High-resolution low-cost tissue space DNA methylomics sequencing method

Abstract

The invention discloses a high-resolution and low-cost tissue space DNA methylomics sequencing method, and relates to the technical field of molecular biology, and the method comprises the following steps: sequentially carrying out cross-linking immobilization, cell permeabilization and transposase transposition on a tissue slice; then tightly covering the micro-fluidic chip on the tissue slice, respectively injecting adaptive sequences containing DNA codes into channels in the X-axis direction and the Y-axis direction of the micro-fluidic chip, adding bar code marks for nucleic acid of the tissue slice, and further combining to form a spatial position coordinate mark with an XiYj form; and then carrying out anti-crosslinking, DNA methylation conversion, library building sequencing and data analysis, and finally successfully obtaining DNA methylation site information of different positions of the tissue slice. According to the method, high genome coverage can be realized by only needing 5X low-depth sequencing data, a highly methylated sample area can be accurately indicated, and the method has the remarkable advantages of low cost and high accuracy, and shows a wide application prospect in the field of biomedicine.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to a high-resolution and low-cost tissue spatial DNA methylome sequencing method. Background Art

[0002] DNA methylation is a key mechanism for regulating transcription, profoundly affecting gene expression and cell fate, and plays a key role in understanding life processes and revealing disease mechanisms. Therefore, in-depth exploration of DNA methylation is crucial for in-depth analysis of cell heterogeneity, elucidation of gene expression regulation mechanisms, and promotion of the development of precision medicine. So far, there is no mature research program for spatial DNA methylome.

[0003] At present, most of the spatial omics technology solutions are focused on transcriptome detection research, and only a small number involve epigenetic regulatory levels such as chromatin open state and histone modification. There is a lack of spatial level detection of DNA methylation, an important epigenetic modification.

[0004] Previous DNA methylome sequencing generally used bisulfite conversion to treat a large number of non-methylated cytosine sites in the genome. This operation resulted in a decrease in the richness of sequencing data and a significant decrease in the unique genome alignment rate. Generally, deep sequencing (30X and above) is required to achieve 80% genome coverage. Problems such as high cost and insufficient coverage have become the main bottlenecks restricting the development of DNA methylome. Summary of the invention

[0005] In order to solve the above technical problems, the present invention aims to provide a high-resolution and low-cost tissue spatial DNA methylome sequencing method. The method introduces a microfluidic system to achieve micrometer-level spatial position marking of tissue sections, integrates the experimental scheme of methylated cytosine conversion, and realizes spatial DNA methylome detection with high genome coverage.

[0006] The technical solution of the present invention to solve the above technical problems is as follows: a high-resolution and low-cost tissue spatial DNA methylome sequencing method is provided, comprising the following steps:

[0007] (1) Cross-linking fixation: cross-linking the tissue sections to be tested using a cross-linking agent;

[0008] (2) Cell permeabilization: permeabilizing the tissue slice to be tested after the treatment in step (1);

[0009] (3) Nucleic acid fragmentation: fragmenting the nucleic acid of the tissue section to be tested after treatment in step (2) and adding nucleic acid headers;

[0010] (4) Overall spatial position marking: Assemble the tissue slices to be tested after the treatment in step (3) into the microfluidic chip in the X direction, and add DNA encoding adapter sequences X1-X1 to the i injection hole channels of the microfluidic chip respectively. i The connection mixture is sucked into the central area of ​​the microfluidic chip by a vacuum pump to add corresponding spatial information marks to the nucleic acid of the tissue slice to be tested in the X-axis direction; the tissue slice to be tested is then assembled into the microfluidic chip in the Y direction, and DNA encoding adapter sequences Y1-Y are respectively added to the injection hole channels of the microfluidic chip. j The ligation mixture is then sucked into the central area of ​​the microfluidic chip by a vacuum pump to add corresponding spatial information marks to the nucleic acid of the tissue slice to be tested in the Y-axis direction. i and Y j The mark can indicate its spatial position on the slice; the X-axis direction is perpendicular to the Y-axis direction;

[0011] (5) Reverse cross-linking: performing reverse cross-linking treatment on the tissue slice to be tested after the treatment in step (4), releasing the cross-linked nucleic acid fragments, and obtaining the nucleic acid fragments to be sequenced;

[0012] (6) DNA methylation labeling: The nucleic acid fragments to be sequenced obtained in step (5) are processed by the TAPS method, and then the nucleic acid fragments are purified;

[0013] (7) Library construction and sequencing: The nucleic acid fragments to be sequenced obtained in step (6) are used as templates, and after enrichment treatment, they are amplified using single-stranded random primers to construct a sequencing library, and the library is sequenced to obtain sequencing data. i Y j The spatial position markers are used to analyze the sequencing data to obtain the DNA methylation information of the tissue section to be tested.

[0014] The beneficial effects of adopting the technical solution of the present invention are:

[0015] The method of the present invention is to mark the DNA encoding X on the X-axis and Y-axis channels of the tissue section. i and Y j , the tissue sections are marked as i×j sample points, and the X bound to the nucleic acid at each sample point i and Y j DNA codes mark the locations of tissue space.

[0016] The method of the present invention innovatively integrates the spatial marking system of tissue sections with the DNA methylation site detection process. The tissue sections are first marked at the micrometer level at the spatial position level, and then the tissue sections are lysed to obtain DNA molecules containing spatial position information, realizing the effective joint analysis of visualized micrometer-level sample observation and non-visualized base molecule capture, breaking through the current technical limitations of molecular detection in tissue sections.

[0017] Furthermore, steps (1)-(3) are carried out in a reaction pool, which is a PDMS (polydimethylsiloxane) chip.

[0018] Furthermore, the reaction pool may be a PDMS (polydimethylsiloxane) single-hole chip, and the size of the central hole may be 0.7 cm×0.7 cm or 0.7 cm×1.2 cm.

[0019] Furthermore, in step (1), the cross-linking agent is formaldehyde at a concentration of 1-2%.

[0020] Furthermore, in step (3), the nucleic acid fragmentation method is Tn5 transposase insertion.

[0021] Further, in step (4), i is a natural number greater than 1 and less than or equal to the number of injection hole channels, and j is a natural number greater than 1 and less than or equal to the number of injection hole channels;

[0022] Furthermore, in step (4), an adapter sequence containing biotin is used.

[0023] Furthermore, in step (6), the DNA methylation labeling method is TAPS.

[0024] Furthermore, in step (7), magnetic beads coated with streptavidin are used to enrich the target sequence.

[0025] Furthermore, in step (7), a random primer method is used to construct a next-generation sequencing library.

[0026] Further, in step (7), the amplification step is: using a single-stranded random primer containing 6 random bases N at the 3' end and a sequencing adapter sequence at the 5' end to randomly bind to the nucleic acid fragment to be sequenced by annealing and renaturing to obtain a renatured product, and then using a DNA polymerase Klenow fragment with strand displacement activity to completely amplify the renatured product to obtain a sequencing library containing two sequencing adapter sequences. The length of the library sequence can be 350-750bp.

[0027] The base N can be any one of A, T, C or G.

[0028] The present invention also provides the application of the above-mentioned high-resolution and low-cost tissue space DNA methylome sequencing method in the development or preparation of related biological research and disease detection products.

[0029] The present invention has the following beneficial effects:

[0030] 1. The method of the present invention realizes the detection of spatial-level DNA methylome for the first time. Compared with DNA methylome at the tissue level and single-cell level, it can provide spatial location information within the tissue and provide a new technical platform for epigenetics.

[0031] 2. The spatial resolution and coverage area of ​​the method of the present invention have been improved, and can cover an area of ​​5mm×5mm to 10mm×5mm. It can be used to detect intact mouse brain tissue slices and embryonic tissue slices within 16 days of development. The area of ​​each sample point is 25μm×25μm to 50μm×50μm.

[0032] 3. The method of the present invention adopts TAPS DNA methylation detection technology, which can greatly improve the library quality and detection effect compared with the traditional bisulfite conversion scheme.

[0033] 4. The method of the present invention can achieve high coverage of the genome and CpG units in sequencing data. The sequencing of mouse embryo sections can achieve coverage of 2,310,449,067bp of the genome region (85.18%) and coverage of 35,639,251 CpG units (82.71%).

[0034] 5. The method of the present invention uses reusable chips and molds, and the required sequencing amount is low, so the cost is low and suitable for large-scale application and promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a flow chart of spatial DNA methylation sequencing of mouse embryonic tissue sections in Example 1;

[0036] Figure 2 Comparison of tissue sections fixed with different concentrations of formaldehyde;

[0037] Figure 3 This is a comparison chart of the DNA shearing effects of restriction endonucleases and commercial Tn5;

[0038] Figure 4 This is a comparison of the DNA shearing effects of laboratory-purified Tn5 and commercial Tn5;

[0039] Figure 5 This is a graph showing the results of a spatial DNA methylome technology experiment using bisulfite sequencing.

[0040] Figure 6 This is the result of the spatial DNA methylome technology experiment using the TAPS method;

[0041] Figure 7 This is a comparison chart of the effects of streptavidin-biotin enrichment schemes;

[0042] Figure 8 A comparison chart of the results of two second-generation library construction schemes;

[0043] Fig. 9 It is the sequencing saturation curve;

[0044] Fig.10 This is a statistical result diagram of genome coverage;

[0045] Fig.11 The spatial methylation data is mapped to the UMAP two-dimensional space result map after K-means clustering;

[0046] Fig.12 This is a diagram showing the spatial information of methylation data in mouse embryo sections. DETAILED DESCRIPTION

[0047] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific conditions are specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0048] The compositions of some test reagents in the embodiments of the present invention are as follows:

[0049] Table 1 Permeabilization buffer 1 (10 mL)

[0050] HEPES 1M 50mM 0.5mL EDTA 0.5M 1mM 0.02mL EGTA 0.5M 1mM 0.02mL NaCl 5M 140mM 0.28mL TritonX-100 5％ 0.25％ 0.5mL NP-40 10％ 0.5％ 0.5mL glycerin 50％ 10％ 2mL

[0051] Table 2 Permeabilization buffer 2 (10 mL)

[0052] HEPES 1M 50mM 0.5mL EDTA 0.5M 1.5mM 0.03mL EGTA 0.5M 1.5mM 0.03mL NaCl 5M 100 mM 0.2mL NLS 20％ 0.5％ 0.25mL DOC 10％ 0.1％ 0.1mL

[0053] Table 3 10× Annealing Buffer (10 mL)

[0054] Tris-HCl (pH = 7.5) 1M 100mM 1mL EDTA 0.5M 1.5mM 0.03mL EGTA 0.5M 0.5mM 0.01mL LiCl 5M 2M 4mL

[0055] Table 4 Channel stop buffer (10mL)

[0056] TritonX-100 10％ 0.1％ 100μL EDTA 0.5M 20mM 400μL EGTA 0.5 M 20mM 400μL DPBS 9.1mL

[0057] Table 5 Channel washing buffer (10mL)

[0058] TritonX-100 10％ 0.1％ 100μL DPBS 9.9mL

[0059] Table 6 Proteinase K buffer (10 mL)

[0060] Tris-HCl (pH = 7.5) 1M 20mM 0,2mL EDTA 0.5M 10mM 0.2mL NLS 20％ 2％ 1mL NaCl 5M 50 mM 0.1mL

[0061] Table 7 1×B&W buffer with Tween-20(50mL)

[0062] Tris-HCl (pH = 8.0) 1M 5mM 0.25mL EDTA 0.5M 0.5mM 0.05mL NaCl 5M 1M 10mL Tween-20 10％ 0.05％ 0.25mL

[0063] Table 8 2×B&W buffer (50mL)

[0064] Tris-HCl (pH = 8.0) 1M 10mM 0.5mL EDTA 0.5M 1mM 0.1mL NaCl 5M 2M 20mL

[0065] Table 9 Tris & Tween buffer (50 mL)

[0066] Tris-HCl (pH = 8.0) 1M 10mM 0.5mL Tween-20 10％ 0.1％ 0.5mL

[0067] The sources of the reagents used in the embodiments of the present invention are as follows:

[0068] Table 10 Reagent list

[0069]

[0070] The mouse breeding and mating process used in the embodiment of the present invention is as follows:

[0071] The culture process is to place 3-4 purchased mice (Vital River Company, about 8 weeks old) in a standard ventilated cage with 12h light / 12h dark. The ambient temperature is 25°C and the relative humidity is 50%. When mating, the male and female mice are caged in a 1:1 ratio at 10 pm and the female mouse vaginal plug is checked at 7 am the next day. The pregnant mice are cultured separately and recorded as 0.5 days of pregnancy. After that, the weight is recorded every day.

[0072] The mouse embryo separation process used in the embodiment of the present invention is as follows:

[0073] After anesthesia, the mice were killed by pulling the neck and the abdomen was disinfected with medical alcohol. The uterus was removed and placed in DPBS solution by cutting along the midline of the abdomen near the diaphragm. The uterus was carefully torn open, the embryos were separated and placed in a new tube and washed several times with DPBS until the washing solution no longer turned red.

[0074] Example 1

[0075] Fresh embryos of CD-1 mice (Vitamin Liva, SPF grade) were placed in an embedding box and embedded with OCT. The embedded tissue blocks were sliced ​​using a freezing microtome with a step size of 10 μm. The frozen tissue sections were used to remove excess OCT embedding agent with ultrapure water, dried in an ultraclean workbench, and used as tissue sections to be tested. The method of the present invention was used to perform tissue spatial DNA methylation sequencing. The experimental process is as follows: Figure 1 shown.

[0076] 1. Cross-linking and fixation

[0077] (1) The tissue slice to be tested is attached to the PDMS reaction pool and fixed with a clamp.

[0078] (2) Use a pipette to add formaldehyde cross-linking system to the reaction pool and incubate at room temperature for 10 minutes.

[0079] Table 11 Formaldehyde cross-linking system

[0080] Reagent name Reagent addition volume / μL (total 200μL) 16% formaldehyde solution 12.5 DPBS (Dulbecco's Phosphate Buffered Saline) 187.5

[0081] (3) Use a vacuum pump to suck out and discard the waste liquid from the reaction pool, add glycine termination system to the PDMS reaction pool and gently shake at room temperature for 5 minutes to terminate the reaction.

[0082] Table 12 Glycine termination system

[0083] Reagent name Reagent addition volume / μL (total 200μL) Glycine(Glycine Solution) 80 Proteinase inhibitor 2 DPBS 118

[0084] 2. Cell Permeabilization

[0085] The cells are permeabilized to permeabilize the cell membrane and nuclear membrane, so that the reaction reagents can fully enter the cell nucleus for reaction. The specific steps are: aspirate and discard the waste liquid, add permeabilization working solution 1 and 2 to the PDMS reaction pool in turn, and react for 10 minutes each at room temperature.

[0086] Table 13 Permeabilization working solution 1 or 2

[0087]

[0088] 3. Nucleic Acid Fragmentation

[0089] (1) Use a vacuum pump to suck and discard the waste liquid from the reaction pool, add SDS system to the PDMS reaction pool (to bind to the protein to deform and precipitate it), and react at 65°C for 10 minutes.

[0090] Table 14 SDS system

[0091] Reagent name Reagent addition volume / μL (total 200μL) Proteinase inhibitor 2 <![CDATA[Ultra-pure dd H2O]]> 192 10% SDS 6

[0092] (2) Aspirate and discard the waste liquid, add TritonX-100 termination system into the PDMS reaction pool and react at 37°C for 10 min.

[0093] (3) Use a vacuum pump to suck and discard the waste liquid from the reaction pool, add the transposition system to the PDMS reaction pool, react at 37°C for 3 hours, obtain the tissue slice with the added nucleic acid linker, and then separate the tissue slice to be tested from the PDMS chip.

[0094] Table 15 Transposase transposition system

[0095] Reagent name Reagent addition volume / μL (total 200μL) Tn5 transposome 20μL 1X DPBS 66μL 5X LM buffer 40μL 10% Tw-20 2μL 1% digitonin 2μL H2O 70μL

[0096] 4. Overall spatial position marking (this process is carried out in a PDMS microfluidic chip, taking a chip with a resolution of 50 μm and a total of 48×48 injection holes as an example)

[0097] After the tissue slice to be tested treated in step 3 is assembled with the central area of ​​the PDMS microfluidic core, X1-X channels are added in the X-axis direction and Y-axis direction, respectively. 48 、Y1-Y 48 The adapter sequence is used to label the nucleic acid of the tissue slice. The reaction is carried out in the center of the PDMS microfluidic chip. After the two sets of chips in the X and Y directions mark the tissue slices in sequence, the tissue slice plane is divided into 48×48 sample points, each of which contains a different combination of X and Y (X i Y j , i is any natural number from 1 to 48, j is any natural number from 1 to 48). The specific steps are as follows:

[0098] (1) Pre-set the spatial X axis X1-X 48 The adapter sequences were distributed in 96-well plates, 1 μL per well.

[0099] (2) Clean the slide with ultrapure water and place it on a clean bench to dry. Prepare the following connection mixture.

[0100] Table 16 Connection Mixture

[0101] Reagent name Reagent addition volume / μL (total 200μL) T4 DNA ligase (rapid) (Novizan) 12.5 2×T4 DNA ligase rapid buffer 100 NP-40(10%) 10 TritonX-100 (10%) 10 Ultra-pure dd H2O 67.5

[0102] (3) 4 μL of the ligation mixture was mixed with the adapter sequences in the 96-well plate, and the total volume was 5 μL.

[0103] (4) Use wafer membrane to clean the surface of the microfluidic chip and the glass slide, fix them with a clamp, and install a drainage device.

[0104] (5) Load the samples into the loading holes of the microfluidic chip in sequence. After loading, use a vacuum pump to suction the sample outlet to allow the liquid to slowly flow through the central area. Let the reaction stand at room temperature for 10 minutes.

[0105] (6) After the reaction is completed, channel cleaning buffer is added to the loading hole of the PDMS microfluidic chip for cleaning.

[0106] (7) Repeat the process (1) to (6) to connect Y1-Y in the Y-axis direction. 48 Adaptation sequence.

[0107] (8) After the ligation reaction is completed, add channel termination buffer to clean the channel.

[0108] 5. Reverse cross-linking

[0109] The tissue slice to be tested after the treatment in step 4 is reverse cross-linked to obtain a cross-linked and non-visualized nucleic acid fragment. The cross-linked nucleic acid fragment carries two kinds of spatial position information (X i Y j ) mark. The specific steps are as follows:

[0110] (1) Reverse cross-link the tissue in proteinase K buffer.

[0111] Table 17 beads reverse cross-linking system (500 μL)

[0112]

[0113]

[0114] (2) Place the reverse cross-linked tube at 55°C with shaking overnight for 12 hours.

[0115] 6. Methylation information marker

[0116] The DNA methylation information of the nucleic acid fragment to be sequenced obtained in step 5 is marked by the TAPS method, and the specific steps are as follows:

[0117] (1) Use a DNA purification kit to purify the nucleic acid fragments from the reverse cross-linking solution.

[0118] (2) The nucleic acid fragments are transformed by TAPS.

[0119] Table 18 TAPS transformation

[0120] Reagent name Reagent addition amount DNA template 16.2μL eTET 2.0Reaction Buffer 1.9 eTET 2.0Enzyme Mix 6 1×Fe2+Solution 0.9 Total volume 25

[0121] Incubate at 37°C for 2 h (heat cover at 60°C).

[0122] (3) Add 1 μL Termination Buffer and 1 μL Termination Enzyme and incubate at 50°C for 30 min (with heated lid at 60°C).

[0123] (4) Prepare the conversion system. Make sure the reagents are completely thawed and placed on ice until ready for use.

[0124] Table 19 Conversion system

[0125] Reagent name Reagent addition amount / μL The above reaction system 27 Conversion Reagent 9 Conversion Enhancer 2 Total volume 38

[0126] (5) Incubate at 37°C overnight for 12-16 h, 4°C, ∞; (heat cover at 60°C).

[0127] (6) After the reaction is completed, add 34 μL Neutralizer and mix well.

[0128] (7) Purify the TAPS reaction products using DNA purification magnetic beads.

[0129] 7. Library Construction and Sequencing

[0130] Use a single-end random amplification system with 6 random bases at the 3' end to build the library and introduce the read1 sequencing adapter: use a single-stranded random primer with 6 random bases N (N is any of A, T, C or G) at the 3' end and a sequencing adapter sequence at the 5' end to randomly bind to the target nucleic acid fragment through annealing and renaturation to obtain a renatured product, then use DNA polymerase to fully amplify the renatured product, and obtain a sequencing library containing two sequencing adapter sequences and a fragment length suitable for the machine (350-750bp) through fragment sorting. The specific steps are:

[0131] (1) Enrich the DNA obtained in step 6 using magnetic beads coated with streptavidin. Resuspend the magnetic beads containing the target DNA fragment using a single-end random amplification system and place at 95°C for 45 seconds and then quickly cool on ice. Then add 1 μL of Klenow enzyme and increase the temperature at a rate of 0.1°C / s until the final temperature reaches 37°C, then incubate for 30 minutes to obtain an incubation product.

[0132] Table 20 Single-end random amplification system (50 μL)

[0133] Reagent name Reagent addition amount / μL KLENOW blue buffer 5 dNTPs 2 Single-stranded amplification primer (Table 1) (10 μm) 10 water 33

[0134] (2) After the magnetic beads in the incubation product are adsorbed using a magnetic stand, the supernatant is added to the following library amplification system for amplification (15 cycles in total) to obtain an amplified product library:

[0135] Table 21 Library amplification system (50 μL)

[0136] Reagent name Reagent addition amount / μL KAPA 2×MIX 25 PCR2 (i1-i4) (10 μM) 2 PCR1 (10 μM) 2 water 21

[0137] (3) The amplified product library was subjected to gel recovery fragment screening, and the bands between 350-750 bp were selected. The quality control was performed using Qubit and fragment analyzer. The total amount of the final library should be at least 50 ng, and the fragment length distribution should be between 350-750 to obtain the sequencing library.

[0138] (4) Sequencing the sequencing library using a BGI DNBSEQ-T7 sequencer to obtain sequencing data, and analyzing the sequencing data to determine the spatial position of the DNA fragment on the tissue section based on the spatial position information on the DNA fragment.

[0139] Example 2

[0140] 1. Selection of fixative concentration for slice samples in step 1

[0141] The fixation intensity of tissue sections is closely related to the subsequent process adjustment of spatial DNA methylome technology experiments. Therefore, exploratory attempts were made on sections with different concentrations of formaldehyde fixation schemes, involving concentrations of 0.2%, 1%, and 2%, respectively. The specific results are as follows: Figure 2 The results showed that in the process of spatial DNA methylome technology experiment, the slices fixed with 0.2% formaldehyde showed tissue shedding; while the slices fixed with 1-2% formaldehyde could maintain the intact tissue morphology in the subsequent experimental links, which provided favorable conditions for the smooth progress of the experiment.

[0142] 2. Testing of nucleic acid fragmentation method in step 3

[0143] The effect of DNA fragmentation is directly related to the amount of information obtained by spatial DNA methylome technology. Therefore, different fragmentation methods were tried on the slices, including restriction endonucleases and transposases. The results are shown in Figure 2. Figure 3-4 As shown, MH is the digestion effect of two restriction endonucleases, MseI and HinP1I, Hn5 is the shearing effect of laboratory-purified Tn5, and Mn5 is the shearing effect of commercial Tn5. The results show that the effect of transposase on DNA fragmentation is better than that of restriction endonucleases, and increasing the concentration of transposase is helpful to improve the shearing effect of transposase.

[0144] 3. Selection of DNA methylation detection technology in step 6

[0145] The balance result of the sequencing library is included in the sequencing data file. Since each round contains a 4-base fixed sequence, there will be 4-base imbalance sites between each round.

[0146] Bisulfite sequencing is a common and sensitive method for detecting DNA methylation. The principle is to convert unmethylated cytosine into uracil, while methylated cytosine is usually not affected. The conversion product can achieve single-base resolution for methylated cytosine through high-throughput sequencing. Figure 5 The results showed that the quality of the library treated with sulfite was poor, with an alignment rate of 18.52%, a sequencing repetition rate of 43.75%, no obvious library structure in the base distribution, and only 26.78% of the reads that met the library structure of spatial DNA methylome technology.

[0147] TET-assisted pyridine borane sequencing, referred to as TAPS, is based on the principle that TET dioxygenase oxidizes methylated cytosine to carboxyl cytosine (5-caC), and pyridine borane converts 5cac into dihydrouracil, while unmethylated cytosine is not affected. It has been reported that TAPS is less destructive to DNA than sulfite treatment, and the library complexity is higher. The TAPS conversion effect was tested, and the internal reference results showed that the methylated cytosine conversion rate was above 90%, and the false positive rate was 0.008%. The results of the spatial DNA methylation genomics technology experiment showed that the results were as follows Figure 6 As shown, the alignment rate is 84.98%, the sequencing repetition rate is 13.42%, the base distribution clearly shows the library structure, and the proportion of reads that conform to the library structure of spatial DNA methylome technology is 82.47%. The experimental results show that the quality of the library produced by TAPS is greatly improved compared with the bisulfite sequencing method.

[0148] 4. Selection of biotin-integrated adapter sequences and streptavidin magnetic beads technology solutions

[0149] The nucleic acid sequence after reverse cross-linking has a variety of structures, including target sequences (fragments with complete spatial information and genomic DNA) and non-target sequences (fragments with missing spatial information and adapter primers that do not contain genomic DNA). When using the random primer method to construct a second-generation library, if total DNA is used as the starting input sequence, the results obtained by library sequencing show that most of the fragments are adapter primers and do not contain inserted genomic DNA sequences. For specific details, please refer to Figure 7 , it is difficult to discern the shape of the tissue from the spatial heat map of the figure. For the analysis of library reads, more than 80% of the reads in the library are fragments that do not contain genomic DNA. Therefore, the presence of a large number of non-target sequences has a very significant negative impact on the quality of the spatial DNA methylome technology library. After adding biotin modification to the adapter primer and then enriching the target sequence with streptavidin, the spatial heat map can preliminarily present the shape of the tissue compared with the scheme without enrichment, which fully demonstrates the effectiveness of this improved method in improving library quality.

[0150] 5. Step 7: Selection of technical solutions for database construction

[0151] The spatial DNA encoding has a second-generation library sequencing adapter P5 at one end. How to bring the sequencing adapter P7 at the other end? We tried different solutions, and the results are as follows Figure 8 As shown. Scheme 1 is a Y-type adapter scheme. During the DNA fragmentation process, a Y-type adapter is connected. However, after sequencing, it is found that the library constructed using this scheme is almost entirely adapter sequences, and the number of effective fragments is extremely small, which is difficult to meet the requirements of subsequent analysis. Scheme 2 uses a random primer method, using a single-end random amplification system containing 6 random bases at the 3' end to carry out library construction operations, and successfully introduces a P7 sequencing adapter. The library sequencing results obtained by this scheme are obviously consistent with the experimental design of the method of the present invention in structure, providing a feasible technical path for subsequent research.

[0152] Example 3 Mouse embryo sample test

[0153] 1. Analysis of base balance of sequencing library and barcode connection efficiency in each round

[0154] The connection efficiency analysis is to count the proportion of read lengths containing different numbers of barcode sequences. Figure 6 As can be seen, the figure shows the library balance results obtained using double-end sequencing and the number of tags connected to all reads. The results show that the connection efficiency of each round is more than 90%.

[0155] 2. According to the read information of library sequencing, the number of sequencing reads of a tissue section reached 168,336,215. The sequencing saturation curve is shown in Fig. 9 As shown in Figure 1, the current sequencing is saturated, with a duplication of 27.19%. The number of reads aligned to the reference genome is 100,248,589, with an alignment of 80.85%. The spatial distribution of genome coverage is shown in Figure 1. Fig.10 As shown, there are 2169 sample points with more than 2000 reads, and their spatial distribution is consistent with the tissue outline. A total of 2,310,449,067bp (85.18%) of the mouse genome is covered, with an average of 5,707,101bp per sample point. There are 2043 sample points with more than 500 CpG units, covering 35,639,251 CpG units (82.71%), with an average of 163,171 CpG units per sample point.

[0156] 3. Spatial methylation distribution and clustering of slices

[0157] Based on the DNA methylation site information, the spatial sample points can be clustered into 5 categories according to the K-means method, and then the clustering results are mapped to the UMAP two-dimensional space. The results are as follows Fig.11The methylation data is displayed according to the spatial information, and the results are as follows Fig.12 As shown, it can be seen that the DNA methylation level in the brain region of mouse embryos is significantly higher than that in other tissue regions, which is consistent with the currently known DNA methylation status of mouse embryos on day 11.5.

[0158] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A high-resolution and low-cost tissue spatial DNA methylome sequencing method, characterized in that: The following steps are involved: (1) Cross-linking fixation: cross-linking the tissue sections to be tested using a cross-linking agent; (2) Cell permeabilization: permeabilizing the tissue slice to be tested after the treatment in step (1); (3) Nucleic acid fragmentation: fragmenting the nucleic acid of the tissue section to be tested after treatment in step (2) and adding nucleic acid headers; (4) Overall spatial position marking: Assemble the tissue slices to be tested after the treatment in step (3) into the microfluidic chip in the X direction, and add DNA encoding adapter sequences X1-X1 to the i injection hole channels of the microfluidic chip respectively. i The connection mixture is added to the central area of ​​the microfluidic chip by a vacuum pump to add corresponding spatial information marks to the nucleic acid of the tissue slice to be tested in the X-axis direction; the tissue slice to be tested is then assembled into the microfluidic chip in the Y direction, and DNA encoding adapter sequences Y1-Y are respectively added to the injection hole channels of the microfluidic chip. j The connection mixture is sucked into the central area of ​​the microfluidic chip by a vacuum pump to add corresponding spatial information marks to the nucleic acid of the tissue section to be tested in the Y-axis direction. i and Y j The markers indicate their spatial location on the slice; The X-axis direction is perpendicular to the Y-axis direction; (5) Reverse cross-linking: performing reverse cross-linking treatment on the tissue slice to be tested after the treatment in step (4), releasing the cross-linked nucleic acid fragments, and obtaining the nucleic acid fragments to be sequenced; (6) DNA methylation labeling: labeling the nucleic acid fragments to be sequenced obtained in step (5) with DNA methylation information by the TAPS method, and then purifying the nucleic acid fragments to be sequenced; (7) Library construction and sequencing: The nucleic acid fragments to be sequenced obtained in step (6) are used as templates, and after enrichment treatment, they are amplified using single-stranded random primers to construct a sequencing library, and the sequencing library is sequenced to obtain sequencing data, and then based on the X i Y j The spatial position markers are used to analyze the sequencing data to obtain DNA methylation information of the tissue section to be tested.

2. The high-resolution and low-cost tissue spatial DNA methylome sequencing method according to claim 1, characterized in that: Steps (1) to (3) are carried out in a reaction pool, which is a PDMS chip.

3. The high-resolution and low-cost tissue spatial DNA methylome sequencing method according to claim 1, characterized in that: In step (1), the cross-linking agent is formaldehyde with a concentration of 1-2%.

4. The high-resolution and low-cost tissue spatial DNA methylome sequencing method according to claim 1, characterized in that: In step (3), the nucleic acid fragmentation method is Tn5 transposase insertion.

5. The high-resolution and low-cost tissue spatial DNA methylome sequencing method according to claim 1, characterized in that: In step (4), i is a natural number greater than 1 and less than or equal to the number of injection hole channels; j is a natural number greater than 1 and less than or equal to the number of injection hole channels.

6. The high-resolution and low-cost tissue spatial DNA methylome sequencing method according to claim 1, characterized in that: In step (6), the DNA methylation labeling method is TAPS.

7. The high-resolution and low-cost tissue spatial DNA methylome sequencing method according to claim 1, characterized in that: In step (7), the target sequence is enriched using magnetic beads coated with streptavidin.

8. The high-resolution and low-cost tissue spatial DNA methylome sequencing method according to claim 1, characterized in that: In step (7), a random primer method is used to construct a next-generation sequencing library.

9. The high-resolution and low-cost tissue spatial DNA methylome sequencing method according to claims 1-8 is used in the development or preparation of products related to biological research and disease detection.

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