CN112592962B

CN112592962B - Detection method suitable for high-throughput transcriptome spatial position information and application thereof

Info

Publication number: CN112592962B
Application number: CN202011291005.6A
Authority: CN
Inventors: 曹罡; 戴金霞; 吴小凤; 徐伟泽; 王忠超; 李月; 王昊棋; 高安然; 张凌凯
Original assignee: Kunyu Biotechnology Jiangmen Co ltd; Huazhong Agricultural University
Current assignee: Kunyu (Shenzhen) Biotechnology Co.,Ltd.
Priority date: 2020-11-18
Filing date: 2020-11-18
Publication date: 2022-11-25
Anticipated expiration: 2040-11-18
Also published as: CN112592962A; CN115198003B; CN115198003A

Abstract

The invention discloses a detection method suitable for high-throughput transcriptome spatial position information and application thereof, and the method can be used for reading in-situ spatial transcription expression profiles of tens of thousands of genes only by 4 rounds of connection sequencing through the design of a hybridization probe, probe pretreatment, sample hybridization, ligation reaction and rolling circle amplification. The probe can be applied to point mutation and methylation site in-situ detection according to the design characteristics of the probe. Can realize single cell, single molecule and single base resolution high flux gene in situ reading. The signal interpretation of the method does not depend on a super-resolution imaging platform, and the popularization and the development of the high-flux in-situ space transcriptome are promoted.

Description

Detection method suitable for high-throughput transcriptome spatial position information and application thereof

Technical Field

The invention relates to the field of high-throughput in-situ hybridization, in particular to a detection method suitable for high-throughput transcriptome spatial position information and application thereof.

Background

The cytology theory lays the foundation for scientists to research the structure and function of the life body and the running mechanism of the life body. With the progress of research, it is found that the cells from the same tissue in the body have genomic, epigenomic and transcriptome differences; therefore, studies based on single cell level are particularly important, and the recent single cell sequencing technology enables amplification and sequencing of transcriptomes or genomes on a single cell level. The Human cell BioMolecular map project (Human BioMolecular Atlas Program HuBMAP) and cellular map project potential await development following the wave of the Human genome project.

The single cell RNA sequencing (scRNA-seq) technique has made a deeper understanding of the heterogeneity of cell populations, the dynamics of developmental processes, the development of diseases, and the mechanisms controlling cellular functions. How a single cell coordinates the functions of a plurality of surrounding cells is far from enough depending on the identity information of the single cell, and a new method is needed to show the spatial position relationship of each cell. And (3) clustering analysis is carried out on the transcriptome of the single cell in the RNAseq data by using a bioinformatics method. The method has a large limitation because the related position information of the cell marker gene has a large blank. On the other hand, the wide application of model biological expression profiles and mouse and human brain profiles of the Allen brain institute have demonstrated the promising value of spatial transcriptome. However, the traditional map flux drawn by traditional in situ hybridization or cell-specific label genes is low, and complex loops and functions cannot be analyzed; in recent years, a plurality of scientists image the spatial transcriptome of the cell by an in situ hybridization method, and really reduce the expression state of the transcriptome in the cell.

At present, the mainstream in-situ transcriptomics method comprises a multiple error correction fluorescence in-situ hybridization (multiplex error-correction fluorescence in-situ hybridization) technology based on single-molecule in-situ hybridization, sequential fluorescence in-situ hybridization (sequence fluorescence in-situ hybridization, seqFISH), STARmap (spread-resolved-transformed amplification reaction mapping. Iterative imaging is carried out on a plurality of different transcriptomes, so that the spatial positions of the transcriptomes are reduced one by one.

STARmap is a method based on in situ sequencing to display a spatial transcriptome, a padlock probe is used for carrying out in situ hybridization on transcripts, corresponding barcode is designed on the padlock probe aiming at different transcripts, then a hybridized signal is amplified in a rolling circle amplification mode, and finally a sequence of the barcode is read in a connection sequencing mode in turn, so that the spatial position information of the transcriptome with single cell resolution is reduced at high flux. The method has the advantages of strong signal, capability of using common confocal imaging, low efficiency, easy occurrence of non-specificity, incapability of detecting gene mutation and capability of only detecting the existence of transcripts.

In-situ transcriptomics are applied to various researches such as high-precision neural loop analysis, individual development and disease development, but the current method still has the following problems:

1. methods based on single molecule hybridization:

1) The probe is complex to prepare and high in cost: more than 24 probes are designed for each gene, and although the probes are synthesized in a primer pool form, each probe is subjected to PCR low-cycle amplification, purification, transcription, reverse transcription, RNA removal, repurification and other processes. The method has large workload for high-throughput polygene hybridization. The reading probes all need amino modification, and the cost is high.

2) Tissue sample processing requirements are high: tissue transparency treatment needs to perform multiple functional treatments such as silanization modification and polylysine on a cover glass and a glass slide, and then perform multiple steps such as tissue hydrogel embedding and amino modification to fix mRNA. The technical requirement is high.

3) The operation steps are complicated, multiple hybridization and iterative imaging are carried out: the whole signal interpretation process needs to be subjected to multiple hybridization-imaging-signal stripping cyclic operations, which not only has high requirements on the stability of the hybridization solution changing system, but also still has a great challenge on maintaining the stability of mRNA in multiple hybridization and signal stripping cycles.

4) The imaging apparatus is demanding: because the number of single-point signal fluorescent molecules is limited, pressure is brought to stable imaging, and imaging needs to be carried out by means of a high-end confocal system or a super-resolution system; repeated imaging of the same tissue for multiple times brings the risk of phototoxicity and light quenching, and the true signal cannot be interpreted.

5) The signal interpretation is cumbersome: in order to realize the purpose of high-flux hybridization, the signals of each round are registered and superposed, the influence of optical distortion such as phase difference and chromatic aberration in the imaging process is not considered, the calibration of the signals of each round is still difficult, and the difficulty is increased along with the increase of the number of hybridization rounds.

6) The detection of multigene point mutation cannot be realized: the specificity of signals of the current hybridization-based mode depends on sequence complementary pairing, and the single base cannot be distinguished.

2. In situ sequencing based methods:

1) Poor probe specificity: the gapped cyclization of the padlock probe is independent of the target sequence mRNA molecules, but is another RCA primer probe matched with the padlock probe, so that the probe generates nonspecific signal amplification after non-target sequence hybridization, and nonspecific is generated.

2) Tissue sample processing requirements are high: similar to the hybridization-based method, accurate display of the spatial transcriptome is challenged by performing hydrogel transparency treatment on thicker tissues, performing functional treatment on glass slides and cover slips, slightly deforming tissues after removing proteins or lipids from the tissues, storing integrity of transcripts and the like.

3) The one-way sequencing efficiency is low: the decoding of the barcode generally adopts a connection-based sequencing mode, only one barcode can be decoded in each cycle of sequencing, 4 steps of hybridization, connection, imaging and signal stripping are required in each cycle, the requirement on the stability of the system is high, at least 5 cycles of connection sequencing are required for performing in-situ space transcriptome display on 1000 genes, the data registration reading difficulty is high, the period is long, and the efficiency is low.

Disclosure of Invention

The invention aims to provide a detection method suitable for high-throughput transcriptome spatial position information. The method can be used for reading the in-situ spatial transcription expression profiles of tens of thousands of genes only by 4 rounds of connecting and sequencing, and overcomes the defects of complex preparation and high cost of a probe in the traditional in-situ spatial transcriptome display technology; the tissue sample treatment requirement is high; point mutation cannot be detected; the operation is complex; multiple hybridization and iterative imaging are carried out, and the requirement on imaging equipment is high; the signal interpretation is complicated; the probe specificity is poor; the method has the defects of low one-way sequencing efficiency, high difficulty in multi-round data registration and reading and the like.

In order to achieve the above object, the present invention provides a method for detecting spatial position information of a high-throughput transcriptome, comprising the following steps:

1) Design of hybridization probes

Screening a target sequence region with high hybridization efficiency, strong specificity and no RNA advanced structure from an RNA sequence of a cell or tissue to be detected to design a hybridization probe, wherein the hybridization probe consists of a padlock probe and an RCA (Rolling circle amplification) priming probe;

2) Probe pretreatment

Carrying out phosphorylation treatment on the 5' end of a lock probe in the hybridization probe;

3) Hybridization of samples

Preparing a reaction chamber on a sample to be detected, fixing the reaction chamber by using paraformaldehyde, dehydrating and denaturing by using methanol, and adding a buffer solution containing a hybridization probe into the reaction chamber for overnight incubation;

4) Ligation reaction

Adding a ligase reaction system into the reaction chamber for a ligation reaction, and enabling the padlock probes to be complementary and paired with target sequences on corresponding RNAs to form a semi-closed annular structure;

5) Rolling circle amplification

The RCA priming probe is simultaneously complementary with a target sequence and a padlock probe on RNA, phi29DNA polymerase takes the RCA priming probe as a primer and takes the annular structure of the padlock probe as a template to carry out rolling circle amplification, and further amplifies signals;

6) In-situ interpretation of spatial information

i. In-situ color development reading spatial information:

adding a fluorescent probe hybridization system which is reversely complementary with the color development sequence of the rolling circle amplification product into the reaction chamber for hybridization in-situ color development, then imaging the research area of the sample to be detected, and reading the RNA space position information;

barcode sequencing (using the principle of sequencing by ligation) and data analysis, interpreting spatial information of high-throughput transcriptome:

a. adding a ligase reaction system into a reaction chamber for performing a connection sequencing reaction, then sequencing a first base sequence of the barcode in a lock probe, performing imaging scanning on a research area layer by layer according to the characteristics of a sample to be detected, superposing signals of each layer for maximum projection, and registering and reading the information of each fluorescence signal corresponding to the barcode information;

b. sequentially reading the second and third base sequence information of the barcode in the padlock probe by the same method;

c. carrying out registration, barcode filtration and cell segmentation on the obtained 3-round sequencing information; then, single cell data analysis, normalization and cluster analysis of expression matrix of the expression value of the gene are sequentially carried out, dimension reduction and clustering are carried out on the gene set through normalization and normalization processing, cell groups are classified and subdivided into sub-groups according to different cell markers, so that special cell groups are determined, and finally expression type analysis is carried out.

Further, in the step 1), the target sequence of the RNA sequence consists of 3 consecutive target sequences a, B and C, and the padlock probe is an a 'sequence, a loop sequence and a B' sequence from 5 'end to 3' end, and the a 'sequence and the B' sequence are complementary to the target sequence a and the target sequence B, respectively, so that the padlock probe and the target sequence form a semi-closed circular structure after complementary pairing (the bases of the 5 'end and the 3' end of the hybridization probe and the target sequence are recognized by ligase, and any end can not be connected and closed into a ring if there is point mutation or RNA methylation. According to the characteristics, the method can be applied to point mutation in-situ detection or methylation site detection); the loop sequence is composed of an anchor sequence and a barcode sequence (the barcode sequence is a general name of two barcode sequences, namely a barcode1 sequence and a barcode2 sequence).

Furthermore, the composition of the loop sequence is divided into the following parts according to different sequencing modes of the double barcode sequence:

when the barcode sequence is double-ended sequencing, the corresponding loop sequence sequentially comprises a barcode1 sequence, a mid-anchor sequence and a barcode2 sequence from the 5 'end to the 3' end

Wherein, the length of the barcode1 sequence and the barcode2 sequence is 3-9bp, the length of mid-anchor sequence is 15-30bp (which is a common connecting sequencing primer sequence);

when the barcode sequence is unidirectional double barcode sequencing, the corresponding loop sequence sequentially comprises a barcode1 sequence, an anchor-1 sequence, a barcode2 sequence and an anchor-2 sequence from the 5 'end to the 3' end.

Wherein, the length of the barcode1 sequence and the barcode2 sequence is 3-9bp, and the length of the anchor-1 sequence and the anchor-2 sequence is 10-20bp (which are common connecting sequencing primer sequences).

Furthermore, the barcode sequence is composed of A, G, C and T.

Furthermore, when double-barcode sequencing is carried out, in the corresponding loop sequence, the bases corresponding to each position of the barcode1 sequence and the barcode2 sequence are combined, and the combination number is

Wherein

In order to select 2 bases from 4 bases for combination, "4" is 4 possibilities that the selected 2 bases are the same base, the number of combinations of the double barcode sequences (barcode 1 sequence and barcode2 sequence) after n rounds of sequencing is 10 ⁿ ；

Furthermore, in the RNA sequence, the lengths of the target sequence A, the target sequence B and the target sequence C are all 11-16 bp.

Still further, in the step 2), the enzyme used in the phosphorylation process is T4 polynucleotide kinase (T4 PNK).

Still further, in the step 4), the ligase of the ligase reaction system is Splint R ligase.

The invention has the beneficial effects that:

1. the specificity is strong: the invention hybridizes different RNAs in a target hybridization way in the form of a padlock probe, the amplification of signals depends on the accurate complementary pairing of target sequences at two ends, the possibility of non-specific amplification is reduced, and the accuracy of the system is improved;

2. the flux is high: the invention mainly generates 10 combined signals in each round through double-end sequencing and bidirectional decoding of barcode, namely, the N round can be decoded by 10 ^N Different RNAs, the 4 system which is interpreted by the previous signals is upgraded into the 10 system;

3. the imaging condition universality is high: confocal imaging systems can be implemented. The invention mainly amplifies signals by a rolling circle amplification method, each signal point is composed of about 1000 fluorescent molecules, and crowded signal clusters can be analyzed by using a confocal imaging system. This will allow the spatial transcriptome to be used more widely;

4. single base mutations of the gene can be detected: two ends of the padlock probe are complementarily paired with a target sequence to form an unclosed ring structure, bases which are accurately complementarily paired at the 5 'end and the 3' end of the opening are identified by virtue of ligase, and any section of the two ends which has point mutation cannot be connected and closed to form a ring. This will allow the spatial transcriptome to be used more widely, such as for tumor mutation detection and the like;

5. suitable for m6A methylation in situ detection: designing a padlock probe aiming at a methylation site, wherein ligase is sensitive to methylated RNA and is difficult to recognize and seal to form a ring;

6. the invention can realize single cell and single molecule resolution high flux gene in situ reading.

7. The invention directly targets RNA through two sets of probes without reverse transcription, thereby reducing cost and operation complexity; different gene transcripts are hybridized in a targeted manner in the form of a padlock probe, the amplification of signals depends on the precise complementary pairing of targeting sequences at two ends, the non-specific amplification is reduced, the accuracy of the system is improved, and the utilization of the splntR enzyme is more efficient compared with the enzyme applied by the existing in-situ sequencing technology.

In conclusion, the signal is amplified by the rolling circle amplification method, each signal point is composed of about 1000 fluorescent molecules, the signal is stronger compared with a hybridization-based method (each signal point is composed of about more than 24 fluorescent molecules), the signal capture is easy, a confocal imaging system is not needed, the signal cluster can be analyzed by using the confocal imaging system, the experiment cost and the operation difficulty are reduced, and the popularization and the development of the high-flux in-situ space transcriptome are promoted.

Drawings

FIG. 1 is a flow diagram of SSIP-Seq operation;

FIG. 2 is a SSIP-Seq probe design;

FIG. 3 is a SSIP-Seq paired end sequencing design;

FIG. 4 is a SSIP-Seq two-end sequencing imaging effect diagram;

FIG. 5 is a SSIP-Seq unidirectional double barcode sequencing design;

FIG. 6 is a graph of SSIP-Seq unidirectional double barcode sequencing imaging effect;

FIG. 7 is a SSIP-Seq graphics processing flow diagram;

FIG. 8 in situ detection of m6A methylation of RNA;

FIG. 9 is a statistical plot of m6A methylation in situ detection 10 visual field counts for RNA;

FIG. 10 shows the spatial distribution of mutant and wild types of 5 genes of c6 glioma;

FIG. 11SSIP-Seq reading 3-round hybridization scheme for 42 genes of hypothalamus;

FIG. 12SSIP-Seq reads the spatial distribution map of 42 genes in the hypothalamus.

Detailed Description

The present invention is described in further detail below with reference to specific examples so as to be understood by those skilled in the art.

The detection method suitable for the high-throughput transcriptome spatial position information comprises the following steps:

1) Design of hybridization probes

Screening out a target sequence region with high hybridization efficiency, strong specificity and no RNA advanced structure from an RNA sequence of a cell or tissue to be detected to design a hybridization probe, wherein the hybridization probe consists of a padlock probe and an RCA (Rolling circle amplification) priming probe;

2) Probe pretreatment

Carrying out phosphorylation treatment on the 5' end of a lock type probe in the hybridization probe;

3) Hybridization of samples

4) Ligation reaction

5) Rolling circle amplification

6) In-situ interpretation of spatial information

i. In-situ color development reading spatial information:

a. adding a ligase reaction system into a reaction chamber for a connecting sequencing reaction, then sequencing a first base sequence of the barcode in a lock probe, performing imaging scanning layer by layer on a research area according to the characteristics of a sample to be detected, superposing signals of each layer for maximum projection, and registering and reading information of each fluorescence signal corresponding to the barcode information;

Based on the method, the following detection is carried out according to the actual situation:

example 1

The detection method of the high-throughput transcriptome spatial position information is utilized to detect the RNA methylation in situ,

1. design of hybridization probes

Designing a hybridization probe according to a target sequence containing a methylation site in RNA, wherein the first base at the 5' end of a lock probe in the hybridization probe is complementary with the methylated base of the target sequence,

2. probe pretreatment

Carrying out phosphorylation treatment on the 5' end of the lock-type probe by using T4 polynucleotide kinase;

3. hybridization of samples

(1) Respectively transfecting RNA target sequence nucleic acid molecules to be detected to contain methylation sites and methylation-free sites into 293T cells cultured in an adherent manner, and culturing for 3 hours; preparing a reaction chamber on a petri dish, quickly washing with PBST (PBS containing 0.1% tween 20) for 2 times, and removing residual culture medium and other impurities;

(2) fixing cells with 4% Paraformaldehyde (PFA) for 10min, absorbing and discarding the solution in the reaction chamber, washing with PBST for 3 times, 5min each time, and fully removing the redundant PFA;

(3) adding methanol pre-cooled at-20 deg.C, immediately reacting at-80 deg.C for 15min, and dehydrating and denaturing tissue; taking out the reaction chamber from-80 ℃, then placing the reaction chamber at room temperature for 5 minutes, sucking and discarding the reaction system, and washing the reaction system for 5 minutes by PBST for 3 times;

(4) the hybridization probe was added to the hybridization buffer at a final concentration of 100 nM; and (3) uniformly mixing the hybridization reaction system, adding the mixture into a reaction chamber, placing the reaction chamber into a wet box, and hybridizing the mixture overnight at 40 ℃.

4. Ligation reaction

Uniformly mixing a Splint R ligase reaction system, adding the mixture into a reaction chamber, and reacting for 2 hours at 25 ℃; the padlock probe and a target sequence on the corresponding RNA are complementarily paired to form a semi-closed annular structure;

5. rolling circle amplification

And (3) uniformly mixing the phi29DNA polymerase rolling circle expansion reaction system, adding the mixture into a reaction chamber, and reacting for 2 hours at the temperature of 30 ℃.

6. In situ interpretation of spatial information (fluorescent probe visualization) and data analysis

(1) Preparing a fluorescent probe hybridization system, adding the fluorescent probe hybridization system into a reaction chamber, and reacting for 3 hours at 25 ℃;

(2) the hybridization reaction system in the chamber is discarded, and washed twice with PBST for 5min each time; then, DAPI staining was performed, and a DAPI staining solution having a concentration of 0.1ug/ml was prepared with PBS, and added to the reaction chamber to react at room temperature for 2min.

(3) The Leica TCS SP8 laser confocal imaging is applied to carry out imaging, corresponding fluorescence channels are respectively arranged, the topmost end and the bottommost end of a signal of a tissue section are determined, the tissue is scanned layer by layer in a step diameter of 0.5 mu m, and an integral signal graph of the maximum projection is obtained by superposing images of all layers; as shown in fig. 9, multiple fields were collected for signal statistics, with statistical differences between methylated and unmethylated sites.

Therefore, the method can be applied to methylation sensitivity examination.

Example 2

The detection method for detecting the spatial position information of the high-throughput transcriptome is used for detecting the mutant type and the wild type of 5 genes in a rat glioma tissue section, and comprises the following steps:

1. design of hybridization probes

Selecting mutation sites of related genes of C6 glioma cells to design hybridization probes, wherein the first base at the 5' end of the padlock probes is complementary with the mutated base of the target sequence, and wild probes corresponding to the padlock probes are designed and respectively allocated with different barcode, and the probe sequences are as follows:

2. probe pretreatment

3. hybridization of samples

(1) Freezing the tissue to slice each brain slice to be 10 μm thick, and making an identification mark; PBST was washed 2 times quickly to remove OCT and other impurities around the remaining tissue.

(2) Brain tissue was fixed with 4% Paraformaldehyde (PFA) for 10min, the solution in the reaction chamber was aspirated and washed with PBST 3 times for 5min each time to remove excess PFA sufficiently.

(3) Adding methanol pre-cooled at-20 deg.C, immediately reacting at-80 deg.C for 15min, and performing tissue dehydration and denaturation treatment; the reaction chamber was removed from-80 ℃ and then allowed to equilibrate at room temperature for 5min, the reaction was aspirated off and washed 3 times with PBST for 5min each.

4. Ligation reaction

Uniformly mixing a ligase reaction system, adding the mixture into a reaction chamber, and reacting for 2 hours at 25 ℃;

5. rolling circle amplification

And (3) uniformly mixing the phi29DNA polymerase rolling ring extension reaction system, adding the mixture into a reaction chamber, and reacting for 2 hours at the temperature of 30 ℃.

Barcode sequencing (applying the principle of sequencing by ligation) and data analysis interpretation high-throughput spatial information:

(1) and preparing a sequencing reaction system, putting the sequencing reaction system into a reaction chamber, and reacting for 3 hours at 25 ℃.

(2) The hybridization reaction system in the chamber was aspirated and washed twice with PBST for 5min each time. Then, DAPI staining was performed, and a DAPI staining solution with a concentration of 0.1ug/ml was prepared with PBS, and added to the reaction chamber to react at room temperature for 2min.

(3) Leica TCS SP8 laser confocal is used for imaging, corresponding fluorescence channels are respectively arranged, the topmost end and the bottommost end of a signal of a tissue section are determined, the tissue is scanned layer by layer in a step diameter of 05 mu m, and images of all layers are superposed to obtain an integral signal diagram of the maximum projection.

As shown in fig. 10, visualization of wild type and mutant transcripts was achieved by reading the corresponding gene barcode, and it was observed that a large amount of mutant genes were greatly enriched in tumor cells.

Example 3

The detection method for detecting 42 genes in-situ space transcriptome in a hypothalamus region by using the high-throughput transcriptome space position information comprises the following steps:

1. design of hybridization probes

The selected genes comprise markers of specific cells and related genes of functions thereof, and corresponding padlock probes and RCA probes are designed aiming at target sequences of the RNAs, wherein the probe sequences are as follows:

2. probe pretreatment

Phosphorylation of the lock probes was performed using T4 PNK.

3. Hybridization of samples

(1) Freezing the tissue to slice each brain slice with the thickness of 10 μm, and making related marks; PBST was washed 2 times quickly to remove OCT and other impurities around the remaining tissue.

(2) Brain tissue was fixed with 4% Paraformaldehyde (PFA) for 10min, the solution in the reaction chamber was aspirated away, washed with PBST 3 times, 5min each time, and the excess PFA was removed sufficiently.

(3) Adding methanol pre-cooled at-20 deg.C, immediately reacting at-80 deg.C for 15min, and performing tissue dehydration and denaturation treatment; the reaction chamber was removed from-80 ℃ and then allowed to equilibrate at room temperature for 5 minutes, the reaction was aspirated and washed 3 times with PBST for 5min each.

4. Ligation reaction

And (3) uniformly mixing the ligase reaction system, adding the mixture into a reaction chamber, and reacting for 2 hours at 25 ℃.

5. Rolling circle amplification

Barcode sequencing (applying the principle of sequencing by ligation) and data analysis, interpretation of high-throughput spatial information:

(1) first barcode sequencing

a. And (3) preparing a sequencing reaction system, adding the sequencing reaction system into a reaction chamber, and reacting for 3 hours at 25 ℃.

b. The hybridization reaction system in the chamber is discarded, and washed twice with PBST for 5min each time; then, DAPI staining is carried out, a DAPI staining solution with the concentration of 0.1ug/ml is prepared by PBS, and the DAPI staining solution is added into a reaction chamber and reacts for 2min at room temperature; imaging buffer was added and the first round of imaging was performed.

c. Leica TCS SP8 laser confocal imaging is applied to imaging, corresponding fluorescence channels are respectively arranged, the topmost end and the bottommost end of a signal of a tissue slice are determined, the tissue is scanned layer by layer in a 0.5-micrometer step diameter, and images of all layers are superposed to obtain an integral signal diagram of a maximum projection.

(2) Second digit barcode sequencing

Washing twice with 60% formamide washing buffer solution at room temperature for 20 minutes each time, stripping a first round of signals, and reading a second-position barcode by using a second round of sequencing primer anchor-2 according to a reaction system and steps in the first round of barcode sequencing; the second imaging round must maintain the same position and parameters as the first imaging round, and a second image acquisition round is performed.

(3) Third digit barcode sequencing

Washing twice with 60% formamide washing buffer solution at room temperature for 20 min, stripping the second round of signals, reading the third barcode with a third round of sequencing primer anchor-3 according to the reaction system and steps in the previous round of barcode sequencing, and keeping the imaging position and related parameters unchanged; a third image acquisition is performed.

(4) Data analysis

As shown in fig. 11, 3 rounds of pictures are registered, barcode is calibrated, the spatial position distribution of each group is determined, and then cell segmentation is performed; finally, analyzing single cell data; reducing the delicate molecular structure of the tissue.

Other parts not described in detail are prior art. Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.

Claims

1. A detection method suitable for high-throughput transcriptome spatial position information is characterized in that: the method comprises the following steps:

1) Design of hybridization probes

Screening a target sequence region with high hybridization efficiency, strong specificity and no RNA advanced structure from an RNA sequence of a cell or tissue to be detected to design a hybridization probe, designing the hybridization probe according to the target sequence containing a methylation site in RNA, wherein a first base at the 5' end of a lock probe in the hybridization probe is complementary with a methylated base of the target sequence, wherein the hybridization probe consists of a lock probe and an RCA (Rolling circle amplification) initiation probe; the target sequence of the RNA sequence consists of 3 sections of continuous target sequences A, B and C, the padlock probe is provided with an A 'sequence, a loop sequence and a B' sequence from 5 'end to 3' end, and the A 'sequence and the B' sequence are respectively complementary with the target sequence A and the target sequence B, so that the padlock probe and the target sequence are complementarily paired to form a semi-closed annular structure;

2) Probe pretreatment

3) Hybridization of samples

4) Ligation reaction

Adding a ligase reaction system into the reaction chamber for a ligation reaction, and enabling the padlock probes to be complementary and paired with target sequences on corresponding RNAs to form a semi-closed annular structure; wherein, the ligase of the ligase reaction system is Splint R ligase;

5) Rolling circle amplification

6) In-situ color development reading spatial information:

and adding a fluorescent probe hybridization system which is reversely complementary with the color development sequence of the rolling circle amplification product into the reaction chamber for hybridization in-situ color development, then imaging the research area of the sample to be detected, and reading the RNA space position information.

2. The method for detecting spatial position information of high-throughput transcriptome according to claim 1, wherein: in the RNA sequence, the lengths of a target sequence A, a target sequence B and a target sequence C are 11-16 bp.

3. The method for detecting spatial position information of high-throughput transcriptome according to claim 1, wherein: in the step 2), the enzyme used in the phosphorylation treatment process is T4 polynucleotide kinase T4 PNK.

4. Use of the method of claim 1 for the detection of point mutations and methylation sites.