Method for multiple cell analysis

Abstract

In some aspects, provided herein are methods, compositions, kits, and systems for multiplex single cell analysis on an in situ platform, providing an alternative with higher cell flux and / or lower cost per cell compared to current single cell analysis techniques. In some embodiments, the methods disclosed herein include tagging a population of single cells with a tagging agent comprising a sample-specific barcode and / or a cell feature-specific barcode (e.g., an analyte-specific barcode), immobilizing tagged cells, or immobilizing tagged cells with a tagging agent comprising a sample-specific barcode and / or a cell feature-specific barcode (e.g., an analyte-specific barcode). And performing in situ detection of the labeling agent and / or other features including the cellular analyte of the labeled cells.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 328,188, filed on April 6, 2022, entitled “METHODS AND COMPOSITIONS FORMULTIPLEX CELL ANALYSIS,” which is incorporated herein by reference in its entirety for all purposes. Technical Field

[0003] The present disclosure relates in some aspects to methods and compositions for multiplexed analysis of dissociated single cell samples on an in situ platform. Background Art

[0004] Analysis of biological targets in samples at the single cell level, such as genome, transcriptome or proteome profiling of single cells, is essential for many purposes, such as understanding the molecular basis of cellular properties and developing treatments for diseases. Current single cell based methods may be costly and have low throughput, but the potential value of such analyses may be enormous. Therefore, new and improved methods are needed. The present disclosure addresses this need and other needs. Summary of the invention

[0005] Microscopic imaging can distinguish multiple analytes in a sample and can provide valuable information, such as analyte abundance and spatial information of analytes in situ (e.g., in a cell or tissue sample). In addition, an in situ analysis platform can be used to analyze multiple cells in a sample. In one aspect, the present disclosure combines the capabilities of single-cell-based assays and in situ-based assays by utilizing an in situ assay platform to merge labeled cells and / or nuclei from multiple samples and perform an analysis that allows single-cell resolution. This multiplex approach allows high-throughput analysis (e.g., 1 cm 2 100,000 cells per substrate area), which is much more cost-effective than single-cell sequencing of a single sample.

[0006] In a preferred embodiment, the present invention provides a method for analysis, which includes (a) providing a cell population comprising labeled cells, wherein the labeled cells are cells labeled with a labeling agent comprising a binding portion and a reporter oligonucleotide, wherein the reporter oligonucleotide comprises a sample-specific barcode sequence corresponding to the sample from which the labeled cells are derived, and wherein the binding portion binds to the cells; (b) depositing the cell population comprising labeled cells on a substrate to provide deposited labeled cells; (c) contacting the deposited labeled cells with: (i) a first probe that directly or indirectly binds to the sample-specific barcode sequence, and (ii) a second probe that directly or indirectly binds to an analyte in and / or on the deposited labeled cells; and (d) detecting, in the deposited labeled cells, a first signal associated with the first probe or its product and a second signal associated with the second probe or its product.

[0007] In any one or more of the aforementioned embodiments, cell colony can include dissociated cells and / or cell nuclei. In any one or more of the aforementioned embodiments, cell colony can be a cell nucleus colony. In any one or more of the aforementioned embodiments, cell colony can include immune cells, such as neutrophils, eosinophils, basophils, mast cells, monocytes, granulocytes, macrophages, dendritic cells, natural killer cells and / or lymphocytes (B cells and / or T cells). In any one or more of the aforementioned embodiments, cell colony can include neuronal cells, such as glial cells, oligodendrocytes, astrocytes, intermediate progenitor cells, immature neurons, mature neurons, glutamatergic neurons, GABAergic neurons, dopaminergic neurons, cholinergic neurons and / or motor neurons.

[0008] In any one or more of the foregoing embodiments, a population of cells can be generated from a sample, such as a tissue sample.

[0009] In any one or more of the foregoing embodiments, the binding moiety may be covalently bound to one or more molecules in and / or on the cell. In any one or more of the foregoing embodiments, the binding moiety may be bound to one or more molecules in and / or on the cell via a linker.

[0010] In any one or more of the foregoing embodiments, the binding moiety may be non-covalently bound to one or more molecules in and / or on the cell.In any one or more of the foregoing embodiments, the binding moiety may be bound to one or more molecules in and / or on the cell via a linker.

[0011] In any one or more of the foregoing embodiments, the binding moiety may bind to the cell surface. In any one or more of the foregoing embodiments, the binding moiety may comprise a polynucleotide or an analog thereof, a polypeptide or an analog thereof, a lipid or an analog thereof, a carbohydrate or an analog thereof, or a combination thereof. In any one or more of the foregoing embodiments, the binding moiety may be selected from the group consisting of: an antibody or an epitope binding fragment thereof; a lipophilic moiety, which is optionally cholesterol; a receptor; a receptor ligand; a small molecule; an aptamer; a monomer; an affimer; a darpin; and a protein scaffold.

[0012] In any one or more of the foregoing embodiments, the labeling agent may further comprise a target-specific barcode sequence that identifies the labeling agent bound to the labeled cells. In any one or more of the foregoing embodiments, the reporter oligonucleotide may further comprise a target-specific barcode sequence. In any one or more of the foregoing embodiments, the target-specific barcode sequence and the sample-specific barcode sequence may overlap at least partially in sequence.

[0013] In any one or more of the foregoing embodiments, before, during, and / or after providing a cell population comprising labeled cells, the labeled cells may, but need not be, in a partition. In any one or more of the foregoing embodiments, during depositing the cell population, the labeled cells may, but need not be, in a partition. In any one or more of the foregoing embodiments, the partition may be an emulsion droplet or a microwell of a microwell array.

[0014] In any one or more of the foregoing embodiments, the labeled cells may, but need not, be lysed prior to and / or during deposition of the cell population.

[0015] In any one or more of the foregoing embodiments, between providing the cell population and depositing the cell population, the nucleic acid that labels the cells can, but need not, be extended by a polymerase and / or reverse transcribed, ligated to another nucleic acid, amplified, and / or sequenced. In any one or more of the foregoing embodiments, the nucleic acid can be a reporter oligonucleotide or a cellular DNA or RNA molecule of the cells.

[0016] In any one or more of the foregoing embodiments, nucleic acids labeling cells may, but need not, be released from cells between providing the cell population and depositing the cell population.In any one or more of the foregoing embodiments, the nucleic acid may be a reporter oligonucleotide or a cellular DNA or RNA molecule of the cell.

[0017] In any one or more of the foregoing embodiments, the first probe may hybridize to a sample-specific barcode sequence. In any one or more of the foregoing embodiments, the first probe may comprise a detectable label, and detecting a first signal associated with the first probe may comprise detecting the detectable label. In any one or more of the foregoing embodiments, the detectable label may be an optical label. In any one or more of the foregoing embodiments, detecting a first signal associated with the first probe may comprise detecting the optical label using imaging.

[0018] In any one or more of the foregoing embodiments, the first probe can be selected from the group consisting of: a probe comprising a 3' overhang or a 5' overhang after hybridization to a sample-specific barcode sequence, optionally wherein the 3' overhang or the 5' overhang comprises one or more additional barcode sequences that identify the sample-specific barcode sequence; a probe comprising a 3' overhang and a 5' overhang after hybridization to a sample-specific barcode sequence, optionally wherein the 3' overhang and the 5' overhang each independently comprise one or more additional barcode sequences that identify the sample-specific barcode sequence; a circularized probe, optionally wherein the circularized probe comprises one or more additional barcode sequences that identify the sample-specific barcode sequence; a circularizable probe or probe set, optionally wherein the circularizable probe or probe set comprises one or more additional barcode sequences that identify the sample-specific barcode sequence; a probe or probe set comprising a segmented hybridization region configured to hybridize to a splint, optionally wherein the segmented hybridization region comprises one or more additional barcode sequences; and combinations thereof, and optionally wherein detecting a first signal associated with the first probe comprises detecting at least a portion of the one or more additional barcode sequences.

[0019] In any one or more of the aforementioned embodiments, the method may also include hybridizing the first detectable probe with the first probe and / or producing the first probe and / or the first product of the first detectable probe. In any one or more of the aforementioned embodiments, the first product can be a hybridization product, a connection product, an amplification product or a combination thereof. In any one or more of the aforementioned embodiments, the first product can include a branched structure, a hybridization chain reaction (HCR) product, a linear-oligomer hybridization chain reaction (LO-HCR) and / or a rolling circle amplification (RCA) product.

[0020] In any one or more of the foregoing embodiments, the analyte may be or include a nucleic acid analyte. In any one or more of the foregoing embodiments, the analyte may be or include a cellular RNA molecule. In any one or more of the foregoing embodiments, the analyte may be or include a polypeptide.

[0021] In any one or more of the foregoing embodiments, the binding portion of the labeling agent may, but need not, bind to the analyte.

[0022] In any one or more of the foregoing embodiments, the second probe can hybridize to a sequence in the analyte or the probe targeting the analyte, optionally wherein the probe targeting the analyte is constructed to bind directly or indirectly to the analyte, and optionally wherein the second probe comprises a detectable label, and optionally wherein detecting a second signal associated with the second probe comprises detecting the detectable label, optionally wherein the detectable label is an optical label, and optionally wherein detecting the second signal associated with the second probe comprises detecting the optical label using imaging.

[0023] In any one or more of the foregoing embodiments, the second probe can be selected from the group consisting of: a probe comprising a 3' overhang or a 5' overhang after hybridization with a sequence in a probe of the analyte or the targeted analyte, optionally wherein the 3' overhang or the 5' overhang comprises one or more additional barcode sequences that identify the analyte; a probe comprising a 3' overhang and a 5' overhang after hybridization with a sequence in a probe of the analyte or the targeted analyte, optionally wherein the 3' overhang and the 5' overhang each independently comprise one or more additional barcode sequences that jointly identify the analyte; a circular probe, optionally wherein the circular probe comprises one or more additional barcode sequences that identify the analyte; a circularizable probe or probe set, optionally wherein the circularizable probe or probe set comprises one or more additional barcode sequences that identify the analyte; a probe or probe set comprising a segmented hybridization region configured to hybridize to a splint, optionally wherein the segmented hybridization region comprises one or more additional barcode sequences that identify the analyte; and combinations thereof, and optionally wherein detecting a second signal associated with the second probe comprises detecting at least a portion of the one or more additional barcode sequences.

[0024] In any one or more of the foregoing embodiments, the method can further include hybridizing a second detectable probe to a second probe and / or producing a second probe and / or a second product of the second detectable probe.

[0025] In any one or more of the foregoing embodiments, the second product can be a hybridization product, a ligation product, an amplification product, or a combination thereof, optionally wherein the second product comprises a branched structure, a hybridization chain reaction (HCR) product, a linear-oligomer hybridization chain reaction (LO-HCR) and / or a rolling circle amplification (RCA) product.

[0026] In any one or more of the foregoing embodiments, the method can further comprise comparing the signal detected in the deposited labeled cells to the signal detected in one or more other cells in the population.

[0027] In any one or more of the foregoing embodiments, the population may comprise at least about 100, at least about 1,000, at least about 10,000, at least about 50,000, at least about 100,000, or at least about 500,000 cells. In any one or more of the foregoing embodiments, optionally wherein the cells are immobilized on a substrate within about 0.1 cm 2 、About 0.5cm 2 , about 1cm 2 , about 2cm 2 、About 5cm 2 or about 10cm 2 and optionally wherein at least about 100,000 cells are immobilized within about 1 cm of the substrate. 2 Inside.

[0028] In any one or more of the foregoing embodiments, the method may further include imaging the deposited labeled cells before, during and / or after detection, optionally wherein the imaging includes imaging the shape, size, morphological characteristics and / or marker of the deposited labeled cells or portions thereof, optionally wherein the marker is a fluorescent marker.

[0029] In any one or more of the foregoing embodiments, the deposited labeled cells may be fixed prior to detection.

[0030] In any one or more of the foregoing embodiments, the method may, but need not, include introducing an exogenous polynucleotide into cells of the cell population prior to labeling the cells with a labeling agent comprising a sample-specific barcode sequence, or does not include introducing an exogenous polynucleotide comprising a barcode sequence into cells of the cell population prior to labeling the cells with a labeling agent comprising a sample-specific barcode sequence.

[0031] In any one or more of the foregoing embodiments, the exogenous polynucleotide may comprise or encode a gene, a modified / damaged / non-natural nucleotide or nucleotide analog, an overexpressed gene, an RNAi-based system, a regulatory RNA, a non-coding RNA, an mRNA, a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), a meganuclease, a computationally designed protein, a computationally designed RNA, and / or a CRISPR-Cas system.

[0032] In any one or more of the foregoing embodiments, the cell population can be or include dissociated cells, a cell suspension, or cell nuclei.

[0033] In any one or more of the foregoing embodiments, the method can be used for cell analysis, comprising: (a) providing a cell population comprising labeled cells, wherein the labeled cells are labeled with a labeling agent comprising (i) a binding portion and (ii) a reporter oligonucleotide comprising a first barcode region, and wherein the binding portion binds to the cells; (b) immobilizing the cell population comprising the labeled cells on a substrate; (c) contacting the immobilized cells with: (i) a first probe that hybridizes to the first barcode region, and (ii) a second probe that directly or indirectly binds to an analyte in and / or on one or more of the immobilized cells, wherein the second probe comprises a second barcode region; and (d) detecting a first signal associated with the first probe or its product and a second signal associated with the second probe or its product on the substrate, thereby detecting the presence or absence of the analyte in the immobilized labeled cells.

[0034] In any one or more of the foregoing embodiments, before, during and / or after providing a cell colony, or during fixing the cell colony, the cells in the cell colony can but need not be divided into a plurality of partitions. In any one or more of the foregoing embodiments, the partitions can be or include emulsion droplets and / or microwells.

[0035] In any one or more of the foregoing embodiments, the first barcode region may comprise a sample-specific barcode sequence corresponding to the sample from which the labeled cell originated. In any one or more of the foregoing embodiments, the first barcode region may comprise a target-specific barcode sequence corresponding to a binding moiety or its target. In any one or more of the foregoing embodiments, the first barcode region may comprise a barcode sequence corresponding to a cell characteristic.

[0036] In any one or more of the foregoing embodiments, the second barcode region may include a binding site for a detectable probe. In any one or more of the foregoing embodiments, the detectable probe may include a fluorescently labeled probe. In any one or more of the foregoing embodiments, the detectable probe may include an intermediate probe comprising a binding site for another detectable probe. In any one or more of the foregoing embodiments, the binding site may be or include a barcode sequence. In any one or more of the foregoing embodiments, the detectable probe may include a circular probe or a circularizable probe or a probe set, wherein the rolling circle amplification (RCA) product comprises multiple binding sites for another detectable probe.

[0037] In any one or more of the foregoing embodiments, the method can be used for sample analysis, comprising: (a) contacting a first sample and a second sample with a first labeling agent and a second labeling agent, respectively, to provide labeled cells in each sample, wherein the first labeling agent and the second labeling agent each comprise (i) a binding portion and (ii) a reporter oligonucleotide comprising a first barcode region, wherein the first barcode region comprises a sample-specific barcode sequence corresponding to the corresponding sample, wherein the binding portion of each labeling agent binds to one or more dissociated cells in the corresponding sample; (b) combining the labeled cells from the first sample and the second sample; (c) immobilizing a cell population comprising labeled cells from the first sample and the second sample on a substrate; (d) contacting the immobilized cells with: (i) a first probe that hybridizes to a sample-specific barcode sequence corresponding to the first sample or the second sample, and (ii) a second probe that directly or indirectly binds to an analyte in and / or on one or more immobilized cells, wherein the second probe comprises a second barcode region; and (e) detecting a first signal associated with the first probe or its product on the substrate and a second signal associated with the second probe or its product, thereby detecting the presence or absence of the analyte in one or more cells from the first sample and / or the second sample.

[0038] In any one or more of the foregoing embodiments, the first sample and / or the second sample may comprise dissociated cells.

[0039] In any one or more of the foregoing embodiments, the method can be used for sample analysis, comprising: (a) contacting a first sample and a second sample with a first labeling agent and a second labeling agent, respectively, to provide labeled cells in each sample, wherein the first labeling agent and the second labeling agent each comprise (i) a binding portion and (ii) a reporter oligonucleotide comprising a first barcode region, wherein the first barcode region comprises a sample-specific barcode sequence corresponding to the corresponding sample, wherein the binding portion of each labeling agent binds to one or more cells in the corresponding sample; (b) combining the first sample and the second sample; (c) fixing a dissociated cell population comprising labeled cells from the first sample and the second sample on a substrate; (d) contacting the fixed cells with: (i) a first probe that hybridizes to a sample-specific barcode sequence corresponding to the first sample or the second sample, and (ii) a second probe that directly or indirectly binds to an analyte in and / or on one or more fixed cells, wherein the second probe comprises a second barcode region; and (e) detecting a first signal associated with the first probe or its product on the substrate and a second signal associated with the second probe or its product, thereby detecting the presence or absence of the analyte in one or more cells from the first sample and / or the second sample.

[0040] In any one or more of the foregoing embodiments, the first sample and the second sample can be combined prior to processing the first sample and the second sample to provide a dissociated cell population.

[0041] In any one or more of the foregoing embodiments, the first sample and the second sample can be separately processed to provide dissociated cells prior to combining the dissociated cells to provide a population of dissociated cells.

[0042] In any one or more of the foregoing embodiments, the first probe and / or the second barcode region may comprise one or more barcode sequences. In any one or more of the foregoing embodiments, the first probe and / or the second probe may be a circularizable probe.

[0043] In any one or more of the foregoing embodiments, the first signal and / or the second signal can be associated with a rolling circle amplification (RCA) product of a circularization probe comprising a circularizable probe sequence, wherein the RCA product comprises multiple copies of a complementary sequence to one or more barcode sequences, and wherein the first signal and / or the second signal is detected using a detectable probe that hybridizes to the complementary sequence. In any one or more of the foregoing embodiments, the detectable probe can be detectably labeled, or comprise a sequence that hybridizes to a detectably labeled probe.

[0044] In any one or more of the foregoing embodiments, the method may comprise detecting the presence or absence of an analyte in one or more cells from the first sample and / or from the second sample using a plurality of detectable probes (e.g., probes that hybridize to an RCA product or probes that hybridize to an intermediate probe that hybridizes to an RCA product) to detect signals associated with one or more barcode sequences in successive cycles.

[0045] In any one or more of the foregoing embodiments, a method can be used for sample analysis, comprising: (a) contacting a first cell population with a first labeling agent to provide a first plurality of labeled cells, and contacting a second cell population with a second labeling agent to provide a second plurality of labeled cells, wherein the first labeling agent comprises (i) a first binding moiety and (ii) a first reporter oligonucleotide comprising a first sample-specific barcode sequence that identifies the first cell population, wherein the second labeling agent comprises (i) a second binding moiety and (ii) a second reporter oligonucleotide comprising a second sample-specific barcode sequence that identifies the second cell population; (b) combining cells from the first plurality of labeled cells and the second plurality of labeled cells to provide a combined plurality of labeled cells; (c) immobilizing the combined plurality of labeled cells on a substrate; (d) contacting the immobilized cells with: i) a first probe that hybridizes to a first sample-specific barcode sequence corresponding to a first sample or a second sample, (ii) a second probe that hybridizes to a second sample-specific barcode sequence corresponding to the first sample or the second sample, and (iii) a third probe or probe set that binds directly or indirectly to one or more analytes in and / or on one or more immobilized cells; and (e) detecting: (i) the first probe that identifies the first sample-specific barcode sequence; (ii) the second probe that identifies the second sample-specific barcode sequence; and (iii) the third probe or probe set that identifies one or more analytes in and / or on one or more immobilized cells; and (f) identifying cells from a first cell population using the first sample-specific barcode sequence and identifying cells from a second cell population using the second sample-specific barcode sequence.

[0046] In any one or more of the foregoing embodiments, the first binding moiety and / or the second binding moiety can be covalently bound to one or more molecules in and / or on cells of the first cell population or the second cell population, optionally wherein the first binding moiety and / or the second binding moiety is bound to one or more molecules in and / or on cells of the first cell population or the second cell population via a linker.

[0047] In any one or more of the foregoing embodiments, the first binding moiety and / or the second binding moiety can non-covalently bind to one or more molecules in and / or on cells of the first cell population or the second cell population, optionally wherein the first binding moiety and / or the second binding moiety binds to one or more molecules in and / or on cells of the first cell population or the second cell population via a linker.

[0048] In any one or more of the foregoing embodiments, the first binding moiety and / or the second binding moiety can bind to the cell surface of the first cell population or the second cell population. In any one or more of the foregoing embodiments, the binding moiety can be selected from the group consisting of: an antibody or an epitope binding fragment thereof; a lipophilic moiety, which is optionally cholesterol; a receptor; a receptor ligand; a small molecule; an aptamer; a monomer; an affimer; a darpin; and a protein scaffold.

[0049] In any one or more of the foregoing embodiments, the first plurality of labeled cells and the second plurality of labeled cells can but need not be in partitions. In any one or more of the foregoing embodiments, the partitions can be or include emulsion droplets or microwells of a microwell array.

[0050] In any one or more of the foregoing embodiments, the first plurality of labeled cells and the second plurality of labeled cells may, but need not, be lysed prior to and / or during immobilization of the combined plurality of labeled cells on the substrate.

[0051] In any one or more of the foregoing embodiments, the first probe may comprise a first detectable label, and wherein detecting the first probe comprises detecting the first detectable label, optionally wherein the first detectable label is a first optical label, and optionally wherein detecting the first probe comprises detecting the first optical label using imaging. In any one or more of the foregoing embodiments, the second probe may comprise a second detectable label, and wherein detecting the second probe comprises detecting the second detectable label, optionally wherein the second detectable label is a second optical label, and optionally wherein detecting the second probe comprises detecting the second optical label using imaging.

[0052] In any one or more of the foregoing embodiments, one or more analytes may be or include one or more nucleic acid analytes. In any one or more of the foregoing embodiments, one or more nucleic acid analytes may be or include cellular RNA molecules. In any one or more of the foregoing embodiments, one or more analytes may be or include one or more polypeptides.

[0053] In any one or more of the foregoing embodiments, the third probe or probe set may comprise: a probe comprising a 3' overhang or a 5' overhang upon hybridization to a sequence in one of the one or more analytes, optionally wherein the 3' overhang or the 5' overhang comprises one or more additional barcode sequences that identify the analyte; a probe comprising a 3' overhang and a 5' overhang upon hybridization to a sequence in one of the one or more analytes, optionally wherein the 3' overhang and the 5' overhang each independently comprise one or more additional barcode sequences that together identify the analyte; a circularized probe that hybridizes to one of the one or more analytes, optionally wherein the circularized probe comprises one or more additional barcode sequences that identify the analyte; a circularizable probe or probe set that hybridizes to one of the one or more analytes, optionally wherein the circularizable probe or probe set comprises one or more additional barcode sequences that identify the analyte; a probe or probe set comprising a segmented hybridization region configured to hybridize to a splint; or any combination thereof, and optionally wherein detecting the third probe or probe set comprises detecting at least a portion of the one or more additional barcode sequences.

[0054] In any one or more of the foregoing embodiments, the first cell population and / or the second cell population can each comprise at least about 100, at least about 1,000, at least about 10,000, at least about 50,000, at least about 100,000, or at least about 500,000 dissociated cells, optionally wherein the combined plurality of labeled cells are immobilized on about 0.1 cm of the substrate. 2 、About 0.5cm 2 , about 1cm 2 , about 2cm 2 、About 5cm 2 or about 10cm 2 and optionally wherein at least about 100,000 of the combined plurality of labeled cells are immobilized within an area of ​​about 1 cm on the substrate. 2 Inside.

[0055] In any one or more of the foregoing embodiments, the method may further include imaging at least some of the fixed cells before, during and / or after the detection in step (e), optionally wherein the imaging includes imaging the shape, size, morphological features and / or markers of the fixed cells, optionally wherein the markers are fluorescent markers.

[0056] In any one or more of the foregoing embodiments, the immobilized cells may be fixed prior to detection.

[0057] In any one or more of the foregoing embodiments, the method may, but need not, include introducing an exogenous polynucleotide into cells of the first cell population or the second cell population prior to contacting, or does not include introducing an exogenous polynucleotide comprising a barcode sequence into cells of the first cell population or the second cell population prior to contacting. In any one or more of the foregoing embodiments, the exogenous polynucleotide may comprise or encode a gene, a modified / damaged / non-natural nucleotide or nucleotide analog, an overexpressed gene, an RNAi-based system, a regulatory RNA, a non-coding RNA, an mRNA, a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), a meganuclease, a computationally designed protein, a computationally designed RNA, and / or a CRISPR-Cas system.

[0058] In any one or more of the foregoing embodiments, the methods herein may include: (a) providing a cell population comprising labeled cells, wherein the cells are labeled with a labeling agent comprising a binding moiety and a reporter oligonucleotide, and wherein the binding moiety is bound to the cells (e.g., the binding moiety is covalently or non-covalently bound to one or more molecules in and / or on the cells); (b) immobilizing the labeled cells on a substrate; (c) contacting the immobilized labeled cells with: (i) a first probe bound to the labeling agent, and (ii) a second probe bound to an analyte in and / or on the immobilized labeled cells; and (d) detecting a first signal associated with the first probe or its product and a second signal associated with the second probe or its product in the immobilized labeled cells. In some embodiments, the first probe is bound to the binding moiety of the labeling agent. In some embodiments, the first probe is bound to the reporter oligonucleotide of the labeling agent. In some embodiments, the first probe is bound to the binding moiety and the reporter oligonucleotide. In some embodiments, the first probe is bound to the binding moiety and the reporter oligonucleotide. In some embodiments, the first probe is a sample identification probe, which is a detectable probe for identifying a sample from which the labeled cells are derived. In some embodiments, the second probe is an analyte detection probe, which is a detectable probe for detecting an analyte of interest in a cell population comprising cells from a different sample.

[0059] In any one or more of the foregoing embodiments, the cell can be labeled with a sample-specific barcode sequence corresponding to the sample from which the labeled cell is derived. The sample-specific barcode sequence can be contained in a labeling agent. For example, the sample-specific barcode sequence can be contained in a reporter oligonucleotide. As an alternative or supplement to a labeling agent containing a sample-specific barcode sequence, a molecule or complex separated from the labeling agent can contain another sample-specific barcode sequence that is identical or different in sequence compared to the sample-specific barcode sequence. For example, the sample-specific barcode sequence can be contained in a separate labeling agent that labels the same cell as the labeling agent.

[0060] In any one or more of the foregoing embodiments, the methods herein may include (a) providing a cell population comprising labeled cells, wherein the cells are labeled with a labeling agent comprising a binding moiety and a reporter oligonucleotide, and wherein the binding moiety is bound to the cells; (b) immobilizing the labeled cells on a substrate; (c) contacting the immobilized labeled cells with: (i) a first probe bound to the labeling agent, such as a binding moiety and / or a reporter oligonucleotide, and (ii) a second probe bound to an analyte in and / or on the immobilized labeled cells; and (d) detecting in the immobilized labeled cells a first signal associated with the first probe or a product thereof and a second signal associated with the second probe or a product thereof, wherein the reporter oligonucleotide of the labeling agent comprises a sample-specific barcode sequence corresponding to a sample from which the labeled cells originated.

[0061] In any one or more of the foregoing embodiments, the methods herein may include (a) providing a cell population comprising labeled cells, wherein the cells are labeled with a labeling agent comprising a binding portion and a reporter oligonucleotide, and wherein the binding portion is bound to the cells; (b) immobilizing the labeled cells on a substrate; (c) contacting the immobilized labeled cells with: (i) a first probe bound to the binding portion and / or the reporter oligonucleotide, and (ii) a second probe bound to an analyte in and / or on the immobilized labeled cells; and (d) detecting in the immobilized labeled cells a first signal associated with the first probe or a product thereof and a second signal associated with the second probe or a product thereof, wherein the first probe is a sample identification probe for identifying a sample from which the labeled cells originate, and the first signal is associated with the sample identity, and the second probe is an analyte detection probe for detecting an analyte of interest in cells or cell populations collected from different samples.

[0062] In any one or more of the aforementioned embodiments, the binding moiety can be covalently or non-covalently bound to one or more molecules in and / or on the cell. In some embodiments, the binding moiety can be combined with one or more molecules through a joint. In some embodiments, the binding moiety can be combined with the cell surface or can be in the cell interior.

[0063] In any one or more of the foregoing embodiments, the binding moiety may comprise a polynucleotide or an analog thereof, a polypeptide or an analog thereof, a lipid or an analog thereof, a carbohydrate or an analog thereof, or a combination thereof. In any one or more of the foregoing embodiments, the binding moiety may be selected from the group consisting of: an antibody or an epitope binding fragment thereof, a lipophilic moiety, a receptor, a receptor ligand, a small molecule, an aptamer, a monomer, an affimer, a darpin, and a protein scaffold. In some embodiments, the lipophilic moiety is cholesterol.

[0064] In any one or more of the aforementioned embodiments, the first probe can be combined with a reporter oligonucleotide or its sequence. In any one or more of the aforementioned embodiments, the reporter oligonucleotide may include a target-specific barcode sequence corresponding to a binding portion (of a labeling agent) or its target. In any one or more of the aforementioned embodiments, the target-specific barcode sequence may be in a reporter oligonucleotide. In any one or more of the aforementioned embodiments, the target-specific barcode sequence and the sample-specific barcode sequence may overlap at least partially in sequence. In some embodiments, the target-specific barcode sequence and the sample-specific barcode sequence do not overlap. In some embodiments, the target-specific barcode sequence and the sample-specific barcode sequence are the same barcode sequence. In some embodiments, the target-specific barcode sequence and the sample-specific barcode sequence are different barcode sequences.

[0065] In any one or more of the foregoing embodiments, the labeled cells may not need to be in a partition before, during, and / or after the providing step in (a) or during the fixing in step (b), optionally wherein the partition is an emulsion droplet or a microwell. In some embodiments, the cells are not separated in a partition (e.g., an emulsion droplet or a microwell) before, during, and / or after labeling with a labeling agent. In some embodiments, the partition is an emulsion droplet or a microwell. In some embodiments, the labeled cells are not separated in a partition (e.g., an emulsion droplet or a microwell) throughout the providing step, fixing step, contacting step, and / or detecting step.

[0066] In any one or more of the foregoing embodiments, before and / or during the fixation of step (b), the labeled cells may not need to be lysed or homogenized. In some embodiments, before, during and / or after labeling with a labeling agent, the cells are not lysed or homogenized. In some embodiments, the labeled cells are not lysed or homogenized throughout the providing step, fixing step, contacting step and / or detecting step, although the labeled cells may be permeabilized, fixed, cross-linked / decross-linked, embedded in a matrix, cleared and / or otherwise processed.

[0067] In any one or more of the foregoing embodiments, between providing the fixation in step (a) and step (b), the nucleic acid of the labeled cell may not need to be extended and / or reverse transcribed by a polymerase, connected to another nucleic acid, amplified and / or sequenced. In any one or more of the foregoing embodiments, the nucleic acid is a reporter oligonucleotide or a cell's cellular DNA or RNA molecule. In some embodiments, in order to detect the labeling agent, the reporter oligonucleotide is not extended and / or reverse transcribed by a polymerase, connected to another nucleic acid, amplified and / or sequenced. In some embodiments, in order to detect the analyte, the cell's cellular DNA or RNA molecule is not extended and / or reverse transcribed by a polymerase, connected to another nucleic acid, amplified and / or sequenced.

[0068] In any one or more of the foregoing embodiments, between providing the fixation in step (a) and step (b), the nucleic acid of the labeled cell may not need to be released from the cell. In some embodiments, the nucleic acid is a reporter oligonucleotide or a cell's cellular DNA or RNA molecule. In some embodiments, in order to detect the labeling agent, the reporter oligonucleotide is not released from the cell. In some embodiments, in order to detect the analyte, the cell's cellular DNA or RNA molecule is not released from the cell.

[0069] In any one or more of the foregoing embodiments, the first probe may hybridize to a sequence in a reporter oligonucleotide. In any one or more of the foregoing embodiments, the first probe may be detectably labeled.

[0070] In any one or more of the foregoing embodiments, the first probe can be selected from the group consisting of: a probe comprising a 3' overhang or a 5' overhang after hybridization with a sequence in a reporter oligonucleotide; a probe comprising a 3' overhang and a 5' overhang after hybridization with a sequence in a reporter oligonucleotide; a circular probe; a circularizable probe or probe set; a probe or probe set comprising a split hybridization region configured to hybridize with a splint; and combinations thereof. In some embodiments, the 3' overhang or the 5' overhang comprises one or more barcode sequences. In some embodiments, the 3' and 5' overhangs independently comprise one or more barcode sequences. In some embodiments, the split hybridization region comprises one or more barcode sequences.

[0071] In any one or more of the aforementioned embodiments, method may also include hybridizing the first detectable probe with the first probe and / or producing the first probe and / or the first product of the first detectable probe. In any one or more of the aforementioned embodiments, the first product can be a hybridization product, a connection product, an amplified product or a combination thereof. In some embodiments, the first product can include a branched structure, a hybridization chain reaction (HCR) product, a linear-oligomer hybridization chain reaction (LO-HCR) and / or a rolling circle amplification (RCA) product. In any one or more of the aforementioned embodiments, the first signal can be associated with the first product, and can be detected in situ.

[0072] In any one or more of the foregoing embodiments, the analyte can be a nucleic acid analyte or a non-nucleic acid analyte.In any one or more of the foregoing embodiments, the binding portion of the labeling agent can, but need not, bind to the analyte.

[0073] In any one or more of the aforementioned embodiments, the second probe can hybridize with the sequence in the analyte.In any one or more of the aforementioned embodiments, the second probe can hybridize with the sequence in the probe of the targeted analyte or the product of the probe of the targeted analyte.For example, the second probe can be a detectable probe that can hybridize with the rolling circle amplification product of a circularized probe or probe group, and the circularized probe or probe group can hybridize with a reporter oligonucleotide of a probe of a cellular nucleic acid (for example, genomic DNA, mRNA or cDNA) or a probe in conjunction with nucleic acid or a non-nucleic acid analyte.In some embodiments, the probe of the targeted analyte is configured to be directly or indirectly combined with the analyte.In any one or more of the aforementioned embodiments, the second probe can be detectably labeled.

[0074] In any one or more of the foregoing embodiments, the second probe can be selected from the group consisting of: a probe comprising a 3' overhang or a 5' overhang after hybridization with an analyte or a probe targeting an analyte; a probe comprising a 3' overhang and a 5' overhang after hybridization with a sequence in a probe targeting an analyte or a probe targeting an analyte; a circular probe; a circularizable probe or probe set; a probe or probe set comprising a split hybridization region configured to hybridize to a splint; and combinations thereof. In some embodiments, the 3' overhang or the 5' overhang comprises one or more barcode sequences. In some embodiments, the 3' overhang and the 5' overhang each independently comprise one or more barcode sequences. In some embodiments, the split hybridization region comprises one or more barcode sequences.

[0075] In any one or more of the foregoing embodiments, the method can further include hybridizing a second detectable probe to a second probe and / or producing a second probe and / or a second product of the second detectable probe.

[0076] In any one or more of the aforementioned embodiments, the second product can be a hybridization product, a connection product, an amplification product or a combination thereof. In some embodiments, the second product comprises a branched structure, a hybridization chain reaction (HCR) product, a linear-oligomer hybridization chain reaction (LO-HCR) and / or a rolling circle amplification (RCA) product. In any one or more of the aforementioned embodiments, the second signal can be associated with the second product and detected in situ.

[0077] In any one or more of the foregoing embodiments, the method may also include comparing the signal detected in the fixed labeled cell with the signal detected in one or more other cells in the cell colony. In some embodiments, the one or more other cells in the cell colony are on the same substrate as the fixed labeled cell. In some embodiments, the fixed labeled cell and the one or more other cells are from the same sample. In some embodiments, the fixed labeled cell and the one or more other cells are from different samples.

[0078] In any one or more of the foregoing embodiments, the cell population can include cells from a plurality of different samples. In any one or more of the foregoing embodiments, the population can include at least about 100, at least about 1,000, at least about 10,000, at least about 50,000, at least about 100,000, or at least about 500,000 dissociated cells. In any one or more of the foregoing embodiments, the dissociated cells can be fixed to about 0.1 cm on the substrate. 2 、About 0.5cm 2 , about 1cm 2 , about 2cm 2 、About 5cm 2 or about 10cm 2 In some embodiments, the cell population comprises at least about 100,000 dissociated cells that are fixed within a space of about 1 cm 2 In any one or more of the foregoing embodiments, the dissociated cells can be from two, three, four, five, six, seven, eight, nine, ten or more different samples, and the sample source of the dissociated cells can be identified in the in situ platform, for example, by detecting a sample-specific barcode sequence in a marker immobilized in the dissociated cells and / or on the substrate.

[0079] In any one or more of the foregoing embodiments, the method may further include imaging the immobilized labeled cells before, during, and / or after the detection step (d). In some embodiments, imaging may include imaging the shape, size, morphological features, and / or markers of the immobilized labeled cells or a portion thereof. In some embodiments, the marker may be a fluorescent marker.

[0080] In some embodiments according to any of the methods described herein, prior to detecting step (d), the immobilized labeled cells can be live or fixed.

[0081] In any one or more of the foregoing embodiments, the methods herein can be used for cell analysis, comprising (a) providing a cell population comprising labeled cells, wherein the cells are labeled with a labeling agent comprising (i) a binding moiety and (ii) a reporter oligonucleotide comprising a first barcode region, and wherein the binding moiety binds to the cells; (b) immobilizing the cell population comprising labeled cells on a substrate; (c) contacting the immobilized cells with: (i) a first probe that hybridizes to the first barcode region, and (ii) a second probe that binds to an analyte in and / or on one or more immobilized cells, wherein the second probe may comprise a second barcode region; and (d) detecting a first signal associated with the first probe or its product and a second signal associated with the second probe or its product on the substrate, thereby detecting the presence or absence of the analyte in the immobilized labeled cells.

[0082] In any one or more of the foregoing embodiments, the cells in the population need not be separated into partitions before, during, and / or after the providing step in step (a) or during the fixing period in step (b). In some embodiments, the partitions may be emulsion droplets and / or microwells. In some embodiments, the cells in the population are not separated in partitions (e.g., emulsion droplets or microwells) before, during, and / or after labeling with a labeling agent. In some embodiments, the cells in the population are not separated in partitions (e.g., emulsion droplets or microwells) throughout the providing step, fixing step, contacting step, and / or detecting step.

[0083] In any one or more of the foregoing embodiments, the first barcode region may include a sample-specific barcode sequence corresponding to the sample from which the labeled cell is derived. In some embodiments, the first barcode region may include (i) a sample-specific barcode sequence corresponding to the sample from which the labeled cell is derived, and (ii) a target-specific barcode sequence corresponding to its binding portion or target. In some embodiments, the first barcode region may include (iii) a barcode sequence corresponding to a cell feature. In some cases, the cell feature includes a cell surface feature. Analytes may include, but are not limited to, proteins, receptors, antigens, surface proteins, transmembrane proteins, differentiation protein clusters, protein channels, protein pumps, carrier proteins, phospholipids, glycoproteins, glycolipids, cell-cell interaction protein complexes, antigen presentation complexes, major histocompatibility complexes, engineered T cell receptors, T cell receptors, B cell receptors, chimeric antigen receptors, gap junctions, adherens junctions, or any combination thereof. In some cases, cell features may include intracellular analytes, such as proteins, protein modifications (e.g., phosphorylation states or other post-translational modifications), nucleoproteins, nuclear membrane proteins, or any combination thereof. Therefore, labeling agents can be used to identify the sample from which the labeled cell is derived. In some aspects, labeling agents can be used to identify the binding moiety or target of the labeling agent. In some aspects, labeling agents can be used to identify the cell characteristics of the labeled cell.

[0084] In any one or more of the foregoing embodiments, the second barcode region may comprise a binding site for a detectable probe. In some embodiments, the detectable probe may comprise (i) a fluorescently labeled probe, (ii) an intermediate probe comprising a binding site for another detectable probe, and / or (iii) a circular probe or a circularizable probe or probe set, wherein the rolling circle amplification (RCA) product may comprise multiple binding sites for yet another detectable probe. In some embodiments, the binding site of the intermediate probe may be or include a barcode sequence.

[0085] In any one or more of the foregoing embodiments, the methods herein can be used for sample analysis, comprising (a) contacting a first sample and a second sample with a first labeling agent and a second labeling agent, respectively, to provide labeled cells in each sample, wherein the first labeling agent and the second labeling agent can each comprise (i) a binding portion and (ii) a reporter oligonucleotide comprising a first barcode region, wherein the first barcode region comprises a sample-specific barcode sequence corresponding to the corresponding sample, wherein the binding portion of each labeling agent binds to one or more dissociated cells in the corresponding sample; (b) combining the labeled cells from the first sample and the second sample; (c) immobilizing a cell population comprising labeled cells from the first sample and the second sample on a substrate; (d) contacting the fixed cells with: (i) a first probe that hybridizes to a sample-specific barcode sequence corresponding to the first sample or the second sample, and (ii) a second probe that binds to an analyte in and / or on one or more fixed cells, wherein the second probe comprises a second barcode region; and (e) detecting a first signal associated with the first probe or its product on the substrate and a second signal associated with the second probe or its product, thereby detecting the presence or absence of the analyte in one or more cells from the first sample and / or the second sample. In some embodiments, the first sample and / or the second sample may comprise dissociated cells.

[0086] In any one or more of the foregoing embodiments, the methods herein can be used for sample analysis, comprising (a) contacting a first sample and a second sample with a first labeling agent and a second labeling agent, respectively, to provide labeled cells in each sample, wherein the first labeling agent and the second labeling agent can each comprise (i) a binding portion and (ii) a reporter oligonucleotide comprising a first barcode region, wherein the first barcode region comprises a sample-specific barcode sequence corresponding to the corresponding sample, wherein the binding portion of each labeling agent binds to one or more cells in the corresponding sample; (b) combining the first sample and the second sample; (c) immobilizing a dissociated cell population comprising labeled cells from the first sample and the second sample on a substrate; (d) contacting the fixed cells with: (i) a first probe that hybridizes to a sample-specific barcode sequence corresponding to the first sample or the second sample, and (ii) a second probe that binds to an analyte in and / or on one or more fixed cells, wherein the second probe can comprise a second barcode region; and (e) detecting a first signal associated with the first probe or its product on the substrate and a second signal associated with the second probe or its product, thereby detecting the presence or absence of the analyte in one or more cells from the first sample and / or the second sample. In some embodiments, the first sample and the second sample can be combined in (b) before processing the first sample and the second sample to provide a dissociated cell population in (c). In some embodiments, the first sample and the second sample can be processed separately to provide dissociated cells before combining the dissociated cells in (b) to provide a dissociated cell population in (c).

[0087] In any one or more of the foregoing embodiments, the first probe and / or the second barcode region may comprise one or more barcode sequences. In some embodiments, the first probe and / or the second probe may be a circularizable probe.

[0088] In any one or more of the foregoing embodiments, the first signal and / or the second signal can be associated with a rolling circle amplification (RCA) product of a circularization probe comprising a circularizable probe sequence, wherein the RCA product can comprise multiple copies of a complementary sequence to one or more barcode sequences, and wherein the first signal and / or the second signal can be detected using a detectable probe that hybridizes to the complementary sequence. In some embodiments, the detectable probe can be detectably labeled, or can comprise a sequence capable of hybridizing to a detectably labeled probe.

[0089] In any one or more of the foregoing embodiments, the methods herein can include detecting the presence or absence of an analyte in one or more cells from the first sample and / or the second sample using a plurality of detectable probes to detect signals associated with one or more barcode sequences in successive cycles.

[0090] In any one or more of the foregoing embodiments, the second probe can: (i) directly or indirectly bind to an analyte (e.g., a nucleic acid analyte or a protein analyte) in and / or on one or more immobilized cells, and (ii) comprise a nucleic acid tag, which can comprise a second barcode region. The second signal can be detected by detecting: (i) the nucleic acid tag of the second probe, (ii) the product of the nucleic acid tag (e.g., an RCA product), (iii) a probe or probe set that hybridizes to the nucleic acid tag, for example at the second barcode region, and (iv) a product of the probe or probe set that hybridizes to the nucleic acid tag (e.g., an RCA product).

[0091] In any one or more of the foregoing embodiments, the nucleus can be isolated from the cell, labeled with a labeling agent, and detected on an in situ platform as described herein. In any one or more of the foregoing embodiments, the cell and / or nucleus population can be in suspension prior to labeling with a labeling agent. In any one or more of the foregoing embodiments, a suspension containing a cell population and / or nucleus can be obtained from a sample labeled with a labeling agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] Figure 1 An exemplary workflow is shown, involving analysis of labeled single cells on a substrate using an in situ assay platform.

[0093] Figure 2 An example of a labeling agent comprising a functional moiety 201 and a reporter oligonucleotide 202 is schematically shown.

[0094] Figure 3 An exemplary method for analyzing dissociated single cells using an in situ assay platform is schematically shown. Multiple biological samples comprising dissociated single cells are labeled with a labeling agent comprising a sample-specific barcode sequence, combined and deposited and / or fixed to a substrate for in situ analysis of analytes in single cells.

[0095] Figure 4 An exemplary assay workflow is shown, where in step 1, a labeling agent binds to a target in and / or on a cell (e.g., on a cell surface), in step 2, a first probe (e.g., for sample identification) hybridizes to a reporter oligonucleotide of the labeling agent, and in step 3, a second probe hybridizes directly or indirectly to an analyte (e.g., RNA or DNA) in the cell. Steps 2 and 3 can be performed simultaneously or in any order.

[0096] Figure 5An example is shown in which a second probe is hybridized directly or indirectly to a nucleic acid (e.g., a nucleic acid analyte or a nucleic acid tag of a probe that binds to a non-nucleic acid analyte) and an RCA product of the second probe is generated in a cell. The second probe may comprise a barcode region, and the RCA product may be detected in situ, for example using an intermediate probe and a detectably labeled probe as shown. DETAILED DESCRIPTION

[0097] In some aspects, multiple determinations using microscopy readings (e.g., optical detection of barcode sequences of probes directly or indirectly bound to analytes) are provided herein, for example, for in situ analysis of multiple analytes in dissociated cells. In some embodiments, the dissociated cells are from different samples, and the method includes labeling cells with a labeling agent comprising a sample-specific barcode, and merging labeled cells from multiple biological samples onto a substrate for in situ detection of analytes in labeled cells. Sample-specific barcodes in labeled cells can be analyzed in situ on a substrate to trace the source of a single cell to the sample before merging. In addition, cell morphology, size, one or more biomarkers (e.g., proteins, nucleic acids, lipids, carbohydrates, etc.), genetic and genomic structures, gene expression, and / or one or more additional cell features of labeled cells can be analyzed under a microscope. For example, expression and spatial information of one or more mRNA species in labeled cells can be detected in situ in cells, and related to the sample source and one or more other features of the cell optionally. Methods, compositions, kits, devices, and systems for in situ determination of these single cells are provided, including genomics and transcriptome determinations. In some embodiments, the method provided is quantitative with single cell resolution in the absence of partitions. Compositions and methods for detecting and / or quantifying nucleic acids in cells, tissues, organs or organisms are also provided herein. In some embodiments, the invention provides methods for in situ high-throughput spectral analysis of a large number of cells, different samples from which a large number of cells are derived, and / or a large number of analytes, such as high-throughput multiple spectral analysis without the need to separate cell populations into separate partitions (e.g., emulsion droplets or microwell arrays).

[0098] All publications (including patent documents, scientific articles, and databases) mentioned in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If definitions set forth herein are contrary to or contradictory to definitions set forth in patents, patent applications, published patent applications, and other publications incorporated herein by reference, the definitions set forth herein take precedence over the definitions incorporated herein by reference.

[0099] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0100] I. Overview

[0101] In some aspects, an in situ platform for analyzing cells and cell populations such as dissociated cells (including blood samples and cell lines) is provided herein, for example, for genomic analysis, transcriptome analysis, analysis of proteins and interactions, and other applications. In some embodiments, the methods, compositions, kits, and systems provided herein may provide at least some advantages over existing single cell assays, for example, the ability to perform higher cell throughput at a lower cost per cell, and the ability to add additional phenotypic information layers to molecular information, such as cell shape and / or expression of certain markers.

[0102] Cells and / or cell nuclei can be relatively densely packed per unit area (e.g., about 100,000 cells / cm 2 ) on a substrate for in situ analysis by imaging cells and / or cell nuclei to analyze the molecular information in cells and / or cell nuclei from different samples. Each sample can independently contain a relatively homogeneous population (e.g., cells of a cell line, or cells purified or enriched based on a specific marker), a mixture of multiple populations (e.g., multiple cell lines) or a heterologous cell population (e.g., a variety of different cell types from organs or tissues such as cancerous tissues or cells with different mutations). Cells and / or cell nuclei from the same sample can share a common sample-specific barcode sequence so that cells and / or cell nuclei from multiple samples can be merged, analyzed by imaging and demultiplexed. In some embodiments, methods and compositions provided herein provide cost-effective analysis methods by merging cells and / or cell nuclei and cell populations from multiple different biological samples. In some embodiments, cells collected from multiple different biological samples can be fixed on a single substrate and analyzed in one or more rounds of in situ analysis.

[0103] In some embodiments, before merging, cells and / or cell nuclei and cell colonies from multiple different biological samples can be stained with multiple analysis reagents such as markers. In some embodiments, the marker can include a detection component, such as a DNA oligonucleotide including a sample-specific barcode sequence. In contrast to other methods involving nucleic acid separation / purification, amplification and sequencing (e.g., on a next-generation sequencing platform), sample-specific barcode sequences can be directly detected in cell samples. In some embodiments, sample-specific barcode sequences can be detected by hybridization of complementary probes of detectable labels, such as complementary probes connected by fluorophores. In some embodiments, the marker can also include a binding component or a binding moiety, such as an antibody, a lipid or an aptamer. In some embodiments, the binding moiety can be combined with the cell surface, or can be absorbed into the cell. In some embodiments, the labeled cells can be combined together and fixed to a substrate, such as a glass slide, for in situ measurement.

[0104] In some embodiments, the labeled immobilized cells can be pre-imaged to obtain information, including non-molecular (e.g., phenotype) or subcellular distribution. In some embodiments, the labeled immobilized cells can be imaged at the end of the in situ analysis. In some embodiments, the in situ analysis includes detecting the sample-specific barcode sequence in the first round by methods such as in situ hybridization and / or in situ sequencing (e.g., in situ sequencing while synthesis or in situ sequencing while connection) to identify the position of the cell on the substrate and the biological sample from which the cell is derived. In some embodiments, the in situ analysis includes other multiple rounds of analysis and disassembly to capture additional molecular information, such as the presence of a target gene or gene expression level, and to associate the molecular information with the sample from which the cell is derived. In some embodiments, sample disassembly includes associating the signal indicating the sample-specific barcode sequence detected on the in situ platform with the source sample of the cell labeled with the sample-specific barcode sequence.

[0105] In some embodiments, the method includes generating a signal code sequence at one or more locations in a dissociated cell on a substrate, wherein the signal code sequence comprises a signal code corresponding to a signal (or the absence thereof) associated with a detectable probe for in situ hybridization, wherein the detectable probe is sequentially hybridized with the dissociated cells, wherein the signal code sequence corresponds to a reporter oligonucleotide (which identifies the sample origin of the dissociated cell labeled with the reporter oligonucleotide) or an analyte in the dissociated cell, thereby detecting the analyte at one or more of a plurality of locations in the dissociated cell and identifying the sample origin of the dissociated cell.

[0106] In some embodiments, a method for cell analysis is provided herein, comprising (a) providing a cell population comprising labeled cells, wherein the cells are labeled with a labeling agent comprising a binding moiety and a reporter oligonucleotide, and wherein the binding moiety binds to the cells; (b) fixing the labeled cells on a substrate; (c) contacting the immobilized labeled cells with: (i) a first probe bound to the labeling agent, and (ii) a second probe bound to an analyte in and / or on the immobilized labeled cells; and (d) detecting a first signal associated with the first probe or its product and a second signal associated with the second probe or its product in the immobilized labeled cells, wherein the labeling agent comprises a sample-specific barcode sequence corresponding to the sample from which the labeled cells are derived. The first signal can be detected by a detectable probe hybridized with the sample-specific barcode sequence, and the second signal can be detected by a detectable probe hybridized with the second probe or the product of the second probe (e.g., a rolling circle amplification product or a hybridization chain reaction complex). The first signal can be used to identify the sample from which the labeled cells are derived, and the second signal can be used to detect and / or decode the analyte in and / or on the immobilized labeled cells. In some embodiments, sequential hybridization of detectable probes can be used to (i) identify the sample from which the labeled single cell is derived and / or (ii) detect multiple analytes in and / or on the labeled single cell. Any suitable detection / decoding method can be used, and exemplary methods, including sequential hybridization of detectable probes, are described in US 2019 / 0161796, US2020 / 0224244, US 2022 / 0010358, US 2021 / 0340618, and WO 2021 / 138676, all of which are incorporated herein by reference.

[0107] On the one hand, a method is provided herein, which includes in situ determination of one or more analytes of interest in one or more cells deposited and / or fixed on a substrate. In some embodiments, determination includes in situ analysis of the presence / absence, distribution, position, amount, level, expression or activity of analytes (e.g., nucleic acid molecules) in one or more single cells. Analytes can include nucleic acid molecules and non-nucleic acid molecules, such as proteins and peptides. Nucleic acid molecules can be derived from cells of any particular type and / or specific subcellular regions or analyzed therein, such as derived from cytosol, nucleus, mitochondria, microsomes, and more generally, derived from a part of any other compartment, organelle or cell. Examples include DNA analytes, such as genomic DNA, methylated DNA, specific methylated DNA sequences, fragmented DNA, mitochondrial DNA and RNA / DNA hybrids. Examples of target nucleic acid molecules also include RNA analytes, such as various types of coding and non-coding RNA. Examples of different types of RNA analytes include messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), pre-mRNA and viral RNA. RNA analytes can be obtained from or analyzed in cells or cellular compartments (eg, the nucleus).

[0108] In some embodiments, the method comprises analyzing the transcriptome or genome, e.g., the whole transcriptome or genome, of one or more single cells deposited and / or fixed on a substrate. In some embodiments, the method comprises a process for in situ high-throughput profiling of a large number of analytes, which enables simultaneous analysis of expression patterns and / or location / distribution patterns of genes or genomic loci expressed or present in one or more single cells deposited and / or fixed on a substrate.

[0109] In some embodiments, the in situ assay is a targeted assay, such as an assay that analyzes pre-designed probes that bind directly or indirectly to a biological analyte of interest, such as an mRNA molecule in one or more single cells deposited and / or fixed on a substrate. In some embodiments, the in situ assay includes providing one or more nucleic acid probes that hybridize with a nucleic acid analyte (or its complementary sequence, amplification product, or derivative), and detecting the one or more nucleic acid probes. In some embodiments, the pre-designed probes include one or more primary probes or probe sets, such as probes that bind to an analyte of interest, and / or one or more secondary probes or probe sets, such as probes that bind to a primary probe or its complementary sequence or its product (e.g., hybridization product, ligation product, extension product (e.g., by DNA or RNA polymerase), replication product, transcription / reverse transcription product, and / or amplification product). In some embodiments, the pre-designed probe includes one or more higher-level probes or probe sets, such as an (n+1)th level probe that binds to an nth level probe or its complementary sequence or its product (e.g., a hybridization product, a ligation product, an extension product (e.g., by a DNA or RNA polymerase), a replication product, a transcription / reverse transcription product, and / or an amplification product), wherein n is an integer of 2 or greater. In some embodiments, the pre-designed probe is a barcoded probe. In some embodiments, the nth level probe comprises a barcode sequence (the nth level barcode sequence), and the (n+1)th level probe binds to the nth level barcode sequence and comprises the (n+1)th level barcode sequence for binding through another barcoded probe or through a detectably labeled probe (e.g., a fluorescently labeled detection oligonucleotide), wherein n is an integer of 1 or greater. In any embodiments disclosed herein, the binding of the probe to another probe or an analyte of interest can be direct (e.g., by direct hybridization of nucleic acid sequences or antigen-antibody binding) or indirect (e.g., by indirect hybridization of one or more bridging oligomers or binding interactions). The binding interaction can be analyzed using microscopy, such as high-resolution optical microscopy, to provide a reading of the presence / absence, distribution, location, amount, level, expression or activity of the target analyte. In some embodiments, the in situ assay includes in situ sequencing and / or in situ hybridization, such as sequential hybridization of probes. In some embodiments, the in situ assay analyzes about 20, about 50, about 100, about 200, about 500, about 1000, about 2000, about 5000, about 10000 or more genes, such as mRNA transcripts of genes from one or more single cells deposited and / or fixed on a substrate. In some embodiments, the in situ analysis is performed on about 100 to about 1,000 mRNA transcripts of genes in one or more single cells deposited and / or fixed on a substrate.

[0110] In some aspects, the method includes the transcriptome or genome of analyzing tissue samples, such as full transcriptome or genome. The cell of tissue sample can be dissociated, and the unicellular of dissociation is deposited and / or fixed on substrate for in situ analysis. In some embodiments, the method includes the process for carrying out transcriptomics and / or genomics, which enables simultaneous analysis of the expression pattern and / or position / distribution pattern of the gene or genomic locus expressed or present in the unicellular of tissue sample. In some embodiments, the method also includes synthetic nucleic acid molecules, which are sequenced, such as with single nucleotide resolution, and analyzed to determine which genes are expressed in one or more unicellular deposited and / or fixed on substrate. In some embodiments, the individual, single and specific transcriptome of each cell can be obtained simultaneously, providing a highly parallel comprehensive transcriptome signature of individual cells in a large number of unicellular cells derived from multiple samples (e.g., tissue sample), without the need to carry out cell partitioning.

[0111] In some embodiments, the methods disclosed herein include sequentially performing one or more in situ assays. The in situ analysis of the first target can be performed before, simultaneously, or after analyzing the second analyte. In some embodiments, the methods disclosed herein include providing a reading, such as a microscopy reading. In some embodiments, the methods disclosed herein include analyzing a microscopy reading of an in situ assay. In some embodiments, the first analyte and the second analyte are different molecules.

[0112] In some embodiments, the methods disclosed herein include contacting dissociated cells deposited and / or fixed on a substrate (e.g., a glass slide) with one or more nucleic acid probes that bind directly or indirectly to a first target nucleic acid or its complementary sequence or product (e.g., a hybridization product, a ligation product, an extension product (e.g., by a DNA or RNA polymerase), a replication product, a transcription / reverse transcription product, and / or an amplification product) in the immobilized cell sample. In some embodiments, the method further includes contacting the dissociated cells on the substrate with a nucleic acid probe that binds directly or indirectly to one or more second target nucleic acids in the immobilized cell sample.

[0113] In some embodiments, the dissociated cells on the substrate are analyzed in situ, such as in situ detection of reporter oligonucleotides indicating the source of the dissociated cell sample, and in situ detection of one or more analytes (e.g., nucleic acid analytes and / or non-nucleic acid analytes) in the immobilized cell sample. In some embodiments, the first target nucleic acid is located in a marker bound to the cell surface. In some embodiments, the first target nucleic acid is a reporter oligonucleotide of a marker, and comprises a sample-specific barcode sequence. In some embodiments, one or more second target nucleic acids are located in and / or on the immobilized cell. In some embodiments, one or more second target nucleic acids include multiple genomic DNA, cellular RNA (e.g., mRNA) and / or cDNA molecules. In some embodiments, one or more second target nucleic acids include transcripts of multiple genes of interest. In some embodiments, one or more second target nucleic acids include multiple nucleic acid tags conjugated directly or indirectly to a binding moiety, and the binding moiety is combined with a non-nucleic acid analyte in the dissociated cells from different samples. In some embodiments, the dissociated single cells are derived from multiple different biological samples, wherein each sample is contacted with a marker, and the marker comprises a reporter molecule that identifies the source of the sample. Thus, using the compositions, systems and methods described herein, cells from different samples (e.g., different cell populations, cell types, experimental conditions, etc.) can be "labeled" with different reporter molecules (e.g., sample-specific barcode sequences), and then combined with a label for later identification of the source of a specific sample when disassembled for in situ multiplex analysis. In some embodiments, the biological sample is a tissue sample, such as a freshly isolated or preserved tissue section. The tissue sample can be labeled with a labeling agent, and then the cells of the tissue sample are dissociated to provide a single cell population. Alternatively, the tissue sample can be dissociated to provide a single cell population, and then the cells in the population are labeled with a labeling agent.

[0114] In some embodiments, the dissociated unicellular is merged and fixed on a substrate. In some embodiments, the dissociated unicellular is fixed by reversible crosslinking. In some embodiments, the dissociated unicellular is processed so that one or more analyte molecules (e.g., RNA molecules) are reversibly or irreversibly locked in place, to retain the analyte in the dissociated unicellular and / or relative to the presence / absence, distribution, position, amount, level, expression or activity of one or more other analytes in the dissociated unicellular. In some embodiments, the unicellular dissociated on the substrate is embedded in a matrix, such as a polymer matrix. In some embodiments, the unicellular dissociated on the substrate is hydrogel-embedded. In some embodiments, one or more analyte molecules (e.g., RNA molecules) are probe-targeted and analyzed using in situ imaging, such as sequencing while connecting, sequencing while hybridizing, sequencing while synthesizing, sequencing while combining and / or sequential hybridization of barcoded probes is subsequently decoded. In some embodiments, the molecules of multiple analytes in the sample are analyzed in situ in a highly multiplex manner.

[0115] In some embodiments, in situ analysis includes detecting the reporter oligonucleotides of the dissociated cells and one or more target nucleic acids in the dissociated cells. In some embodiments, before detecting the one or more signals associated with the one or more target nucleic acids in the dissociated cells, one or more signals associated with the reporter oligonucleotides of the dissociated cells are detected. In some embodiments, after detecting the one or more signals associated with the one or more target nucleic acids in the dissociated cells, one or more signals associated with the reporter oligonucleotides of the dissociated cells are detected. In some embodiments, while detecting the one or more signals associated with the one or more target nucleic acids in the dissociated cells, one or more signals associated with the reporter oligonucleotides of the dissociated cells are detected.

[0116] For example, dissociated cells can be derived from four different samples, and each sample is identified by a reporter oligonucleotide comprising a sample-specific barcode sequence. In this example, the specific dissociated cells on the substrate are marked with one of four different reporter oligonucleotides. The dissociated cells can be contacted with a variety of detectable probes, and each probe is constructed to be directly or indirectly combined with one of four different reporter oligonucleotides, and the signal associated with the multiple detectable probes at the dissociated cell position on the substrate can be detected to identify the sample source of the dissociated cells. For example, one of four different reporter oligonucleotides can be detected by a fluorescently labeled probe of different colors. In the case where the number of different samples is very large, a combined method can be used to increase the ability of sample multiplex analysis. For example, reporter oligonucleotides and corresponding detectable probes can be designed so that each reporter oligonucleotide includes two regions, and each region can be detected by a detectable probe. Using four different colors, at least sixteen different samples can be identified by reporter oligonucleotides of different combinations of four colors.

[0117] In some cases, the in situ assay uses a targeted approach, using target binding probes and microscopy-based optical signal reading and analysis to interrogate a set of target analytes, such as about 50 to about 1,000 genes and their mRNA transcripts, for target analysis and / or sequence determination. In some embodiments, the in situ assay includes in situ detection of one or more barcode sequences of a probe that directly or indirectly binds to an analyte. In some embodiments, the in situ assay includes detecting a signal indicating hybridization of one or more detectably labeled oligonucleotides to one or more barcode sequences of a probe (or its amplified product), the probe directly or indirectly hybridizing with the nucleic acid portion of the analyte, and using the spatial and / or temporal pattern of the signal from sequential hybridization to determine the analyte. In some embodiments, an in situ probe (e.g., a circular or circularizable probe for RCA and / or a primary probe for in situ hybridization, the in situ hybridization involving sequential hybridization of a detectable probe) is combined with a target analyte (e.g., a nucleic acid molecule) in a sample for in situ analysis. The in situ probe may comprise two or more probes that assemble to form a larger probe after binding to a target molecule and ligation of the two or more probes (e.g., by ligation, such as DNA-templated ligation and / or RNA-templated dependent ligation of a DNA probe comprising ribonucleotides). The in situ probe or its product may be amplified, for example, using rolling circle amplification, to produce an amplification product that can be detected in situ, e.g., Figure 1 shown.

[0118] In another aspect, the present disclosure provides a method for sample analysis, comprising (a) contacting a first sample and a second sample with a first labeling agent and a second labeling agent, respectively, to provide labeled cells in each sample, wherein the first labeling agent and the second labeling agent each comprise (i) a binding portion and (ii) a reporter oligonucleotide comprising a first barcode region, wherein the first barcode region comprises a sample-specific barcode sequence corresponding to the respective sample, wherein the binding portion of each labeling agent binds to one or more cells in the respective sample; (b) combining the first sample and the second sample; (c) dissociating the first sample and the second sample into dissociated cells; and (d) ) fix a cell population comprising labeled cells from a first sample and a second sample on a substrate; (e) contact the fixed cells with: (i) a first probe that hybridizes to a sample-specific barcode sequence corresponding to the first sample or the second sample, and (ii) a second probe that binds to an analyte in and / or on one or more fixed cells, wherein the second probe comprises a second barcode region; and (f) detect a first signal associated with the first probe or its product on the substrate and a second signal associated with the second probe or its product, thereby detecting the presence or absence of the analyte in one or more cells from the first sample and / or the second sample. In some embodiments, the first sample and the second sample are combined in (b) before being dissociated into dissociated cells in (c). In some embodiments, the first sample and the second sample are dissociated into dissociated cells in (c) before being combined in (b).

[0119] The following provides additional details of the present disclosure by way of example.

[0120] II. Samples, Analytes, and Target Sequences

[0121] A.Sample

[0122] Samples disclosed herein can be or are derived from any biological sample. Methods and compositions disclosed herein can be used to analyze biological samples, which can be obtained from a subject using any of a variety of techniques (including but not limited to biopsy, surgery, and laser capture microscopy (LCM)), and generally include cells and / or other biological materials from a subject. In addition to the subject described above, biological samples can be obtained from prokaryotes (such as bacteria, archaea, viruses, or viroids). Biological samples can also be obtained from non-mammalian organisms (e.g., plants, insects, arthropods, nematodes, fungi, or amphibians). Biological samples can also be obtained from eukaryotic organisms, such as tissue samples, patient-derived organoids (PDO) or patient-derived xenografts (PDX). Biological samples from organisms can include one or more other organisms or components thereof. For example, in addition to mammalian cells and non-cellular tissue components, mammalian tissue sections can include prions, viroids, viruses, bacteria, fungi, or components from other organisms. The subject from whom a biological sample can be obtained can be a healthy or asymptomatic individual, an individual having or suspected of having a disease (e.g., a patient having a disease such as cancer) or susceptible to a disease, and / or an individual in need of therapy or suspected of being in need of therapy.

[0123] Biological samples can include any number of macromolecules, for example, cellular macromolecules and organelles (for example, mitochondria and nuclei). Biological samples can include nucleic acids (such as DNA or RNA), proteins / polypeptides, carbohydrates and / or lipids. Biological samples can be obtained as tissue samples (such as tissue sections, biopsy samples, core needle biopsy samples, needle aspirates or fine needle aspirates). The sample can be a fluid sample, such as a blood sample, a urine sample or a saliva sample. The sample can be a skin sample, a colon sample, a cheek swab, a histological sample, a histopathological sample, a plasma or serum sample, a tumor sample, a living cell, a cultured cell, a clinical sample (for example, whole blood or blood-derived products, hemocytes or cultured tissues or cells, including cell suspensions). In some embodiments, the biological sample can include cells deposited on the surface.

[0124] The biological sample may be derived from a homogenous culture or population of the subjects or organisms mentioned herein, or alternatively from a collection of several different organisms, for example, in a community or ecosystem.

[0125] Biological samples can include one or more diseased cells. Diseased cells may have altered metabolic properties, gene expression, protein expression and / or morphological characteristics. Examples of diseases include inflammatory disorders, metabolic disorders, nervous system disorders and cancer. Cancer cells can be derived from solid tumors, hematological malignancies, cell lines, or obtained as circulating tumor cells. Biological samples can also include fetal cells and immune cells.

[0126] The biological sample may include an analyte (e.g., protein, RNA, and / or DNA) embedded in a 3D matrix. In some embodiments, an amplicon (e.g., rolling circle amplification product) derived from an analyte (e.g., protein, RNA, and / or DNA) or associated therewith may be embedded in a 3D matrix. In some embodiments, the 3D matrix may include natural molecules and / or synthetic molecular networks chemically and / or enzymatically connected (e.g., by crosslinking). In some embodiments, the 3D matrix may include a synthetic polymer. In some embodiments, the 3D matrix includes a hydrogel.

[0127] In some embodiments, the substrate herein can be any support that is insoluble in aqueous liquids and allows positioning of biological samples, analytes, features and / or reagents (e.g., probes) on the support. In some embodiments, the biological sample can be attached to the substrate as described elsewhere herein.

[0128] Various steps may be performed to prepare or process biological samples for and / or during an assay. Unless otherwise indicated, the preparation or processing steps described below may generally be combined in any manner and in any order to appropriately prepare or process a particular sample for and / or analysis.

[0129] (i) Cell disaggregation

[0130] In some embodiments, biological samples correspond to cells (e.g., cells derived from cell culture, tissue samples, or cells deposited on a surface). In a cell sample with multiple cells, individual cells may not aggregate naturally. For example, cells may be derived from a cell suspension (e.g., a body fluid, such as blood) and / or from a dissociated or disaggregated cell of a tissue or tissue section. The number of cells in a biological sample may vary. Some biological samples include a large number of cells, such as a blood sample, while other biological samples include fewer or only a small amount of cells, or may only be suspected of containing cells, such as plasma, serum, urine, saliva, synovial fluid, amniotic fluid, tears, lymph, liquid, cerebrospinal fluid, etc.

[0131] In some embodiments, the biological sample containing cells can include body fluid or a cell-containing sample derived from body fluid, such as whole blood, a sample derived from blood (e.g., plasma or serum), a buffy coat, urine, sputum, tears, lymph, sweat, liquid, cerebrospinal fluid, ascites, milk, feces, bronchial lavage fluid, saliva, amniotic fluid, nasal secretions, vaginal secretions, semen (semen / seminal fluid), wound secretions, cell culture and swab samples, or any cell-containing sample derived from the above-mentioned samples. In some embodiments, the biological sample containing cells can be body fluid, body secretions or body excretions, such as lymph, blood, buffy coat, plasma or serum. In some embodiments, the biological sample containing cells can be a circulating body fluid, such as blood or lymph, for example, peripheral blood obtained from mammals such as humans.

[0132] In some embodiments, the cells in the biological sample can be aggregated and can be depolymerized into single cells using, for example, enzymes or mechanical techniques. Examples of enzymes for enzymatic depolymerization include, but are not limited to, dispase, collagenase, trypsin, and combinations thereof. For example, a tissue homogenizer can be used for mechanical depolymerization. The biological sample can include depolymerized cells (e.g., non-adherent or suspended cells) that are deposited on a surface and subjected to in situ assays disclosed herein.

[0133] In addition to separating biological samples into single cells, methods for separating biological samples into cell groups, cell types, or one or more regions of interest are described herein. For example, before contacting with a labeling agent (e.g., attached to a binding moiety of a reporter oligonucleotide), the biological sample can be dissociated and / or separated into single cells, cell groups, cell types, or one or more regions of interest. In other examples, the biological sample is first contacted with one or more labeling agents and then separated into single cells, cell groups, cell types, or one or more regions of interest.

[0134] In some embodiments, pixelation can be used to divide the biological sample into cartridges.Pixelation can include the steps of providing a biological sample and punching out one or more portions of the biological sample.

[0135] Non-limiting, non-exhaustive steps for "pixelating" a sample include cutting, punching, microdissection, or transfer through a hollow needle or microneedle, and / or moving a small portion of the sample into a separate compartment or well. In one example, an array of needles is passed through the sample on a support and into a nanowell containing a reagent.

[0136] In any embodiments herein, a cell population may comprise cells derived from a plurality of different samples. In any embodiments herein, a population may comprise at least about 100, at least about 1,000, at least about 10,000, at least about 50,000, at least about 100,000, or at least about 500,000 dissociated cells.

[0137] In some aspects, the methods disclosed herein provide an alternative to partition-based analysis, and the methods can be used to analyze multiple samples (e.g., multiple different cell suspensions) in bulk without separating individual cells into separate partitions or reaction chambers. Although some single-cell assays such as single-cell RNA sequencing techniques may require the generation of singlets and the removal of multiplets, the methods disclosed herein use an in-situ platform for multiplet detection and can tolerate multiplets (e.g., doublets), as long as when deposited on a substrate, cells or nuclei in the multiplets can be segmented, and analytes in individual cells or nuclei can be detected without interference from adjacent cells or nuclei in the multiplets.

[0138] In some embodiments, samples such as tissue samples can be dissociated to produce singlet states contacted with sample-specific labeling agents. In some embodiments, samples can be contacted with sample-specific labeling agents and dissociated to produce singlet states, wherein the singlet states remain labeled by sample-specific labeling agents. Singlet states may include single cells or nuclei captured after removing multiple states. Single cells or nuclei dissociated from multiple biological samples can be labeled with sample-specific labeling agents so that the sample source of a specific single cell or nucleus can be identified based on its sample-specific labeling agent. In some embodiments, at least or about 100, at least or about 200, at least or about 500, at least or about 1,000, at least or about 2,000, at least or about 5,000, at least or about 10,000, at least or about 20,000, at least or about 50,000, at least or about 100,000, at least or about 200,000, at least or about 500,000, at least or about 1,000,000, at least or about 2,000,000, or at least or about 5,000,000 singlets from a first sample are labeled with the same first sample-specific labeling agent or combination of sample-specific labeling agents, and at least or about 100,000, at least or about 200,000, at least or about 500,000, at least or about 1,000,000, at least or about 2,000,000, or at least or about 5,000,000 singlets from a first sample are labeled with the same second sample-specific labeling agent or combination of sample-specific labeling agents. At least or about 1,000, at least or about 2,000, at least or about 5,000, at least or about 10,000, at least or about 20,000, at least or about 50,000, at least or about 100,000, at least or about 200,000, at least or about 500,000, at least or about 1,000,000, at least or about 2,000,000, or at least or about 5,000,000 singlet states from a second sample, wherein the first sample-specific labeling agent and the second sample-specific labeling agent (or a combination of sample-specific labeling agents) are different, such that when the samples are combined for multiplex detection on an in situ platform, the sample source of a particular singlet state can be identified. In some embodiments, at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, at least or about 90%, at least or about 95%, or at least or about 99% of the cells or nuclei in a sample (e.g., a suspension of cells and / or nuclei) labeled with the same sample-specific barcode sequence can be singlets, while the remainder can be multiplets (e.g., doublets), which may or may not be removed prior to depositing the labeled cells and / or nuclei for analysis on an in situ platform.In some embodiments, the sample-specific barcode sequence of a sample is shared by at least or about 100, at least or about 200, at least or about 500, at least or about 1,000, at least or about 2,000, at least or about 5,000, at least or about 10,000, at least or about 20,000, at least or about 50,000, at least or about 100,000, at least or about 200,000, at least or about 500,000, at least or about 1,000,000, at least or about 2,000,000, or at least or about 5,000,000 cells and / or nuclei from the sample and is not specific for any particular endogenous polynucleotide or exogenous polynucleotide introduced into one or more cells or nuclei of the sample.

[0139] (ii) Substrate attachment

[0140] In some embodiments, the biological sample can be attached to a substrate. Examples of substrates suitable for this purpose are described in detail below. The attachment of the biological sample can be irreversible or reversible, depending on the nature of the sample and the subsequent steps in the analytical method.

[0141] In certain embodiments, the sample can be reversibly attached to the substrate by applying a suitable polymer coating to the substrate and contacting the sample with the polymer coating. The sample can then be separated from the substrate using an organic solvent that at least partially dissolves the polymer coating. Hydrogels are examples of polymers suitable for this purpose.

[0142] More generally, in some embodiments, the substrate can be coated or functionalized with one or more substances to facilitate sample attachment to the substrate. Suitable substances that can be used to coat or functionalize the substrate include, but are not limited to, lectins, polylysine, antibodies, and polysaccharides.

[0143] (iii) Staining and immunohistochemistry (IHC)

[0144] For the convenience of visualization, various staining agents and staining techniques can be used to stain biological samples. For example, in some embodiments, any number of staining agents and / or immunohistochemical reagents can be used to stain samples. One or more staining steps can be performed to prepare or process biological samples for determinations described herein, or can be performed during and / or after determination. In some embodiments, samples can be contacted with one or more nucleic acid stains, membrane stains (for example, cell membrane or nuclear membrane), cytological stains, or a combination thereof. In some instances, staining can be specific to the compartment of proteins, phospholipids, DNA (for example, dsDNA, ssDNA), RNA, organelles, or cells. Samples can be contacted with one or more labeled antibodies (for example, a first antibody specific to an analyte of interest and a second antibody specific to a label of the first antibody). In some embodiments, one or more images taken of the stained sample can be used to segment the cells in the sample.

[0145] In some embodiments, lipophilic dye is used for dyeing. In some instances, lipophilic carbocyanine or aminostyrene dye or their analog (for example, DiI, DiO, DiR, DiD) are used for dyeing. Other cell membrane stains can include FM and RH dye or immunohistochemical reagents specific to cell membrane proteins. In some instances, staining agent can include but is not limited to acridine orange, acid fuchsin, Bismarck brown, carmine, Coomassie blue, cresyl violet, DAPI, eosin, ethidium bromide, acid fuchsin, hematoxylin, Hoechst stain, iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red, osmium tetroxide, ruthenium red, propidium iodide, rhodamine (for example, rhodamine B) or safranin or their derivatives. In some embodiments, sample can be dyed with hematoxylin and eosin (H&E).

[0146] Samples can be stained using hematoxylin and eosin (H&E) staining techniques, using Papanicolaou staining techniques, Masson's trichrome staining techniques, silver staining techniques, Sudan staining techniques, and / or using periodic acid Schiff (PAS) staining techniques. PAS staining is usually performed after formalin or acetone fixation. In some embodiments, Romanovsky stains (including Wright's stain, Jenner's stain, Can-Grunwaldstain, Leishman stain, and Giemsa stain) can be used to stain samples.

[0147] In some embodiments, the biological sample can be decolorized. Any suitable method for destaining or decolorizing the biological sample can be used, and these methods generally depend on the nature of the stain applied to the sample. For example, in some embodiments, one or more immunofluorescent stains are applied to the sample by antibody coupling. Such stains can be removed using techniques such as cleavage of disulfide bonds by washing with a reducing agent and a detergent, treatment with a chaotropic salt, treatment with an antigen retrieval solution, and treatment with an acidic glycine buffer. Methods for multiple staining and destaining are described, for example, in Bolognesi et al., J. Histochem. Cytochem. 2017; 65(8): 431-444; Lin et al., Nat Commun. 2015; 6: 8390; Pirici et al., J. Histochem. Cytochem. 2009; 57: 567–75; and Glass et al., J. Histochem. Cytochem. 2009; 57: 899–905, the entire contents of each of which are incorporated herein by reference.

[0148] (iv) Isovolumetric expansion

[0149] In some embodiments, the biological sample embedded in the matrix (e.g., hydrogel) can be isochoric expanded. The isochoric expansion method that can be used includes hydration, which is a preparation step in expansion microscopy, such as (e.g.) Chen et al., Science 347 (6221): 543–548, 2015 and described in U.S. Patent No. 10,059,990. Isochoric expansion can be carried out in the following manner: one or more components of the biological sample are anchored to the gel, followed by gel formation, protein hydrolysis and swelling. In some embodiments, the analyte in the sample, the product of the analyte and / or the probe associated with the analyte in the sample can be anchored to the matrix (e.g., hydrogel).

[0150] (v) Cross-linking and de-cross-linking

[0151] In some embodiments, before or during the original position determination, biological sample is reversibly cross-linked. In some respects, the amplified product (for example, amplicon) of analyte, polynucleotide and / or analyte or the probe combined therewith can be anchored on the polymer matrix. For example, the polymer matrix can be a hydrogel. In some embodiments, one or more polynucleotide probes and / or its amplified product (for example, amplicon) can be modified to contain a functional group, and this functional group can be used as the anchoring point that polynucleotide probe and / or amplified product are connected to the polymer matrix. In some embodiments, biological sample is fixed in the hydrogel by the crosslinking of the polymer material forming the hydrogel.

[0152] In some embodiments, the hydrogel can include hydrogel subunits such as, but not limited to, acrylamide, bisacrylamide, polyacrylamide and its derivatives, poly(ethylene glycol) and its derivatives (e.g., PEG-acrylate (PEG-DA), PEG-RGD), methacrylated gelatin (GelMA), methacrylated hyaluronic acid (MeHA), polyaliphatic polyurethanes, polyether polyurethanes, polyester polyurethanes, polyethylene copolymers, polyamides, polyvinyl alcohol, polypropylene glycol, polytetramethylene oxide, polyvinyl pyrrolidone, polyacrylamide, poly(hydroxyethyl acrylate) and poly(hydroxyethyl methacrylate), collagen, hyaluronic acid, chitosan, dextran, agarose, gelatin, alginate, protein polymers, methylcellulose, and the like, and combinations thereof. Examples of suitable hydrogels are described, for example, in U.S. Pat. Nos. 6,391,937, 9,512,422, and 9,889,422 and U.S. Patent Application Publication Nos. 2017 / 0253918, 2018 / 0052081, and 2010 / 0055733, the entire contents of each of which are incorporated herein by reference.

[0153] In some embodiments, the hydrogel can form a substrate. In some embodiments, the substrate includes a hydrogel and one or more second materials. In some embodiments, the hydrogel is placed on the top of the one or more second materials. For example, the hydrogel can be preformed and then placed on the top, bottom or any other configuration with the one or more second materials. In some embodiments, the hydrogel forms after contacting the one or more second materials during the formation of the substrate. The hydrogel formation can also occur in the structure (e.g., hole, ridge, protrusion and / or texture) located on the substrate.

[0154] In some embodiments, hydrogel formation on the substrate occurs before, simultaneously with, or after providing the probe to the sample. For example, hydrogel formation can be performed on a substrate that already contains the probe.

[0155] In some embodiments, hydrogel formation occurs within a biological sample. In some embodiments, a biological sample (e.g., a tissue section) is embedded in a hydrogel. In some embodiments, a hydrogel subunit is injected into a biological sample, and polymerization of the hydrogel is initiated by an external or internal stimulus.

[0156] In the embodiment of forming hydrogel in biological sample, functionalization chemistry can be used.In some embodiments, functionalization chemistry includes hydrogel-tissue chemistry (HTC).Any hydrogel-tissue skeleton (for example, synthetic or natural) suitable for HTC can be used for anchoring biomacromolecules and regulating functionalization.Non-limiting examples of methods using HTC skeleton variants include CLARITY, PACT, ExM, SWITCH and ePACT.In some embodiments, the hydrogel formation in biological sample is permanent.In some embodiments, the hydrogel formation in biological sample is reversible.

[0157] In some embodiments, HTC and / or additional reagents are added to the hydrogel subunits before, simultaneously and / or after polymerization. For example, additional reagents may include, but are not limited to, oligonucleotides (e.g., probes), endonucleases of fragmented DNA, fragmentation buffers of DNA, DNA polymerases, dNTPs for amplifying nucleic acids and connecting barcodes to amplified fragments. Other enzymes may be used, including but not limited to RNA polymerases, ligases, proteinase K and DNA enzymes. Additional reagents may also include reverse transcriptases (including enzymes with terminal transferase activity), primers and switch oligonucleotides. In some embodiments, cell markers including optical markers are added to the hydrogel subunits before, simultaneously and / or after polymerization.

[0158] Any suitable method can be used to make the hydrogel embedded in the biological sample transparent. For example, the electrophoretic tissue clearing method can be used to remove biomacromolecules from the hydrogel-embedded sample. In some embodiments, the hydrogel-embedded sample is stored in a medium (e.g., a fixing medium, methylcellulose or other semi-solid medium) before or after the hydrogel is cleared.

[0159] In some embodiments, the methods disclosed herein include decrosslinking a reversibly crosslinked biological sample. Decrosslinking does not need to be complete. In some embodiments, only a portion of the crosslinked molecules in the reversibly crosslinked biological sample are decrosslinked and allowed to migrate.

[0160] (vi) Cell permeabilization and treatment

[0161] In some embodiments, a biological sample (e.g., dissociated cells on a substrate) can be permeabilized to facilitate transfer of a substance (e.g., a labeling agent and / or a detectable probe) into the sample. Typically, a biological sample can be permeabilized by exposing the sample to one or more permeabilizing agents. Suitable agents for this purpose include, but are not limited to, organic solvents (e.g., acetone, ethanol, and methanol), cross-linking agents (e.g., paraformaldehyde), detergents (e.g., saponin, Triton X-100, TM or Tween-20 TM) and enzymes (e.g., trypsin, proteases). In some embodiments, the biological sample can be incubated with a cell permeabilizing agent to promote permeabilization of the sample. Additional methods for sample permeabilization are described in, for example, Jamur et al., Method Mol. Biol. 588: 63-66, 2010, the entire contents of which are incorporated herein by reference. Any suitable sample permeabilization method can generally be used in conjunction with the samples described herein.

[0162] In some embodiments, the biological sample can be permeabilized by adding one or more lysing agents to the sample. Examples of suitable lysing agents include, but are not limited to, bioactive agents such as lysing enzymes for lysing different cell types (e.g., Gram-positive or negative bacteria, plants, yeasts, mammals), such as lysozymes, colorless peptidases, lysostaphin, labiase, kitalase, lysomycin, and various other commercially available lysing enzymes.

[0163] Other lysing agents can be added to the biological sample additionally or alternatively to promote permeabilization. For example, a lysing solution based on a surfactant can be used for lysing sample cells. The lysing solution can include an ionic surfactant, such as sodium lauryl sarcosinate and sodium lauryl sulfate (SDS). More generally, chemical lysing agents can include but are not limited to organic solvents, chelating agents, detergents, surfactants and chaotropic agents.

[0164] In some embodiments, biological samples can be permeabilized by non-chemical permeabilization methods, such as physical lysis techniques such as electroporation, mechanical permeabilization methods (e.g., bead beating using a homogenizer and grinding balls to mechanically disrupt sample tissue structure), acoustic permeabilization (e.g., sonication), and thermal lysis techniques such as heating that induce thermal permeabilization of the sample.

[0165] Before analyzing the sample, additional reagents can be added to the biological sample to perform various functions. In some embodiments, DNA enzyme and RNA enzyme inactivators or inhibitors (such as proteinase K) and / or chelating agents (such as EDTA) can be added to the sample. For example, the method disclosed herein can include a step of increasing the accessibility of the nucleic acid for binding, for example, opening the DNA in the cell for the denaturation step of probe hybridization. For example, proteinase K treatment can be used to release DNA bound to proteins.

[0166] B. Labeling agent

[0167] In some embodiments, provided herein are methods and compositions for cell analysis using one or more labeling agents. In some embodiments, labeling agents can be combined with or otherwise coupled to one or more cells or cell features (e.g., molecules on and / or in cells). For example, labeling agents can include lipophilic moieties (e.g., cholesterol), which can interact with all cells in one or more samples in a non-analyte-specific manner. In some embodiments, labeling agents do not bind analytes. In some embodiments, labeling agents include binding moieties and / or reporter oligonucleotides. In some embodiments, the binding moieties of labeling agents are not combined with analytes. In some embodiments, reporter oligonucleotides include one or more barcodes (e.g., target-specific barcodes and / or sample-specific barcodes). Exemplary labeling agents for binding or coupling to one or more cells or cell features are described in Section III-A.

[0168] In some embodiments, provided herein are methods and compositions for analyzing endogenous analytes (e.g., RNA, ssDNA, cell surface or intracellular proteins and / or metabolites) in a sample using one or more labeling agents. In some embodiments, a labeling agent may include an agent that interacts with an analyte (e.g., an endogenous analyte in a sample).

[0169] In some embodiments, the labeling agent comprises a probe that targets the analyte. For example, the labeling agent comprises a probe that binds to a housekeeping gene transcript present in substantially all cells in the sample. In some embodiments, the labeling agent may comprise a sample-specific barcode. In some embodiments, the labeling agent may comprise a sample-specific barcode and / or a target-specific barcode.

[0170] In some embodiments, the labeling agent comprises a probe that is combined with an analyte. In some embodiments, the probe can be directly or indirectly combined with a nucleic acid analyte (e.g., mRNA). In some embodiments, the probe can include one or more barcode sequences (e.g., target-specific barcode sequences and / or sample-specific barcode sequences). The probe can include a sample-specific barcode sequence corresponding to a sample from which a biological cell is derived. The probe can include a target-specific barcode that includes a nucleic acid analyte that is combined with the probe to associate or otherwise identify a barcode for the nucleic acid analyte. In some embodiments, the analyte combined with the labeling agent is detected in situ.

[0171] In some embodiments, the labeling agent includes an agent that is bound to a non-nucleic acid analyte (e.g., an intracellular protein). In some embodiments, the non-nucleic acid analyte bound to the labeling agent is detected in situ. In some embodiments, the labeling agent may include a binding moiety and a reporter oligonucleotide. The reporter oligonucleotide indicates a target analyte or a portion thereof that interacts with the binding moiety. For example, the reporter oligonucleotide may include a barcode sequence that allows identification of the binding moiety. In some cases, the sample contacted by the labeling agent may be further contacted with a probe (e.g., a single-stranded probe sequence), which is hybridized with the reporter oligonucleotide of the labeling agent to identify the analyte associated with the labeling agent. In some embodiments, the labeling agent includes a binding moiety and a reporter oligonucleotide, and the reporter oligonucleotide includes one or more barcode sequences, such as a barcode sequence corresponding to a binding moiety and / or a target analyte. A binding moiety barcode or a target-specific barcode includes a barcode that associates with a binding moiety or otherwise identifies a binding moiety. In some embodiments, the binding moiety is identified by identifying the associated target-specific barcode of the binding moiety, and the target analyte bound to the binding moiety may also be identified. In some embodiments, the labeling agent comprises one or more barcode sequences, such as a sample-specific barcode sequence corresponding to the sample from which the biological cell is derived. Sample-specific or target-specific barcodes can be nucleic acid sequences of a given length and / or sequences associated with a binding moiety. Sample-specific or target-specific barcodes can generally include any of the various aspects of barcodes described herein. In some embodiments, the method includes one or more post-fixation steps after contacting the sample with one or more labeling agents.

[0172] In the methods and systems described herein, one or more markers that can be combined with one or more features or otherwise coupled can be used to characterize analytes, cells and / or cell features, including cell surface features. Analytes can include but are not limited to proteins, receptors, antigens, surface proteins, transmembrane proteins, differentiation protein clusters, protein channels, protein pumps, carrier proteins, phospholipids, glycoproteins, glycolipids, cell-cell interaction protein complexes, antigen presentation complexes, major histocompatibility complexes, engineered T cell receptors, T cell receptors, B cell receptors, chimeric antigen receptors, gap connections, adhesion connections or any combination thereof. In some cases, cell features can include intracellular analytes, such as proteins, protein modifications (e.g., phosphorylation status or other post-translational modifications), nucleoproteins, nuclear membrane proteins or any combination thereof.

[0173] In some embodiments, the binding moiety may include any molecule or part that can be combined with an analyte (e.g., a biological analyte, e.g., a macromolecular component) in a cell and / or on a cell. The binding moiety may include, but is not limited to, proteins, peptides, antibodies (or their epitope binding fragments), lipophilic moieties (such as cholesterol), cell surface receptor binding molecules, receptor ligands, small molecules, bispecific antibodies, bispecific T cell adapters, T cell receptor adapters, B cell receptor adapters, antibody prodrugs, aptamers, monomers, affimers, darpins and protein scaffolds or any combination thereof. The analyte binding moiety may be attached to the reporter oligonucleotides of the sample from which the indicator cell originates. For example, the same analyte binding moiety (e.g., an antibody or lipid moiety configured to be combined with the same cell surface feature) may be used to combine with cells from different samples, wherein the same analyte binding moiety is attached to different reporter oligonucleotides, and each reporter oligonucleotide identifies one of the different samples. For example, the reporter oligonucleotide may include a barcode sequence that allows identification of the sample from which the cell labeled with the reporter oligonucleotide originates. For example, a first sample can be contacted with a binding moiety coupled to a first reporter oligonucleotide that identifies the first sample, and a second sample can be contacted with the same binding moiety coupled to a different reporter oligonucleotide that identifies the second sample.

[0174] Optionally, the reporter oligonucleotide can indicate a cell surface feature bound to a binding moiety. For example, a binding moiety specific for one type of cell feature (e.g., a first cell surface feature) can have a first reporter oligonucleotide coupled thereto, while a binding moiety specific for different cell features (e.g., a second cell surface feature) can have different reporter oligonucleotides coupled thereto. For a description of exemplary binding moieties, including antibody-based markers, reporter oligonucleotides, and methods of use, see, for example, U.S. Patent No. 10,550,429; PCT Publication No. WO2019113533A1; U.S. Patent Publication No. 20190177800; and U.S. Patent Publication No. 20190367969, all of which are incorporated herein by reference in their entirety.

[0175] In some embodiments, the binding moiety includes one or more antibodies or epitope binding fragments thereof. Antibodies or epitope binding fragments including binding moieties can specifically bind to target analytes. In some embodiments, analytes are proteins (e.g., proteins on the surface of biological samples (e.g., cells) or intracellular proteins). In some embodiments, multiple labeling agents comprising multiple binding moieties bind to multiple analytes present in biological samples. In some embodiments, multiple analytes include a single species of analyte (e.g., a single species of polypeptide). In some embodiments in which multiple analytes include a single species of analyte, the binding moieties of multiple labeling agents are the same. In some embodiments in which multiple analytes include a single species of analyte, the binding moieties of multiple labeling agents are different (e.g., members of multiple analyte labeling agents can have two or more species of binding moieties, wherein each species in the two or more species of analyte binding moieties binds to a single species of analyte, such as in different binding sites). In some embodiments, multiple analytes include multiple different species of analytes (e.g., multiple different species of polypeptides).

[0176] In other cases, for example, to facilitate multiplex analysis of samples, a labeling agent specific for a particular cellular characteristic (e.g., a molecule on and / or in a cell) can have a first plurality of binding moieties (e.g., antibodies or lipophilic moieties) coupled to a first reporter oligonucleotide and a second plurality of binding moieties coupled to a second reporter oligonucleotide.

[0177] In some aspects, these reporter oligonucleotides can include nucleic acid barcode sequences that allow identification of the sample source of cells labeled with the reporter oligonucleotides. Selecting oligonucleotides as reporters can provide the following advantages: the ability to generate significant diversity in sequence, while also being easily attached to most biomolecules (e.g., antibodies, etc.), and being easily detected (e.g., using in situ detection techniques described herein).

[0178] The reporter oligonucleotide can be linked (coupled) to the binding moiety by any of a variety of direct or indirect, covalent or non-covalent associations or linkages. For example, the oligonucleotide can be linked (coupled) using chemical conjugation techniques (e.g., available from InnovaBiosciences). Antibody labeling kits) are covalently linked to a portion of a labeling agent (such as a protein, such as an antibody or antibody fragment), as well as using other non-covalent attachment mechanisms, such as using a biotinylated antibody and an oligonucleotide with an avidin or streptavidin linker (or an agent comprising one or more biotinylated linkers coupled to an oligonucleotide). Antibody and oligonucleotide biotinylation techniques are available. See, for example, Fang et al., "Fluoride-Cleavable Biotinylation Phosphoramidite for 5′-end-Labelling and Affinity Purification of Synthetic Oligonucleotides," Nucleic Acids Res. 2003 Jan 15; 31(2):708-715, all of which are incorporated herein by reference in their entirety. Similarly, protein and peptide biotinylation techniques have been developed and are readily available. See, for example, U.S. Pat. No. 6,265,552, the contents of which are incorporated herein by reference in their entirety. In addition, click reaction chemistry can be used to couple reporter oligonucleotides to labeling agents. Commercially available kits (such as those from Thunderlink and Abcam) and common techniques in the art can be used to couple the reporter oligonucleotide to the labeling agent when appropriate. In another example, the labeling agent is indirectly (e.g., via hybridization) coupled to the reporter oligonucleotide, and the reporter oligonucleotide includes a barcode sequence for identifying the labeling agent. For example, the labeling agent can be directly coupled (e.g., covalently bound) to a hybrid oligonucleotide, which includes a sequence hybridized with the sequence of the reporter oligonucleotide. The hybridization of the hybrid oligonucleotide and the reporter oligonucleotide couples the labeling agent to the reporter oligonucleotide. In some cases, in addition to the sample-specific barcode sequence, the reporter oligonucleotide described herein may include one or more other sequences, such as additional barcode sequences, aptamer sequences, unique molecular identifiers (UMI) sequences and / or primers or primer binding sequences.

[0179] C. Analyte

[0180] The biological sample may contain one or more analytes of interest. Methods are provided for performing multiplex assays to analyze two or more different analytes in a single biological sample.

[0181] A variety of different analytes can be detected and analyzed using methods and compositions disclosed herein. In some aspects, the analyte can include any biological substance, structure, part or component to be analyzed. In some aspects, the target disclosed herein can similarly include any analyte of interest. In some instances, the target or analyte can be detected directly or indirectly.

[0182] The analyte can be derived from a particular type of cell and / or a particular subcellular region. For example, the analyte can be derived from the cytosol, from the nucleus, from the mitochondria, from the microsomes, and more generally, from any other compartment, organelle or part of the cell. Permeabilizing agents that specifically target certain cell compartments and organelles can be used to selectively release the analyte from the cell for analysis, and / or allow one or more reagents (e.g., probes for analyte detection) to approach the analyte in the cell or cell compartment or organelle.

[0183] Analyte can comprise any biomolecule or chemical compound, including macromolecule (such as protein or peptide, lipid or nucleic acid molecule) or small molecule (including organic or inorganic molecule). Analyte can be cell or microorganism, including virus or its fragment or product. Analyte can be any substance or entity, can develop specific binding partner (such as affinity binding partner) for it. Such specific binding partner can be nucleic acid probe (for nucleic acid analyte) and can directly lead to the generation of RCA (rolling circle amplification) template (for example, padlock or other circularizable probe). Alternatively, specific binding partner can be coupled to nucleic acid, and described nucleic acid can be detected using RCA strategy, for example, in the determination of circular nucleic acid molecule that can be used as RCA template using or generating.

[0184] Analytes of particular interest may include nucleic acid molecules such as DNA (e.g., genomic DNA, mitochondrial DNA, plastid DNA, viral DNA, etc.) and RNA (e.g., mRNA, microRNA, rRNA, snRNA, viral RNA, etc.); and synthetic and / or modified nucleic acid molecules (e.g., including nucleic acid domains comprising synthetic or modified nucleotides such as LNA, PNA, morpholino, etc. or consisting of these substances); protein molecules such as peptides, polypeptides, proteins or prions, or any molecule comprising a protein or polypeptide component, etc., or a fragment thereof; or lipid or carbohydrate molecules or any molecule comprising a lipid or carbohydrate component. The analyte may be a single molecule or a complex containing two or more molecular subunits, including combinations of the above-mentioned types of molecules.

[0185] (i) Endogenous analytes

[0186] In some embodiments, the analyte herein is endogenous to the biological sample, and may include nucleic acid analytes and non-nucleic acid analytes. The methods and compositions disclosed herein may be used in any suitable combination to analyze nucleic acid analytes (e.g., using nucleic acid probes or probe groups that are directly or indirectly hybridized with nucleic acid analytes) and / or non-nucleic acid analytes (e.g., using a labeling agent that includes a reporter oligonucleotide and is directly or indirectly combined with a non-nucleic acid analyte).

[0187] Examples of non-nucleic acid analytes include, but are not limited to, lipids, carbohydrates, peptides, proteins, glycoproteins (N-connected or O-connected), lipoproteins, phosphoproteins, specific phosphorylation or acetylation variants of proteins, amidation variants of proteins, hydroxylation variants of proteins, methylation variants of proteins, ubiquitination variants of proteins, sulfated variants of proteins, viral capsid proteins, extracellular proteins and intracellular proteins, antibodies and epitope binding fragments. In some embodiments, the analyte is inside the cell or on the cell surface, such as a transmembrane analyte or an analyte attached to the cell membrane. In some embodiments, the analyte can be an organelle (e.g., a nucleus or mitochondria). In some embodiments, the analyte is an extracellular analyte, such as a secreted analyte. Exemplary analytes include, but are not limited to, receptors, antigens, surface proteins, transmembrane proteins, clusters of differentiation proteins, protein channels, protein pumps, carrier proteins, phospholipids, glycoproteins, glycolipids, cell-cell interaction protein complexes, antigen presentation complexes, major histocompatibility complexes, engineered T cell receptors, T cell receptors, B cell receptors, chimeric antigen receptors, extracellular matrix proteins, the post-translational modification (e.g., phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, or lipidation) state of cell surface proteins, nick junctions, or adherens junctions.

[0188] Examples of nucleic acid analytes include DNA analytes, such as single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), genomic DNA, methylated DNA, specific methylated DNA sequences, fragmented DNA, mitochondrial DNA, PCR products synthesized in situ, and RNA / DNA hybrids. A DNA analyte can be a transcript of another nucleic acid molecule (e.g., DNA or RNA (such as mRNA)) present in a cell or tissue sample.

[0189] Examples of nucleic acid analytes also include RNA analytes, such as various types of coding and non-coding RNA. Examples of different types of RNA analytes include messenger RNA (mRNA), including nascent RNA, pre-mRNA, primary transcription RNA, and processed RNA (such as capped mRNA (e.g., with a 5'7-methylguanosine cap), polyadenylated mRNA (poly A tail at the 3' end), and spliced ​​mRNA with one or more introns removed). Uncapped mRNA, unpolyadenylated mRNA, and unspliced ​​mRNA are also included in the analytes disclosed herein. RNA analytes can be transcripts of another nucleic acid molecule (e.g., DNA or RNA (such as viral RNA)) present in a cell or tissue sample. Examples of non-coding RNA (ncRNA) that is not translated into protein include transfer RNA (tRNA) and ribosomal RNA (rRNA), as well as small non-coding RNAs, such as microRNA (miRNA), small interfering RNA (siRNA), etc.

[0190] In some embodiments described herein, the analyte can be a denatured nucleic acid, wherein the resulting denatured nucleic acid is single-stranded. The nucleic acid can be denatured, for example, optionally denatured using formamide, heat, or both formamide and heat. In some embodiments, the nucleic acid is not denatured and is used in the methods disclosed herein.

[0191] In certain embodiments, the analyte may be derived from or detected in living cells. Processing conditions may be adjusted to ensure that the biological sample remains active during analysis. Analytes derived from living cells may be obtained only once from a sample, or may be obtained at intervals from a sample that is continuously kept alive.

[0192] The methods and compositions disclosed herein can be used to analyze any number of analytes. For example, the number of analytes analyzed can be at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 25, at least about 30, at least about 40, at least about 50, at least about 100, at least about 1,000, at least about 10,000, at least about 100,000 or more different analytes present in one region of a sample or in a separate feature of a substrate.

[0193] In some embodiments described herein, the analyte comprises a target sequence. In some embodiments, the target sequence can be an endogenous sequence of the sample, a sequence generated in the sample, a sequence added to the sample, or a sequence associated with the analyte in the sample. In some embodiments, the target sequence is a single-stranded target sequence (e.g., a sequence in a rolling circle amplification product). In some embodiments, the analyte comprises one or more single-stranded target sequences. In one aspect, the first single-stranded target sequence is different from the second single-stranded target sequence. In another aspect, the first single-stranded target sequence is the same as the one or more second single-stranded target sequences. In some embodiments, one or more second single-stranded target sequences are contained in the same analyte (e.g., nucleic acid) as the first single-stranded target sequence. Alternatively, one or more second single-stranded target sequences are contained in an analyte (e.g., nucleic acid) different from the first single-stranded target sequence.

[0194] In situ (ii) Products of endogenous analyte and / or labeling agent

[0195] In some embodiments, provided herein are methods and compositions for analyzing one or more products of endogenous analytes and / or labeling agents in biological samples. In some embodiments, endogenous analytes (e.g., viral or cellular DNA or RNA) or their products (e.g., hybridization products, connection products, extension products (e.g., by DNA or RNA polymerase), replication products, transcription / reverse transcription products and / or amplification products (such as rolling circle amplification (RCA) products)) are analyzed. In some embodiments, the labeling agent directly or indirectly combined with the analyte in the biological sample is analyzed. In some embodiments, the product of the labeling agent directly or indirectly combined with the analyte in the biological sample is analyzed (e.g., hybridization products, connection products, extension products (e.g., by DNA or RNA polymerase), replication products, transcription / reverse transcription products and / or amplification products (such as rolling circle amplification (RCA) products)).

[0196] (a) Hybridization

[0197] In some embodiments, the product of an endogenous analyte and / or a labeling agent is a hybridization product comprising a pairing of substantially complementary or complementary nucleic acid sequences within two different molecules, wherein one molecule is an endogenous analyte or a labeling agent (e.g., a reporter oligonucleotide connected thereto). The other molecule may be another endogenous molecule or another labeling agent, such as a probe. Pairing may be achieved by any method, wherein the nucleic acid sequence is combined with a substantially or completely complementary sequence by base pairing to form a hybridization complex. For the purpose of hybridization, if at least 60% (e.g., at least 70%, at least 80%, or at least 90%) of the bases of the two nucleic acid sequences are each complementary to each other, the two nucleic acid sequences are "substantially complementary".

[0198] Various probes and probe sets can hybridize to endogenous analytes and / or labeling agents, and each probe can contain one or more barcode sequences. Exemplary barcoded probes or probe sets can be based on circularizable probes or probe sets (e.g., padlock probes), gapped padlock probes, SNAIL (splint nucleotide assisted intramolecular ligation) probe sets, PLAYR ( R The specific probe or probe set design can vary.

[0199] (b) Connection

[0200] In some embodiments, the product of endogenous analyte and / or labeling agent is a connection product. In some embodiments, a connection product is formed between two or more endogenous analytes. In some embodiments, a connection product is formed between two or more labeling agents. In some embodiments, the connection product is the intramolecular connection of endogenous analytes. In some embodiments, the connection product is an intramolecular connection product or an intermolecular connection product, for example, the connection product can be produced by the cyclization of a cyclizable probe or probe group after hybridization with a target sequence. The target sequence can be included in an endogenous analyte (for example, nucleic acid, such as genomic DNA or mRNA) or its product (for example, cDNA from a cell mRNA transcript), or included in a labeling agent (for example, a reporter oligonucleotide) or its product.

[0201] In some embodiments, probes or probe groups capable of DNA templated connection (such as from cDNA molecules) are provided herein. See, for example, U.S. Patent No. 8,551,710, the contents of which are incorporated herein by reference in their entirety. In some embodiments, circularizable DNA probes or probe groups are provided herein, which can hybridize with target nucleic acids (e.g., mRNA molecules) and connect in a target-dependent manner (e.g., RNA template connection). See, for example, U.S. Patent Publication 2020 / 0224244, the contents of which are incorporated herein by reference in their entirety. In some embodiments, probe groups are SNAIL probe groups. See, for example, U.S. Patent Publication 20190055594, the contents of which are incorporated herein by reference in their entirety. In some embodiments, multiple proximity connection assays are provided herein. See, for example, U.S. Patent Publication 20140194311, the contents of which are incorporated herein by reference in their entirety. In some embodiments, probes or probe groups capable of proximity connection are provided herein, such as RNA proximity connection assays (e.g., PLAYR) probe groups. See, for example, U.S. Patent Publication 20160108458, which is hereby incorporated by reference in its entirety. In some embodiments, the circular probe can hybridize indirectly with the target nucleic acid. In some embodiments, the circular construct is formed by a probe group capable of adjacent ligation (e.g., adjacent ligation in situ hybridization (PLISH) probe group). See, for example, U.S. Patent Publication 2020 / 0224243, the contents of which are incorporated herein by reference in their entirety.

[0202] In some embodiments, the connection includes chemical connection (e.g., click chemical connection). Exemplary click chemical reactions are described in Section III-C-vi. In some embodiments, chemical connection involves template-dependent connection. In some embodiments, chemical connection involves non-template-dependent connection. In some embodiments, the click reaction is a non-template-dependent reaction (see, for example, Xiong and Seela (2011), J.Org.Chem.76 (14): 5584-5597, the contents of which are incorporated herein by reference in their entirety). In some embodiments, the click reaction is a template-dependent reaction or a template-directed reaction. In some embodiments, the template-dependent reaction is sensitive to base pair mismatches, so that the reaction rate of the matching template is significantly higher than that of the unmatched template. In some embodiments, the click reaction is a nucleophilic addition template-dependent reaction. In some embodiments, the click reaction is a cyclopropane-tetrazine template-dependent reaction.

[0203] In some embodiments, the connection involves enzymatic connection. In some embodiments, the enzymatic connection involves the use of a ligase. In some aspects, the ligase used herein includes an enzyme that is commonly used to connect polynucleotides together or connect the ends of a single polynucleotide. RNA ligase, DNA ligase or another ligase can be used to connect two nucleotide sequences together. Ligase includes ATP-dependent double-stranded polynucleotide ligase, NAD-i-dependent double-stranded DNA or RNA ligase and single-stranded polynucleotide ligase, such as any ligase described in EC 6.5.1.1 (ATP-dependent ligase), EC 6.5.1.2 (NAD+-dependent ligase), EC 6.5.1.3 (RNA ligase). Specific examples of ligases include bacterial ligases (such as Escherichia coli DNA ligase), Tth DNA ligase, Thermococcus species (strain 9°N) DNA ligase (9°N TM DNA ligase, New England Biolabs), Taq DNA ligase, Ampligase TM (Epicentre Biotechnologies) and bacteriophage ligases (such as T3 DNA ligase, T4 DNA ligase and T7 DNA ligase) and their mutants. In some embodiments, the ligase is T4 RNA ligase. In some embodiments, the ligase is splintR ligase. In some embodiments, the ligase is a single-stranded DNA ligase. In some embodiments, the ligase is T4 DNA ligase. In some embodiments, the ligase is a ligase with DNA splinting DNA ligase activity. In some embodiments, the ligase is a ligase with RNA splinting DNA ligase activity.

[0204] In some embodiments, the connection herein is directly connected. In some embodiments, the connection herein is indirectly connected. "Direct connection" means that the ends of polynucleotides are hybridized in close proximity to each other to form a substrate for a ligase, thereby causing them to be connected to each other (intramolecular connection). Alternatively, "indirectly" means that the ends of polynucleotides are hybridized non-adjacently to each other, for example, separated by one or more insertion nucleotides or "gaps". In some embodiments, the ends are not directly connected to each other, but by one or more insertions (so-called "gaps" or "filling gaps" (oligo) nucleotides) of intermediates or by extending the 3' end of the probe to "fill" the "gap" corresponding to the insertion nucleotide and occur (intermolecular connection). In some cases, the gap of one or more nucleotides between the polynucleotide hybridization ends can be "filled" with one or more "gap" (oligo) nucleotides complementary to a splint, a circularizable probe or a probe group (e.g., a padlock probe) or a target nucleic acid. The gap can be a gap of 1 to 60 nucleotides or a gap of 1 to 40 nucleotides or a gap of 3 to 40 nucleotides. In a specific embodiment, the gap can be a gap of about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleotides, a gap of any integer (or integer range) of nucleotides between the values ​​shown. In some embodiments, the gap between the terminal regions can be filled by a gap oligonucleotide or by extending the 3' end of the polynucleotide. In some cases, the connection involves connecting the end of the probe to at least one gap (oligo) nucleotide so that the gap (oligo) nucleotide is incorporated into the resulting polynucleotide. In some embodiments, gap filling is performed before the connection herein. In other embodiments, the connection herein does not require gap filling.

[0205] A variety of different methods can be used to adjacently connect nucleic acid molecules, including (but not limited to) "sticky end" and "blunt end" ligation. In addition, single-stranded ligation can be used to perform adjacent ligation on single-stranded nucleic acid molecules.

[0206] (c) Primer extension and amplification

[0207] In some embodiments, the product is a primer extension product of an analyte, a labeling agent, a probe or probe set bound to an analyte (e.g., a circularizable probe or probe set (e.g., a padlock probe) that binds to genomic DNA, mRNA, or cDNA), or a probe or probe set bound to a labeling agent (e.g., a circularizable probe or probe set (e.g., a padlock probe) that binds to one or more reporter oligonucleotides from the same or different labeling agent).

[0208] Primer is generally a single-stranded nucleic acid sequence with 3' end that can be used as a nucleic acid polymerase substrate in a nucleic acid extension reaction. RNA primers are formed by RNA nucleotides and are used for RNA synthesis, while DNA primers are formed by DNA nucleotides and are used for DNA synthesis. Primers may also include RNA nucleotides and DNA nucleotides (e.g., in a random or designed pattern), other natural or synthetic nucleotides described herein that may have additional functions. When two nucleic acid sequences are hybridized by overlapping complementary nucleic acid sequences (e.g., 3' ends) at their respective ends, a primer extension reaction may occur. Such a connection may be followed by nucleic acid extension (e.g., enzymatic extension) of one or both ends using another nucleic acid sequence as an extension template. Enzymatic extension may be performed by enzymes including, but not limited to, polymerases and / or reverse transcriptases.

[0209] In some embodiments, the product of endogenous analyte and / or labeling agent is an amplification product of one or more polynucleotides (e.g., circular probe or circularizable probe or probe group). In some embodiments, amplification is achieved by performing rolling circle amplification (RCA). In other embodiments, a primer hybridized with a circular probe or circularized probe is added, and is used for amplification as is. In some embodiments, RCA includes linear RCA, branched RCA, dendritic RCA or any combination thereof.

[0210] In some embodiments, the RCA template may include a target analyte or a portion thereof, wherein the target analyte is a nucleic acid, or the RCA template may be provided or generated as a substitute or marker for an analyte. In some embodiments, a detection system based on RCA is used to detect different analytes in situ in one or more cells, for example, wherein a signal is provided by generating an RCA product from a circular RCA template provided or generated in the assay, and the RCA product is detected to detect the corresponding analyte. Therefore, the RCA product can be regarded as a reporter molecule, which is detected to detect the target analyte. However, the RCA template may also be regarded as a reporter molecule of the target analyte; the RCA product is generated based on the RCA template, and comprises a complementary copy of the RCA template. The RCA template determines the detected signal, and therefore indicates the target analyte. As will be described in more detail below, the RCA template may be a probe or a part or component of a probe, or may be generated by a probe, or may be a component of a detection assay (e.g., a reagent in a detection assay), which is used as a reporter molecule, or a part of a reporter molecule, or a signal generating system for the assay. Thus, the RCA template used to generate the RCP may be a circular (e.g., circularized) reporter nucleic acid molecule, i.e. from any RCA-based detection assay that uses or generates a circular nucleic acid molecule as a reporter molecule for the assay. Because the RCA template generates the RCP reporter molecule, it may be considered as part of the reporter system of the assay.

[0211] In some embodiments, the products herein include molecules or complexes produced in a series of reactions in any suitable combination, such as hybridization, connection, extension, replication, transcription / reverse transcription and / or amplification (e.g., rolling circle amplification). For example, the product of the target sequence of the nucleic acid probe disclosed herein can be a hybridization complex formed by the cell nucleic acid in the sample and the exogenously added nucleic acid probe. The exogenously added nucleic acid probe may include an overhang that is not hybridized with the cell nucleic acid but hybridized with another probe (e.g., a nucleic acid probe described herein). The exogenously added nucleic acid probe may be optionally connected to a cell nucleic acid molecule or another exogenous nucleic acid molecule. In other examples, the product of the target sequence of the nucleic acid probe disclosed herein can be a circularizable probe or RCP of a probe set, which is hybridized with a cell nucleic acid molecule (e.g., genomic DNA or mRNA) or a product thereof (e.g., a transcript such as cDNA, a DNA template connection product of two probes, or a connection product of two DNA probes generated in an RNA template-dependent manner (e.g., RNA template connection)). In some embodiments, the detectable probe may include an overhang that is not hybridized with the RCA product but hybridized with another probe (e.g., a fluorescently labeled probe).

[0212] C. Target sequence

[0213] The target sequence of the probes disclosed herein can be contained in any analyte disclosed herein, including an endogenous analyte (eg, a viral or cellular nucleic acid), a labeling agent, or a product of an endogenous analyte and / or a labeling agent.

[0214] In some aspects, one or more of the target sequences comprises one or more barcodes, such as at least two, three, four, five, six, seven, eight, nine, ten or more barcodes. The barcodes can spatially resolve molecular components found in biological samples, such as molecular components within a cell or tissue sample. The barcodes can be attached to an analyte or another part or structure in a reversible or irreversible manner. The barcodes can be added to fragments of, for example, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) samples before or during sample sequencing. The barcodes can allow identification and / or quantification of individual sequencing reads (e.g., the barcodes can be or can include unique molecular identifiers or "UMIs"). In some aspects, the barcode comprises about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides.

[0215] In some embodiments, the barcode includes two or more sub-barcodes that act together as a single barcode. For example, a polynucleotide barcode can include two or more polynucleotide sequences (e.g., sub-barcodes) separated by one or more non-barcode sequences. In some embodiments, one or more barcodes can also provide a platform for targeting functions, such as oligonucleotides, oligonucleotide-antibody conjugates, oligonucleotide-streptavidin conjugates, modified oligonucleotides, affinity purification, detectable moieties, enzymes, enzymes for detection assays or other functions, and / or enzymes for detection and identification of polynucleotides. In any of the foregoing embodiments, the methods provided herein can include analyzing barcodes by sequential hybridization and detection with multiple labeled probes (e.g., detection oligos).

[0216] In some embodiments, in a barcode sequencing method, a barcode sequence is detected to identify other molecules that contain a nucleic acid molecule (DNA or RNA) that is longer than the barcode sequence itself, as opposed to directly sequencing a longer nucleic acid molecule. In some embodiments, given a sequenced read of N bases, an N-mer barcode sequence contains 4 N The complexity of barcode sequencing is greatly increased, and molecular identification may require much shorter sequencing reads compared to non-barcode sequencing methods such as direct sequencing. For example, 1024 molecular species can be identified using a 5-nucleotide barcode sequence (4 5 =1024), and an 8-nucleotide barcode can be used to identify up to 65,536 molecular species, a number greater than the total number of different genes in the human genome. In some embodiments, the barcode sequence contained in the probe or RCP is detected, rather than the endogenous sequence, which can be an effective read in terms of information for each sequencing cycle. Because the barcode sequences are predetermined, they can also be designed to characterize error detection and correction mechanisms, see, for example, U.S. Patent Publication Nos. 20190055594 and US20210164039A1, all of which are incorporated herein by reference in their entirety.

[0217] III. General Cell-Based Analysis Methods

[0218] A. Labeling agents for multiple samples and multiplexed analytical approaches

[0219] The present disclosure provides methods and systems for multiple analysis and otherwise increasing the throughput of samples for analysis.For example, a single or integrated processing workflow can allow processing, identification and / or analysis of more or multiple analytes, more or multiple types of analytes, and / or more or multiple types of analyte characterization.For example, in the methods and systems described herein, one or more labeling agents that can be combined or coupled with one or more cells or cell features (for example, molecules on and / or in cells) can be used to characterize cells (including cell features and / or cells and / or analytes other than cell features). In some cases, cell features include cell surface features. Exemplary cell surface features are described in Section II-B. In some embodiments, cell features may include intracellular analytes, such as proteins, protein modifications (for example, phosphorylation states or other post-translational modifications), nucleoproteins, nuclear membrane proteins, or any combination thereof. Labeling agents may include, but are not limited to, proteins, peptides, antibodies (or epitope-binding fragments thereof), lipophilic moieties (such as cholesterol), cell surface receptor binding molecules, receptor ligands, small molecules, bispecific antibodies, bispecific T cell engagers, T cell receptor engagers, B cell receptor engagers, antibody prodrugs, aptamers, monomers, affimers, darpins, and protein scaffolds, or any combination thereof.

[0220] In a specific example, a library of potential cell feature markers can be provided, wherein the corresponding cell feature markers are associated with nucleic acid reporters so that different reporter oligonucleotide sequences are associated with each marker that can bind to a specific cell feature. In other aspects, different members of the library can be characterized by the presence of different oligonucleotide sequence tags. For example, an antibody that can bind to a first protein may have a first reporter oligonucleotide sequence associated therewith, while an antibody that can bind to a second protein may have a different reporter oligonucleotide sequence associated therewith. The presence of a specific oligonucleotide sequence can indicate the presence of a specific antibody or a cell feature that can be recognized or bound by a specific antibody.

[0221] The labeling agent that can be combined with one or more cells or otherwise coupled can be used to characterize cells as belonging to a specific cell group. For example, a labeling agent can be used to mark a cell sample or a group of cells. In this way, a group of cells can be labeled to be different from another group of cells. In an example, a first group of cells can be derived from a first sample, and a second group of cells can be derived from a second sample. Labeling agents can allow the first group and the second group to have different labeling agents (or reporter oligonucleotides associated with labeling agents). This can, for example, promote multiple analysis, in which the cells of the first group and the cells of the second group can be labeled respectively, and then merged together for downstream analysis. When disassembling to associate analyte with different sample sources, the downstream detection of labeling can indicate that the analyte belongs to a specific group.

[0222] For example, the reporter oligonucleotide can be attached to an antibody or an epitope binding fragment thereof, and labeling the cell can include subjecting the antibody-attached barcode molecule or the epitope binding fragment-attached barcode molecule to conditions suitable for binding the antibody to a molecule present on the surface of the cell. The binding affinity between the antibody or its epitope binding fragment and the molecule present on the surface can be within a desired range to ensure that the antibody or its epitope binding fragment remains bound to the molecule. For example, the binding affinity can be within a desired range to ensure that the antibody or its epitope binding fragment remains bound to the molecule during various sample processing steps (e.g., nucleic acid amplification and / or extension). The dissociation constant (K) between the antibody or its epitope binding fragment and the molecule to which it binds can be determined. d ) can be less than about 100μM, 90μM, 80μM, 70μM, 60μM, 50μM, 40μM, 30μM, 20μM, 10μM, 9μM, 8μM, 7μM, 6μM, 5μM, 4μM, 3μM, 2μM, 1μM, 900nM, 800nM, 700nM, 600nM, 500nM, 400nM, 300nM, 200nM, 100nM, 90nM, 80nM, 70nM, 60nM, 50nM, 40nM, 30nM The dissociation constant may be less than about 10 μM.

[0223] In another example, the reporter oligonucleotide can be coupled to a cell penetrating peptide (CPP), and labeling cells can include delivering the CPP-coupled reporter oligonucleotide to an analyte carrier. Labeling the analyte carrier can include delivering the CPP-conjugated oligonucleotide to the cell via a cell penetrating peptide. The CPP that can be used in the methods provided herein can include at least one non-functional cysteine ​​residue, which can be free or derived, for forming a disulfide bond with an oligonucleotide that has been modified for such bonding. Non-limiting examples of CPPs that can be used in embodiments herein include permeabilin, transporter, plsl, TAT (48-60), pVEC, MTS, and MAP. Cell penetrating peptides that can be used in the methods provided herein can have the ability to induce cell penetration of at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of cells in a cell population. The CPP can be an arginine-rich peptide transporter. The CPP can be a permeabilin or a Tat peptide. In another example, the reporter oligonucleotide can be coupled to a fluorophore or a dye, and labeling the cell can include subjecting the fluorophore-linked barcode molecule to conditions suitable for binding the fluorophore to the surface of the cell. In some cases, the fluorophore can interact strongly with the lipid bilayer, and labeling the cell can include subjecting the fluorophore-linked barcode molecule to conditions such that the fluorophore binds to or inserts into the cell membrane. In some cases, the fluorophore is a water-soluble organic fluorophore. In some cases, the fluorophore is Alexa 532 maleimide, tetramethylrhodamine-5-maleimide (TMR maleimide), BODIPY-TMR maleimide, sulfo-Cy3 maleimide, Alexa 546 carboxylic acid / succinimidyl ester, Atto 550 maleimide, Cy3 carboxylic acid / succinimidyl ester, Cy3B carboxylic acid / succinimidyl ester, Atto 565 biotin, sulforhodamine B, Alexa 594 maleimide, TexasRed maleimide, Alexa 633 maleimide, Abberior STAR 635P azide, Atto 647N maleimide, Atto 647 SE, or sulfo-Cy5 maleimide. See, e.g., Hughes LD et al., PLoS One. 2014 Feb 4;9(2):e87649, which is hereby incorporated by reference in its entirety for all purposes, for a description of organic fluorophores.

[0224] The reporter oligonucleotide can be a part of a nucleic acid molecule comprising any number of functional sequences. In other cases, for example, in order to promote sample multiplex analysis, a specific cell feature can have a first multiple labeling agent (for example, an antibody or a lipophilic part) coupled to the first reporter oligonucleotide and a second multiple labeling agent coupled to the second reporter oligonucleotide. For example, a first group of multiple labeling agents and a second group of multiple labeling agents can interact with different cells, cell colonies or samples, thereby allowing specific reporter oligonucleotides to indicate specific cell colonies (or cells or samples) and cell features. In this way, different samples or groups can be processed independently and then combined together for combined analysis.

[0225] As described elsewhere herein, the labeling agent library can be associated with a specific cell feature, and can also be used to identify that an analyte is derived from a specific cell population or sample. A cell population can be incubated with multiple libraries so that one or more cells include a variety of labeling agents. For example, a cell can include a lipophilic labeling agent and an antibody coupled thereto. The lipophilic labeling agent can indicate that the cell is a member of a specific cell sample, and the antibody can indicate that the cell contains a specific analyte. In this way, reporting oligonucleotides and labeling agents can allow multiple analytes to be multiplexed.

[0226] In some cases, the reporter oligonucleotides may comprise nucleic acid barcode sequences that allow identification of the labeling agent to which the reporter oligonucleotide is coupled. The use of oligonucleotides as reporters can provide the advantages of being able to generate significant diversity in sequence while also being easily attached to most biomolecules (e.g., antibodies) and being detectable.

[0227] In some cases, the labeling agent can include a reporter oligonucleotide and a label. The label can be a fluorophore, a radioisotope, a molecule capable of colorimetric reaction, a magnetic particle, or any other suitable molecule or compound capable of detection. The label can be directly or indirectly conjugated to a labeling agent (or a reporter oligonucleotide) (for example, the label can be conjugated to a molecule that can be combined with a labeling agent or a reporter oligonucleotide). In some cases, the label is conjugated to an oligonucleotide complementary to the sequence of the reporter oligonucleotide, and the oligonucleotide can be allowed to hybridize with the reporter oligonucleotide.

[0228] Figure 2A labeling agent comprising an exemplary functional portion 201 and a reporter oligonucleotide 202 attached thereto is described. The functional portion can react and / or bind to one or more molecules on and / or in a cell. The functional portion can be attached (directly, such as covalently attached, or indirectly, such as through a linker) to a reporter oligonucleotide. The reporter oligonucleotide 202 may include a barcode region comprising one or more barcode sequences, including a sample-specific barcode sequence 203 that identifies the sample source of a cell labeled with the labeling agent. The reporter oligonucleotide 202 may also include one or more other sequences 204, such as one or more other barcode sequences and / or functional sequences that can be used for subsequent processing, such as an aptamer sequence, a unique molecular identifier (UMI) sequence, and / or a primer or primer binding sequence.

[0229] refer to Figure 2 In the above figure, the functional part can be covalently attached to the reporter oligonucleotide. As shown, one or more other sequences 204 can be between the sample-specific barcode sequence 203 and the functional part 201. However, these components can be attached to each other in any suitable configuration. For example, the sample-specific barcode sequence can be between one or more other sequences and the functional part, or between any two other sequences in the reporter oligonucleotide. Alternatively, the sample-specific barcode sequence and one or more other sequences can be attached to the functional part separately.

[0230] refer to Figure 2 In the figure below, the reporter oligonucleotide also includes an aptamer sequence 205 complementary to the aptamer sequence 206 attached to the functional portion 201. The reporter oligonucleotide is attached to the functional portion by nucleic acid hybridization between the aptamer sequences 205 and 206. These components can be attached to each other in any suitable configuration. For example, the sample-specific barcode sequence can be between the aptamer sequence 205 and one or more other sequences, or between any two other sequences in the reporter oligonucleotide.

[0231] The functional part can be any part on the first molecule that can interact with another functional part on the same molecule and / or the second molecule. The interaction between the functional parts can include physical interactions (e.g., direct or indirect binding) and / or chemical or enzymatic reactions, such as reactions forming covalent or ionic bonds between the first molecule and the second molecule. In some cases, the functional part is a binding part. In some cases, the binding part is a protein or polypeptide (e.g., an antigen or an expected antigen) attached to the reporting oligonucleotide. In some cases, the sample-specific barcode sequence identifies the sample from which the target (e.g., cell) is derived. In some cases, the binding part is a lipophilic part (e.g., cholesterol) attached to the reporting oligonucleotide, wherein the lipophilic part is selected so that the binding part is integrated into the membrane (e.g., cell membrane) of the cell or nucleus. The reporting oligonucleotide can optionally include a target-specific barcode sequence identifying the binding part and can be used for multiple analysis described elsewhere herein, and the binding part is used to label cells (e.g., cell colonies, cell samples, etc.) in some cases. In some cases, the sample-specific barcode sequence identifies the sample from which the target (e.g., cell) is derived. In some cases, the binding moiety is an antibody (or an epitope-binding fragment thereof) attached to a reporter oligonucleotide.

[0232] One or more markers for targeting cells can be used to produce cells comprising multiple markers. In some cases, multiple markers are the same. In some cases, multiple markers are different. For example, a cell can include a first marker (for example, including an antibody) and a second marker (for example, including a lipophilic molecule) coupled to a cell surface. Exemplary lipophilic molecules are disclosed in Section III-Ci. As another example, a cell can include a first class marker including a first lipophilic molecule coupled to a cell surface and a second marker including a second lipophilic molecule, wherein the first lipophilic molecule and the second lipophilic molecule are different. In some cases, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different markers are used. In some cases, multiple markers including different lipophilic molecules and / or antibodies can be used to label cells in a sample, and various combinations of lipophilic molecules and / or antibodies can be used as functional parts in the marker to perform multiple analysis samples. Exemplary cell labeling methods and compositions that can be used in the present disclosure include those disclosed in WO 2021046475 A1, the contents of which are incorporated herein by reference in their entirety.

[0233] Target-specific barcodes and binding moiety barcodes are barcodes that associate with a binding moiety and / or a binding moiety's target or otherwise identify a binding moiety and / or a binding moiety's target. In some embodiments, the binding moiety is identified by identifying the associated binding moiety barcode (e.g., a target-specific barcode) of the binding moiety, and the target bound to the binding moiety can also be identified. The binding moiety barcode can be a nucleic acid sequence of a given length and / or a sequence associated with the binding moiety. The binding moiety barcode can generally include any of the aspects of the barcode described herein. For example, a marker having specificity for one type of analyte can have a first barcode domain coupled thereto (e.g., it includes a first analyte binding moiety barcode), while a marker having specificity for different analytes can have different barcode domains coupled thereto (e.g., it includes a second barcode analyte binding moiety barcode). In some aspects, such a barcode domain can include a binding moiety barcode (e.g., a target-specific barcode), which allows identification of the binding moiety coupled to the marker. The choice of barcode domains can allow for significant diversity in terms of sequence while also being easily attached to most binding moieties (e.g., antibodies) and easily detectable. In some embodiments, the labeling agent can include a binding moiety having a target-specific barcode domain attached thereto. For example, the labeling agent can include a first binding moiety (e.g., an antibody that binds to a target, such as a first cell surface feature), the first binding moiety having a target-specific barcode domain associated therewith, the target-specific barcode domain comprising a first binding moiety barcode.

[0234] Sample-specific barcodes are barcodes associated with samples from which labeled cells are derived, or barcodes that identify the sample. In some embodiments, by identifying sample-specific barcodes, samples from which labeled cells are derived are identified. Sample-specific barcodes can be nucleic acid sequences of a given length and / or sequences associated with samples. Sample-specific barcodes can generally include any of the aspects of barcodes described herein. For example, a marker having specificity for a type of sample can have a first barcode domain coupled thereto (e.g., it includes a first sample-specific barcode), while a marker having specificity for different samples can have different barcode domains coupled thereto (e.g., it includes a second sample-specific barcode). In some aspects, this barcode domain can include a sample-specific barcode that allows identification of samples from which cells are derived. The selection of barcode domains can allow for significant diversity in terms of sequence, while also being easily attached to most binding moieties (e.g., antibodies) and being easy to detect.

[0235] In some embodiments, the target-specific barcode domains and / or sample-specific barcode domains can be directly coupled to a binding moiety, or they can be attached to a bead, a molecular lattice (e.g., a linear, globular, cross-linked or other polymer), or other framework to which a binding moiety is attached or otherwise associated, which allows multiple target-specific barcode domains and / or sample-specific barcode domains to be attached to a single binding moiety. Attachment (coupling) of the target-specific barcode domains and / or sample-specific barcode domains to a binding moiety can be achieved by any of a variety of direct or indirect, covalent or non-covalent associations or attachments. For example, in the case of a target-specific barcode domain and / or sample-specific barcode domain coupled to a binding moiety comprising an antibody or epitope-binding fragment, such target-specific barcode domain and / or sample-specific barcode domain can be conjugated using chemical conjugation techniques (e.g., Lightning-Tran available from Innova Biosciences). Antibody Labeling Kit) is covalently attached to a portion of the antibody or epitope-binding fragment.

[0236] In some embodiments, the target-specific barcode domain and / or the sample-specific barcode domain can be attached to the antibody or epitope binding fragment using a non-covalent attachment mechanism (e.g., using a biotinylated antibody and oligonucleotide or a reagent comprising one or more biotinylated linkers, coupled to the oligonucleotide with an avidin or streptavidin linker). Antibody and oligonucleotide biotinylation techniques can be used, and are described, for example, in Fang et al., Nucleic Acids Res. (2003), 31 (2): 708-715, the entire contents of which are incorporated herein by reference. Similarly, protein and peptide biotinylation techniques have been developed and can be used, and are described, for example, in U.S. Pat. No. 6,265,552, the entire contents of which are incorporated herein by reference. In addition, click reaction chemistry, such as methyl tetrazine-PEG5-NHS ester reaction, TCO-PEG4-NHS ester reaction, etc., can be used to couple the target-specific barcode domain and / or the sample-specific barcode domain to the binding moiety. The reactive moieties on the binding moiety can also include amines for targeting aldehydes, amines for targeting maleimides (e.g., free thiols), azides for targeting click chemistry compounds (e.g., alkynes), biotin for targeting streptavidin, phosphates for targeting EDC and then active esters (e.g., NH2). The reactive moiety on the binding moiety can be a chemical compound or group that binds to the reactive moiety on the analyte binding moiety. Exemplary strategies for conjugating the binding moiety to the target-specific barcode domain and / or sample-specific barcode domain include the use of commercial kits (e.g., Solulink, Thunder link), mild reduction of the hinge region and conjugation of maleimide labels, dye-promoted click chemistry reactions with labeled amides (e.g., copper-free), and periodate oxidation of sugar chains and amine conjugation. In the case where the binding moiety is an antibody, the antibody can be modified before or simultaneously with oligonucleotide conjugation.

[0237] In some embodiments, the barcode domain coupled to the labeling agent may include a modification that makes it non-extendable by a polymerase. In some embodiments, the barcode domain may be used as a template. The modification that blocks primer extension by a polymerase may be a carbon spacer or dideoxynucleotide of different lengths. In some embodiments, the modification may be an abasic site having an analog of apurine-free or pyrimidine-free structure, a base analog, or a phosphate backbone, such as an N-(2-aminoethyl)-glycine backbone connected by an amide bond, tetrahydrofuran, or 1',2'-dideoxyribose. The modification may also be a uracil base, 2'OMe-modified RNA, a C3-18 spacer (e.g., a structure having 3-18 consecutive carbon atoms, such as a C3 spacer), an ethylene glycol polymer spacer (e.g., a spacer 18 (hexaethylene glycol spacer), biotin, dideoxynucleotide triphosphates, ethylene glycol, amines, or phosphates.

[0238] In some embodiments, the barcode domain coupled to the binding moiety includes a cleavable domain. For example, after the labeling agent is bound to the target (e.g., a cell surface molecule), the barcode domain can be cut and collected according to the method as described herein for downstream analysis. In some embodiments, the cleavable domain of the barcode domain includes a U-excision element that allows the substance to be released from the bead. In some embodiments, the U-excision element can include a single-stranded DNA (ssDNA) sequence containing at least one uracil. The species can be attached to the bead by the ssDNA sequence. The species can be released by a combination of uracil-DNA glycosylase (e.g., to remove uracil) and an endonuclease (e.g., to induce ssDNA breakage). If the endonuclease produces a 5' phosphate group from the cutting, then additional enzyme treatment can be included in the downstream processing to eliminate the phosphate group, such as before connecting the additional element.

[0239] In some embodiments, the binding portion of the labeling agent includes one or more antibodies or epitope binding fragments thereof. Antibodies or epitope binding fragments including binding portions can be specifically bound to a target. In some embodiments, the target is a protein (e.g., a protein on the surface of a biological sample (e.g., a cell) or an intracellular protein). In some embodiments, multiple labeling agents comprising multiple binding portions bind to a variety of targets present in a biological sample. In some embodiments, a variety of targets include a single species of a target (e.g., a single species of a polypeptide). In some embodiments in which a variety of targets include a single species of a target, the binding portion of a variety of labeling agents is the same. In some embodiments in which a variety of targets include a single species of a target, the binding portion of a variety of labeling agents is different (e.g., a member of a variety of labeling agents can have two or more species of a binding portion, wherein each species in the two or more species of a binding portion binds a single species of a target, such as in different binding sites). In some embodiments, a variety of targets include a variety of different species of a target (e.g., a variety of different species of a polypeptide).

[0240] In some embodiments, a variety of different species of targets (e.g., polypeptides) from biological samples can then be associated with one or more physical properties of the biological sample. For example, a variety of different species of targets can be associated with the location of the target in the biological sample. Such information (e.g., proteome information when the binding moiety recognizes the polypeptide) can be used in combination with other spatial information (e.g., genetic information from the biological sample, such as DNA sequence information, transcriptome information (e.g., transcript sequence) or both). For example, the cell surface protein of a cell can be associated with one or more physical properties of the cell (e.g., the shape, size, activity or type of the cell). The one or more physical properties can be characterized by imaging the cell. The cell can be bound by a marker, the marker comprising a binding moiety that binds to the cell surface protein and a target-specific barcode that identifies the binding moiety, and the cell can be analyzed in situ. The distribution of a single cell or a cell population can be compared with the distribution from other cells (e.g., "normal" cells) to identify changes in the analyte, which can provide diagnostic related information. In some embodiments, these atlases can be used to diagnose a variety of diseases characterized by cell surface receptor mutations, such as cancer and other diseases.

[0241] B. Barcoding biological samples

[0242] In some embodiments, provided herein are methods and materials for attaching and / or introducing molecules (e.g., reporter oligonucleotide molecules) having barcodes (e.g., sample-specific barcodes) into biological samples (e.g., cells in a biological sample) for in situ analysis. In some embodiments, multiple molecules (e.g., multiple reporter oligonucleotide molecules) having multiple barcodes (e.g., multiple sample-specific and / or target-specific barcodes) are introduced into a biological sample (e.g., multiple cells introduced into a biological sample) for in situ analysis.

[0243] The cells in biological samples can be marked using covalent conjugation of labeling agents to cell surfaces or non-covalent interactions with cell membrane components. A non-exhaustive list of covalent labeling agents / cell surface interactions includes protein targeting, amine conjugation using NHS chemistry, cyanuric chloride, thiol conjugation by maleimide addition, and glycoprotein / glycolipid expressed on the cell surface by click chemistry targeting. Non-exhaustive examples of non-covalent interactions with cell membrane components include lipid-modified oligomers, biocompatible anchors (oil-PEG-NHS) of cell membranes, lipid-modified positive neutral polymers, and antibodies to membrane proteins. In some embodiments, physical forces are used to promote attachment to binding moieties and molecules (e.g., reporter oligonucleotide molecules) with barcodes to be attached to or introduced into biological samples (e.g., cells present in biological samples).

[0244] In some embodiments, the binding moiety can directly bind to one or more molecules in the cell. In some embodiments, the binding moiety can indirectly bind to one or more molecules in and / or on the cell. In some embodiments, the antibody or epitope binding fragment comprising the binding moiety binds to the cell surface or the interior of the cell.

[0245] In some embodiments, a biological sample (e.g., a cell in a biological sample) can be labeled with a labeling agent, wherein the labeling agent includes a binding moiety (e.g., a polynucleotide), a molecule (e.g., a reporter oligonucleotide) with a barcode (e.g., a sample-specific barcode). In some embodiments, the labeling agent comprises a barcode (e.g., a sample-specific barcode or a target-specific barcode). The barcode of the barcoded labeling agent can be any type of barcode described herein. In some embodiments, the barcode of the barcoded labeling agent is a sample-specific barcode corresponding to the sample from which the labeled cell is derived. In some embodiments, the barcode of the barcoded labeling agent is a sample-specific barcode corresponding to a sample containing cells before labeling. In some embodiments, the sample-specific barcode sequence is in a reporter oligonucleotide. In some embodiments, the barcode of the barcoded labeling agent is a target-specific barcode. In some embodiments, the barcode of the barcoded labeling agent identifies associated molecules, wherein each target-specific barcode is associated with a specific molecule. In some embodiments, the target-specific barcode corresponds to a binding moiety or a target. In some embodiments, the target-specific barcode sequence is in a reporter oligonucleotide. In some embodiments, the sample-specific barcode and the target-specific barcode can be the same or different. In some embodiments, the sample-specific barcode and the target-specific barcode are in the same oligonucleotide, or in different oligonucleotides that are conjugated to the binding moiety, respectively. In some embodiments, the target-specific barcode sequence and the sample-specific barcode sequence overlap at least partially in sequence.

[0246] In some embodiments, the barcode of the barcoded labeling agent corresponds to a cell feature. In some embodiments, the cell feature includes a cell surface feature. Analyte can include but is not limited to protein, receptor, antigen, surface protein, transmembrane protein, differentiation protein cluster, protein channel, protein pump, carrier protein, phospholipid, glycoprotein, glycolipid, cell-cell interaction protein complex, antigen presentation complex, major histocompatibility complex, engineered T cell receptor, T cell receptor, B cell receptor, chimeric antigen receptor, gap connection, adhesion connection or any combination thereof. In some embodiments, the cell feature may include intracellular analyte, such as protein, protein modification (e.g., phosphorylation state or other post-translational modifications), nucleoprotein, nuclear membrane protein or any combination thereof.

[0247] In some embodiments, one or more molecules are applied to a sample. In some embodiments, a nucleic acid molecule (e.g., a reporter oligonucleotide molecule) comprising a barcode is covalently attached to a binding portion. In some embodiments, a nucleic acid molecule (e.g., a reporter oligonucleotide molecule) comprising a barcode is non-covalently attached to a binding portion. Non-limiting examples of non-covalent attachment include hybridization of a nucleic acid molecule (e.g., a reporter oligonucleotide molecule) comprising a barcode with a nucleic acid molecule on a binding portion. In some embodiments, the nucleic acid molecule attached to the binding portion comprising a barcode also includes one or more additional domains. Such additional domains include, but are not limited to, a PCR handle, a sequencing initiation site, a domain for hybridizing with another nucleic acid molecule, and a combination thereof.

[0248] In some embodiments, the binding moiety is attached to the cell surface. When the binding moiety includes a barcode (for example, a reporter oligonucleotide molecule including a sample-specific barcode), the barcode is also attached to the cell surface. In some embodiments, the binding moiety is covalently attached to the cell surface to facilitate the introduction of the barcode sequence. In some embodiments, the binding moiety is non-covalently attached to the cell surface to facilitate the introduction of the barcode sequence. In some embodiments, the binding moiety can be combined with one or more molecules on the cell surface by a joint. In some embodiments, the joint includes one or more of N-hydroxysuccinimide (NHS) joints, bifunctional NHS joints, azides, alkynes, ethylene glycol chitosan, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly (ethylene glycol) (DSPE-PEG) and succinimidyl-3-(2-pyridyl dithio) propionate (SPDP). Exemplary joints include those described in US 2021 / 0332425, which are incorporated herein by reference in their entirety for all purposes.

[0249] In some embodiments, before being labeled with a labeling agent, the biological sample is dissociated into a single cell with any method described herein. In some embodiments, before being dissociated into a single cell, the biological sample is labeled. Once one or more cells in the biological sample are labeled with a labeling agent, cells from multiple biological samples are combined for in situ analysis. In some embodiments, this in situ analysis includes fixing the labeled cells of multiple biological samples (or a subset of biological samples), and analyzing the analyte present in those cells with single cell resolution. Any of the various methods of analyzing the analyte present in the cell with single cell resolution can be used. Cells can be permeabilized as needed (for example, using a permeabilizing agent and method as described herein), reagents (for example, reverse transcriptase, polymerase, nucleotides, etc.) for analyte analysis can be provided to cells, and analytes can be measured in situ.

[0250] In any embodiment herein, the method for cell analysis includes (a) providing a cell colony comprising labeled cells, and (b) depositing the labeled cells on a substrate. In any embodiment herein, before, during and / or after the providing step in step (a) or during the deposition in step (b), the labeled cells are not in a partition. In some embodiments, throughout the providing step, fixing step, contacting step and / or detecting step, the labeled cells are not separated in a partition (e.g., an emulsion droplet or micropore). In some embodiments, a partition is an emulsion droplet or micropore. In any embodiment herein, before and / or during the deposition of step (b), the labeled cells are not lysed or homogenized. In some embodiments, after being labeled with a labeling agent, the cells are not lysed or homogenized. In some embodiments, the labeled cells are not lysed or homogenized throughout the providing step, fixing step, contacting step and / or detecting step, although the labeled cells can be permeabilized, fixed, cross-linked / de-cross-linked, embedded in a matrix, removed and / or otherwise processed. In some embodiments, the nucleic acid labeling the cell is not released from the cell between providing step (a) and depositing in step (b).In some embodiments, the nucleic acid is a reporter oligonucleotide or a cellular DNA or RNA molecule of the cell.

[0251] In any embodiment herein, between providing step (a) and the deposition in step (b), the nucleic acid of the labeled cell is not extended by a polymerase and / or reverse transcribed, not connected to another nucleic acid, not amplified and / or sequenced. In any embodiment herein, wherein between providing step (a) and the deposition in step (b), the nucleic acid of the labeled cell is not released from the cell.

[0252] C. Introducing the binding moiety to the cell surface

[0253] Non-limiting examples of binding moieties and systems attached to the surface of cells (e.g., thereby introducing the binding moiety and any barcode attached thereto to the exterior of the cell) that can be used according to the materials and methods provided herein to analyze one or more analytes in a biological sample include lipophilic moieties conjugated to oligonucleotides, polymers conjugated to positive or neutral oligomers, antibody-conjugated oligonucleotides, streptavidin-conjugated oligonucleotides, dye-labeled oligonucleotides, click chemistry, receptor-ligand systems, covalent binding systems via amine or thiol functional groups, azide-based systems, lectin-based systems, and combinations thereof.

[0254] i. Labeling agents comprising a lipophilic moiety

[0255] In some embodiments, the binding moiety in the labeling agent may comprise a lipid moiety, such as a lipid anchor that is independent of the antigen and universally inserts into the cell membrane or nuclear membrane regardless of the cell or sample type. In some embodiments, labeling agents comprising such lipid-based binding moieties can be used to barcode samples in a sample-agnostic manner. In some embodiments, the binding moiety comprises cholesterol or a derivative thereof.

[0256] In some embodiments, a molecule (e.g., a reporter oligonucleotide molecule) with a barcode (e.g., a sample-specific barcode) is coupled to a lipophilic molecule. In some embodiments, a lipophilic molecule can deliver a molecule to a cell membrane or a nuclear membrane. In some embodiments, a molecule (e.g., a reporter oligonucleotide molecule) with a barcode (e.g., a sample-specific barcode) coupled to a lipophilic molecule can be associated with a lipid membrane such as a cell membrane and a nuclear membrane and / or inserted therein. In some cases, insertion can be reversible. In some cases, the combination between a lipophilic molecule and a cell can cause the cell to retain a lipophilic molecule (e.g., and an associated component, such as a reporter oligonucleotide and a barcode sequence) in subsequent treatments (e.g., cell permeabilization, amplification, merging, etc.). In some embodiments, a molecule (e.g., a reporter oligonucleotide molecule) with a barcode (e.g., a sample-specific barcode) coupled to a lipophilic molecule can enter the intracellular space and / or the nucleus. By delivering the nucleic acid barcode molecule to the cell membrane or nuclear membrane by the lipophilic molecule, the sample-specific nucleic acid barcode coupled to the lipophilic molecule can be used to label the cell. The lipophilic molecule can be associated with a lipid membrane (such as a cell membrane and a nuclear membrane) and / or inserted into the lipid membrane. In some cases, the insertion can be reversible. In some cases, the association between the lipophilic molecule and the cell can be such that the cell retains the lipophilic molecule (e.g., and its combined components, such as nucleic acid barcode molecules) during subsequent processing (e.g., cell permeabilization, amplification, merging, etc.).

[0257] Non-limiting examples of lipophilic molecules that can be used in the embodiments described herein include sterol lipids, such as cholesterol, tocopherol, sterol, palmitic acid, tetracosanoic acid and their derivatives. In some embodiments, the lipophilic molecule is a neutral lipid conjugated to a hydrophobic portion (e.g., cholesterol, squalene or fatty acid) (see Raouane et al. Bioconjugate Chem., 23 (6): 1091-1104 (2012), the contents of which are incorporated herein by reference in their entirety). In some embodiments, a molecule (e.g., a nucleic acid molecule) with a barcode (e.g., a sample-specific barcode) can be attached to the lipophilic portion by a joint, such as a tetraethylene glycol (TEG) joint. Other exemplary joints include, but are not limited to, amino joint C6, amino joint C12, spacer C3, spacer C6, spacer C12, spacer 9 and spacer 18. In some embodiments, molecules (e.g., nucleic acid molecules) with barcodes (e.g., sample-specific barcodes) are indirectly coupled to lipophilic molecules (e.g., by hybridization or ligand-ligand interactions, such as biotin-streptavidin). Other lipophilic molecules that can be used according to the methods provided herein include amphiphilic molecules in which the head group (e.g., charge, aliphatic content, and / or aromatic content) and / or fatty acid chain length (e.g., C12, C14, C16, or C18) can vary. For example, fatty acid side chains (e.g., C12, C14, C16, or C18) can be coupled to glycerol or glycerol derivatives (e.g., 3-tert-butyldiphenylsilylglycerol), which can also include, for example, cationic head groups. In some embodiments, molecules (e.g., reporter oligonucleotide molecules) with barcodes (e.g., sample-specific barcodes) disclosed herein can then be coupled (directly or indirectly) to these amphiphilic molecules. In some embodiments, a molecule (e.g., a reporter oligonucleotide molecule) having a barcode (e.g., a sample-specific barcode) coupled to an amphiphilic molecule can be associated with and / or inserted into a membrane (e.g., a cell, a cell bead, or a nuclear membrane). In some cases, an amphiphilic or lipophilic moiety can pass through the cell membrane and provide a molecule (e.g., a reporter oligonucleotide molecule) having a barcode (e.g., a sample-specific barcode) to the interior region of a cell and / or a cell bead.

[0258] In some embodiments, where a molecule (e.g., a reporter oligonucleotide molecule) has an amino group within the molecule, a molecule (e.g., a reporter oligonucleotide molecule) having a barcode (e.g., a sample-specific barcode) and an amino group can be coupled to an amine-reactive lipophilic molecule. For example, a molecule (e.g., a reporter oligonucleotide molecule) having a barcode and an amino group can be conjugated to DSPE-PEG (2000)-cyanuric chloride (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[cyanuric acid (polyethylene glycol)-2000]).

[0259] In some embodiments, binding moieties can be attached to the cell surface by a combination of lipophilic attachment and covalent attachment. For example, attached to lipids to target oligonucleotides to cell membranes include amine groups, which can be covalently linked to cell surface proteins by any number of chemical methods described herein. In these embodiments, lipids can increase the surface concentration of oligonucleotides, and covalent reactions can be promoted. In some cases, lipophilic molecules can include labels, such as optical labels. Such labels can, for example, enable detection of the part associated with the lipophilic molecule. For example, the lipophilic molecule can include a fluorescent moiety. The fluorescent moiety can allow optical detection of the lipophilic molecule and the part associated with it.

[0260] ii. Positive or neutral oligomer conjugated polymer

[0261] In some embodiments, a molecule (e.g., a reporter oligonucleotide molecule) having a barcode (e.g., a sample-specific barcode) can be coupled to an ethylene glycol chitosan derivative. An ethylene glycol chitosan derivative (e.g., ethylene glycol chitosan-cholesterol) can be used as a hydrophobic anchor (see Wang et al. J. Mater. Chem. B., 30: 6165 (2015), the contents of which are incorporated herein by reference in their entirety). Non-limiting examples of chitosan derivatives that can be coupled to a molecule (e.g., a reporter oligonucleotide molecule) having a barcode (e.g., a sample-specific barcode) can be found in Cheung et al., Marine Drugs, 13 (8): 5156-5186 (2015), the entire document of which is incorporated herein by reference in its entirety.

[0262] iii. Antibody-based labeling agents

[0263] In some embodiments, a molecule (e.g., a sample-specific barcode) with a barcode (e.g., a reporter oligonucleotide molecule) can be coupled to an antibody or its epitope binding fragment in a manner that promotes the attachment of a molecule (e.g., a sample-specific barcode) with a barcode (e.g., a reporter oligonucleotide molecule) to a cell surface. In some embodiments, promoting attachment to a cell surface helps introduce a molecule (e.g., a reporter oligonucleotide molecule) with a barcode (e.g., a sample-specific barcode) into a cell. In some embodiments, a molecule (e.g., a reporter oligonucleotide molecule) with a barcode (e.g., a sample-specific barcode) can be coupled to an antibody for an antigen present on a cell surface. In some embodiments, a molecule (e.g., a reporter oligonucleotide molecule) with a barcode (e.g., a sample-specific barcode) can be coupled to an antibody for an antigen present on a plurality of cells (e.g., a plurality of cells in a biological sample) surface. In some embodiments, a molecule (e.g., a reporter oligonucleotide molecule) with a barcode (e.g., a sample-specific barcode) can be coupled to an antibody for an antigen present on all or substantially all cell surfaces present in a biological sample. Any of the exemplary methods described herein for attaching a molecule (eg, a reporter oligonucleotide molecule) having a barcode (eg, a sample-specific barcode) to another molecule (eg, a binding moiety) can be used.

[0264] In some embodiments, a reporter oligonucleotide comprising a barcode (e.g., a sample-specific barcode) can be coupled to an antibody or its epitope-binding fragment, which is coupled to an antigen commonly expressed on and / or in sample cells, such as cell surface markers CD298 and / or β2-microglobulin. In some embodiments, the antigen is commonly expressed on and / or in sample cells. In some embodiments, the antigen is expressed on and / or in at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of cells in the sample.

[0265] iv. Streptavidin-conjugated oligonucleotides

[0266] In some embodiments, molecules (e.g., reporter oligonucleotide molecules) with barcodes (e.g., sample-specific barcodes) can be attached to cell surfaces using biotin-streptavidin. In some embodiments, primary amines in the side chains of lysine residues of cell surface polypeptides are labeled with NHS-activated biotin reagents. For example, the N-terminus of a polypeptide can react with an NHS-activated biotin reagent to form a stable amide bond. In some embodiments, the binding moiety includes a molecule (e.g., reporter oligonucleotide molecule) having a barcode (e.g., sample-specific barcode) conjugated to streptavidin. In some cases, streptavidin can be conjugated to a molecule (e.g., reporter oligonucleotide molecule) with a barcode using click chemistry (e.g., maleimide modification) as described herein. In some embodiments, cells containing NHS-activated biotin incorporated into the side chains of lysines of cell surface proteins form non-covalent bonds with streptavidin conjugated to a molecule (e.g., reporter oligonucleotide molecule) having a barcode (e.g., sample-specific barcode). In some embodiments, a molecule (eg, a reporter oligonucleotide molecule) having a barcode (eg, a sample-specific barcode) conjugated to streptavidin is itself part of the binding moiety.

[0267] v. Dye-labeled oligonucleotides

[0268] In some embodiments, the molecule (e.g., reporter oligonucleotide molecule) with a barcode (e.g., sample-specific barcode) is directly connected to a fluorescent tag. In some embodiments, the physical property (e.g., hydrophobic property) of the fluorescent tag can overcome the hydrophilic property of the molecule (e.g., reporter oligonucleotide molecule) with a barcode (e.g., sample-specific barcode). For example, in some embodiments, wherein the molecule is a reporter oligonucleotide molecule, a fluorescent tag (e.g., BODIPY, Cy3, Atto647N and rhodamine red C2) can be coupled to the 5' end of the reporter oligonucleotide molecule with a barcode (e.g., sample-specific barcode). In some embodiments, wherein the molecule is a reporter oligonucleotide molecule, any fluorescent tag with hydrophobic property can overcome the hydrophilic property of nucleic acid molecules and couple with the reporter oligonucleotide molecule with a barcode (e.g., sample-specific barcode). Non-limiting examples of hydrophobic fluorescent labels include BODIPY, Cy3, Atto647N and rhodamine red C2. In some embodiments, the fluorescent tag is a part of a binding moiety.

[0269] vi. Chemical conjugation including click chemistry conjugation

[0270] The method of producing surface indexed cells can generally include functionalizing the cell surface with a labeling agent, which can be (1) directly attached to the oligonucleotide sequence or (2) indirectly attached to the oligonucleotide sequence by using a joint. In other embodiments, the oligonucleotide can be directly attached to the cell surface by a covalent bond. In some embodiments, the primary amine in the side chain of the lysine residue of the cell surface polypeptide is labeled with a functionalized molecule containing an oligonucleotide, such as an NHS activated molecule. For example, the N-terminus of the polypeptide can react with an NHS activated reagent to form a stable amide bond. In some cases, the cell is contacted with an agent (e.g., a chemical agent) that produces a functional group on the cell surface (e.g., on a protein on the cell surface). In some embodiments, the component on the cell surface (e.g., surface protein or carbohydrate) includes or is modified to include -COOH (carboxylic acid), -NH2 (amine), -CH2Cl (chloromethyl), -CONH2 (amide), -CONHNH2 (hydrazide), -CHO (aldehyde), -OH (hydroxyl), -SH (thiol), -COC- (epoxy), click chemistry functional group or maleimide group. The oligonucleotide can then react with the functional group to attach the oligonucleotide to the cell surface. In some cases, the oligonucleotide comprises a functional group that reacts with a functional group on the cell surface. In some embodiments, the oligonucleotide to be conjugated to the cell surface comprises an amino modification, a thiol modification, or an acrylate (acrydite) modification. In some embodiments, the oligonucleotide and the cell surface are each functionalized with a click chemistry functional group (e.g., a component of copper-free click chemistry, such as SPAAC), as described in, for example, Akayama et al., ClickChemistry as a Tool for Cell Engineering and Drug Delivery Molecules 2019, 24 (1), 172. In some embodiments, a chemical crosslinker is used to couple the oligonucleotide to a component on the cell surface. In some cases, commercial conjugation kits and chemical reagents (e.g., Or protein-oligonucleotide conjugation kit (HyNic / 4FB conjugation-Solulink) to attach oligonucleotides to components on the cell surface.

[0271] In some embodiments, a molecule (e.g., a reporter oligonucleotide molecule) having a barcode (e.g., a sample-specific barcode) is coupled to a click chemistry moiety. In some embodiments, click chemistry includes modular, wide-ranging, high-yield reactions that produce only harmless byproducts (such as those that can be removed by non-chromatographic methods) and are stereospecific (but not necessarily enantioselective) (see, e.g., Angew. Chem. Int. Ed., 2001, 40 (11): 2004-2021, the contents of which are incorporated herein by reference in their entirety). In some cases, click chemistry can describe paired functional groups that can selectively react with each other under mild aqueous conditions.

[0272] An example of a click chemistry reaction is the Huisgen 1,3-dipolar cycloaddition of azide and alkyne, such as the copper-catalyzed reaction of azide and alkyne to form a 5-membered heteroatom ring 1,2,3-triazole. The reaction is also referred to as Cu(I)-catalyzed azide-alkyne cycloaddition reaction (CuAAC), Cu(I) click chemistry, or Cu+ click chemistry. Catalysts for click chemistry include, but are not limited to, Cu(I) salts or Cu(I) salts prepared in situ by reducing a Cu(II) reagent to a Cu(I) reagent with a reducing agent (Pharm Res. 2008, 25(10): 2216–2230, the entire document is incorporated herein by reference). Cu(II) reagents for click chemistry may include, but are not limited to, Cu(II)-(TBTA) complexes and Cu(II)(THPTA) complexes. TBTA, tris-[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, also known as tris-(benzyltriazolylmethyl)amine, can be a stabilizing ligand for Cu(I) salts. THPTA, tris-(hydroxypropyltriazolylmethyl)amine, is another example of a Cu(I) stabilizer. Other conditions can also be used to construct 1,2,3-triazole rings from azides and alkynes using copper-free click chemistry, such as strain-promoted azide-alkyne click chemistry (SPAAC) (see, e.g., Chem. Commun., 2011, 47:6257-6259 and Nature, 2015, 519(7544):486-90, each of which is incorporated herein by reference in its entirety).

[0273] vii. Receptor-ligand system

[0274] In some embodiments, a molecule (e.g., a reporter oligonucleotide molecule) having a barcode (e.g., a sample-specific barcode) can be coupled to a ligand, wherein the ligand is part of a receptor-ligand interaction on a cell surface. For example, a molecule (e.g., a reporter oligonucleotide molecule) having a barcode (e.g., a sample-specific barcode) can be coupled to a ligand, which selectively interacts with a cell surface receptor, thereby targeting a molecule (e.g., a reporter oligonucleotide molecule) having a barcode (e.g., a sample-specific barcode) to a specific cell. Non-limiting examples of receptor-ligand systems that can be used include integrin receptor-ligand interactions, GPCR receptor-ligand interactions, RTK receptor-ligand interactions, and TLR-ligand interactions (see Juliano, Nucleic Acids Res., 44 (14): 6518-6548 (2016), the contents of which are incorporated herein by reference in their entirety). Any of the methods described herein for attaching a molecule (e.g., a reporter oligonucleotide molecule) having a barcode (e.g., a sample-specific barcode) to a ligand (e.g., any of the methods described herein involving attaching a molecule (e.g., a reporter oligonucleotide molecule) having a barcode (e.g., a sample-specific barcode) to an antibody) can be used. In some embodiments, the ligand is part of a binding moiety.

[0275] viii. Covalent bonding systems via amine or thiol functional groups

[0276] In some embodiments, a molecule (e.g., a reporter oligonucleotide molecule) having a barcode (e.g., a sample-specific barcode) can incorporate a reactive functional group at a site within the molecule (e.g., a reporter oligonucleotide molecule). In such cases, the reactive functional group can promote conjugation with a ligand and / or surface. In some embodiments, a molecule (e.g., a reporter oligonucleotide molecule) having a barcode (e.g., a sample-specific barcode) can include a thiol modifier that is designed to react with a variety of activated accepting groups (e.g., maleimide and gold microspheres). For example, a molecule (e.g., a reporter oligonucleotide molecule) having a barcode (e.g., a sample-specific barcode) with a thiol modifier can interact with a maleimide-conjugated peptide, resulting in the labeling of the peptide. In some embodiments, the maleimide-conjugated peptide is present on the cell surface, thereby interacting with a thiol-modified molecule (e.g., a reporter oligonucleotide molecule) having a barcode (e.g., a sample-specific barcode), and the molecule (e.g., a reporter oligonucleotide molecule) having a barcode (e.g., a sample-specific barcode) is coupled to the cell surface. Non-limiting examples of thiol modifiers include: 5' thiol modifier C6 SS, 3' thiol modifier C3 SS, dithiol, 3' thiol modifier oxa-6-SS and dithiol serine alcohol. In some embodiments, the reactive functional group is part of the binding moiety.

[0277] In some embodiments, a molecule (e.g., a reporter oligonucleotide molecule) with a barcode (e.g., a sample-specific barcode) may include an amine modifier, such as an amine modifier designed to be attached to another molecule in the presence of an acylating agent. In some embodiments, a molecule (e.g., a reporter oligonucleotide molecule) with a barcode (e.g., a sample-specific barcode) may include an amine modifier, which is designed to be attached to a wide range of linking groups (e.g., carbonyl amides, thioureas, sulfonamides, and carboxamides). For example, a molecule (e.g., a reporter oligonucleotide molecule) with a barcode (e.g., a sample-specific barcode) and an amine modifier may interact with a sulfonamide-conjugated peptide, thereby resulting in a marker for the peptide. In some embodiments, a sulfonamide-conjugated peptide is present on the cell surface, thereby interacting with an amine-modified molecule (e.g., a reporter oligonucleotide molecule) with a barcode (e.g., a sample-specific barcode), and a molecule (e.g., a reporter oligonucleotide molecule) with a barcode (e.g., a sample-specific barcode) is coupled to the cell surface. Non-limiting examples of amine modifiers include: DMS(O)MT-aminomodifier-C6, aminomodifier-C3-TFA, aminomodifier-C12, aminomodifier-C6-TFA, amino-dT, aminomodifier-5, aminomodifier-C2-dT, aminomodifier-C6-dT, and 3'-aminomodifier-C7. In some embodiments, the amine modifier is part of a binding moiety.

[0278] As another example, a molecule (e.g., a reporter oligonucleotide molecule) with a barcode (e.g., a sample-specific barcode) can introduce a reactive functional group, such as N-hydroxysuccinimide (NHS) at a site within the molecule (e.g., a reporter oligonucleotide molecule). In some embodiments, an amine (e.g., an amine-containing peptide) is present on the cell surface, thereby interacting with a molecule (e.g., a reporter oligonucleotide molecule) with a barcode (e.g., a sample-specific barcode), and the molecule (e.g., a reporter oligonucleotide molecule) with a barcode (e.g., a sample-specific barcode) is coupled to the cell surface. In some embodiments, a molecule (e.g., a reporter oligonucleotide molecule) with a barcode (e.g., a sample-specific barcode) reacts with a bifunctional NHS connector to form a molecule (e.g., a nucleic acid molecule) with a barcode.

[0279] In some embodiments, molecules (e.g., reporter oligonucleotide molecules) with barcodes (e.g., sample-specific barcodes) can be coupled to biocompatible anchors (BAMs) of cell membranes. For example, BAMs can include molecules containing oily groups and PEGs. Oily groups can promote the anchoring of molecules (e.g., nucleic acid molecules) with barcodes (e.g., sample-specific barcodes) to cells, and PEGs can increase water solubility. In some embodiments, oily groups-PEG-NHS can be coupled to molecules (e.g., reporter oligonucleotide molecules) with barcodes (e.g., sample-specific barcodes) using NHS chemistry.

[0280] ix. Azide-based systems

[0281] In some embodiments, wherein a molecule (e.g., a reporter oligonucleotide molecule) incorporates a reactive functional group at a site within the molecule, a molecule (e.g., a reporter oligonucleotide molecule) with a barcode (e.g., a sample-specific barcode) can be coupled to an azide group on a cell surface. In some embodiments, the reactive functional group is an alkynyl group. In some embodiments, click chemistry as described herein can be used to attach an alkynyl-modified molecule (e.g., a reporter oligonucleotide molecule) with a barcode (e.g., a sample-specific barcode) to an azide group on a cell surface. An azide group can be attached to a cell surface by a variety of methods. For example, NHS chemistry can be used to attach an azide group to a cell surface. In some embodiments, N-azidoacetylmannosamine-tetraacylates (Ac4ManNAz) containing an azide group can react with sialic acid on a cell surface to attach an azide to a cell surface. In some embodiments, an azide is attached to a cell surface by the bioorthogonal expression of an azide.

[0282] x. Lectin-based systems

[0283] In some embodiments, a molecule (e.g., a reporter oligonucleotide molecule) with a barcode (e.g., a sample-specific barcode) can be coupled to a lectin, which promotes the attachment of a molecule (e.g., a reporter oligonucleotide molecule) with a barcode (e.g., a sample-specific barcode) to a cell surface. Lectins can bind to polysaccharides, such as polysaccharides on the cell surface. In some embodiments, a molecule (e.g., a reporter oligonucleotide molecule) with a barcode (e.g., a sample-specific barcode) has a reactive functional group incorporated, such as an azide group. In some embodiments, a molecule (e.g., a reporter oligonucleotide molecule) with a barcode (e.g., a sample-specific barcode) and an azide group reacts with a modified lectin (e.g., a lectin modified using NHS chemistry) to introduce an azide reactive group. In some embodiments, living cells are labeled with a lectin-modified molecule (e.g., a reporter oligonucleotide molecule) with a barcode (e.g., a sample-specific barcode). In some embodiments, fixed cells are labeled with lectin-modified molecules (e.g., reporter oligonucleotide molecules) with barcodes (e.g., sample-specific barcodes). In some embodiments, permeabilized cells are labeled with lectin-modified molecules (e.g., reporter oligonucleotide molecules) with barcodes (e.g., sample-specific barcodes). In some embodiments, organelles in the secretory pathway can be labeled with lectin-modified molecules (e.g., reporter oligonucleotide molecules) with barcodes (e.g., sample-specific barcodes).

[0284] D. Introducing the binding moiety into the interior of the cell

[0285] Non-limiting examples of binding moieties and systems that can permeate and / or cross a cell membrane (e.g., thereby introducing the binding moiety, reporter oligonucleotide, and any barcode attached thereto into the interior of the cell) and are used to analyze one or more analytes in a biological sample according to the materials and methods provided herein include: cell penetrating agents (e.g., cell penetrating peptides), nanoparticles, liposomes, polymer vesicles, peptide-based chemical carriers, electroporation, sonoporation, lentiviral vectors, retroviral vectors, and combinations thereof.

[0286] Non-exhaustive examples of oligomer delivery vehicles may include cell penetrating peptides or nanoparticles. Non-exhaustive examples of delivery systems may include lipid-based polymers and metal nanoparticles or oligomers, which may be conjugated or encapsulated within the delivery system.

[0287] i. Cell penetrating agents

[0288] In some embodiments, a cell penetrating agent promotes the binding of a molecule (e.g., a labeling agent) with a barcode (e.g., a sample-specific barcode) and a binding moiety to a cell feature (e.g., a molecule on and / or in a cell). In some embodiments, a molecule (e.g., a reporter oligonucleotide molecule) with a barcode (e.g., a sample-specific barcode) and a binding moiety is coupled to a cell penetrating agent, and the cell penetrating agent allows the molecule to interact with an analyte in the cell. In some embodiments, a cell penetrating agent is an agent that can promote the introduction of a molecule (e.g., a reporter oligonucleotide molecule) with a barcode (e.g., a sample-specific barcode) and a binding moiety into a cell of a biological sample (see, e.g., Lovatt et al. Nat Methods. 2014 Feb; 11 (2): 190-6, the contents of which are incorporated herein by reference in their entirety). In some embodiments, a cell penetrating agent is a cell penetrating peptide. In some embodiments, a "cell penetrating peptide" comprises a peptide (e.g., a short peptide, e.g., a peptide generally not exceeding 30 residues) having the ability to pass through a cell membrane.

[0289] In some embodiments, a cell penetrating peptide coupled to a molecule having a barcode (e.g., a sample-specific barcode) and a binding moiety (e.g., a reporter oligonucleotide molecule) can cross the cell membrane using an energy-dependent mechanism or an energy-independent mechanism. For example, the cell penetrating peptide can directly translocate across the cell membrane by physical perturbation of the plasma membrane, endocytosis, adaptive translocation, pore formation, electroporation-like permeabilization, and / or entry into microdomain boundaries. Non-limiting examples of cell penetrating peptides include penetratin, tat peptide, pVEC, transporter, MPG, Pep-1, polyarginine peptide, MAP, R6W3, (D-Arg)9, Cys(Npys)-(D-Arg)9, anti-βγ(MPS-light sensor-like protein C-terminal), Cys(Npys)antennapedia, Cys(Npys)-(Arg)9, Cys(Npys)-TAT(47-57), HIV-1Tat(48-60), KALA, wasp toxin, penetratin-Arg, pep-1-cysteamine, TAT(47-57)GGG-Cys(Npys), Tat-NR2Bct, transdermal peptide, SynB1, SynB3, PTD-4, PTD-5, FHV Coat-(35-49), BMV Gag-(7-25), HTLV-II Rex-(4-16), R9-tat, SBP, FBP, MPG, MPG (ΔNLS), Pep-2, MTS, plsl and polylysine peptides (see, e.g., Bechara et al. FEBS Lett. 2013 Jun 19; 587(12): 1693-702, the contents of which are incorporated herein by reference in their entirety).

[0290] ii. Nanoparticles

[0291] In some embodiments, inorganic particles (e.g., nanoparticles) promote the binding of molecules (e.g., markers) with barcodes (e.g., sample-specific barcodes) and binding moieties to cell features (e.g., molecules on and / or in cells). In some embodiments, molecules (e.g., reporter oligonucleotide molecules) with barcodes (e.g., sample-specific barcodes) and binding moieties are coupled to inorganic particles (e.g., nanoparticles), and molecules (e.g., reporter oligonucleotide molecules) with barcodes (e.g., sample-specific barcodes) and binding moieties approach analytes within cells using nanoparticles. Non-limiting examples of nanoparticles that can be used in embodiments herein for delivering molecules (e.g., reporter oligonucleotide molecules) with barcodes (e.g., sample-specific barcodes) and binding moieties to cells and / or cell beads include inorganic nanoparticles prepared from metals (e.g., iron, gold, and silver), inorganic salts, and ceramics (e.g., phosphates or carbonates of calcium, magnesium, or silicon). The surface of the nanoparticle can be coated to promote the binding of molecules having barcodes (e.g., sample-specific barcodes) and binding moieties (e.g., reporter oligonucleotide molecules), or the surface can be chemically modified to promote the attachment of molecules having barcodes (e.g., sample-specific barcodes) and binding moieties (e.g., reporter oligonucleotide molecules). Magnetic nanoparticles (e.g., supermagnetic iron oxide), fullerenes (e.g., soluble carbon molecules), carbon nanotubes (e.g., cylindrical fullerenes), quantum dots, and supramolecular systems can also be used.

[0292] iii. Liposomes

[0293] In some embodiments, liposomes promote the binding of molecules (e.g., labeling agents) with barcodes (e.g., sample-specific barcodes) and binding moieties to cell features (e.g., molecules on and / or in cells). Various types of lipids, including cationic lipids, can be used for liposome delivery. In some cases, molecules (e.g., reporter oligonucleotide molecules) with barcodes (e.g., sample-specific barcodes) and binding moieties are delivered to cells by lipid nanoemulsions. In some embodiments, lipid emulsions include a dispersion of an immiscible liquid stabilized by an emulsifier in another immiscible liquid. Labeling cells can include the use of solid lipid nanoparticles.

[0294] iv. Polymer vesicles

[0295] In some embodiments, polymer vesicles promote the binding of molecules (e.g., markers) with barcodes (e.g., sample-specific barcodes) and binding moieties to cell features (e.g., molecules on and / or in cells). In some embodiments, molecules (e.g., reporter oligonucleotide molecules) with barcodes (e.g., sample-specific barcodes) and binding moieties are contained in polymer vesicles, and molecules (e.g., nucleic acid molecules) with barcodes (e.g., sample-specific barcodes) and binding moieties approach intracellular analytes using polymer vesicles. In some embodiments, "polymer vesicles" include artificial vesicles. For example, polymer vesicles can be vesicles similar to liposomes, but the membrane includes amphiphilic synthetic block copolymers (see, e.g., Rideau et al. Chem. Soc. Rev., 2018, 47, 8572-8610, the contents of which are incorporated herein by reference in their entirety). In some embodiments, the polymersome comprises a di-(AB) or triblock copolymer (e.g., ABA or ABC), wherein A and C are hydrophilic blocks and B is a hydrophobic block. In some embodiments, the polymersome comprises poly(butadiene)-b-poly(ethylene oxide), poly(ethylethylene)-b-poly(ethylene oxide), polystyrene-b-poly(ethylene oxide), poly(2-vinylpyridine)-b-poly(ethylene oxide), polydimethylsiloxane-b-poly(ethylene oxide), polydimethylsiloxane-g-poly(ethylene oxide), polycaprolactone-b-poly(ethylene oxide), polyisobutylene-b-poly(ethylene oxide), polystyrene-b-polyacrylic acid, polydimethylsiloxane-b-poly-2-methyl-2-oxazoline, or a combination thereof (where b=block and g=grafted).

[0296] v. Peptide-based chemical carriers

[0297] In some embodiments, chemical carriers based on peptides, such as chemical carriers based on cationic peptides, promote the binding of molecules (e.g., markers) with barcodes (e.g., sample-specific barcodes) and binding moieties to cell features (e.g., molecules on and / or in cells). Cationic peptides can be rich in basic residues, such as lysine and / or arginine. In some embodiments, chemical carriers based on polymers promote the binding of molecules to biological analytes. When cationic polymers are mixed with molecules (e.g., reporter oligonucleotide molecules) with barcodes (e.g., sample-specific barcodes) and binding moieties, nanocomplexes referred to as polycomplexes can be formed. Polymer-based carriers can include natural proteins, peptides and / or polysaccharides. Polymer-based carriers can include synthetic polymers. In some embodiments, polymer-based carriers include polyethyleneimine (PEI). PEI can condense DNA into positively charged particles, which bind to anionic cell surface residues and are brought into cells by endocytosis. In some embodiments, polymer-based chemical carriers include poly (L) -lysine (PLL), poly (DL-lactic acid) (PLA), poly (DL-lactide-co-glycoside) (PLGA), polyornithine, polyarginine, histones, protamine, or a combination thereof. Polymer-based carriers can include mixtures of polymers such as PEG and PLL. Other non-limiting examples of polymers include dendrimers, chitosan, synthetic amino derivatives of dextran, and cationic acrylic polymers.

[0298] vi. Electroporation

[0299] In some embodiments, electroporation promotes the binding of molecules (e.g., markers) with barcodes (e.g., sample-specific barcodes) and binding moieties to cell features (e.g., molecules on and / or in cells). Through electroporation, biological analytes with barcodes (e.g., sample-specific barcodes) and binding moieties (e.g., reporter oligonucleotide molecules) can enter cells through one or more holes in the cell membrane formed by the applied electricity. Based on the applied field strength and pulse duration, the pores of the membrane can be reversible.

[0300] vii. Acoustic perforation

[0301] In some embodiments, sonoporation promotes the binding of a molecule (e.g., a labeling agent) having a barcode (e.g., a sample-specific barcode) and a binding moiety to a cell feature (e.g., a molecule on and / or in a cell). Acoustic waves can be used to temporarily permeabilize the cell membrane, thereby allowing the cell to take up a biological analyte through a molecule (e.g., a sample-specific barcode) and a binding moiety (e.g., a reporter oligonucleotide molecule).

[0302] viii. Lentiviral and Retroviral Vectors

[0303] In some embodiments, the vector facilitates the binding of a molecule (e.g., a labeling agent) having a barcode (e.g., a sample-specific barcode) and a binding moiety to a cell feature (e.g., a molecule on and / or in a cell). For example, the vector described herein can be an expression vector, wherein the expression vector comprises a promoter sequence operably linked to a sequence encoding a molecule (e.g., a reporter oligonucleotide molecule) having a barcode (e.g., a sample-specific barcode) and a binding moiety. Non-limiting examples of vectors include plasmids, transposons, cosmids, and viral vectors (e.g., any adenoviral vector (e.g., pSV or pCMV vector), adeno-associated virus (AAV) vector, lentiviral vector, and retroviral vector) and any Vector. For example, the vector can include sufficient cis-acting elements for expression, wherein other elements for expression can be provided by the host mammalian cell or in an in vitro expression system. A skilled practitioner will be able to select appropriate vectors and mammalian cells to introduce any spatially distributed agent described herein.

[0304] E. Other Methods and Agents for Intracellular Introduction of Binding Moieties

[0305] In some embodiments, needles, such as needles for injection (e.g., microinjection), particle bombardment, photoporation, magnetic infection and / or hydroporation (hydroporation), promote the binding of molecules (e.g., markers) with barcodes (e.g., sample-specific barcodes) and binding moieties to cell features (e.g., molecules on and / or in cells). For example, by particle bombardment, molecules (e.g., reporter oligonucleotide molecules) with barcodes (e.g., sample-specific barcodes) and binding moieties can be coated with heavy metal particles and delivered to cells at high speed. In photoporation, laser pulses can be used to create transient holes in the cell membrane, allowing cells to take in molecules (e.g., reporter oligonucleotide molecules) with barcodes (e.g., sample-specific barcodes) and binding moieties. In magnetic transfection, molecules (e.g., reporter oligonucleotide molecules) with barcodes (e.g., sample-specific barcodes) and binding moieties can be coupled to magnetic particles (e.g., magnetic nanoparticles, nanowires, etc.) and positioned in target cells by an applied magnetic field. In water pores, molecules with barcodes (eg, sample-specific barcodes) and binding moieties (eg, reporter oligonucleotide molecules) can be delivered to cells and / or cell beads by hydrodynamic pressure.

[0306] IV. Base

[0307] In some embodiments, the substrate herein can be any support that is insoluble in aqueous liquids, and allows to locate biological samples, analytes, features and / or reagents on the support. In some embodiments, biological samples can be attached to the substrate. The attachment of biological samples can be irreversible or reversible, depending on the subsequent steps in the properties of the sample and the analytical method. In certain embodiments, by applying a suitable polymer coating to the substrate and contacting the sample with the polymer coating, the sample can be reversibly attached to the substrate. Then, for example, using an organic solvent that at least partially dissolves the polymer coating, the sample can be separated from the substrate. Hydrogel is an example of a polymer suitable for this purpose.

[0308] In some embodiments, the substrate can be coated or functionalized with one or more substances to promote cell attachment to the substrate. Suitable substances that can be used to coat or functionalize the substrate include, but are not limited to, lectins, polylysine, antibodies, and polysaccharides.

[0309] In some embodiments, the substrate is used as a support for direct or indirect attachment of cells. In addition, in some embodiments, a substrate (e.g., the same substrate or a different substrate) can be used to provide a support for cells, such as a single cell layer deposited and / or fixed on a substrate. Thus, a "substrate" is a support that is insoluble in aqueous liquids and allows cells to be positioned on a substrate.

[0310] A wide variety of different substrates can be used for the aforementioned purposes. In general, the substrate can be any suitable support material. Exemplary substrates include, but are not limited to, glass, modified and / or functionalized glass, hydrogels, films / membranes, plastics (including, for example, acrylic plastics, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, Teflon TM , cyclic olefins, polyimides, etc.), nylon, ceramics, resins, Zeonor, silica or silica-based materials (including silicon and modified silicon), carbon, metals, inorganic glasses, optical fiber bundles, and polymers such as polystyrene, cyclic olefin copolymers (COC), cyclic olefin polymers (COP), polypropylene, polyethylene, and polycarbonate.

[0311] The substrate may also correspond to a flow cell. A flow cell may be formed from any of the aforementioned materials and may include channels that allow reagents, solvents, features, and molecules to pass through the cell.

[0312] In the examples of substrate materials discussed above, polystyrene is a hydrophobic material suitable for binding negatively charged macromolecules because it generally contains few hydrophilic groups. For nucleic acids immobilized on glass slides, the immobilization of nucleic acids can be increased by increasing the hydrophobicity of the glass surface. Such enhancement can allow the formation of relatively denser packing (e.g., providing improved specificity and resolution).

[0313] In some embodiments, the substrate is coated with a surface treatment agent such as poly(L)-lysine. Additionally or alternatively, the substrate can be treated by silanization, for example with epoxysilanes, aminosilanes and / or with polyacrylamide.

[0314] The substrate can generally have any suitable form or format. For example, the substrate can be flat, curved, such as convex or concave toward the area where the interaction occurs between the biological sample (e.g., cell or tissue sample) and the substrate. In some embodiments, the substrate is flat, such as a planar, chip or slide. The substrate can contain one or more patterned surfaces (e.g., channels, holes, protrusions, ridges, pits, etc.) within the substrate.

[0315] The substrate can be any desired shape. For example, the substrate can be generally a thin flat shape (e.g., a square or a rectangle). In some embodiments, the substrate structure has rounded corners (e.g., to increase safety or robustness). In some embodiments, the substrate structure has one or more cut corners (e.g., to be used with a sliding fixture or a cross table). In some embodiments in which the substrate structure is flat, the substrate structure can be a support of any suitable type with a flat surface (e.g., a chip or a slide such as a microscope slide).

[0316] The substrate can optionally include various structures, such as but not limited to projections, ridges and channels. The substrate can be micropatterned to limit lateral diffusion (e.g., to prevent the overlapping of spatial barcodes). The substrate modified with such a structure can be modified to allow the association of probes at analytes, features or each site. For example, the site wherein the substrate is modified with various structures can be adjacent or not adjacent to other sites.

[0317] In some embodiments, the surface of the substrate can be modified so that discrete sites are formed that have or accommodate only a single feature. In some embodiments, the surface of the substrate can be modified so that features are attached to random locations.

[0318] In some embodiments, the surface of the substrate is modified to include one or more holes using techniques such as, but not limited to, stamping techniques, micro-etching techniques, and molding techniques. In some embodiments where the substrate includes one or more holes, the substrate can be a concave slide or a cavity slide. For example, the hole can be formed by one or more shallow depressions on the substrate surface. In some embodiments where the substrate includes one or more holes, the hole can be formed by attaching a box (e.g., a box containing one or more chambers) to the surface of the substrate structure.

[0319] In some embodiments, the structure of the substrate can include pores that house cells.

[0320] In some embodiments, the substrate includes one or more marks on the substrate surface, for example, to provide guidance for relating spatial information to the characteristics of cells and / or analytes of interest in cells. For example, the substrate can be marked with a wire grid (e.g., to allow the size of an object seen under magnification to be easily estimated and / or to provide a reference area for counting objects). In some embodiments, a fiducial marker can be included on the substrate. Such marks can be made using techniques including but not limited to printing, sandblasting, and deposition on the surface.

[0321] In some embodiments where the substrate is modified to contain one or more structures (including but not limited to holes, protrusions, ridges, features or markings), the structures may include physically altered sites. For example, substrates modified with various structures may include physical properties including but not limited to physical configurations, magnetic or compressive forces, chemically functionalized sites, chemically altered sites, and / or electrostatically altered sites.

[0322] In some embodiments where the substrate is modified to contain various structures (including but not limited to holes, protrusions, ridges, features or markings), the structures are applied in a pattern. Alternatively, the structures may be randomly distributed.

[0323] In some embodiments, the substrate is treated to minimize or reduce non-specific analyte hybridization within or between features. For example, the treatment may include coating the substrate with a hydrogel, a film, and / or a film that forms a physical barrier to non-specific hybridization. Any suitable hydrogel may be used. For example, a hydrogel matrix prepared according to the method stated in U.S. Patent Nos. 6,391,937, 9,512,422 and 9,889,422 and U.S. Patent Application Publication Nos. US2017 / 0253918 and US2018 / 0052081 may be used. The entire contents of each of the aforementioned documents are incorporated herein by reference.

[0324] Treatment can include adding a functional group that is reactive or can be activated so that it becomes reactive (e.g., photoreactive) upon receiving a stimulus. Treatment can include treatment with a polymer having one or more physical properties (e.g., mechanical, electrical, magnetic, and / or thermal) that minimize nonspecific binding (e.g., activating the substrate at certain locations to allow hybridization of the analyte at those locations).

[0325] A substrate (e.g., features on an array) can include tens to hundreds of thousands or millions of individual oligonucleotide molecules (e.g., at least about 10,000, 50,000, 100,000, 500,000, 1,000,000, 10,000,000, 100,000,000, 1,000,000,000 or 10,000,000,000 oligonucleotide molecules).

[0326] In some embodiments, the surface of the substrate is coated with a cell-permissive coating to allow living cells to attach. In some embodiments, a cell-permissive coating is a coating that allows or helps cells to maintain cell viability (e.g., maintain viability) on a substrate. For example, a cell-permissive coating can enhance cell attachment, cell growth, and / or cell differentiation, e.g., a cell-permissive coating can provide nutrients to living cells. A cell-permissive coating can include biomaterials and / or synthetic materials. Non-limiting examples of cell-permissive coatings include those characterized by one or more extracellular matrix (ECM) components (e.g., proteoglycans and fibrous proteins such as collagen, elastin, fibronectin, and laminin), polylysine, poly(L)-ornithine, and / or biocompatible silicones (e.g., ). For example, a cell-permissive coating comprising one or more extracellular matrix components may include type I collagen, type II collagen, type IV collagen, elastin, fibronectin, laminin, and / or vitronectin. In some embodiments, the cell-permissive coating comprises a dissolved basement membrane preparation extracted from an Engelbreth-Holm-Swarm (EHS) mouse sarcoma (e.g., In some embodiments, the cell-permissive coating comprises collagen. The cell-permissive coating can be used to culture adherent cells on a spatially barcoded array or to maintain cell viability of a sample when in contact with a spatially barcoded array.

[0327] Substrates for in situ detection of analytes in cells

[0328] In some embodiments, multiple labeled cells from different samples are deposited and / or fixed on a substrate for one or more in situ determinations, such as using one or more nucleic acid probes to detect analytes in the labeled cells. The one or more nucleic acid probes can hybridize directly or indirectly with a target nucleic acid or its complementary sequence or amplification product in the labeled cells.

[0329] A variety of different substrates can be used for in-situ determination, as long as the substrate is compatible with sample and sample treatment, in-situ reagents and reactions and in-situ signal detection (e.g., optical imaging such as fluorescence microscopy). The substrate can be any suitable support material, and is generally transparent. For example, a slide, such as a cover glass, can be used. The substrate can include but is not limited to glass, modified and / or functionalized glass, hydrogel, film (film / membrane), plastic, nylon, ceramic, resin, Zeonor, silicon dioxide or silicon dioxide-based materials (including silicon and modified silicon), carbon, metal, inorganic glass, fiber bundles and polymers, such as polystyrene, cycloolefin copolymer (COC), cycloolefin polymer (COP), polypropylene, polyethylene and polycarbonate. The substrate can also correspond to a flow cell.

[0330] In some embodiments, the thickness of the substrate is between about 0.01 mm and about 5 mm, such as between about 0.05 mm and about 3 mm, between about 0.1 mm and about 2.5 mm, between about 0.2 mm and about 2 mm, between about 0.5 mm and about 1.5 mm, or about 1 mm. In some embodiments, the thickness of the substrate is or is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 mm, or the thickness is between any of the above values.

[0331] V. In-situ Detection

[0332] In some embodiments, the method provided herein includes using microscopy as the in-situ determination of reading, such as nucleic acid sequencing, hybridization or other detection or determination methods related to optical reading. In some aspects, one, two, three, four, five or more nucleotide sequences of nucleic acid analytes are detected or determined in situ in cells on substrates. In some embodiments, the determination includes the presence or absence of detection amplification products (e.g., RCA products). In some embodiments, the disclosure provides for a large number of targets and analytes (such as transcripts and / or DNA loci) to be carried out in-situ high-throughput spectrometry, for example, for detecting and / or quantitatively analyzing nucleic acids and / or proteins in cells, tissues, organs or organisms.

[0333] In some aspects, a method is provided herein, which includes analyzing a biological target based on in situ hybridization of a probe comprising a nucleic acid sequence. In some embodiments, the method includes continuous hybridization of a detectably labeled oligonucleotide with a barcoded probe, which directly or indirectly binds to a biological target in a sample. In some embodiments, the detectably labeled oligonucleotide directly binds to one or more barcoded probes. In some embodiments, the detectably labeled oligonucleotide indirectly binds to one or more barcoded probes, such as through one or more bridging nucleic acid molecules.

[0334] In some aspects, an in situ hybridization-based assay is used to locate and measure nucleic acid sequences (e.g., DNA or RNA molecules containing one or more specific sequences of interest) within a natural biological sample (e.g., a single cell). In some embodiments, an in situ assay is used to analyze the presence, absence, amount, or level of mRNA transcripts (e.g., a transcriptome or a subset thereof, or an mRNA molecule of interest) in a biological sample while retaining spatial context. In some embodiments, the disclosure provides compositions and methods for in situ hybridization using directly or indirectly labeled molecules (e.g., complementary DNA or RNA or modified nucleic acids) as probes that bind or hybridize to a target nucleic acid in a biological sample of interest.

[0335] In some instances, nucleic acid probes can be labeled with radioisotopes, epitopes, haptens, biotin or fluorophores to detect the position of specific nucleic acid sequences on chromosomes or in cells. In some embodiments, probes are locus-specific (e.g., gene-specific) and are incorporated into or coupled to a specific region of a chromosome. In alternative embodiments, probes are centromere (alphoid or centromeric) repeat probes that are combined with or coupled to the repetitive sequences in each chromosome. Probes can also be complete chromosome probes (e.g., multiple smaller probes) that are combined with or coupled to sequences along the entire chromosome.

[0336] In some embodiments, provided herein is a method including DNA in situ hybridization to measure and locate DNA. In some embodiments, provided herein is a method including RNA in situ hybridization to measure and locate RNA (e.g., mRNA, lncRNA and miRNA) in a biological sample (e.g., a dissociated cell sample). In some embodiments, RNA in situ hybridization relates to single molecule RNA fluorescence in situ hybridization (FISH). In some embodiments, a fluorescently labeled nucleic acid probe is hybridized with a predetermined RNA target to visualize gene expression in a biological sample. In some embodiments, the FISH method includes using a single nucleic acid probe specific for each target, such as single molecule FISH (smFISH). Using smFISH, a fluorescent signal allowing quantitative measurement of RNA transcripts can be generated. In some embodiments, smFISH includes a nucleic acid probe having a length of about 50 base pairs, wherein each probe is coupled to a group of fluorophores. For example, the group of nucleic acid probes can include five probes, wherein each probe is coupled to five fluorophores. In some embodiments, the nucleic acid probes are each coupled to a fluorophore alternatively. For example, the smFISH scheme can use a group of about 40 nucleic acid probes, about 20 base pairs in length, each probe coupled to a single fluorophore. In some embodiments, the length of the nucleic acid probe can vary, comprising 10 to 100 base pairs, such as 30 to 60 base pairs. Alternatively, multiple nucleic acid probes targeting different regions of the same RNA transcript can be used. It will be appreciated by those skilled in the art that the type of nucleic acid probe, the number of nucleic acid probes, the number of fluorophores coupled to the probe, and the length of the probe can vary to adapt to the specifications of a single assay.

[0337] In other embodiments, smFISH is applied to multiple workflows, where continuous / sequential hybridization (e.g., as in seqFISH or seqFISH+) is used to assign time barcodes on target transcripts. Continuous cycles of fluorescence in situ hybridization can be accompanied by imaging and probe elution to detect individual transcripts (e.g., RNA transcripts) in biological samples of interest (e.g., dissociated cell samples or extracted RNA). In some embodiments, each round of hybridization comprises a predefined probe set (e.g., about 10 to about 50 probes, such as 24 to 32 probes) targeting a unique RNA transcript. In some instances, the predefined probe set is multicolor. Optionally, multiple nucleic acid probes are attached to the sample, wherein each probe comprises a starting sequence for amplification, thereby allowing autofluorescence (e.g., as in single molecule hybridization chain reaction (smHCR)) to be reduced. In some embodiments, the multiple smFISH method described herein can be multiplexed to more than 10,000 mRNAs from 10s, optionally accompanied by imaging, to effectively and accurately analyze the entire transcriptome. In situ hybridization methods may also include using two probes to bind to target transcripts (e.g., RNA transcripts) that serve as binding targets for amplification primers. In some embodiments, this process results in signal amplification (e.g., in RNAscope). In some embodiments, in situ hybridization methods may use metal tags instead of fluorophores (e.g., imaging mass spectrometry cytometry). Metal-conjugated antibodies can be coupled to metal tags that hybridize to transcripts on biological samples. In some embodiments, mass spectrometry cytometry can be used to quantify metal abundance, allowing for simultaneous assessment of RNA and protein in biological samples.

[0338] In some embodiments, the methods described herein include error-robust multiplex FISH schemes (e.g., MERFISH). In some embodiments, the scheme includes a non-reading nucleic acid probe (e.g., a primary probe) comprising a binding region (e.g., a region bound to a target such as an RNA transcript) coupled to one or more flanking regions. In some embodiments, each non-reading nucleic acid probe is coupled to two flanking regions. The non-reading nucleic acid probe can be hybridized with a transcript (e.g., an RNA transcript) in a biological sample (e.g., a dissociated cell sample or a single cell) so that the fluorescent reading nucleic acid probe can be subsequently hybridized continuously with the flanking regions of the non-reading nucleic acid probe. In some embodiments, each round of hybridization includes continuous imaging and probe elution to quench the signal of the reading nucleic acid probe from the previous rounds. RNA can be imaged by FISH, and errors accumulated during multiple rounds of imaging (e.g., incomplete hybridization) are detected and / or corrected. In some embodiments, amplification microscopy is used to increase the number of RNA targets detected without signal overlap. In similar embodiments, the non-reading nucleic acid probe is cross-linked to the target transcript before imaging. Cross-linking can be performed by any suitable method. In a preferred embodiment, cross-linking is performed using hydrogel embedding. After the cross-linking step, it can be barcoded, including using sequential hybridization of readout probes coupled to predetermined colors to generate unique barcodes (e.g., generating false colors from sequential hybridization). In some embodiments, one or more barcodes of the probe are targeted by a detectably labeled detection oligonucleotide, such as a fluorescently labeled oligonucleotide.

[0339] The methods described herein can employ similar strategies for in situ hybridization using a variant of the FISH technique. In some embodiments, the methods include multiplex FISH protocols without barcodes (e.g., ouroboros sm-FISH (osmFISH)). Methods without barcodes may be limited to detecting a specific number of targets, which is limited by the number of hybridization rounds performed. In some embodiments, imaging is performed after each round of hybridization, wherein the probes are eluted after imaging, allowing subsequent hybridization and imaging rounds.

[0340] Other embodiments of the present disclosure may include the use of an in situ hybridization scheme that does not rely on the capture of transcripts from a predetermined position probe. In some embodiments, optical-free spatial mapping of transcripts in biological samples (e.g., a chemically encoded microscopy system) can be used. In some embodiments, transcripts are first labeled in situ with unique nucleotide tags (e.g., unique molecular identifiers). This first reaction can be followed by a second in situ amplification reaction, labeled by a new set of unique nucleotide tags (e.g., unique event identifiers). In some embodiments, RNA or DNA sequencing can be used to identify each molecular chain sequence (e.g., concatemer). In other embodiments, algorithms can be used to evaluate the proximity of sequences and generate a combination of images of target transcripts and sequence information.

[0341] In some embodiments, a method is provided herein, which includes linking the sequencing information and spatial information of the target in the endogenous environment. For example, the analysis of the nucleic acid sequence can be directly carried out on the DNA or RNA in the complete biological sample of interest, such as by in situ detection of the nucleic acid sequence, including sequential hybridization of detectable probes. In some embodiments, in addition to the spatial resolution of the transcript, the disclosure allows simultaneous identification and quantification of multiple targets, such as 100, 1000 or more transcripts (e.g., mRNA transcripts). In some aspects, the spatial resolution of the transcript can be subcellular. Optionally, the signal amplification strategy described herein can be used to increase the spatial resolution.

[0342] In some embodiments, fluorescent dyes are used to target nucleic acid bases, and circularized probes or probe groups (e.g., padlock probes) are used to target RNA of interest in situ. In some embodiments, mRNA is reverse transcribed into cDNA, and circularized probes or probe groups (e.g., padlock probes) can be combined with or coupled to cDNA. In some embodiments, circularized probes or probe groups (e.g., padlock probes) comprise oligonucleotides whose ends are complementary to target sequences (e.g., target cDNA transcripts). After the padlock probes hybridize to the target sequence, the ends of the padlock probes can be connected with enzymes, and the formation of circularized DNA can be catalyzed.

[0343] In some embodiments, the ends of the circularizable probe or probe set (e.g., padlock probe) are very close to each other after hybridization with the target RNA or cDNA to allow the connection and circularization of the padlock probe. The padlock probe may additionally include one or more barcode sequences. In alternative embodiments, after hybridization with the target RNA or cDNA, there may be a gap between the ends of the padlock probe, and the gap must be filled with nucleic acid (e.g., by DNA polymerization) before the connection and circularization of the padlock probe ends. In some embodiments, the gap between the two ends of the padlock probe has a variable length, such as up to four base pairs, and can allow the actual RNA or cDNA sequence to be read out. In some embodiments, the DNA polymerase has strand displacement activity. In some embodiments, the DNA polymerase may not have strand displacement activity, such as the polymerase used in barcode in situ target sequencing (BaristaSeq), which uses a gap filling padlock probe method to provide a read length of up to 15 bases. See, for example, Chen et al., Nucleic Acids Res.2018, 46, e22, the contents of which are incorporated herein by reference in their entirety..

[0344] The methods described herein may include performing DNA cyclization and amplification (e.g., rolling circle amplification) at the location of a cyclizable probe or probe set (e.g., a padlock probe). In some embodiments, amplification results in multiple repetitions of the padlock probe sequence. The sequencing and / or decoding of the amplified padlock probe can be performed while sequencing while connected. In an alternative approach, sequencing by hybridization or sequencing by synthesis is used. In some embodiments, during the sequencing process, the amplicon is stabilized by crosslinking as described herein. In some embodiments, the method proposed in the present disclosure, including an in situ detection step, can be automated on a microfluidic platform.

[0345] Those skilled in the art will appreciate other methods for in situ detection. For example, in some embodiments, the barcode padlock probe may not be reverse transcribed. Instead, the second primer directly binds to (e.g., ligates) the RNA sequence of an adjacent circularizable probe or probe set (e.g., padlock probe). In some embodiments, amplification (e.g., rolling circle amplification) is performed, wherein the amplified product is embedded in a hydrogel by any suitable method (e.g., hydrogel-histochemistry), and then unbound proteins and lipids are removed. For example, a variation of the sequencing-by-ligation method can be used to sequence the embedded amplified product to determine the barcode sequence of each padlock probe. In some embodiments, a combination of chemistry and sequencing described herein can be used to analyze the spatial orientation of target transcripts in 3D.

[0346] In some embodiments, the in situ detection method described in the present disclosure may be non-targeted. In some embodiments, non-targeted in situ detection can include the distribution of the whole genome / transcriptome of gene expression in the biological sample of interest, such as in fluorescent in situ RNA sequencing (FISSEQ). In some embodiments, RNA species are converted into cross-linked cDNA amplicons (e.g., cDNA cross-linked with the cell protein matrix of the sample). In some instances, cDNA synthesis is performed using modified amine bases to facilitate the cross-linking process. After the synthesis of the cross-linked cDNA amplicon, amplification (e.g., rolling circle amplification) as described elsewhere herein can be performed. In some embodiments, sequencing while connecting can be used to sequence the amplified product. In some embodiments, the sequencing step includes partition sequencing to selectively sequence a subset of the amplified product. In some embodiments, the strategy described herein allows serial detection of RNA, DNA and / or protein. In some embodiments, in situ detection can be combined with ectopic sequencing, such as in situ transcriptome accessibility sequencing (INSTA-Seq).

[0347] In some embodiments, in situ detection involves incorporating labeled nucleotides (e.g., fluorescently labeled mononucleotides or dinucleotides) or hybridizing labeled primers (e.g., labeled random hexamers) to nucleic acid templates in a sequential, template-dependent manner so that the identity (nucleotide sequence) of the incorporated nucleotides or labeled primer extension products can be determined, and thus the nucleotide sequence of the corresponding template nucleic acid can be determined. Aspects of in situ detection are described in, for example, Mitra et al., (2003) Anal. Biochem. 320, 55-65; and Lee et al., (2014) Science, 343 (6177), 1360-1363. In addition, examples of methods and systems for performing in situ detection are described in US 2021 / 0340618; US 2019 / 0032121, US 2019 / 0330617; US 2019 / 0194709; US 10,138,509; US 10,179,932; Wang et al., (2018) Science, 361(6499)5691); and Moffitt, (2016) Methods in Enzymology, 572, 1-49.

[0348] i. Probes and probe hybridization

[0349] In some aspects, the methods disclosed herein include the use of one or more probes or probe sets that hybridize to an analyte, such as an RNA molecule.

[0350] Exemplary probes or probe sets can be based on circularizable probes or probe sets (e.g., padlock probes), gapped padlock probes, SNAIL (splint nucleotide assisted intramolecular ligation) probe sets, PLAYR ( R

[00136] The specific probe or probe set design may vary, for example, a proximity ligation assay for RNA (e.g., a PLISH (proximity ligation in situ hybridization) probe set, and a circularizable DNA that binds to an RNA target (e.g., an RNA template ligation probe).

[0351] In some embodiments, the primary probe (e.g., a DNA probe that binds directly to an RNA target) is amplified by rolling circle, e.g., using a circular probe or a circularized probe from a padlock connection as a template. In some embodiments, the primary probe, such as a padlock probe or a probe set including a padlock probe, contains one or more barcodes. In some embodiments, the one or more barcodes indicate a sequence in the target nucleic acid, such as a single nucleotide of interest (e.g., a SNP or a point mutation), a dinucleotide sequence, a short sequence of about 5 nucleotides in length, or a sequence of any suitable length.

[0352] In some embodiments, a probe or probe set capable of DNA templated connection (such as from a cDNA molecule) is provided herein. See, for example, U.S. Patent 8,551,710, the contents of which are incorporated herein by reference in their entirety. In some embodiments, a DNA probe or probe set is provided herein, which comprises a ribonucleotide capable of hybridizing with a target nucleic acid (e.g., an mRNA molecule) and connecting in a target-dependent manner at a specific position. See, for example, US 2020 / 0224244, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the probe set is a SNAIL probe set. See, for example, U.S. Patent Publication 2019 / 0055594, the contents of which are incorporated herein by reference in their entirety. In some embodiments, a probe or probe set capable of adjacent connection is provided herein, such as RNA adjacent connection determination (e.g., PLAYR) probe set. See, for example, U.S. Patent Publication 20160108458, the contents of which are incorporated herein by reference in their entirety.

[0353] In some embodiments, the circular probe can be indirectly hybridized with the target nucleic acid. In some embodiments, the circular construct is formed by a probe group (e.g., a proximity connection in situ hybridization (PLISH) probe group) capable of adjacent connection. See, for example, US 2020 / 0224243, the contents of which are incorporated herein by reference in their entirety.

[0354] Exemplary probe groups and hybridization complexes include circularized probes or probe groups (e.g., padlock probes) or circular probes that directly hybridize to RNA transcripts. Splint primers can be used to promote DNA template padlock connections. Padlocks or circular probes can include targeting (e.g., target hybridization) sequences and one or more barcode regions, such as primary barcode sequences BC1 and BC2. After probe hybridization and / or any circularization step to provide a circular probe, in some embodiments, the circular probe is amplified, such as amplified in an RCA reaction, to produce an amplified molecule comprising a primary barcode (e.g., BC1) or its complementary sequence. In some embodiments, after amplification, the method also includes detecting the amplified product using a detectably labeled oligonucleotide (such as a fluorescently labeled detection oligonucleotide), which can hybridize with one or more barcode sequences (such as BC1 or BC2) or its complementary sequence.

[0355] Another exemplary probe group and hybridization complex include a circularized probe or probe group (e.g., a padlock probe) or a circular probe that directly hybridizes to an RNA transcript. A splint primer can be used to promote padlock connection of a DNA template. A padlock or circular probe can include a targeting (e.g., target hybridization) sequence and one or more barcode regions, such as primary barcode sequences BC1 and BC2. After probe hybridization and / or any circularization step to provide a circular probe, in some embodiments, the circular probe is amplified, such as amplified in an RCA reaction, to produce an amplified molecule comprising a primary barcode (e.g., BC1) or its complementary sequence. In some embodiments, after amplification, the method also includes using a detectable probe (e.g., a secondary probe), which includes (1) a barcode binding region that directly or indirectly hybridizes to the primary barcode region of the targeting probe, and (2) two or more detection barcode regions (e.g., SBC11 and SBC12), each detection barcode region hybridizing with a detectably labeled oligonucleotide, secondary probe 1, and secondary probe 2. Secondary probe 1 comprises a barcode binding region that directly or indirectly hybridizes with BC1 of the targeting probe, and four detection barcode regions, namely secondary barcodes (SBC) 11, SBC12, SBC13 and SBC14. Each of SBC11, SBC12, SBC13 and SBC14 can hybridize with a detectably labeled oligonucleotide, such as a fluorescently labeled detection oligonucleotide. Similarly, secondary probe 2 comprises a barcode binding region that directly or indirectly hybridizes with BC2 of the targeting probe, and four detection barcode regions, namely secondary barcodes (SBC) 21, SBC22, SBC23 and SBC24, each of which can hybridize with a detectably labeled oligonucleotide, such as a fluorescently labeled detection oligonucleotide. In some embodiments, two or more secondary barcodes are different from each other. For example, all secondary barcodes of the secondary probes combined with the same primary probe can be different, for example, each secondary barcode can specifically hybridize with the detection oligonucleotide and be uniquely identified by the detection oligonucleotide sequence.

[0356] In any of the embodiments disclosed herein, multiplex assays are performed in which multiple target nucleic acids (e.g., genes or RNA transcripts) are probed with multiple primary probes (e.g., padlock primary probes), and optionally, multiple secondary probes hybridized to primary barcodes (or their complements) are all hybridized at once, followed by sequential secondary barcode detection and signal decoding. In any of the embodiments disclosed herein, multiplex assays further include hybridizing a labeling agent to the probes and / or detectably labeled probes, followed by barcode detection and signal decoding.

[0357] ii. Connection

[0358] In some embodiments, the methods provided include connecting one or more polynucleotides that are part of a hybridization complex comprising a reporter oligonucleotide and / or a target nucleic acid for in situ analysis. In some embodiments, the connection involves chemical connection. In some embodiments, the connection involves template-dependent connection. In some embodiments, the connection involves non-template-dependent connection. In some embodiments, the connection involves enzymatic connection.

[0359] In some embodiments, enzymatic ligation involves the use of a ligase. In some aspects, the ligase used herein includes enzymes that are commonly used to connect polynucleotides together or to connect the ends of a single polynucleotide. RNA ligase, DNA ligase, or another ligase can be used to connect two nucleotide sequences together. Ligase includes ATP-dependent double-stranded polynucleotide ligases, NAD-i-dependent double-stranded DNA or RNA ligases, and single-stranded polynucleotide ligases, such as any of the ligases described in EC 6.5.1.1 (ATP-dependent ligases), EC 6.5.1.2 (NAD+-dependent ligases), and EC 6.5.1.3 (RNA ligases). Specific examples of ligases include bacterial ligases (such as Escherichia coli DNA ligases), Tth DNA ligases, Thermococcus species (strain 9°N) DNA ligases (9°N TM DNA ligase, NewEngland Biolabs), Taq DNA ligase, Ampligase TM (Epicentre Biotechnologies) and bacteriophage ligases (such as T3 DNA ligase, T4 DNA ligase and T7 DNA ligase) and their mutants. In some embodiments, the ligase is T4 RNA ligase. In some embodiments, the ligase is splintR ligase. In some embodiments, the ligase is a single-stranded DNA ligase. In some embodiments, the ligase is T4 DNA ligase. In some embodiments, the ligase is a ligase with DNA splinting DNA ligase activity. In some embodiments, the ligase is a ligase with RNA splinting DNA ligase activity.

[0360] In some embodiments, the connection herein is directly connected. In some embodiments, the connection herein is indirectly connected. "Direct connection" means that the ends of polynucleotides are hybridized in close proximity to each other to form a substrate for a ligase, thereby causing them to be connected to each other (intramolecular connection). Alternatively, "indirectly" means that the ends of polynucleotides are hybridized non-adjacently to each other, for example, separated by one or more insertion nucleotides or "gaps". In some embodiments, the ends are not directly connected to each other, but by one or more insertions (so-called "gaps" or "filling gaps" (oligo) nucleotides) of intermediates or by extending the 3' end of the probe to "fill" the "gap" corresponding to the insertion nucleotide and occur (intermolecular connection). In some cases, the gap of one or more nucleotides between the polynucleotide hybridization ends can be "filled" with one or more "gap" (oligo) nucleotides complementary to a splint, a circularizable probe or a probe group (e.g., a padlock probe) or a target nucleic acid. The gap can be a gap of 1 to 60 nucleotides or a gap of 1 to 40 nucleotides or a gap of 3 to 40 nucleotides. In a specific embodiment, the gap can be a gap of about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleotides, a gap of any integer (or integer range) of nucleotides between the values ​​shown. In some embodiments, the gap between the terminal regions can be filled by a gap oligonucleotide or by extending the 3' end of the polynucleotide. In some cases, the connection involves connecting the end of the probe to at least one gap (oligo) nucleotide so that the gap (oligo) nucleotide is incorporated into the resulting polynucleotide. In some embodiments, gap filling is performed before the connection herein. In other embodiments, the connection herein does not require gap filling.

[0361] In some embodiments, the melting temperature of the polynucleotide generated by polynucleotide ligation is higher than the melting temperature of the unligated polynucleotide.Thus, in some aspects, ligation stabilizes the hybridization complex containing the ligated polynucleotide prior to subsequent steps (including amplification and detection).

[0362] In some aspects, a high-fidelity ligase is used, such as a thermostable DNA ligase (e.g., Taq DNA ligase). The thermostable DNA ligase is active at elevated temperatures, allowing for the ligation of DNA strands at temperatures close to the melting temperature (T m This selectively reduces the concentration of annealed mismatched substrates (expected to have a slightly lower T around mismatches) compared to annealed fully base-paired substrates. m ). Therefore, high-fidelity ligation can be achieved through a combination of the intrinsic selectivity of the ligase active site and equilibrium conditions to reduce the incidence of annealing mismatched dsDNA.

[0363] iii. Amplification

[0364] In some embodiments, the methods of the present disclosure include the step of amplifying one or more polynucleotides, such as a circularizable probe or probe set (e.g., a padlock probe) or a circular probe formed by a padlock probe. In some embodiments, amplification is achieved by performing rolling circle amplification (RCA). In other embodiments, primers that hybridize to the padlock probe are added and also used for amplification.

[0365] In some embodiments, a removal step is performed to remove molecules that are not specifically hybridized to the target nucleic acid, reporter oligonucleotide, and / or circular probe. In some embodiments, a removal step is performed to remove unconnected probes. In some embodiments, a removal step is performed after connection and before amplification.

[0366] In some embodiments, the amplification is performed at a temperature equal to or approximately between 20° C. and 60° C. In some embodiments, the amplification is performed at a temperature between about 30° C. and about 40° C. In some aspects, the amplification step (such as rolling circle amplification (RCA)) is performed at a temperature between about 25° C. and about 50° C. (such as about 25° C., 27° C., 29° C., 31° C., 33° C., 35° C., 37° C., 39° C., 41° C., 43° C., 45° C., 47° C., or 49° C.).

[0367] In some embodiments, after adding DNA polymerase in the presence of appropriate dNTP precursors and other cofactors, primers are extended to produce multiple copies of circular templates. This amplification step can utilize isothermal amplification or non-isothermal amplification. In some embodiments, after hybridization complex formation and amplification probe combination, hybridization complex rolling circle amplification occurs and generates cDNA nano balls (e.g., amplicon) comprising multi-copy cDNA. Available rolling circle amplification (RCA) technology includes linear RCA, branched RCA, tree-like RCA or any combination thereof. See, e.g., Baner et al., Nucleic Acids Research, 26:5073-5078, 1998; Lizardi et al., Nature Genetics 19:226, 1998; Mohsen et al., Acc Chem Res. 2016 Nov 15;49(11):2540–2550; Schweitzer et al., Proc. Natl Acad. Sci. USA 97:101 13-1 19, 2000; Faruqi et al., BMC Genomics 2:4, 2000; Nallur et al., Nucl. Acids Res. 29:el 18, 2001; Dean et al., Genome Res. 11:1095-1099, 2001; Schweitzer et al., Nature Biotech.20:359-365,2002; U.S. Pat. Nos. 6,054,274, 6,291,187, 6,323,009, 6,344,329 and 6,368,801, all of which are incorporated herein by reference in their entirety. Exemplary polymerases for RCA include DNA polymerases such as phi29 (φ29) polymerase, Klenow fragment, Bacillus stearothermophilus DNA polymerase (BST), T4 DNA polymerase, T7 DNA polymerase or DNA polymerase I. In some aspects, a DNA polymerase that has been engineered or mutated to have the desired characteristics can be used. In some embodiments, the polymerase is phi29 DNA polymerase.

[0368] In some aspects, during the amplification step, modified nucleotides can be added to the reaction to incorporate modified nucleotides into the amplified product (e.g., nanospheres). Examples of modified nucleotides include amine-modified nucleotides. In some aspects of the method, for example, for anchoring or cross-linking the amplified product (e.g., nanospheres) produced to a support, a cell structure, and / or other amplified products (e.g., other nanospheres). In some aspects, the amplified product comprises modified nucleotides, such as amine-modified nucleotides. In some embodiments, amine-modified nucleotides include acrylic acid N-hydroxysuccinimide partial modifications. Examples of other amine-modified nucleotides include, but are not limited to, 5-aminoallyl-dUTP partial modifications, 5-propargylamino-dCTP partial modifications, N6-6-aminohexyl-dATP partial modifications, or 7-deaza-7-propargylamino-dATP partial modifications.

[0369] In some respects, polynucleotide and / or amplified product (e.g., amplicon) can be anchored to a polymer matrix. For example, the polymer matrix can be a hydrogel. In some embodiments, one or more polynucleotide probes can be modified to contain a functional group that can be used as an anchoring site for attaching polynucleotide probes and / or amplified product to the polymer matrix.

[0370] Exemplary modifications and polymer matrices that can be used according to the provided embodiments include, for example, those described in WO 2014 / 163886, US 10138509, WO2014 / 025392, US 10545075, WO 2017 / 079406, US 2016 / 0024555, US 2018 / 0251833, US2019 / 0276881, US2020 / 0071751, WO2020 / 076976, WO2020 / 076979, and WO2020 / 096687, which are incorporated herein by reference in their entirety. In some instances, the support also includes a modification or functional group that can react with a modification or functional group of a probe set or amplification product or incorporate a modification or functional group of a probe set or amplification product. In some examples, a scaffold may comprise oligonucleotides, polymers, or chemical groups to provide a matrix and / or support structure.

[0371] The amplified product can be fixed in the matrix, usually in the position where the nucleic acid is amplified, thereby producing a local colony of the amplicon. The amplified product can be fixed in the matrix by spatial factors. The amplified product can also be fixed in the matrix by covalent bonds or non-covalent bonds. In this way, it can be considered that the amplified product is connected to the matrix. By being fixed on the matrix, such as by covalent bonds or crosslinking, the size and spatial relationship of the original amplicon are maintained. By being fixed on the matrix, such as by covalent bonds or crosslinking, the amplified product is resistant to moving or spreading under mechanical stress.

[0372] In some aspects, the amplification products are copolymerized and / or covalently linked to the surrounding matrix, thereby retaining their spatial relationship and any inherent information thereof. For example, if the amplification products are those generated by DNA or RNA in cells embedded in the matrix, the amplification products can also be functionalized to form covalent connections with the matrix, retaining their spatial information in the cell, thereby providing a subcellular localization distribution pattern. In some embodiments, the provided method involves embedding one or more polynucleotide probe groups and / or amplification products in the presence of hydrogel subunits to form one or more hydrogel-embedded amplification products. In some embodiments, the described hydrogel-tissue chemistry includes covalently linking nucleic acids to hydrogels synthesized in situ for tissue clearance, enzyme diffusion, and multi-cycle sequencing, while existing hydrogel-tissue chemistry methods cannot. In some embodiments, in order to enable the amplification products to be embedded in the tissue-hydrogel setting, amine-modified nucleotides are included in the amplification step (e.g., RCA), functionalized to acrylamide moieties using acrylic acid N-hydroxysuccinimide esters, and copolymerized with acrylamide monomers to form a hydrogel.

[0373] iv. Detection and analysis

[0374] In some embodiments, the method disclosed herein includes using a plurality of primary probes to detect reporter oligonucleotides and / or one or more target nucleic acids in dissociated cells on a substrate, wherein the primary probes are configured to hybridize with reporter oligonucleotides and / or one or more target nucleic acids, wherein each primary probe comprises (i) a target hybridization region configured to hybridize with different target regions in the corresponding target nucleic acid, and (ii) a barcode region. In some embodiments, the dissociated cells on the substrate are contacted with a plurality of detectable probes, wherein each detectable probe is configured to hybridize with: (i) a barcode sequence in the barcode region of a plurality of primary probes, or (ii) a complementary sequence of a barcode sequence. In some embodiments, the method further includes detecting a signal or its absence associated with a plurality of detectable probes at one or more locations in the dissociated cells. In some embodiments, the dissociated cells on the substrate are contacted with a plurality of subsequent detectable probes, wherein each of the subsequent multiple detectable probes is configured to hybridize with: (i) a subsequent barcode sequence in the barcode region of a plurality of primary probes, or (ii) a complementary sequence of a subsequent barcode sequence. In some embodiments, the method also includes detecting subsequent signals or their absence associated with subsequent multiple detectable probes at one or more positions of the dissociated cell. In some embodiments, the method also includes generating a signal code sequence, the signal code sequence includes a signal code corresponding to a signal or its absence and a subsequent signal or its absence at one or more positions, respectively, wherein the signal code sequence corresponds to one of a reporter oligonucleotide or one or more target nucleic acids, thereby identifying a reporter oligonucleotide or target nucleic acid at one or more positions in the dissociated cell. Therefore, in situ detection of target nucleic acids in a specific dissociated cell can be associated with the sample source of the dissociated cell, because the reporter oligonucleotide detected in situ in a specific dissociated cell identifies the sample from which the specific dissociated cell is derived. In some embodiments, the method disclosed herein includes comparing the signal detected in the deposited labeled cell with the signal detected in one or more other cells in the colony. In some embodiments, before, during and / or after detecting a signal associated with a plurality of probes or their products, the dissociated cells deposited on the substrate are imaged. In some embodiments, before detecting a signal associated with a plurality of probes or their products, the deposited labeled cells are alive or fixed.

[0375] In some embodiments, in situ detection of a reporter oligonucleotide and / or one or more target nucleic acids in a dissociated cell is performed by sequential hybridization of a detectable probe with a plurality of primary probes, and using a signal decoding signal code sequence associated with the sequentially hybridized detectable probes, each signal code sequence being assigned to one of the reporter oligonucleotide or one or more target nucleic acids in the dissociated cell. Each primary probe can be selected from the group consisting of: a probe comprising a 3' overhang or a 5' overhang (e.g., an L-shaped probe), optionally wherein the 3' overhang or the 5' overhang comprises one or more barcode sequences; a probe comprising a 3' overhang and a 5' overhang (e.g., a U-shaped probe), optionally wherein the 3' overhang and the 5' overhang each independently comprise one or more barcode sequences; a circularized probe; a circularizable probe or probe set; a probe or probe set comprising a segmented hybridization region configured to hybridize to a splint, optionally wherein the segmented hybridization region comprises one or more barcode sequences; and combinations thereof.

[0376] In some embodiments, the present disclosure relates to in situ detection of nucleic acid sequences using probe hybridization and generating an amplified signal associated with the probe, wherein background signal is reduced and sensitivity is increased.

[0377] Exemplary in situ detection methods include targeted deposition of detectable reactive molecules around probe hybridization sites, targeted assembly of branched structures (e.g., bDNA or branched assays using locked nucleic acids (LNA)), programmed in situ growth of concatemers by enzymatic rolling circle amplification (RCA) (e.g., as described in US 2019 / 0055594, which is incorporated herein by reference), hybridization chain reaction, assembly of topological concatenated DNA structures using successive rounds of chemical ligation (clampFISH), signal amplification by hairpin-mediated concatenation (e.g., as described in US 2020 / 0362398, which is incorporated herein by reference), such as primer exchange reactions, such as signal amplification by exchange reactions (SABER) or SABER with DNA exchange (exchange-SABER). In some embodiments, non-enzymatic signal amplification methods can be used.

[0378] In some embodiments, the method disclosed herein includes generating a rolling circle amplification (RCA) product associated with a reporter oligonucleotide and / or one or more target nucleic acids in a dissociated cell on a substrate. In some embodiments, the RCA product is detected in situ in a dissociated cell on a substrate, thereby detecting a reporter oligonucleotide and / or one or more target nucleic acids. In some embodiments, each RCA product comprises multiple complementary copies of a barcode sequence, wherein the barcode sequence is associated with a reporter oligonucleotide or a target nucleic acid in a dissociated cell, and a signal code sequence is assigned. In some embodiments, the method includes contacting the dissociated cell with a first detectable probe, wherein the first detectable probe comprises (i) a recognition sequence complementary to a sequence in a complementary copy of the barcode sequence and (ii) a reporter molecule. In some embodiments, the method includes detecting a first signal or its absence from a reporter molecule of a first detectable probe hybridized to a corresponding sequence of a complementary copy of a barcode sequence in an RCA product, wherein the first signal or its absence corresponds to a first signal code in a signal code sequence. In some embodiments, the method includes contacting the dissociated cell with a subsequent detectable probe, the subsequent detectable probe comprising (i) a recognition sequence complementary to the sequence of the complementary copy of the barcode sequence and (ii) a reporter molecule. In some embodiments, the method includes detecting a subsequent signal or its absence from a reporter molecule of a subsequent detectable probe hybridized with a corresponding sequence of a complementary copy of the barcode sequence in the RCA product, wherein the subsequent signal or its absence corresponds to a subsequent signal code in a signal code sequence. In some embodiments, a signal code sequence comprising a first signal code and a subsequent signal code is determined at a position in the dissociated cell, thereby decoding the barcode sequence and identifying the target analyte at the position in the dissociated cell. In this way, in situ detection of a target analyte (e.g., a target nucleic acid) in a specific dissociated cell can be associated with the sample source of the dissociated cell, because the reporter oligonucleotide detected in situ in the specific dissociated cell identifies the sample from which the specific dissociated cell is derived.

[0379] In some embodiments, the barcode sequence comprises one or more barcode positions, each position comprising one or more barcode subunits. In some embodiments, the barcode position in the barcode sequence partially overlaps with the adjacent barcode position in the barcode sequence. In some embodiments, the first detectable probe and the subsequent detectable probe are in a group of detectable probes, each probe comprising the same recognition sequence and reporter molecule. In some embodiments, the reporter molecule of each detectable probe in the group comprises a binding site for the reporter probe containing a detectable portion. In some embodiments, the reporter probe binding site of the first detectable probe and the reporter probe binding site of the subsequent detectable probe are the same. In some embodiments, the reporter probe binding site of the first detectable probe and the reporter probe binding site of the subsequent detectable probe are different. In some embodiments, the detectable portion is a fluorophore, and the signal code sequence is a fluorophore sequence specifically assigned to the target analyte. In some embodiments, the detectable probes in the group are sequentially contacted with the dissociated cells in a predetermined order, and the predetermined order corresponds to the signal code sequence assigned to the barcode sequence. In some embodiments, the detectable probes in the panel are contacted with the dissociated cells to determine the signal codes in the signal code sequence until sufficient signal codes have been determined to decode the barcode sequence, thereby identifying the target analyte.

[0380] The detectable reactive molecule may include tyramide, such as for tyramide signal amplification (TSA) or multiple catalytic reporter deposition (CARD)-FISH. In some embodiments, the detectable reactive molecule may be released and / or cut from a detectable label such as a fluorophore. In some embodiments, the methods disclosed herein include multiplex analysis of biological samples, including probe hybridization, continuous cycles of fluorescence imaging and signal removal, wherein signal removal includes removing the fluorophore from a reactive molecule (e.g., tyramide) labeled with a fluorophore. Exemplary detectable reaction reagents and methods are described in US 6,828,109, US 2019 / 0376956, WO2019 / 236841, WO 2020 / 102094, US 20220026433, WO 2020 / 163397, US 20220128565 and WO2021 / 067475, US 20210222234, all of which are incorporated herein by reference in their entirety.

[0381] In some embodiments, hybridization chain reaction (HCR) can be used for in situ signal detection.HCR is an enzyme-free nucleic acid amplification based on the hybridization trigger chain of nucleic acid molecules, starting from HCR monomers, these monomers hybridize to form a nicked nucleic acid polymer.The polymer is the product of HCR reactions, which is ultimately detected to indicate the presence of a target analyte. HCR is described in detail in the following literature: Dirks and Pierce, 2004, PNAS, 101 (43), 15275-15278; and US 7,632,641 and US 7,721,721 (see also US 2006 / 00234261; Chemeris et al., 2008 Doklady Biochemistry and Biophysics, 419, 53-55; Niu et al., 2010, 46, 3089-3091; Choi et al., 2010, Nat. Biotechnol. 28 (11), 1208-1212; and Song et al., 2012, Analyst, 137, 1396-1401). HCR monomers typically contain hairpins or other metastable nucleic acid structures. In the simplest HCR form, when introducing " initiator " nucleic acid molecule, two different types of stable hairpin monomers (here referred to as the first and second HCR monomers) experience hybridization chain reaction events, forming a double-stranded DNA molecule with long nicks.HCR monomer has a hairpin structure, which comprises a double-stranded stem region, a loop region connecting two chains in the stem region and a single-stranded region at one end of the double-stranded stem region.When monomer is in a hairpin structure, the single-stranded region exposed (and therefore can be used for hybridization with another molecule such as initiator or other HCR monomers) can be referred to as "foothold region" (or "input domain").The first HCR monomer also includes a sequence complementary to the sequence in the foothold region exposed with the second HCR monomer.This complementary sequence in the first HCR monomer can be referred to as "interaction region" (or "output domain").Similarly, the second HCR monomer includes an interaction region (output domain) each, such as a sequence complementary to the foothold region (input domain) exposed with the first HCR monomer. In the absence of HCR initiators, these interaction regions are protected by secondary structures (e.g., they are not exposed), so hairpin monomers are stable or dynamically trapped (also referred to as "metastable"), and remain as monomers (e.g., to prevent rapid system equilibrium), because the first and second groups of HCR monomers cannot hybridize with each other. However, once an initiator is introduced, it is able to hybridize with the exposed foothold region of the first HCR monomer, and invade it, causing it to open. This will expose the interaction region of the first HCR monomer (e.g., a sequence complementary to the foothold region of the second HCR monomer), thereby allowing it to hybridize with the second HCR monomer at the foothold region and invade the second HCR monomer.This hybridization and invasion and then open the second HCR monomer, expose its interaction zone (it is complementary to the foothold zone of the first HCR monomer), and allow it to hybridize with another first HCR monomer and invade another first HCR monomer.Reaction continues in this way, until all HCR monomers are exhausted (for example, all HCR monomers are incorporated into polymer chain).Finally, this chain reaction causes the notched chain of the alternating unit forming the first and second monomer species.Therefore, it is necessary to have the presence of HCR initiator in order to trigger HCR reaction by hybridizing with the first HCR monomer and invading the first HCR monomer.The first and second HCR monomers are designed to hybridize each other and therefore can be defined as homologous to each other.They are also homologous to given HCR initiator sequence.The HCR monomer interacting with each other (hybridization) can be described as a group of HCR monomer or HCR monomer or hairpin system.

[0382] The HCR reaction can be carried out with more than two kinds or types of HCR monomers.For example, a system involving three HCR monomers can be used.In such a system, each first HCR monomer can include an interaction zone combined with the foothold area of ​​the second HCR monomer; Each second HCR can include an interaction zone combined with the foothold area of ​​the 3rd HCR monomer; and each 3rd HCR monomer can include an interaction zone combined with the foothold area of ​​the first HCR monomer.Then, the HCR polymerization reaction will be carried out as described above, except that the product therefrom will be a polymer with a continuous first, second and third monomer repeating unit.It is easy to think of a corresponding system with a large amount of HCR monomer groups.

[0383] In some embodiments, similar to the HCR reaction using hairpin monomers, linear oligo hybridization chain reaction (LO-HCR) can be used for in situ signal detection. In some embodiments, a method for detecting an analyte in a sample is provided herein, comprising: (i) performing a linear oligonucleotide hybridization chain reaction (LO-HCR), wherein an initiator is contacted with a plurality of LO-HCR monomers of at least the first and second species to generate a polymerized LO-HCR product hybridized with a target nucleic acid molecule, wherein the first species comprises a first hybridization region complementary to the initiator and a second hybridization region complementary to the second species, wherein the first species and the second species are linear single-stranded nucleic acid molecules; wherein the initiator is provided in one or more parts and is directly or indirectly hybridized with the target nucleic acid molecule or is contained in the target nucleic acid molecule; and (ii) detecting the polymer product, thereby detecting the analyte. In some embodiments, the first species and / or the second species may not contain a hairpin structure. In some embodiments, the plurality of LO-HCR monomers may not contain a metastable secondary structure. In some embodiments, the LO-HCR polymer may not contain a branched structure. In some embodiments, performing a linear oligonucleotide hybridization chain reaction comprises contacting the target nucleic acid molecule with an initiator to provide the initiator to hybridize to the target nucleic acid molecule.In any of the embodiments herein, the target nucleic acid molecule and / or the analyte may be an RCA product.

[0384] In some embodiments, the in situ detection of nucleic acid sequences includes a combination of RCA and a component for branch signal amplification. In some embodiments, the component complex includes an amplifier that directly or indirectly (through one or more oligonucleotides) hybridizes with the sequence of the RCA product. In some embodiments, the component includes one or more amplifiers, each amplifier including an amplifier repeat sequence. In some aspects, the one or more amplifiers are labeled. This article describes a method using the aforementioned components, which includes, for example, using the component in a multiple error robust fluorescence in situ hybridization (MERFISH) application, wherein branch DNA amplification is used for signal readout. In some embodiments, the amplifier repeat sequence is about 5-30 nucleotides, and is repeated N times in the amplifier. In some embodiments, the amplifier repeat sequence is about 20 nucleotides, and is repeated at least twice in the amplifier. In some aspects, the one or more amplifier repeat sequences are labeled. For exemplary branched signal amplification, see, e.g., U.S. Patent Publication Nos. US20200399689A1, WO 2020 / 123742, US 20220064697, and Xia et al., Multiplexed Detection of RNA using MERFISH and branched DNA amplification. Scientific Reports (2019), each of which is incorporated herein by reference in its entirety.

[0385] In some embodiments, the in situ detection of nucleic acid sequence includes primer exchange reaction (PER). In various embodiments, a primer with a domain at its 3' end is combined with a catalytic hairpin, and a new domain is extended by a strand displacement polymerase. For example, a primer with domain 1 at its 3' end is combined with a catalytic hairpin, and a new domain 1 is extended by a strand displacement polymerase, and a concatemer of a repeating domain 1 sequence is generated by repeated cycles. In various embodiments, the strand displacement polymerase is Bst. In various embodiments, the catalytic hairpin includes a stopper for releasing a strand displacement polymerase. In various embodiments, branch migration displaces the extended primer, which can then dissociate. In various embodiments, primers undergo repeated cycles to form concatemer primers. In various embodiments, a variety of concatemer primers are contacted with a sample comprising an RCA product produced using the method described herein. In various embodiments, RCA products can be contacted with multiple concatemer primers and multiple labeled probes. Regarding exemplary molecules and PER reaction components, see, for example, U.S. Patent Publication No. US20190106733, which is incorporated herein by reference.

[0386] In some embodiments, the reporter oligonucleotide and the sample identification probe directly or indirectly combined therewith may include a region configured to hybridize with a detectable probe or probe group. In some embodiments, the detectable probe or probe group is circular or cyclizable to produce a circularized template. In some embodiments, the circular probe or circular template is used to produce an RCA product. In some embodiments, the reporter oligonucleotide and the sample identification probe directly or indirectly combined therewith may include a region as an initiator of a hybridization chain reaction (HCR) or hybridized with an initiator of HCR. In some embodiments, the reporter oligonucleotide and the sample identification probe directly or indirectly combined therewith may include a region as an initiator of a linear oligonucleotide hybridization chain reaction (LO-HCR) or hybridized with an initiator of LO-HCR. In some embodiments, the reporter oligonucleotide and the sample identification probe directly or indirectly combined therewith may include a region as a primer of a primer exchange reaction (PER) or hybridized with a primer of PER. In some embodiments, the reporter oligonucleotide and the sample identification probe directly or indirectly combined therewith may include a region as a preamplifier of a branched DNA (bDNA) or hybridized with a preamplifier of a bDNA.

[0387] In some embodiments, the analyte detection probe may include a region configured to hybridize with a detectable probe or probe groups. In some embodiments, detectable probes or probe groups are circular or cyclizable, to produce a cyclized template. In some embodiments, circular probes or circular templates are used to produce RCA products. In some embodiments, the analyte detection probe may include a region as an initiator of hybridization chain reaction (HCR) or hybridized with an initiator of HCR. In some embodiments, the analyte detection probe may include an initiator of linear oligonucleotide hybridization chain reaction (LO-HCR) or hybridized with an initiator of LO-HCR. In some embodiments, the analyte detection probe may include a primer as a primer exchange reaction (PER) or hybridized with a primer of PER. In some embodiments, the analyte detection probe may include a region as a preamplifier of branched DNA (bDNA) or hybridized with a preamplifier of bDNA.

[0388] exist Figure 4 In, the second probe (e.g., analyte detection probe) is shown as a detectably labeled probe. However, it should be understood that the second probe does not need to be detectably labeled. In some instances, the second probe or its product can be detected by a detectable probe that hybridizes to the detectably labeled probe. For example, the second probe can be a circularizable probe that is circularized and amplified in situ (e.g., by rolling circle amplification, RCA) to produce an amplification product that is detected by a detectable probe (e.g., an intermediate probe) that hybridizes to the amplification product and a fluorescently labeled probe. In Figure 5 In, the RCA product can include multiple copies of the hybridization region (for example, one or more barcode sequences) bound by the intermediate probe. The intermediate probe can include 3' overhangs and / or 5' overhangs, which include hybridization regions hybridized with a detectably labeled probe providing a second signal. The reporter oligonucleotide (for example, by producing a reporter oligonucleotide or a probe or probe group hybridized therewith) can be detected similarly, or by detecting the first signal associated with the first probe hybridized with the reporter oligonucleotide, as shown in the figure.

[0389] In some cases, the first probe (e.g., a sample identification probe) and / or the second probe (e.g., an analyte detection probe) may comprise one or more features and / or be modified based on: a split FISH probe or probe set, as described in WO2021 / 167526A1 or Goh et al., "Highly specific multiplexed RNA imaging in tissues with split-FISH," Nat Methods 17(7):689-693 (2020), the entirety of which is incorporated herein by reference; a Z-probe or probe set, such as US 7,709,198 B2, US 8,604,182 B2, US 8,951,726 B2, US 8,658,361 B2, or Tripathi et al., "Z Probe, An Efficient Tool for Characterizing Long Non-Coding RNA in FFPE Tissues," Noncoding RNA 4(3):20(2018), which is incorporated herein by reference in its entirety;HCR initiators or amplifiers, such as those described in US 7,632,641 B2, US 2017 / 0009278 A1, US 10,450,599 B2, Dirks and Pierce, "Triggered amplification by hybridization chain reaction," PNAS 101(43):15275-15278 (2004), Chemeris et al., "Real-time hybridization chain reaction," Dokl. Biochem 419:53-55 (2008), Niu et al., "Fluorescence detection for DNA using hybridization chain reaction with enzyme-amplification," Chem Commun (Camb) 46(18):3089-91 (2010), Choi et al., "Programmable in situ amplification for multiplexed imaging of mRNA expression," Nat Biotechnol 28(11):1208-12(2010), Song et al., "Hybridization chain reaction-based aptameric system for the highly selective and sensitive detection of protein," Analyst 137(6):1396-401(2012), Choi et al., "Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust," Development 145(12):dev165753(2018), or Tsuneoka and Funato, "Modified in situ Hybridization Chain Reaction Using Short HairpinDNAs," Front Mol Neurosci 13:75(2020), the entire text of which is incorporated herein by reference;PLAYR probes or probe sets, such as those described in US 2016 / 0108458 A1 or Frei et al., "Highly multiplexed simultaneous detection of RNAs and proteins in single cells," Nat Methods 13(3):269-75 (2016), which are incorporated herein by reference in their entirety; PLISH probes or probe sets, such as those described in US 2020 / 0224243 A1 or Nagendran et al., "Automated cell-type classification in intact tissues by single-cell molecular profiling," eLife 7:e30510 (2018), which are incorporated herein...

Claims

1. A method for analysis, comprising: (a) providing a cell population comprising labeled cells, wherein the labeled cells are cells labeled with a labeling agent comprising a binding moiety and a reporter oligonucleotide, wherein the reporter oligonucleotide comprises a sample-specific barcode sequence corresponding to a sample from which the labeled cells are derived, and wherein the binding moiety binds to the cells; (b) depositing the cell population comprising the labeled cells on a substrate to provide deposited labeled cells; (c) contacting the deposited labeled cells with: (i) a first probe that directly or indirectly binds to the sample-specific barcode sequence, and (ii) a second probe that directly or indirectly binds to an analyte in and / or on the deposited labeled cells; as well as (d) detecting a first signal associated with the first probe or its product and a second signal associated with the second probe or its product in the deposited labeled cells.

2. The method of claim 1, wherein the binding moiety is covalently bound to one or more molecules in and / or on the cell, optionally wherein the binding moiety is bound to one or more molecules in and / or on the cell via a linker.

3. The method of claim 1 or 2, wherein the binding moiety non-covalently binds to one or more molecules in and / or on the cell, optionally wherein the binding moiety binds to one or more molecules in and / or on the cell via a linker.

4. The method of any one of claims 1 to 3, wherein the binding moiety binds to the surface of the cell.

5. The method of any one of claims 1 to 4, wherein the binding moiety comprises a polynucleotide or an analog thereof, a polypeptide or an analog thereof, a lipid or an analog thereof, a carbohydrate or an analog thereof, or a combination thereof.

6. The method of any one of claims 1 to 5, wherein the binding moiety is selected from the group consisting of: an antibody or an epitope-binding fragment thereof; a lipophilic moiety, which is optionally cholesterol; a receptor; a receptor ligand; a small molecule; an aptamer; a monomer; an affimer; a darpin; and a protein scaffold.

7. The method of any one of claims 1 to 6, wherein the marker further comprises a target-specific barcode sequence for identifying the marker bound to the labeled cells, optionally wherein the reporter oligonucleotide further comprises the target-specific barcode sequence, and optionally wherein the target-specific barcode sequence and the sample-specific barcode sequence at least partially overlap in sequence.

8. The method of any one of claims 1 to 7, (i) wherein the labeled cells are not in a partition before, during and / or after the providing in (a); or (ii) wherein the labeled cells are not in a partition during the depositing in (b), optionally wherein the partition is an emulsion droplet or a microwell of a microwell array.

9. The method of any one of claims 1 to 8, wherein the labeled cells are not lysed before and / or during the depositing in (b).

10. The method of any one of claims 1 to 9, wherein between the providing in (a) and the depositing in (b), the nucleic acid of the labeled cell is not extended by a polymerase and / or reverse transcribed, not linked to another nucleic acid, not amplified and / or sequenced, optionally wherein the nucleic acid is the reporter oligonucleotide or a cellular DNA or RNA molecule of the cell.

11. The method of any one of claims 1 to 10, wherein between the providing in (a) and the depositing in (b), the nucleic acid marking the cell is not released from the cell, optionally wherein the nucleic acid is the reporter oligonucleotide or a cellular DNA or RNA molecule of the cell.

12. The method of any one of claims 1 to 11, wherein the first probe hybridizes to the sample-specific barcode sequence, optionally wherein the first probe comprises a detectable label, and wherein detecting the first signal associated with the first probe comprises detecting the detectable label, optionally wherein the detectable label is an optical label, and optionally wherein detecting the first signal associated with the first probe comprises detecting the optical label using imaging.

13. The method of claim 12, wherein the first probe is selected from the group consisting of: a probe comprising a 3' overhang or a 5' overhang after hybridization with the sample-specific barcode sequence, optionally wherein the 3' overhang or the 5' overhang comprises one or more additional barcode sequences that identify the sample-specific barcode sequence; a probe comprising a 3' overhang and a 5' overhang after hybridization with the sample-specific barcode sequence, optionally wherein the 3' overhang and the 5' overhang each independently comprise one or more additional barcode sequences that identify the sample-specific barcode sequence; a circle probe, Optionally, the circularized probe comprises one or more additional barcode sequences that identify the sample-specific barcode sequence; a circularizable probe or probe set, optionally wherein the circularizable probe or probe set comprises one or more additional barcode sequences that identify the sample-specific barcode sequence; a probe or probe set comprising a segmented hybridization region configured to hybridize to a splint, optionally wherein the segmented hybridization region comprises one or more additional barcode sequences; and combinations thereof, and optionally wherein detecting the first signal associated with the first probe comprises detecting at least a portion of the one or more additional barcode sequences.

14. The method of any one of claims 1 to 13, further comprising hybridizing a first detectable probe to the first probe and / or generating a first product of the first probe and / or the first detectable probe.

15. The method of claim 14, wherein the first product is a hybridization product, a ligation product, an amplification product or a combination thereof, optionally wherein the first product comprises a branched structure, a hybridization chain reaction (HCR) product, a linear-oligomer hybridization chain reaction (LO-HCR) and / or a rolling circle amplification (RCA) product.

16. The method of any one of claims 1 to 15, wherein the analyte is a nucleic acid analyte.

17. The method of claim 16, wherein the analyte is a cellular RNA molecule.

18. The method of any one of claims 1 to 15, wherein the analyte is a polypeptide.

19. The method of any one of claims 1 to 18, wherein the binding portion of the labeling agent does not bind to the analyte.

20. A method as described in any one of claims 1 to 19, wherein the second probe hybridizes to the analyte or a sequence in a probe targeting the analyte, optionally wherein the probe targeting the analyte is constructed to bind directly or indirectly to the analyte, and optionally wherein the second probe comprises a detectable label, and optionally wherein detecting the second signal associated with the second probe comprises detecting the detectable label, optionally wherein the detectable label is an optical label, and optionally wherein detecting the second signal associated with the second probe comprises detecting the optical label using imaging.

21. The method of claim 20, wherein the second probe is selected from the group consisting of: a probe comprising a 3' overhang or a 5' overhang after hybridization with the analyte or a sequence in the probe targeting the analyte, optionally wherein the 3' overhang or the 5' overhang comprises one or more additional barcode sequences that identify the analyte; a probe comprising a 3' overhang and a 5' overhang after hybridization with the analyte or a sequence in the probe targeting the analyte, optionally wherein the 3' overhang and the 5' overhang each independently comprise one or more additional barcode sequences that collectively identify the analyte; a circular probe, optionally wherein the circular probe comprises one or more additional barcode sequences that identify the analyte; a circularizable probe or probe set, optionally wherein the circularizable probe or probe set comprises one or more additional barcode sequences that identify the analyte; a probe or probe set comprising a segmented hybridization region configured to hybridize to a splint, optionally wherein the segmented hybridization region comprises one or more additional barcode sequences that identify the analyte; and combinations thereof, and optionally wherein detecting the second signal associated with the second probe comprises detecting at least a portion of the one or more additional barcode sequences.

22. The method of any one of claims 1 to 21, further comprising hybridizing a second detectable probe to the second probe and / or generating a second product of the second probe and / or the second detectable probe.

23. The method of claim 22, wherein the second product is a hybridization product, a ligation product, an amplification product or a combination thereof, optionally wherein the second product comprises a branched structure, a hybridization chain reaction (HCR) product, a linear-oligomer hybridization chain reaction (LO-HCR) and / or a rolling circle amplification (RCA) product.

24. The method of any one of claims 1 to 23, further comprising comparing a signal detected in the deposited labeled cell to a signal detected in one or more other cells in the population.

25. The method of any one of claims 1 to 24, wherein the population comprises at least about 100, at least about 1,000, at least about 10,000, at least about 50,000, at least about 100,000, or at least about 500,000 dissociated cells, optionally wherein the dissociated cells are immobilized within about 0.1 cm of the substrate. 2 、About 0.5cm 2 , about 1cm 2 , about 2cm 2 、About 5cm 2 or about 10cm 2 and optionally wherein at least about 100,000 dissociated cells are immobilized within an area of ​​about 1 cm on the substrate. 2 Inside.

26. The method of any one of claims 1 to 25, further comprising imaging the deposited labeled cells before, during and / or after the detection in (d), optionally wherein the imaging comprises imaging the shape, size, morphological features and / or markers of the deposited labeled cells or a portion thereof, optionally wherein the markers are fluorescent markers.

27. The method of any one of claims 1 to 26, wherein prior to said detecting in (d), said deposited labeled cells are fixed.

28. The method of any one of claims 1 to 27, which does not comprise introducing an exogenous polynucleotide into the cells of the cell population prior to labeling the cells with the labeling agent comprising the sample-specific barcode sequence, or which does not comprise introducing an exogenous polynucleotide comprising a barcode sequence into the cells of the cell population prior to labeling the cells with the labeling agent comprising the sample-specific barcode sequence.

29. The method of claim 28, wherein the exogenous polynucleotide comprises or encodes a gene, a modified / damaged / non-natural nucleotide or nucleotide analog, an overexpressed gene, an RNAi-based system, a regulatory RNA, a non-coding RNA, an mRNA, a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), a meganuclease, a computationally designed protein, a computationally designed RNA, and / or a CRISPR-Cas system.

30. The method of any one of claims 1 to 29, wherein the cell population is dissociated cells or a cell suspension.

31. A method for cell analysis, comprising: (a) providing a cell population comprising labeled cells, wherein the labeled cells are labeled with a labeling agent comprising (i) a binding moiety and (ii) a reporter oligonucleotide comprising a first barcode region, and wherein the binding moiety binds to the cells; (b) fixing the cell population comprising the labeled cells on a substrate; (c) contacting the immobilized cells with: (i) a first probe that hybridizes to the first barcode region, and (ii) a second probe that directly or indirectly binds to an analyte in and / or on one or more of the immobilized cells, wherein the second probe comprises a second barcode region; and (d) detecting a first signal associated with the first probe or its product and a second signal associated with the second probe or its product on the substrate, thereby detecting the presence or absence of the analyte in the immobilized labeled cells.

32. A method as claimed in claim 31, wherein before, during and / or after the providing in step (a) or during the fixation in step (b), the cells in the population are not separated into partitions, optionally wherein the partitions are emulsion droplets and / or microwells.

33. The method of claim 31 or 32, wherein the first barcode region comprises (i) a sample-specific barcode sequence corresponding to the sample from which the labeled cell originates, and optionally (ii) a target-specific barcode sequence corresponding to the binding moiety or its target, and / or optionally (iii) a barcode sequence corresponding to a cellular characteristic.

34. The method of any one of claims 31 to 33, wherein the second barcode region comprises a binding site for a detectable probe, optionally wherein the detectable probe comprises: (i) Fluorescently labeled probes, (ii) an intermediate probe comprising a binding site for another detectable probe, optionally wherein the binding site is a barcode sequence, and / or (iii) a circular probe or circularizable probe or probe set, wherein the rolling circle amplification (RCA) product comprises multiple binding sites for a further detectable probe.

35. A method for sample analysis, comprising: (a) contacting a first sample and a second sample with a first labeling agent and a second labeling agent, respectively, to provide labeled cells in each sample, wherein the first labeling agent and the second labeling agent each comprise (i) a binding moiety and (ii) a reporter oligonucleotide comprising a first barcode region, wherein the first barcode region comprises a sample-specific barcode sequence corresponding to the respective sample, wherein the binding portion of each labeling agent binds to one or more dissociated cells in the corresponding sample; (b) combining labeled cells from the first sample and the second sample; (c) fixing a cell population comprising labeled cells from the first sample and the second sample on a substrate; (d) contacting the immobilized cells with: (i) a first probe that hybridizes to the sample-specific barcode sequence corresponding to the first sample or the second sample, and (ii) a second probe that directly or indirectly binds to an analyte in and / or on one or more of the immobilized cells, wherein the second probe comprises a second barcode region; and (e) detecting a first signal associated with the first probe or its product and a second signal associated with the second probe or its product on the substrate, thereby detecting the presence or absence of the analyte in one or more cells from the first sample and / or the second sample.

36. The method of claim 35, wherein the first sample and / or the second sample comprises dissociated cells.

37. A method for sample analysis, comprising: (a) contacting a first sample and a second sample with a first labeling agent and a second labeling agent, respectively, to provide labeled cells in each sample, wherein the first labeling agent and the second labeling agent each comprise (i) a binding moiety and (ii) a reporter oligonucleotide comprising a first barcode region, wherein the first barcode region comprises a sample-specific barcode sequence corresponding to the respective sample, wherein the binding portion of each labeling agent binds to one or more cells in the corresponding sample; (b) combining the first sample and the second sample; (c) fixing a dissociated cell population comprising labeled cells from the first sample and the second sample on a substrate; (d) contacting the immobilized cells with: (i) a first probe that hybridizes to the sample-specific barcode sequence corresponding to the first sample or the second sample, and (ii) a second probe that directly or indirectly binds to an analyte in and / or on one or more of the immobilized cells, wherein the second probe comprises a second barcode region; and (e) detecting a first signal associated with the first probe or its product and a second signal associated with the second probe or its product on the substrate, thereby detecting the presence or absence of the analyte in one or more cells from the first sample and / or the second sample.

38. The method of claim 37, wherein the first sample and the second sample are combined prior to processing the first sample and the second sample in (b) to provide the dissociated cell population in (c).

39. The method of claim 37, wherein the first sample and the second sample are separately processed to provide dissociated cells prior to combining the dissociated cells in (b) to provide the dissociated cell population in (c).

40. The method of any one of claims 35 to 39, wherein the first probe and / or the second barcode region comprises one or more barcode sequences, optionally wherein the first probe and / or the second probe is a circularizable probe.

41. A method as claimed in claim 40, wherein the first signal and / or the second signal is associated with a rolling circle amplification (RCA) product of a circularization probe comprising the sequence of the circularizable probe, wherein the RCA product comprises multiple copies of a complementary sequence to the one or more barcode sequences, and wherein the first signal and / or the second signal is detected using a detectable probe that hybridizes to the complementary sequence, optionally wherein the detectable probe is detectably labeled or comprises a sequence that hybridizes to a detectably labeled probe.

42. A method as described in claim 40 or claim 41, comprising using multiple detectable probes to detect signals associated with the one or more barcode sequences in continuous cycles to detect the presence or absence of the analyte in one or more cells from the first sample and / or the second sample.

43. A method for sample analysis, comprising: (a) contacting a first cell population with a first labeling agent to provide a first plurality of labeled cells, and contacting a second cell population with a second labeling agent to provide a second plurality of labeled cells, wherein the first labeling agent comprises (i) a first binding moiety and (ii) a first reporter oligonucleotide, the first reporter oligonucleotide comprising a first sample-specific barcode sequence that identifies the first cell population, wherein the second labeling agent comprises (i) a second binding moiety and (ii) a second reporter oligonucleotide, the second reporter oligonucleotide comprising a second sample-specific barcode sequence that identifies the second cell population; (b) combining cells from the first plurality of labeled cells and the second plurality of labeled cells to provide a combined plurality of labeled cells; (c) fixing the merged plurality of labeled cells on a substrate; (d) contacting the immobilized cells with: (i) a first probe that hybridizes to the first sample-specific barcode sequence corresponding to the first sample or the second sample, (ii) a second probe that hybridizes to the second sample-specific barcode sequence corresponding to the first sample or the second sample, and (iii) a third probe or probe set that binds directly or indirectly to one or more analytes in and / or on one or more of the immobilized cells; and (e) Detection: (i) identifying the first probe of the first sample-specific barcode sequence; (ii) identifying the second probe that is specific to the second sample and that is specific to the barcode sequence; as well as (iii) a third probe or probe set identifying the one or more analytes in and / or on one or more of the immobilized cells; as well as (f) using the first sample-specific barcode sequence to identify cells from the first population of cells, and using the second sample-specific barcode sequence to identify cells from the second population of cells.

44. A method as claimed in claim 43, wherein the first binding moiety and / or the second binding moiety are covalently bound to one or more molecules in and / or on cells of the first cell colony or the second cell colony, optionally wherein the first binding moiety and / or the second binding moiety are bound to one or more molecules in and / or on cells of the first cell colony or the second cell colony via a linker.

45. A method as described in claim 43 or 44, wherein the first binding moiety and / or the second binding moiety non-covalently binds to one or more molecules in and / or on cells of the first cell colony or the second cell colony, optionally wherein the first binding moiety and / or the second binding moiety binds to one or more molecules in and / or on cells of the first cell colony or the second cell colony via a linker.

46. ​​The method of any one of claims 43 to 45, wherein the first binding moiety and / or the second binding moiety binds to the cell surface of the first cell population or the second cell population.

47. The method of any one of claims 43 to 46, wherein the binding moiety is selected from the group consisting of: an antibody or epitope-binding fragment thereof; a lipophilic moiety, which is optionally cholesterol; a receptor; a receptor ligand; a small molecule; an aptamer; a monomer; an affimer; a darpin; and a protein scaffold.

48. The method of any one of claims 43 to 47, wherein the first plurality of labeled cells and the second plurality of labeled cells are not in partitions, optionally wherein the partitions are emulsion droplets or microwells of a microwell array.

49. The method of any one of claims 43 to 48, wherein the first plurality of labeled cells and the second plurality of labeled cells are not lysed prior to and / or during the fixation in (c).

50. The method of any one of claims 43 to 49, (i) wherein the first probe comprises a first detectable label, and wherein detecting the first probe comprises detecting the first detectable label, optionally wherein the first detectable label is a first optical label, and optionally wherein detecting the first probe comprises detecting the first optical label using imaging; and / or (ii) wherein the second probe comprises a second detectable label, and wherein detecting the second probe comprises detecting the second detectable label, optionally wherein the second detectable label is a second optical label, and optionally wherein detecting the second probe comprises detecting the second optical label using imaging.

51. The method of any one of claims 43 to 50, wherein the one or more analytes are one or more nucleic acid analytes.

52. The method of claim 51, wherein the one or more nucleic acid analytes are cellular RNA molecules.

53. The method of any one of claims 43 to 50, wherein the one or more analytes are one or more polypeptides.

54. The method of any one of claims 43 to 53, wherein the third probe or probe set comprises: a probe comprising a 3' overhang or a 5' overhang upon hybridization to a sequence in one of the one or more analytes, optionally wherein the 3' overhang or the 5' overhang comprises one or more additional barcode sequences that identify the analyte; a probe comprising a 3' overhang and a 5' overhang upon hybridization to a sequence in one of the one or more analytes, optionally wherein the 3' overhang and the 5' overhang each independently comprise one or more additional barcode sequences that jointly identify the analyte. a barcode sequence; a circular probe that hybridizes to one of the one or more analytes, optionally wherein the circular probe comprises one or more additional barcode sequences that identify the analyte; a circularizable probe or probe set that hybridizes to one of the one or more analytes, optionally wherein the circularizable probe or probe set comprises one or more additional barcode sequences that identify the analyte; a probe or probe set comprising a segmented hybridization region configured to hybridize to a splint; or any combination thereof, and optionally wherein detecting the third probe or probe set comprises detecting at least a portion of the one or more additional barcode sequences.

55. The method of any one of claims 43 to 54, wherein the first cell population and / or the second cell population each comprises at least about 100, at least about 1,000, at least about 10,000, at least about 50,000, at least about 100,000, or at least about 500,000 dissociated cells, optionally wherein the combined plurality of labeled cells are immobilized within about 0.1 cm of the substrate. 2 、About 0.5cm 2 , about 1cm 2 , about 2cm 2 、About 5cm 2 or about 10cm 2 and optionally wherein at least about 100,000 of said merged plurality of labeled cells are immobilized within an area of ​​about 1 cm on said substrate. 2 Inside.

56. The method of any one of claims 43 to 55, further comprising imaging at least some of the immobilized cells before, during and / or after the detecting in (e), optionally wherein the imaging comprises imaging the shape, size, morphological features and / or markers of the immobilized cells, optionally wherein the markers are fluorescent markers.

57. The method of any one of claims 43 to 56, wherein the immobilized cells are fixed prior to the detecting in (e).

58. The method of any one of claims 43 to 57, which does not comprise introducing an exogenous polynucleotide into the cells of the first cell population or the second cell population prior to the contacting in step (a), or does not comprise introducing an exogenous polynucleotide comprising a barcode sequence into the cells of the first cell population or the second cell population prior to the contacting in step (a).

59. The method of claim 58, wherein the exogenous polynucleotide comprises or encodes a gene, a modified / damaged / non-natural nucleotide or nucleotide analog, an overexpressed gene, an RNAi-based system, a regulatory RNA, a non-coding RNA, an mRNA, a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), a meganuclease, a computationally designed protein, a computationally designed RNA, and / or a CRISPR-Cas system.

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