A method combining single-cell gene expression mapping with target RNA or c-DNA sequencing using padlock oligonucleotides containing barcode regions.

Abstract

To provide methods for single-cell gene expression mapping and target RNA or c-DNA sequencing of samples containing at least one RNA or c-DNA strand.SOLUTION: Provided is a method with less restrictions on the amount of transcripts that can be analyzed and the length of the sequence (Fly FISH), in which the use of padlock oligonucleotides as fluorescent in situ hybridization (FISH) probes for detection is combined with sequencing of the targeted moieties of RNA or cDNA transcripts at a cellular level. Padlock probes containing various barcode regions in the center are utilized as FISH probes and even to capture RNA moieties that can be sequenced. The same barcode can be used to selectively prime rolling circle amplification and amplify a subset of transcripts derived from a particular region tagged as a target during the probing process.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for sequencing and localizing RNA or cDNA strands by selective amplification of padlock oligonucleotides containing barcode regions.

[0002] Background of the Invention Padlock oligonucleotides have proven to be highly successful in polymerizing short portions of hybridized nucleic acids. Most padlock approaches begin by reverse-transcribing 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 inclusive assay for targeted multiplex amplification of human DNA sequences is disclosed in Sujatha Krishnakumar et al.; PNAS sent for review February 19, 2008.

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

[0006] The publicly available Padlock method enables DNA or RNA sequencing, but it does not provide any information about the specific cell or tissue location from which the sequenced DNA or RNA originates.

[0007] Microscopic imaging, which enables the analysis of numerous mRNAs at the single-cell level, provides valuable information about transcriptional volume and localization, which is crucial for understanding tissue heterogeneity, molecular development, and disease treatment.

[0008] Methods based on fluorescence in situ hybridization (FISH) allow for direct labeling of transcripts in tissue sections, enabling the acquisition of spatial information. However, the number of probes that can be used is limited, and overlapping fluorescence signals are often a problem. Furthermore, the optical resolution of confocal microscopy is often limited, reducing the amount of probe that can be detected simultaneously. SeqFISH+ is an approach that uses transcript-specific probes containing barcode sequences that serve as target sites for fluorescently labeled secondary probes, rather than using probes that are already labeled with fluorophores. Various target-specific probes are identified using secondary probes that bind to these barcode sites in a sequential series of probing. By limiting the amount of probe detected by the secondary probes, a limited amount will fluoresce, and as a result, the signal may be discernible. By collecting multiple separate images and integrating them by computer, a synthetic high-resolution image can be created without the need for high-resolution microscopy equipment.

[0009] However, while these approaches allow for the simultaneous evaluation of several genes, they do not provide sequence information for transcripts. Other methods based on single-cell RNA sequencing (scRNA-seq) can profile the entire transcriptome and provide sequence information. However, the original locations at the tissue or single-cell level are often similarly unknown. A method that provides both sequence and spatial information at near single-cell resolution remains a challenging task. Several approaches employ FISSEQ and BaristaSeq (another gap-filling padlock-based approach that achieves this challenge with a limited read length of approximately 15 bases).

[0010] Summary of the Invention The object of the present invention is a method for single-cell gene expression mapping and target RNA or c-DNA sequencing of a sample containing at least one RNA or c-DNA strand, a. A step of preparing oligonucleotides having 5' and 3' ends by combining them with 50 to 1000 nucleic acids complementary to at least one RNA or c-DNA strand of a sample, wherein the oligonucleotide comprises at least one barcode region of 2 to 20 nucleotides. b. A step of hybridizing the 5' and 3' ends of an oligonucleotide to a complementary portion of at least one RNA or c-DNA strand to create a padlock with a gap between the 5' and 3' ends of the padlock. c. A step of filling the gaps in a padlock with complementary nucleotides and ligating them to generate a single-stranded circular template, wherein at least one detection probe capable of binding to at least a portion of the barcode region is supplied to the single-stranded circular template. d. A process of increasing a single-stranded circular template into multiple DNA concatemers that form Rollonie using a polymerase capable of rolling circle amplification. e. Process for determining the spatial localization of a single-chain annular template. f. The process of determining the arrangement of the single-strand annular template. The method is characterized in that the detection probe comprises an oligonucleotide of 2 to 20 nucleotides capable of binding to at least a portion of the barcode region, and a detection region selected from the group comprising magnetic particles, fluorescent dyes, radiolabeling, or antigen-binding moieties.

[0011] This invention modifies known gap-filling padlock techniques by using a padlock anchored to a detection probe, preferably at its antigen-binding site. The detection probe can optionally be crosslinked with tissue. The padlock can then be released from the detection probe and directly hybridize to a specific portion of messenger RNA on an immobilized and permeabilized tissue section.

[0012] In a modified version of the present invention, steps e) and f) can be performed using a padlock instead of a single-chain annular mold.

[0013] The drawings illustrate the method and embodiments of the present invention without limiting the scope of the claims. [Brief explanation of the drawing]

[0014] [Figure 1]This figure shows an example of a padlock of the present invention that can also be used as a FISH (fluorescence in situ hybridization) probe. The center of the padlock incorporates a series of barcodes or barcode regions that can be used to identify the actual padlock bound to mRNA by a series of hybridizations of a fluorescently labeled detection probe that specifically binds to at least one of the barcode regions (A). The center of the padlock, i.e., oligonucleotide, may include a universal binding site (B) for primer binding used in a rolling circle amplification step (RCA) by polymerase. When selective RCA is used, a specific primer that binds to one of the barcode regions is used. Furthermore, potential priming sites for non-selective RCA (C and D), and the 5' and 3' ends of the oligonucleotide that hybridize to a complementary portion of at least one RNA or c-DNA strand are shown in Figure 1. The gap created between the 5' and 3' ends of the padlock (E) may be between 0 and 500, preferably less than 200. [Figure 2] This figure shows that a pool of various cyclized padlock oligonucleotides with spatial information is used for selective / targeted RCA using oligonucleotides that recognize a specific set of barcodes. These oligonucleotides act as primers for the RCA reaction, allowing DNA nanoballs / Roronie to be amplified only from selected cyclized padlocks. Figure 2 shows a specific amplification step in which a pool of various single-stranded circular templates with already obtained spatial information is used for selective / targeted RCA using short oligonucleotide primers that recognize a specific set of barcodes. These short oligonucleotides act as primers for the RCA reaction, and DNA nanoballs / Roronie are generated only from selected and primed cyclized padlocks. In the example shown, only the single-stranded circular template marked (B) is amplified. [Figure 3]This figure illustrates a gap-filling padlock probe technique using a probe that directly hybridizes to a specific portion of messenger RNA on a fixed, permeabilized tissue section. (A) Oligonucleotides that form the padlock after hybridization and (B) Identification of the actual padlock bound to mRNA using a fluorescently labeled detection probe complementary to the barcode region. (C) The gap-filling region is the target sequence on the mRNA, and the gap between the two ends of the probe is filled from the 5' end using reverse polymerase with the target mRNA as a guide. After gap filling, the elongated, hybridized padlock is probed by sequential hybridization of a fluorescently labeled detection probe that binds to a specific internal barcode (1-4). Each end of the padlock ligates to form a circular molecule. (D) The circularized DNA is extracted and amplified in tubular extraction or amplified directly on fixed tissue using specific Rollonie primers that selectively generate Rollonie sequences based on expression profiling. [Figure 4] This figure illustrates an example of a process that enables the identification of hybridized padlock oligonucleotides following gap filling. The gap-filled, hybridized padlocks are probed by sequential hybridization of fluorescently labeled detection probes that bind to a specific internal barcode. This probing can be performed using a subset of padlock oligonucleotides, each containing a different barcode region. Binding of the detection probe to the barcode region allows for the identification of the actual padlock bound to mRNA. In this example, the detection probes and subsets of padlock oligonucleotides are identified by padlock probes using three different fluorophores and subdivided into three groups, each detected by a different luminescence channel. Individual probes are identified based on the actual order in which the hybridization steps of a given detection probe are performed. [Figure 5]This figure shows padlock oligonucleotides, either anchored to beads or linked to antibodies, placed in microdroplets containing single cells (A) and padlocks (B). The padlocks can also be incubated in the presence of immobilized and permeabilized tissue sections. [Figure 6] This figure shows arbitrary fragmentation of a single-stranded circular template, and subsequent amplification of the fragments by rolling circle amplification into a second set of DNA concatemers that form Rollonie. [Figure 7] This figure shows the arbitrary removal of a detection probe from a chain-like annular template using light-cuttable linker units.

[0015] Detailed explanation This method combines the use of oligonucleotides that form padlocks during hybridization (padlock probes), sequential hybridization for the detection of bound probes, and sequencing of targeted regions of RNA or DNA transcripts at the cellular level, resulting in fewer limitations on the amount of transcripts and sequence lengths that can be analyzed.

[0016] In this approach, gap-filling padlock probes containing one or more barcode regions at their center are used as FISH probes and also to capture RNA portions that can be sequenced. Padlock probes have proven highly successful in polymerization of short portions of the nucleic acids they hybridize. Most padlock approaches begin with reverse transcription of the target into cDNA.

[0017] Following hybridization of the padlock probe to the DNA or RNA strand, a gap filling step is performed, where the reverse polymerase fills the opening between the anchor and the extending side of the padlock using the target mRNA as a guide from the 5’ part of the hybridized probe, which is subsequently ligated to form a circular DNA molecule. Alternatively, the padlock may be hybridized to cDNA, but this requires additional steps that can be avoided by directly targeting the mRNA. This technique is known, for example, from the prior art described above.

[0018] The padlock probe with the gap filled and ligated to form a circular template (the probe is filled but may not be ligated until later in the process) is first used as a FISH probe or Seq FISH probe using a barcoded region incorporated into the actual non-hybridized part of the padlock that is detected by a labeled oligonucleotide. Finally, the circularized padlock probe is used as a template for rolling circle amplification (RCA) to generate a DNA strand for sequencing. The DNA strand thus obtained is hereinafter referred to as a "rolony" or "DNA nanoball". The padlock probe can be detected directly on the tissue or after amplification into a rolony.

[0019] The present invention describes a method of using a padlock oligomer having a barcode region used to obtain unique information or a gap filling padlock that enables further downstream processing of the rolony and / or its combination obtained.

[0020] Detection probe The detection probe used in the method of the present invention can be composed of a) an oligonucleotide of 2 to 20 nucleotides capable of binding to at least a part of the barcode region, and b) a detection region selected from the group including magnetic particles, fluorescent dyes, and radioactive labels.

[0021] The detection probe preferably contains a fluorescent dye known in the technical field of immunofluorescence techniques, such as flow cytometry or fluorescence microscopy techniques. Useful fluorescent moieties can be protein-based such as phycobiliprotein, polymers such as polyfluorene, xanthenes such as fluorescein or rhodamine, cyanines, oxazines, coumarins, acridines, oxadiazoles, pyrenes, pyromethenes and other small organic molecular dyes, or organometallic complexes such as Ru, Eu, Pt complexes. In addition to single molecular entities, clusters of fluorescent proteins or small organic molecular dyes, and nanoparticles such as quantum dots, upconversion nanoparticles, gold nanoparticles, stained polymer nanoparticles can also be used as fluorescent moieties.

[0022] Furthermore, the detection region can include a radiolabel in the form of radiolabeling by exchanging a non-radioactive isotope with a radioactive counterpart such as tritium, 32P, 35S or 14C, or introducing a covalent label such as 125I bound to tyrosine, 18F in fluorodeoxyglucose, or an organometallic complex, namely 99Tc-DTPA.

[0023] In a variant of the present invention, the detection probe comprises a photocrosslinkable unit capable of photocrosslinking a single-stranded circular template with a sample by light irradiation during or after step c).

[0024] The crosslinking agent used in the present invention can be a crosslinking agent known in the technical fields of oligonucleotide capture on solid surfaces or tissues, such as microarray generation, protein-protein interactions, isolation of cell surface proteins and preparation of labeled probes. Useful moieties can include crosslinking agents and labeling reagents containing aryl azide or diazirine functional groups that can be activated by ultraviolet light to react to form covalent bonds between antigen-binding moieties and other molecules.

[0025] Padlock oligonucleotide As shown in Figure 1, the oligonucleotide has 5' and 3' ends that recognize a target region, each containing approximately 50 to 1000 nucleic acids, preferably 50 to 200 nucleic acids, and at least one, preferably 1 to 4, barcode regions, each containing 2 to 20 nucleotides. Each barcode region contains a different sequence.

[0026] The padlock probe can, for example, be attached to an antigen-recognizing portion on a tissue section mounted on a solid surface that can bind to a cell-specific antigen, and can be crosslinked before tissue permeabilization, as shown in Figure 5.

[0027] Optionally, after step c), the detection probe can be removed from the single-strand annular template by, for example, chemical cutting or optical cutting.

[0028] In the method of the present invention, a padlock oligonucleotide is cyclic, and the resulting single-stranded cyclic template is replicated by a polymerase capable of rolling circle amplification into multiple DNA concatemers that form DNA nanoballs or loroney. For this purpose, the oligonucleotide used in the present invention may contain at least one primer region of 5 to 50 nucleotides for rolling circle amplification.

[0029] In one embodiment of the present invention, at least one primer region is located between the barcode region and the 5' and / or 3' end of the oligonucleotide. This embodiment is used to non-selectively replicate a single-stranded cyclic template using an oligonucleotide complementary to the padlock primer region as the priming site for a rolling circle amplification polymerase.

[0030] In another embodiment of the present invention, at least one barcode region is used as a primer region. This embodiment is used when selectively replicating a single-stranded cyclic template using an oligonucleotide complementary to the barcode region as the priming site for a rolling circle amplification polymerase.

[0031] method As shown at the top of Figure 6, the antigen-binding moiety containing the padlock oligonucleotide can be crosslinked to the tissue, allowing the padlock to be subsequently released from the tissue.

[0032] Gap-filling padlock probe technology is used for probes that directly hybridize to a specific portion of messenger RNA on immobilized and permeabilized tissue sections. The gap-filling region is the target sequence on the mRNA, and the gap between the two ends of the probe is filled from the 5' end by reverse polymerase (POL) using the target mRNA as a guide (Section A).

[0033] Padlock probes are either extracted and amplified intratubularly by RCA, or amplified directly on immobilized and permeabilized tissue using specific primers that selectively generate loroney probes that are sequenced based on the detection of probes actually bound to the target of interest.

[0034] In one embodiment, after step e), the single-stranded circular template is sheared into fragments, and the fragments are amplified into a second set of DNA concatemers that form Rollonie using a polymerase capable of rolling circle amplification.

[0035] When amplifying on tissue, the extracted loroney can be sheared into smaller fragments and re-amplified before sequencing (right side of section C and section E).

[0036] The shearing process is known to those skilled in the art and may include ultrasonic or random shearing with uracil-containing Rorony and USER enzymes (uracil DNA glycosylase (UDG) and DNA glycosylase-lyase endonuclease VIII or site-specific restriction sites).

[0037] Before or after gap filling, the extended, hybridized padlock is probed by sequential hybridization of fluorescently labeled detection probes bound to specific internal barcodes (1-4).

[0038] Ligation of each end of the padlock that creates the cyclic molecule can be performed before or after probing.

[0039] Padlock probes are either extracted and amplified in a tube, or amplified directly on immobilized and permeabilized tissue using specific primers that selectively generate loroney probes that are sequenced based on the detection of probes actually bound to the target of interest.

[0040] The basic steps of the present invention are shown in Figure 1. Here, the gap-filling padlock probe technique was applied to a probe that directly hybridizes to a specific portion of messenger RNA on an immobilized and permeabilized tissue section. The gap-filling region is the target sequence on the mRNA, and the gap between the two ends of the probe is filled from the 5' end by reverse polymerase (POL) using the target mRNA as a guide (Section A).

[0041] After gap filling, the extended, hybridized padlock is probed by sequential hybridization of a fluorescently labeled detection probe that binds to specific internal barcodes (1-4) (Section B). Visualization of the detection probe by fluorescence microscopy allows for confirmation of the physical localization of the padlock oligonucleotide on the tissue, and the actual identity of the padlock bound to the mRNA or cDNA transcript is determined (the padlock is used as a fluorescent in situ hybridization probe) (Section C).

[0042] Finally, the ends of the padlock are ligated to create a circular molecule. The circularized DNA is extracted and replicated in the tube by rolling circle amplification, or replicated directly on fixed tissue using specific oligonucleotide primers that bind to the barcode region and selectively generate only the roloney to be sequenced (Section D).

[0043] Figure 3 shows an example of sequential steps used in an embodiment of a method for detecting and identifying padlocks. Hybridized oligonucleotides forming a gap-filled padlock are probed by sequential hybridization of detection probes that bind to a specific internal barcode. This probing can be performed using a subset of padlock oligonucleotides, each containing a different barcode region. The binding of the detection probes to the barcode regions is sequential, enabling the identification of the actual padlock bound to the mRNA or cDNA transcript.

[0044] In this example, padlock oligonucleotides targeting various mRNA regions are subdivided into three groups, each identified by three sets of detection probes labeled with three different detection regions, preferably fluorophores. Each of these fluorophores can be detected by its respective luminescence channel.

[0045] In the second embodiment, multiple detection probes are supplied sequentially, which can be coupled to various parts of the barcode area. Here, the first detection probe is removed after detection before the next detection probe is supplied.

[0046] The detection probe is supplied sequentially in multiple rounds to the identified padlock. Three rounds are shown in this example. In round 1, a mixture of detection probes complementary to portions I, II-II, or IV of the padlock barcode region is supplied. Some of the detection probes hybridize to one of the barcode regions, while others do not. The bound probes are detected in channels corresponding to the emission spectra by their respective fluorescent dyes after each round of hybridization. In subsequent rounds, the detection probe mixture is modified to bind the detection probes to different portions of the barcode region. Three individual examples are shown, but for the first individual padlock, the detection probe binds to portion I of the padlock barcode region in round 1, portions II-III in round 2, and finally portion IV in round 3. Thus, detection is positive in rounds 1, 2, and 3, indicating the actual padlocks present for the known barcode. The other two individual probes showed positive binding with different fluorophore-labeled probes (different emission spectra and channel detection) in rounds 2, 3, 1 and 3, 1, 2, respectively, allowing for the identity of the padlock-binding sequence and the determination of the sequence of the targeted DNA (DNA in the filled gap) after sequencing.

[0047] This embodiment is performed while the actual padlock is attached to the sample (tissue), allowing for the identification of the specific distribution of the padlock on the tissue. Furthermore, three different channels are used for detection, and multiple probes can be supplied at various rounds of hybridization, thus reducing the amount of information per channel and increasing the accuracy and / or speed of detection of specific information.

[0048] In a third embodiment, a gap-filled and / or cyclic padlock, i.e., a single-stranded cyclic template, is released from the tissue after detection, i.e., after being used as a seq FISH probe and visualized. In a variation thereof, the padlock probe can be anchored to a paramagnetic bead or antigen-recognizing portion by a severable (e.g., photocleavable) linker, and the padlock can be targeted into a specific region or microdroplet of a tissue section.

[0049] The present invention is shown in Figure 5. Here, a padlock oligonucleotide is anchored to a bead or linked to an antigen recognition portion and placed in a microdroplet containing a single cell (A) and a padlock (B) (top of Figure 5) or on tissue (bottom of Figure 5). The technique of creating droplets of aqueous suspension in a hydrophobic liquid is generally known as "single-cell sequencing technique".

[0050] The term “antigen-recognition moiety” refers to any type of antibody, fragmented antibody, or fragmented antibody derivative against a marker expressed on cells in a cell sample. This term relates to 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, including covalent and non-covalent conjugates containing these types of molecules, can be synthesized by recombinant procedures. Further examples of antigen-recognition moieties are peptide / MHC complexes targeting TCR molecules, cell adhesion receptor molecules, receptors for co-stimulatory molecules, artificially modified binding molecules, peptides, or aptamers targeting cell surface molecules, etc. Such antigen-recognition moieties may be antibodies against intracellular antigens expressed by a biological sample (target cells), e.g., IL2, FoxP3, CD154, or extracellular antigens, e.g., CD3, CD14, CD4, CD8, CD25, CD34, CD56, and CD133.

[0051] In another embodiment, an array can be generated on a solid surface using probes anchored to beads or antibodies.

[0052] In a variation of this embodiment, a sample, preferably a solid tissue such as a fresh frozen tissue section, is placed on a coated glass slide containing a padlock-forming oligonucleotide anchored to beads or an antibody. The tissue is fixed and imaged for histological purposes and then permeabilized to release mRNA that can bind to adjacent padlock probes, thereby enabling the acquisition of gene expression information and later sequence information.

[0053] In another example, an oligonucleotide with an antigen-binding moiety binds to a region of the sample that presents the appropriate antigen; however, in this case, only mRNA derived from the sample located near the target region binds to the padlock oligonucleotide and is subjected to steps a) to f).

[0054] For example, the padlock can be used to generate loroney (DNA nanoballs) for sequencing (in situ) as described above, or it can be used for sequencing (ex situ). In that case, the padlock is circularized and removed from the tissue-containing flow cell, and the loroney generated from the RNA-captured padlock is sequenced. When sequencing is performed ex situ, there are fewer limitations in terms of read length.

[0055] In a modified version of this embodiment, specific cyclic padlocks released when anchored to beads or antibodies can be specifically isolated and sorted.

[0056] In this modification, specific lorony can be generated ex vivo from bulk-release padlocks using specific primers corresponding to specific barcodes recognized by the Phi29 enzyme used, for example, in RCA, enabling selective amplification of a subset of transcripts originating from a specific region or tagged as target during the probing process (seq FISH). Finally, the sequencing data is reassociated with the tissue region where the mRNA or cDNA transcript first interacted with the padlock.

[0057] The padlock region that recognizes the target region can also be designed to be more universal in order to recognize the 3' portion of the mRNA on the padlock binding site, which is composed of polyT, and the 5' portion, which is composed of random n-mers (e.g., random hexamers). The padlock region that recognizes the target region can also be designed to have modified nucleotides such as LNAs that help bind to the target with higher specificity.

[0058] Samples analyzed using the disclosed methods may originate from any specimen, such as whole bodies, organs, tissue fragments, cell aggregates, or single cells, of animals including invertebrates (e.g., nematodes (Caenorhabditis elegans), fruit flies (Drosophila melanogaster)), vertebrates (e.g., zebrafish (Danio rerio), African clawed frogs (Xenopus laevis)), and mammals (e.g., mice (Mus musculus), humans (Homo sapiens)). Biological samples may take the form of tissue fragments, cell aggregates, suspension cells, or adherent cells. Cells may be living or dead.

[0059] The spatial information of the loroni on the sample, i.e., the position of the loroni, is determined, for example, by an imaging step. In yet another variation of the method according to the present invention, the sample is converted into isolated cells and then immobilized by capture or adhesion to a microcavity.

[0060] Imaging can be performed using techniques known as "Multi Epitope Ligand Cartography," "Chip-based Cytometry," or "Multiomyx," as described, for example, in European Patent No. 0810428, European Patent No. 1181525, European Patent No. 1136822, or European Patent No. 1224472. In this technique, cells are immobilized and brought into contact with antibodies coupled to fluorescent moieties. The antibodies are recognized by each antigen on the biological sample (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 modifications, antibodies coupled to moieties detectable by MALDI-Imaging or CyTOF can be used instead of antibodies coupled to fluorescent moieties. Methods for modifying the technique based on the fluorescent moieties that cooperate with these detectable moieties are known to those skilled in the art. The location of the target moiety is obtained by a digital imaging device with sufficient resolution and sensitivity at the wavelength of fluorescence emission. The digital imaging device can be used, for example, with or without optical magnification using a fluorescence microscope. The resulting images are saved to a suitable storage device, such as a hard drive, in a format such as RAW, TIF, JPEG, or HDF5.

Claims

1. A method for single-cell gene expression mapping and target RNA or c-DNA sequencing of a sample containing at least one RNA or c-DNA strand, a. A step of preparing an oligonucleotide having 5' and 3' ends by combining it with 50 to 1000 nucleic acids complementary to the at least one RNA or c-DNA strand of the sample, wherein the oligonucleotide comprises at least one barcode region of 2 to 20 nucleotides, the at least one barcode region being used as a primer region for rolling circle amplification, or the oligonucleotide comprises at least one barcode region of 2 to 20 nucleotides and at least one primer region of 5 to 50 nucleotides for rolling circle amplification. b. A step of hybridizing the 5' and 3' ends of the oligonucleotide to the complementary portion of the at least one RNA or c-DNA strand to create a padlock having a gap between the 5' and 3' ends of the padlock. c. A step of filling the gap in the padlock with complementary nucleotides and ligating them to generate a single-stranded circular template, wherein at least one detection probe capable of binding to at least a portion of the barcode region is supplied to the single-stranded circular template. d. A step of amplifying the single-stranded circular template into multiple first DNA concatemers that form Rollonie using a polymerase capable of rolling circle amplification. e. Steps for determining the spatial localization of the single-chain annular template. f. Step of determining the arrangement of the single-strand annular template. Includes, The above process is carried out in the order of steps a) to f), The detection probe of step c) comprises an oligonucleotide of 2 to 20 nucleotides capable of binding to at least a portion of the barcode region, and a detection region selected from the group including magnetic particles, fluorescent dyes, radiolabeling, or antigen-binding moieties. The method is characterized by, after step e), shearing the single-stranded annular template to form fragments, annularizing the fragments, and amplifying them into multiple second DNA concatemers that form Rollonie using a polymerase capable of rolling circle amplification. ,method.

2. The method according to claim 1, characterized in that the detection probe comprises photocrosslinkable units capable of photocrosslinking the single-stranded annular template with the sample by light irradiation during or after step c).

3. The method according to claim 1 or 2, characterized in that the detection probe is removed from the single-chain annular mold after step c).

4. The method according to any one of claims 1 to 3, characterized in that the at least one primer region is located between the barcode region and the 5' and / or 3' end of the oligonucleotide.

5. The method according to any one of claims 1 to 4, characterized in that the single-stranded cyclic template is non-selectively replicated using an oligonucleotide complementary to the padlock primer region as the priming site for a rolling circle amplification polymerase.

6. The method according to any one of claims 1 to 5, characterized in that the at least one barcode region is used as a primer region.

7. The method according to any one of claims 1 to 4, characterized in that the single-stranded cyclic template is selectively replicated using an oligonucleotide complementary to the barcode region as the priming site for the rolling circle amplification polymerase.

8. The method according to any one of claims 1 to 7, characterized by supplying a plurality of detection probes capable of binding to at least one barcode region of the oligonucleotide.

9. The method according to any one of claims 1 to 8, characterized in that a plurality of detection probes capable of being coupled to different parts of the barcode area are supplied sequentially, and the first detection probe is removed after detection before the next detection probe is supplied.

10. The method according to any one of claims 1 to 9, characterized in that the sample comprising at least one RNA or c-DNA strand is prepared from a plurality of cell or tissue sections, and the sample is lysed or permeabilized before or after step a).

11. The method according to any one of claims 1 to 10, characterized in that the oligonucleotide comprises an antigen-binding portion that binds to a region of the sample that presents a suitable antigen, and only mRNA located near that region binds to the padlock oligonucleotide and is subjected to steps a) to f).

12. The method according to any one of claims 1 to 10, characterized in that the antigen-binding portion or the detection probe comprises a fluorescent dye, and only the region of the sample bound to the fluorescent dye is subjected to steps a) to f).

13. The method according to any one of claims 1 to 12, characterized in that a region of the sample that has released mRNA or cDNA bound to an antibody and hybridized to a padlock is subjected to imaging of the resulting emission with light capable of exciting a fluorescent dye, and the spatial localization of the single-stranded circular template is determined.

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