Method combining in situ target amplification and spatial unique molecular identifier (SUMI) identification using RT-pcr

Abstract

To provide methods for obtaining spatial position and sequence information of a target sequence in RNA or single stranded DNA.SOLUTION: Disclosed is a method comprising the steps of: using a first oligonucleotide amplified by reverse transcription-polymerase chain reaction (RT-PCR) as a template to amplify a second oligonucleotide by RT-PCR to thereby obtain a third oligonucleotide comprising sequences as first and second PCR handles and a target sequence; providing a fourth oligonucleotide comprising a sequence complementary to the second PCR handle and a sequence as the spatial unique location molecular identifier (SUMI); determining the sequence of SUMI of a fourth oligonucleotide; extending the third oligonucleotide with a polymerase using nucleotides complementary to the fourth oligonucleotide as a template; incorporating the SUMI into the extended third oligonucleotide; and determining the sequence.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for sequencing and in situ localization of loronies, including spatially specific molecular identification (SUMI) combined with targeted capture of RNA or DNA by hybridization and / or amplification from tissue sections for high-density spatial analysis by in vitro sequencing. Modifications to the sequencing method allow for the localization of protein and metabolite molecules.

[0002] Background technology Padlock oligonucleotides have proven extremely successful in polymerizing short segments of hybridized nucleic acid. Most padlock approaches begin with reverse transcribing the target into cDNA.

[0003] The Padlock method is disclosed, for example, in "Highly multiplexed subcellular RNA sequencing in situ," 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," Nils Schneider and Matthias Meier; February 5, 2020 - Cold Spring Harbor Laboratory Press.

[0004] A universal assay for targeted multiplex amplification of human DNA sequences was published by Sujatha Krishnakumar et al; PNAS sent for review on February 19, 2008.

[0005] Furthermore, WO2017143155 discloses multiplex modification of cells and analysis thereof using pooled nucleic acid libraries, and WO2018045181 discloses methods for generating libraries of nucleic acid sequences for detection by fluorescence in situ sequencing.

[0006] The published padlock method allows for sequencing of DNA or RNA, but does not provide any spatial information about the intracellular and intratissue location of origin of the sequenced DNA or RNA.

[0007] Microscopic imaging, which allows for single-cell resolution of multiple mRNAs, provides valuable information on transcript abundance and localization, which is crucial for understanding tissue heterogeneity, molecular development, and disease treatment.

[0008] Fluorescence in situ hybridization (FISH)-based methods allow for direct labeling of transcripts in tissue sections and capture of spatial information. However, the number of probes that can be used is limited, and overlapping fluorescent signals is often problematic. Furthermore, the optical resolution limit of confocal microscopes is often reached, thus reducing the number of probes that can be simultaneously detected. SeqFISH+ is a sequential fluorescence in situ hybridization approach that uses transcript-specific probes containing barcode sequences that serve as target sites for fluorescently labeled secondary probes, rather than probes already labeled with fluorophores. Diverse target-specific probes are identified using secondary probes that bind to these barcode sites during sequential rounds of probing. Due to the limited amount of probe detected by the secondary probe, only a limited amount fluoresces, thereby enabling signal differentiation. Multiple separate images are collected and aggregated by computer to create a composite high-resolution image without the need for a high-resolution instrument microscope.

[0009] However, while this approach allows for the simultaneous evaluation of a large number of genes, it does not capture sequence information of the transcripts. Other methods based on single-cell RNA sequencing (scRNA-seq) can profile the entire transcriptome and capture sequence information. However, the original location at the tissue or single-cell level is often lost. Capturing both sequence and spatial information at near-single-cell resolution remains a challenging task. Some approaches use FISSEQ and BaristaSeq (other gap-filling padlock-based approaches) to accomplish the task with a limited read length of approximately 15 bases.

[0010] Recently, in situ genome sequencing (IGS) has been described as a method for simultaneously sequencing and imaging the genome within a sample. This method, published by A.C. Payne et al. in Science 10.1126 / science.aay3446 (2020) (first online release December 31, 2020), describes a workflow for localizing unique molecular identifiers (UMIs) by short-read in situ sequencing, followed by ex situ sequencing of amplicons associated with the UMI-bearing gene sequence via PCR and paired-end sequencing.

[0011] More recently, a "method for combining in vitro and in situ sequencing of target RNA or c-DNA using padlock oligonucleotides containing spatially unique molecular identifiers (SUMIs)" has been described (EP 22154712.8). This method describes spatial identification of target sequences incorporated into padlocks by a combination of in situ sequencing (SUMI) and in vitro sequencing (SUMI and target sequence). Because the target sequence and SUMI sequence are part of the same padlock and resulting lorony, the density of target information is limited by the number of lorony that can be sequenced in situ within a region of a cell. The following invention overcomes this limitation.

[0012] Summary of the Invention The subject of the present invention is 1. A method for obtaining spatial location and sequence information of a target sequence within at least one RNA or single-stranded DNA, comprising: a. hybridizing a first oligonucleotide to a complementary section of at least one RNA or single-stranded DNA, wherein the first oligonucleotide is provided with a sequence as a first PCR handle; b. amplifying the first oligonucleotide using at least one RNA or single-stranded DNA as a template using reverse transcription polymerase chain reaction (RT-PCR); c. removing at least one RNA or single-stranded DNA from the amplified first oligonucleotide; d. hybridizing a second oligonucleotide to the complementary section of the amplified first oligonucleotide, wherein the second oligonucleotide is provided with a sequence as a second PCR handle; e. amplifying a second oligonucleotide using the amplified first oligonucleotide as a template using reverse transcription polymerase chain reaction (RT-PCR), thereby obtaining a third oligonucleotide having sequences as the first and second PCR handles and a target sequence; f. removing the third oligonucleotide from the amplified second oligonucleotide; g. providing a fourth oligonucleotide at a dedicated spatial location on the sample, the fourth oligonucleotide having a plurality of concatemers each comprising a sequence complementary to the second PCR handle and at least one sequence as a spatially unique molecular identifier (SUMI) having at least two nucleic acids; h. determining the sequence of the SUMI of the fourth oligonucleotide by a first sequencing step to determine the spatial location of the fourth oligonucleotide, thereby linking the spatial location with the SUMI sequence; i. hybridizing a third oligonucleotide to the complementary sequence of a fourth oligonucleotide at the second PCR handle; j. extending the third oligonucleotide with a polymerase using the nucleotide complementary to the fourth oligonucleotide as a template, thereby incorporating a SUMI into the extended third oligonucleotide; k. dehybridizing the extended third oligonucleotide and determining the sequence of the extended third oligonucleotide by a second sequencing step; l. Linking the sequence information of the extended third oligonucleotide with the spatial location information obtained in the first sequencing step. The method includes:

[0013] The method of the present invention can also be used to obtain the spatial location of a protein or metabolite within a sample. For protein localization, a third oligonucleotide contains a barcode tag adjacent to the first and second PCR handles. The barcode tag encodes the protein. The third oligonucleotide is linked to an antibody that binds to the protein.

[0014] Detailed Description All embodiments and variations of the method for obtaining spatial location and sequence information of a target sequence in a sample having at least one RNA or single-stranded DNA strand can also be applied in the method for spatial single-cell protein expression.

[0015] Preferably, the spatially unique molecular identifier (SUMI) has 2 to 500 bp.

[0016] The target sequence comprises at least the nucleic acid 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 present invention for single-stranded DNA and RNA targets, but may also include the sequence of the region of the oligonucleotide that fills the gap after hybridization with RNA or single-stranded DNA.

[0017] In another embodiment of this method, the fourth oligonucleotide may be generated by rolling circle amplification of a circular oligonucleotide, and may have a sequence that allows the extended third oligonucleotide to be chemically segmented with a restriction enzyme.

[0018] In the present invention, the extended target SUMI sequence may be amplified by a general PCR reaction before collecting the extended target SUMI sequence or the extended barcode tag SUMI sequence in step f) and before or after determining the spatial location of the LOI by in situ sequencing of the SUMI or sequential fluorescent in situ hybridization.

[0019] As an alternative to general-purpose PCR amplification, the extended target SUMI sequence or the 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 the extended barcode tag SUMI sequence bind to the 5' and 3' regions complementary to the SUMI sequence of the SUMI rolony, thereby generating a padlock. After padlock gap filling and ligation of the SUMI sequence, the target sequence is linked to the SUMI sequence to form a circle. The gap-filled and ligated padlock probe (the padlock can be filled, but can also only be further ligated in this process) forms a circular template and is used to encode the SUMI within 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 a DNA strand used for defined sequencing.

[0020] In the present invention, the workflow can be modified to enable spatial localization by SUMI sequencing for other classes of biomolecules. Here, oligonucleotides are linked to biomolecule binders. The oligonucleotides contain sequences (barcode tags) that encode the biomolecule binder (e.g., an antibody specific for a particular protein). After linking the SUMI sequence with the SUMI sequence barcode tag sequence, spatial multi-omics results of in situ and in vitro sequencing of the binder-linked biomolecules are obtained.

[0021] In the present invention, the formation of rolone as the fourth oligonucleotide can be initiated by an external force (e.g., light or heat) that can be linked to a potential digital pathology imaging process.

[0022] In the present invention, cells may be harvested and subjected to single cell sequencing analysis.

[0023] The present invention describes a method to overcome the spatial resolution limitations from in situ sequencing or sequential in situ hybridization by using SUMI loci as templates for the spatial localization of target nucleic acids (SUMIs and targets) identified by in vitro sequencing.

[0024] The method of the present invention and its embodiments will now be further explained with reference to the drawings. [Brief explanation of the drawings]

[0025] [Figure 1] FIG. 1 shows oligonucleotide designs for high-density (HD) spatially unique molecular identifier (SUMI) sequencing workflow for nucleic acid analysis. [Figure 2] FIG. 1 illustrates the high-density (HD) spatially unique molecular identifier (SUMI) sequencing workflow for nucleic acid analysis. [Figure 3] FIG. 1 illustrates the high-density (HD) spatially unique molecular identifier (SUMI) sequencing workflow for protein and metabolite analysis. [Figure 4] FIG. 1 illustrates the oligonucleotide design for high-density (HD) spatially unique molecular identifier (SUMI) hybridization and sequencing workflow for nucleic acid, protein, or metabolite analysis. [Figure 5] FIG. 1 shows isothermal amplification of SUMI containing amplified probes. [Figure 6]FIG. 1 shows an example of an in situ sequenced rolony generated from a circular oligonucleotide that can serve as a template for a target amplification product extended by a SUMI sequence.

[0026] Figure 1 shows oligonucleotide design for high-density (HD) spatially unique molecular identifier (SUMI) sequencing workflow for nucleic acid analysis. (A) Circular first sequence (universal sequence 5' of SUMI sequence). (B) Spatially unique molecular identifier (SUMI) sequence (unique sequence for each circular molecule). (C) Circular second sequence (universal sequence 3' of SUMI sequence). (D) Priming region used for universal rolling circle amplification. (E) Nucleic acid extended by polymerase after initiation of rolling circle amplification. (F) Rolling circle generated after several rounds of rolling circle amplification.

[0027] Figure 2 shows the high-density (HD) spatially unique molecular identifier (SUMI) sequencing workflow for nucleic acid analysis. (A) A SUMI circular nucleic acid template is added to a fixed and permeabilized tissue section. The 5' (1st) generic sequence of the SUMI sequence, the 3' (2nd) generic sequence of the SUMI sequence, and the spatially unique molecular identifier (SUMI) are all part of the SUMI circular nucleic acid template. (B) A LONY generated from the SUMI circular nucleic acid template. The sequence from the SUMI circular nucleic acid template molecule is converted into multiple concatemers of the template sequence after rolling circle amplification. The LONY (circled within the cell representation) is sequenced in situ to derive the spatially recorded SUMI sequence information (e.g., 1, 2, and 3). Note: Only a single cell and three SUMI LONYs are shown for illustrative purposes. (C) In situ target capture is performed by a targeted RT-PCR reaction in which a target-specific pair of oligonucleotides is applied directly to fixed and permeabilized tissue sections to first reverse transcribe and then amplify a specific portion of messenger RNA. The oligonucleotides contain a primary sequence that directly binds to the target nucleic acid or its complementary sequence, and also contain a set of sequences for universal PCR handles (1st and 2nd). (D) The resulting double-stranded product contains the universal PCR handles (1st and 2nd) and the desired target. Further, in situ amplification of the target sequence (if necessary) is achieved by labeling the first (1st) PCR handle and the second (2nd) PCR handle. (E) The rDNA serves as a template for the universal target amplification product. The target sequence product is then extended to include a SUMI sequence and link to the target amplification product. More specifically, the double-stranded amplified target sequence is denatured into a single-stranded oligonucleotide, and the second PCR handle (2nd handle) of one strand of the amplified target product hybridizes to the complementary Rolony universal sequence 5' of the SUMI sequence (1st handle). After hybridization to Rolony, the 3' end of the target amplification product is extended in situ beyond the SUMI sequence.A single LOLONY 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 optionally amplified with PCR primers complementary to the (1st) and (2nd) handles and finally removed from the tissue section for further processing in vitro (additional PCR may be performed in vitro). The extended target SUMI sequence molecule is circularized and amplified to form a LOLONY in vitro. The LOLONY is loaded into a flow cell and serves as a template for in vitro sequencing. The flow cell is inserted into an instrument, and in vitro sequencing of the LOLONY is performed. (G) Sequence information of the SUMI and target sequences is obtained (multiple target sequences may be linked to the same SUMI sequence). The in vivo location of the SUMI sequence and the linked (SUMI sequence-target amplicon) in vitro sequence are paired to obtain spatial locations.

[0028] Figure 3 shows the high-density (HD) spatially unique molecular identifier (SUMI) sequencing workflow for protein and metabolite analysis. (A) An antibody is bound to an oligonucleotide containing a barcode tag region for antibody identification. (B) A binder is bound to an oligonucleotide containing a barcode tag region for metabolite identification. (C) Further, in situ amplification of the barcode tag sequence is achieved by a universal PCR handle using the respective universal primers (1st & 2nd). (D) The barcode tag sequence is linked to the SUMI sequence as shown in Figure 2.

[0029] Figure 4 shows the oligonucleotide design for high-density (HD) spatially unique molecular identifier (SUMI) hybridization and sequencing workflow for nucleic acid, protein, or metabolite analysis. (A) The generic sequence 5' (1st) of the SUMI sequence. (B) The unique sequence with four detection barcode regions shown here as an example. Each of the four barcode regions has 2-20 nucleotides. The barcode regions can be >4 to increase coding capacity, and all barcodes have unique sequences (not shown). (C) The generic sequence 3' (2nd) of the SUMI sequence may contain a restriction site for monomerization. (D) The priming region used for global rolling circle amplification.

[0030] Figure 5 shows the isothermal amplification of SUMIs containing amplified probes. (A) Fixed and permeabilized tissue sections are placed on a SUMI LOLONY. The LOLONY (black dot on the glass flow cell) is sequenced in situ. (B) Target portions of messenger RNA are amplified directly on the section using proximity ligation probes. (C) The LOLONY serves as a template for the generic target capture product and contains the SUMI, the generic sequence 3' (2nd) of the SUMI sequence, and, in this example, an optional restriction site. This allows the target sequence to be extended by the SUMI sequence and cleaved into unique monomers. The second PCR handle (2nd) of the target product, complementary to the LOLONY generic sequence 5' (1st) of the SUMI sequence, contains a 3'OH blocking nucleotide (azide or disulfide group) and can only be extended beyond the SUMI sequence in situ after deprotection using a reducing agent, such as phosphine. A single rDNA serves as a template for many target sequences, providing the basis for a high-density (HD) spatially unique molecular identifier (SUMI) sequencing workflow. The extended target SUMI sequence can be amplified (exponential RCA) by PCR primers (1st & 2nd) using a highly processive enzyme, such as Phi29 enzyme, as shown, and optionally subsequently cleaved with a restriction enzyme before or after extraction from the flow cell, and further processed and amplified by PCR (see workflow in Figure 2).

[0031] Figure 6 shows an example of an in situ sequenced LORONY generated from a circular oligonucleotide that can serve as a template for a target amplicon extended by a SUMI sequence. The LORONY is placed on a flow cell and subjected to sequencing by synthesis (SBS). One cycle showing all of the fluorescent channels for each of the interrogated bases and the unique incorporation of a T is shown (cycle 6). The target amplicon is then extended in situ by the SUMI sequence. The SUMI-extended target amplicon is then removed from the tissue section and subjected to direct in vitro sequencing.

[0032] As shown in Figures 1 and 4, the oligonucleotide has at least one SUMI with at least two nucleotides. At the 5' and 3' ends of the SUMI, the circular oligonucleotide has a universal sequence (1st & 2nd) with 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 have at least one primer region with 5 to 50 nucleotides for rolling circle amplification.

[0033] In one embodiment of the present invention, the universal sequence may not directly follow the SUMI sequence, but may be located at variable distances from the SUMI sequence.

[0034] In another embodiment of the invention, the primer regions that initiate rolling circle amplification may be identical.

[0035] In other embodiments, the SUMI-containing oligonucleotide may not be circular when provided on the tissue section, but circularization may be performed directly on the tissue.

[0036] In one embodiment of the present invention, target capture may start with DNA as the target. Each design adjustment of the linear oligonucleotide needs to be taken into account.

[0037] Linear oligos may be used to encode proteins or metabolites, as shown in Figure 3. Again, the 3' end of the linear oligonucleotide is complementary to the sequence of the LONGITID generated from the circular oligonucleotide to combine the barcode tag and SUMI sequence into one nucleic acid molecule.

[0038] In a first embodiment of the present invention, as shown in Figure 1, circular oligonucleotides containing spatially unique molecular identifiers (SUMIs) are used to generate Lolonies on tissue sections. The Lolonies serve as sequencing templates for in situ sequencing to identify the Spatial Unique Molecular Identifiers (SUMIs). The intracellular spatial information for every Lolony is recorded and linked to the SUMI sequence. In situ sequencing can be performed before or after in situ target capture.

[0039] The general steps of the present invention are shown in Figure 2. Here, a target portion of messenger RNA is amplified directly on a section of fixed and permeabilized tissue. The sequence of interest on the mRNA is amplified using an RT-PCR reaction using two specific oligonucleotides that directly target the messenger RNA. The oligonucleotides contain primary sequences flanking the target region for amplification and a set of universal sequences as PCR handles. A rolony is generated from the circularized oligonucleotide and serves as a sequencing template for in situ sequencing and as a template for a target amplicon that is extended with a SUMI sequence. After the target amplicon is extended in situ with the SUMI sequence and the SUMI sequence is determined by in situ sequencing, the SUMI-extended target amplicon is removed from the tissue section and subjected directly to in vitro sequencing. It may be desirable to amplify the SUMI-extended target amplicon prior to circularization, rolony, and in vitro sequencing.

[0040] The subcellular location of the target sequence containing the identified mutation is revealed by linking the target sequence / SUMI obtained in vitro to the SUMI sequence from in situ sequencing. Because hundreds of unique SUMI sequences can be spatially resolved within a single cell, and each sequence provides thousands of concatemerized SUMI sequences as templates, hundreds of thousands of target amplified mRNA sequences can theoretically be spatially identified within a single cell at cellular resolution, thereby providing a high-density (HD) method.

[0041] In a second embodiment of the present invention, the subcellular location of proteins and metabolites is revealed by a sequencing workflow. The linear oligonucleotide design shown in Figure 3 uses an "antigen recognition moiety" as the binding principle for proteins to determine subcellular protein location.

[0042] The term "antigen recognition moiety" refers to any type of antibody, antibody fragment, or antibody fragment derivative directed against a marker expressed on cells of a cell sample. This term relates to fully intact antibodies, antibody fragments, or antibody fragment derivatives, such as Fab, Fab¢, F(ab¢)2, sdAb, scFv, di-scFv, and nanobodies. Such antibody fragment derivatives may be synthesized by recombinant procedures involving covalent or non-covalent bonds involving these types of molecules. Further examples of antigen recognition moieties are peptide / MHC complexes targeting TCR molecules, cell adhesion receptor molecules, receptors for costimulatory molecules, and artificially engineered binding molecules, such as peptides or aptamers targeting cell surface molecules. Such antigen recognition moieties for antibodies may be directed against antigens expressed by or intracellularly in the biological sample (target cells), such as IL2, FoxP3, CD154, or against antigens expressed extracellularly, such as CD3, CD14, CD4, CD8, CD25, CD34, CD56, and CD133.

[0043] As a variation of the first embodiment, a circular oligonucleotide for the hybridization-based SUMI decoding principle for spatial localization of Lolony is shown in Figure 4. When in situ sequencing of SUMI is not available, a hybridization-based method may be advantageous. In this case, a multicolor decoding scheme may be used to decode Lolony. The detection probe used in the method of the present invention may have an oligonucleotide having 2 to 20 nucleotides capable of binding to at least a portion of the barcode region. The generated Lolony continues to serve 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, a relatively long sequencing read length is required for in vitro sequencing to decode SUMI sequences.

[0044] In a second embodiment, the method should be limited to a tissue region of interest. The tissue region of interest is identified by conventional imaging techniques such as microscopy. To focus the in situ sequencing method on the region of interest, the formation of Lolony should be controlled by an external force (e.g., light or heat). Because Lolony serves as a sequencing template, sequencing cannot occur without Lolony. The initiation of polymerization and Lolony formation can be inhibited by blocking the polymerase or by blocking the primer. This blocking principle can be removed by an external force, such as light or heat, which can conceptually be directed by imaging techniques.

[0045] In a third embodiment, after SUMI decoding (e.g., in situ sequencing), tissue sections may be digested and individual cells isolated. Rolony-containing cells are selected and ultimately subjected to single-cell sequencing. Selection of Rolony-containing cells may be achieved by their increased nucleic acid content as a result of rolling circle amplification or by the fluorescence intensity derived from hybridization probes directed to the Rolony sequence. The SUMI sequences from in situ sequencing may be identified by single-cell sequencing, so that the information content from single-cell sequencing can be linked to spatial location via the SUMIs derived from in situ sequencing.

[0046] In this third variant, specific loci can be generated from the circular oligonucleotide by using specific primers corresponding to the target gene (Figure 2) or the barcode tag of the target antibody or target molecule binding moiety (Figure 3), which are recognized by the Phi29 enzyme used for RCA, allowing selective amplification of a subset of amplicons. Finally, the sequencing data can be linked back to the region on the tissue where the target mRNA or cDNA transcript or antibody or molecule binding moiety originally interacted with the circular oligonucleotide.

[0047] In a fourth embodiment, as visualized in Figure 5, a target SUMI sequence is established and amplified by exponential RCA using a highly processive polymerase, such as Phi29. Again, the LORONY serves as a template for a generic target capture product, containing the SUMI. One LORONY may serve as a template for many target sequences, providing the basis for a high-density (HD) spatially unique molecular identifier (SUMI) sequencing workflow.

[0048] Samples analyzed using the disclosed methods can be derived from any specimen, such as whole animals, organs, tissue sections, cell aggregates, or single cells from invertebrates (e.g., Caenorhabditis elegans, Drosophila melanogaster), vertebrates (e.g., zebrafish (Danio rerio), Xenopus laevis), and mammals (e.g., Mus musculus, Homo sapiens). Biological samples can be in the form of tissue sections, cell aggregates, suspension cells, or adherent cells. Cells can be live or dead.

[0049] The spatial information of the Rolony, i.e. the position of the Rolony on the sample, is determined, for example, by an imaging step. In yet another variant of the method according to the invention, the sample is converted into isolated cells, which are then immobilized by trapping or adhering in microcavities.

[0050] Imaging can be performed using a technique known as "Multi-Epitope Ligand Cartography," "Chip-Based Cytometry," or "Multiomics," as described, for example, in EP 0810428, EP 1181525, EP 1136822, or EP 1224472. In this technique, cells are immobilized and contacted with antibodies conjugated to fluorescent moieties. The antibodies are recognized by the respective antigens on the biological sample (e.g., on the cell surface), and after removing unbound marker and exciting the fluorescent moiety, the location of the antigen is detected by the fluorescence emission of the fluorescent moiety. In certain variants, antibodies conjugated to moieties detectable in MALDI imaging or CyTOF can be used instead of antibodies conjugated to fluorescent moieties. Those skilled in the art know how to modify fluorescent moiety-based techniques to incorporate these detection moieties. The location of the target moiety is achieved using a digital imaging device with sufficient resolution and sensitivity for the wavelength of fluorescent radiation. A digital imaging device, such as a fluorescence microscope, may be used with or without optical magnification. The resulting image is stored on a suitable storage device, such as a hard drive, in, for example, RAW, TIF, JPEG, or HDF5 format.

Claims

1. A method for obtaining spatial location and sequence information of a target sequence within at least one RNA or single-stranded DNA, a. A step of hybridizing a first oligonucleotide to a complementary compartment of at least one RNA or single-stranded DNA, wherein the first oligonucleotide is provided with a sequence as a first PCR handle. b. A step of amplifying the first oligonucleotide using the at least one RNA or single-stranded DNA as a template by reverse transcription polymerase chain reaction (RT-PCR), c. The step of removing the at least one RNA or single-stranded DNA from the amplified first oligonucleotide. d. A step of hybridizing a second oligonucleotide to a complementary compartment of the amplified first oligonucleotide, wherein the second oligonucleotide is provided with a sequence as a second PCR handle. e. A step of amplifying the second oligonucleotide using the amplified first oligonucleotide as a template by reverse transcription polymerase chain reaction (RT-PCR), thereby obtaining a third oligonucleotide having the sequences of the first and second PCR handles and the target sequence. f. A step of removing the third oligonucleotide from the amplified second oligonucleotide, g. A step of providing a fourth oligonucleotide at a specific spatial position on a sample, wherein the fourth oligonucleotide has a plurality of concatemers each comprising a sequence complementary to the second PCR handle and at least one sequence as a spatially specific molecular identifier (SUMI) having at least two nucleic acids. h. To determine the spatial position of the fourth oligonucleotide, the sequence of the SUMI of the fourth oligonucleotide is determined by the first sequencing step, thereby linking the spatial position with the SUMI sequence. i. A step of hybridizing the third oligonucleotide with the complementary sequence of the fourth oligonucleotide using the second PCR handle, j. 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. k. A step of dehybridizing the extended third oligonucleotide and determining the sequence of the extended third oligonucleotide by a second sequencing step. l. 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, wherein the fourth oligonucleotide further has a sequence that enables the extended third oligonucleotide to be segmented by restriction enzymes or chemically.

3. The method according to claim 1 or 2, characterized in that the fourth oligonucleotide is provided by rolling circle amplification (RCA) of a cyclic oligonucleotide having a sequence complementary to the second PCR handle and at least one sequence as a spatially specific molecular identifier (SUMI).

4. The method according to claim 3, characterized in that the rolling circle amplification (RCA) is activated by light and / or heat.

5. The method according to claim 1 or 2, characterized in that the first sequencing step is performed after incorporating the SUMI sequence into the extended third oligonucleotide.

6. The method according to claim 1 or 2, characterized in that the third oligonucleotide has an antigen-recognizing portion capable of binding to a protein.

7. The method according to claim 6, characterized in that the third oligonucleotide has a barcode tag sequence to which the antigen recognition portion is linked.

8. The method according to claim 1 or 2, characterized in that the cells of the sample are further subjected to single-cell sequencing.

9. The method according to claim 1 or 2, characterized in that the cells are subjected to single-cell sequencing after in situ sequencing.

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