Method combining in situ target capture and spatial unique molecular identifier (SUMI) identification with in vitro sequencing for high density spatial multiomics

Abstract

To provide a method of combining in situ target capture and spatial unique molecular identifier (SUMI) identification with in vitro sequencing.SOLUTION: The present invention describes a method (High Density - SUMI-Seq) which combines the use of Spatial Unique Molecular Identifier in situ localization and identification (by in situ sequencing or sequential fluorescence hybridization) of rolonies derived from rolling circle amplification of circular oligonucleotides and in vitro sequencing of target captured RNA or DNA in combination with SUMI identification at a subcellular level with no optical diffraction limitation in the amount of captured target information that can be analyzed per cell. Apart from captured RNA or DNA, the High Density - SUMI-Seq method can also be applied using linear oligonucleotides to spatially resolve proteins and metabolites to provide multiomics results.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to methods for sequencing and in situ localization of rolonies containing spatially unique molecular identifiers (SUMIs), combined with target capture of RNA or DNA by hybridization and / or amplification from tissue sections, for high-density spatial analysis by in vitro sequencing. Modification of the sequencing method enables the localization of protein and metabolite molecules.

[0002] Background of the Invention Padlock oligonucleotides have proven very successful in polymerizing short portions of hybridized nucleic acids. Most padlock approaches are initiated by reverse transcription of the target into cDNA.

[0003] The padlock method is disclosed, for example, in "Highly multiplexed subcellular RNA sequencing in situ" by Lee et al., Science. 2014 March 21; 343(6177): 1360-1363. doi:10.1126 / science.1250212 or "Efficient In Situ Detection of mRNAs using the Chlorella virus DNA ligase for Padlock Probe Ligation" by Nils Schneider and Matthias Meier; February 5, 2020 - Cold Spring Harbor Laboratory Press.

[0004] An integrated assay for targeted multiplex amplification of the human DNA sequence has been published by Sujatha Krishnakumar et al.; sent to PNAS for review on February 19, 2008.

[0005] Furthermore, International Publication No. 2017143155 discloses multiple modification and analysis of cells using a pooled nucleic acid library. International Publication No. 2018045181 discloses a method for generating a library of nucleic acid sequences for detection by fluorescence in situ sequencing.

[0006] The published padlock method allows for the sequencing of DNA or RNA, but the sequenced DNA or RNA does not provide any spatial information about its origin within the cell or tissue.

[0007] Microscopic imaging that enables the degradation of multiple mRNAs at the single-cell level provides valuable information about the quantity and localization of transcripts. This is a crucial factor in understanding the tissue heterogeneity, molecular development, and therapeutic aspects of disease.

[0008] Fluorescence in situ hybridization (FISH)-based methods allow for direct labeling of transcripts in tissue sections, capturing spatial information. However, the number of probes that can be used is limited, and overlapping fluorescence signals are often problematic. Furthermore, the optical resolution limits of confocal microscopy are often reached. Consequently, the amount of probe that can be detected simultaneously is small. SeqFISH+ is a sequential fluorescence in situ hybridization approach that does not use probes already labeled with a phosphor, but rather uses transcript-specific probes containing barcode sequences that function as target sites for fluorescently labeled secondary probes. Various target-specific probes are identified using secondary probes that bind to these barcode sites during a sequential round of probes. By limiting the amount of probe detected by the secondary probes, a limited amount of fluorescence is emitted, and therefore the signal can be identified. Multiple separated images are computationally collected and aggregated to create a composite high-resolution image without requiring high-resolution instrumental microscopy.

[0009] However, while these approaches allow for the simultaneous evaluation of several genes, they do not capture transcript sequence information. Other methods based on single-cell RNA sequencing (scRNA-seq) can profile the entire transcriptome and capture sequence information. However, they often lack the original location at the tissue or single-cell level. A method that captures both sequence and spatial information with a resolution approaching that of a single cell remains a challenging task. Several approaches utilize FISSEQ and BaristaSeq (another gap-filling padlock-based approach to achieve its task with a limited read length of approximately 15 bases).

[0010] In recent years, in situ genome sequencing (IGS) has been described as a method for simultaneously sequencing and imaging the genome within a sample. This method describes a workflow for localizing unique molecular identifiers (UMIs) by short-read in situ sequencing, followed by amplicon dissociation of amplicons associated with UMI-containing genome sequences using paired-end sequencing, as published in AC Payne et al., Science 10.1126 / science.aay3446 (2020), first online release 31st of December 2020, PCR, and ex situ sequencing.

[0011] Very recently, a method combining in vitro and in situ sequencing of target RNA or c-DNA using a padlock oligonucleotide containing a spatially unique molecular identifier (SUMI) was described (European Patent Application No. 22154712.8). This method describes the spatial identification of a target sequence embedded in a padlock by combining in situ sequencing (SUMI) and in vitro sequencing (SUMI and target sequence). Since the target sequence and SUMI sequence are part of the same padlock and the resulting rolony, the density of target information is limited by the number of rolony that can be sequenced in situ within a region of the cell. The following invention overcomes this limitation.

[0012] Summary of the Invention The present invention relates to a method for obtaining spatial location and sequence information of a target sequence in a sample containing at least one RNA or single-stranded DNA, a. A step of hybridizing a first oligonucleotide, which has a sequence as a first PCR handle at its 5' end, to a complementary section of at least one RNA or single-stranded DNA at its 3' end, b. A step of hybridizing a second oligonucleotide, which has a sequence as a second PCR handle at its 3' end, to a complementary section of at least one RNA or single-stranded DNA at its 5' end, c. A step of ligating the 3' end of the first oligonucleotide with the 5' end of the second oligonucleotide to obtain a third oligonucleotide containing the target sequence, and removing the target RNA or single-stranded DNA from the third oligonucleotide. d. A step of providing a fourth oligonucleotide comprising multiple concatemers, each containing a sequence complementary to the second PCR handle and at least one sequence as a spatially unique molecular identifier (SUMI) containing at least two nucleic acids, at a dedicated spatial location on the sample; e. A first sequencing step for determining the spatial position of the fourth oligonucleotide, preferably by a first in situ sequencing step, to determine the SUMI sequence of the fourth oligonucleotide and thereby link the spatial position to the SUMI sequence. f. A step of hybridizing the third oligonucleotide to the complementary sequence of the fourth oligonucleotide using the second PCR handle, g. A step in which a nucleotide complementary to the fourth oligonucleotide is used as a template to extend the third oligonucleotide with polymerase, thereby incorporating SUMI into the extended third oligonucleotide. h. Dehybridizing the extended third oligonucleotide and determining the sequence of the extended third oligonucleotide by a second sequencing step, preferably a second in vitro sequencing step. i. A step of linking the sequence information of the extended third oligonucleotide to the spatial position information obtained in the first sequencing step. This method includes [something].

[0013] To obtain spatial position, a solid surface such as tissue or a fixed sample is required. Therefore, the method of the present invention preferably aims to obtain spatial position and sequence information of a target sequence on the surface of a sample, and provides a fourth oligonucleotide to a specific spatial position on the surface of the sample.

[0014] The method of the present invention can be further used to obtain the spatial location of proteins or metabolites in a sample. In this modified form, the third oligonucleotide may include an antigen-recognizing moiety capable of binding to a protein.

[0015] Modes for carrying out the invention All embodiments and variations of the method for obtaining spatial location and sequence information of a target sequence in a sample containing at least one RNA or single-stranded DNA strand can also be applied to methods for spatial single-cell protein expression.

[0016] Preferably, the spatially unique molecular identifier (SUMI) contains 2 to 500 bp.

[0017] In the first embodiment of the present invention, a second oligonucleotide is hybridized adjacent to the first oligonucleotide, thereby enabling direct ligation of the first and second oligonucleotides.

[0018] In a second embodiment of the present invention, a second oligonucleotide is hybridized upstream with a first oligonucleotide, thereby creating a gap of 2 to 100 nucleotides between the first and second oligonucleotides. A third oligonucleotide is obtained by filling this gap with RNA or nucleotides complementary to a single DNA strand.

[0019] The target sequence includes, at least, the nucleic acids of the hybridized 3' end of the first oligonucleotide and the hybridized 5' end of the second oligonucleotide, as defined in steps a and b of the objective of the present invention, for single-stranded DNA and RNA targets, but may also include the sequence of the oligonucleotide region that fills the gap after hybridization with RNA or single-stranded DNA.

[0020] In a further embodiment of this method, a fourth oligonucleotide can be generated by rolling circle amplification of a cyclic oligonucleotide. The fourth oligonucleotide may also include sequences that allow the extended third oligonucleotide to be segmented by restriction enzymes or chemically.

[0021] In the present invention, the extended target SUMI sequence can be amplified by general-purpose PCR before the collection of the extended target SUMI sequence or the extended barcode tag SUMI sequence in step g), and before or after in situ sequencing of the SUMI or determination of the spatial position of rolony by serial fluorescence in situ hybridization.

[0022] As an alternative embodiment to general-purpose PCR amplification, the extended target SUMI sequence or extended barcode tag SUMI sequence can be part of the padlock probe itself. Here, the 5' and 3' ends of the extended target SUMI sequence or extended barcode tag SUMI sequence will bind to the 5' and 3' regions complementary to the SUMI sequence of the SUMI rolony, thereby forming a padlock. After filling and ligating the padlock gap of the SUMI sequence, the target sequence will be ligated to the SUMI sequence forming the ring. The padlock probe, with the gap filled and ligated to form a circular template (the padlock is also filled, but may only be ligated in a further process), is used to encode the SUMI in the gap-filled portion of the padlock. Finally, the circularized padlock probe can be used as a template for rolling circle amplification (RCA) to generate the DNA strand used for sequencing as defined in step h.

[0023] In the present invention, the workflow can be modified to enable spatial localization by SUMI sequencing for other classes of biomolecules. Here, the oligonucleotide will be linked to a biomolecule binder. The oligonucleotide will contain a sequence (barcode tag) encoding a binder for the biomolecule (e.g., a specific antibody as a binder for a particular protein). After ligating the SUMI sequence to the barcode-tag sequence of the SUMI rolony, spatial multi-omics results for the biomolecule linked to the binder will be obtained as a result of step i) by in situ and in vitro sequencing.

[0024] In the present invention, prior to step d), the formation of rolony as the fourth oligonucleotide can be initiated by an external force (e.g., light or heat) that links it to a potential digital pathology imaging process.

[0025] In the present invention, after step d), the cells can be harvested and subjected to single-cell sequencing analysis.

[0026] The present invention describes a method of overcoming the spatial resolution constraints from in situ sequencing or sequential in situ hybridization by using SUMI rolony as a template for the spatial localization of target nucleic acids (SUMI and targets) identified by in vitro sequencing.

[0027] The method of the present invention can include performing the first sequencing step after incorporating the SUMI sequence into the extended third oligonucleotide.

[0028] Preferably, the third oligonucleotide includes a barcode-tag sequence to which the antigen recognition moiety is linked. For protein localization, the third oligonucleotide may include a barcode tag adjacent to the first and second PCR handles. The barcode tag encodes a protein. The third oligonucleotide is linked to an antibody that binds to the protein.

[0029] Therefore, the third oligonucleotide includes an antigen-recognition moiety that can bind to a protein. Preferably, the third oligonucleotide includes a barcode-tag sequence to which the antigen-recognition moiety is linked.

[0030] The term "antigen recognition moiety" refers to any type of antibody, fragmented antibody, or fragmented antibody derivative directed toward a marker expressed on cells in a cell sample. This term applies to fully intact antibodies, fragmented antibodies, or fragmented antibody derivatives, e.g., Fab, Fab', F(ab')2, sdAb, scFv, di-scFv, and nanobodies. Such fragmented antibody derivatives can be synthesized by recombinant techniques involving covalent and non-covalent conjugates containing these types of molecules. Further examples of antigen recognition moieties include peptide / MHC complexes targeting TCR molecules, cell adhesion receptor molecules, receptors for costimulatory molecules, and peptides or aptamers targeting artificially engineered binding molecules, such as cell surface molecules. Antibodies that are such targeted antigen recognition moieties can be directed toward antigens expressed intracellularly, such as IL2, FoxP3, and CD154, or extracellularly by biological specimens (target cells), such as CD3, CD14, CD4, CD8, CD25, CD34, CD56, and CD133.

[0031] The method and embodiments of the present invention will be further described with reference to the drawings. [Brief explanation of the drawing]

[0032] [Figure 1]Figure 1 shows an oligonucleotide design for a high-density (HD) spatially unique molecular identifier (SUMI) sequencing workflow for nucleic acid analysis. [Figure 2] Figure 2 shows a high-density (HD) spatially unique molecular identifier (SUMI) sequencing workflow for nucleic acid analysis. [Figure 3] Figure 3 shows a high-density (HD) spatially unique molecular identifier (SUMI) sequencing workflow for protein and metabolite analysis. [Figure 4] Figure 4 shows oligonucleotide designs for high-density (HD) spatially unique molecular identifier (SUMI) hybridization and sequencing workflows for nucleic acid, protein, or metabolite analysis. [Figure 5] Figure 5 shows the isothermal amplification of SUMI containing the captured probe.

[0033] Figure 1 shows oligonucleotide designs for a high-density (HD) spatially unique molecular identifier (SUMI) sequencing workflow for nucleic acid analysis. (A) First circular sequence (generic sequence 5' of the SUMI sequence). (B) Spatially unique molecular identifier (SUMI) sequence (sequence specific to each circular molecule). (C) Second circular sequence (generic sequence 3' of the SUMI sequence). (D) Priming region used for universal rolling circle amplification. (E) Nucleic acid extended by polymerase after the start of rolling circle amplification. (F) Rollony generated after several rounds of rolling circle amplification.

[0034] Figure 2 shows the high-density (HD) spatially unique molecular identifier (SUMI) sequencing workflow for nucleic acid analysis. (A) The SUMI circular nucleic acid template is added to a fixed and permeabilized tissue section. The generic sequence 5' (1st) and generic sequence 3' (2nd) of the SUMI sequence, as well as the spatially unique molecular identifier (SUMI), are all parts of the SUMI circular nucleic acid template. (B) Rolony generated from the SUMI circular nucleic acid template. Sequences from the SUMI circular nucleic acid template molecule are converted to multiple concatemers of the template sequence after rolling circle amplification. The rolony (circled inside the cell representation) is sequenced in situ to derive spatially registered SUMI sequence information (1, 2, and 3 as examples). Note: Only a single cell and three SUMI rolony are shown for illustrative purposes. (C) In situ target capture is performed by close-proximity ligation of messenger RNA in the fixed and permeabilized tissue section. (D) The resulting target capture product contains universal PCR handles (first and second). Further in situ amplification of the captured target sequence (if necessary) is achieved by the universal PCR handle labeled with the first PCR handle (first) and the second PCR handle (second). (E) The rolony serves as a template for the universal target capture product. Thereafter, the target sequence product is extended to include the SUMI sequence and to ligate the SUMI sequence to the target capture product. More specifically, the second PCR handle (second) of the target capture product is complementary to the rolony universal sequence 5' (first) of the SUMI sequence. After hybridization to the rolony, the 3' end of the target capture product is extended in situ beyond the SUMI sequence. One rolony serves as a template for many target sequences, providing the basis for a high-density (HD) spatially unique molecular identifier (SUMI) sequencing workflow. (F) The extended target SUMI sequence is amplified, if applicable, with PCR primers complementary to the (first) and (second) handles, and finally isolated from the tissue section for further processing in vitro (optional PCR may also be performed in vitro).The extended target SUMI sequence molecule is cyclized and amplified to form a rolony in vitro. The rolony is placed in a flow cell to serve as a template for in vitro sequencing. The flow cell is loaded into the instrument, and the rolony is sequenced in vitro. (G) Next, sequence information of the SUMI and target sequence is obtained (some target sequences may be ligated to the same SUMI sequence). The in vitro sequencing (SUMI sequence-target capture product) ligated to the in vivo position of the SUMI sequence is paired to obtain its spatial position.

[0035] Figure 3 shows a high-density (HD) spatially unique molecular identifier (SUMI) sequencing workflow for protein and metabolite analysis. (A) Antibody ligated to an oligonucleotide containing a barcode-tag region for antibody identification. (B) Binder ligated to an oligonucleotide containing a barcode-tag region for metabolite identification. (C) Further in situ amplification of the barcode-tag sequence is achieved using a general-purpose PCR handle with the respective general-purpose primers (1st and 2nd). (D) The barcode-tag sequence is ligated to the SUMI sequence, as shown in Figure 2.

[0036] Figure 4 shows oligonucleotide designs for high-density (HD) spatially unique molecular identifier (SUMI) hybridization and sequencing workflows for nucleic acid, protein, or metabolite analysis. (A) Universal sequence 5' (1st) of the SUMI sequence. (B) Spatially unique molecular identifier (SUMI) sequence (a unique sequence consisting of four detection barcode regions, as shown here as an example). Each of the four barcode regions contains 2 to 20 nucleotides. There may be more than four barcode regions to improve coding ability, and all barcodes have a unique sequence (not shown). (C) Universal sequence 3' (2nd) of the SUMI sequence. This may also contain a restriction site for monomerization. (D) Priming region used for universal rolling circle amplification.

[0037] Figure 5 shows isothermal amplification of SUMI containing the captured probe. (A) A fixed and permeabilized tissue section is placed on the SUMI rolony. The rolony (black dot on the glass flow cell) is sequenced in situ. (B) The target portion of the messenger RNA is captured directly onto the section by the proximity ligation probe. (C) The rolony serves as a template for a general-purpose target capture product and contains SUMI, the general-purpose sequence 3' (second) of the SUMI sequence, and in this example, also a restriction site (optional). This allows the target sequence to be extended by the SUMI sequence and cleaved into specific monomers. The second PCR handle (second) of the target product, complementary to the general-purpose sequence 5' (first) of the rolony of the SUMI sequence, contains a 3'OH blocking nucleotide (azide or disulfide group) and is extended beyond the SUMI sequence in situ only after being deprotected with a reducing agent, e.g., phosphine. A single rolony serves as a template for many target sequences, providing the foundation for a high-density (HD) spatially unique molecular identifier (SUMI) sequencing workflow. The extended target SUMI sequence can be amplified by PCR primers (1st and 2nd) using a high-capacity enzyme such as the Phi29 enzyme (exponential RCA), and optionally subsequently cleaved with restriction enzymes before or after extraction from the flow cell, further processed, and amplified by PCR (see workflow in Figure 2).

[0038] As shown in Figures 1 and 4, the oligonucleotide has one SUMI containing at least two nucleotides. The 5' and 3' of the SUMI circular oligonucleotide contain general-purpose sequences (first and second) having 5 to 50 nucleotides. In the method of the present invention, the single-stranded circular template is replicated by a polymerase capable of rolling circle amplification into multiple DNA concatemers that form DNA nanoballs or rolony. For this purpose, the oligonucleotide used in the present invention may include at least one primer region having 5 to 50 nucleotides for rolling circle amplification.

[0039] In one embodiment of the present invention, the general-purpose array does not have to directly follow the SUMI array, and may be located at a variable distance from the SUMI array.

[0040] In another embodiment of the present invention, the primer region for initiating rolling circle amplification can be identical to the general sequence.

[0041] In further embodiments, the oligonucleotide containing SUMI may not be cyclic at the time it is provided on the tissue section, but cyclization may occur directly on the tissue.

[0042] In one embodiment of the present invention, target capture can be initiated by target DNA or proximity ligation. Each design adjustment of the linear oligonucleotide must be considered.

[0043] As shown in Figure 3, linear oligonucleotides can be used to encode proteins or metabolites. Here, the 3' end of the linear oligonucleotide is complementary to the rolony sequence generated from the cyclic oligonucleotide in order to combine the barcode tag and SUMI sequence into a single nucleic acid molecule.

[0044] In a further embodiment of the present invention, rolony is generated on a tissue section using a cyclic oligonucleotide containing a spatially unique molecular identifier (SUMI) as shown in Figure 1. The rolony serves as a sequencing template for in situ sequencing to identify the spatially unique molecular identifier (SUMI). Intracellular spatial information for all rolony is registered and linked to the SUMI sequence. In situ sequencing can be performed before or after in situ target capture.

[0045] The overall process of the present invention is shown in Figure 2. Here, messenger RNA is directly captured onto a fixed and permeabilized tissue section. The target sequence in the mRNA is captured by proximity ligation. Multiple ligation reactions are performed according to each multiple oligonucleotide target capture design in order to capture multiple mRNA types in parallel. A rolony is generated from a cyclic oligonucleotide and functions as a sequencing template for in situ sequencing, and also as a template for the target capture product extended by the SUMI sequence. The target capture product is extended in situ by the SUMI sequence, and after the SUMI sequence is determined by in situ sequencing, the extended SUMI target capture product is removed from the tissue section and subjected directly to in vitro sequencing. It may be preferable to amplify the extended SUMI target capture product before cyclization, rolonyization, and in vitro sequencing.

[0046] The intracellular location of target sequences, including identified mutations, is revealed by linking the in vitro target sequence / SUMI to the SUMI sequence from in situ sequencing. Since hundreds of unique SUMI rolony can be spatially degraded within a single cell, and each rolony provides thousands of concatemerized SUMI sequences as templates, hundreds of thousands of target capture mRNA sequences can theoretically be spatially identified at a degree of intracellular degradation within a single cell, thus providing a high-density (HD) method.

[0047] In a second embodiment of the present invention, the intracellular locations of proteins and metabolites will be revealed by a sequencing workflow. The linear oligonucleotide design shown in Figure 3 will use an "antigen recognition moiety" as the binding principle for the protein to determine the intracellular protein location.

[0048] As a variation of the first embodiment, a cyclic oligonucleotide for a hybridization-based SUMI decoding principle for spatial localization of rolony is shown in Figure 4. Hybridization-based methods may be advantageous when in situ sequencing of SUMI is not possible. In such cases, rolony can be decoded using a multicolor decoding scheme. The detection probe used in the method of the present invention may consist of an oligonucleotide having 2 to 20 nucleotides that can bind to at least a portion of the barcode region. The resulting rolony continues to function as a template for target capture extension, as described above (see Figure 2). For high-density (HD) spatially unique molecular identifier (SUMI) hybridization and sequencing workflows for nucleic acid analysis, longer sequencing read lengths are required for in vitro sequencing to decode SUMI sequences.

[0049] In further embodiments, the method is limited to a target tissue region. The target tissue region is identified by classical imaging techniques, such as microscopy. To concentrate the in situ sequencing method on the target region, rolony formation is controlled by an external force (such as light or heat). Since rolony functions as a sequencing template, sequencing would not occur without rolony. Polymerization and the initiation of rolony formation can be inhibited by blocking polymerase or blocking primers. The blocking principle can be removed by an external force, such as light or heat, which can be conceptually directed by imaging techniques.

[0050] In a further embodiment, after decoding the SUMI (e.g., by in situ sequencing), the tissue section can be digested to isolate individual cells. The rolony-containing cells are then sorted and finally subjected to single-cell sequencing. Sorting of rolony-containing cells can be achieved by increasing nucleic acid content as a result of rolling circle amplification or by fluorescence intensity derived from a hybridization probe against the rolony sequence. Since the SUMI sequence from in situ sequencing can also be identified by single-cell sequencing, the information from single-cell sequencing can be linked to the spatial location via the SUMI derived from in situ sequencing.

[0051] In this embodiment, a specific rolony can be generated from a cyclic oligonucleotide by using a specific primer recognized by the Phi29 enzyme used in RCA, which enables selective amplification of a subset of amplicons, corresponding to a targeted gene (Figure 2) or barcode-tag (Figure 3) for a targeted antibody or targeted molecular binder. Finally, the sequenced data is linked to a region on the tissue where the mRNA or cDNA transcript or the antibody or molecular binder of interest originally interacted with the cyclic oligonucleotide.

[0052] In further embodiments, as visualized in Figure 5, the target SUMI sequence is established and amplified by exponential RCA using a high-capacity polymerase, such as Phi29. Here again, the rolony serves as a template for a general-purpose target capture product containing the SUMI. One rolony serves as a template for many target sequences, providing the basis for a high-density (HD) spatially unique molecular identifier (SUMI) sequencing workflow.

[0053] Samples analyzed by the disclosed methods may be derived from any specimen, such as whole animals, organs, tissue sections, cell aggregates, or single cells of invertebrates (e.g., nematodes (Caenorhabditis elegans), fruit flies (Drosophila melanogaster)), vertebrates (e.g., zebrafish (Danio rerio), clawed frogs (Xenopus laevis)), and mammals (e.g., mice (Mus musculus), humans (Homo sapiens)). Biological samples may be in the form of tissue slices, cell aggregates, suspension cells, or adherent cells. Cells may be alive or dead.

[0054] In yet another variation of the method according to the present invention, the sample is converted into isolated cells, which are then immobilized by trapping them in a microcavity or by adhesion.

[0055] Next, the sample cells can be subjected to single-cell sequencing.

[0056] The spatial information of a fourth oligomer, for example, rolony, i.e., their positions on the sample, is determined, for example, by an imaging process.

[0057] Imaging can be performed by techniques known, for example, "multiepitope ligand mapping," "tip-based cytometry," or "Multiomics," as described in, for example, European Patent No. 0810428, No. 1181525, No. 1136822, or European Patent Application Publication No. 1224472. In this technique, cells are immobilized and brought into contact with antibodies bound to fluorescent moieties. The antibodies are recognized by each antigen on the biological specimen (e.g., on the cell surface), unbound markers are removed, and the fluorescent moieties are excited, after which the location of the antigens is detected by the fluorescence emission of the fluorescent moieties. In certain modified forms, instead of antibodies bound to fluorescent moieties, antibodies bound to moieties detectable for MALDI imaging or CyTOF can be used. Those skilled in the art will know how to modify this technique to work using these detection moieties based on the fluorescent moieties. The location of the target moieties is achieved by a digital imaging device with sufficient resolution and sensitivity to the wavelength of fluorescence emission. Digital imaging devices can be used, for example, with or without optical magnification, using a fluorescence microscope. The resulting images can be stored in a suitable storage device, such as a hard drive, in a format such as RAW, TIF, JPEG, or HDF5.

Claims

1. A method for obtaining spatial location and sequence information of a target sequence in a sample containing at least one RNA or single-stranded DNA, a. A step of hybridizing a first oligonucleotide, which has a sequence as a first PCR handle at its 5' end, to a complementary section of at least one RNA or single-stranded DNA at its 3' end, b. A step of hybridizing a second oligonucleotide, which has a sequence as a second PCR handle at its 3' end, to a complementary section of at least one RNA or single-stranded DNA at its 5' end, c. Ligating the 3' end of the first oligonucleotide with the 5' end of the second oligonucleotide to obtain a third oligonucleotide containing the target sequence, and removing the target RNA or single-stranded DNA from the third oligonucleotide. d. A step of providing a fourth oligonucleotide, which comprises a plurality of concatemers, each containing a sequence complementary to the second PCR handle and at least one sequence as a spatially unique molecular identifier (SUMI) containing at least two nucleic acids, at a dedicated spatial position on the sample; e. A step of determining the sequence of the SUMI of the fourth oligonucleotide by a first sequence determination step for determining the spatial position of the fourth oligonucleotide, thereby linking the spatial position to the SUMI sequence, f. A step of hybridizing the third oligonucleotide to the complementary sequence of the fourth oligonucleotide using the second PCR handle, g. A step of extending the third oligonucleotide with polymerase using a nucleotide complementary to the fourth oligonucleotide as a template, thereby incorporating the SUMI into the extended third oligonucleotide, h. A step of dehybridizing the extended third oligonucleotide and determining the sequence of the extended third oligonucleotide by a second sequencing step, i. A step of linking the sequence information of the extended third oligonucleotide with the spatial position information obtained in the first sequencing step. Methods that include...

2. The method according to claim 1, characterized in that the second oligonucleotide is hybridized adjacent to the first oligonucleotide, thereby enabling direct ligation of the first and second oligonucleotides.

3. The method according to claim 1, characterized in that the second oligonucleotide is hybridized upstream with the first oligonucleotide, thereby forming a gap of 2 to 100 nucleotides between the first oligonucleotide and the second oligonucleotide, and the gap between the first oligonucleotide and the second oligonucleotide is filled with nucleotides complementary to the RNA or single DNA strand to obtain the third oligonucleotide.

4. The method according to claim 1, characterized in that the fourth oligonucleotide further comprises a sequence that enables the extended third oligonucleotide to be segmented by restriction enzymes or chemically.

5. The method according to claim 1, characterized in that the fourth oligonucleotide is provided by rolling circle amplification of a cyclic oligonucleotide containing a sequence complementary to the second PCR handle and at least one sequence as a spatially specific molecular identifier (SUMI).

6. The method according to claim 5, characterized in that rolling circle amplification (RCA) is activated by light and / or heat.

7. The method according to claim 1, characterized in that the first sequencing step is performed after incorporating the SUMI sequence into the extended third oligonucleotide.

8. The method according to claim 1, characterized in that the third oligonucleotide includes an antigen-recognizing portion capable of binding to a protein.

9. The method according to claim 8, characterized in that the third oligonucleotide includes a barcode-tag sequence to which the antigen recognition portion is linked.

10. The method according to claim 1, characterized in that the cells of the sample are further subjected to single-cell sequencing.

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