.

55. The method according to claim 34, characterized in that: If the tissue analyte in step (11) needs to spatially locate RNA, the polyT on X-DNA is directly used to hybridize with the 3'-end polyA tail of mRNA.

56. The method according to claim 34, wherein: If the tissue analyte in step (11) needs to spatially locate protein, multiple DNA-conjugated antibodies are pre-incubated with the tissue to bind to different proteins; the DNA-conjugated antibody consists of an antibody, a PCR amplification adaptor, an antibody encoding and a 3'-end constant sequence, where the constant sequence is complementary to the capture domain of X-DNA described in step (3), and the constant sequence is a specific DNA sequence or polyA.

57. The method according to claim 34, characterized in that: If the tissue analyte in step (11) needs to spatially locate protein, after co-incubating the tissue with the DNA-conjugated antibody, the excess DNA-conjugated antibody is washed away, and X-DNA is driven into the tissue by voltage to hybridize with the constant sequence at the 3'-end of the DNA-conjugated antibody using the capture domain on X-DNA.

58. The method according to claim 34, wherein: If the tissue analyte in step (11) needs to spatially locate carbohydrates, multiple DNA-conjugated lectins are pre-incubated with the tissue to bind to different carbohydrates; the DNA-conjugated lectin consists of a lectin, a PCR amplification adaptor, a lectin encoding and a 3'-end constant sequence, where the constant sequence is complementary to the capture domain of X-DNA described in step (3), and the constant sequence is a specific DNA sequence or polyA.

59. The method according to claim 34, characterized in that, If the tissue analyte described in step (11) requires spatial localization of carbohydrates, after co-incubating the tissue with DNA-conjugated lectin, the excess DNA-conjugated lectin is washed away, and voltage-driven X-DNA is introduced into the tissue. The capture domain on X-DNA hybridizes with the constant sequence at the 3' end of DNA-conjugated lectin.

60. The method according to claim 34, wherein: For the construction of the sequencing library described in step (12), next-generation sequencing is performed on the machine to identify the analyte that specifically binds to the capture domain and its spatial position information, which can be used for individual spatial genome, transcriptome, proteome, epigenome, and glycomics sequencing, or for combined genome and transcriptome, transcriptome and proteome for spatial multi-omics sequencing.

61. The method according to claim 34, wherein: For the construction of the sequencing library described in step (12), next-generation sequencing is performed on the machine to identify the analyte that specifically binds to the capture domain and its spatial position information. The specific steps include analyte capture, reverse transcription, collection and amplification of cDNA, library construction using a DNA library construction kit, and sequencing on the machine.

62. The method according to claim 34, characterized in that: For the development of the corresponding bioinformatics algorithm described in step (13), to determine the spatial position of the analyte in the tissue sample and reconstruct the three-dimensional spatial analysis map of the tissue, including analyzing the sequencing library, comparing the measured XYZ spatial barcodes with the pre-designed spatial barcode library, and obtaining the three-dimensional coordinates (xi, yj, zk) based on the X-DNA, Y-DNA, and Z-DNA spatial barcodes; combining the DNA library, cDNA library, antibody coding, and lectin coding to achieve spatial genome, transcriptome, proteome, epigenome, glycomics, and spatial multi-omics sequencing analysis.

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