A decoding method for spatial transcriptome chip and application thereof

Abstract

The application relates to a decoding method of a spatial transcriptome chip and application thereof. The application designs a decoding scheme based on an auxiliary primer, uses the auxiliary primer to selectively connect a corresponding barcode and a decoding probe, and does not add the corresponding auxiliary primer, so that the corresponding barcode and the decoding probe cannot be connected, no fluorescence is generated, and conversely, fluorescence is generated. Decoding can be sequentially performed. Compared with a traditional decoding scheme of simply adjusting a decoding probe, the auxiliary primer does not need to be fluorescently modified, is convenient to synthesize, has a lower cost, can be freely combined into a pool, is convenient for later updating and iteration of a hybridization scheme, a smaller number of decoding fluorescent probes are required, decoding cost can be greatly reduced, in addition, the two sides of the auxiliary primer can increase the binding region of the decoding probe, so that the imaging brightness is doubled, and the operation success rate of decoding is greatly improved.

Description

Technical Field

[0001] This application belongs to the field of space omics technology and relates to a decoding method for space transcriptome chips. Background Technology

[0002] In recent years, spatial transcriptomics technology has developed rapidly, achieving breakthroughs from low-resolution multicellular to single-cell and even subcellular high-resolution levels. This is thanks to the upgrading and optimization of biochemical reaction enzyme systems and the advancement of chip manufacturing processes.

[0003] Mainstream spatial transcriptome chips are divided into three types. The first type is solid-phase barcode chips, such as the 10×GenomicsVisium and Visium HD platforms, with a resolution of 2μm-55μm / spot. The second type is microsphere array chips, such as Slide-seq, HDST, and BMKMANU S series, with a resolution of 2-10μm. The third type is DNA nanosphere array chips, with BGI Stereo-seq being a typical example, with a maximum resolution of 500 nm.

[0004] Spatial transcriptome chip decoding refers to mapping spatial barcode sequences to specific physical locations on the chip. This step is the foundation for obtaining gene expression location information. Specifically, it may include predicting the sequence of the chip to obtain a unique spatial barcode sequence for each capture unit. After sequencing, the coordinates of the data can be mapped using the barcode tags to restore the original location of the mRNA.

[0005] Multi-round hybridization imaging decoding can achieve ultra-high resolution decoding, enabling the analysis of fine tissue structures and the developmental and tumor microenvironment analyses. This decoding method also boasts high localization accuracy and strong anti-interference capabilities. Through a multi-color + multi-round approach, it achieves decoding of a pool of hundreds of millions of microspheres, thus achieving a larger field of view while maintaining capture resolution. Taking the BMKMANU S3000 series as an example, a 0.68mm × 0.68mm capture area contains 4.1 million capture microspheres, allowing for unbiased mRNA capture and spatial expression analysis of the entire transcriptome. However, traditional methods for this decoding require the synthesis of complementary probe pools for different barcodes, placing extremely high demands on the stability of hybridization imaging, resulting in high technical barriers, high experimental costs, and long decoding cycles.

[0006] In summary, traditional biochip decoding methods are inefficient and costly. Therefore, developing a novel decoding method for subcellular precision spatial transcriptome chips is beneficial for better studying tissue spatial location information. Summary of the Invention

[0007] To address the shortcomings of existing technologies and practical needs, this application provides a decoding method for spatial transcriptome chips and its application, aiming to improve decoding efficiency and reduce decoding costs.

[0008] To achieve the above objectives, this application adopts the following technical solution:

[0009] In a first aspect, this application provides a method for decoding a spatial transcriptome chip, the method comprising the following steps:

[0010] (1) The capture microspheres connected with the first barcode, the second barcode and the third barcode in sequence are fixed at the well positions of the multi-well plate to obtain a biochip;

[0011] (2) Hybridize the auxiliary primer pool with the decoding probe to obtain the decoding primer pool; the auxiliary primer pool includes auxiliary primers that are complementary to the first barcode, the second barcode and the third barcode respectively, and the auxiliary primers also contain at least one sequence that is complementary to the decoding probe; the decoding probe carries a fluorescent group;

[0012] (3) The decoding primer pool is brought into contact with the biochip to perform a hybridization reaction, the fluorescence signal is detected, and the corresponding barcode is determined based on the fluorescence signal;

[0013] (4) The biochip is unchained, and after being washed and dried, it is hybridized with the next round of decoding primer pool.

[0014] (5) Repeat steps (3)-(4) to achieve chip decoding.

[0015] This application presents a decoding scheme based on auxiliary primers. The core of this scheme lies in utilizing auxiliary primers as selective bridging bridges to achieve precise decoding of specific bars. Specifically, during the decoding process, only when an auxiliary primer paired with the target barcode sequence is added to the system can the auxiliary primer mediate a specific connection between the corresponding barcode and a fluorescently labeled decoding probe, thereby generating a detectable fluorescent signal. Conversely, if the corresponding auxiliary primer is not added, an effective connection cannot be formed between the corresponding barcode and the decoding probe, and no fluorescence is generated. This achieves "present or absent" signal reading, allowing us to decode different bars sequentially according to a preset order.

[0016] Compared to traditional methods that rely solely on adjusting decoding conditions or probes, the key advantages of this application's method are mainly reflected in the following aspects: First, it offers significant advantages in cost control. The auxiliary primer pool used in this application does not require any fluorescent modification, which greatly reduces synthesis and preparation costs. Simultaneously, the auxiliary primers can be freely designed and mixed in advance according to different barcode combinations. This modular design greatly facilitates flexible updates and iterations of subsequent hybridization schemes. Second, it simplifies the use of decoding probes. Since the specificity of the ligation reaction is determined by the auxiliary primers, the actual number of decoding probes with fluorescent groups required can be significantly reduced. Typically, only a few general-purpose fluorescent probes are needed to cover the entire decoding system, which fundamentally reduces the reagent costs for decoding. Finally, it significantly improves signal intensity and operational success rate. This application allows for the design of additional decoding probe binding regions at both ends of the auxiliary primers, enabling one auxiliary primer to bind two or more decoding probes simultaneously, thereby multiplying the intensity of the imaging fluorescence signal. This signal amplification effect not only improves detection sensitivity and reduces background interference, but more importantly, it greatly enhances the robustness and success rate of the decoding process, making the entire process more tolerant of fluctuations in experimental conditions and resulting in more stable and reliable results.

[0017] In one embodiment of this application, the auxiliary primer contains two sequences complementary to the decoding probe, located at both ends of the sequence complementary to the barcode.

[0018] In one embodiment of this application, the first barcode, the second barcode, and the third barcode each independently include 4-768 barcode sequences.

[0019] In one embodiment of this application, the first barcode, the second barcode, and the third barcode are tandemly linked by a linker in a 5'-3' sequence. In another embodiment of this application, the linker can be a nucleic acid sequence of 1-20 bp.

[0020] In one embodiment of this application, the capturing microspheres are selected from any one of silica microspheres, polystyrene microspheres, or magnetic beads.

[0021] In one embodiment of this application, the silica microspheres are selected from carboxylated silica microspheres.

[0022] In one embodiment of this application, the method for preparing the capture microspheres sequentially connected with a first barcode, a second barcode, and a third barcode includes: connecting the first barcode to carboxylated silica microspheres respectively; after the connection is completed, mixing and washing the microspheres, dividing them evenly into 'a' portions; performing a connection reaction on each portion of the second barcode; after the connection is completed, mixing and washing the microspheres, dividing them evenly into 'b' portions; and adding a third barcode to each portion for a connection reaction, wherein 'a' and 'b' are the number of types of second and third barcodes used, respectively.

[0023] In one embodiment of this application, a and b are each independently selected from 4-768.

[0024] In one embodiment of this application, the fluorescent group is selected from at least one of DAPI, FITC, FAM, Alexa fluor 488, Cy2, Cy3, Cy5, Cy5.5, TRITC, or Cy7.

[0025] In one embodiment of this application, the hybridization reaction in step (3) is carried out in a hybridization buffer; the components of the hybridization buffer include: 1~10 mM NaCl, 2~5 mM Tris-HCl, 1~3 mM MgCl2 and 0.5~5 mM DTT (dithiothreitol).

[0026] In one embodiment of this application, in step (3), the concentration of the decoding probe pool is 1~50 nM (e.g., it can be 2, 3, 4, 5, 6, 10, 15, 20, 25, 30, 35, 40, 45, 46, 47, 48 or 49 nM, etc.). 10 µL of the decoding probe pool is mixed with 40~190 µL of hybridization buffer (e.g., it can be 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170 or 180 µL, etc.) and added to the biochip wells to carry out the hybridization reaction.

[0027] In one embodiment of this application, the hybridization reaction conditions in step (3) are: 37~60℃ (e.g., 38, 39, 40, 45, 50, 55, 56, 57, 58 or 59℃, etc.) for 5~20 min (e.g., 6, 7, 8, 9, 10, 15, 16, 17, 18 or 19 min, etc.).

[0028] In one embodiment of this application, the unwinding process in step (4) includes: contacting the unwinding reagent with the biochip, or treating the biochip at 93~98°C (preferably 94~96°C).

[0029] In one embodiment of this application, the chain-breaking agent is selected from sodium hydroxide solution or urea solution.

[0030] In one embodiment of this application, the concentration of the sodium hydroxide solution is 0.1~2 M, for example, it can be 0.2, 0.5, 1, 1.5, 1.6, 1.7, 1.8 or 1.9 M, etc.

[0031] Preferably, step (4) specifically includes:

[0032] (4-1) Add a melting reagent (e.g., 50-20 µL) to each well of the biochip and let it stand (e.g., 1-10 min), then discard the solution in the well.

[0033] (4-2) Repeat step (4-1) 2-3 times;

[0034] (4-3) Wash the biochip with water 1-3 times, dry it, and then hybridize it with the next round of decoding primer pool.

[0035] In one embodiment of this application, steps (3)-(4) are repeated 2 to 9 times in step (5).

[0036] Secondly, this application provides the application of the spatial transcriptome chip decoding method described in the first aspect in spatial transcriptomics research.

[0037] In this application, the spatial transcriptomics research includes related studies based on spatial transcriptome capture and sequencing.

[0038] Compared with the prior art, this application has at least the following beneficial effects:

[0039] This application designs a decoding scheme based on auxiliary primers. The auxiliary primers selectively connect the corresponding barcode and decoding probe. Without the auxiliary primer, the barcode and decoding probe cannot connect, and no fluorescence is generated. Conversely, fluorescence is generated, allowing for sequential decoding. Compared to traditional decoding schemes that simply adjust the decoding probe, the auxiliary primers do not require fluorescence modification, are easy to synthesize, have lower costs, and can be freely combined and mixed, facilitating subsequent updates and iterations of the hybridization scheme. Fewer types of decoding fluorescent probes are required, significantly reducing decoding costs. Furthermore, the auxiliary primers can increase the binding area of ​​the decoding probe on both sides, thereby doubling the imaging brightness and greatly improving the success rate of decoding. Attached Figure Description

[0040] Figure 1This diagram illustrates the hybridization of auxiliary primers, decoding probes, and barcodes. 1, 8, and 5 represent three types of barcodes, 2 is the linker, 3, 7, and 10 are auxiliary primers that are complementary to the three types of barcodes, and 4, 6, and 9 are decoding probes carrying different fluorescent groups.

[0041] Figure 2 This diagram illustrates the hybridization of the auxiliary primers and barcodes used to combine the two decoding probes.

[0042] Figure 3 The images shown are decoded fluorescence patterns from the examples and comparative examples. Detailed Implementation

[0043] The technical solution of this application will be further described below with reference to the accompanying drawings and specific embodiments. However, the examples below are merely simplified examples of this application and do not represent or limit the scope of protection of this application. The scope of protection of this application shall be determined by the claims.

[0044] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0045] This application designs a novel decoding scheme for a spatial transcriptome chip. For a capture microsphere sequentially connected to three different types of bars, an auxiliary primer pool is designed for each barcode, along with decoding probes carrying different fluorescent groups. Each auxiliary primer contains a sequence complementary to the corresponding barcode and a sequence complementary to the decoding probe. A schematic diagram illustrating the binding of the auxiliary primers, decoding probes, and bars on the capture microsphere is shown below. Figure 1 As shown, decoding probes carrying different fluorescent groups can bind to corresponding bars using auxiliary primers. The specific decoding principle is as follows: different bars emit fluorescence in different rounds depending on whether hybridization occurs (adding the corresponding auxiliary primer allows hybridization with the decoding probe; without it, hybridization does not occur). Finally, the fluorescence images are analyzed to obtain the fluorescence coding sequence of different microbead bars, thereby deducing the barcode number for subsequent experimental analysis. In addition, sequences complementary to the decoding probe can be designed at both ends of the auxiliary primers, thereby doubling the imaging brightness and greatly improving the success rate of decoding. Figure 2 Compared with traditional decoding schemes that only use decoding probes, the method in this application does not require the synthesis of fluorescently modified probes for each type of barcode. It only requires the synthesis of ordinary primers that are complementary to the barcode and a few fluorescently modified decoding probes, which reduces the cost by more than 50%. Furthermore, the synthesis of a large quantity of complementary primer pools is convenient for later recombination, upgrading and expansion.

[0046] Specifically, the three types of bars sequentially attached to the capture microspheres can be named the first barcode, the second barcode, and the third barcode, starting from the capture microsphere. The number of each barcode can be designed according to actual needs, and the sequences in each barcode are also different to accommodate different numbers of microspheres. For example, each barcode can be designed with 4-768 sequences. Specifically, the design of the specific sequence bases and length can be selected according to actual needs. The first barcode may include a sequence that binds to the sequencing primer, and the remaining part may be a random sequence. The second barcode may include a random sequence, and the third barcode may include a random sequence and a polyT sequence for capturing RNA from the 5'-3' end.

[0047] Specifically, the first barcode, the second barcode, and the third barcode can be connected in series via a linker in a 5'-3' sequence.

[0048] Specifically, the fluorescent group can be selected from DAPI, FITC, FAM, Alexa fluor 488, CY2, Cy3, Cy5, CY5.5, TRITC or Cy7.

[0049] In one specific embodiment of this application, a method for decoding a spatial transcriptome chip is provided, the method comprising the following steps:

[0050] A biochip with a load-capturing microspheres, wherein a first barcode, a second barcode, and a third barcode are sequentially connected to the capturing microspheres;

[0051] The auxiliary primer pool and the decoding probe are mixed and hybridized to obtain the decoding primer pool;

[0052] The decoding primer pool is brought into contact with the biochip to perform a hybridization reaction, and then the fluorescence signal is detected. The corresponding barcode is determined based on the fluorescence signal.

[0053] The biochip is contacted with a melting reagent to perform a melting treatment. After the melting treatment, the biochip is washed, dried, and then hybridized with the next round of decoding primer pool.

[0054] Repeat the decoding process 2 to 9 times to achieve chip decoding.

[0055] In a specific embodiment of this application, the decoding scheme of this application is verified by taking the design of four sequences for each type of barcode.

[0056] Example

[0057] 1. Chip fabrication

[0058] Three sets of barcode sequences were designed and synthesized: the first barcode, the second barcode, and the third barcode. Each set of primers included four sequences, as shown in Table 1. After the barcode sequences were synthesized, the first barcode sequence was first ligated to carboxylated silica microspheres. After ligation, the microspheres were mixed and washed, then evenly divided into four portions. The second barcode sequence was added to each portion for the ligation reaction, and this process was repeated until the third barcode sequence was ligated. The prepared microspheres were then evenly spread onto a microporous glass plate and fixed in place at the pores to obtain the space chip.

[0059] 2. Decoding Primer Pool Design

[0060] The prepared chip requires multiple rounds of decoding to finally determine the sequence structure of each microsphere. The auxiliary primers consist of single-stranded oligonucleotide structures (Table 1) complementary to the first barcode (auxiliary primers I-1 to I-4), second barcode (auxiliary primers II-1 to II-4), and third barcode (auxiliary primers III-1 to III-4) on the microsphere. Universal binding regions for the decoding probes are added to both ends. Three fluorescently labeled decoding probes, which are anticomplementary to the universal binding regions, are selected from DAPI, FITC, FAM, Alexa fluor 488, Cy2, Cy3, Cy5, Cy5.5, TRITC, and Cy7. These three fluorescent dyes are used to label the first, second, and third barcodes, respectively. Two to nine decoding primer pools are designed according to permutations and combinations. Each primer pool contains some auxiliary primers and decoding probes. Barcodes without auxiliary primers are identified as dark.

[0061] Table 1 shows the specific sequences of the barcode, auxiliary primers, and decoding probe.

[0062] Table 1

[0063]

[0064] Where V represents A, G, or C, and N represents A, T, G, or C.

[0065] 3. Chip hybridization

[0066] The chip hybridization process begins with the preparation of the decoding hybridization solution. Each well contains 200 µL of hybridization solution per round, including 190 µL of hybridization buffer (5 mM NaCl, 3 mM Tris-HCl, 2 mM MgCl2, and 1 mM DTT) and 10 µL (20 nM) of three-color decoding primer pools. After preparing the hybridization solution, the plated chip is placed in the clip, and the hybridization solution is added to the wells requiring decoding, ensuring the solution covers the entire surface of the well. The chip is then placed in a 48°C metal bath and reacted for 20 min. After the reaction, the hybridization solution is removed, excess probes are washed with nuclease-free water (NFW), the chip is dried, and then fluorescence signals from the wells are acquired using a fluorescence scanner.

[0067] 4. Chip unchaining and cleaning

[0068] After fluorescence acquisition from the chip, 100 µL of 0.5M NaOH was placed on the well and allowed to stand for 5 min to ensure complete dissociation. This process was repeated twice. The chip was then cleaned three times with NFW to ensure the hybridization probes were thoroughly cleaned and to avoid affecting the next reaction. After cleaning and drying the chip, the next round of decoding probes was hybridized again. This process was repeated to complete two rounds of decoding.

[0069] 5. Chip Decoding

[0070] After the chip completes two rounds of decoding, the fluorescence signal of each well position in each round of fluorescence image is extracted. Based on the arrangement of the fluorescence label colors of each well position in each round, the sequence structure corresponding to each well position is obtained, thereby realizing chip decoding.

[0071] Table 2 shows the fluorescently labeled probes corresponding to two rounds of decoding loop imaging for 64 types of microsphere pool chips.

[0072] Table 2

[0073]

[0074] “√” indicates that the corresponding mark is present, and “ / ” indicates that it is absent.

[0075] Table 3 shows the decoding combinations for each barcode in the 64 types of microsphere pools.

[0076] Table 3

[0077]

[0078] As shown in Tables 2 and 3, Table 2 represents the fluorescence status of different barscodes in each round of decoding; Table 3 represents the final fluorescence encoding order of different barscodes, used to deduce the barcode number from the analysis data of fluorescence decoding. The above results demonstrate that the decoding method of this application can achieve efficient decoding.

[0079] Comparative Example

[0080] This comparative example uses the traditional method without auxiliary primer pools for decoding.

[0081] The specific experimental procedure includes:

[0082] 1. Chip fabrication

[0083] The first, second, and third bars were designed and synthesized, each containing four sequences (Table 6). After primer sequence synthesis, the first barcode sequence was first ligated to carboxylated silica microspheres via an amino-carboxyl condensation reaction. After ligation, the microspheres were mixed and washed, then evenly divided into four portions. The second barcode sequence was added to each portion for ligation, and this process was repeated until the third barcode sequence was ligated. The prepared microspheres were then evenly spread onto a microporous glass plate and fixed in place at the pores to obtain the space chip.

[0084] 2. Decoding probe design

[0085] The fabricated chip requires multiple rounds of decoding to ultimately determine the sequence structure of each microsphere. The decoding probes are oligonucleotide single-stranded structures complementary to the first, second, and third bars of the microsphere. Three fluorescently labeled decoding probes are selected from DAPI, FITC, Alexa fluor 488, CY2, Cy3, Cy5, CY5.5, TRITC, and Cy7. Unlabeled probes are left as dark. The three fluorescent dyes label the first, second, and third bars, respectively.

[0086] 3. Chip hybridization

[0087] The chip hybridization process begins with the preparation of a decoding hybridization solution. Each well contains 200 µL of hybridization solution per round, including 190 µL of hybridization buffer (5 mM NaCl, 3 mM Tris-HCl, 2 mM MgCl2, and 1 mM DTT) and 10 µL (20 nM) of a three-color mixed probe. After preparing the hybridization solution, the chip is placed in the holder, and the hybridization solution is added to the wells requiring decoding, ensuring the solution covers the entire surface of the well. The chip is then placed in a 48°C metal bath and reacted for 10 min. After the reaction, the hybridization solution is removed, excess probes are cleaned with NFW (Non-Fluorescent Wave Wrapper), the chip is dried, and then fluorescence signals from the wells are acquired using a fluorescence scanner.

[0088] 4. Chip unchaining and cleaning

[0089] After fluorescence acquisition from the chip, 100µL of 0.5M NaOH was placed on the well and allowed to stand for 5 minutes to ensure complete dissociation. This process was repeated twice. Then, the chip was cleaned three times with NFW to ensure the hybridization probes were thoroughly cleaned and to avoid affecting the next reaction. After cleaning and drying the chip, the next round of decoding probes was hybridized again. This process was repeated to complete two rounds of decoding.

[0090] 5. Chip Decoding

[0091] After the chip completes 2-9 rounds of decoding, the fluorescence signal of each well position in each round of fluorescence image is extracted. According to the arrangement of the fluorescence label color of each well position in each round, as shown in Tables 4 and 5, the sequence structure corresponding to each well position is obtained, thereby realizing chip decoding.

[0092] Table 4 shows the fluorescently labeled probes corresponding to each decoding cycle.

[0093] Table 4

[0094]

[0095] Table 5 shows the combination of fluorescence signals corresponding to each primer sequence.

[0096] Table 5

[0097]

[0098] Table 6 shows the specific sequences of the barcode and decoding probes.

[0099] Table 6

[0100]

[0101] Where V represents A, G, or C, and N represents A, T, G, or C.

[0102] The results above show that, to decode the same number of bars, traditional methods require more decoding probes and need to be designed specifically for the barcode sequence. For different batches of bars, the decoding probes need to be adjusted and resynthesized. In contrast, the design of this application uses auxiliary primers, which requires fewer decoding probes. For different bars, only the auxiliary primers need to be adjusted, without adjusting the decoding probes (the binding of the decoding probe and the auxiliary primer can remain unchanged; only the binding part of the auxiliary primer and the barcode needs to be adjusted). The auxiliary primers do not require fluorescent modification, are easy to synthesize, and have low cost, which can greatly reduce the decoding cost.

[0103] In addition, the fluorescence detection results of the decoding process in the examples and comparative examples are as follows: Figure 3 As shown, the decoding method of this application can significantly improve the brightness of fluorescence decoding, which is beneficial to improving the success rate of decoding operations.

[0104] In summary, this application designs a decoding scheme based on auxiliary primers. The auxiliary primers selectively connect the corresponding barcode and decoding probe. Without the auxiliary primers, the barcode and decoding probe cannot connect, and no fluorescence is generated. Conversely, fluorescence is generated, allowing for sequential decoding. Compared to traditional decoding schemes that simply adjust the decoding probes, the auxiliary primers do not require fluorescence modification, are easy to synthesize, have lower costs, and can be freely combined and mixed, facilitating subsequent updates and iterations of the hybridization scheme. Fewer types of decoding fluorescent probes are required, significantly reducing decoding costs. Furthermore, the auxiliary primers can increase the binding area of ​​the decoding probe on both sides, thereby doubling the imaging brightness and greatly improving the success rate of decoding.

[0105] The above description is only a specific embodiment of this application, but the protection scope of this application is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in this application fall within the protection and disclosure scope of this application.

Claims

1. A decoding method for a spatial transcriptome chip, characterized in that, The method includes the following steps: (1) The capture microspheres connected with the first barcode, the second barcode and the third barcode in sequence are fixed at the well positions of the multi-well plate to obtain a biochip; (2) Hybridize the auxiliary primer pool with the decoding probe to obtain the decoding primer pool; the auxiliary primer pool includes auxiliary primers that are complementary to the first barcode, the second barcode and the third barcode respectively, and the auxiliary primers also contain at least one sequence that is complementary to the decoding probe; the decoding probe carries a fluorescent group; (3) The decoding primer pool is brought into contact with the biochip to perform a hybridization reaction, the fluorescence signal is detected, and the corresponding barcode is determined based on the fluorescence signal; (4) The biochip is unchained, and after being washed and dried, it is hybridized with the next round of decoding primer pool. (5) Repeat steps (3)-(4) to achieve chip decoding.

2. The decoding method for a spatial transcriptome chip according to claim 1, characterized in that, The first, second, and third barcodes each independently include 4-768 barcode sequences; And / or, the first barcode, the second barcode, and the third barcode are connected in series via a linker in a 5'-3' sequence.

3. The decoding method for a spatial transcriptome chip according to claim 1, characterized in that, The capturing microspheres are selected from any one of silica microspheres, polystyrene microspheres, or magnetic beads; And / or, the silica microspheres are selected from carboxylated silica microspheres.

4. The decoding method for a spatial transcriptome chip according to claim 1, characterized in that, The method for preparing the capture microspheres sequentially connected with a first barcode, a second barcode, and a third barcode includes: connecting the first barcode to carboxylated silica microspheres respectively; after the connection is completed, mixing and washing the microspheres, dividing them evenly into a portions, and performing a connection reaction with a second barcode in each portion; after the connection is completed, mixing and washing the microspheres, dividing them evenly into b portions, and adding a third barcode to each portion for a connection reaction, wherein a and b are the number of types of second and third barcodes used, respectively; And / or, a and b are each independently selected from 4-768.

5. The decoding method for a spatial transcriptome chip according to claim 1, characterized in that, The fluorescent group is selected from at least one of DAPI, FITC, FAM, Alexa fluor 488, Cy2, Cy3, Cy5, Cy5.5, TRITC or Cy7.

6. The decoding method for a spatial transcriptome chip according to claim 1, characterized in that, The hybridization reaction in step (3) is carried out in a hybridization buffer; the components of the hybridization buffer include: 1~10 mM NaCl, 2~5 mM Tris-HCl, 1~3 mM MgCl2 and 0.5~5 mM DTT.

7. The decoding method for a spatial transcriptome chip according to claim 6, characterized in that, In step (3), the concentration of the decoding probe cell is 1~50 nM. 10 µL of the decoding probe cell is mixed with 40~190 µL of hybridization buffer and added to the wells of the biochip for hybridization reaction. And / or, the conditions for the hybridization reaction in step (3) are: 37~60℃ for 5~20 min.

8. The decoding method for a spatial transcriptome chip according to claim 1, characterized in that, The chain-breaking process in step (4) includes: contacting the chain-breaking reagent with the biochip, or treating the biochip at 93~98℃; And / or, the chain-breaking agent is selected from sodium hydroxide solution or urea solution; And / or, the concentration of the sodium hydroxide solution is 0.1~2 M; And / or, step (4) specifically includes: (4-1) Add the melting reagent to each well of the biochip and let it stand, then discard the solution in the well; (4-2) Repeat step (4-1) 2-3 times; (4-3) Wash the biochip with water 1-3 times, dry it, and then hybridize it with the next round of decoding primer pool.

9. The decoding method for a spatial transcriptome chip according to claim 1, characterized in that, Repeat steps (3)-(4) 2-9 times in step (5).

10. The application of the spatial transcriptome chip decoding method according to any one of claims 1-8 in spatial transcriptomics research.

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