CN116445594A - Sequencing method suitable for in-situ detection of continuous multiple nucleotide sites and application thereof - Google Patents

Abstract

The invention discloses a sequencing method suitable for in situ detection of multiple consecutive nucleotide sites and its application. The method designs a semi-closed locking probe that captures the target region, and fills in the gaps on the probe with ligase. Vacancies, the sequencing of the unknown sequence to be detected is realized by rolling circle amplification, and X number of bases to be detected can be resolved by multiple rounds of ligation sequencing. According to its probe design characteristics, it can sequence unknown nucleic acid sequences, and can be applied to tumor point mutation detection, continuous mutation detection, etc. It can realize in situ interpretation of gene sequences with single-cell, single-molecule, and single-base resolution. This method can perform high-throughput tissue in situ detection, and obtain in situ spatial spectra of target sequences in different cells of tissue samples, without the need for difficult in vitro oligo-specific cell enrichment library construction and sequencing. Moreover, the signal interpretation does not depend on the super-resolution imaging platform, and has good application prospects in the fields of individual development, genetic breeding, tumor microenvironment, and clinical pathological detection.

Description

Sequencing method suitable for in situ detection of multiple nucleotide sites and its application

technical field

The invention relates to the field of in situ sequencing, in particular to a sequencing method suitable for in situ detection of multiple consecutive nucleotide sites and its application.

Background technique

Cells are the basic units of structure and function in living organisms. Since the establishment of the cell theory in 1839, countless scientists have devoted themselves to studying the deconstruction, function and relationship between cells, in order to ascertain the essence of life. In 1958, Watson and Crick put forward the central dogma, explaining the order in which genetic information is transmitted between biological macromolecules, and the era of molecular biology kicked off. On this basis, modern biology disciplines such as genomics, transcriptomics, and proteomics are flourishing, and scientists are setting sail to explore the mysteries of life activities. Among them, mRNA is transcribed from DNA and synthesizes protein with the assistance of ribosomes. It is the carrier of genetic information, the blueprint of protein translation, and plays a vital role in life activities. So far, tens of thousands of researchers around the world are devoted to the research of mRNA.

Technological change is fundamental to promote the development of research. The early research on mRNA was restricted by experimental techniques. It was necessary to extract mRNA from cells or tissues first, and then through Northern blot hybridization (Northern blot), fluorescent quantitative real time PCR (qPCR), the first Generational sequencing technology, etc. to study. Such methods are time-consuming and laborious, and the information that can be obtained is also very limited. With the promotion of next-generation sequencing technology, scientists have gradually realized large-scale parallel sequencing of mRNA and developed a series of RNA sequencing technologies (RNA-seq). Over the past decade, RNA-seq has emerged as an important tool for transcriptome-wide analysis of differential gene expression and differential mRNA splicing, including single-cell gene expression, translation, and RNA structure.

Traditional RNA sequencing (such as bulk RNA-seq) is to extract RNA from a whole tissue for library construction and sequencing to obtain the average expression level of various cell transcripts in this region. However, different cells in tissues have different functions and mRNA expression levels are also different. The normalization strategy of conventional RNA-seq will cover up many important information. In order to explore the characteristics and functions of different cells in tissues, in 2009, Professor Tang Fuchou improved based on the previous technology and formed the first real single cell RNA sequencing technology (single cell RNA sequencing, scRNA-seq).

In 2013, Nature Method awarded Technology of the Year to single-cell sequencing. This technology can provide the RNA expression profile of each cell and identify rare cells in heterogeneous cell populations, which greatly promotes the research of developmental biology, neurobiology, cancer mechanism and so on. The scRNA-seq method and bulk RNA-seq technology work together to provide researchers with highly detailed data about a tissue or cell population, improving people's understanding of the identity and status of cells within the tissue. However, the common disadvantage of such methods is the loss of spatial information of mRNA in cells. This type of information is important because specific cell types and tissues are closely related to life activities.

In order to study the spatial information of transcripts and analyze the close relationship between cell environment and gene expression, scientists proposed spatial transcriptomics (spatial transcriptomics, ST). Among them, the imaging-based RNA in situ sequencing method (in situ sequencing, ISS) can directly sequence the mRNA in cells in the tissue environment, so that not only the specific sequence of a specific transcript of a single cell can be obtained, but also its The spatial position and the corresponding expression level can be used to calibrate the obtained mRNA information to its real place, which is a relatively ideal way.

Currently, commonly used mRNA in situ sequencing can be divided into two categories according to the method used to capture transcripts:

1) One category is the method of non-targeted capture of transcripts, representative technologies include FISSEQ and INSTA-Seq. This type of method can theoretically capture all transcripts and detect the information of all transcripts in the cell, but in practice this type of technology has poor specificity, there are more rRNA signals in the obtained data, and the detection efficiency of mRNA is low, and often The steps are cumbersome, and there are not many applications at present.

2) Another method is the method of targeted capture of transcripts, and the representative technology is STARmap (Spatially-resolved Transcript Amplicon Readout Mapping). This type of method usually relies on a padlock probe, which is used to capture a specific transcript in situ. This type of method is usually more sensitive and specific, but the sequencing throughput is low. A padlock probe The needle can only detect one mutation at one site, and it is necessary to design multiple probes for slightly longer sequencing, which is cumbersome and costly.

As shown in Figure 1: when performing in situ sequencing with classical padlock probes, once there is a mutation in the sequence to be tested, it is necessary to design and synthesize a variety of probes based on prior knowledge. When there is a point mutation at the site to be tested, because the mutation result may be any of A\T\C\G, it is necessary to synthesize corresponding probes, at least 4 kinds, and it is also necessary to know the specificity of the mutated base position to be captured with a classic padlock probe. Once there are several consecutive mutations, or even multiple mutations appearing at the same time, it is necessary to design a large number of probes for detection. Using a variety of classical probes in one experiment not only greatly increases the cost, but also reduces hybridization efficiency and increases non-specificity.

How to directly, targeted and simply perform in situ sequencing of nucleic acids is a top priority for current technology development.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art, and to provide a sequencing method suitable for in situ detection of multiple nucleotide sites and its application. The present invention uses a semi-closed padlock probe for specific hybridization The target nucleic acid is filled with a single-stranded random deoxyribonucleotide into the gap formed by the padlock probe by ligation to form a complete circular structure, and the captured information is amplified in situ by rolling circle amplification. Then, the corresponding sequence in the Gap was interpreted by ligation sequencing, and finally the image analysis was performed to obtain the spatial information of the transcript. This method only needs 4 rounds of ligation sequencing to measure the sequence of 4 consecutive bases on the nucleic acid. Using ordinary padlock probes, 4 ⁴ =256 probes are required to measure 4 base sequences, but only one probe is required in this method, which greatly reduces the workload of probe design and the cost of preparation. The invention can directly sequence the RNA in situ without reverse transcribing it into cDNA, and retains the spatial information, reduces the operation steps and improves the detection efficiency. The method can detect an unknown sequence without prior knowledge, and has strong specificity and high error tolerance.

In order to achieve the above object, the technical scheme designed by the present invention is as follows:

The present invention provides a sequencing method suitable for in situ detection of multiple consecutive nucleotide sites, comprising the following steps:

1) Design of the probe

According to the sequence to be detected, a target region with high hybridization efficiency, good specificity, and no advanced structure is screened out, and hybridization probes are set; wherein, the hybridization probes include semi-closed lock probes and RCA priming probes;

After the half-closed lock probe is complementary to the target sequence of the sequence to be detected, a gap will be formed between the 5' and 3' ends of the half-closed lock probe,

The gap is filled by a random fragment of single-stranded DNA of X bases (5'-NNNN-3') and is complementary to the target sequence of the sequence to be detected (ie, the sequencing target); wherein, X is 2, 3 , 4, 5, 6, 7, 8...;

2) Probe pretreatment

Phosphorylate the 5' end of the semi-closed padlock probe, a random fragment of single-stranded deoxyribose nucleic acid of X bases in the hybridization probe;

3) Sample hybridization

Prepare a reaction chamber on the sample to be tested, fix the sample with 4% paraformaldehyde (PFA), then dehydrate and denature it with methanol, then add the system containing the hybridization probe into it and incubate for a period of time;

4) Ligation reaction

Add the ligation system of random fragments of single-stranded deoxyribonucleic acid containing X bases into the reaction chamber, so that the gap (Gap) on the semi-closed locking probe that is complementary to the sequence to be detected is filled to form a closed ring structure;

5) Rolling circle amplification

The RCA priming probe is complementary to the target sequence of the sequence to be detected and the semi-closed lock probe at the same time. Under the action of Phi29 DNA polymerase, the RCA priming probe is used as a primer, and the semi-closed lock probe forms a circular structure As a template, rolling circle amplification is performed to amplify the target signal;

6) Sequencing the target sequence in situ (applying the principle of ligation sequencing)

Ⅰ. In situ sequencing

Add the sequencing system containing anchor primers and sequencing primers into the reaction chamber for in situ sequencing reaction, sequence and image the sequence corresponding to the first base in the gap (Gap) between semi-closed probes, and interpret The spatial position and sequence information of the target sequence;

Ⅱ. Interpret the base sequence information at other positions in the gap (Gap) between the half-closed padlock probes sequentially in the same way;

III. Match the multiple rounds of sequencing information obtained above, and interpret the target sequence corresponding to the gap (Gap) formed between the half-closed probes.

Further, in the step 1), the target sequence of the sequence to be detected is composed of 4 consecutive target sequences A, D, B and C, and the semi-closed lock probe is from the 5' end to the 3 end'. A' sequence, loop sequence and B' sequence,

The A' sequence and the B' sequence are complementary to the target sequence A and the target sequence B respectively, and the semi-closed lock probe forms a semi-closed loop structure after complementary pairing with the target sequence; the loop sequence consists of an anchor sequence Composed of barcode sequences;

The vacant sequence D' is complementary to the target sequence D.

Still further, in the step 1), the vacant sequence D' is composed of a sequence Y, wherein Y is less than X and Y is 1, 2, 3, 4, 5, 6, 7, 8...( That is: X=x ₁ +x ₂ +...+x _Y , X is the number of all bases in the gap, Y is the number of segments forming the random sequence in the gap, and x is the number of bases in each sequence).

Still further, in the vacant sequence D', X is 2-8, and Y is 1-6.

Still further, in the step 1), X is 4 (the sequence D' of the vacancy is composed of random fragments of single-stranded deoxyribonucleic acid of 4 bases), and Y is 1.

Further, in the step 2), the 5' end of the random fragment of single-stranded deoxyribose nucleic acid is phosphorylated.

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

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

Further, in the step 6), the target sequence is sequenced in situ

Ⅰ. In situ sequencing

Add the sequencing system containing anchor primers and sequencing primers into the reaction chamber for in situ sequencing reaction, sequence and image the sequence corresponding to the first base in the gap (Gap) between semi-closed probes, and interpret The number, spatial location and sequence information of the target sequence;

Ⅱ. Interpret the information of the second, third, and fourth base sequences in the gaps (Gap) between semi-closed locked probes in the same way;

III. Match the four rounds of sequencing information obtained above, and interpret the target sequence corresponding to the gap (Gap) formed between the semi-closed probes.

The present invention also provides an application of the above-mentioned sequencing method suitable for in situ detection of multiple consecutive nucleotide sites in the detection of point mutations.

Principle of the present invention:

In the present invention, after the half-closed lock probe is complementary to the target sequence of the sequence to be detected, a gap will be formed between the 5' and 3' ends of the half-closed lock probe,

The vacancy can be filled by random fragments of single-stranded deoxyribonucleic acid of X base length, and connected with padlock probes to form a complete circular structure. This process relies on the ligase to recognize the precise complementary pairing of the bases at the 5' and 3' ends of the random fragments and the target sequence. If any end does not match, it cannot be ligated by the ligase to form a closed loop. At the same time, when the middle part of the random fragment does not completely match the target sequence, the random fragment is easily dissociated from the target sequence due to the weak hydrogen bond force formed by hybridization. Only fragments that are completely complementary can be stably filled into the gap (Gap) and connected to the padlock probe by ligase. Target sequences include, but are not limited to, DNA or RNA.

Beneficial effects of the present invention:

1. High specificity: The present invention uses a semi-closed padlock probe to capture specific transcripts, and uses random fragments of single-stranded DNA to fill in the Gap, and its signal amplification depends on two parts: the padlock probe capture area is the same as Precise complementary pairing of target sequences, and precise filling of random fragments. Only target fragments that meet the above conditions at the same time can be finally detected, which improves the specificity of the method.

2. The probe is simple in design and low in cost. Traditional in situ sequencing technology requires 4 ⁴ = 256 probes to measure consecutive 4 base sequences. The present invention only needs 1 padlock probe, combined with random fragments of 4-base single-stranded deoxyribonucleic acid. Measure the sequence of 4 consecutive bases on the RNA. The probe design is simplified and the cost is greatly reduced.

3. It can be used to detect gene mutations. The present invention can directly sequence point mutations or continuous mutations of a certain length on the sequence, and has good application prospects in tumor mutation detection. The present invention can also detect the deletion mutation of a single site, for example: use a padlock probe that is expected to form a Gap of 4 bases to detect a site that may have a deletion mutation, and add an additional 3 bases to the connection system If a deletion mutation occurs in a random fragment of strand deoxyribonucleic acid, the padlock probe forms a 3-base gap, which can also be filled in, and the information of the deletion mutation can be obtained through subsequent sequencing.

4. This method can directly detect a nucleic acid with an unknown sequence, and only need to know the fragments on both sides when designing the probe.

5. High universality of imaging conditions: the confocal imaging system can realize signal interpretation. The invention amplifies the signal through the method of rolling circle amplification, and the result has high brightness and can be detected by a confocal imaging system.

6. The present invention can realize in situ sequencing at the single cell level.

7. The present invention directly targets mRNA without reverse transcription into cDNA, which can improve accuracy, save cost, and is easy to operate.

8. The connection step uses SplintR enzyme, which is more specific and efficient.

In summary, the present invention uses a semi-closed padlock probe to accurately fill in random fragments of 4-base single-stranded deoxyribonucleic acid in the Gap under the action of the SplintR enzyme to form a complete circular structure, and then pass Rolling circle amplification amplifies the signal and reads the sequencing results. Compared with the traditional method, the probe design of the present invention is simpler, saves time and effort, and is low in cost, and can directly capture the sequence on the mRNA, and has a good application prospect for detecting mutations. Moreover, the present invention uses a confocal imaging system to capture signals without requiring a super-resolution imaging system, which reduces the cost and difficulty of experiments and is easy to popularize.

Description of drawings

Figure 1 is a design diagram of a classic padlock probe;

Fig. 2 is the design drawing of the hybridization probe of the present invention;

Fig. 3 is the technical flowchart of the Fill-in method of the present invention;

Fig. 4 is the effect diagram of sequencing imaging of the Fill-in method of the present invention;

In the figure, the red dot represents the detected sequence is guanine G, and the indigo dot represents cytosine C.

Detailed ways

The present invention will be described in further detail below in conjunction with specific embodiments, so that those skilled in the art can understand.

The present invention provides a sequencing method suitable for in situ detection of multiple consecutive nucleotide sites, comprising the following steps:

1) Design of the probe

According to the sequence to be detected, a target region with high hybridization efficiency, good specificity, and no advanced structure is screened out, and hybridization probes are set; wherein, the hybridization probes include semi-closed lock probes and RCA priming probes;

After the half-closed lock probe is complementary to the target sequence of the sequence to be detected, a gap will be formed between the 5' and 3' ends of the half-closed lock probe,

The gap is filled by a random fragment of single-stranded DNA of X bases (5'-NNNN-3') and is complementary to the target sequence of the sequence to be detected (ie, the sequencing target); wherein, X is 2, 3 , 4, 5, 6, 7, 8...;

2) Probe pretreatment

Phosphorylate the 5' end of the semi-closed padlock probe, a random fragment of single-stranded deoxyribose nucleic acid of X bases in the hybridization probe;

3) Sample hybridization

Prepare a reaction chamber on the sample to be tested, fix the sample with 4% paraformaldehyde (PFA), then dehydrate and denature it with methanol, then add the system containing the hybridization probe into it and incubate for a period of time;

4) Ligation reaction

Add the ligation system of random fragments of single-stranded deoxyribonucleic acid containing X bases into the reaction chamber, so that the gap (Gap) on the semi-closed locking probe that is complementary to the sequence to be detected is filled to form a closed ring structure;

5) Rolling circle amplification

The RCA priming probe is complementary to the target sequence of the sequence to be detected and the semi-closed lock probe at the same time. Under the action of Phi29 DNA polymerase, the RCA priming probe is used as a primer, and the semi-closed lock probe forms a circular structure As a template, rolling circle amplification is performed to amplify the target signal;

6) Sequencing the target sequence in situ (applying the principle of ligation sequencing)

Ⅰ. In situ sequencing

Add the sequencing system containing anchor primers and sequencing primers into the reaction chamber for in situ sequencing reaction, sequence and image the sequence corresponding to the first base in the gap (Gap) between semi-closed probes, and interpret Spatial location and sequence information of the target sequence.

Ⅱ. Interpret the base sequence information of other positions in the gap (Gap) between the semi-closed padlock probes sequentially in the same way.

III. Match the multiple rounds of sequencing information obtained above, and interpret the target sequence corresponding to the gap (Gap) formed between the half-closed probes.

Based on the above method, the following tests are carried out according to the actual situation:

Example 1

In situ sequencing of 4-base-long sequences on β-actin transcripts using the above-mentioned sequencing method for in situ detection of consecutive multiple nucleotide sites

1. Design of Hybridization Probes

According to the target sequence of the sequence to be detected, a segment with high hybridization efficiency, good specificity, and no advanced structure is screened out, and a hybridization probe is designed, which targets a sequence on the mRNA. Table 1

2. Probe Pretreatment

Apply T4 polynucleotide kinase (PNK) to phosphorylate the 5' end of the semi-closed padlock probe, a random fragment of 4-base single-stranded deoxyribonucleic acid;

3. Sample hybridization

① This method is tested on cell samples. To culture cells to a sufficient number for experiments, and to facilitate subsequent operations, first, culture cells in a glass-bottomed Petri dish to 80% full, discard the medium before the experiment, and prepare a reaction chamber on the glass-bottomed Petri dish . Wash with PBST (PBS containing 0.1% Tween-20) 3 times to remove residual medium and other impurities.

②In order to maintain the shape of the cells and keep the mRNA in the original spatial position, fix the cells with 4% paraformaldehyde for 10 minutes, discard the liquid after the reaction is completed, wash 3 times with PBST at room temperature, 5 minutes each time, remove excess PFA.

③In order to remove the original water in the cells and facilitate the entry and reaction of reagents, pre-cooled 100% methanol was added to the fixed cells, and immediately incubated at -80°C for 15 minutes to dehydrate and denature the tissues. After the reaction was completed, the sample was taken out from -80°C, placed at room temperature for 5 minutes, and then the liquid was discarded, and washed 3 times with PBST at room temperature, 5 minutes each time.

④ In order to increase cell permeability and allow macromolecular reagents in subsequent reaction reagents to easily enter the cells, use 0.1M hydrochloric acid solution to permeabilize the cell membrane to form a hole structure on the cell membrane. In this process, the cells were treated at room temperature for 5 minutes, the liquid was discarded after the reaction was completed, and the cells were washed 3 times with PBST at room temperature, 5 minutes each time.

⑤ After the cell treatment is completed, the hybridization solution containing the padlock probe is added to the cell sample, and the final concentration of the probe is 100nM. After overnight incubation at 37°C, the padlock probes bind to the target mRNA and form a complementary structure. Discard the liquid after the reaction is complete, wash with PBST 3 times at room temperature, 5 minutes each time.

4. Ligation reaction

After the padlock probe hybridizes to the mRNA, the gap formed needs to be filled. At this time, the ligation system containing 4-base random fragments was added into the reaction chamber, and the final concentration of the random fragments was 100 nM. Incubate at 25°C for 2 hours, under the action of ligase, the random fragments that are complementary to the RNA are connected to the semi-closed padlock probe to form a complete circular structure. Discard the liquid after the reaction is complete, wash with PBST 3 times at room temperature, 5 minutes each time.

5. Rolling Circle Amplification

After the lock probe is formed into a circle, in order to read the target sequence information, the captured information must be amplified. This step uses rolling circle amplification to amplify the lock probe. Configure a rolling circle amplification system containing Phi29 DNA polymerase, use the RCA priming probe as a primer, and the half-closed locking probe that completes the ligation reaction as a template, mix well and add to the reaction chamber, and incubate at 30°C for 2 hours. Discard the liquid after the reaction is complete, wash with PBST 3 times at room temperature, 5 minutes each time.

6. In situ sequencing (application of the principle of ligation sequencing)

① The first round of sequencing

a. Add the ligation sequencing system containing anchor primer Anchor-1 and 4 kinds of sequencing primers into the reaction chamber for in situ sequencing reaction, and incubate at 25°C for 2 hours. The sequences of anchor primers and sequencing primers are shown in Table 2.

Table 2

Discard the liquid after the reaction is complete, wash with PBST 3 times at room temperature, 5 minutes each time. Add 0.1 μg/mL DAPI to stain cell nuclei and incubate at room temperature for 5 minutes. Discard the liquid after the reaction is complete, wash with PBST 3 times at room temperature, 5 minutes each time.

b. Apply Leica TCS SP8 laser confocal imaging, read the sequencing result of the first base to be tested, and interpret the quantity, spatial position and sequence information of the target mRNA at the same time. Set the channels corresponding to different fluorescence, observe and set the range of cell signal, scan the sample with a step of 0.4 μm, and then superimpose the images of each layer to obtain the image of the overall signal.

②The second round of sequencing

a. Prepare 70% formamide elution buffer, add it to the reaction chamber, and incubate at room temperature for 5 minutes to elute the first-round sequencing signal. Discard the liquid after the reaction is complete, wash with PBST 3 times at room temperature, 5 minutes each time.

b. Add the ligation sequencing system containing the anchor primer Anchor-2 and 4 kinds of sequencing primers into the reaction chamber, and read the sequencing results of the second base to be tested according to the reaction time and process of the first round of sequencing. In the second round of imaging, the same position and parameters as the first imaging must be maintained.

③ The third round of sequencing

a. Prepare 70% formamide elution buffer, add it to the reaction chamber, and incubate at room temperature for 5 minutes to elute the second-round sequencing signal. Discard the liquid after the reaction is complete, wash with PBST 3 times at room temperature, 5 minutes each time.

b. Add the ligation sequencing system containing the anchor primer Anchor-3 and 4 kinds of sequencing primers into the reaction chamber, and read the sequencing results of the third base to be tested according to the reaction time and process of the first round of sequencing. In the third round of imaging, the same position and parameters during imaging must be maintained.

③ The fourth round of sequencing

a. Prepare 70% formamide elution buffer, add it to the reaction chamber, and incubate at room temperature for 5 minutes to elute the sequencing signal of the third round. Discard the liquid after the reaction is complete, wash with PBST 3 times at room temperature, 5 minutes each time.

b. Add the ligation sequencing system containing anchor primer Anchor-4 and 4 kinds of sequencing primers into the reaction chamber, and read the sequencing results of the fourth base to be tested according to the reaction time and process of the first round of sequencing. In the fourth round of imaging, the same position and parameters during imaging must be maintained.

7. Data analysis

The four rounds of sequencing information obtained above were matched, and the mRNA sequence captured in the Gap formed by the padlock probe was read out.

As shown in Figure 4: the target sequence is located on the mRNA transcribed by the β-Actin gene, and the part to be tested is a sequence of 4 bases long on it. According to the existing data, the sequence is 5'-GGCC-3'. During the experiment, the sequencing probes used emit different fluorescent colors, corresponding to different bases sequenced. The corresponding relationship is as follows: green - sequenced to A (adenine); red - sequenced to C (cytosine); magenta - sequenced to T (thymine); indigo - sequenced to G (guanine) . Sequencing from the 5' end to the 3' end of the sequence to be tested, according to our experimental results, the color of the image obtained is: red-red-indigo-indigo, that is, the corresponding sequencing result is: 5'-GGCC-3' , exactly as expected.

Other parts not specified in detail are all prior art. Although the foregoing embodiment has described the present invention in detail, it is only a part of the embodiments of the present invention, rather than all embodiments, and people can also obtain other embodiments according to the present embodiment without inventive step, these embodiments All belong to the protection scope of the present invention.

Claims (10)

1. A sequencing method suitable for in situ detection of a plurality of consecutive nucleotide sites, characterized by: the method comprises the following steps:

1) Design of probes

Screening out a target area with high hybridization efficiency, good specificity and no advanced structure according to a sequence to be detected, and setting a hybridization probe; wherein the hybridization probe comprises a semi-closed lock-type probe and an RCA initiation probe;

after the semi-closed lock probe is complemented with a target sequence of a sequence to be detected, a gap is formed between the 5 'end and the 3' end of the semi-closed lock probe,

the gap is filled by a single-stranded deoxyribonucleic acid random fragment with X bases and is complementary with a targeting sequence of a sequence to be detected; wherein X is 2, 3, 4, 5, 6, 7, 8 and … …;

2) Probe pretreatment

Phosphorylating the 5' end of a semi-closed lock probe and a single-stranded deoxyribonucleic acid random fragment of X bases in the hybridization probe;

3) Sample hybridization

Preparing a reaction chamber on a sample to be detected, fixing the sample by using 4% Paraformaldehyde (PFA), dehydrating and denaturing the sample by using methanol, and adding a system containing a hybridization probe into the reaction chamber for incubation for a period of time;

4) Ligation reaction

Adding a connection system containing single-stranded deoxyribonucleic acid random fragments with X bases into a reaction chamber, filling gaps (Gap) on a semi-closed lock-type probe complementarily paired with a sequence to be detected, and forming a closed annular structure;

5) Rolling circle amplification

The RCA initiating probe is complementary with the targeting sequence of the sequence to be detected and the semi-closed lock probe at the same time, and under the action of Phi29DNA polymerase, rolling circle amplification is carried out by taking the RCA initiating probe as a primer and the annular structure formed by the semi-closed lock probe as a template, so that a target signal is amplified;

6) In situ sequencing of target sequences

In situ sequencing

Adding a sequencing system containing an anchor primer and a sequencing primer into a reaction chamber for in-situ sequencing reaction, sequencing and imaging a sequence corresponding to a first base in a gap between semi-closed lock probes, and reading the spatial position and sequence information of a target sequence of a sequence to be detected;

sequentially reading information of base sequences at other positions in the gaps between the semi-closed lock probes by the same method;

and III, matching the obtained multiple rounds of sequencing information, and reading a target sequence corresponding to the gap formed between the semi-closed lock probes.

2. The sequencing method of claim 1 adapted for in situ detection of a plurality of consecutive nucleotide sites, wherein: in the step (1) of the above-mentioned process,

the targeting sequence of the sequence to be detected consists of 4 segments of continuous target sequences A, D, B and C, the semi-closed lock probe is A 'sequence from 5' end to 3 'end, loop sequence and B' sequence,

the A 'sequence and the B' sequence are respectively complementary with the target sequence A and the target sequence B, and the semi-closed lock type probe and the target sequence are complementary and paired to form a semi-closed annular structure; the loop sequence consists of an anchor sequence and a barcode sequence respectively;

the empty sequence D' is complementary to the target sequence D.

3. The sequencing method suitable for in situ detection of a plurality of consecutive nucleotide sites according to claim 2, wherein: in step 1), the vacant sequence D' is composed of a Y segment sequence, Y is smaller than X and is 1, 2, 3, 4, 5, 6, 7 and 8 … ….

4. A sequencing method suitable for in situ detection of a plurality of consecutive nucleotide sites according to claim 3, wherein: in the vacant sequence D', X is 2-8 and Y is 1-6.

5. The sequencing method of claim 4 adapted for in situ detection of a plurality of consecutive nucleotide sites, wherein: in the step 1), X is 4, and Y is 1.

6. The sequencing method of claim 1 adapted for in situ detection of a plurality of consecutive nucleotide sites, wherein: in the step 2), 5' -end of the random fragment of single-stranded deoxyribonucleic acid is phosphorylated.

7. The sequencing method of claim 1 adapted for in situ detection of a plurality of consecutive nucleotide sites, wherein: in the step 2), the enzyme used in the phosphorylation process is T4 polynucleotide kinase.

8. The sequencing method of claim 1 adapted for in situ detection of a plurality of consecutive nucleotide sites, wherein: in the step 4), the ligase of the ligase reaction system is a Splint R ligase.

9. The sequencing method of claim 5 adapted for in situ detection of a plurality of consecutive nucleotide sites, wherein: in step 6), the target sequence is sequenced in situ

In situ sequencing

Adding a sequencing system containing an anchor primer and a sequencing primer into a reaction chamber for in-situ sequencing reaction, sequencing and imaging a sequence corresponding to a first base in a gap between semi-closed lock probes, and reading the number, the spatial position and the sequence information of a target sequence;

sequentially reading the information of the base sequences of the second position, the third position and the fourth position in the gap between the semi-closed lock probes by the same method;

and III, matching the obtained four-wheel sequencing information, and reading a targeting sequence corresponding to the gap formed between the semi-closed lock probes.

10. Use of a sequencing method suitable for in situ detection of consecutive nucleotide sites according to claim 1 in point mutation detection.