Spacial sequencing with mictag

Abstract

To provide methods for determining the distribution of mRNA in areas of a tissue or in individual cells within the tissue.SOLUTION: The invention is directed to a method to obtain a spatial location and sequence information of at least a part of an RNA or cDNA strand in a sample, the method comprising the steps of: a. hybridizing a first detection probe oligonucleotide to the complementary part of the at least one RNA or cDNA strand, where the detection probe oligonucleotide is partially hybridized to a bridge oligonucleotide to create a gap region capable of binding to the oligonucleotide; b. filling the gap region in part with barcode oligonucleotides where the barcode oligonucleotides determine the spatial information of the RNA or cDNA strand in the sample; c. partially hybridizing a second detection probe oligonucleotide to create a circular template; d. multiplying the circular template by a polymerase capable of rolling circle amplification into rolonies comprising a plurality of concatemers; e. determining the sequence of nucleotides of the rolonies.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to a technique for spatial sequencing. Its purpose is to determine the distribution of mRNA in a region of tissue or in individual cells within a tissue.

[0002] Spatial sequencing is a general term for methods that allow for the direct sequencing of cellular mRNA content in relation to its tissue context. These methods can, on the one hand, be useful for analyzing cellular mRNA expression profiles in a type of highly multiplexed fluorescence in situ hybridization (FISH) assay.

[0003] On the other hand, in situ sequencing can sometimes read mRNA sequence information using specific mRNA-binding probes. These probes can block copies of predetermined portions of specific mRNA or cDNA sequences ("Gap-fill padlock probes", Ke et al., Nature Methods 2013, doi:10.1038 / nmeth.2563). All in situ sequencing methods require a signal amplification step, which in most cases is performed by cyclization of the mRNA or cDNA-binding probe and subsequent rolling circle amplification (RCA), generating DNA molecules containing multiple copies of the probe sequence, so-called nanoballs, rollonies, or rolling circle amplification products (RCPs). Because these are large molecules with sizes on the nm or μm scale, the number of rollonies that can be formed in a single cell is strictly limited by the size of that cell.

[0004] Furthermore, if the density of loroni cells within the cell is too high, the identification of a single mRNA signal during the optical detection step of the sequencing method is severely impaired. This is a major drawback of the technique, and to circumvent it, various techniques have been developed, such as designing smaller loroni cells or generating and removing tissue-hydrogel complexes (Asp et al., BioEssays 2020, DOI: 10.1002 / bies.201900221). However, these methods still cannot completely circumvent the inherent spatial limitations of in situ sequencing.

[0005] Another approach avoids in situ signal amplification. In situ capture involves the migration of mRNA molecules from tissue to a surface coated with barcoded primer spots, allowing for backtracking of ex situ-acquired sequence information to the specific tissue region from which the sequenced mRNA was extracted. Nevertheless, this method is also limited due to its limited RNA capture efficiency and insufficient resolution (unable to perform single-cell analysis) caused by the relatively large size of the barcoded capture spots (Asp et al., BioEssays 2020, DOI:10.1002 / bies.201900221).

[0006] Summary of the Invention The present invention relates to a method for inserting a gene code into a sequence using an optical method. This code can be used to locate the site where the encoding was performed. The objective herein is to largely overcome the limitations of existing in situ sequencing methods regarding the number of mRNA sequences measurable in situ and their expression dynamics. Optical encoding can have a resolution in the range of 1 μm and coding variability sufficient for each cell to receive its own code in a tissue section of typical size.

[0007] Furthermore, it is possible to optically encode specific mRNA-binding probes and reverse-transcribe the mRNA regions encapsulated within these probes. Therefore, this method can be used not only to profile mRNA distribution (expression profiling of a single cell) but also to determine the sequence information of specific mRNA sections (e.g., single nucleotide polymorphisms, SNPs; single-cell sequencing).

[0008] The basic principle of the methods disclosed herein is based on the binding of padlock probes to mRNA present in cells. These are referred to below as MIC tag probes. Mi smatch C ell Tag ging (mismatch cell tag) or Mi smatch C ode Tag This shall be referred to as ging (mismatch code tag).

[0009] The proposed MIC tagging workflow is shown in Figure 1: Sample preparation, tissue staining, and imaging (transmission, fluorescence) are followed by calculation of masks for segmentation or cluster analysis and structured illumination. The MIC tag probe binds to specific mRNA molecules within the cell, enabling reverse transcriptase-mediated gap filling (copying of mRNA sequences to the MIC tag probe) and periodic incorporation of the MIC tag code, rolling circle amplification of the MIC tag probe, and sequencing of the MIC tag code. The sequences obtained by gap filling and the MIC tag code are annotated to the image. Bioinformatics analysis and the relationship between the results and the initial sample source complete the workflow.

[0010] Providing MIC-tagged probes to mRNA is an essential part of the present invention, and this can be achieved through two common modifications.

[0011] A first object of the present invention is a method for obtaining spatial position and sequence information of at least a portion of an RNA or cDNA strand (006) in a sample, a. A step of hybridizing a first detection probe oligonucleotide (204) containing 50 to 1000 nucleotides, which is partially hybridized to a bridge oligonucleotide (205) containing 5 to 100 nucleotides and forms a gap region (206) that can bind to the oligonucleotide, to a complementary portion of at least one RNA or cDNA strand at its 3' and / or 5' end, b. A step of partially filling the gap region (206) with 1 to 16 barcode oligonucleotides containing 4 to 20 nucleotides, which determine the spatial information of the RNA or cDNA strand in the sample. c. A step of forming a circular template by partially hybridizing a second detection probe oligonucleotide (204') containing 50 to 1000 nucleotides at its 3' and / or 5' ends to the same or complementary portion of the cDNA strand and partially hybridizing the other ends to a bridge oligonucleotide (205), d. A step of amplifying a cyclic template with a polymerase capable of rolling circle amplification in a Rollonie containing multiple concatemers, e. The process of determining the nucleotide sequence of Loroney This method includes [something].

[0012] An MIC-tagged probe may consist of two probes (204,204') each containing one of the aforementioned specific mRNA binding sites, linked by a partially complementary "bridge" (bridge oligonucleotide (205) in Figure 2). The sequence of this bridge primer may be the same for all MIC-tagged probes. The affinity and binding efficiency of bridge oligonucleotide (205) to its binding region on the probe can be improved by incorporating locked nucleic acid (LNA) or peptide nucleic acid (PNA) into the bridge oligomer sequence.

[0013] A second object of the present invention is a method for obtaining at least partial spatial position and sequence information of RNA or cDNA strands ((006)) in a sample, comprising: f. hybridizing the 3' and 5' ends of a detection probe oligonucleotide, comprising a first oligonucleotide (204) and a second oligonucleotide (204') each containing 50 to 1000 nucleotides, which are linked by a partially hybridized bridge oligonucleotide (205) containing 5 to 100 nucleotides, and a bridge gap region (206) is formed between the first oligonucleotide (204) and the second oligonucleotide (204'), to complementary portions of at least one RNA or cDNA strand; g. filling the bridge gap region (206) with 1 to 16 barcode oligonucleotides, each containing 4 to 20 nucleotides, for determining the spatial information of RNA or cDNA in the sample to form a circular template; h. amplifying the circular template with a polymerase capable of rolling circle amplification within a rolling circle containing a plurality of concatemers; i. determining the nucleotide sequence of the rolling circle. A method comprising the above steps. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] [Figure 1] A diagram showing the MIC tag workflow. Sample preparation, tissue staining, and imaging. [Figure 2] A diagram showing a modification of padlock formation and gap filling of the MIC tag probe. [Figure 3] A diagram showing MIC tag coding. [Figure 4] A diagram showing the MIC tag probe after gap filling, coding, and bridge primer removal. [Figure 5] A diagram showing sequencing of the MIC tag rolling circle. [Figure 6] A diagram showing an accelerated sequencing technique for the MIC tag code. [Figure 7] It is a diagram showing a parallel coding modification example of a MIC tag probe. [Figure 8] It is a diagram showing the incorporation of a unique molecular identifier (UMI) into a MIC tag probe array. [Figure 9] It is a diagram showing primer-controlled MIC tag coding.

[0015] Modes for Carrying Out the Invention Hereinafter, the detection probe oligonucleotide is referred to as a "MIC tag probe", the bridge oligonucleotide (205) contains a plurality of "MIC tag digits" or shorter "digits", and the gap region (206) is filled with a barcode oligonucleotide referred to as a "MIC tag snippet" or shorter "snippet".

[0016] Modification examples for generating a detection probe oligonucleotide are shown in FIGS. 2A to 2D.

[0017] FIG. 2A shows a padlock. The detection probe oligonucleotide (204) has a binding region (203) that binds to a complementary portion (006) of at least one RNA or cDNA strand (005). The detection probe oligonucleotide is partially hybridized and includes a bridge oligonucleotide (205) that forms a gap region (206) capable of binding to the oligonucleotide.

[0018] Figure 2B shows the general concept and shape of a padlock. The first (204) and second (204') detection probe oligonucleotides have binding regions (203) and (203') that bind to complementary portions (006) and (006') of at least one RNA or cDNA strand (005). The final detection probe oligonucleotide is produced by partially hybridizing the first (204) and second (204') detection probe oligonucleotides with a bridge oligonucleotide (205), where a gap region (206) that can bind to the oligonucleotide is formed.

[0019] In the first modified embodiment shown in Figure 2B, the detection probe oligonucleotide is hybridized to at least one RNA or cDNA strand by hybridizing a first detection probe oligonucleotide (204) and a second detection probe oligonucleotide (204'), each containing 50 to 1000 nucleotides, at their respective 3' and 5' ends to a complementary portion of at least one RNA or cDNA strand, and then partially hybridizing the first (204) and the second oligonucleotide (204') to a bridge oligonucleotide (205) to link them together.

[0020] In a second modification of the embodiment shown in Figure 2B, the detection probe oligonucleotide is hybridized to at least one RNA or cDNA strand by ligating the first oligonucleotide (204) to the second oligonucleotide (204') (parts 203 and 203'), then hybridizing the resulting oligonucleotide to a complementary portion of at least one RNA or cDNA strand, and subsequently ligating the unbound end of the resulting oligonucleotide to a bridge oligonucleotide (205) by partial hybridization.

[0021] In the modified example shown in Figure 2A, a portion of the first and / or second oligonucleotides hybridized to the detection probe oligonucleotide and / or at least one RNA or cDNA is used to obtain the second target sequence.

[0022] Unlike the embodiment shown in Figure 2A, the embodiment shown in Figure 2B, when hybridized to mRNA(005), has a gap (207') between the first oligonucleotide (204) and the second oligonucleotide (204').

[0023] This modified example is shown in Figure 2C, where the detection probe oligonucleotide is hybridized to a complementary portion of at least one RNA or cDNA strand, thereby creating a gap of 1 to 150 nucleotides (207') between the first oligonucleotide (204) and the second oligonucleotide (204') of the detection probe oligonucleotide.

[0024] Next, the gap (207') is filled with a nucleotide complementary to at least one adjacent portion of the RNA or cDNA strand using reverse transcriptase (208) to obtain the first target sequence (207). Then, the final ligation of the first oligonucleotide (204) and the second oligonucleotide (204') is performed using DNA ligase (209).

[0025] Figure 2D shows the method of the present invention relating to its first objective. In this specification, the first detection probe oligonucleotide (204) is hybridized at its 3' and / or 5' end (203) to a complementary portion ((006)) of at least one RNA or cDNA strand (005). The first detection probe oligonucleotide (204) can be provided in advance together with a bridge oligonucleotide (205) and hybridized to the mRNA strand (005).

[0026] Alternatively, the first detection probe oligonucleotide (204) is first hybridized to a complementary portion ((006)) of at least one RNA or cDNA strand (005), and then partially hybridized to the bridge oligonucleotide (205).

[0027] In both cases, a gap region (206) capable of binding to the oligonucleotide is formed.

[0028] Specifically, Figure 2(A) shows an MIC tag probe with one mRNA binding site, Figure 2(B) shows an MIC tag probe with two mRNA binding sites hybridized to two directly adjacent mRNA regions, and Figure 2(C) shows gap filling of the MIC tag probe. After the MIC tag probe binds to two non-directly adjacent mRNA regions of a specific mRNA species, cDNA synthesis for gap filling is performed by reverse transcriptase. The strands are then joined by DNA ligase.

[0029] This type of single-stranded DNA probe contains two specific mRNA-binding regions that hybridize with two adjacent or adjacent but not directly adjacent regions of mRNA. The gap created when probe binding occurs at the adjacent but not directly adjacent regions can then be "filled" ("gap-filled") with a sequence complementary to the encapsulated mRNA using reverse transcriptase.

[0030] Next, the probe is cyclized by a DNA ligase (209 in Figure 2) to enable subsequent rolling circle amplification.

[0031] In the process steps described below, the gap region (206) is filled stepwise, and fragments of complementary sequences (MIC tag snips) are inserted into the complementary MIC tag digit at each step and fixed with light. For example, in contrast to the cyclic chemistry used for sequencing by synthetic processes, in one embodiment, a single deoxynucleotide is not inserted, but a small oligonucleotide (MIC tag snip) is inserted.

[0032] Therefore, the bridge gap region (206) can be at least partially filled by hybridizing a barcode oligonucleotide containing the same or different photocleavable protecting groups to the complementary portion of the bridge oligonucleotide (205) by removing the photocleavable protecting groups with light after hybridization.

[0033] Mismatch MIC tag snips (barcode oligonucleotides) may have one or more mismatches with the corresponding MIC tag digit depending on their length, but should still have a sufficient sequence length to specifically bind to the (nearly) complementary portion of the bridge oligonucleotide.

[0034] The term "mismatch" refers to a barcode oligonucleotide that has at least one non-complementary nucleotide.

[0035] MIC tag snips may contain one or two mismatches having a preferred ratio of MIC tag snips:mismatch = 10:1 bp. In addition to the mismatch version, MIC tag snips may also contain matching codes, which can function as another variation.

[0036] To ensure specific hybridization with the associated MIC tag digit, the snips must be designed so that they cannot bind to other digits. As an alternative to mismatches in the snips, bridge primers can also be designed to contain a universal base in a position complementary to the snip-smith match.

[0037] The annealing of these MIC tag snips is performed within a periodic process, which is triggered in each period by structured illumination of the tissue sample by treating the tissue sample with a different spatially structured light pattern. "Structured illumination" and "spatially structured light pattern" refer to illuminating only a portion or selected area of ​​the sample. For example, Figure 1 shows a (optionally stained) tissue section 002 obtained from a tissue donor 001 subjected to imaging 100. This imaging 100 allows for segmentation or clustering analysis, i.e., selection of the portion of the sample to be further investigated by the method of the present invention. Such segmentation / clustering / selection allows for the calculation of a mask for structured illumination and / or spatially structured light patterns.

[0038] Further downstream in the method of the present invention, information obtained for structured illumination and / or spatially structured light patterns is utilized during photoprocessing (102) for MIC tag code generation. This enables cyclization of the padlock probe 201, and ultimately, enables the formation of Rollony 202 after RCA. With the help of structured illumination, only selected regions / cells of sample 202 are subjected to next-generation sequencing (100) and sequence analysis (101) of Rollony.

[0039] The binding kinetics of MIC tag snips are regulated so that, if the photocleavage groups are not removed, the snips can be separated and washed away, for example, by increasing the temperature. When exposure is performed, the MIC tag snips hybridized with their corresponding MIC tag digits in the bridge oligonucleotide are immobilized within the probe. This is because the photocleavage groups are separated by patterned illumination, allowing for ligase reactions at this site. Finally, the individual combinations of MIC tag snips determine the MIC tag code that encodes the location of the MIC tag probe, whether in a tissue section or even within an cell.

[0040] The MIC tag encoding process is shown in Figure 3: one of four specific gap-filling oligos is added to the bound and gap-filled MIC tag probe. This oligo either has no base mismatches or has one or two base mismatches. This scheme allows for the design of 16 different mismatch probes for each MIC tag digit. These mismatch probes bind to one of the four MIC tag digit binding regions (I-IV). Illumination of a single cell induces photocleavage, enabling the incorporation of the MIC tag snips by DNA ligase.

[0041] Starting from an MIC-tagged probe whose target chain gaps have already been filled with reverse transcriptase, the MIC-tagged snips are constructed in a periodic process until a code consisting of several snips is written. In the example shown in Figure 3, the snip variability is 16, which can be achieved by two mismatches and four binding regions (MIC-tagged digits) within the bridge. Therefore, 4 × 16 cycles are required to write a code that can have 65,536 different values.

[0042] After coding is complete, all MIC tag probes, i.e., the circular templates, are isolated, i.e., removed, from the tissue sample. The bridge oligonucleotide can be separated from the circular template, for example, by thermally induced double-strand fusion. In an alternative modification, the bridge oligonucleotide may remain on the circular template / MIC tag probe because it contains a primer sequence for inducing rolling circle amplification.

[0043] Figure 4 shows an isolated MIC tag probe carrying an MIC tag snip (217). The MIC tag snip (217) provides information from tissue structure, cell, or intracellular region originating from its mRNA strand. In addition, cDNA synthesis by gap filling (207) between the mRNA binding sites ((006), (006')) of the MIC tag probe allows for the inclusion of information about the final mutation or SNP (single nucleotide polymorphism) of the target mRNA (Figure 4, 207).

[0044] In principle, this method is not limited by the number of mRNA types being tested simultaneously, nor by the expression levels of individual mRNAs. As a result, high levels of expression of housekeeping genes will not disrupt the process. The number of cells individually tested simultaneously is limited by the selected coding length. This limitation is solely due to the sequencer's capacity and throughput.

[0045] Sequence determination One step in the method of the present invention is directed towards sequencing the nucleotides of Loroney, i.e., reading the information encoded on the MIC tag probe by sequencing. One method for sequencing may be sequencing by synthesis (SBS). Amplification of the MIC tag probe sequence can be performed to increase the readout signal. One method for clonal amplification may be rolling circle amplification (RCA) of the isolated MIC tag probe. This amplification is performed before initiating the sequencing process for Loroney.

[0046] Figure 5 shows the sequencing of the MIC tag lorony. Two sequencing primers lead to the sequential sequencing of the gap-filling cDNA sequence (218,218') and the MIC tag code (219,219').

[0047] In a modified version of the present invention, the sequence of the MIC tag code can be read separately from the sequence of the target gene (gap-filling sequence). This can be achieved by dividing the sequencing method into two runs using two different sequencing primers.

[0048] Accelerating MIC tag code sequence determination The sequencing of MIC tag snips can be accelerated if the matching codes before and after the mismatch region consist of only three of the four bases (e.g., G, T, C). In the sequence below, M is a placeholder for one of the bases G, A, or C, and X is a placeholder for the mismatch code.

[0049] MMMMMTXXMMMMM(MIC tag snips, M={G,A,C} and X={A,G,T,C})

[0050] In the context of cyclic chemicals applied to synthetic sequencing, the incorporated nucleotides are typically base-specific fluorophores and terminators (A in this specification). * , G * , C * , T * Modified with (indicated by ), thereby blocking further polymerase-mediated chain elongation until its position is read. When reading MIC tag snips in the modified example given above, nucleotides T, G, C and A that do not contain dyes and terminators are read. * A mixture of these can be applied. Then, these are incorporated until T occurs, always preceding a mismatch, A * Incorporating this would hinder further growth.

[0051] Next, mismatched regions of the sequence can be detected by using a mixture of nucleotides modified with terminators and fluorescent dyes. Then, T, G, C, A * The mixture is added again to advance chain elongation to the next T, which is before the next mismatch position in the next MIC tag snip.

[0052] This can essentially reduce the number of cycles required to determine the mismatch positions in the MIC tag code. Using this strategy, periodic incorporation stuttering is avoided and corrected. The first MIC tag snippet array to be read is designed such that the sequencing primer binds just prior to the mismatch array, thus eliminating the need for the first padding by T, G, C, A * as well.

[0053] This variant of the present invention is shown in FIG. 6. In this case, for example, two consecutive mismatches are present in the MIC tag snippet. FIG. 6 shows an accelerated sequencing method for the MIC tag code. Induced by a specific sequencing primer, nucleotides fluorescently labeled with terminators (T * C * G * A * ) are added in 3 cycles, enabling mismatch detection in sequencing by the synthesis approach. In the next step, unlabeled and non-terminating nucleotides (T, C, G) and A fluorescently labeled with a terminator (A * ) are added, and the strand is filled by DNA polymerase. The binding of A * stops this step, which is to indicate reaching the next mismatch position. Again, nucleotides fluorescently labeled with terminators (T * C * G * A * ) are added in 3 cycles, enabling mismatch detection in sequencing by the synthesis approach. This approach is repeated until the entire MIC tag code is sequenced and a specially designed MIC tag snippet that does not contain thymidine in its sequence except for the mismatch region is required.

[0054] In this variant, for example, the following sequences are utilized.

[0055] Sequence ID 1: 3' end of sequencing primer 221: 5'-...TGATCATG-3' (less than 10 nucleotides, no sequence listing available) Sequence ID 2: First MIC tag snip (with mismatched nucleotide 224): 3'-GCTAGTACTCGAGCC-5' Sequence ID 3: Second MIC tag snip (with mismatched nucleotide 224'): 3'-GCAACGGCCTAGCCTG-5' Sequence ID 4: 3' end of the third MIC tag snip: 3'-CGCAAAGGCT...-5' Sequence ID 5: 5' end of the complementary sequence generated by the synthetic sequencing method (using labeled nucleotide 222): 5'-...TGATCATGAGCTCGGCGTTGCCGGATCGGTCGCGTTTCCGA...-3'

[0056] Various MIC tag codes MIC tag codes can be generated by various combinations of MIC tag snips. In this specification, snip variability N represents the number of different snips. Preferably, the number of different snips is N = 2, 4, or 16. The number of MIC tag digits M (number of binding regions) can be in the range of 1 to 32. Then, the total variability of the MIC tag code is NM. The number of cycles required to write the code is N × M.

[0057] Accelerating code generation After incorporating (N-1) types of MIC tag snips into each MIC tag digit (binding site), the last type of MIC tag snip can be incorporated without applying light. This is because it relates to a complementary entity in a region already illuminated in the first N-1 steps. This can be achieved by providing a snip that does not have a photocleavable group. This non-photocleavable snip can then be provided along with the first (photocleavable) MIC tag snip for the next MIC tag digit. Counting this as one cycle, the required cycles are reduced to N×M-M+1=(N-1)×M+1. In the final cycle, the MIC tag coding will be completed without applying light. This method is particularly useful when N=2, in which case N×M-M+1=M+1.

[0058] The table below shows examples of modifications of the present invention method and their effect on the number of cycles required for MIC tag coding.

[0059] [Table 1]

[0060] Error handling When the MIC tag code is written, an error may occur when the MIC tag snips are incorporated. If one of the MIC tag snips is not inserted, the probe will not be circulated, RCA cannot be performed, and subsequent sequencing information will not be generated.

[0061] If an incorrect MIC tag snip is inserted, the code will not point to the correct cell. This limitation is only valid if the code generation acceleration outlined in the previous section is not applied.

[0062] In other words, if MIC tag snips are not inserted, a padlock will not be formed and will not be subjected to sequencing within the specified period. To that extent, the method of the present invention has built-in error handling.

[0063] In addition, it can be beneficial to include other error correction methods. For example, a second identical code can be inserted into the bridge at different locations simultaneously, allowing for internal verification of the code. It is also possible to increase the code length and add redundancy for error correction.

[0064] In this modified example, the bridge oligonucleotide contains at least two identical binding regions for the same MIC tag snips, which are simultaneously accessible to ensure a specific binding reaction. Even in this case, if one MIC tag snip is not correctly inserted, each padlock will not be formed and will not be available for sequencing.

[0065] In this regard, Figure 7 shows an MIC tag probe having two identical regions written in parallel. In this case, it is possible to assign only those sequences to locations where both codes are identical. This prevents the contrast from being smoothed out by inaccurate coding, especially in samples with very different mRNA expression profiles across different sample regions.

[0066] The reading of the genetic code is subject to reading errors. These errors can occur in both the MIC tag code and the mRNA sequence. Therefore, it may be useful to enzymatically cleave the strands produced by RCA and then generate multiple strands from each strand again using RCA for sequencing. Subsequently, it is useful to include a unique molecular identifier (UMI) in the MIC tag probe so that error correction can be performed on both the MIC tag code and the mRNA sequence. This modification is shown in Figure 8, with the unique molecular identifier (UMI) 226 incorporated into the MIC tag probe sequence. The UMI can be used to identify the original MIC tag probe after enzymatic digestion of Roronie.

[0067] Alternative coding approaches In addition to the proposed MIC tagging method, alternative methods can be used to program MIC tag probes in situ. One variation of the MIC tagging method described herein is shown in Figure 9. In this specification, the incorporation of ligase-mediated MIC tag snips can be controlled by additional primers that need to be pre-incorporated. As MIC tag snips, these primers have photoinstability groups that are removed by illumination with light of a specific wavelength, and these photoinstability groups enable subsequent incorporation into the strand by DNA ligase. The release of fluorophores optionally bound to the photoinstability groups can be detected and used as an internal control.

[0068] Instead of the additional primers mentioned in Figure 9, a single nucleotide with a photocleavable group can be used. In contrast to MIC tag snips, which hybridize with the complementary strands of the primer and bridge probe and are incorporated into the probe by DNA ligase, the nucleotide needs to be incorporated by DNA polymerase.

[0069] Figure 9 shows a modified version of the present invention involving primer-controlled MIC tag encoding. In this specification, a primer modified with a photoinstability group is hybridized to a single-stranded region adjacent to a first MIC tag digit. Light induces cleavage of the photoinstability group bound to the primer. The primer is incorporated into the strand by DNA ligase, and then the first snips are added and hybridized with the first digit. This process is repeated until all different snips at digit I are incorporated. Here, the first step is repeated to prepare the incorporation of different types of snips II at digit II. After multiple cycles, MIC tag encoding is complete, and the MIC tag probe is finally ligated. [Explanation of Symbols]

[0070] Glossary of terms used in drawings Organisms, tissues, and tissue contents 001 Tissue donor 002 Stained tissue section 003 Cell 004 Cell nucleus 005 mRNA 006 MIC tag probe binding site on mRNA technical method 100 images 101 Segmentation or cluster analysis, calculation of masks for structured illumination 102 Optical processing for MIC tag code generation 103. Next-generation sequencing of Loroney 104 Sequence Analysis 105 Periodic barcoding Reagents and Products 200 cDNA gap-filling and individual pre-coding MIC tag probes 201 cDNA gap filling and individual post-encoding MIC tag probes 202 Rollonie or Rolling Circle Amplification Product (RCP) 203 Specific mRNA binding site 204 MIC tag probe skeleton 205 MIC tag probe skeleton hybridized bridge oligonucleotide 206 Gap formed on the MIC tag probe skeleton by bridge oligonucleotide hybridization 207 cDNA (or cDNA gap, 207') 208 Reverse transcriptase 209 DNA ligase 210 MIC tag probe after gap filling 211 Bridge primer with MIC tag digits (I-IV) 212 Oligonucleotides or MIC tag snips having photoinstability protecting groups Focused light to cleave photoinstability protecting groups from 213 MIC tag snips 214 Cleavable Photolabile Protecting Group 215 MIC tag snips that do not contain photoinstability protecting groups MIC tag snips integrated into 216 MIC tag probes 217 MIC tag code 218 Sequencing primer binding sites 219 mRNA binding site and cDNA sequencing methods 220 MIC Tag Code Sequence Determination Method 221 Sequencing primers 222 (Photoinstability) Nucleotides with Protecting Groups 223 nucleotides 224 Variable nucleotides ("mismatches") in MIC tag codes 225 Splint oligonucleotides that support DNA ligase activity 226 Unique Molecular Identifier (UMI) 227 Restricted areas 228 restriction enzymes

Claims

1. A method for obtaining at least partial spatial position and sequence information of an RNA or cDNA strand ((006)) in a sample, comprising: a. Hybridizing a first detection probe oligonucleotide (204) comprising 50 to 1000 nucleotides, which is partially hybridized to a bridging oligonucleotide (205) comprising 5 to 100 nucleotides and forms a gap region (206) capable of binding to the oligonucleotide, at its 3' and / or 5' end to a complementary portion of at least one of the RNA or cDNA strands; b. Partially filling the gap region (206) with 1 to 16 barcode oligonucleotides comprising 4 to 20 nucleotides for determining the spatial information of the RNA or cDNA strand in the sample; c. Partially hybridizing a second detection probe oligonucleotide (204') comprising 50 to 1000 nucleotides at its 3' and / or 5' end to the same or a complementary portion of the cDNA strand and partially hybridizing to the bridging oligonucleotide (205) at each other end to form a circular template; d. Amplifying the circular template with a polymerase capable of rolling circle amplification within a rolling circle containing a plurality of concatemers; e. Determining the nucleotide sequence of the rolling circle and comprising a method.

2. A method for obtaining at least partial spatial position and sequence information of an RNA or cDNA strand ((006)) in a sample, comprising: f. Hybridizing the 3' and 5' ends of a detection probe oligonucleotide, which comprises a first oligonucleotide (204) and a second oligonucleotide (204') each comprising 50 to 1000 nucleotides and are linked by a partially hybridized bridging oligonucleotide (205) comprising 5 to 100 nucleotides and a bridging gap region (206) is formed between the first oligonucleotide (204) and the second oligonucleotide (204'), to the complementary portion of the at least one RNA or cDNA strand; g. 1 to 16 barcode oligonucleotides, containing 4 to 20 nucleotides, for determining the spatial information of the RNA or cDNA in the sample, filling the bridge gap region (206) to form a circular template; h. amplifying the circular template with a polymerase capable of rolling circle amplification in a rolling circle containing a plurality of concatemers; i. determining the nucleotide sequence of the rolling circle The method includes.

3. The detection probe oligonucleotides hybridize the first detection probe oligonucleotide (204) and the second detection probe oligonucleotide (204'), each containing 50 to 1000 nucleotides, to the complementary portions of the at least one RNA or cDNA strand at their respective 3' and 5' ends, and then the first (204) and the second oligonucleotide (204') are partially hybridized to and ligated to the bridge oligonucleotide (205), thereby hybridizing to the at least one RNA or cDNA strand. The method according to claim 2, characterized in that.

4. The detection probe oligonucleotides are ligating the first oligonucleotide (204) to the second oligonucleotide (204'), then hybridizing the resulting oligonucleotide to the complementary portion of the at least one RNA or cDNA strand, and then partially hybridizing and ligating the unbound end of the resulting oligonucleotide to the bridge oligonucleotide (205) Thereby, the method according to claim 2, characterized in that it hybridizes to the at least one RNA or cDNA strand.

5. The detection probe oligonucleotides hybridize to the complementary portion of the at least one RNA or cDNA strand, whereby a gap (207') of 1 to 150 nucleotides is formed between the first oligonucleotide (204) and the second oligonucleotide (204') of the detection probe oligonucleotides. The method according to claim 1 or 2, characterized in that.

6. The method according to claim 5, characterized in that the gap (207') is filled with nucleotides complementary to adjacent portions of the at least one RNA or cDNA strand, whereby a first target sequence (207) is obtained.

7. The method according to claim 1 or 2, characterized in that a second target sequence is obtained using a portion of the first and / or second oligonucleotide hybridized to the at least one RNA or cDNA.

8. The method according to claim 7, characterized in that the spatial information of the circular template in the sample is linked to the first and / or second target sequence.

9. The method according to claim 1 or 2, characterized in that the bridge gap region (206) is at least partially filled by removing the photocleavable protecting group with light after hybridization and hybridizing a barcode oligonucleotide containing the same or a different photocleavable protecting group to a complementary portion of the bridge oligonucleotide (205).

10. The method according to claim 1 or 2, characterized in that the circular template is selectively propagated by providing (hybridizing) a primer oligonucleotide complementary to one of the barcode oligonucleotides as a priming site for rolling circle amplification polymerase.

11. The method according to claim 1 or 2, characterized in that the sample is fixed to a surface and subjected to a permeation treatment.

12. The method according to claim 1 or 2, characterized in that the sample is provided as a tissue, a single-stranded circular template is isolated from the sample, and replicated ex situ by rolling circle amplification.

13. The method according to claim 1 or 2, characterized in that the sample is provided as a tissue, and a single-stranded circular template is replicated on the tissue by rolling circle amplification.

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