Increase the spatial array resolution

Abstract

The present invention relates to methods for fabricating spatial arrays and determining the location of analytes present in a biological sample (e.g., by using cellular markers).

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 724,561, filed on August 29, 2018; U.S. Provisional Patent Application No. 62 / 723,950, filed on August 28, 2018; U.S. Provisional Patent Application No. 62 / 723,957, filed on August 28, 2018; U.S. Provisional Patent Application No. 62 / 723,960, filed on August 28, 2018; U.S. Provisional Patent Application No. 62 / 723,964, filed on August 28, 2018; U.S. Provisional Patent Application No. 62 / 723,970, filed on August 28, 2018; U.S. Provisional Patent Application No. 62 / 723,972, filed on August 28, 2018; U.S. Provisional Patent Application No. 62 / 724,483, filed on August 29, 2018; U.S. Provisional Patent Application No. 62 / 724,487, filed on August 29, 2018; U.S. Provisional Patent Application No. 62 / 724,489, filed on August 29, 2018; U.S. Provisional Patent Application No. 62 / 788,905, filed on January 6, 2019; U.S. Provisional Patent Application No. 62 / 788,867, filed on January 6, 2019; U.S. Provisional Patent Application No. 62 / 788,871, filed on January 6, 2019; U.S. Provisional Patent Application No. 62 / 788,897, filed on January 6, 2019; U.S. Provisional Patent Application No. 62 / 788,885, filed on January 6, 2019; U.S. Provisional Patent Application No. 62 / 779,342, filed on December 13, 2018; U.S. Provisional Patent Application No. 62 / 822,565, filed on March 22, 2019; U.S. Provisional Patent Application No. 62 / 819,496, filed on March 15, 2019; U.S. Provisional Patent Application No. 62 / 819,486, filed on March 15, 2019; U.S. Provisional Patent Application No. 62 / 819,467, filed on March 15, 2019; U.S. Provisional Patent Application No. 62 / 822,632, filed on March 22, 2019; U.S. Provisional Patent Application No. 62 / 822,618, filed on March 22, 2019; U.S. Provisional Patent Application No. 62 / 822,592, filed on March 22, 2019; U.S. Provisional Patent Application No. 62 / 819,468, filed on March 15, 2019; U.S. Provisional Patent Application No. 62 / 822,627, filed on March 22, 2019; U.S. Provisional Patent Application No. 62 / 819,448, filed on March 15, 2019; U.S. Provisional Patent Application No. 62 / 822,649, filed on March 22, 2019; U.S. Provisional Patent Application No. 62 / 819, filed on March 15, 2019Priority is claimed to U.S. Provisional Patent Application No. 62 / 819,478, filed Mar. 15, 2019; U.S. Provisional Patent Application No. 62 / 819,449, filed Mar. 15, 2019; U.S. Provisional Patent Application No. 62 / 822,554, filed Mar. 22, 2019; U.S. Provisional Patent Application No. 62 / 822,575, filed Mar. 22, 2019; U.S. Provisional Patent Application No. 62 / 822,605, filed Mar. 22, 2019; U.S. Provisional Patent Application No. 62 / 812,219, filed Feb. 28, 2019; U.S. Provisional Patent Application No. 62 / 819,458, filed Mar. 15, 2019; U.S. Provisional Patent Application No. 62 / 839,223, filed Apr. 26, 2019; U.S. Provisional Patent Application No. 62 / 839,320, filed Apr. 26, 2019; U.S. Provisional Patent Application No. 62 / 839,346, filed Apr. 26, 2019; U.S. Provisional Patent Application No. 62 / 842,463, filed May 2, 2019; U.S. Provisional Patent Application No. 62 / 860,993, filed Jun. 13, 2019; U.S. Provisional Patent Application No. 62 / 839,526, filed Apr. 26, 2019; and U.S. Provisional Patent Application No. 62 / 858,331, filed Jun. 7, 2019. The contents of each of these applications are hereby incorporated by reference in their entireties., BACKGROUND OF THE INVENTION

[0003] Cells within an object tissue differ in cell morphology and / or function due to different analyte levels (e.g., gene and / or protein expression) within different cells. The specific location of a cell within a tissue (e.g., the cell's position relative to neighboring cells or the cell's position relative to the tissue microenvironment) can affect, e.g., the cell's morphology, differentiation, fate, viability, proliferation, behavior, and signaling and crosstalk with other cells in the tissue.

[0004] Spatial heterogeneity has been previously studied using techniques that provide data on only a small number of analytes in whole or partial tissue contacts, or provide data on a large number of analytes in single cells, but do not provide information on the location of single cells within a parent biological sample (e.g., a tissue sample).

[0005] The spatial organization of gene expression can be observed in individual cells, tissues, or organisms. The genetic material and associated gene and protein expression influence the fate and behavior of cells. Spatial heterogeneity in developmental systems is typically studied by RNA hybridization, immunohistochemistry, fluorescent reporters, purification or induction of predefined subsets, and subsequent genomic profiling (e.g., RNA-seq). However, such methods rely on a small set of predefined markers, thus introducing selection biases that limit discovery and making it costly and laborious to map across the entire RNA transcriptome. SUMMARY OF THE INVENTION

[0006] Provided herein are methods for fabricating spatial arrays and determining the location of analytes present in a biological sample (e.g., by using a cell-tagging agent).

[0007] In one aspect, a method for determining the location of an analyte present in a biological sample includes (a) providing a substrate comprising a plurality of spatially barcoded oligonucleotides arrayed thereon, wherein the spatially barcoded oligonucleotides among the plurality of spatially barcoded oligonucleotides comprise a spatial barcode, a priming domain, and a first hybridization domain; (b) conjugating a cell-tagging agent to the spatially barcoded oligonucleotide; (c) contacting the biological sample with the cell-tagging agent such that cells in the biological sample are labeled with the spatially barcoded oligonucleotide; (d) providing the cells comprising the spatially barcoded oligonucleotide to beads, wherein the beads comprise (1) a first bead-binding oligonucleotide, wherein the first bead-binding oligonucleotide comprises a cell barcode and a second hybridization domain, and (2) a second bead-binding oligonucleotide, wherein the second bead-binding oligonucleotide comprises the cell barcode and a capture domain; (e) allowing an analyte from the cells to interact with the capture domain of the second bead-binding oligonucleotide; (f) associating the analyte bound to the capture domain of the second bead-binding oligonucleotide with the cell barcode; and (g) associating the cell barcode with the spatial barcode, thereby determining the location of the analyte present in the biological sample.

[0008] In some embodiments, the method for determining the location of an analyte present in a biological sample includes dissociating cells from the biological sample.

[0009] In some embodiments, providing the cell-tagging agent includes hybridizing a priming oligonucleotide to the priming domain, wherein the priming oligonucleotide is conjugated to the cell-tagging agent. In some embodiments, the priming oligonucleotide conjugated to the cell-tagging agent is substantially complementary to the priming domain of the spatially barcoded oligonucleotide. In some embodiments, the priming oligonucleotide conjugated to the cell-tagging agent is substantially complementary to the priming domain, the spatial barcode, and the first hybridization domain of the spatially barcoded oligonucleotide.

[0010] In some embodiments, methods for determining the location of an analyte present in a biological sample include determining the sequence of at least a portion of a spatially barcoded oligonucleotide. In some embodiments, determining the sequence includes in situ sequencing. In some embodiments, in situ sequencing includes one or more of sequencing by synthesis, sequencing by ligation, rolling circle amplification sequencing, fluorescence in situ sequencing (FISSEQ), and spatially resolved transcript amplicon readout mapping (STARmap).

[0011] In some embodiments, providing a cell comprising a spatially barcoded oligonucleotide to a bead includes hybridization. In some embodiments, hybridization includes a first hybridization domain that hybridizes with a second hybridization domain.

[0012] In some embodiments, a spatially barcoded oligonucleotide comprises one or more of a unique molecular identifier, an attachment sequence, a cleavage domain, and a functional domain. In some embodiments, the attachment sequence includes one or more of a flow cell attachment sequence and a substrate attachment sequence.

[0013] In some embodiments, allowing an analyte from a cell to interact with a capture domain includes releasing the analyte from the cell. In some embodiments, the release includes permeabilization of the cell. In some embodiments, associating an analyte bound to a capture domain with a cell domain includes identifying the analyte. In some embodiments, associating a cell barcode with a spatial barcode includes extending a first bead-binding oligonucleotide that hybridizes and a spatially barcoded oligonucleotide. In some embodiments, associating a cell barcode with a spatial barcode includes determining the sequences of the extended bead-binding oligonucleotide and the spatially barcoded oligonucleotide.

[0014] In another aspect, a method for determining the location of an analyte present in a biological sample includes (a) providing a substrate comprising a plurality of spatially barcoded oligonucleotides arrayed thereon, wherein the spatially barcoded oligonucleotides of the plurality of spatially barcoded oligonucleotides comprise a spatial barcode, a first hybridization domain, and a cleavage domain; (b) coupling a cell marker to the spatially barcoded oligonucleotide; and (c) determining the location of the analyte present in the biological sample. In some embodiments, the cell marker includes a first association domain that hybridizes with the spatially barcoded oligonucleotide, wherein the coupling includes hybridizing the first association domain with the spatially barcoded oligonucleotide. In some embodiments, the spatially barcoded oligonucleotide comprises a second association domain, wherein the first association domain of the cell marker hybridizes with the second association domain.

[0015] In some embodiments, the cell labeling agent is conjugated to the spatially barcoded oligonucleotide via a linker. In some embodiments, the spatially barcoded oligonucleotide comprises one or more of a unique molecular identifier, an attachment sequence, a restriction endonuclease sequence, and a functional domain. In some embodiments, the attachment sequence comprises one or more of a flow cell attachment sequence and a substrate attachment sequence.

[0016] In some embodiments, a method for determining the location of an analyte present in a biological sample comprises (a) contacting the biological sample with a cell labeling agent such that cells in the biological sample are labeled with a spatially barcoded oligonucleotide; (b) cleaving the spatially barcoded oligonucleotide from the substrate; (c) providing the cells comprising the spatially barcoded oligonucleotide to beads, wherein the beads comprise (1) a first bead-binding oligonucleotide, wherein the first bead-binding oligonucleotide comprises a cell barcode and a second hybridization domain, and (2) a second bead-binding oligonucleotide, wherein the second bead-binding oligonucleotide comprises the cell barcode and a capture domain; (d) allowing the analyte from the cells to interact with the capture domain of the second bead-binding oligonucleotide; (e) associating the analyte bound to the capture domain of the second bead-binding oligonucleotide with the cell barcode; and (f) associating the cell barcode with the spatially barcoded oligonucleotide.

[0017] In some embodiments, the biological sample is a tissue sample. In some embodiments, the tissue sample is a fresh frozen tissue sample. In some embodiments, the tissue sample is a formalin-fixed paraffin-embedded (FFPE) tissue sample. In some embodiments, the tissue sample comprises tumor cells. In some embodiments, the tissue sample comprises tissue sections. In some embodiments, a method for determining the location of an analyte present in a biological sample comprises imaging the biological sample. In some embodiments, the analyte comprises at least one of RNA, DNA, protein, lipid, peptide, metabolite, small molecule, and cell labeling agent. In some embodiments, the analyte comprises RNA.

[0018] In another aspect, a method for generating an array comprises (a) providing a plurality of spatially barcoded oligonucleotides to a substrate, wherein two or more of the plurality of spatially barcoded oligonucleotides comprise a first attachment sequence and a second attachment sequence, and wherein the substrate comprises a plurality of functional domains, wherein at least two of the plurality of functional domains hybridize with at least two of the spatially barcoded oligonucleotides, thereby generating an array of spatially barcoded oligonucleotides; and (b) amplifying the spatially barcoded oligonucleotides on the substrate, thereby generating the array.

[0019] In some embodiments, the two or more spatially barcoded oligonucleotides on the amplification substrate include bridge amplification. In some embodiments, the method of generating an array includes determining the identity of two or more spatially barcoded oligonucleotides of the array. In some embodiments, determining the identity of two or more spatially barcoded oligonucleotides includes sequencing the spatially barcoded oligonucleotides. In some embodiments, the sequencing includes in situ sequencing. In some embodiments, the in situ sequencing includes hybridizing a primer oligonucleotide to the two or more spatially barcoded oligonucleotides. In some embodiments, the primer oligonucleotide is conjugated to a cell label. In some embodiments, the in situ sequencing includes one or more of sequencing by synthesis, sequencing by ligation, and rolling circle amplification sequencing. In some embodiments, the in situ sequencing includes sequencing by synthesis. In some embodiments, sequencing by synthesis includes hybridizing a primer oligonucleotide to the two or more spatially barcoded oligonucleotides. In some embodiments, the primer oligonucleotide is conjugated to a cell label.

[0020] In some embodiments, the two or more spatially barcoded oligonucleotides include one or more of a spatial barcode, a primer domain, a hybridization domain, a unique molecular identifier, a functional domain, and a cleavage domain. In some embodiments, the two or more spatially barcoded oligonucleotides include a spatial barcode. In some embodiments, the two or more spatially barcoded oligonucleotides include a cleavage domain.

[0021] In some embodiments, the cell label includes an extracellular cell label. In some embodiments, the cell label includes an intracellular cell label. In some embodiments, the cell label is localized to an internal component of the cell. In some embodiments, the internal components of the cell include one or more of mitochondria, Golgi apparatus, smooth endoplasmic reticulum, rough endoplasmic reticulum, nucleus, nucleolus, and lysosome. In some embodiments, the cell label includes one or more of a lipid, an antibody, chitosan, lectin, streptavidin, a click chemistry modification moiety, a cell penetrating peptide, a nanoparticle, a TIVA tag, and a liposome / polysome. In some embodiments, the cell label is amphiphilic. In some embodiments, the cell label is lipophilic. In some embodiments, the cell label is a cholesterol moiety. In some embodiments, the cell label is conjugated to the primer oligonucleotide via a linker. In some embodiments, the linker includes one or more of the following: N-hydroxysuccinimide (NHS) linker, bifunctional NHS linker, azide, alkyne, glycol chitosan, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG), and succinimidyl 3-(2-pyridyldithio)propionate (SPDP).

[0022] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference as if each individual publication, patent, patent application, and information item were specifically and individually incorporated by reference. With respect to any content of the incorporated publications, patents, patent applications, and information items that is inconsistent with the disclosure contained in this specification, this specification is intended to prevail and / or this specification takes precedence over any conflicting material.

[0023] In cases where values are described in ranges, it should be understood that such description includes disclosure of all possible sub-ranges within that range, as well as disclosure of specific numerical values within that range, regardless of whether the specific numerical values or specific sub-ranges are explicitly stated.

[0024] The term "each" when referring to a group of items is intended to identify an individual item within that collection, but does not necessarily refer to every item within that collection, unless explicitly stated otherwise or unless the context of the usage otherwise clearly indicates.

[0025] Various embodiments of the features of the present invention are described herein. However, it should be understood that these embodiments are provided only as examples, and that those skilled in the art may make many variations, changes, and substitutions without departing from the scope of the present disclosure. It should also be understood that various alternatives to the specific embodiments described herein are also within the scope of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The following drawings illustrate certain embodiments of the features and advantages of the present invention. These embodiments are not intended in any way to limit the scope of the appended claims. Like reference numerals in the drawings represent like elements.

[0027] Figure 1 An exemplary spatial analysis workflow is shown.

[0028] Figure 2 An exemplary spatial analysis workflow is shown.

[0029] Figure 3 An exemplary spatial analysis workflow is shown.

[0030] Figure 4 An exemplary spatial analysis workflow is shown.

[0031] Figure 5 An exemplary spatial analysis workflow is shown.

[0032] Figure 6 is a schematic diagram showing an example of a barcoded capture probe as described herein.

[0033] Figure 7It is a schematic diagram illustrating a cleavable capture probe, where the cleaved capture probe can enter non-permeabilized cells and bind to target analytes within a sample.

[0034] Figure 8 It is a schematic diagram of an exemplary multiplexed spatial barcoding feature.

[0035] Figure 9 It is a schematic diagram of an exemplary analyte capture agent.

[0036] Figure 10 It is a schematic diagram depicting an exemplary interaction between a feature-immobilized capture probe 1024 and an analyte capture agent 1026.

[0037] Figure 11A 、 11B and 11C are schematic diagrams illustrating how streptavidin cell tags are utilized in an array-based system to generate spatially barcoded cells or cell contents.

[0038] Figure 12 It is a schematic diagram showing the arrangement of barcoded features within an array.

[0039] Figure 13 It is a schematic diagram showing a side view of an anti-diffusion medium (such as a lid).

[0040] Figure 14A and 14B It is a schematic diagram illustrating the deployment Figure 14A and side view Figure 14B of an electrophoresis transfer system configured to direct transcript analytes to a spatially barcoded capture probe array.

[0041] Figure 15 It is a schematic diagram showing an exemplary workflow scenario utilizing an electrophoresis transfer system.

[0042] Figure 16 Shows an example of a microfluidic channel structure 1600 for partitioning a dissociated sample (such as biological particles or individual cells from a sample).

[0043] Figure 17A Shows an example of a microfluidic channel structure 1700 for delivering beads carrying spatial barcodes to droplets.

[0044] Figure 17B Shows a cross-sectional view of another example of a microfluidic channel structure 1750 having geometric features for controlled partitioning.

[0045] Figure 17C Shows a workflow schematic diagram.

[0046] Figure 18 Schematic illustration of cell labeling using covalent coupling of an analyte-binding moiety to the cell surface or non-covalent interaction with cell membrane components.

[0047] Figure 19 Schematic illustration of cell labeling using a cell-penetrating peptide or delivery system.

[0048] Figure 20A Workflow schematic illustration showing exemplary, non-limiting, non-exhaustive steps for "pixelating" a sample, where the sample is cut, imprinted, microdissected, or transferred through a hollow or microneedle, and small portions of the sample are moved to individual partitions or wells.

[0049] Figure 20B Schematic illustration of multi-needle pixelation, where a set of needles passes through a sample on a support and into nanopores below containing gel beads and reagents. Once the needles are in the nanopores, cells are ejected.

[0050] Figure 21 Workflow schematic illustration showing exemplary, non-limiting, non-exhaustive steps for dissociating a spatially barcoded sample for analysis by droplet or flow cell analysis methods.

[0051] Figure 22 Schematic illustration showing exemplary spatially barcoded oligonucleotides on a substrate (depicted as gray rectangles). Exemplary components of the spatially barcoded oligonucleotides are as follows: attachment sequences (depicted as P5 / P7), primer domains (depicted as R1 / R2), hybridization domains (depicted as Cap), and spatial barcodes (depicted as SpBC).

[0052] Figure 23 Schematic illustration showing an exemplary spatially barcoded oligonucleotide attached to a cell labeling agent. In the Figure 23 illustrated embodiment, the cell labeling agent is a lipid. As Figure 23 illustrated, the spatially barcoded oligonucleotide attached to the cell labeling agent is complementary to the spatially barcoded oligonucleotide attached to the substrate. Exemplary components of the spatially barcoded oligonucleotides are as follows: attachment sequences (depicted as P5 / P7), primer domains (depicted as R1 / R2), hybridization domains (depicted as Cap), and spatial barcodes (depicted as SpBC).

[0053] Figure 24 Schematic illustration showing an exemplary biological sample (depicted as a light gray rectangle above the substrate) in contact with a spatially barcoded oligonucleotide attached to a cell labeling agent. In the Figure 24 illustrated embodiment, the cell labeling agent is a lipid. As Figure 24As shown, the spatially barcoded oligonucleotide attached to the cell labeler is complementary to the spatially barcoded oligonucleotide attached to the substrate. Exemplary components of the spatially barcoded oligonucleotide are as follows: attachment sequences (depicted as P5 / P7), primer domains (depicted as R1 / R2), hybridization domains (depicted as Cap), and spatial barcodes (depicted as SpBC).

[0054] Figure 25 is a schematic diagram showing exemplary gel emulsion (GEM) droplets, in which cells labeled with spatially barcoded oligonucleotides hybridize to beads through complementary hybridization domains. In Figure 25 the illustrated embodiment, the cell labeler is a lipid, and the beads are gel beads.

[0055] Figure 26 is a schematic diagram showing an exemplary spatially barcoded oligonucleotide on a substrate (depicted as a gray rectangle). An exemplary oligonucleotide that is partially complementary to the spatially barcoded oligonucleotide is depicted as a means for determining at least part of the sequence of the spatially barcoded oligonucleotide. Exemplary components of the spatially barcoded oligonucleotide are as follows: attachment sequences (depicted as P5 / P7), primer domains (depicted as R1 / R2), association domains (depicted as hyb), hybridization domains (depicted as Cap), and spatial barcodes (depicted as SpBC).

[0056] Figure 27 is a schematic diagram showing an exemplary spatially barcoded oligonucleotide attached to a cell labeler. In Figure 27 the illustrated embodiment, the cell labeler is a lipid. Exemplary components of the spatially barcoded oligonucleotide are as follows: attachment sequence (depicted as P7), primer domain (depicted as R2), association domain (depicted as hyb), hybridization domain (depicted as Cap), and spatial barcode (depicted as SpBC).

[0057] Figure 28A is a schematic diagram showing an exemplary sample processing device that can be used to implement the various steps and methods described herein.

[0058] Figure 28B is a schematic diagram showing an exemplary imaging device that can be used to obtain images of biological samples, analytes, and feature arrays.

[0059] Figure 28C is Figure 28A and Figure 28B a schematic diagram of an example of the control unit of the device. Detailed Description

[0060] I. Introduction

[0061] The present invention describes devices, systems, methods, and compositions for the spatial analysis of biological samples. This section specifically describes certain general terms, analytes, sample types, and preparation steps that will be referred to in later parts of the present invention.

[0062] (a) Spatial analysis

[0063] Tissues and cells can be obtained from any source. For example, tissues and cells can be obtained from single-celled or multi-celled organisms (e.g., mammals). Tissues and cells obtained from mammals (e.g., humans) typically have different analyte levels (e.g., gene and / or protein expression), which can lead to differences in cell morphology and / or function. The position of a cell within a tissue can affect the cell's fate, behavior, morphology, as well as signal transduction and crosstalk with other cells in the tissue. Information regarding differences in analyte levels (gene and / or protein expression) within different cells in mammalian tissues can also help a physician select or administer a treatment effective for a single-celled or multi-celled organism (e.g., a mammal) based on the detected differences in analyte levels within different cells in the tissue. Differences in analyte levels within different cells in mammalian tissues can also provide information on how tissues (e.g., healthy and diseased tissues) function and / or develop. Differences in analyte levels within different cells in mammalian tissues can also provide different information on disease pathogenesis within the tissue as well as information on the mechanism of action of a treatment within the tissue. Differences in analyte levels within different cells in mammalian tissues can also provide information regarding resistance mechanisms and the development of resistance mechanisms in mammalian tissues. Differences in the presence or absence of analytes within different cells in tissues of a multi-celled organism (e.g., a mammal) can provide information on resistance mechanisms and their development in tissues of the multi-celled organism.

[0064] Spatial analysis methods are used to detect differences in analyte levels (e.g., gene and / or protein expression) within different cells in mammalian tissues or within a single cell of a mammal. For example, spatial analysis methods can be used to detect differences in analyte levels (e.g., gene and / or protein expression) within different cells in a tissue section sample, and data therefrom can be recombined to generate a three-dimensional map of analyte levels (e.g., gene and / or protein expression) of a tissue sample obtained from a mammal, for example, with a certain degree of spatial resolution (e.g., single-cell resolution).

[0065] Spatial heterogeneity in developmental systems is typically studied by RNA hybridization, immunohistochemistry, fluorescent reporters, purification or induction of predefined subpopulations, and subsequent genomic profiling (e.g., RNA-seq). However, such methods rely on a relatively small set of predefined markers and thus introduce selection biases that limit discovery. These previous methods also rely on prior knowledge. Traditionally, spatial RNA analysis has relied on the staining of a limited number of RNA species. In contrast, single-cell RNA sequencing allows for deep profiling of cellular gene expression (including non-coding RNAs), but established methods isolate cells from their native spatial context.

[0066] Current spatial analysis methods provide a large amount of analyte level and / or expression data for multiple analytes in a sample at high spatial resolution, e.g., while preserving the native spatial context. Spatial analysis methods include, for example, using capture probes that include spatial barcodes (e.g., nucleic acid sequences that provide information about the location of the capture probe within a cell or tissue sample (e.g., a mammalian cell or mammalian tissue sample)) and a capture domain capable of binding to an analyte (e.g., a protein and / or nucleic acid) produced by or present in a cell. As described herein, the spatial barcode can be a nucleic acid having a unique sequence, a unique fluorophore or unique combination of fluorophores, a unique amino acid sequence, a unique heavy metal or unique combination of heavy metals, or any other uniquely detectable reagent. The capture domain can be any reagent capable of binding to an analyte produced and / or present in a cell (e.g., a nucleic acid capable of hybridizing to a nucleic acid from a cell (e.g., mRNA, genomic DNA, mitochondrial DNA, or miRNA)), a substrate or binding partner for the analyte, or an antibody that specifically binds to the analyte). The capture probe can also include a nucleic acid sequence complementary to a universal forward and / or universal reverse primer sequence. The capture probe can also include a cleavage site (e.g., a cleavage recognition site for a restriction endonuclease), a photo-labile bond, a thermosensitive bond, or a chemically sensitive bond.

[0067] A variety of different methods can be used to detect the binding of an analyte to a capture probe, e.g., nucleic acid sequencing, fluorophore detection, nucleic acid amplification, nucleic acid ligation detection, and / or nucleic acid cleavage product detection. In some examples, the detection is used to associate a specific spatial barcode with a specific analyte produced and / or present in a cell (e.g., a mammalian cell).

[0068] Capture probes can be attached, for example, to a surface such as a solid array, bead, or coverslip. In certain examples, the capture probes are not attached to a surface. In some instances, the capture probes can be encapsulated within, embedded within, or layered on the surface of a permeabilizable composition (e.g., any of the substrates described herein). For example, the capture probes can be encapsulated or disposed within a permeabilizable bead (e.g., a gel bead). In some instances, the capture probes can be encapsulated within, embedded within, or layered on the surface of a substrate (e.g., any of the exemplary substrates described herein, such as a hydrogel or a porous membrane).

[0069] In some embodiments, a cell or a tissue sample comprising cells is contacted with a capture probe attached to a substrate (e.g., the surface of the substrate), and the cell or tissue sample is permeabilized to release an analyte from the cell and bind it to the capture probe attached to the substrate. In some examples, various methods (e.g., electrophoresis, chemical gradient, pressure gradient, fluid flow, or magnetic field) can be used to actively direct the analyte released from the cell to the capture probe attached to the substrate.

[0070] In other examples, various methods can be used to direct the interaction of the capture probe with the cell or tissue sample, including, for example, a lipid anchor in the capture probe, a reagent that can specifically bind to or form a covalent bond with a membrane protein in the capture probe, fluid flow, pressure gradient, chemical gradient, or magnetic field.

[0071] Non-limiting aspects of spatial analysis methods are described in WO 2011 / 127099, WO 2014 / 210233, WO 2014 / 210225, WO 2016 / 162309, WO 2018 / 091676, WO 2012 / 140224, WO 2014 / 060483, U.S. Patent No. 10,002,316, U.S. Patent No. 9,727,810, U.S. Patent Application Publication No. 2017 / 0016053, Rodriques et al., Science 363(6434):1463 - 1467, 2019; WO 2018 / 045186, Lee et al., Nat.Protoc.10(3):442 - 458, 2015; WO 2016 / 007839, WO 2018 / 045181, WO 2014 / 163886, Trejo et al., PLoS ONE 14(2):e0212031, 2019, U.S. Patent Application Publication No. 2018 / 0245142, Chen et al., Science 348(6233):aaa6090, 2015, Gao et al., BMC Biol.15:50, 2017, WO 2017 / 144338, WO 2018 / 107054, WO 2017 / 222453, WO 2019 / 068880, WO 201I / 094669, U.S. Patent No. 7,709,198, U.S. Patent No. 8,604,182, U.S. Patent No. 8,951,726, U.S. Patent No. 9,783,841, U.S. Patent No. 10,041,949, WO 2016 / 057552, WO 2017 / 147483, WO 2018 / 022809, WO 2016 / 166128, WO 2017 / 027367, WO 2017 / 027368, WO2018 / 136856, WO 2019 / 075091, U.S. Patent No. 10,059,990, WO 2018 / 057999, WO 2015 / 161173, and Gupta et al., Nature Biotechnol.36:1197 - 1202, 2018 and can be used in any combination herein. Further non-limiting aspects of spatial analysis methods are described herein.

[0072] (b) General terms

[0073] Certain terms are used in this invention to explain various aspects of the described devices, systems, methods, and compositions. This subsection includes an explanation of certain terms that appear in later sections of this invention. If the description in this section conflicts significantly with the usage in other sections of this invention, the definition in this section shall prevail.

[0074] (i) Barcode

[0075] A “barcode” is a label or identifier that conveys or is capable of conveying information (e.g., information about an analyte, bead, and / or capture probe in a sample). A barcode can be part of an analyte or independent of the analyte. A barcode can be attached to an analyte. A particular barcode may be unique relative to other barcodes.

[0076] Barcodes can have a variety of different formats. For example, a barcode can include a polynucleotide barcode, a random nucleic acid and / or amino acid sequence, and a synthetic nucleic acid and / or amino acid sequence. A barcode can be attached to an analyte or another part or structure in a reversible or irreversible manner. For example, a barcode can be added to a fragment of a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before or during sequencing of the sample. A barcode can allow for the identification and / or quantification of individual sequencing reads (e.g., a barcode can be or can include a unique molecular identifier or “UMI”).

[0077] Barcodes can spatially resolve molecular components found in a biological sample, e.g., at single-cell resolution (e.g., a barcode can be or can include a “spatial barcode”). In some embodiments, a barcode includes both a UMI and a spatial barcode. In some embodiments, a barcode includes two or more sub-barcodes that together function 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.

[0078] (ii) Nucleic Acids and Nucleotides

[0079] The terms “nucleic acid” and “nucleotide” are intended to be consistent with their use in the art and include naturally occurring substances or functional analogs thereof. Particularly useful nucleic acid functional analogs are capable of hybridizing to nucleic acids in a sequence-specific manner (e.g., capable of hybridizing to two nucleic acids such that ligation can occur between the two hybridized nucleic acids), or are capable of serving as a template for replicating a specific nucleotide sequence. Naturally occurring nucleic acids typically have a backbone containing phosphodiester bonds. Analog structures can have other backbone linkages, including any of the various linkages known in the art. Naturally occurring nucleic acids typically have deoxyribose (e.g., present in deoxyribonucleic acid (DNA)) or ribose (e.g., present in ribonucleic acid (RNA)).

[0080] The nucleic acid may comprise nucleotides having any of a variety of analogs of these sugar moieties known in the art. Nucleic acids can include natural or unnatural nucleotides. In this regard, natural deoxyribonucleic acid can have one or more bases selected from adenine (A), thymine (T), cytosine (C), or guanine (G), and ribonucleic acid can have one or more bases selected from uracil (U), adenine (A), cytosine (C), or guanine (G). Useful unnatural bases are known in the art that can be included in nucleic acids or nucleotides.

[0081] (iii) Probes and targets

[0082] "Probe" or "target", when used in reference to a nucleic acid or nucleic acid sequence, means a semantic identifier of the nucleic acid or sequence in the context of a method or composition, and does not limit the structure or function of the nucleic acid or sequence beyond what is expressly indicated.

[0083] (iv) Oligonucleotides and polynucleotides

[0084] The terms "oligonucleotide" and "polynucleotide" are used interchangeably to refer to single-stranded polynucleotides of nucleotides that are about 2 to about 500 nucleotides in length. Oligonucleotides can be synthesized, prepared enzymatically (e.g., by polymerization), or prepared using "split-pool" methods. Oligonucleotides can include ribonucleotide monomers (i.e., can be oligoribonucleotides) and / or deoxyribonucleotide monomers (i.e., oligodeoxyribonucleotides). In some examples, oligonucleotides can include a combination of deoxyribonucleotide monomers and ribonucleotide monomers in the oligonucleotide (e.g., a random or ordered combination of deoxyribonucleotide monomers and ribonucleotide monomers). For example, the length of an oligonucleotide can be 4 to 10, 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 80 to 100, 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 350, 350 to 400, or 400 to 500 nucleotides. Oligonucleotides can include one or more functional moieties attached (e.g., covalently or non-covalently) to the polymeric structure. For example, oligonucleotides can include one or more detectable labels (e.g., a radioisotope or fluorophore).

[0085] (v) Subject

[0086] "Subject" is an animal, such as a mammal (e.g., a human or non-human ape), or a bird (e.g., a poultry), or other organisms, such as a plant. Examples of subjects include, but are not limited to, mammals such as rodents, mice, rats, rabbits, guinea pigs, ungulates, horses, sheep, pigs, goats, cows, cats, dogs, primates (i.e., humans or non-human primates); plants such as Arabidopsis thaliana, corn, sorghum, oats, wheat, rice, rapeseed or soybean; algae such as Chlamydomonas reinhardtii; nematodes such as Caenorhabditis elegans; insects such as Drosophila melanogaster, mosquitoes, fruit flies or bees; arachnids such as spiders; fish such as zebrafish; reptiles; amphibians such as frogs or Xenopus laevis; Dictyostelium discoideum; fungi such as Pneumocystis carinii, Takifugu rubripes, yeast, Saccharamoyces cerevisiae or Schizosaccharomyces pombe; or Plasmodium falciparum.

[0087] (vi) Genome

[0088] "Genome" generally refers to genomic information from a subject, for example, it can be at least part or all of the genetic information encoded by the subject's genes. The genome can include coding regions (e.g., regions encoding proteins) and non-coding regions. The genome can include the sequences of some or all of the subject's chromosomes. For example, the human genome usually has a total of 46 chromosomes. The sequences of some or all of these can constitute the genome.

[0089] (vii) Adaptor, Adapter and Tag

[0090] "Adaptor", "Adapter" and "Tag" are terms used interchangeably in the present disclosure and refer to substances that can be coupled to a polynucleotide sequence (in a process called "tagging") using any of a number of different techniques including, but not limited to, ligation, hybridization and tagging. An adaptor can also be a nucleic acid sequence that adds a function, such as a spacer sequence, a primer sequence / site, a barcode sequence, a unique molecular identifier sequence.

[0091] (viii) Hybridization, cross-hybridization, annealing, and strand annealing

[0092] The terms “hybridization,” “cross-hybridization,” “annealing,” and “strand annealing” are used interchangeably in the present disclosure and refer to the pairing of substantially complementary or complementary nucleic acid sequences within two different molecules. The pairing can be achieved by any process in which the nucleic acid sequences are joined by base pairing to a substantially complementary or fully complementary sequence to form a hybridization complex. For the purposes of hybridization, two nucleic acid sequences are “substantially complementary” if at least 60% (e.g., at least 70%, at least 80%, or at least 90%) of the individual bases in the two nucleic acid sequences are complementary to each other.

[0093] (ix) Primer

[0094] A “primer” is a single-stranded nucleic acid sequence having a 3′ end that can be used as a substrate for a nucleic acid polymerase in a nucleic acid extension reaction. RNA primers are formed from RNA nucleotides and are used for RNA synthesis, while DNA primers are composed of DNA nucleotides and are used for DNA synthesis. Primers can also include RNA nucleotides and DNA nucleotides (e.g., in a random or designed pattern). Primers can also include other natural or synthetic nucleotides having other functionality as described herein. In some examples, DNA primers can be used to prime RNA synthesis and vice versa (e.g., RNA primers can be used to prime DNA synthesis). The length of the primer can vary. For example, a primer can be from about 6 bases to about 120 bases. For example, a primer can include up to about 25 bases.

[0095] (x) Primer extension

[0096] “Primer extension” refers to any method in which two nucleic acid sequences (e.g., constant regions from each of two different capture probes) are joined (e.g., hybridized) by an overlap of their respective terminal complementary nucleic acid sequences (e.g., 3′ ends). After such joining, nucleic acid extension (e.g., enzymatic extension) of one or both ends can be performed using another nucleic acid sequence as an extension template. Enzymatic extension can be performed by enzymes including but not limited to polymerase and / or reverse transcriptase.

[0097] (xi) Proximity ligation

[0098] “Proximity ligation” is a method of joining two (or more) nucleic acid sequences that are in proximity to each other by enzymatic means (e.g., ligase). In some embodiments, proximity ligation can include a “gap filling” step that involves incorporation of one or more nucleic acids by a polymerase across the distance between two nucleic acid molecules of interest based on the nucleic acid sequence of a template nucleic acid molecule (see, e.g., U.S. Patent No. 7,264,929, the entire contents of which are incorporated herein by reference).

[0099] A variety of different methods can be used to ligate nucleic acid molecules in proximity, including (but not limited to) "sticky-end" and "blunt-end" ligation. Additionally, single-strand ligation can be used for proximity ligation of single-stranded nucleic acid molecules. Sticky-end proximity ligation involves hybridization of complementary single-stranded sequences between two nucleic acid molecules to be ligated prior to the ligation event. Blunt-end proximity ligation generally does not involve hybridization of complementary regions from each nucleic acid molecule, as both nucleic acid molecules lack single-stranded overhangs at the ligation site.

[0100] (xii) Nucleic acid extension

[0101] "Nucleic acid extension" generally involves incorporating one or more nucleic acids (e.g., A, G, C, T, U, nucleotide analogs or derivatives thereof) into a molecule (e.g., but not limited to a nucleic acid sequence) in a template-dependent manner such that successive nucleic acids are incorporated by an enzyme (e.g., polymerase or reverse transcriptase), thereby generating a newly synthesized nucleic acid molecule. For example, by using a complementary nucleic acid sequence as a template for nucleic acid synthesis, a primer hybridized to the complementary nucleic acid sequence can be used to synthesize a new nucleic acid molecule. Similarly, the 3′ polyadenylated tail of an mRNA transcript hybridized to a poly(dT) sequence (e.g., a capture domain) can be used as a template for the synthesis of a single strand of the corresponding cDNA molecule.

[0102] (xiii) PCR amplification

[0103] "PCR amplification" refers to generating copies of genetic material, including DNA and RNA sequences, using the polymerase chain reaction (PCR). For example, suitable reagents and conditions for performing PCR are described in U.S. Patent Nos. 4,683,202, 4,683,195, 4,800,159, 4,965,188, and 5,512,462, the entire contents of which are incorporated herein by reference. In a typical PCR amplification, the reaction mixture includes the genetic material to be amplified, an enzyme, one or more primers for primer extension reactions, and reagents for the reaction. The oligonucleotide primers are of sufficient length to hybridize to the complementary genetic material under annealing conditions. The length of the primers generally depends on the length of the amplification domain but is typically at least 4 bases, at least 5 bases, at least 6 bases, at least 8 bases, at least 9 bases, at least 10 base pairs (bp), at least 11 bp, at least 12 bp, at least 13 bp, at least 14 bp, at least 15 bp, at least 16 bp, at least 17 bp, at least 18 bp, at least 19 bp, at least 20 bp, at least 25 bp, at least 30 bp, at least 35 bp, and can be up to 40 bp or longer, where the length of the primers generally ranges between 18 - 50 bp. The genetic material can be contacted with a single primer or a set of two primers (forward and reverse primers), depending on whether primer extension, linear or exponential amplification of the genetic material is desired.

[0104] In some embodiments, the PCR amplification process uses a DNA polymerase. The DNA polymerase activity can be provided by one or more different DNA polymerases. In certain embodiments, the DNA polymerase is from bacteria. For example, the DNA polymerase is a bacterial DNA polymerase. For example, the DNA polymerase can be from bacteria of the genus Escherichia, Bacillus, Thermus, or Pyrococcus.

[0105] Suitable examples of DNA polymerases that can be used include, but are not limited to: Escherichia coli DNA polymerase I, Bsu DNA polymerase, Bst DNA polymerase, Taq DNA polymerase, VENT TM DNA polymerase, DEEPVENT TM DNA polymerase, Taq DNA polymerase, Hot start Taq DNA polymerase, Crimson Taq DNA polymerase, Crimson Taq DNA polymerase, DNA polymerase, Quick DNA polymerase, Hemo DNA polymerase, DNA polymerase, DNA polymerase, High-fidelity DNA polymerases, Platinum Pfx DNA polymerase, AccuPrime Pfx DNA polymerase, Phi29 DNA polymerase, Klenow fragment, Pwo DNA polymerase, Pfu DNA polymerase, T4 DNA polymerase, and T7 DNA polymerase.

[0106] The term "DNA polymerase" includes not only naturally occurring enzymes, but also all of their modified derivatives, and also derivatives of naturally occurring DNA polymerases. For example, in some embodiments, the DNA polymerase can be modified to remove 5′-3′ exonuclease activity. Sequence-modified derivatives or mutants of DNA polymerases that can be used include, but are not limited to, mutants that retain at least part of the function, such as the DNA polymerase activity of the wild-type sequence. Under different reaction conditions, such as temperature, template concentration, primer concentration, etc., the mutation can affect the activity profile of the enzyme, such as increasing or decreasing the polymerization rate. The mutation or sequence modification may also affect the exonuclease activity and / or thermal stability of the enzyme.

[0107] In some embodiments, the PCR amplification can include reactions such as, but not limited to, strand displacement amplification reaction, rolling circle amplification reaction, ligase chain reaction, transcription-mediated amplification reaction, isothermal amplification reaction, and / or loop-mediated amplification reaction.

[0108] In some embodiments, PCR amplification uses a single primer that is complementary to the 3′ tag of the target DNA fragment. In some embodiments, PCR amplification uses a first and a second primer, wherein at least the 3′ terminal portion of the first primer is at least partially complementary to the 3′ tag of the target nucleic acid fragment, and wherein at least the 3′ terminal portion of the second primer exhibits the sequence of at least a portion of the 5′ tag of the target nucleic acid fragment. In some embodiments, the 5′ terminal portion of the first primer is not complementary to the 3′ tag of the target nucleic acid fragment, and the 5′ terminal portion of the second primer does not exhibit the sequence of at least a portion of the 5′ tag of the target nucleic acid fragment. In some embodiments, the first primer includes a first universal sequence and / or the second primer includes a second universal sequence.

[0109] In some embodiments (e.g., when PCR amplification is performed on captured DNA), a DNA ligase can be used to ligate the PCR amplification product to other sequences. The DNA ligase activity can be provided by one or more different DNA ligases. In some embodiments, the DNA ligase is from bacteria, e.g., the DNA ligase is a bacterial DNA ligase. In some embodiments, the DNA ligase is from a virus (e.g., a bacteriophage). For example, the DNA ligase can be T4 DNA ligase. Other enzymes suitable for the ligation step include, but are not limited to, Tth DNA ligase, Taq DNA ligase, Pyrococcus sp. (strain 9oN) DNA ligase (9oNTM DNA ligase, available from New England Biolabs, Ipswich, MA) and Ampligase TM (available from Epicentre Biotechnologies, Madison, WI). Derivatives, such as sequence-modified derivatives and / or mutants thereof, can also be used.

[0110] In some embodiments, genetic material is amplified by reverse transcription polymerase chain reaction (RT-PCR). The desired reverse transcriptase activity can be provided by one or more different reverse transcriptases, suitable examples of which include, but are not limited to: M-MLV, MuLV, AMV, HIV, ArrayScript TM 、MultiScribe TM 、ThermoScript TM 、and types I, II, III, and IV enzymes. "Reverse transcriptase" includes not only naturally occurring enzymes, but also all such modified derivatives thereof, and also derivatives of naturally occurring reverse transcriptases.

[0111] In addition, sequence-modified derivatives or mutants of M-MLV, MuLV, AMV, and HIV reverse transcriptases can be used for reverse transcription, including mutants that retain at least some functional (e.g., reverse transcriptase) activity of the wild-type sequence. The reverse transcriptase can be provided as part of a composition that includes other components, such as stabilizing components that enhance or improve the reverse transcriptase activity, such as ribonuclease inhibitors, inhibitors of DNA-dependent DNA synthesis, e.g., actinomycin D. Sequence-modified derivatives or mutants of many reverse transcriptases, such as M-MLV, and compositions that include unmodified and modified enzymes, such as ArrayScript TM 、MultiScribe TM 、ThermoScript TM 、and enzymes I, II, III, and IV are commercially available.

[0112] Certain reverse transcriptases (e.g., avian myeloblastosis virus (AMV) reverse transcriptase and Moloney murine leukemia virus (M-MuLV, MMLV) reverse transcriptase) can use RNA (cDNA synthesis) and single-stranded DNA (ssDNA) as templates to synthesize complementary DNA strands. Thus, in some embodiments, the reverse transcription reaction can use an enzyme (reverse transcriptase) that is capable of using both RNA and ssDNA as templates for the extension reaction, such as AMV or MMLV reverse transcriptase.

[0113] In some embodiments, the quantification of RNA and / or DNA is performed by real-time PCR (also known as quantitative PCR or qPCR), using techniques well known in the art, such as but not limited to "TAQMAN TM " or or on a capillary (" capillary"). In some embodiments, the quantification of genetic material is determined by light absorbance and real-time PCR. In some embodiments, the quantification of genetic material is determined by digital PCR. In some embodiments, the gene analyzed can correspond to the expression (mRNA) and quantity (DNA) and be compared to a reference nucleic acid extract (DNA and RNA) in order to compare the expression levels of the target nucleic acid.

[0114] (xiv) Antibodies

[0115] "Antibody" is a polypeptide molecule that recognizes and binds to a complementary target antigen. Antibodies typically have a molecular structure shape similar to a Y. Naturally occurring antibodies, known as immunoglobulins, belong to one of the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE. Antibodies can also be synthetically produced. For example, recombinant antibodies, such as monoclonal antibodies, can be synthesized using synthetic genes by recovering the antibody gene from a source cell, amplifying it into an appropriate vector, and introducing the vector into a host to cause the host to express the recombinant antibody. Generally, recombinant antibodies can be cloned from any antibody-producing animal species using appropriate oligonucleotide primers and / or hybridization probes. Recombinant techniques can be used to generate antibodies and antibody fragments, including non-endogenous substances.

[0116] Synthetic antibodies can be obtained from non-immunoglobulin sources. For example, antibodies can be produced from nucleic acids (such as aptamers) and non-immunoglobulin scaffolds (such as peptide aptamers), where hypervariable loops are inserted to form an antigen-binding site. Synthetic antibodies based on nucleic acid or peptide structures can be smaller than immunoglobulin-derived antibodies, resulting in greater tissue penetration.

[0117] Antibodies can also include affimer proteins, which are affinity reagents typically having a molecular weight of about 12 - 14 kDa. Affimer proteins typically bind to a target (such as a target protein) with high affinity and specificity. Examples of such targets include, but are not limited to, ubiquitin chains, immunoglobulins, and C-reactive protein. In some embodiments, the affimer protein is derived from a cysteine protease inhibitor and includes a peptide loop and a variable N-terminal sequence that provides a binding site.

[0118] Antibodies can also include single-domain antibodies (V H H domains and VNAR domains), scFv, and Fab fragments.

[0119] (xv) Affinity group

[0120] "Affinity group" is a molecule or molecular moiety that has a high affinity or preference for associating or binding to another specific or particular molecule or moiety. The association or binding to another specific or particular molecule or moiety can occur through non-covalent interactions, such as hydrogen bonds, ionic forces, and van der Waals interactions. For example, an affinity group can be biotin, which has a high affinity or preference for associating or binding to avidin or streptavidin. For example, an affinity group can also refer to avidin or streptavidin, which has an affinity for biotin. Other examples of an affinity group and the specific or particular molecule or moiety to which it binds or associates include, but are not limited to, an antibody or antibody fragment and its respective antigen, such as digoxin and anti-digoxin antibody, lectin and carbohydrate (such as sugar, monosaccharide, disaccharide, or polysaccharide), and receptor and receptor ligand.

[0121] The action of any pair of affinity groups and the specific or particular molecule or moiety to which they bind or associate therewith can be reversed. For example, between a first molecule and a second molecule, in a first instance, the first molecule is characterized as an affinity group for the second molecule, and in a second instance, the second molecule is characterized as an affinity group for the first molecule.

[0122] (xvi) Labels, detectable labels and optical labels

[0123] The terms “detectable label”, “optical label” and “label” are used interchangeably herein to refer to a directly or indirectly detectable moiety associated (e.g., conjugated) with a molecule to be detected (e.g., a capture probe or an analyte). A detectable label can be directly detectable by itself (e.g., a radioisotope label or a fluorescent label), or, in the case of an enzyme label, can be indirectly detectable, e.g., by catalyzing a chemical change in a substrate compound or composition that is directly detectable. A detectable label can be suitable for small-scale detection and / or suitable for high-throughput screening. Accordingly, suitable detectable labels include, but are not limited to, radioisotopes, fluorophores, chemiluminescent compounds, bioluminescent compounds and dyes.

[0124] A detectable label can be detected qualitatively (e.g., optically or spectroscopically), or can be quantified. Qualitative detection generally includes detection methods that confirm the presence or occurrence of a detectable label, while quantifiable detection generally includes detection methods having a quantifiable (e.g., numerically reportable) value, such as intensity, duration, polarization and / or other properties. In some embodiments, a detectable label is bound to or associated with a feature of a capture probe. For example, a feature labeled with a detectable label can include a fluorescent, colorimetric or chemiluminescent label attached to a bead (see, e.g., Rajeswari et al., J. Microbiol Methods 139:22-28, 2017, and Forcucci et al., J. Biomed Opt. 10:105010, 2015, the entire contents of which are incorporated herein by reference).

[0125] In some embodiments, multiple detectable labels can be attached to a feature, capture probe or composition to be detected. For example, detectable labels can be incorporated during nucleic acid polymerization or amplification (e.g., -labeled nucleotides, such as -dCTP). Any suitable detectable label can be used. In some embodiments, the detectable label is a fluorophore. For example, fluorophores can be from the group: 7-AAD (7-aminoactinomycin D), acridine orange (+DNA), acridine orange (+RNA), Alexa- 350, Alexa- 430, Alexa- 488, Alexa- 532, Alexa- 546, Alexa- 555, Alexa- 568, Alexa- 594, Alexa- 633, Alexa- 647, Alexa 660, Alexa 680, Alexa 700, Alexa 750, Allophycocyanin (APC), AMCA / AMCA-X, 7-Aminoactinomycin D (7-AAD), 7-Amino-4-methylcoumarin, 6-Aminoquinoline, Aniline Blue, ANS, APC-Cy7, ATTO-TAG TM CBQCA, ATTO-TAG TM FQ, Auramine O-Feulgen, BCECF (high pH), BFP (Blue Fluorescent Protein), BFP / GFP FRET, BOBO TM -1 / BO-PRO TM -1, BOBO TM -3 / BO-PRO TM -3, FL, TMR, TR-X, 530 / 550, 558 / 568, 564 / 570, 581 / 591, 630 / 650-X, 650-665-X, BTC, Calcein, Calcein Blue, Calcium Crimson TM , Calcium Green-1 TM , Calcium Orange TM , White, 5-Carboxyfluorescein (5-FAM), 5-Carboxynaphthofluorescein, 6-Carboxyrhodamine 6G, 5-Carboxytetramethylrhodamine (5-TAMRA), Carboxy-X-rhodamine (5-ROX), Cascade CascadeYellow TM , CCF2 (GeneBLAzer TM) CFP (Cyan Fluorescent Protein), CFP / YFP FRET, Chromomycin A3, C1-NERF (low pH), CPM, 6-CR 6G, CTC-methyl , Cychrome (PE-Cy5), Dansylamide, Dansylcadaverine, Dansyl chloride, DAPI, Dapoxyl, DCFH, DHR, DiA (4-Di-16-ASP), DiD (DilC18(5)), DIDS, Dil (DilC18(3)), DiO (DiOC18(3)), DiR (DilC18(7)), Di-4ANEPPS, Di-8 ANEPPS, DM-NERF (4.5 - 6.5 pH), DsRed (Red Fluorescent Protein), EBFP, ECFP, EGFP, -97 alcohol, Eosin, Erythromycin, Ethidium bromide, Ethidium homodimer-1 (EthD-1), Europium(III) chloride, 5-FAM (5-carboxyfluorescein), Fast Blue, Fluorescein dT phosphoramidite, FITC, Fluo-3, Fluo-4, Fluoro-Gold TM (high pH), Fluoro-Gold TM (low pH), Fluoro-Jade, 1 - 43, Fura-2 (high calcium), Fura-2 / BCECF, FuraRed TM (high calcium), Fura Red TM / Fluo-3, GeneBLAzer TM (CCF2), GFP red shift (rsGFP), GFP wild type, GFP / BFP-FRET, GFP / DsRed-FRET, Hoechst 33342&33258, 7-hydroxy-4-methylcoumarin (pH9), 1,5-indoleacetic acid, Indo-1 (high calcium), Indo-1 (low calcium), Indodicarbocyanine, Indotricarbocyanine, JC-1, 6-JOE, JOJO TM -1 / JO-PRO TM -1, LDS 751 (+DNA), LDS 751 (+RNA), LOLO TM -1 / LO-PRO TM -1, Lucifer Yellow, LysoSensor TM Blue (pH5), LysoSensor TM Green (pH5), LysoSensor TM Yellow / Blue (pH 4.2), Green, Red, Yellow, Mag-Fura-2, Mag-Indo-1, Magnesium Green TM , Marina 4-Methylumbelliferone, Mitramycin, Green, Orange, Mito Red, NBD (amine), Nile Red, Oregon 488, Oregon 500, Oregon 514, Pacific Blue, PBF1, PE (R-phycoerythrin), PE-Cy5, PE-Cy7, PE-Texas Red, PerCP (Peridinin-Chlorophyll-Protein), PerCP-Cy5.5 (TruRed), PharRed (APC-Cy7), C-Phycocyanin, R-Phycocyanin, R-Phycoerythrin (PE), PI (Propidium Iodide), PKH26, PKH67, POPO TM -1 / PO-PRO TM -1, POPO TM -3 / PO-PRO TM -3, Propidium Iodide (PI), PyMPO, Pyrene, Pyrrolin Y, Quantum Red (PE-Cy5), Quinacrine Mustard, R670 (PE-Cy5), Red 613 (PE-Texas Red), Red Fluorescent Protein (DsRed), Resorufin, RH 414, Rhod-2, Rhodamine B, Rhodamine Green TM , Rhodamine Red TM , Rhodamine Phalloidin, Rhodamine 110, Rhodamine 123, 5-ROX (Carboxy-X-Rhodamine), S65A, S65C, S65L, S65T, SBFI, SITS, -1 (High pH), -2, -1 (High pH), -1 (Low pH), Sodium Green TM , #1, #2, 11, 13, 17, 45, Blue, Green, Orange, 5-TAMRA (5-Carboxytetramethylrhodamine), Tetramethylrhodamine (TRITC), Texas / Texas -X, Texas -X (NHS ester), thiodicarbocyanine, thiazole orange, -1 / TO- -1, -3 / TO- -3, TO- -5, triple color (PE-Cy5), TRITC (tetramethylrhodamine), TruRed (PerCP-Cy5.5), WW 781, X-rhodamine (XRITC), Y66F, Y66H, Y66W, YFP (yellow fluorescent protein), -1 / YO- -1, -3 / YO- -3, 6-FAM (fluorescein), 6-FAM (NHS ester), 6-FAM (azide), HEX, TAMRA (NHS ester), Yakima yellow, MAX, TET, TEX615, ATTO 488, ATTO 532, ATTO 550, ATTO 565, ATTO Rho101, ATTO 590, ATTO 633, ATTO 647N, TYE 563, TYE 665, TYE 705, 700, 800, 800CW (NHS ester), WellRED D4 dye, WellRED D3 dye, WellRED D2 dye, 640 (NHS ester) and Dy 750 (NHS ester).

[0126] As described above, in some embodiments, the detectable label is or includes a luminescent or chemiluminescent moiety. Common luminescent / chemiluminescent moieties include, but are not limited to, peroxidases such as horseradish peroxidase (HRP), soybean peroxidase (SP), alkaline phosphatase, and luciferase. These protein moieties can catalyze chemiluminescent reactions given an appropriate substrate (e.g., an oxidizing reagent plus a chemiluminescent compound). Many families of compounds are known to provide chemiluminescence under various conditions. Non-limiting examples of families of chemiluminescent compounds include 2,3-dihydro-1,4-phthalazinedione luminol, 5-amino-6,7,8-trimethoxy- and dimethylamino [ca] benzene analogs. These compounds can emit light in the presence of alkaline hydrogen peroxide or calcium hypochlorite and base. Other examples of families of chemiluminescent compounds include, for example, 2,4,5-triphenylimidazole, p-dimethylamino and -methoxy substituents, oxalates (e.g., oxalyl active esters), p-nitrophenyl, N-alkyl acridinium esters, fluorescein, lucigenin, or acridinium esters.

[0127] (xvii) Template-switching oligonucleotide

[0128] A "template-switching oligonucleotide" is an oligonucleotide that hybridizes to untemplated nucleotides added by a reverse transcriptase (e.g., an enzyme with terminal transferase activity) during reverse transcription. In some embodiments, the template-switching oligonucleotide hybridizes to an untemplated poly(C) nucleotide added by a reverse transcriptase. In some embodiments, the template-switching oligonucleotide adds a common 5′ sequence to full-length cDNA for cDNA amplification.

[0129] In some embodiments, the template-switching oligonucleotide adds a common sequence to the 5′ end of the RNA being reverse-transcribed. For example, the template-switching oligonucleotide can hybridize to an untemplated poly(C) nucleotide added to the end of a cDNA molecule and provide a template for the reverse transcriptase to continue copying to the 5′ end of the template-switching oligonucleotide, thereby generating full-length cDNA capable of further amplification. In some embodiments, once the full-length cDNA molecule is generated, the template-switching oligonucleotide can serve as a primer in a cDNA amplification reaction.

[0130] In some embodiments, the template-switching oligonucleotide is added before, simultaneously with, or after reverse transcription or other terminal transferase-based reactions. In some embodiments, the template-switching oligonucleotide is included in a capture probe. In certain embodiments, a sample analysis method using a template-switching oligonucleotide can involve generating a nucleic acid product from an analyte of a tissue sample and subsequently further processing the nucleic acid product using the template-switching oligonucleotide.

[0131] The template-switching oligonucleotide can include a hybridization region and a template region. The hybridization region can include any sequence capable of hybridizing to a target. In some embodiments, the hybridization region can include, for example, consecutive G bases to be complementary to the protruding C bases at the 3′ end of a cDNA molecule. The consecutive G bases can include 1 G base, 2 G bases, 3 G bases, 4 G bases, 5 G bases, or more than 5 G bases. The template sequence can include any sequence to be incorporated into the cDNA. In other embodiments, in addition to at least one G base, the hybridization region can include at least one base. In other embodiments, the hybridization can include bases other than G bases. In some embodiments, the template region includes at least 1 (e.g., at least 2, 3, 4, 5, or more) tag sequences and / or functional sequences. In some embodiments, the template region and the hybridization region are separated by a spacer.

[0132] In some embodiments, the template region includes a barcode sequence. The barcode sequence can function as a spatial barcode and / or as a unique molecular identifier. The template conversion oligonucleotide can include deoxyribonucleic acid; ribonucleic acid; modified nucleic acids, including 2-aminopurine, 2,6-diaminopurine (2-amino-dA), inverted dT, 5-methyl dC, 2′-deoxyinosine, super T (5-hydroxybutyl-2′-deoxyuridine), super G (8-aza-7-deazaguanosine), locked nucleic acid (LNA), unlocked nucleic acid (UNA, e.g., UNA-A, UNA-U, UNA-C, UNA-g), Iso-dG, Iso-dC, 2′-fluorobases (e.g., fluoro-C, fluoro-U, fluoro-A, and fluoro-G), or any combination of the above.

[0133] In some embodiments, the length of the template conversion oligonucleotide can be at least about 1, 2, 10, 20, 50, 75, 100, 150, 200, or 250 nucleotides or longer. In some embodiments, the length of the template conversion oligonucleotide can be at most about 2, 10, 20, 50, 100, 150, 200, or 250 nucleotides or longer.

[0134] (xviii) Splint oligonucleotide

[0135] A "splint oligonucleotide" is an oligonucleotide that, when hybridized to other polynucleotides, functions as a "splint" to hold the polynucleotides adjacent to each other so that they can be ligated together. In some embodiments, the splint oligonucleotide is DNA or RNA. The splint oligonucleotide can include a nucleotide sequence that is partially complementary to nucleotide sequences from two or more different oligonucleotides. In some embodiments, the splint oligonucleotide assists in ligating a "donor" oligonucleotide and an "acceptor" oligonucleotide. Generally, RNA ligase, DNA ligase, or other types of ligases are used to ligate two nucleotide sequences together.

[0136] In some embodiments, the length of the splint oligonucleotide is between 10 and 50 oligonucleotides, e.g., between 10 and 45, 10 and 40, 10 and 35, 10 and 30, 10 and 25, or 10 and 20 oligonucleotides. In some embodiments, the length of the splint oligonucleotide is between 15 and 50, 15 and 45, 15 and 40, 15 and 35, 15 and 30, 15 and 30, or 15 and 25 nucleotides.

[0137] (c) Analyte

[0138] The devices, systems, methods, and compositions described in this disclosure can be used to detect and analyze a variety of different analytes. For the purposes of this invention, an "analyte" can include any biological substance, structure, part, or component to be analyzed. The term "target" can similarly refer to an analyte of interest.

[0139] Analytes can be broadly classified into two categories: nucleic acid analytes and non-nucleic acid analytes. Examples of non-nucleic acid analytes include, but are not limited to, lipids, carbohydrates, peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins, phosphoproteins, specific phosphorylated or acetylated variants of proteins, amidated variants of proteins, hydroxylated variants of proteins, methylated variants of proteins, ubiquitinated variants of proteins, sulfated variants of proteins, viral coat proteins, extracellular and intracellular proteins, antibodies, and antigen-binding fragments. In some embodiments, the analyte can be an organelle (e.g., the nucleus or mitochondria).

[0140] Cell surface features corresponding to analytes can 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) status of cell surface proteins, gap junctions, and adherens junctions.

[0141] Analytes can be from a specific type of cell and / or a specific subcellular region. For example, analytes can be from the cytoplasm, nucleus, mitochondria, microsomes, and more generally, from any other compartment, organelle, or part of a cell. Permeabilizing agents that specifically target certain cell compartments and organelles can be used to selectively release analytes from cells for analysis.

[0142] Examples of nucleic acid analytes include DNA analytes, such as genomic DNA, methylated DNA, specific methylated DNA sequences, fragmented DNA, mitochondrial DNA, in situ synthesized PCR products, and RNA / DNA hybrids.

[0143] Examples of nucleic acid analytes also include RNA analytes, such as various types of coding and non-coding RNAs. Different types of RNA analytes include messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), small RNA (miRNA), and viral RNA. RNA can be a transcript (e.g., present in a tissue section). RNA can be small (e.g., less than 200 nucleic acid bases in length) or large (e.g., RNA greater than 200 nucleic acid bases in length). Small RNAs mainly include 5.8S ribosomal RNA (rRNA), 5S rRNA, transfer RNA (tRNA), microRNA (miRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), Piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA), and small rDNA-derived RNA (srRNA). RNA can be double-stranded RNA or single-stranded RNA. RNA can be circular RNA. RNA can be bacterial rRNA (e.g., 16S rRNA or 23S rRNA).

[0144] Other examples of analytes include mRNA and cell surface features (e.g., using the labeling agents described herein), mRNA and intracellular proteins (e.g., transcription factors), mRNA and cell methylation status, mRNA and accessible chromatin (e.g., ATAC-seq, DNase-seq, and / or micrococcal nuclease-seq), mRNA and metabolites (e.g., using the labeling agents described herein), barcoded labeling agents (e.g., the oligonucleotide-labeled antibodies described herein), and the V(D)J sequences of immune cell receptors (e.g., T cell receptors), mRNA and perturbing agents (e.g., CRISPR crRNA / sgRNA, TALEN, zinc finger nucleases, and / or antisense oligonucleotides as described herein).

[0145] An analyte can include a nucleic acid molecule having at least a portion of a nucleic acid sequence encoding a V(D)J sequence of an immune cell receptor (e.g., TCR or BCR). In some embodiments, the nucleic acid molecule is cDNA generated by reverse transcription of the corresponding mRNA using a poly(T)-containing primer. The generated cDNA can then be barcoded using a capture probe that has a barcode sequence (and optional UMI sequence) that hybridizes to at least a portion of the generated cDNA. In some embodiments, a template-switching oligonucleotide hybridizes to a poly(C) tail added to the 3′ end of the cDNA by reverse transcriptase. The original mRNA template and the template-switching oligonucleotide can be denatured from the cDNA, and then the barcoded capture probe can hybridize to the cDNA and generate a complement of the cDNA. Other methods and compositions suitable for barcoding cDNA generated from mRNA transcripts, including those encoding V(D)J regions of immune cell receptors, and / or compositions and barcoding methods including template-switching oligonucleotides are described in PCT patent application PCT / US2017 / 057269, filed Oct. 18, 2017, and U.S. patent application Ser. No. 15 / 825,740, filed Nov. 29, 2017, both of which are incorporated herein by reference in their entireties. V(D)J analysis can also be done by using one or more labeling agents that bind to specific surface features of immune cells and are associated with barcode sequences. The one or more labeling agents can include MHC or MHC multimers.

[0146] As described above, the analyte can include nucleic acids capable of functioning as components of a gene editing reaction, e.g., CRISPR-based gene editing. Thus, the capture probe can include a nucleic acid sequence complementary to the analyte (e.g., a sequence that can hybridize to a CRISPR RNA (crRNA), single guide RNA (sgRNA), or an adaptor sequence engineered into a crRNA or sgRNA).

[0147] In certain embodiments, the analyte can be extracted from a live cell. Processing conditions can be adjusted to ensure that the biological sample remains viable during the assay and the analyte is extracted (or released) from the live cells of the sample. Analytes derived from live cells can only be obtained once from a sample, or can be obtained at intervals from a sample that continues to remain viable.

[0148] Generally, the systems, devices, methods, and compositions can be used to analyze any number of analytes. For example, the number of analytes being 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 1000, at least about 10000, at least about 100000, or more different analytes are present in a sample area or within an individual feature of a substrate. Methods for performing multiplex assays to analyze two or more different analytes will be discussed in subsequent portions of the present invention.

[0149] (d) Biological sample

[0150] (i) Type of biological sample

[0151] A "biological sample" is obtained from a subject for analysis using any of a variety of techniques, including but not limited to biopsy, surgery, and laser capture microscopy (LCM), and generally includes cells and / or other biological materials from the subject. In addition to the above subjects, biological samples can also be obtained from prokaryotes, such as bacteria, e.g., Escherichia coli, Staphylococcus aureus, or Mycoplasma pneumoniae; archaea; viruses, such as hepatitis C virus or human immunodeficiency virus; or viroids. Biological samples can be obtained from non-mammalian organisms (such as plants, insects, arachnids, nematodes, fungi, or amphibians). Biological samples can also be obtained from eukaryotes, such as patient-derived organoids (PDOs) or patient-derived xenografts (PDXs). The subjects from whom biological samples can be obtained can be healthy or asymptomatic individuals, individuals with or suspected of having a disease (e.g., patients with diseases such as cancer), or individuals with a predisposition to a disease, and / or individuals in need of treatment or suspected of needing treatment.

[0152] Biological samples can include any number of macromolecules, such as cellular macromolecules and cell organelles (e.g., mitochondria and nuclei). Biological samples can be nucleic acid samples and / or protein samples. Biological samples can be carbohydrate samples or lipid samples. Biological samples can be obtained as tissue samples, such as tissue sections, biopsies, core biopsies, needle aspirations, or fine needle aspirations. Samples can be liquid samples, such as blood samples, urine samples, or saliva samples. Samples can be skin samples, colon samples, buccal swabs, histological samples, histopathological samples, plasma or serum samples, tumor samples, live cells, cultured cells, clinical samples, such as whole blood or blood-derived products, blood cells, or cultured tissues or cells, including cell suspensions.

[0153] Cell-free biological samples can include extracellular polynucleotides. Extracellular polynucleotides can be isolated from body samples such as blood, plasma, serum, urine, saliva, mucosal excretions, sputum, feces, and tears.

[0154] Biological samples can be from a homogeneous culture or population of the subject or organism described herein, or alternatively from a collection of several different organisms in, for example, a community or ecosystem.

[0155] Biological samples can include one or more diseased cells. Diseased cells can have altered metabolic properties, gene expression, protein expression, and / or morphological characteristics. Examples of diseases include inflammatory disorders, metabolic disorders, neurological disorders, and cancer. Cancer cells can be derived from solid tumors, hematological malignancies, cell lines, or can be obtained in the form of circulating tumor cells.

[0156] Biological samples can also include fetal cells. For example, procedures such as amniocentesis can be performed to obtain fetal cell samples from the maternal circulation. Fetal cell sequencing can be used to identify any of a number of genetic diseases, including, for example, aneuploidies such as Down syndrome, Edwards syndrome, and Patau syndrome. In addition, the cell surface characteristics of fetal cells can be used to identify any of a variety of diseases or disorders.

[0157] Biological samples can also include immune cells. Sequential analysis of the immune function of such cells, including genomics, proteomics, and cell surface characteristics, can provide rich information that helps to understand the state and function of the immune system. For example, determining the status (e.g., negative or positive) of minimal residual disease (MRD) in multiple myeloma (MM) patients after autologous stem cell transplantation is considered a predictor of MRD in MM patients (see, e.g., U.S. Patent Application Publication No. 2018 / 0156784, which is incorporated herein by reference in its entirety).

[0158] Examples of immune cells in biological samples include, but are not limited to, B cells, T cells (e.g., cytotoxic T cells, natural killer T cells, regulatory T cells, and helper T cells), natural killer cells, cytokine-induced killer (CIK) cells, myeloid cells such as granulocytes (basophils, eosinophils, neutrophils / polymorphonuclear neutrophils), monocytes / macrophages, mast cells, platelets / megakaryocytes, and dendritic cells.

[0159] As described above, biological samples can include a single analyte of interest or multiple analytes of interest. Methods for performing multiplex analysis to analyze two or more different analytes in a single biological sample will be discussed in subsequent sections of the present invention.

[0160] (ii) Preparation of Biological Samples

[0161] Various steps can be carried out to prepare a biological sample for analysis. Unless otherwise specified, the preparation steps described below can generally be combined in any manner to appropriately prepare a specific sample for analysis.

[0162] (1) Tissue section

[0163] A biological sample can be obtained from a subject (e.g., by surgical biopsy, whole subject sectioning) or grown in vitro as a cell population on a growth substrate or culture dish and prepared as a tissue cross-section or tissue section for analysis. The grown sample can be thin enough to be analyzed without further processing steps. Alternatively, the grown sample and the sample obtained by biopsy or sectioning can be prepared as thin tissue sections using a mechanical cutting device (e.g., a vibrating blade microtome). As another alternative, in some embodiments, thin tissue sections can be prepared by applying a biological sample toughimprint to a suitable substrate material.

[0164] The thickness of the tissue section can be a fraction of the maximum cross-sectional dimension of the cell (e.g., less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1). However, tissue sections with a thickness greater than the maximum cross-sectional cell dimension can also be used. For example, cryosections with a thickness of, for example, 10 - 20 microns can be used.

[0165] More generally, the thickness of the tissue section typically depends on the method used to prepare the section and the physical properties of the tissue, and thus sections with a variety of different thicknesses can be prepared and used. For example, the thickness of the tissue section can be at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, 1.0, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 20, 30, 40, or 50 microns. If needed or convenient, thicker sections can also be used, e.g., at least 70, 80, 90, or 100 microns or greater. Generally, the thickness of the tissue section is between 1 - 100 microns, 1 - 50 microns, 1 - 30 microns, 1 - 25 microns, 1 - 20 microns, 1 - 15 microns, 1 - 10 microns, 2 - 8 microns, 3 - 7 microns, or 4 - 6 microns, but as noted above, sections with a thickness greater than or less than these ranges can also be analyzed.

[0166] Multiple sections can also be obtained from a single biological sample. For example, multiple tissue sections can be obtained from a surgical biopsy sample by serially sectioning the biopsy sample using a sectioning blade. In this way, the spatial information between consecutive sections can be preserved, and the sections can be analyzed serially to obtain three-dimensional information about the biological sample.

[0167] (2) Freezing

[0168] In some embodiments, a biological sample (e.g., a tissue section as described above) can be prepared by cryopreservation at a temperature suitable for maintaining or preserving the integrity of the tissue structure (e.g., physical properties). For example, the temperature can be below -20 °C, or below -25 °C, -30 °C, -40 °C, -50 °C, -60 °C, -70 °C, -80 °C, -90 °C, -100 °C, -110 °C, -120 °C, -130 °C, -140 °C, -150 °C, -160 °C, -170 °C, -180 °C, -190 °C or -200 °C. The frozen tissue sample can be cut (e.g., sectioned thinly) onto the surface of a substrate using any number of suitable methods. For example, a tissue sample can be prepared using a cryostat (e.g., a microtome), which is set at a temperature suitable for maintaining the structural integrity of the tissue sample and the chemical properties of the nucleic acids in the sample. For example, such a temperature can be below -15 °C, below -20 °C or below -25 °C.

[0169] (3) Formaldehyde fixation and paraffin embedding

[0170] In some embodiments, formaldehyde fixation and paraffin embedding (FFPE) can be used to prepare biological samples, which is an established method. In some embodiments, formaldehyde fixation and paraffin embedding can be used to prepare cell suspensions and other non-tissue samples. After fixing the sample and embedding it in a paraffin or resin block, the sample can be sectioned as described above. Before analysis, the paraffin-embedded material can be removed (e.g., deparaffinized) from the tissue section by incubating the tissue section in a suitable solvent (e.g., xylene) and then rinsing (e.g., 99.5% ethanol for 2 minutes, 96% ethanol for 2 minutes and 70% ethanol for 2 minutes).

[0171] (4) Fixation

[0172] As an alternative to the above formaldehyde fixation, a biological sample can be fixed in any of a variety of other fixatives to maintain the biological structure of the sample prior to analysis. For example, the sample can be fixed by immersing the sample in ethanol, methanol, acetone, paraformaldehyde-Triton and combinations thereof.

[0173] In some embodiments, acetone fixation is used with fresh frozen samples, which can include but are not limited to cortical tissue, mouse olfactory bulb, human brain tumor, human postmortem brain and breast cancer samples. When acetone fixation is performed, a pre-permeabilization step (described below) can be omitted. Alternatively, acetone fixation can be combined with a permeabilization step.

[0174] (5) Embedding

[0175] As an alternative to the above paraffin embedding, biological samples can be embedded in any of a variety of other embedding materials to provide a structural substrate for the sample prior to sectioning and other processing steps. Generally, the embedding material is removed prior to analyzing tissue sections obtained from the sample. Suitable embedding materials include, but are not limited to, waxes, resins (such as methacrylate resins), epoxy resins, and agar.

[0176] (6) Staining

[0177] For ease of visualization, biological samples can be stained using a variety of stains and staining techniques. In some embodiments, for example, the sample can be stained with any number of stains, including but not limited to acridine orange, 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, propidium iodide, rhodamine, or safranin.

[0178] The sample can be stained using hematoxylin-eosin (H&E) staining technique, Papanicolaou staining technique, Masson's trichrome staining technique, silver staining technique, Sudan red staining technique, and / or periodic acid Schiff (PAS) staining technique. PAS staining is usually performed after fixation with formaldehyde or acetone. In some embodiments, the sample can be stained using Romanowsky staining methods, including Wright staining, Giemsa staining, Can-Grünwald staining, Leishman staining, and Giemsa staining.

[0179] In some embodiments, biological samples can be decolorized. Methods for decontaminating or decolorizing biological samples are known in the art and 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. These stains can be removed using techniques such as cleavage of disulfide bonds by treatment with reducing agents and detergents, chaotropic salt treatment, antigen retrieval solution treatment, and acidic glycine buffer treatment. For example, methods for multiple staining and decolorization are described 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 which are incorporated herein by reference.

[0180] (7) Hydrogel embedding

[0181] In some embodiments, a biological sample can be embedded in a hydrogel substrate. Embedding the sample in this way generally involves contacting the biological sample with the hydrogel such that the biological sample is surrounded by the hydrogel. For example, the sample can be embedded by contacting the sample with a suitable polymeric material and activating the polymeric material to form a hydrogel. In some embodiments, the hydrogel is formed such that the hydrogel is internalized within the biological sample.

[0182] In some embodiments, the biological sample is fixed in the hydrogel by crosslinking of the polymeric material that forms the hydrogel. Crosslinking can be carried out chemically and / or photochemically, or by any other hydrogel formation method known in the art.

[0183] The composition and application of the hydrogel substrate to the biological sample generally depend on the nature and preparation of the biological sample (e.g., sectioned, non-sectioned, type of fixation). As an example, where the biological sample is a tissue section, the hydrogel substrate can include a monomer solution and an ammonium persulfate (APS) initiator / tetramethylethylenediamine (TEMED) accelerator solution. As another example, when the biological sample consists of cells (e.g., cultured cells or cells isolated from a tissue sample), the cells can be incubated with the monomer solution and the APS / TEMED solution. For cells, the hydrogel substrate gel is formed in a compartment, including but not limited to a device for culturing, maintaining, or transporting cells. For example, the hydrogel substrate can be formed using a monomer solution plus APS / TEMED, which is added to a depth in the compartment between about 0.1 μm and about 2 mm.

[0184] Other methods and aspects of hydrogel embedding of biological samples are described, for example, in Chen et al., Science 347(6221):543 - 548, 2015, the entire content of which is incorporated herein by reference.

[0185] (8) Isometric Expansion

[0186] In some embodiments, a biological sample embedded in a hydrogel can be isometrically expanded. Isometric expansion methods that can be used include hydration, which is a preparatory step in expansion microscopy, as described in Chen et al., Science 347(6221):543 - 548 2015.

[0187] Isometric expansion can be achieved by anchoring one or more components of the biological sample to the gel and then performing gel formation, proteolysis, and swelling. Isometric expansion of the biological sample can occur before the biological sample is fixed to the substrate, or after the biological sample is fixed to the substrate. In some embodiments, the isometrically expanded biological sample can be removed from the substrate before contacting the substrate with capture probes, which will be discussed in more detail in subsequent sections.

[0188] Generally, the steps for performing equidistant expansion of a biological sample can depend on the characteristics of the sample (e.g., thickness of tissue section, fixation, crosslinking) and / or the analyte of interest (e.g., different conditions for anchoring RNA, DNA, and proteins to the gel).

[0189] In some embodiments, proteins in a biological sample are anchored to a swellable gel (e.g., a polyelectrolyte gel). Antibodies can be directed to the proteins before, after, or while being anchored to the swellable gel. DNA and / or RNA in the biological sample can also be anchored to the swellable gel via a suitable linker. Examples of such linkers include, but are not limited to, 6-((acryloyl)amino)hexanoic acid (acryloyl-X-SE) (available from ThermoFisher, Waltham, MA), Label IT Amine (available from MirusBio, Madison, WI), and Label X (e.g., described in Chen et al., Nat. Methods 13: 679-684, 2016, the entire content of which is incorporated herein by reference).

[0190] Equidistant expansion of the sample can improve the spatial resolution of subsequent analysis of the sample. The increased resolution in the spatial profile can be determined by comparing the equidistant-expanded sample and the non-equidistant-expanded sample.

[0191] In some embodiments, the biological sample is equidistantly expanded to at least 2x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3x, 3.1x, 3.2x, 3.3x, 3.4x, 3.5x, 3.6x, 3.7x, 3.8x, 3.9x, 4x, 4.1x, 4.2x, 4.4x, 4.5x, 4.6x, 4.7x, 4.8x, or 4.9x its non-expanded size. In some embodiments, the sample is equidistantly expanded to at least 2 times and less than 20 times its non-expanded size.

[0192] (9) Substrate attachment

[0193] In some embodiments, the biological sample can be attached to a hydrogel 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 analysis method.

[0194] In certain embodiments, the sample can be reversibly attached to the substrate by applying a suitable polymer coating to the substrate and bringing the sample into contact 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.

[0195] More generally, in some embodiments, a substrate can be coated or functionalized with one or more substances to facilitate attachment of a sample to the substrate. Suitable substances for coating or functionalizing the substrate include, but are not limited to, lectins, polylysine, antibodies, and polysaccharides.

[0196] (10) Cell disaggregation

[0197] In some embodiments, the biological sample corresponds to cells (e.g., from a cell culture or a tissue sample). In a cell sample having multiple cells, the multiple individual cells can be naturally non-aggregated. For example, the cells can be from a cell suspension and / or isolated or disaggregated cells from a tissue or tissue section.

[0198] Alternatively, the cells in the sample can be aggregated and can be disaggregated into multiple individual cells using, for example, enzymatic or mechanical techniques. Examples of enzymes for enzymatic disaggregation include, but are not limited to, dispase, collagenase, trypsin, and combinations thereof. For example, a tissue homogenizer can be used for mechanical disaggregation.

[0199] (11) Suspended and adherent cells

[0200] In some embodiments, the biological sample can be obtained from a cell culture grown in vitro. A sample from a cell culture can include one or more suspended cells that are anchorage-independent within the cell culture. Examples of such cells include, but are not limited to, cells derived from hematopoietic cells and cell lines derived from the following cell lines: Colo205, CCRF-CEM, HL-60, K562, MOLT-4, RPMI-8226, SR, HOP-92, NCI-H322M, and MALME-3M.

[0201] Samples from cell cultures can include one or more adherent cells growing on the surface of a container containing a culture medium. Non-limiting examples of adherent cells include DU145 (prostate cancer) cells, H295R (adrenocortical carcinoma) cells, HeLa (cervical cancer) cells, KBM-7 (chronic myelogenous leukemia) cells, LNCaP (prostate cancer) cells, MCF-7 (breast cancer) cells, MDA-MB-468 (breast cancer) cells, PC3 (prostate cancer) cells, SaOS-2 (bone cancer) cells, SH-SY5Y (neuroblastoma, clone white myeloma) cells, T-47D (breast cancer) cells, THP-1 (acute myeloid leukemia) cells, U87 (glioblastoma) cells, the National Cancer Institute 60 cancer cell line panel (NCI60), vero (African green monkey kidney epithelial cell line) cells, MC3T3 (embryonic calvaria) cells, GH3 (pituitary tumor) cells, PC12 (pheochromocytoma) cells, dog MDCK kidney epithelial cells, Xenopus laevis A6 kidney epithelial cells, zebrafish AB9 cells, and Sf9 insect epithelial cells.

[0202] Other examples of adherent cells are shown in Table 1 and are classified, for example, in "A Catalog of in Vitro Cell Lines, Transplantable Animal and Human Tumors and Yeast," Division of Cancer Treatment and Diagnosis (DCTD), National Cancer Institute, USA (2013) and in "The exomes of the NCI-60 panel: a genomic resource for cancer biology and systems pharmacology" by Abaan et al., Cancer Research 73(14):4372 - 82, 2013, the entire contents of which are incorporated herein by reference.

[0203] Table 1: Examples of adherent cells

[0204]

[0205]

[0206]

[0207] In some embodiments, the adherent cells are cells corresponding to one or more of the following cell lines: BT549, HS578T, MCF7, MDA-MB-231, MDA-MB-468, T-47D, SF268, SF295, SF539, SNB-19, SNB-75, U251, Colo205, HCC 2998, HCT-116, HCT-15, HT29, KM12, SW620, 786-O, A498, ACHN, CAKI, RXF 393, SN12C, TK-10, UO-31, A549, EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-H460, NCI-H522, LOX IMVI, M14, MALME-3M, MDA-MB-435, SK-, EL-2, SK-MEL-28, SK-MEL-5, UACC-257, UACC-62, IGROV1, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, SK-OV-3, NCI-ADR-RES, DU145, PC-3, DU145, H295R, HeLa, KBM-7, LNCaP, MCF-7, MDA-MB-468, PC3, SaOS-2, SH-SY5Y, T-47D, THP-1, U87, vero, MC3T3, GH3, PC12, dog MDCK renal epithelial, Xenopus laevis A6 renal epithelial, zebrafish AB9, and Sf9 insect epithelial cell lines.

[0208] (12) Tissue permeabilization

[0209] In some embodiments, the biological sample can be permeabilized to facilitate the transfer of analytes out of the sample and / or to facilitate the transfer of substances (such as capture probes) into the sample. If the sample is not permeabilized enough, the amount of analyte captured from the sample may be too low for sufficient analysis. Conversely, if the tissue sample is too permeable, the relative spatial relationship of the analytes within the tissue sample may be lost. Therefore, it is desirable to balance sufficient permeabilization of the tissue sample to obtain good signal intensity while still maintaining the spatial resolution of the analyte distribution in the sample.

[0210] Generally, a biological sample can be permeabilized by exposing the sample to one or more permeabilizing agents. Suitable reagents for this purpose include, but are not limited to, organic solvents (such as acetone, ethanol, and methanol), crosslinking agents (such as paraformaldehyde), detergents (such as saponin, Triton X-100 TM or Tween-20 TM) and enzymes (such as trypsin, protease). In some embodiments, the biological sample can be incubated with a cell permeabilizer to facilitate permeabilization of the sample. For example, other methods of sample permeabilization are described in Jamur et al., Method Mol. Biol. 588: 63-66, 2010, the entire content of which is incorporated herein by reference. Any suitable method for sample permeabilization can generally be used in conjunction with the samples described herein.

[0211] In some embodiments, where an anti-diffusion medium is used during an assay procedure to restrict the migration of an analyte or other substance, the anti-diffusion medium can include at least one permeabilizing reagent. For example, the anti-diffusion medium can include pores (e.g., micropores, nanopores, or picopores) containing a permeabilization buffer or reagent. In some embodiments, where the anti-diffusion medium is a hydrogel, the hydrogel can include a permeabilization buffer. In some embodiments, the hydrogel is soaked in a permeabilization buffer prior to contact with the sample. In some embodiments, when the anti-diffusion medium is applied to a biological sample, the hydrogel or other anti-diffusion medium can contain a dry reagent or monomer to deliver the permeabilizing reagent. In some embodiments, the anti-diffusion medium (i.e., the hydrogel) is covalently attached to a solid substrate (i.e., a plexiglass slide). In some embodiments, the hydrogel can be modified to both contain capture probes and deliver a permeabilizing reagent. For example, a hydrogel film can be modified to include spatially barcoded capture probes. The spatially barcoded hydrogel film is then soaked in a permeabilization buffer prior to contact with the sample. Thus, the spatially barcoded hydrogel film delivers the permeabilizing reagent to the sample surface in contact with the spatially barcoded hydrogel, enhancing analyte migration and capture. In some embodiments, the spatially barcoded hydrogel is applied to the sample and placed in a large volume of permeabilization solution. In some embodiments, a hydrogel film soaked in a permeabilizing reagent is sandwiched between the sample and a spatially barcoded array. In some embodiments, the target analyte is able to diffuse through the permeabilizing reagent-soaked hydrogel and hybridize or bind to capture probes on the other side of the hydrogel. In some embodiments, the thickness of the hydrogel is proportional to the loss of resolution. In some embodiments, pores (e.g., micrometer pores, nanometer pores, or picometer pores) can contain spatially barcoded capture probes and a permeabilizing reagent and / or buffer. In some embodiments, the spatially barcoded capture probes and the permeabilizing reagent are held between spacers. In some embodiments, the sample is punched, cut, or transferred into the pores, where the target analyte diffuses through the permeabilizing reagent / buffer to the spatially barcoded capture probes. In some embodiments, the loss of resolution can be proportional to the gap thickness (e.g., the amount of permeabilization buffer between the sample and the capture probes). In some embodiments, the thickness of the anti-diffusion medium (e.g., the hydrogel) is between about 50 - 500 micrometers, including thicknesses of 500, 450, 400, 350, 300, 250, 200, 150, 100, or 50 micrometers, or any thickness within 50 and 500 micrometers.

[0212] In some embodiments, the permeabilization solution can be delivered to the sample through a porous membrane. In some embodiments, the porous membrane is used to limit the loss of diffusible analytes while allowing the permeabilization reagent to reach the sample. The membrane chemistry and pore size can be controlled to minimize analyte loss. In some embodiments, the porous membrane can be made of glass, silicon, paper, hydrogel, polymer monolith, or other materials. In some embodiments, the material can be naturally porous. In some embodiments, the material can have pores or holes etched into the solid material. In some embodiments, the permeabilization reagent flows through microfluidic chambers or channels on the porous membrane. In some embodiments, the flow controls the accessibility of the sample to the permeabilization reagent. In some embodiments, the porous membrane is sandwiched between a spatially barcoded array and the sample, where the permeabilization solution is applied to the porous membrane. The permeabilization reagent diffuses through the membrane pores into the tissue.

[0213] In some embodiments, a biological sample can be permeabilized by adding one or more lysis agents to the sample. Examples of suitable lysis agents include, but are not limited to, bioactive reagents such as lytic enzymes for lysing different cell types, such as Gram-positive or Gram-negative bacteria, plants, yeast, mammals, such as lysozyme, achromopeptidase, lysostaphin, Labiase enzyme, kitalase, lyticase, and a variety of other commercially available lytic enzymes.

[0214] Other lysis agents can be added to the biological sample additionally or alternatively to facilitate permeabilization. For example, surfactant-based lysis solutions can be used to lyse sample cells. The lysis solution can include ionic surfactants such as sarcosyl and sodium dodecyl sulfate (SDS). More generally, chemical lysis agents can include, but are not limited to, organic solvents, chelating agents, detergents, surfactants, and chaotropes.

[0215] In some embodiments, a biological sample can be permeabilized by non-chemical permeabilization methods. Non-chemical permeabilization methods are known in the art. For example, non-chemical permeabilization methods that can be used include, but are not limited to, physical lysis techniques such as electroporation, mechanical permeabilization methods (e.g., bead milling using a homogenizer and grinding beads to mechanically disrupt the tissue structure of the sample), acoustic permeabilization (e.g., ultrasound), and thermal decomposition techniques such as heating to induce thermal permeabilization of the sample.

[0216] (13) Selective enrichment of RNA species

[0217] In some embodiments where the RNA is the analyte, one or more RNA analytes of interest can be selectively enriched. For example, one or more RNAs of interest can be selected by adding one or more oligonucleotides to a sample. In some embodiments, other oligonucleotides are sequences for polymerase-initiated reactions. For example, one or more primer sequences that are sequence-complementary to one or more RNAs of interest can be used to amplify one or more RNAs of interest, thereby selectively enriching these RNAs. In some embodiments, oligonucleotides that are sequence-complementary to the complementary strand of the captured RNA (e.g., cDNA) can bind to the cDNA. For example, biotinylated oligonucleotides having sequences complementary to one or more cDNAs of interest can bind to the cDNA and can be selected using any of a variety of methods known in the art (e.g., streptavidin beads) that utilize the biotin-streptavidin affinity.

[0218] Alternatively, any of a variety of methods can be used to down-select (e.g., deplete) one or more RNAs. For example, a probe can be administered to the sample that selectively hybridizes to ribosomal RNA (rRNA), thereby reducing the pool and concentration of rRNA in the sample. Subsequently applying a capture probe to the sample can result in improved capture of other types of RNA due to the reduction of non-specific RNA present in the sample. Additionally or alternatively, double-stranded specific nuclease (DSN) treatment can deplete rRNA (see, e.g., Archer et al., Selective and flexible depletion of problematic sequences from RNA-seq libraries at the cDNA stage, BMC Genomics, 15:401, (2014), the entire content of which is incorporated herein by reference). In addition, hydroxyapatite chromatography can deplete abundant species (e.g., rRNA) (see, e.g., Vandernoot, V.A., cDNA normalization by hydroxyapatite chromatography to enrich transcriptome diversity in RNA-seq applications, Biotechniques, 53(6):373-80, (2012), the entire content of which is incorporated herein by reference).

[0219] (14) Other reagents

[0220] Prior to analyzing the sample, other reagents may be added to the biological sample for various functions. In some embodiments, DNase and RNase inactivators or inhibitors (such as Proteinase K) and / or chelating agents (such as EDTA) may be added to the sample.

[0221] In some embodiments, the sample may be treated with one or more enzymes. For example, one or more endonucleases for fragmenting DNA, DNA polymerases, and dNTPs for amplifying nucleic acids may be added. Other enzymes that may also be added to the sample include, but are not limited to, polymerases, transposases, ligases, DNases, and RNases.

[0222] In some embodiments, reverse transcriptase may be added to the sample, including enzymes with terminal transferase activity, primers, and switch oligonucleotides. Template switching can be used to increase the length of cDNA, for example, by appending a predefined nucleic acid sequence to the cDNA.

[0223] (15) Pretreatment for capturing probe interaction

[0224] In some embodiments, analytes in the biological sample may be pretreated prior to interaction with the capture probe. For example, a polymerization reaction catalyzed by a polymerase (such as DNA polymerase or reverse transcriptase) is performed in the biological sample prior to interaction with the capture probe. In some embodiments, the primers for the polymerization reaction include functional groups that enhance hybridization with the capture probe. The capture probe may include an appropriate capture domain to capture the biological analyte of interest (e.g., a poly(dT) sequence to capture poly(A) mRNA).

[0225] In some embodiments, biological analytes are pretreated to generate a library by next-generation sequencing. For example, analytes can be pretreated by adding modifiers (such as ligating sequences that interact with the capture probe). In some embodiments, fragmentation techniques (such as using transposases and / or fragmentation buffers) are used to fragment analytes (such as DNA or RNA).

[0226] The analyte can be modified after fragmentation. For example, the modification can be adding an adapter sequence that hybridizes with the capture probe by ligation. In some embodiments, where the analyte of interest is RNA, poly(A) tailing is performed. Adding a poly(A) tail to RNA that does not contain a poly(A) tail can facilitate hybridization with a capture probe that includes a capture domain with a functional amount of poly(dT) sequence.

[0227] In some embodiments, a ligase-catalyzed ligation reaction is performed in a biological sample prior to interaction with a capture probe. In some embodiments, the ligation can be performed by chemical ligation. In some embodiments, click chemistry can be used to perform the ligation, as described below. In some embodiments, the capture domain comprises a DNA sequence that is complementary to an RNA molecule, wherein the RNA molecule is complementary to a second DNA sequence, and wherein the RNA-DNA sequence complementarity is used to ligate the second DNA sequence to the DNA sequence in the capture domain. In these embodiments, the RNA molecule can be directly detected.

[0228] In some embodiments, a target-specific reaction is performed in a biological sample prior to interaction with a capture probe. Examples of target-specific reactions include, but are not limited to, the ligation of target-specific adaptors, probes, and / or other oligonucleotides, target-specific amplification using primers specific for one or more analytes, and target-specific detection using in situ hybridization, DNA microscopy, and / or antibodies. In some embodiments, the capture probe comprises a capture domain (e.g., amplification or ligation) that is targeted to a target-specific product.

[0229] II. General spatially array-based analysis methods

[0230] This section of the invention describes methods, devices, systems, and compositions for the analysis of biological samples based on spatially arrays.

[0231] (a) Spatial analysis method

[0232] Array-based spatially analysis methods involve transferring one or more analytes from a biological sample to a feature array on a substrate, each associated with a unique spatial position on the array. Subsequent analysis of the transferred analytes includes determining the identity of the analytes and the spatial position of each analyte in the sample. The spatial position of each analyte in the sample is determined based on the features to which each analyte binds in the array and the relative spatial positions of the features in the array.

[0233] There are at least two general methods for associating a spatial barcode with one or more adjacent cells such that the spatial barcode identifies one or more cells and / or the contents of one or more cells as being associated with a particular spatial position. One general method is to drive a target analyte out of the cell and toward a spatially barcoded array. Figure 1 An exemplary embodiment of this general method is depicted. In Figure 1In this method, a spatially barcoded array that aggregates capture probes (as further described herein) is contacted with a sample 101, and the sample is permeabilized to allow the target analyte to migrate from the sample to the array. The target analyte interacts with the capture probes on the spatially barcoded array 102. Once the target analyte hybridizes / binds to the capture probes, the sample is optionally removed from the array and the capture probes are analyzed to obtain spatially resolved analyte information 103.

[0234] Another general method is to lyse the spatially barcoded capture probes from the array and drive the spatially barcoded capture probes towards and / or into or onto the sample. Figure 2 An exemplary embodiment of this general method is depicted where a spatially barcoded array that aggregates capture probes (as further described herein) can be contacted with a sample 201. The spatially barcoded capture probes are lysed and then interact with the cells within the provided sample 202. This interaction can be a covalent or non-covalent cell surface interaction. The interaction can be an intracellular interaction facilitated by a delivery system or a cell-penetrating peptide. Once the spatially barcoded capture probes are associated with specific cells, the sample can be optionally removed for analysis. The sample can be selectively separated prior to analysis. Once the labeled cells are associated with the spatially barcoded capture probes, the capture probes can be analyzed to obtain spatially resolved information about the labeled cells 203.

[0235] Figure 3 An exemplary workflow for preparing a sample on a spatially barcoded array is shown. Sample preparation can include placing the sample on a slide, fixing the sample, and / or staining the sample for imaging. The stained sample is then imaged on the array 302 using bright field (imaging the sample stained with hematoxylin and eosin) and fluorescence (imaging features) modes. In some embodiments, the target analyte is then released from the sample, and the capture probes forming the spatially barcoded array hybridize or bind to the released target analyte 303. The sample is then removed from the array 304, and the capture probes are lysed from the array 305. The sample and the array are then optionally imaged a second time in two modes 305B while reverse transcribing the analyte into cDNA, preparing an amplicon library 306, and sequencing 307. These two sets of images are then spatially overlapped in order to correlate the spatially identified sample information 308. When there is no second imaging 305B of the sample and the array, a spot coordinate file is provided by the manufacturer. The spot coordinate file replaces the second imaging step 305B. Additionally, unique PCR adapters and sequencing 307 can be used to perform amplicon library preparation 306.

[0236] Figure 4Shows another exemplary workflow that utilizes an array of spatial markers on a substrate, where capture probes labeled with spatial barcodes aggregate in regions called features. The spatially labeled capture probes can include a cleavage domain, one or more functional sequences, a spatial barcode, a unique molecular identifier, and a capture domain. The spatially labeled capture probes can also include a 5′-end modification for reversible attachment to the substrate. The spatial barcode array is contacted with sample 401, and the sample is permeabilized by application of a permeabilization reagent 402. The permeabilization reagent can be administered by placing the array / sample assembly in a bulk solution. Alternatively, the permeabilization reagent can be administered to the sample via an anti-diffusion medium and / or a physical barrier (e.g., a lid), where the sample is sandwiched between the anti-diffusion medium and / or barrier and the substrate containing the array. The analyte is migrated to the spatially barcoded capture array using any number of techniques disclosed herein. For example, analyte migration can be performed using an anti-diffusion medium lid and passive migration. As another example, analyte migration can be active migration, e.g., using an electrophoresis transfer system. Once the analyte is in proximity to the spatially barcoded capture probes, the capture probes can hybridize or otherwise bind to the target analyte 403. The sample can optionally be removed from the array 404.

[0237] The capture probes can optionally be cleaved from the array 405, and the captured analyte can be spatially labeled by performing a reverse transcriptase first-strand cDNA reaction. The first-strand cDNA reaction can optionally be performed using a template-switching oligonucleotide. For example, the template-switching oligonucleotide can hybridize to a poly(C) tail added to the 3′-end of the cDNA by reverse transcriptase. The original mRNA template and the template-switching oligonucleotide can be denatured from the cDNA, and then the barcoded capture probes can hybridize to the cDNA and can generate a complement of the cDNA. The first-strand cDNA can then be purified and collected for downstream amplification steps. The first-strand cDNA can be amplified using PCR 406, where forward and reverse primers flank the spatial barcode and the targeted analyte region of interest, generating a library associated with a particular spatial barcode. In some embodiments, the cDNA contains a sequencing-by-synthesis (SBS) primer sequence. The library amplicons are sequenced and analyzed to decode the spatial information 407.

[0238] Figure 5An exemplary workflow is described where a sample is removed from a spatially barcoded array and spatially barcoded capture probes are removed from the array for barcoded analyte amplification and library preparation. Another embodiment includes performing first strand synthesis on the spatially barcoded array using template switching oligonucleotides without cleaving the capture probes. In this embodiment, sample preparation 501 and permeabilization 502 are performed as described elsewhere herein. Once the capture probes capture the target analyte, the first strand cDNA generated by template switching and reverse transcriptase 503 is then denatured and the second strand is subsequently extended 504. The second strand cDNA is then denatured, neutralized, and transferred to a tube 505 from the first strand cDNA. cDNA quantification and amplification can be performed using standard techniques discussed herein. The cDNA can then undergo library preparation 506 and indexing 507, including fragmentation, end repair, A-tailing, and indexing PCR steps.

[0239] In some non-limiting examples of the above workflow, the sample can be immersed in 100% chilled methanol and incubated at -20 °C for 30 minutes. After 20 minutes, the sample can be removed and rinsed in ultrapure water. After rinsing the sample, a fresh eosin solution is prepared and the sample is covered with isopropanol. After incubating the sample in isopropanol for 1 minute, the reagent can be removed by holding the slide at an angle (the bottom edge of the slide can contact a laboratory wipe and air dry). The sample can be evenly covered with hematoxylin solution and incubated at room temperature for 7 minutes. After incubating the sample in hematoxylin for 7 minutes, the reagent can be removed by holding the slide at an angle (the bottom edge of the slide can contact a laboratory wipe). The slide containing the sample can be immersed in water and the excess liquid can be removed. The sample is then covered with bluing buffer and incubated at room temperature for 2 minutes. The slide containing the sample is immersed in water again, evenly covered with eosin solution, and incubated at room temperature for 1 minute. The slide can be air dried and incubated at 37 °C for 5 minutes. The sample can be imaged using the methods disclosed herein.

[0240] The following are non-limiting exemplary steps for sample permeabilization and cDNA generation. The sample can be exposed to permeabilase and incubated at 37 °C for 6 minutes. Other permeabilization methods are described herein. By adding SSC buffer, the permeabilase can be removed and the sample can be prepared for analyte capture. The sample is then subjected to a pre-equilibration thermal cycling protocol and the SSC buffer is removed. A master mix containing nuclease-free water, reverse transcriptase reagent, template-switching oligonucleotide, reducing agent, and reverse transcriptase can be added, and the sample with the master mix can be subjected to a thermal cycling protocol. The reagents can be removed from the sample, and NaOH can be applied and incubated at room temperature for 5 minutes. The sodium hydroxide can be removed, and an elution buffer can be added and removed from the sample. A second-strand mix, including second-strand reagent, second-strand primer, and second-strand enzyme, can be added to the sample, and the sample can be sealed and incubated. After incubation, the reagents are removed, an elution buffer is added and removed from the sample, NaOH is added to the sample again, and the sample is incubated at room temperature for 10 minutes. Tris-HCl can be added and the reagents are mixed.

[0241] The following steps are non-limiting exemplary steps for cDNA amplification and quality control. A qPCR mix can be prepared, including nuclease-free water, qPCR master mix, and cDNA primers, and a NaOH / Tris-HCl mix can be mixed with the qPCR mix and the sample and thermal cycling can be performed according to a predetermined thermal cycling protocol. After completion of thermal cycling, a cDNA amplification mix can be prepared and combined and mixed with the sample. The sample is then incubated and thermally cycled. Then, the sample can be resuspended in SPRIselect reagent and pipetted to ensure proper mixing. The sample can then be incubated at room temperature for 5 minutes and the sample can be placed on a magnet (e.g., the magnet is in the high position) for cleanup. The supernatant is removed, 80% ethanol is added to the pellet, and incubated for 30 seconds. The ethanol can be removed and the pellet can be washed again. The sample is then centrifuged and placed on a magnet (e.g., the magnet is in the low position). Any residual ethanol can be removed and the sample can be air-dried. The magnet can be removed, an elution buffer is added to the sample, mixed, and incubated at room temperature for 2 minutes. The sample is then placed on a magnet (e.g., in the high position) until the solution is clear. A portion of the sample can be run on an Agilent Bioanalyzer High Sensitivity Chip, where an area can be selected and the cDNA concentration can be measured to calculate the total cDNA yield. Alternatively, quantification can be performed by an Agilent Bioanalyzer or Agilent TapeStation.

[0242] The following steps are non-limiting exemplary steps for constructing a spatial gene expression library. The fragmentation mixture, including fragmentation buffer and fragmentation enzyme, can be prepared on ice. The elution buffer and fragmentation mixture can be added to each sample, mixed, and centrifuged. Then the sample mixture can be placed in a thermal cycler and cycled according to a predetermined protocol. SPRIselect reagent can be added to the sample and incubated at room temperature for 5 minutes. The sample can be placed on a magnet (e.g., in the high position) until the solution is clear, and the supernatant can be transferred to a new tube strip. SPRIselect reagent can be added to the sample, mixed, and incubated at room temperature for 5 minutes. The sample can be placed on a magnet (e.g., in the high position) until the solution is clear. The supernatant is removed, 80% ethanol is added to the pellet, and the pellet is incubated for 30 seconds, and the ethanol can be removed. The ethanol wash can be repeated, and the sample can be placed on a magnet (e.g., in the low position) until the solution is clear. The remaining ethanol can be removed, elution buffer is added to the sample, mixed, and incubated at room temperature for 2 minutes. The sample can be placed on a magnet (e.g., in the high position) until the solution is clear, and a portion of the sample is transferred to a new tube strip. An adapter ligation mixture, including ligation buffer, DNA ligase, and adapter oligonucleotides, can be prepared and centrifuged. The adapter ligation mixture can be added to the sample, pipetted to mix, and briefly centrifuged. Then the sample can be thermally cycled according to a predetermined protocol. SPRIselect reagent can be added to the sample, incubated at room temperature for 5 minutes, and placed on a magnet (e.g., in the high position) until the solution is clear. The supernatant is removed, the pellet is washed with 80% ethanol, incubated for 30 seconds, and the ethanol can be removed. The ethanol wash can be repeated, and the sample can be briefly centrifuged before placing the sample on a magnet (e.g., in the low position). Any remaining ethanol can be removed, and the sample can be air-dried. Elution buffer can be added to the sample, the sample is removed from the magnet, then the sample is pipetted to mix, incubated at room temperature for 2 minutes, and placed on a magnet (e.g., in the low position) until the solution is clear. A portion of the sample can be transferred to a new tube strip. A sample index PCR mixture, including amplification mixture and SI primers, can be prepared and combined with the sample. The sample / sample index PCR mixture can be loaded into individual chromium i7 sample index wells and a thermal cycling protocol can be used. SPRIselect reagent can be added to each sample, mixed, and incubated at room temperature for 5 minutes. The sample can be placed on a magnet (e.g., in the high position) until the solution is clear, and the supernatant can be transferred to a new tube strip. SPRIselect reagent can be added to each sample, pipetted to mix, and incubated at room temperature for 5 minutes. Then the sample can be placed on a magnet (e.g., in the high position) until the solution is clear. The supernatant is removed, the pellet is washed with 80% ethanol, incubated for 30 seconds, and then the ethanol can be removed. The ethanol wash can be repeated, the sample is centrifuged, and placed on a magnet (e.g., in the low position) to remove any remaining ethanol.The sample can be removed from the magnet, the elution buffer can be added to the sample, pipetted to mix, and incubated at room temperature for 2 minutes. The sample can be placed on the magnet (e.g., at a high position) until the solution is clear, and a portion of the sample can be transferred to a new tube strip. The average fragment size can be determined using a Bioanalyzer trace or an Agilent TapeStation.

[0243] In some embodiments, performing a correlation analysis on the data generated by this workflow and other workflows described herein can result in a correlation of more than 95% (e.g., 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher) of the genes expressed across two capture regions. When single-cell RNA sequencing of the cell nucleus is used to perform the workflow, in some embodiments, the correlation analysis of the data can result in a correlation of more than 90% (e.g., more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) of the genes expressed across two capture regions.

[0244] (b) Capture probe

[0245] A "capture probe" refers to any molecule that can capture (directly or indirectly) and / or label an analyte of interest in a biological sample. In some embodiments, the capture probe is a nucleic acid or a polypeptide. In some embodiments, the capture probe is a conjugate (e.g., an oligonucleotide-antibody conjugate). In some embodiments, the capture probe includes barcodes (e.g., spatial barcodes and / or unique molecular identifiers (UMIs)) and a capture domain.

[0246] Figure 6Is a schematic diagram showing an example of a capture probe as described herein. As shown, the capture probe 602 is optionally coupled to the feature 601 through a cleavage domain 603 such as a disulfide bond linker. The capture probe may include functional sequences useful for subsequent processing, such as the functional sequence 604, which may include a sequencer-specific flow cell attachment sequence, such as a P5 sequence, and the functional sequence 606, which may include a sequencing primer sequence, such as an R1 primer binding site. In some embodiments, the sequence 604 is a P7 sequence and the sequence 606 is an R2 primer binding site. The spatial barcode 605 may be included within the capture probe for barcoding the target analyte. The functional sequences are typically selected to be compatible with any of a variety of different sequencing systems, such as 454 sequencing, Ion Torrent Proton or PGM, Illumina X10, PacBio, Nanopore, etc. and their requirements. In some embodiments, the functional sequences are selected to be compatible with non-commercial sequencing systems. Examples of such sequencing systems and techniques that may use appropriate functional sequences include (but are not limited to) Roche 454 sequencing, Ion Torrent Proton or PGM sequencing, Illumina X10 sequencing, PacBio SMRT sequencing, and Oxford Nanopore sequencing. Additionally, in some embodiments, the functional sequences may be selected to be compatible with other sequencing systems, including non-commercial sequencing systems.

[0247] In some embodiments, the spatial barcode 605, the functional sequences 604 (e.g., the flow cell attachment sequence) and 606 (e.g., the sequencing primer sequence) may be common to all probes attached to a given feature. The spatial barcode may also include a capture domain 607 to facilitate the capture of the target analyte.

[0248] Capture domain

[0249] As described above, each capture probe includes at least one capture domain. A "capture domain" is an oligonucleotide, polypeptide, small molecule, or any combination thereof that specifically binds to a desired analyte. In some embodiments, the capture domain may be used to capture or detect the desired analyte.

[0250] In some embodiments, the capture domain is a functional nucleic acid sequence configured to interact with one or more analytes, such as one or more different types of nucleic acids (e.g., RNA molecules and DNA molecules). In some embodiments, the functional nucleic acid sequence may include an N-mer sequence (e.g., a random N-mer sequence), wherein the N-mer sequence is configured to interact with multiple DNA molecules. In some embodiments, the functional sequence may include a poly(T) sequence, which is configured to interact with messenger RNA (mRNA) molecules via the poly(A) tail of the mRNA transcript. In some embodiments, the functional nucleic acid sequence is a binding target for a protein (e.g., a transcription factor, a DNA-binding protein, or an RNA-binding protein), wherein the analyte of interest is a protein.

[0251] The capture probe may include ribonucleotides and / or deoxyribonucleotides and synthetic nucleotide residues capable of participating in Watson-Crick type or similar base pair interactions. In some embodiments, the capture domain is capable of initiating a reverse transcription reaction to produce cDNA complementary to the captured RNA molecule. In some embodiments, the capture domain of the capture probe may initiate a DNA extension (polymerase) reaction to produce DNA complementary to the captured DNA molecule. In some embodiments, the capture domain may serve as a template for a ligation reaction between the captured DNA molecule and a surface probe directly or indirectly immobilized on a substrate. In some embodiments, the capture domain may be ligated to one strand of the captured DNA molecule. For example, SplintR ligase together with an RNA or DNA sequence (e.g., a degenerate RNA) can be used to ligate single-stranded DNA or RNA to the capture domain. In some embodiments, a ligase having RNA-templated ligase activity (e.g., SplintR ligase, T4 RNA ligase 2, or KOD ligase) can be used to ligate single-stranded DNA or RNA to the capture domain. In some embodiments, the capture domain includes a splint oligonucleotide. In some embodiments, the capture domain captures the splint oligonucleotide.

[0252] In some embodiments, the capture domain is located at the 3′ end of the capture probe and includes a free 3′ end, which can be extended, for example, by template-dependent polymerization to form an extended capture probe as described herein. In some embodiments, the capture domain includes a nucleotide sequence capable of hybridizing to a nucleic acid (e.g., RNA or other analyte) present in a cell of a tissue sample contacting the array. In some embodiments, the capture domain can be selected or designed to selectively or specifically bind to a target nucleic acid. For example, the capture domain can be selected or designed to capture mRNA by hybridizing to the mRNA poly(A) tail. Thus, in some embodiments, the capture domain includes a poly(T) DNA oligonucleotide, i.e., a series of consecutive deoxythymidine residues linked by phosphodiester bonds, which is capable of hybridizing to the poly(A) tail of mRNA. In some embodiments, the capture domain can include nucleotides that are functionally or structurally similar to the poly(T) tail. For example, a poly(U) oligonucleotide or an oligonucleotide containing deoxythymidine analogs. In some embodiments, the capture domain includes at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides. In some embodiments, the capture domain includes at least 25, 30, or 35 nucleotides.

[0253] In some embodiments, a random sequence (e.g., a random hexamer or a similar sequence) can be used to form all or part of the capture domain. For example, a random sequence can be used in combination with a poly(T) (or poly(T)-like) sequence. Thus, in cases where the capture domain includes a poly(T) (or “poly(T)-like”) oligonucleotide, it can also include a random oligonucleotide sequence (e.g., a “poly(T)-random sequence” probe). For example, this can be located at the 5′ or 3′ of the poly(T) sequence, e.g., at the 3′ end of the capture domain. The poly(T)-random sequence probe can facilitate the capture of the mRNA poly(A) tail. In some embodiments, the capture domain can be a completely random sequence. In some embodiments, a degenerate capture domain can be used.

[0254] In some embodiments, a set of two or more capture probes forms a mixture, wherein the capture domain of one or more capture probes comprises a poly(T) sequence, and the capture domain of one or more capture probes comprises a random sequence. In some embodiments, a set of two or more capture probes forms a mixture, wherein the capture domain of one or more capture probes comprises a poly(T)-like sequence, and the capture domain of one or more capture probes comprises a random sequence. In some embodiments, a set of two or more capture probes forms a mixture, wherein the capture domain of one or more capture probes comprises a poly(T) random sequence, and the capture domain of one or more capture probes comprises a random sequence. In some embodiments, probes having degenerate capture domains can be added to any of the foregoing combinations listed herein. In some embodiments, probes having degenerate capture domains can replace one of the probes in each pair described herein.

[0255] The capture domain can be based on a particular gene sequence or a particular motif sequence or a consensus / conserved sequence (i.e., a sequence-specific capture domain) that it is designed to capture. Thus, in some embodiments, the capture domain is capable of selectively binding to a desired nucleic acid subtype or subset, such as a particular type of RNA, such as mRNA, rRNA, tRNA, SRP RNA, tRNA, snRNA, snRNA, SmY RNA, sARNA, gRNA, ribozyme P, ribozyme MRP, TERC, SL RNA, aRNA, cis-NAT, crRNA, lncRNA, miRNA, piRNA, siRNA, shRNA, tasiRNA, rasiRNA, 7SK, eRNA, ncRNA, or other types of RNA. In a non-limiting example, the capture domain is capable of selectively binding to a desired subset of ribonucleic acids, such as microbiome RNA, such as 16S rRNA.

[0256] In some embodiments, the capture domain comprises an "anchor" or "anchor sequence", which is a nucleotide sequence designed to ensure hybridization of the capture domain to the expected bioanalyte. In some embodiments, the anchor sequence comprises a nucleotide sequence, including a 1-mer, 2-mer, 3-mer, or longer sequence. In some embodiments, the short sequence is random. For example, a capture domain comprising a poly(T) sequence can be designed to capture mRNA. In such embodiments, the anchor sequence can comprise a random 3-mer (e.g., GGG) that helps ensure hybridization of the poly(T) capture domain to mRNA. In some embodiments, the anchor sequence can be VN, N, or NN. Alternatively, a specific nucleotide sequence can be used to design the sequence. In some embodiments, the anchor sequence is located at the 3' end of the capture domain. In some embodiments, the anchor sequence is located at the 5' end of the capture domain.

[0257] In some embodiments, the capture domain of the capture probe is blocked prior to contact of the biological sample with the array, and a blocking probe is used when the nucleic acid in the biological sample is modified prior to its capture onto the array. In some embodiments, the blocking probe is used to block or modify the free 3′ end of the capture domain. In some embodiments, the blocking probe can hybridize to the capture probe to mask the free 3′ end of the capture domain, such as a hairpin probe or a partial duplex probe. In some embodiments, the free 3′ end of the capture domain can be blocked by chemical modification, e.g., adding an azidomethyl group as a chemically reversible capping moiety so that the capture probe does not include a free 3′ end. Blocking or modifying the capture probe, particularly at the free 3′ end of the capture domain, prior to contact of the biological sample with the array prevents modification of the capture probe, e.g., prevents addition of a poly(A) tail to the free 3′ end of the capture probe.

[0258] Non-limiting examples of 3′ modifications include dideoxy C-3′ (3′-ddC), 3′ inverted dT, 3′ C3 spacer, 3′ amino, and 3′ phosphorylation. In some embodiments, the nucleic acid in the biological sample can be modified such that it can be captured by the capture domain. For example, an adaptor sequence (including a binding domain capable of binding to the capture domain of the capture probe) can be added to the ends of the nucleic acid (e.g., fragmented genomic DNA). In some embodiments, this is accomplished by ligating the adaptor sequence or extending the nucleic acid. In some embodiments, enzymes are used to incorporate additional nucleotides at the end of the nucleic acid sequence, such as a poly(A) tail. In some embodiments, the capture probe can be reversibly masked or modified such that the capture domain of the capture probe does not include a free 3′ end. In some embodiments, the 3′ end is removed, modified, or made inaccessible such that the capture domain is not susceptible to processes (e.g., ligation or extension) used to modify the nucleic acid of the biological sample.

[0259] In some embodiments, the capture domain of the capture probe is modified to remove any modification of the capture probe that occurs during modification of the nucleic acid molecule of the biological sample. In some embodiments, the capture probe can include additional sequences downstream of the capture domain, i.e., 3′ to the capture domain, i.e., a blocking domain.

[0260] In some embodiments, the capture domain of the capture probe can be a non-nucleic acid domain. Examples of suitable capture domains that are not entirely nucleic acid-based include, but are not limited to, proteins, peptides, aptamers, antigens, antibodies, and molecular analogs that mimic the function of any of the capture domains described herein.

[0261] Cleavage domain

[0262] Each capture probe may optionally include at least one cleavage domain. The cleavage domain represents the probe portion for reversibly attaching the probe to the array feature, as described below. Additionally, one or more segments or regions of the capture probe may optionally be released from the array feature by cleaving the cleavage domain. For example, a spatial barcode and / or a universal molecular identifier (UMI) may be released by cleaving the cleavage domain.

[0263] Figure 7 is a schematic illustration of a cleavable capture probe, where the cleaved capture probe can enter non-permeabilized cells and bind to a target analyte within the sample. The capture probe 701 includes a cleavage domain 702, a cell-penetrating peptide 703, a reporter molecule 704, and a disulfide bond (-S-S-). 705 represents all other portions of the capture probe, such as the spatial barcode and the capture domain.

[0264] In some embodiments, the cleavage domain that attaches the capture probe to the feature is a disulfide bond. A reducing agent can be added to break the disulfide bond, thereby releasing the capture probe from the feature. As another example, heating can also cause degradation of the cleavage domain and release of the attached capture probe from the array feature. In some embodiments, laser radiation is used to heat and degrade the cleavage domain of the capture probe at a specific location. In some embodiments, the cleavage domain is a photosensitive chemical bond (i.e., a chemical bond that dissociates when exposed to light such as ultraviolet light).

[0265] Other examples of cleavage domains include labile chemical bonds such as, but not limited to, ester bonds (e.g., cleavable with acid, base, or hydroxylamine), vicinal diol bonds (e.g., cleavable with sodium periodate), Diels-Alder bonds (e.g., cleavable with heat), sulfone bonds (e.g., cleavable with base), silyl ether bonds (e.g., cleavable with acid), glycosidic bonds (e.g., cleavable with amylase), peptide bonds (e.g., cleavable with protease), or phosphodiester bonds (e.g., cleavable with nuclease (e.g., DNase)).

[0266] In some embodiments, the cleavage domain includes a sequence recognized by one or more enzymes capable of cleaving nucleic acid molecules, e.g., capable of breaking the phosphodiester bond between two or more nucleotides. The bond can be cleaved by other nucleic acid molecule-targeting enzymes such as restriction enzymes (e.g., restriction endonucleases). For example, the cleavage domain can include a restriction enzyme (restriction endonuclease) recognition sequence. Restriction endonucleases cut double-stranded or single-stranded DNA at specific recognition nucleotide sequences called restriction sites. In some embodiments, rare-cutting restriction enzymes, i.e., enzymes with long recognition sites (length at least 8 base pairs), are used to reduce the likelihood of cleavage at other positions in the capture probe.

[0267] In some embodiments, the cleavage domain includes a poly(U) sequence that can be cleaved by a mixture of uracil DNA glycosylase (UDG) and DNA glycosylase-lyase endonuclease VIII (commercially known as USER TM enzyme). Once released, the releasable capture probe can be used in the reaction. Thus, for example, an activatable capture probe can be activated by releasing the capture probe from the feature.

[0268] In some embodiments, when the capture probe is indirectly attached to a substrate, e.g., via a surface probe, the cleavage domain includes one or more mismatched nucleotides such that the complementary portions of the surface probe and the capture probe are not 100% complementary (e.g., the number of mismatched base pairs can be one, two, or three base pairs). For example, such mismatches are recognized by MutY and T7 endonuclease I enzymes, which results in cleavage of the nucleic acid molecule at the mismatch position.

[0269] In some embodiments, when the capture probe is indirectly attached to a feature, e.g., via a surface probe, the cleavage domain includes a nickase recognition site or sequence. A nickase is a type of endonuclease that can cleave only one strand of a DNA duplex. Thus, the cleavage domain can include a nickase recognition site near the 5′ end of the surface probe (and / or the 5′ end of the capture probe) such that cleavage of the surface probe or the capture probe destabilizes the duplex between the surface probe and the capture probe, thereby releasing the capture probe from the feature.

[0270] Nickases can also be used in some embodiments where the capture probe is directly attached to a feature. For example, the substrate can be contacted with a nucleic acid molecule that hybridizes to the cleavage domain of the capture probe to provide or reconstitute a nickase recognition site, e.g., a cleavage assist probe. Thus, contacting with a nickase will result in cleavage of the cleavage domain, thereby releasing the capture probe from the feature. Such a cleavage assist probe can also be used to provide or reconstitute a cleavage recognition site for other lyases (e.g., restriction endonucleases).

[0271] Some nickases introduce single-strand nicks only at specific sites in a DNA molecule by binding and recognizing a specific nucleotide recognition sequence. Many natural nickases have been discovered, and the sequence recognition properties of at least four nickases have been determined. Nickases are described in U.S. Patent No. 6,867,028, which is incorporated herein by reference in its entirety. Generally, any suitable nickase can be used to bind to the complementary nickase recognition site in the cleavage domain. After use, the nickase can be removed from the assay or inactivated after the capture probe is released to prevent unnecessary cleavage of the capture probe.

[0272] Suitable capture domains include, but are not limited to, proteins, peptides, aptamers, antigens, antibodies, and molecular analogs that mimic the function of any of the capture domains described herein.

[0273] In some embodiments, the cleavage domain is absent from the capture probe. For example, instances of substrates with attached capture probes lacking a cleavage domain are described in Macosko et al., (2015) Cell 161, 1202-1214, the entire content of which is incorporated herein by reference.

[0274] In some embodiments, the capture probe region corresponding to the cleavage domain can be used for certain other functions. For example, other regions for nucleic acid extension or amplification can be included at the location that would typically position the cleavage domain. In such embodiments, the region can complement a functional domain or even exist as another functional domain. In some embodiments, a cleavage domain is present, but its use is optional.

[0275] Functional domain

[0276] Each capture probe can optionally include at least one functional domain. Each functional domain typically includes functional nucleotide sequences for downstream analysis steps in the overall assay procedure.

[0277] In some embodiments, the capture probe can include a functional domain for attachment to a sequencing flow cell, e.g., for the P5 sequence for sequencing. In some embodiments, the capture probe or its derivative can include another functional domain, e.g., for attachment to the P7 sequence of the sequencing flow cell for sequencing. The functional domains can be selected to be compatible with any of a variety of different sequencing systems, such as 454 sequencing, Ion Torrent Proton or PGM, Illumina X10, etc. and their requirements.

[0278] In some embodiments, the functional domain includes a primer. The primer can include the R1 primer sequence for sequencing, and in some embodiments, includes the R2 primer sequence for sequencing. Examples of such capture probes and their uses are described in U.S. Patent Publication Nos. 2014 / 0378345 and 2015 / 0376609, the entire content of which is incorporated herein by reference.

[0279] Spatial barcode

[0280] As described above, the capture probe can include one or more spatial barcodes (e.g., two or more, three or more, four or more, five or more) spatial barcodes. A "spatial barcode" is a continuous nucleic acid segment or two or more non - continuous nucleic acid segments that serves as a tag or identifier that conveys or is capable of conveying spatial information. In some embodiments, the capture probe includes a spatial barcode with a spatial aspect, where the barcode is associated with a specific location within the array or on the substrate.

[0281] A spatial barcode can be part of an analyte or independent of the analyte (i.e., part of a capture probe). A spatial barcode can be a tag attached to an analyte (e.g., a nucleic acid molecule), or a combination of tags in addition to endogenous features of the analyte (e.g., the size or end sequence of the analyte). The spatial barcode can be unique. In some embodiments where the spatial barcode is unique, the spatial barcode functions both as a spatial barcode and as a unique molecular identifier (UMI) associated with a particular capture probe.

[0282] Spatial barcodes can have a variety of different formats. For example, a spatial barcode can include a polynucleotide spatial barcode, a random nucleic acid and / or amino acid sequence, and a synthetic nucleic acid and / or amino acid sequence. In some embodiments, the spatial barcode is attached to the analyte in a reversible or irreversible manner. In some embodiments, the spatial barcode is added to a fragment of, for example, a DNA or RNA sample before, during, and / or after sample sequencing. In some embodiments, the spatial barcode enables the identification and / or quantification of individual sequence reads. In some embodiments, the spatial barcode serves as a fluorescent barcode, where a fluorescently labeled oligonucleotide probe hybridizes to the spatial barcode.

[0283] In some embodiments, the spatial barcode is a nucleic acid sequence that substantially does not hybridize to the analyte nucleic acid molecules in a biological sample. In some embodiments, the spatial barcode has a sequence identity of less than 80% (e.g., less than 70%, 60%, 50%, or less than 40% sequence identity) to the nucleic acid sequence over a substantial portion (e.g., 80% or more) of the nucleic acid molecules in the biological sample.

[0284] The spatial barcode sequence can include from about 6 to about 20 or more nucleotides within the capture probe sequence. In some embodiments, the length of the spatial barcode sequence can be about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some embodiments, the length of the spatial barcode sequence can be at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some embodiments, the length of the spatial barcode sequence can be at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or shorter.

[0285] These nucleotides can be completely contiguous, i.e., in a single stretch of adjacent nucleotides, or they can be divided into two or more separate subsequences that are separated by one or more nucleotides. The length of the isolated spatial barcode subsequences is about 4 to 16 nucleotides. In some embodiments, the spatial barcode subsequences can be about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some embodiments, the spatial barcode subsequences can be at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some embodiments, the spatial barcode subsequences can be at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or shorter.

[0286] For a plurality of capture probes attached to a common array feature, one or more spatial barcode sequences of the plurality of capture probes can include sequences that are the same for all capture probes coupled to the feature and / or sequences that are different between all capture probes coupled to the feature.

[0287] Figure 8 is a schematic diagram of an exemplary multiplexed spatial tagging feature. In Figure 8 , feature 801 can be coupled to spatially barcoded capture probes, where the spatially barcoded probes for a particular feature can have the same spatial barcode but different capture domains designed to associate the spatial barcode of the feature with multiple target analytes. For example, the feature can be coupled to four different types of spatially barcoded capture probes, each type of spatially barcoded capture probe having spatial barcode 802. One type of capture probe associated with this feature includes the combination of spatial barcode 802 and a poly(T) capture domain 803, which is designed to capture mRNA target analytes. A second type of capture probe associated with this feature includes the combination of spatial barcode 802 and a random N-mer capture domain 804 for gDNA analysis. A third type of capture probe associated with this feature includes the combination of spatial barcode 802 and a capture domain complementary to a capture domain on an analyte capture agent barcode domain 805. A fourth type of capture probe associated with this feature includes the combination of spatial barcode 802 and a capture probe that can specifically bind a nucleic acid molecule 806 that can function in a CRISPR assay (e.g., CRISPR / Cas9). While Figure 8 only four different capture probe barcoding constructs are shown in, the capture probe barcoding constructs can be customized for the analysis of any given analyte related to nucleic acids and are capable of binding to such constructs. For example, Figure 8The schemes shown can also be used for the simultaneous analysis of other analytes disclosed herein, including but not limited to: (a) mRNA, lineage tracing constructs, cell surface or intracellular proteins and metabolites, and gDNA; (b) mRNA, accessible chromatin (e.g., ATAC-seq, DNase-seq, and / or MNase-seq), cell surface or intracellular proteins and metabolites, and perturbagens (e.g., CRISPR-crRNA / sgRNA, TALEN, zinc finger nucleases, and / or antisense oligonucleotides as described herein); (c) mRNA, cell surface or intracellular proteins and / or metabolites, barcoded labels (e.g., MHC multimers as described herein), and V(D)J sequences of immune cell receptors (e.g., T cell receptors).

[0288] Capture probes attached to a single array feature can include the same (or common) spatial barcode sequences, different spatial barcode sequences, or a combination of both. The capture probes attached to a feature can include multiple sets of capture probes. The capture probes of a given set can include the same spatial barcode sequence. The same spatial barcode sequence can be different from the spatial barcode sequence of another set of capture probes.

[0289] Multiple capture probes can include spatial barcode sequences (e.g., nucleic acid barcode sequences) associated with a specific location on the spatial array. For example, a first plurality of capture probes can be associated with a first region based on a spatial barcode sequence common to the capture probes within the first region, and a second plurality of capture probes can be associated with a second region based on a spatial barcode sequence common to the capture probes within the second region. The second region may or may not be associated with the first region. Other pluralities of capture probes can be associated with spatial barcode sequences common to the capture probes within other regions. In some embodiments, the spatial barcode sequences can be the same among multiple capture probe molecules.

[0290] In some embodiments, multiple different spatial barcodes are incorporated into a single array capture probe. For example, a mixed but known set of spatial barcode sequences can provide stronger addressing or attribution of the spatial barcode to a given point or location by providing repeated or independent confirmation of the location characteristics. In some embodiments, multiple spatial barcodes represent increased specificity of the location of a particular array point.

[0291] Unique molecular identifier

[0292] The capture probe may include one or more (e.g., two or more, three or more, four or more, five or more) unique molecular identifiers (UMIs). A unique molecular identifier is a contiguous nucleic acid segment or two or more non-contiguous nucleic acid segments that serve as a label or identifier for a particular analyte or a capture probe that binds to a particular analyte (e.g., via a capture domain).

[0293] The UMI can be unique. The UMI may include one or more specific polynucleotide sequences, one or more random nucleic acid and / or amino acid sequences, and / or one or more synthetic nucleic acid and / or amino acid sequences.

[0294] In some embodiments, the UMI is a nucleic acid sequence that does not substantially hybridize to analyte nucleic acid molecules in a biological sample. In some embodiments, the UMI has less than 80% sequence identity (e.g., less than 70%, 60%, 50%, or less than 40% sequence identity) to the nucleic acid sequences on a substantial portion (e.g., 80% or more) of the nucleic acid molecules in the biological sample.

[0295] The UMI may include from about 6 to about 20 or more nucleotides within the capture probe sequence. In some embodiments, the length of the UMI can be about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some embodiments, the length of the UMI can be at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some embodiments, the length of the UMI can be at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or shorter.

[0296] These nucleotides can be completely contiguous, i.e., in a single sequence segment of adjacent nucleotides, or they can be divided into two or more separate sub-sequences that are separated by 1 or more nucleotides. The length of the separated UMI sub-sequences is about 4 to 16 nucleotides. In some embodiments, the UMI sub-sequence can be about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some embodiments, the UMI sub-sequence can be at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some embodiments, the UMI sub-sequence can be at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or shorter.

[0297] In some embodiments, the UMI is attached to the analyte in a reversible or irreversible manner. In some embodiments, the UMI is added to fragments of, for example, a DNA or RNA sample before, during, and / or after analyte sequencing. In some embodiments, the UMI enables the identification and / or quantification of individual sequence reads. In some embodiments, the UMI serves as a fluorescent barcode, where a fluorescently labeled oligonucleotide probe hybridizes to the UMI.

[0298] Other aspects of capture probes

[0299] For capture probes attached to array features, an individual array feature can include one or more capture probes. In some embodiments, an individual array feature includes hundreds or thousands of capture probes. In some embodiments, the capture probes are associated with a specific individual feature, where the individual feature contains a capture probe that includes a spatial barcode unique to a defined region or location on the array.

[0300] In some embodiments, a specific feature can contain capture probes that include more than one spatial barcode (e.g., one capture probe at a specific feature can include a spatial barcode different from the spatial barcode included in another capture probe at the same specific feature, while both capture probes include a second common spatial barcode), where each spatial barcode corresponds to a specific defined region or location on the array. For example, multiple spatial barcode sequences associated with a specific feature on the array can provide a stronger address or attribute to a given location by providing a duplicate or independent confirmation of the location. In some embodiments, multiple spatial barcodes represent an increased specificity of the location of a specific array spot. In a non-limiting example, two different spatial barcodes can be used to encode a specific array spot, where each spatial barcode identifies a specific defined region within the array, and an array spot with two spatial barcodes identifies a sub-region where the two defined regions overlap, e.g., like the overlapping portion of a Venn diagram.

[0301] In another non-limiting example, a specific array spot can be encoded with three different spatial barcodes, where the first spatial barcode identifies a first region within the array, the second spatial barcode identifies a second region, where the second region is a sub-region entirely within the first region, and the third spatial barcode identifies a third region, where the third region is a sub-region entirely within the first and second sub-regions.

[0302] In some embodiments, the capture probes attached to the array features are released from the array features for sequencing. Alternatively, in some embodiments, the capture probes remain attached to the array features and the probes are sequenced while remaining attached to the array features (e.g., by in situ sequencing). Other aspects of sequencing the capture probes are described in subsequent portions of the present invention.

[0303] In some embodiments, the array features can include different types of capture probes attached to the features. For example, the array features can include a first type of capture probe and a second type of capture probe, the first type of capture probe having a capture domain designed to bind to a first class of analytes and the second type of capture probe having a capture domain designed to bind to a second class of analytes. Generally, the array features can include one or more (e.g., two or more, three or more, four or more, five or more, six or more, eight or more, ten or more, 12 or more, 15 or more, 20 or more, 30 or more, 50 or more) different types of capture probes attached to a single array feature.

[0304] In some embodiments, the capture probes are nucleic acids. In some embodiments, the capture probes are attached to the array features by their 5′ ends. In some embodiments, the capture probes include, from the 5′ end to the 3′ end: one or more barcodes (e.g., spatial barcodes and / or UMIs) and one or more capture domains. In some embodiments, the capture probes include, from the 5′ end to the 3′ end: one barcode (e.g., a spatial barcode or UMI) and one capture domain. In some embodiments, the capture probes include, from the 5′ end to the 3′ end: a cleavage domain, a functional domain, one or more barcodes (e.g., spatial barcodes and / or UMIs), and a capture domain. In some embodiments, the capture probes include, from the 5′ end to the 3′ end: a cleavage domain, a functional domain, one or more barcodes (e.g., spatial barcodes and / or UMIs), a second functional domain, and a capture domain. In some embodiments, the capture probes include, from the 5′ end to the 3′ end: a cleavage domain, a functional domain, a spatial barcode, a UMI, and a capture domain. In some embodiments, the capture probes do not include spatial barcodes. In some embodiments, the capture probes do not include UMIs. In some embodiments, the capture probes include a sequence for initiating a sequencing reaction.

[0305] In some embodiments, the capture probe is immobilized on the feature by its 3′ end. In some embodiments, the capture probe from the 3′ end to the 5′ end includes: one or more barcodes (e.g., spatial barcodes and / or UMIs) and one or more capture domains. In some embodiments, the capture probe from the 3′ end to the 5′ end includes: one barcode (e.g., a spatial barcode or UMI) and one capture domain. In some embodiments, the capture probe from the 3′ end to the 5′ end includes: a cleavage domain, a functional domain, one or more barcodes (e.g., spatial barcodes and / or UMIs), and a capture domain. In some embodiments, the capture probe from the 3′ end to the 5′ end includes: a cleavage domain, a functional domain, a spatial barcode, a UMI, and a capture domain.

[0306] In some embodiments, the capture probe comprises an in-situ synthesized oligonucleotide. In some embodiments, the in-situ synthesized oligonucleotide comprises one or more constant sequences, wherein one or more are used as primer sequences (e.g., primers for amplifying target nucleic acids). In some embodiments, the constant sequence is a cleavable sequence. In some embodiments, the in-situ synthesized oligonucleotide comprises a barcode sequence, e.g., a variable barcode sequence. In some embodiments, the in-situ synthesized oligonucleotide is attached to a feature of the array.

[0307] In some embodiments, the capture probe is the product of two or more oligonucleotide sequences, e.g., two or more oligonucleotide sequences ligated together. In some embodiments, one of the oligonucleotide sequences is an in-situ synthesized oligonucleotide.

[0308] In some embodiments, the capture probe comprises a splint oligonucleotide. Two or more oligonucleotides can be ligated together using a splint oligonucleotide and any type of ligase known in the art or described herein (e.g., a splint ligase).

[0309] In some embodiments, one of the oligonucleotides includes: a constant sequence (e.g., a sequence complementary to a portion of the splint oligonucleotide), a degenerate sequence, and a capture domain (e.g., as described herein). In some embodiments, the capture probe is produced by adding a polynucleotide to the end of the oligonucleotide sequence using an enzyme. The capture probe can include a degenerate sequence that can serve as a unique molecular identifier.

[0310] The capture probe can include a degenerate sequence, which is a sequence in which certain positions of the nucleotide sequence contain many possible bases. The degenerate sequence can be a degenerate nucleotide sequence, including about or at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45 or 50 nucleotides. In some embodiments, the nucleotide sequence contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more degenerate positions within the nucleotide sequence. In some embodiments, the degenerate sequence is used as a UMI.

[0311] In some embodiments, the capture probe includes a restriction endonuclease recognition sequence or a nucleotide sequence that can be cleaved by a specific enzymatic activity. For example, a uracil sequence can be cleaved by a specific enzymatic activity. As another example, other modified bases (e.g., methylated modifications) can be recognized and cleaved by a specific endonuclease. The capture probe can be subjected to enzymatic cleavage, which removes the blocking domain and any other nucleotides added to the 3′ end of the capture probe during the modification process. Removal of the blocking domain will expose and / or restore the free 3′ end of the capture domain of the capture probe. In some embodiments, other nucleotides can be removed to expose and / or restore the 3′ end of the capture domain of the capture probe.

[0312] In some embodiments, the blocking domain can be incorporated into the capture probe during or after its synthesis. The terminal nucleotide of the capture domain is a reversible terminator nucleotide (e.g., 3′-O-blocked reversible terminator and 3′-unblocked reversible terminator), and can be included in the capture probe during or after probe synthesis.

[0313] Extended capture probe

[0314] An "extended capture probe" is a capture probe with an extended nucleic acid sequence. For example, in the case where the capture probe includes a nucleic acid, an "extended 3′ end" means that additional nucleotides are added to the most terminal 3′ nucleotide of the capture probe to extend the length of the capture probe, e.g., by extending the nucleic acid molecule through a standard polymerization reaction, including template polymerization catalyzed by a polymerase (e.g., DNA polymerase or reverse transcriptase).

[0315] In some embodiments, extending the capture probe includes generating cDNA from the captured (hybridized) RNA. This process involves synthesizing the complementary strand of the hybridized nucleic acid, e.g., generating cDNA based on the captured RNA template (RNA hybridized to the capture domain of the capture probe). Thus, in the initial step of extending the capture probe (e.g., cDNA generation), the captured (hybridized) nucleic acid (e.g., RNA) serves as the template for extension (e.g., reverse transcription step).

[0316] In some embodiments, capture probes use reverse transcription extension. For example, reverse transcription involves using reverse transcriptase to synthesize cDNA (complementary or copy DNA) from RNA (such as messenger RNA). In some embodiments, reverse transcription is performed while the tissue is still in situ, generating an analyte library, where the analyte library includes spatial barcodes from adjacent capture probes. In some embodiments, capture probes are extended using one or more DNA polymerases.

[0317] In some embodiments, the capture domain of the capture probe includes a primer for generating a nucleic acid complementary strand that hybridizes to the capture probe, e.g., a primer for DNA polymerase and / or reverse transcription. The nucleic acid (e.g., DNA and / or cDNA) molecules produced by the extension reaction contain the sequence of the capture probe. Extension of the capture probe, e.g., DNA polymerase and / or reverse transcription reactions, can be carried out using a variety of suitable enzymes and protocols.

[0318] In some embodiments, full-length DNA (e.g., cDNA) molecules are generated. In some embodiments, a "full-length" DNA molecule refers to the entire captured nucleic acid molecule. However, if the nucleic acid (e.g., RNA) is partially degraded in the tissue sample, the captured nucleic acid molecule will be of a different length than the initial RNA in the tissue sample. In some embodiments, the 3′ end of the extended probe (e.g., the first-strand cDNA molecule) is modified. For example, an adapter or linker can be ligated to the 3′ end of the extended probe. This can be achieved by using a single-strand ligase such as T4 RNA ligase or CircleGase TM (available from Epicentre Biotechnologies, Madison, Wisconsin). In some embodiments, template-switching oligonucleotides are used to extend cDNA to generate full-length cDNA (or as close to full-length cDNA as possible). In some embodiments, a second-strand synthesis helper probe (a partially double-stranded DNA molecule capable of hybridizing to the 3′ end of the extended capture probe) can be ligated to the 3′ end of the extended probe, e.g., the first-strand cDNA molecule, using a double-strand ligase (such as T4 DNA ligase). Other enzymes suitable for the ligation step are known in the art and include, for example, Tth DNA ligase, Taq DNA ligase, Pyrococcus sp. (strain 9°N) DNA ligase (9°N TM DNA ligase, New England Biolabs), Ampligase TM(Available from Epicentre Biotechnologies, Madison, WI) and SplintR (available from New England Biolabs, Ipswich, MA). In some embodiments, a polynucleotide tail (e.g., a poly(A) tail) is incorporated at the 3′ end of the extended probe molecule. In some embodiments, a polynucleotide tail is incorporated using a terminal transferase activity enzyme.

[0319] In some embodiments, the double-stranded extended capture probe is processed to remove any unextended capture probe prior to amplification and / or analysis (e.g., sequence analysis). This can be achieved by a variety of methods, e.g., using an enzyme to degrade the unextended probe, such as an exonuclease, or using a purification column.

[0320] In some embodiments, the extended capture probe is amplified to produce an amount sufficient for analysis, e.g., by DNA sequencing. In some embodiments, the first strand (e.g., a DNA and / or cDNA molecule) of the extended capture probe is used as a template for an amplification reaction (e.g., a polymerase chain reaction).

[0321] In some embodiments, the amplification reaction uses primers that include an affinity group to incorporate the affinity group onto the extended capture probe (e.g., an RNA-cDNA hybrid). In some embodiments, the primer includes an affinity group and the extended capture probe includes an affinity group. The affinity group can correspond to any of the affinity groups described previously.

[0322] In some embodiments, the extended capture probe that includes an affinity group can be coupled to an array feature specific for the affinity group. In some embodiments, the substrate can include an antibody or an antibody fragment. In some embodiments, the array feature includes avidin or streptavidin and the affinity group includes biotin. In some embodiments, the array feature includes maltose and the affinity group includes maltose binding protein. In some embodiments, the array feature includes maltose binding protein and the affinity group includes maltose. In some embodiments, amplification of the extended capture probe can serve to release the amplified probe from the array feature as long as the copies of the extended probe are not attached to the array feature.

[0323] In some embodiments, the extended capture probe or its complement or amplicon is released from the array feature. The step of releasing the extended capture probe or its complement or amplicon from the array feature can be achieved in a variety of ways. In some embodiments, the extended capture probe or its complement is released from the feature by nucleic acid cleavage and / or denaturation (e.g., by heating to denature a double-stranded molecule).

[0324] In some embodiments, the extended capture probes or their complements or amplicons are released from the array features by physical means. For example, methods for inducing physical release include denaturing double-stranded nucleic acid molecules. Another method for releasing extended capture probes is to use a solution that disrupts the hydrogen bonds of double-stranded molecules. In some embodiments, the extended capture probes are released by applying heated water (e.g., water at least 85°C or a buffer, e.g., water at least 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, or 99°C). In some embodiments, a solution including salts, surfactants, etc., which can further destabilize the interactions between nucleic acid molecules, is added to release the extended capture probes from the array features. In some embodiments, a formamide solution can be used to destabilize the interactions between nucleic acid molecules to release the extended capture probes from the array features.

[0325] Analyte capturant

[0326] The present invention also provides methods and materials for spatially profiling biological analytes (e.g., mRNA, genomic DNA, accessible chromatin, and cell surface or intracellular proteins and / or metabolites) using analyte capture agents. As used herein, an "analyte capture agent" (sometimes previously also referred to as a "cell labeler") refers to a substance that interacts with an analyte (e.g., an analyte in a sample) and a capture probe (e.g., a capture probe attached to a substrate) to identify the analyte. In some embodiments, the analyte capture agent includes an analyte binding portion and a capture agent barcode domain.

[0327] Figure 9 is a schematic diagram of an exemplary analyte capture agent 902 composed of an analyte binding portion 904 and a capture agent barcode domain 908. The analyte binding portion 904 is a molecule capable of binding to an analyte 906 and interacting with a spatially barcoded capture probe. The analyte binding portion can bind to the analyte 906 with high affinity and / or high specificity. The analyte capture agent can include a capture agent barcode domain 908, a nucleotide sequence (e.g., an oligonucleotide), which can hybridize with at least a part or all of the capture domain of the capture probe. The analyte binding portion 904 can include a polypeptide and / or an aptamer (e.g., an oligonucleotide or peptide molecule that binds to a specific target analyte). The analyte binding portion 904 can include an antibody or an antibody fragment (e.g., an antigen-binding fragment).

[0328] As used herein, the term "analyte binding portion" refers to a molecule or portion thereof that is capable of binding to a macromolecular component (e.g., an analyte, e.g., a bioanalyte). In some embodiments of any of the spatial profiling methods described herein, the analyte binding portion of an analyte capture agent that binds to a bioanalyte can include, but is not limited to, an antibody or an epitope-binding fragment thereof, a cell surface receptor-binding molecule, a receptor ligand, a small molecule, a bispecific antibody, a bispecific T cell conjugate, a T cell receptor conjugate, a B cell receptor conjugate, a precursor, an aptamer, a monomer, an integrin, a DARPin, and a protein scaffold, or any combination thereof. The analyte binding portion can bind to the macromolecular component (e.g., an analyte) with high affinity and / or high specificity. The analyte binding portion can include a nucleotide sequence (e.g., an oligonucleotide), which can correspond to at least a portion or all of the analyte binding portion. The analyte binding portion can include a polypeptide and / or an aptamer (e.g., a polypeptide and / or an aptamer that binds to a specific target molecule, e.g., an analyte). The analyte binding portion can include an antibody or an antibody fragment (e.g., an antigen-binding fragment) that binds to a specific analyte (e.g., a polypeptide).

[0329] In some embodiments, the analyte capture agent is capable of binding to an analyte present within a cell. In some embodiments, the analyte capture agent is capable of binding to a cell surface analyte, which can include, but is not limited to, a receptor, an antigen, a surface protein, a transmembrane protein, a cluster of differentiation protein, a protein channel, a protein pump, a carrier protein, a phospholipid, a glycoprotein, a glycolipid, a cell-cell interaction protein complex, an antigen presentation complex, a major histocompatibility complex, an engineered T cell receptor, a T cell receptor, a B cell receptor, a chimeric antigen receptor, an extracellular matrix protein, the post-translational modification (e.g., phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, or lipidation) state of a cell surface protein, a gap junction, and an adhesion junction. In some embodiments, the analyte capture agent is capable of binding to a post-translationally modified cell surface analyte. In such embodiments, the analyte capture agent can be specific for the cell surface analyte based on a given state of the post-translational modification (e.g., phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, or lipidation), such that the cell surface analyte profile can include information on the post-translational modification of one or more analytes.

[0330] In some embodiments, the analyte capture agent includes a capture agent barcode domain that is conjugated to or otherwise attached to the analyte binding portion. In some embodiments, the capture agent barcode domain is covalently linked to the analyte binding portion. In some embodiments, the capture agent barcode domain is a nucleic acid sequence. In some embodiments, the capture agent barcode domain includes an analyte binding portion barcode and an analyte capture sequence.

[0331] As used herein, the term "analyte-binding portion barcode" refers to a barcode that is associated with or otherwise identifies an analyte-binding portion. In some embodiments, by identifying the analyte-binding portion (by identifying its associated analyte-binding portion barcode), the analyte bound by the analyte-binding portion can also be identified. An analyte-binding portion barcode can be a nucleic acid sequence of a given length and / or a sequence associated with the analyte-binding portion. An analyte-binding portion barcode can generally include any aspect of the barcodes described herein. For example, an analyte-specific analyte capture agent of one type can have a first capture agent barcode domain (e.g., which includes a first analyte-binding portion barcode) coupled thereto, while an analyte capture agent of a different analyte specificity can have a different capture agent barcode domain (e.g., which includes a second barcode analyte-binding portion barcode) coupled thereto. In some aspects, such a capture agent barcode domain can include an analyte-binding portion barcode that enables the identification of the analyte-binding portion to which the capture agent barcode domain is coupled. The selection of the capture agent barcode domain can result in significant sequence diversity while also being readily attachable to most analyte-binding portions (e.g., antibodies) and readily detectable (e.g., using sequencing or array technologies). In some embodiments, an analyte capture agent can include an analyte-binding portion having a capture agent barcode domain attached thereto. For example, an analyte capture agent can include a first analyte-binding portion (e.g., an antibody that binds to an analyte, e.g., a first cell surface feature) having an associated capture agent barcode domain that includes a first analyte-binding portion barcode.

[0332] In some embodiments, the capture agent barcode domain of an analyte capture agent includes an analyte capture sequence. As used herein, the term "analyte capture sequence" refers to a region or portion that is configured to hybridize to, bind to, be coupled to, or otherwise interact with a capture domain of a capture probe. In some embodiments, an analyte capture sequence includes a nucleic acid sequence that is complementary or substantially complementary to the capture domain of a capture probe such that the analyte capture sequence hybridizes to the capture domain of the capture probe. In some embodiments, an analyte capture sequence includes a poly(A) nucleic acid sequence that hybridizes to a capture domain that includes a poly(T) nucleic acid sequence. In some embodiments, an analyte capture sequence includes a poly(T) nucleic acid sequence that hybridizes to a capture domain that includes a poly(A) nucleic acid sequence. In some embodiments, an analyte capture sequence includes a non-homopolymeric nucleic acid sequence that hybridizes to a capture domain that includes a non-homopolymeric nucleic acid sequence that is complementary (or substantially complementary) to the non-homopolymeric nucleic acid sequence of the analyte capture region.

[0333] In some embodiments of any of the spatial analysis methods described herein that use an analyte capture agent, the capture agent barcode domain can be directly coupled to the analyte binding portion, or they can be attached to beads, molecular lattices such as linear, globular, cross-linked or other polymers, or other frameworks attached to or otherwise associated with the analyte binding portion, which enables multiple capture agent barcode domains to be attached to a single analyte binding portion. The attachment (coupling) of the capture agent barcode domain to the analyte binding portion can be achieved by any of a variety of direct or indirect, covalent or non-covalent binding or attachment methods. For example, in the case where the capture agent barcode domain is coupled to an analyte binding portion comprising an antibody or antigen-binding fragment, chemical coupling techniques can be used (e.g., Lightning available from InnovaBiosciences Antibody labeling kits) covalently attach these capture agent barcode domains to a portion of an antibody or antigen-binding fragment. In some embodiments, the capture agent barcode domain can be coupled to an antibody or antigen-binding fragment using a non-covalent attachment mechanism (e.g., using a biotinylated antibody and an oligonucleotide or bead 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 content of which is 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. Patent No. 6,265,552, the entire content of which is incorporated herein by reference. In addition, click reaction chemistry (e.g., methyltetrazine-PEG5-NHS ester reaction, TCO-PEG4-NHS ester reaction, etc.) can be used to couple the capture agent barcode domain to the analyte-binding portion. The reactive moiety on the analyte-binding portion can also include an amine for targeting aldehydes, an amine for targeting maleimides (e.g., free thiols), an azide for targeting click chemistry compounds (e.g., alkynes), a biotin for targeting streptavidin, a phosphate for targeting EDC, which in turn targets activated esters (e.g., NH2). The reactive moiety on the analyte-binding portion can be a compound or group that binds to the reactive moiety on the analyte-binding portion. Exemplary strategies for conjugating the analyte-binding portion to the capture agent barcode domain include using commercial kits (e.g., Solulink, Thunder link), mild reduction of the hinge region and maleimide labeling conjugation, copper-free click chemical reactions facilitated by staining of labeled amides, conjugation by periodate oxidation of sugar chains and conjugation of amines. In the case where the analyte-binding portion is an antibody, the antibody can be modified before or simultaneously with oligonucleotide conjugation. For example, the antibody can be glycosylated with a mutant GalT (Y289L) of β-1,4-galactosyltransferase allowed by the substrate and an azide-containing uridine diphosphate-N-acetylgalactosamine analogue, uridine diphosphate-GalNAz. The modified antibody can be conjugated to an oligonucleotide having a dibenzocyclooctyne-PEG4-NHS group. In some embodiments, certain steps (e.g., COOH activation (e.g., EDC) and homobifunctional crosslinkers) can be avoided to prevent the analyte-binding portion from conjugating to itself.In some embodiments of any of the spatial profiling methods described herein, an analyte capture agent (e.g., an analyte binding moiety coupled to an oligonucleotide) can be delivered into a cell by transfection (e.g., using transfection amines, cationic polymers, calcium phosphate, or electroporation), transduction (e.g., using phage or recombinant viral vectors), by mechanical delivery (e.g., magnetic beads), by lipids (e.g., 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)), or by transporters. The analyte capture agent can be delivered into a cell by exosomes. For example, a first cell that releases exosomes containing the analyte capture agent can be generated. The analyte capture agent can be attached to the exosome membrane. The analyte capture agent can be contained in the cytoplasm of the exosome. The released exosomes can be harvested and provided to a second cell, thereby delivering the analyte capture agent into the second cell. The analyte capture agent can be released from the exosome membrane before, during, or after delivery to the cell. In some embodiments, the cell is permeabilized to couple the analyte capture agent to intracellular cell components (such as, but not limited to, intracellular proteins, metabolites, and nuclear membrane proteins). After intracellular delivery, the analyte capture agent can be used to analyze intracellular components as described herein.

[0334] In some embodiments of any of the spatial profiling methods described herein, the capture agent barcode domain coupled to an analyte capture agent can include modifications that render it non-amplifiable by polymerase. In some embodiments, the capture agent barcode domain can serve as a template rather than a primer when bound to a nucleic acid or the capture domain of a capture probe in a sample for a primer extension reaction. When the capture agent barcode domain further includes a barcode (e.g., an analyte binding moiety barcode), such a design can improve the efficiency of molecular barcoding by increasing the affinity between the capture agent barcode domain and the uncoded sample nucleic acid and eliminating the potential formation of adapter artifacts. In some embodiments, the capture agent barcode domain can include a random N-mer sequence that is capped with modifications that render it non-extendable by polymerase. In certain cases, the composition of the random N-mer sequence can be designed to maximize the binding efficiency to free, uncoded ssDNA molecules. The design can include a random sequence composition with a higher GC content, a partially random sequence with a fixed G or C at specific positions, the use of guanosine, the use of locked nucleic acids, or any combination thereof.

[0335] Modifications that block primer extension by polymerase can be carbon spacer groups of different lengths or dideoxynucleotides. In some embodiments, the modification can be an abasic site, a base analog, or an analog of the phosphate backbone having an apurinic or apyrimidinic structure, such as an N-(2-aminoethyl)-glycine backbone, tetrahydrofuran, or 1′,2′-dideoxyribose linked by an amide bond. The modification can also be a uracil base, 2′OMe-modified RNA, a C3-18 spacer (e.g., a structure having 3-18 contiguous carbon atoms, such as a C3 spacer), an ethylene glycol polymer spacer (e.g., spacer 18 (hexaethylene glycol spacer)), biotin, a dideoxynucleotide triphosphate, ethylene glycol, an amine, or a phosphate.

[0336] In some embodiments of any of the spatial profiling methods described herein, the capture agent barcode domain coupled to the analyte binding portion includes a cleavable domain. For example, after the analyte capture agent binds to an analyte (e.g., a cell surface analyte), the capture agent barcode domain can be cleaved and collected for downstream analysis according to the methods described herein. In some embodiments, the cleavable domain of the capture agent barcode domain includes a U-excision element that releases the material from the bead. In some embodiments, the U-excision element can include a single-stranded DNA (ssDNA) sequence containing at least one uracil. The material can be attached to the bead via the ssDNA sequence. The material 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 generates a 5′ phosphate group from the cleavage, additional enzymatic treatment can be included in the downstream processing to remove the phosphate group, e.g., prior to ligation of other sequencing handle elements (e.g., an Illumina full P5 sequence, a partial P5 sequence, a full R1 sequence, and / or a partial R1 sequence).

[0337] In some embodiments, the analyte-binding portion of the analyte capture agent includes one or more antibodies or antigen-binding fragments thereof. The antibody or antigen-binding fragment including the analyte-binding portion can specifically bind to a target analyte. In some embodiments, the analyte 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 analyte capture agents each including multiple analyte-binding portions bind to multiple analytes present in a biological sample. In some embodiments, the multiple analytes include a single species of analyte (e.g., a single species of polypeptide). In some embodiments where the multiple analytes include a single species of analyte, the analyte-binding portions of the multiple analyte capture agents are the same. In some embodiments where the multiple analytes include a single species of analyte, the analyte-binding portions of the multiple analyte capture agents are different (e.g., members of the multiple analyte capture agents can have two or more analyte-binding portions, where each of the two or more analyte-binding portions binds a single species of analyte (e.g., at different binding sites)). In some embodiments, the multiple analytes include multiple different species of analytes (e.g., multiple different species of polypeptides).

[0338] In some embodiments, multiple different classes of analytes (e.g., polypeptides) from a biological sample can subsequently be associated with one or more physical properties of the biological sample. For example, multiple different classes of analytes can be associated with the location of the analytes within the biological sample. Such information (e.g., proteomic information when an analyte binding moiety recognizes a polypeptide) can be used in conjunction with other spatial information (e.g., genetic information from the biological sample, such as DNA sequence information, transcriptomic information (i.e., transcript sequences), or both). For example, cell surface proteins 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). One or more physical properties can be characterized by imaging the cell. The cell can be bound by an analyte capture agent that includes an analyte binding moiety that binds to the cell surface protein and an analyte binding moiety barcode that recognizes the analyte binding moiety, and the cell can be subjected to spatial analysis (e.g., any of the various spatial analysis methods described herein). For example, the analyte capture agent that binds to the cell surface protein can bind to a capture probe (e.g., a capture probe on an array) that includes a capture domain that interacts with an analyte capture sequence present on the capture agent barcode domain of the analyte capture agent. All or part of the capture agent barcode domain (including the analyte binding moiety barcode) can be replicated using a polymerase, using the 3′ end of the capture domain as a primer site, to generate an extended capture probe that includes all or part of the capture probe (including the spatial barcode present on the capture probe) and a copy of the analyte binding moiety barcode. In some embodiments, a spatial array having the extended capture probe can be contacted with a sample, wherein the analyte capture agent associated with the spatial array captures a target analyte. The analyte capture agent that includes the extended capture probe (which includes the spatial barcode of the capture probe and the analyte binding moiety barcode) can then be denatured from the capture probe of the spatial array. This allows the spatial array to be reused. The sample can be separated into non-aggregated cells (e.g., single cells) and analyzed by the single cell / droplet methods described herein. The extended capture probe can be sequenced to obtain a nucleic acid sequence, wherein the spatial barcode of the capture probe is associated with the analyte binding moiety barcode of the analyte capture agent. Thus, the nucleic acid sequence of the extended capture probe can be associated with the analyte (e.g., cell surface protein) and, in turn, with one or more physical properties of the cell (e.g., shape or cell type). In some embodiments, the nucleic acid sequence of the extended capture probe can be associated with intracellular analytes of nearby cells, wherein the intracellular analytes are released using any of the cell permeabilization or analyte migration techniques described herein.

[0339] In some embodiments of any of the spatial profiling methods described herein, the capture agent barcode domains released from the analyte capture agents can then be sequenced to identify which analyte capture agents have bound to an analyte. Based on the presence of the capture agent barcode domains and analyte binding portion barcode sequences associated with features (e.g., features at specific locations) on a spatial array, an analyte profile can be created for a biological sample. Profiles of individual cells or cell populations can be compared to profiles of other cells (e.g., “normal” cells) to determine analyte variations, thereby providing diagnostically relevant information. In some embodiments, these profiles can be used to diagnose various diseases characterized by changes in cell surface receptors, such as cancer and other diseases.

[0340] Figure 10 FIG. 4 is a schematic diagram depicting an exemplary interaction between a feature-fixed capture probe 1024 and an analyte capture agent 1026. The feature-fixed capture probe 1024 can include a spatial barcode 1008 and one or more functional sequences 1006 and 1010, as described elsewhere herein. The capture probe can also include a capture domain 1012 capable of binding to the analyte capture agent 1026. The analyte capture agent 1026 can include a functional sequence 1018, a capture agent barcode domain 1016, and an analyte capture sequence 1014 capable of binding to the capture domain 1012 of the capture probe 1024. The analyte capture agent can also include a linker 1020 that couples the capture agent barcode domain 1016 to the analyte binding portion 1022.

[0341] In some embodiments of any of the spatial profiling methods described herein, the method is used to identify immune cell profiles. Immune cells express various adaptive immune receptors associated with immune function, such as the T cell receptor (TCR) and the B cell receptor (BCR). The T cell receptor and the B cell receptor play a role in the immune response by specifically recognizing and binding antigens and assisting in their destruction.

[0342] The T cell receptor (TCR) is a molecule present on the surface of T cells that is generally responsible for recognizing antigen fragments as peptides bound to major histocompatibility complex (MHC) molecules. The TCR is typically a heterodimer of two chains, each of which is a member of the immunoglobulin superfamily and has an N-terminal variable (V) domain and a C-terminal constant domain. In humans, in 95% of T cells, the TCR is composed of α and β chains, while in 5% of T cells, the TCR is composed of γ and δ (γ / δ) chains. This ratio varies during ontogeny, in disease states, and among different species. When the TCR binds to an antigen peptide and MHC (peptide / MHC or pMHC), the T lymphocyte is activated through signal transduction.

[0343] Each of the two chains of the TCR contains multiple copies of gene segments - variable "V" gene segments, diversity "D" gene segments, and joining "J" gene segments. The TCRα chain (TCRa) is generated by recombination of V and J segments, while the β chain (TCRb) is generated by recombination of V, D, and J segments. Similarly, the generation of the TCRγ chain involves recombination of V and J gene segments, and the generation of the TCRδ chain is achieved through recombination of V, D, and J gene segments. The junctions of these specific regions (V and J for the α or γ chain, V, D, and J for the β or δ chain) correspond to the CDR3 region that is important for antigen MHC recognition. Complementary determining regions (e.g., CDR1, CDR2, and CDR3), or hypervariable regions, are sequences in the variable domains of antigen receptors (e.g., T cell receptors and immunoglobulins) that can be complementary to antigens. Most of the diversity of the CDR exists in CDR3, and this diversity is generated by somatic recombination events during T lymphocyte development. The unique nucleotide sequences generated during gene rearrangement can be called clonotypes.

[0344] The B cell receptor (BCR) is a molecule present on the surface of B cells. The antigen-binding portion of the BCR consists of membrane-bound antibodies, which, like most antibodies (e.g., immunoglobulins), have unique and randomly determined antigen-binding sites. The antigen-binding portion of the BCR includes membrane-bound immunoglobulin molecules of an isotype (e.g., IgD, IgM, IgA, IgG, or IgE). When a B cell is activated by the homologous antigen it encounters for the first time, the cell proliferates and differentiates to produce antibody-secreting plasma B cells and a population of memory B cells. Different immunoglobulin isotypes differ in biological properties, structure, target specificity, and distribution. There are multiple molecular mechanisms to generate initial diversity, including gene recombination at multiple loci.

[0345] The BCR consists of two genes, IgH and IgK (or IgL), which encode the heavy and light chains of the antibody. Immunoglobulins are formed by recombination between gene segments, sequence diversification at the junctions of these segments, and point mutations throughout the genes. Each heavy chain gene contains multiple copies of three different gene segments - variable "V" gene segments, diversity "D" gene segments, and joining "J" gene segments. Each light chain gene contains multiple copies of two different gene segments of the variable region of the protein - variable "V" gene segments and joining "J" gene segments.

[0346] Recombination can generate a molecule with one V, D, and J segment each. In addition, several bases can be deleted at each of the two junctions of the two ligations, and other bases (termed N and P nucleotides) can be added, resulting in further diversity. After B cell activation, the affinity maturation process occurs through somatic hypermutation. In this process, the progeny cells of the activated B cells accumulate different somatic mutations throughout the gene, with a higher mutation concentration in the CDR regions, thus generating antibodies with higher affinity for the antigen.

[0347] In addition to somatic hypermutation, activated B cells also undergo the process of isotype switching. Antibodies with the same variable segments can have different forms (isotypes) according to the constant segments. While all naive B cells express IgM (or IgD), most activated B cells express IgG, but also express IgM, IgA, and IgE. This expression switch from IgM (and / or IgD) to IgG, IgA, or IgE occurs through recombination events that result in a cell producing a specific isotype exclusively. The unique nucleotide sequences generated during gene rearrangement can similarly be termed clonotypes.

[0348] Certain methods described herein are used to analyze various sequences of TCR and BCR from immune cells, such as various clonotypes. In some embodiments, the methods are used to analyze the sequences of TCR alpha chain, TCR beta chain, TCR delta chain, TCR gamma chain, or any fragment thereof (e.g., variable regions including V(D)J or VJ regions, constant regions, transmembrane regions, fragments thereof, combinations thereof, and combinations of fragments thereof). In some embodiments, the methods described herein can be used to analyze the sequences of B cell receptor heavy chain, B cell receptor light chain, or any fragment thereof (e.g., variable regions including V(D)J or VJ regions, constant regions, transmembrane regions, fragments thereof, combinations thereof, and combinations of fragments thereof).

[0349] In the case of immune cells to be analyzed, primer sequences for attaching barcode sequences and / or for any of the various operations in an amplification reaction can include gene-specific sequences targeting genes or gene regions of immune cell proteins (such as immune receptors). These gene sequences include, but are not limited to, sequences of various T cell receptor alpha variable genes (TRAV genes), T cell receptor alpha joining genes (TRAJ genes), T cell receptor alpha constant genes (TRAC genes), T cell receptor beta variable genes (TRBV genes), T cell receptor beta diversity genes (TRBD genes), T cell receptor beta joining genes (TRBJ genes), T cell receptor beta constant genes (TRBC genes), T cell receptor gamma variable genes (TRGV genes), T cell receptor gamma joining genes (TRGJ genes), T cell receptor gamma constant genes (TRGC genes), T cell receptor delta variable genes (TRDV genes), T cell receptor delta diversity genes (TRDD genes), T cell receptor delta joining genes (TRDJ genes), and T cell receptor delta constant genes (TRDC genes).

[0350] In some embodiments, the analyte-binding portion is based on class I or class II major histocompatibility complex (MHC). In some embodiments, the analyte-binding portion is an MHC multimer, including but not limited to MHC dextramers, MHC tetramers, and MHC pentamers (e.g., see U.S. Patent Application Publication Nos. US 2018 / 0180601 and US 2017 / 0343545, the entire contents of which are incorporated herein by reference). MHCs that include all or part of an MHC peptide (such as soluble MHC monomer molecules) can be used as the analyte-binding portion of an analyte capture agent that is coupled to a capture agent barcode domain that includes an analyte-binding portion barcode that identifies its associated MHC (thus, for example, the TCR-binding partner of the MHC). In some embodiments, MHC is used to analyze one or more cell surface features of T cells, such as TCR. In certain cases, multiple MHCs are bound together in a larger complex (MHC multimer) to increase the binding affinity of MHC for TCR through multiligand binding cooperativity.

[0351] Figure 11A 、 11BAnd 11C is a schematic diagram showing how streptavidin cell tags can be utilized in an array-based system to generate spatially barcoded cells or cell contents. For example, as shown in FIG. 11, peptide-bound major histocompatibility complex (pMHC) can be individually biotinylated and bound to the streptavidin moiety such that the streptavidin moiety contains multiple pMHC moieties. Each of these moieties can bind to a TCR such that streptavidin binds to a target T cell through multiple MCH / TCR binding interactions. The multiple interactions act synergistically and can greatly increase the binding affinity. This improved affinity can enhance the labeling of T cells and also reduce the likelihood of the label dissociating from the T cell surface. As Figure 11B shown, the capture agent barcode domain 1101 can be modified by streptavidin 1102 and contacted with multiple molecules of biotinylated MHC 1103 (e.g., pMHC) such that the biotinylated MHC 1103 molecules are coupled to the streptavidin-coupled capture agent barcode domain 1101. The result is a barcoded MHC multimer complex 1105. As Figure 11B shown, the capture agent barcode domain sequence 1101 can recognize the MHC as its associated tag and also includes optional functional sequences, such as sequences for hybridizing with other oligonucleotides. As Figure 11CAs shown, an exemplary oligonucleotide is capture probe 1106, which includes a complementary sequence (e.g., rGrGrG corresponding to CCC), a barcode sequence, and other functional sequences, e.g., UMI, adaptor sequences (e.g., including a sequencing primer sequence (e.g., R1 or part of R1 (“pR1”)), a flow cell attachment sequence (e.g., P5 or P7 or a partial sequence thereof), etc.). In some cases, capture probe 1106 may first be associated with a feature (e.g., a gel bead) and released from the feature. In other embodiments, capture probe 1106 may hybridize to the capture agent barcode domain 1101 of the MHC-oligonucleotide complex 1105. The hybridized oligonucleotides (spacer CCC and spacer rGrGrG) may then be extended in a primer extension reaction such that a construct is generated that contains sequences corresponding to each of two spatial barcode sequences (the spatial barcode associated with the capture probe and the barcode associated with the MHC-oligonucleotide complex). In some cases, one or both of these corresponding sequences may be complementary to the original sequences in capture probe 1106 or capture agent barcode domain 1101. In other embodiments, the capture probe and the capture agent barcode domain are linked together. The resulting construct may optionally be further processed (e.g., to add any other sequences and / or for cleanup) and sequenced. As described elsewhere herein, sequences derived from the spatial barcode sequence of capture probe 1106 can be used to identify features, and sequences derived from the spatial barcode sequence on capture agent barcode domain 1101 can be used to identify specific peptide-MHC complexes 1104 bound to the cell surface (e.g., when using an MHC peptide library for screening immune cells or populations of immune cells).

[0352] (c) Substrate

[0353] For the spatially array-based analysis methods described in this section, the substrate serves to support the direct or indirect attachment of capture probes to array features. Additionally, in some embodiments, the substrate (e.g., the same substrate or a different substrate) can be used to provide support for a biological sample, particularly, e.g., a thin tissue section. Thus, a “substrate” is a support that is insoluble in aqueous solutions and positions a biological sample, analyte, feature, and / or capture probe on the substrate.

[0354] A variety of different substrates can be used for the above purposes. Generally, the substrate can be any suitable support material. Exemplary substrates include, but are not limited to, glass, modified and / or functionalized glass, hydrogels, membranes, thin films, plastics (including, for example, copolymers of acrylic, polystyrene, styrene, and other materials, polypropylene, polyethylene, polybutene, polyurethane, Teflon TM, cycloolefins, polyimides, etc.), nylon, ceramics, resins, Zeonor, silica or silica-based materials, including silicon and modified silicon, carbon, metals, inorganic glass, fiber optic bundles, and polymers such as polystyrene, cycloolefin copolymers (COC), cycloolefin polymers (COP), polypropylene, polyethylene, and polycarbonate.

[0355] The substrate can also correspond to a flow cell. The flow cell can be formed from any of the above materials and can include channels for allowing reagents, solvents, features, and molecules to pass through the flow cell.

[0356] Among 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 a glass slide, the immobilization of nucleic acids can be increased by increasing the hydrophobicity of the glass surface. This enhancement can enable the formation of relatively denser stacks (e.g., providing improved specificity and resolution).

[0357] 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 (such as using epoxy silane, amino silane) and / or by polyacrylamide treatment.

[0358] The substrate can generally have any suitable form or morphology. For example, the substrate can be flat, curved, such as convexly or concavely curved towards the region where interaction occurs between a biological sample (such as a tissue sample) and the substrate. In some embodiments, the substrate is flat, e.g., a plane, a chip, or a glass slide. The substrate can include one or more patterned surfaces (such as channels, pores, protrusions, ridges, pits, etc.) within the substrate.

[0359] The substrate can be of any desired shape. For example, the substrate can generally be of a thin flat shape (such as square or rectangular). In some embodiments, the substrate structure has rounded corners (e.g., for increased safety or robustness). In some embodiments, the substrate structure has one or more cut-off corners (e.g., for a slide clamp or a cross stage). In some embodiments, when the substrate structure is flat, the substrate structure can be any suitable type of support with a flat surface (such as a chip or a glass slide, such as a microscope slide).

[0360] The substrate can optionally include various structures, such as but not limited to protrusions, ridges, and channels. The substrate can be micro-patterned to limit lateral diffusion (e.g., to prevent overlap of spatial barcodes). The substrate modified with such structures can be modified to enable the binding of analytes, features (such as beads), or probes at various positions. For example, the sites on the substrate modified with various structures can be continuous or discontinuous with other sites.

[0361] In some embodiments, the surface of the substrate can be modified to form discrete locations that can only have or accommodate a single feature. In some embodiments, the surface of the substrate can be modified such that the features adhere to random sites.

[0362] In some embodiments, techniques such as (but not limited to) stamping techniques, micro-etching techniques, and molding techniques are used to modify the surface of the substrate to include one or more holes. In some embodiments where the substrate includes one or more holes, the substrate can be a concave slide or a well slide. For example, the holes can be formed by one or more shallow depressions on the surface of the substrate. In some embodiments, in the case where the substrate includes one or more holes, the holes can be formed by attaching a cartridge (e.g., a cartridge containing one or more chambers) to the surface of the substrate structure.

[0363] In some embodiments, the structures of the substrate (e.g., holes) can each carry different capture probes. The different capture probes attached to each structure can be identified based on the position of the structure in or on the surface of the substrate. Exemplary substrates include arrays where separate structures are located on the substrate, including for example an array of holes having accommodation features.

[0364] In some embodiments, the substrate includes one or more markings on the surface of the substrate, e.g., to provide guidance for correlating spatial information with the characterization of an analyte of interest. For example, the substrate can be marked with a grid of lines (e.g., to facilitate estimating the size of an object seen at magnification and / or to provide a reference area for counting objects). In some embodiments, fiducial marks can be included on the substrate. Such markings can be made using techniques including but not limited to printing, sandblasting, and surface deposition.

[0365] In some embodiments, where the substrate is modified to include one or more structures, including but not limited to holes, protrusions, ridges, or markings, the structures can include physically altered sites. For example, a substrate modified with various structures can include physical properties including but not limited to physical configuration, magnetic or compressive forces, chemically functionalized sites, chemically altered sites, and / or electrostatically altered sites.

[0366] In some embodiments, where the substrate is modified to include various structures, including but not limited to holes, protrusions, ridges, or markings, the structures are applied in a pattern. Alternatively, the structures can be randomly distributed.

[0367] In some embodiments, the substrate is treated to minimize or reduce non-specific analyte hybridization within or between features. For example, the treatment can include coating the substrate with a hydrogel, membrane, and / or film that forms a physical barrier to non-specific hybridization. Any suitable hydrogel can be used. For example, a hydrogel substrate prepared according to the methods described in U.S. Patent Nos. 6,391,937, 9,512,422, and 9,889,422, and U.S. Patent Application Publication Nos. U.S. 2017 / 0253918 and U.S. 2018 / 0052081 can be used. The entire contents of each of the above documents are incorporated herein by reference.

[0368] The treatment can include adding functional groups that are reactive or capable of being activated such that they become reactive upon receiving a stimulus (e.g., photoactive). The treatment can include treating with a polymer having one or more physical properties (e.g., mechanical, electrical, magnetic, and / or thermal) to minimize non-specific binding (e.g., activating the substrate at certain locations to enable analyte hybridization at those locations).

[0369] The substrate (e.g., features on beads or arrays) can include tens of thousands 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, 10,000,000, 100,000,000, 100,000,000, 100,000,000, or 1,000,000,000 oligonucleotide molecules).

[0370] In some embodiments, the surface of the substrate is coated with a cell-permissive coating to enable adhesion of live cells. A "cell-permissive coating" is a coating that enables cells or helps cells maintain cell viability (e.g., remain viable) on the substrate. For example, a cell-permissive coating can enhance cell attachment, cell growth, and / or cell differentiation. For example, a cell-permissive coating can provide nutrients to live cells. A cell-permissive coating can include biomaterials and / or synthetic materials. Non-limiting examples of cell-permissive coatings include coatings characterized by having one or more extracellular matrix (ECM) components (e.g., proteoglycans and fibrin, e.g., collagen, elastin, fibronectin, and laminin), polylysine, poly(L)-ornithine, and / or biocompatible silicone (e.g., )). For example, a cell-permissive coating that includes one or more extracellular substrate components can include type I collagen, type II collagen, type IV collagen, elastin, fibronectin, laminin, and / or vitronectin. In some embodiments, the cell-permissive coating includes material from Engelbreth-Holm-Swarm (EHS) mouse sarcoma (e.g., )A soluble basement membrane preparation extracted therefrom. In some embodiments, the cell-receptive coating comprises collagen. The cell-receptive coating can be used to culture adherent cells on a spatially barcoded array or to maintain the cell viability of a tissue sample or section when in contact with the spatially barcoded array.

[0371] When the substrate comprises a gel (e.g., a hydrogel or a gel substrate), the oligonucleotides within the gel can be attached to the substrate. The terms "hydrogel" and "hydrogel substrate" are used interchangeably herein to refer to a macromolecular polymer gel comprising a network. In the network, some polymer chains can be selectively crosslinked, although crosslinking does not always occur.

[0372] In some embodiments, the hydrogel can comprise hydrogel subunits. A "hydrogel subunit" is a hydrophilic monomer, molecular precursor, or polymer that can be polymerized (e.g., crosslinked) to form a three-dimensional (3D) hydrogel network. The hydrogel subunits can include any convenient hydrogel subunits, such as, but not limited to, acrylamide, bisacrylamide, polyacrylamide and its derivatives, polyethylene glycol and its derivatives (e.g., PEG-acrylate (PEG-DA), PEG-RGD), gelatin-methacryloyl (GelMA), methacrylated hyaluronic acid (MeHA), polyaliphatic polyurethane, polyether-based polyurethane, polyester-based polyurethane, polyethylene copolymer, polyamide, polyvinyl alcohol, polypropylene glycol, polyoxytetramethylene, polyvinylpyrrolidone, polyacrylamide, 2-hydroxyethyl acrylate and 2-hydroxyethyl methacrylate, collagen, hyaluronic acid, chitosan, dextran, agarose, gelatin, alginate, protein polymer, methylcellulose, etc. and combinations thereof.

[0373] In some embodiments, the hydrogel comprises a hybrid material, e.g., the hydrogel material comprises elements of synthetic polymers and natural polymers. For example, examples of suitable hydrogels are described in U.S. Patent 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 which are incorporated herein by reference.

[0374] In some embodiments, a crosslinking agent and / or an initiator are added to the hydrogel subunits. Examples of crosslinking agents include, but are not limited to, bisacrylamide and bisaziridine. Examples of initiators include, but are not limited to, azobisisobutyronitrile (AIBN), riboflavin, and L-arginine. The inclusion of a crosslinking agent and / or an initiator can result in an increase in covalent bonding between biomacromolecules that interact during a later polymerization step.

[0375] In some embodiments, the hydrogel can have a colloidal structure (e.g., agarose) or a polymer network structure (e.g., gelatin).

[0376] In some embodiments, some hydrogel subunits are covalently or physically crosslinked and polymerized (e.g., undergo “formation”) to form a hydrogel network. For example, the hydrogel subunits can be polymerized by any method, including but not limited to thermal crosslinking, chemical crosslinking, physical crosslinking, ionic crosslinking, photo-crosslinking, radiation crosslinking (e.g., x-rays, electron beam), and combinations thereof. Techniques such as photolithographic polymerization can also be used to form hydrogels.

[0377] The polymerization method of the hydrogel subunits can be selected to form hydrogels with different properties (e.g., pore size, swelling properties, biodegradability, conductivity, transparency, and / or permeability of the hydrogel). For example, the hydrogel can include pores of a sufficient size to allow macromolecules (e.g., nucleic acids, proteins, chromatin, metabolites, gRNAs, antibodies, carbohydrates, peptides, metabolites, and / or small molecules) to enter a sample (e.g., a tissue section). It is well known that the pore size generally decreases with an increase in the concentration of the hydrogel subunits and generally increases with an increase in the ratio of the hydrogel subunits to the crosslinker. Thus, a fixative / hydrogel composition can be prepared that includes a certain concentration of hydrogel subunits that allow such biological macromolecules to pass through.

[0378] In some embodiments, the hydrogel can form a substrate. In some embodiments, the substrate includes the hydrogel and one or more second materials. In some embodiments, the hydrogel is placed on top of the one or more second materials. For example, the hydrogel can be pre-formed and then placed on top, at the bottom, or in any other configuration with the one or more second materials. In some embodiments, the hydrogel formation occurs after contacting the one or more second materials during the substrate formation. The hydrogel formation can also occur within structures (e.g., pores, ridges, protrusions, and / or markings) located on the substrate.

[0379] In some embodiments, the hydrogel formation on the substrate occurs before, simultaneously with, or after a feature (e.g., a bead) is attached to the substrate. For example, when a capture probe is attached (e.g., directly or indirectly) to the substrate, the hydrogel formation can be performed on the substrate that already contains the capture probe.

[0380] In some embodiments, the hydrogel formation occurs within a biological sample. In some embodiments, a biological sample (e.g., a tissue section) is embedded in the hydrogel. In some embodiments, the hydrogel subunits are injected into the biological sample, and the polymerization of the hydrogel is triggered by an external or internal stimulus.

[0381] In embodiments where a hydrogel is formed within a biological sample, functionalized chemistry can be used. In some embodiments, the functionalized chemistry includes hydrogel tissue chemistry (HTC). Any hydrogel tissue scaffold (e.g., synthetic or natural) suitable for HTC can be used to anchor biomacromolecules and regulate functionalization. Non-limiting examples of methods using HTC scaffold variants include CLARITY, PACT, ExM, SWITCH, and ePACT. In some embodiments, the hydrogel formation within the biological sample is permanent. For example, biomacromolecules can be permanently adhered to the hydrogel, enabling multiple rounds of interrogation. In some embodiments, the hydrogel formation within the biological sample is reversible.

[0382] In some embodiments, other reagents are added to the hydrogel subunits before, during, and / or after polymerization. For example, other reagents can include, but are not limited to, oligonucleotides (e.g., capture probes), endonucleases for fragmenting DNA, fragmentation buffers for DNA, DNA polymerases, dNTPs for amplifying nucleic acids and attaching barcodes to amplified fragments. Other enzymes can be used, including but not limited to RNA polymerase, transposase, ligase, proteinase K, and DNase. Other reagents can also include reverse transcriptase, including enzymes with terminal transferase activity, primers, and conversion oligonucleotides. In some embodiments, optical labels are added to the hydrogel subunits before, during, and / or after polymerization.

[0383] In some embodiments, HTC reagents are added to the hydrogel before, during, and / or after polymerization. In some embodiments, cell labeling agents are added to the hydrogel before, during, and / or after polymerization. In some embodiments, cell permeabilizing agents are added to the hydrogel before, during, and / or after polymerization.

[0384] The hydrogel embedded in the biological sample can be cleared by any suitable method. For example, electrophoretic tissue clearing methods 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., fixation medium, methylcellulose, or other semi-solid medium) before or after hydrogel clearing.

[0385] A "conditionally removable coating" refers to a coating that can be removed from the surface of a substrate after application of a release agent. In some embodiments, the conditionally removable coating includes a hydrogel as described herein, e.g., a hydrogel comprising a polypeptide-based material. Non-limiting examples of hydrogels with polypeptide-based materials include synthetic peptide-based materials having a combination of spider silk and transmembrane segments of the human muscle L-type calcium channel (e.g., ) and materials containing a repeated arginine-alanine-aspartic acid-alanine sequence (RADARADARADARADA) (e.g., ) EAK16 (AEAEAKAKAEAEAKAK), KLD12 (KLDLKLDLKLDLKLDL), and PGMATRIX TM are amphiphilic 16-residue peptides.

[0386] In some embodiments, the conditionally removable coating is a stimuli-responsive hydrogel. The stimuli-responsive hydrogel can undergo a gel-to-solution and / or gel-to-solid transition upon application of one or more external triggers (e.g., release agents). See, e.g., Willner, Acc. Chem. Res. 50: 657-658, 2017, which is incorporated herein by reference in its entirety. Non-limiting examples of stimuli-responsive hydrogels include thermoresponsive hydrogels, pH-responsive hydrogels, photoresponsive hydrogels, redox-responsive hydrogels, analyte-responsive hydrogels, or combinations thereof. In some embodiments, the stimuli-responsive hydrogel can be a multi-stimuli-responsive hydrogel.

[0387] A "release agent" or "external trigger" is a reagent that, when applied to a conditionally removable coating, causes the conditionally removable coating to be removed from the substrate. The external trigger or release agent can include physical triggers such as heat, magnetism, ultrasound, electrochemistry, and / or light stimuli, as well as chemical triggers such as pH, redox reactions, supramolecular complexes, and / or biocatalytic-driven reactions. See, e.g., Echeverria et al., Gels (2018), 4, 54; doi:10.3390 / gels4020054, which is incorporated herein by reference in its entirety. The type of "release agent" or "external trigger" can depend on the type of conditionally removable coating. For example, a conditionally removable coating characterized by a redox-responsive hydrogel can be removed upon application of a release agent comprising a reducing agent (e.g., dithiothreitol (DTT)). As another example, a pH-responsive hydrogel can be removed upon application of a release agent that changes the pH.

[0388] (d) Array

[0389] In many of the methods described herein, features (as further described below) are collectively positioned on a substrate. An "array" is a particular arrangement of multiple features that are irregular or form a regular pattern. The individual features in an array are different from one another based on their relative spatial positions. Generally, at least two of the multiple features in an array include different capture probes (e.g., any example of a capture probe described herein).

[0390] The array can be used to simultaneously measure a large number of analytes. In some embodiments, oligonucleotides are at least partially used to create the array. For example, one or more copies of a single type of oligonucleotide (e.g., a capture probe) can correspond to or be directly or indirectly attached to a given feature in the array. In some embodiments, a given feature in the array includes two or more oligonucleotides (e.g., capture probes). In some embodiments, two or more oligonucleotides (e.g., capture probes) that are directly or indirectly linked to a given feature on the array include a common (e.g., identical) spatial barcode.

[0391] A "feature" is an entity that serves as a support or repository for various molecular entities used in sample analysis. Examples of features include, but are not limited to, beads, points of any two- or three-dimensional geometry (e.g., inkjet dots, mask dots, squares on a grid), wells, and hydrogel pads. In some embodiments, the feature is directly or indirectly attached or fixed to a substrate. In some embodiments, the feature is not directly or indirectly attached or fixed to the substrate, but is instead disposed, for example, within an enclosed or partially enclosed three-dimensional space (e.g., a well or a pit).

[0392] In addition to the above features, a variety of other features can be used to form the arrays described herein. For example, in some embodiments, features formed from polymers and / or biopolymers that are inkjet printed, screen printed, or electrostatically deposited onto a substrate can be used to form the array. For example, the inkjet printing of biopolymers is described in PCT patent application publication no. WO 2014 / 085725. For example, the inkjet printing of polymers is described in de Gans et al., Adv Mater. 16(3):203-213 (2004). Methods for the electrostatic deposition of polymers and biopolymers are described in, for example, Hoyer et al., Anal. Chem. 68(21):3840-3844 (1996). The entire contents of each of the above references are incorporated herein by reference.

[0393] As another example, in some embodiments, the feature is formed from metal microparticles or nanoparticles. For example, suitable methods for depositing such particles to form an array are described in Lee et al., Beilstein J. Nanotechnol. 8:1049-1055 (2017), the entire contents of which are incorporated herein by reference.

[0394] As another example, in some embodiments, the feature is formed from magnetic particles assembled on a substrate. Examples of methods for such assembled arrays and particles are described in Ye et al., Scientific Reports 6:23145 (2016), the entire contents of which are incorporated herein by reference.

[0395] As another example, in some embodiments, the feature corresponds to a substrate region in which one or more optical markers have been incorporated and / or that has been altered by a process such as permanent photo-bleaching. Suitable substrates for implementing the feature in this way include, for example, a variety of polymers. For example, a method of forming such a feature is described in Moshrefzadeh et al., Appl. Phys. Lett. 62:16 (1993), the entire content of which is incorporated herein by reference.

[0396] As yet another example, in some embodiments, the feature can correspond to colloidal particles that are assembled (e.g., via self-assembly) to form an array. Suitable colloidal particles are described, for example, in Sharma, Resonance 23(3):263 - 275 (2018), the entire content of which is incorporated herein by reference.

[0397] As another example, in some embodiments, features can be formed by point array photopolymerization of a monomer solution on a substrate. In particular, two-photon and three-photon polymerization can be used to fabricate features of relatively small (e.g., sub-micron) size. Suitable methods of preparing features on a substrate in this way are described in Nguyen et al., Materials Today 20(6):314 - 322 (2017), the entire content of which is incorporated herein by reference.

[0398] In some embodiments, the feature is directly or indirectly attached or fixed to a liquid-permeable substrate. In some embodiments, the feature is directly or indirectly attached or fixed to a biocompatible substrate. In some embodiments, the feature is directly or indirectly attached or fixed to a substrate that is a hydrogel.

[0399] Figure 12 An exemplary arrangement of barcoded features in an array is described. From left to right, Figure 12 shown are (L) a slide including six spatially barcoded arrays, (C) an enlarged schematic of one of the six spatially barcoded arrays showing a grid of barcoded features related to a biological sample, and (R) an enlarged schematic of a portion of the array showing specific identifications (labeled ID578, ID579, ID560, etc.) of multiple features in the array.

[0400] As used herein, the term "bead array" refers to an array that includes a plurality of beads as features in the array. In some embodiments, the beads are attached to a substrate. For example, the beads can optionally be attached to a substrate such as a microscope slide and be close to a biological sample (e.g., a tissue section including cells). The beads can also be suspended in a solution and deposited on a surface (e.g., a membrane, a tissue section, or a substrate (e.g., a microscope slide)).

[0401] Examples of bead arrays on or within a substrate include beads located in wells, such as BeadChip arrays (available from Illumina Inc., San Diego, California), arrays used in the 454 LifeSciences (a subsidiary of Roche, Basel, Switzerland) sequencing platform, and arrays for the Ion Torrent sequencing platform (a subsidiary of Life Technologies, Carlsbad, California). For example, bead array examples are described in U.S. Patent Nos. 6,266,459, 6,355,431, 6,770,441, 6,859,570, 6,210,891, 6,258,568, and 6,274,320; U.S. Patent Application Publication Nos. 2009 / 0026082; 2009 / 0127589; 2010 / 0137143; and 2010 / 0282617; and PCT Patent Application Publication Nos. WO 00 / 063437 and WO2016 / 162309, the entire contents of which are incorporated herein by reference.

[0402] In some embodiments, the bead array includes a plurality of beads. For example, the bead array can include at least 10,000 beads (e.g., at least 100,000 beads, at least 1,000,000 beads, at least 5,000,000 beads, at least 10,000,000 beads). In some embodiments, the plurality of beads includes a single type of bead (e.g., substantially uniform in size, shape, and other physical properties, such as translucent). In some embodiments, the plurality of beads includes two or more different types of beads.

[0403] In some embodiments, a bead array is formed when beads are embedded in a hydrogel layer, where the hydrogel polymerizes and fixes the relative bead positions. The bead array can be pre-equilibrated and combined with a reaction buffer and enzymes (e.g., reverse transcription mixture). In some embodiments, the bead array is frozen.

[0404] "Flexible array" includes a plurality of spatially barcoded features that are attached to or embedded in a flexible substrate (e.g., a membrane or a strip) placed on a biological sample. In some embodiments, the flexible array includes a plurality of spatially barcoded features embedded in a hydrogel substrate. To form such an array, the features of a microarray are replicated into the hydrogel and the size of the hydrogel is reduced by removing water. These steps can be performed multiple times. For example, in some embodiments, a method for preparing a high-density spatially barcoded array can include replicating a plurality of features from a microarray into a first hydrogel, where the first hydrogel is in contact with the microarray; reducing the size of the first hydrogel including the replicated features by removing water to form a first shrunk hydrogel including the replicated features; replicating the features in the first shrunk hydrogel into a second hydrogel, where the second hydrogel is in contact with the first hydrogel; and reducing the size of the second hydrogel including the replicated features by removing water to form a second shrunk hydrogel including the replicated features, thereby generating a high-density spatially barcoded array. The result is a high-density flexible array including spatially barcoded features.

[0405] In some embodiments, spatially barcoded beads can be loaded onto a substrate (e.g., a hydrogel) to produce a high-density self-assembled bead array.

[0406] The flexible array can be pre-equilibrated with a reaction buffer and a functional concentration of an enzyme (e.g., a reverse transcription mixture). In some embodiments, the flexible bead array can be stored or frozen for a long time (e.g., for days) until use. In some embodiments, permeabilization of a biological sample (e.g., a tissue section) can be performed by adding an enzyme / detergent before contacting with the flexible array. The flexible array can be placed directly on the sample or in indirect contact with the biological sample (e.g., having an intermediate layer or substance between the biological sample and the flexible bead array). In some embodiments, once the flexible array is applied to the sample, reverse transcription and target capture of an analyte can be performed on solid microspheres or circular beads of a first size and circular beads of a second size.

[0407] "Microrod array" is a series of array features delineated by microrods. "Microrod channel" is a separate partition created by microrods. For example, microrod channels can be fluidically isolated from other microrod channels such that the fluid or other contents in one microrod channel in the array are separated from the fluid or other contents in an adjacent microrod channel in the array. The density and arrangement order of the microrods can be the density or arrangement order of any suitable discrete sites.

[0408] In some embodiments, the microtube arrays are processed to create conditions conducive to loading. An example is using a corona rod (BD-20AC, Electro Technic Products) to create a hydrophilic surface. In some embodiments, features (e.g., beads to which capture probes are attached) are loaded onto the microtube arrays such that the exact position of the features within the array is known. For example, capture probes containing spatial barcodes can be placed into the microtube channels such that the spatial barcodes can identify the position of the barcode sequences from which the barcoded nucleic acid molecules are derived.

[0409] In some embodiments, when using a random distribution to distribute features, empirical testing can be performed to generate loading / distribution conditions conducive to a single feature per microtube. In some embodiments, it may be desirable to achieve distribution conditions that promote only a single feature (e.g., bead) per microtube channel. In some embodiments, it may be desirable to achieve distribution conditions that promote more than one feature (e.g., beads) per microtube channel by flowing the features through the microtube channels.

[0410] In some embodiments, the microtube arrays are placed in a position in contact with the sample (e.g., on top or below) such that the microtubes containing the features (e.g., beads that may include capture probes) are in contact with the biological sample. In some embodiments, the biological sample is placed on the exposed side of the microtube array and mechanical pressure is applied to move the biological sample into the microtube channels to create fluidically isolated reaction chambers containing the biological sample.

[0411] In some embodiments, the biological sample is partitioned by contacting the microtube array with the biological sample, thereby creating microtube channels that include portions of the beads and the biological sample. In some embodiments, the portion of the biological sample contained within the microtube channels is one or more cells. In some embodiments, after adding one or more cells to the microtube channels, the features are introduced into the microtube array by flow.

[0412] In some embodiments, reagents are added to the microtube arrays. The added reagents can include enzyme reagents and reagent mixtures for performing nucleic acid amplification. In some embodiments, the reagents include reverse transcriptase, ligase, one or more nucleotides, and any combination thereof. After adding the reagents to the microtube channels, one or more microcapillary channels can be sealed, for example, using silicone oil, mineral oil, non-porous materials, or lids.

[0413] In some embodiments, after incubation for a certain time and at a certain temperature or temperature range (e.g., after a hybridization or amplification reaction), the reagent solution is removed from each microcapillary channel. The reagent solutions can be processed individually for sequencing or combined for sequencing analysis.

[0414] In some embodiments, some or all of the features in the array include capture probes. In some embodiments, the array may include capture probes directly or indirectly attached to a substrate.

[0415] The capture probe includes a capture domain (e.g., a nucleotide sequence) that can specifically bind (e.g., hybridize) to a target analyte (e.g., mRNA, DNA, or protein) within a sample. In some embodiments, the binding (e.g., hybridization) of the capture probe to the target can be detected and quantified by detecting a visual signal (e.g., a fluorophore, a heavy metal (e.g., silver ions), or a chemiluminescent label) incorporated into the target. In some embodiments, the intensity of the visual signal is related to the relative abundance of each analyte in the biological sample. Since an array can contain thousands or millions of capture probes (or more), an array of features with capture probes can interrogate many analytes in parallel.

[0416] In some embodiments, the substrate includes one or more capture probes designed to capture analytes from one or more organisms. In a non-limiting example, the substrate may contain one or more capture probes designed to capture mRNA from one organism (e.g., a human) and one or more capture probes designed to capture DNA from a second organism (e.g., a bacterium).

[0417] The capture probes can be attached to the substrate or feature using a variety of techniques. In some embodiments, the capture probe is directly attached to a feature immobilized on the array. In some embodiments, the capture probe is immobilized on the substrate by chemical fixation. For example, chemical fixation can occur between a functional group on the substrate and a corresponding functional element on the capture probe. Exemplary corresponding functional elements in the capture probe can be an inherent chemical group of the capture probe, such as a hydroxyl group, or a functional element can be introduced onto the capture probe. Examples of functional groups on the substrate are amine groups. In some embodiments, the capture probe to be immobilized includes a functional amine group or is chemically modified to include a functional amine group. Means and methods for such chemical modification are well known in the art.

[0418] In some embodiments, the capture probe is a nucleic acid. In some embodiments, the capture probe is immobilized on the feature or substrate by its 5′ end. In some embodiments, the capture probe is immobilized on the feature or substrate by its 5′ end and comprises, from the 5′ end to the 3′ end: one or more barcodes (e.g., spatial barcodes and / or UMIs) and one or more capture domains. In some embodiments, the capture probe is immobilized on the feature by its 5′ end and comprises, from the 5′ end to the 3′ end: one barcode (e.g., a spatial barcode or UMI) and one capture domain. In some embodiments, the capture probe is immobilized on the feature or substrate by its 5′ end and comprises, from the 5′ end to the 3′ end: a cleavage domain, a functional domain, one or more barcodes (e.g., spatial barcodes and / or UMIs), and a capture domain.

[0419] In some embodiments, the capture probe is immobilized on the feature or substrate by its 5′ end and comprises, from the 5′ end to the 3′ end: a cleavage domain, a functional domain, one or more barcodes (e.g., spatial barcodes and / or UMIs), a second functional domain, and a capture domain. In some embodiments, the capture probe is immobilized on the feature or substrate by its 5′ end and comprises, from the 5′ end to the 3′ end: a cleavage domain, a functional domain, a spatial barcode, a UMI, and a capture domain. In some embodiments, the capture probe is immobilized on the feature or substrate by its 5′ end and does not include a spatial barcode. In some embodiments, the capture probe is immobilized on the feature or substrate by its 5′ end and does not include a UMI. In some embodiments, the capture probe comprises a sequence for initiating a sequencing reaction.

[0420] In some embodiments, the capture probe is immobilized on the feature or substrate by its 3′ end. In some embodiments, the capture probe is immobilized on the feature or substrate by its 3′ end and comprises, from the 3′ end to the 5′ end: one or more barcodes (e.g., spatial barcodes and / or UMIs) and one or more capture domains. In some embodiments, the capture probe is immobilized on the feature or substrate by its 3′ end and comprises, from the 3′ end to the 5′ end: one barcode (e.g., a spatial barcode or UMI) and one capture domain. In some embodiments, the capture probe is immobilized on the feature or substrate by its 3′ end and comprises, from the 3′ end to the 5′ end: a cleavage domain, a functional domain, one or more barcodes (e.g., spatial barcodes and / or UMIs), and a capture domain. In some embodiments, the capture probe is immobilized on the feature or substrate by its 3′ end and comprises, from the 3′ end to the 5′ end: a cleavage domain, a functional domain, a spatial barcode, a UMI, and a capture domain.

[0421] The positioning of functional groups within a capture probe to be immobilized can be used to control and shape the binding behavior and / or orientation of the capture probe. For example, the functional groups can be placed at the 5′ or 3′ end of the capture probe or within the sequence of the capture probe. In some embodiments, the capture probe can also include a substrate (e.g., a support attached to the capture probe, a support attached to a feature, or a support attached to a substrate). Typical substrates for the capture probe to be immobilized include moieties capable of binding to the capture probe (e.g., binding to amine-functionalized nucleic acids). Examples of such substrates are carboxyl, aldehyde, or epoxy supports.

[0422] In some embodiments, the substrate on which the capture probe can be immobilized can be chemically activated, e.g., by activating available functional groups on the substrate. The term “activated (substrate)” refers to a material in which an interaction or reactive chemical functional group is established or achieved through a chemical modification procedure. For example, a substrate including carboxyl can be activated prior to use. Additionally, certain substrates contain functional groups that can react with specific moieties already present in the capture probe.

[0423] In some embodiments, covalent bonds are used to directly couple the capture probe to the substrate. In some embodiments, the capture probe is indirectly coupled to the substrate via a linker (i.e., a chemical linker) that separates the “first” nucleotide of the capture probe from the substrate. In some embodiments, the capture probe does not directly bind to the array but instead interacts indirectly, e.g., by binding to a molecule that itself directly or indirectly binds to the array. In some embodiments, the capture probe is indirectly attached to the substrate (e.g., via a solution including a polymer).

[0424] In some embodiments where the capture probe is indirectly immobilized on an array feature, e.g., by hybridization to a surface probe capable of binding the capture probe, the capture probe can also include an upstream sequence capable of hybridizing to the 5′ end of the surface probe (in the 5′ direction of the sequence hybridizing to nucleic acid, e.g., RNA of a tissue sample). Separately, the capture domain of the capture probe can be regarded as a capture domain oligonucleotide, which can be used for the synthesis of the capture probe in embodiments where the capture probe is indirectly immobilized on the array.

[0425] In some embodiments, the substrate consists of an inert material or matrix (e.g., a glass slide) that has been functionalized by treatment with, for example, a material containing reactive groups capable of immobilizing the capture probe. See, for example, WO2017 / 019456, the entire content of which is incorporated herein by reference. Non-limiting examples include polyacrylamide hydrogels supported on an inert substrate (e.g., a glass slide; see WO 2005 / 065814 and U.S. Patent Application No. 2008 / 0280773, the entire contents of which are incorporated herein by reference).

[0426] In some embodiments, functionalized biomolecules (e.g., capture probes) are immobilized on a functionalized substrate using covalent methods. Methods of covalent attachment include, for example, condensation of amines and activated carboxylic acid esters (e.g., N-hydroxysuccinimide esters); condensation of amines and aldehydes under reductive amination conditions; and cycloaddition reactions such as Diels-Alder [4+2] reactions, 1,3-dipolar cycloaddition reactions, and [2+2] cycloaddition reactions. Methods of covalent attachment also include, for example, click chemistry reactions, including [3+2] cycloaddition reactions (e.g., Huisgen 1,3-dipolar cycloaddition reaction and copper(I)-catalyzed azide-alkyne cycloaddition reaction (CuAAC)); thiol-ene reactions; Diels-Alder reactions and inverse electron demand Diels-Alder reactions; [4+1] cycloaddition of isonitriles and tetrazines; nucleophilic ring opening of small carbon rings (e.g., epoxide ring opening of amino oligonucleotides). Methods of covalent attachment also include, for example, maleimide and thiol; and p-nitrophenyl ester-functionalized oligonucleotides and polylysine-functionalized substrates. Methods of covalent attachment also include, for example, disulfide bond reactions; radical reactions (see, e.g., U.S. Patent No. 5,919,626, the entire content of which is incorporated herein by reference); and hydrazide-functionalized substrates (e.g., where the hydrazide functional group is directly or indirectly attached to the substrate) and aldehyde-functionalized oligonucleotides (see, e.g., Yershov et al., (1996) Proc. Natl. Acad. Sci. USA 93, 4913-4918, the entire content of which is incorporated herein by reference).

[0427] In some embodiments, functionalized biomolecules (e.g., capture probes) are immobilized on a functionalized substrate using photochemical covalent methods. Methods of photochemical covalent attachment include, for example, immobilization of anthraquinone-conjugated oligonucleotides (see, e.g., Koch et al., (2000) Bioconjugate Chem. 11, 474-483, the entire content of which is incorporated herein by reference).

[0428] In some embodiments, functionalized biomolecules (e.g., capture probes) are immobilized on a functionalized substrate using non-covalent methods. Methods for non-covalent attachment include, for example, biotin-functionalized oligonucleotides and streptavidin-treated substrates (see, e.g., et al., (1993) Analytical Biochemistry 209, 278-283 and Gilles et al., (1999) Nature Biotechnology 17, 365-370, the entire content of which is incorporated herein by reference).

[0429] In some embodiments, oligonucleotides (e.g., capture probes) can be attached to substrates or features according to the methods described in the following: U.S. Patent Nos. 6,737,236; 7,259,258; 7,375,234; 7,427,678; 5,610,287; 5,807,522; 5,837,860; and 5,472,881; U.S. Patent Application Publication Nos. 2008 / 0280773 and 2011 / 0059865; Shalon et al., (1996) Genome Research, 639-645; Rogers et al., (1999) Analytical Biochemistry 266, 23-30; Stimpson et al., (1995) Proc. Natl. Acad. Sci. USA 92, 6379-6383; Beattie et al., (1995) Clin. Chem. 45, 700-706; Lamture et al., (1994) Nucleic Acids Research 22, 2121-2125; Beier et al., (1999) Nucleic Acids Research 27, 1970-1977; Joos et al., (1997) Analytical Biochemistry 247, 96-101; Nikiforov et al., (1995) Analytical Biochemistry 227, 201-209; Timofeev et al., (1996) Nucleic Acids Research 24, 3142-3148; Chrisey et al., (1996) Nucleic Acids Research 24, 3031-3039; Guo et al., (1994) Nucleic Acids Research 22, 5456-5465; Running and Urdea (1990) BioTechniques 8, 276-279; Fahy et al., (1993) Nucleic Acids Research 21, 1819-1826; Zhang et al., (1991) 19, 3929-3933; and Rogers et al., (1997) Gene Therapy 4, 1387-1392. The entire content of each of the above documents is incorporated herein by reference.

[0430] Arrays can be prepared by a variety of methods. In some embodiments, the array is prepared by synthesizing (e.g., in situ synthesizing) oligonucleotides on the array or by inkjet printing or lithography. For example, photodirected synthesis of high-density DNA oligonucleotides can be achieved by photolithography or solid-phase DNA synthesis. To achieve photolithographic synthesis, a synthesis linker modified with a photochemical protecting group can be attached to the substrate, and a photolithographic mask (applied to a specific area of the substrate) and light can be used to modify the photochemical protecting group, thereby generating an array with local photodeprotection. Many of these methods are known in the art and are described, for example, in Miller et al., "Basic concepts of microarrays and potential applications in clinical microbiology," Clinical microbiology reviews 22.4 (2009): 611-633; US201314111482A; US9593365B2; US2019203275; and WO2018091676, which are all incorporated herein by reference in their entirety.

[0431] In some embodiments, the array is "spotted" or "printed" with oligonucleotides, and then these oligonucleotides (e.g., capture probes) are attached to the substrate. The oligonucleotides can be applied by non-contact or contact printing. A non-contact printer can use the same method as a computer printer (e.g., bubble jet or inkjet) to eject small droplets of the probe solution onto the substrate. Such a dedicated inkjet printer can eject nanoliter to picoliter volumes of oligonucleotide solution droplets (instead of ink) onto the substrate. In contact printing, each printing pin directly applies the oligonucleotide solution to a specific location on the surface. The oligonucleotides can be attached to the substrate surface by electrostatic interaction between the negative charge of the DNA phosphate backbone and the positively charged coating on the substrate surface or by ultraviolet light cross-linking covalent bonds between the thymine bases in the DNA and the amine groups on the treated substrate surface. In some embodiments, the substrate is a glass slide. In some embodiments, the oligonucleotides (e.g., capture probes) are attached to a chemical substrate, such as epoxy silane, amino silane, lysine, polyacrylamide, etc., by covalent bonds.

[0432] These arrays can also be prepared by in-situ synthesis methods. In some embodiments, photolithography can be used to prepare these arrays. This method typically relies on the combination of ultraviolet light masking and light-directed combinatorial chemistry synthesis on a substrate to directly synthesize probes selectively on the array surface, one nucleotide at a time per spot, while being used for multiple spots simultaneously. In some embodiments, the substrate contains a covalent linker molecule that has a photo-removable protecting group at the free end. Ultraviolet light is directed through a photolithography mask to deprotect and activate selected sites bearing hydroxyl groups, which initiates the coupling of incoming protected nucleotides attached to the activated sites. The mask is designed such that the exposed sites can be selected, thereby specifying the coordinates on the array where each nucleotide can be attached. This process can be repeated, with a new mask being applied to activate different sets of sites and couple different bases, enabling the construction of arbitrary oligonucleotides at each site. This process can be used to synthesize thousands of different oligonucleotides. In some embodiments, maskless array synthesizer technology can be used. It uses a programmable micro-mirror array to create a digital mask that reflects the required ultraviolet light pattern to remove the protection of features.

[0433] In some embodiments, an inkjet spotting process can also be used for in-situ oligonucleotide synthesis. Different nucleotide precursors plus catalysts can be printed on the substrate, followed by coupling and deprotection steps. This method relies on printing picoliter volumes of nucleotides in a base-by-base printing manner in repeated rounds on the array surface, which extends the length of oligonucleotide probes on the array.

[0434] Arrays can also be prepared by active electrokinetic hybridization to control the transport of nucleic acids. When a positive current is applied to one or more test sites on the array, negatively charged nucleic acids can be transported to specific sites or features. The surface of the array can contain binding molecules, such as streptavidin, which enables the formation of bonds (e.g., streptavidin-biotin bonds) when electronically addressed biotinylated probes reach their targeted positions. Then the positive current is removed from the activated feature, and new test sites can be activated by the targeted application of a positive current. This process is repeated until all sites on the array are covered.

[0435] Arrays for spatial analysis can be generated by various methods as described herein. In some embodiments, the array has multiple capture probes containing spatial barcodes. These spatial barcodes and their relationship to the positions on the array can be determined. In some cases, such information is readily available because the oligonucleotides are spotted, printed, or synthesized on the array in a pre-determined pattern. In some cases, the spatial barcodes can be decoded by the methods described herein (e.g., by in-situ sequencing, by various tags associated with the spatial barcodes, etc.). In some embodiments, the array can be used as a template to generate sub-arrays. Thus, the spatial barcodes can be transferred to sub-arrays with known patterns.

[0436] In some embodiments, an array comprising barcoded probes can be generated by ligating a plurality of oligonucleotides. In some instances, the plurality of oligonucleotides comprise portions of a barcode and a complete barcode is generated upon ligation of the plurality of oligonucleotides. For example, a first oligonucleotide comprising a first portion of a barcode can be attached to a substrate (e.g., using any method for attaching an oligonucleotide to a substrate described herein), and then, a second oligonucleotide comprising a second portion of the barcode can be ligated to the first oligonucleotide to generate a complete barcode. Different combinations of the first, second, and any other portions of the barcode can be used to increase the diversity of the barcode. In instances where the second oligonucleotide is also attached to the substrate prior to ligation, the first and / or second oligonucleotide can be attached to the substrate via a surface linker comprising a cleavage site. After ligation, the ligated oligonucleotides are linearized by cleavage at the cleavage site.

[0437] To increase the diversity of the barcode, a plurality of second oligonucleotides comprising two or more different barcode sequences can be ligated to a plurality of first oligonucleotides comprising the same barcode sequence, thereby generating two or more different types of barcodes. To effect selective ligation, the first oligonucleotide attached to the substrate comprising the first portion of the barcode can initially be protected with a protecting group (e.g., a photocleavable protecting group), and the protecting group can be removed prior to ligation between the first and second oligonucleotides. In cases where the barcoded probes on the array are generated by ligating two or more oligonucleotides, a concentration gradient of the oligonucleotides can be applied to the substrate such that different combinations of the oligonucleotides are incorporated into the barcoded probes depending on their position on the substrate.

[0438] Barcoded probes on the array can also be generated by adding individual nucleotides to existing oligonucleotides on the array, e.g., using a polymerase that acts in a template-independent manner. The individual nucleotides can be added to the existing oligonucleotides in a concentration gradient, thereby generating probes of different lengths depending on the position of the probe on the array.

[0439] Arrays can also be prepared by modifying existing arrays, e.g., by modifying oligonucleotides attached to the array. For example, probes can be generated on an array containing oligonucleotides that are attached to the array at the 3′ end and have a free 5′ end. The oligonucleotides can be oligonucleotides synthesized in situ and can include barcodes. The length of the oligonucleotides can be less than 50 nucleotides (nt) (e.g., less than 45, 40, 35, 30, 25, 20, 15, or 10 nt). To generate probes using these oligonucleotides, a primer complementary to a portion of the oligonucleotide (e.g., a constant sequence common to the oligonucleotides) can be hybridized to the oligonucleotide and extended (using the oligonucleotide as a template) to form a duplex and generate a 3′ overhang. Thus, the 3′ overhang allows other nucleotides or oligonucleotides to be added to the duplex. Capture probes can be generated, for example, by adding one or more oligonucleotides to the end of the 3′ overhang (e.g., by splint oligonucleotide-mediated ligation), where the added oligonucleotides can include the sequence of the capture domain or a portion of that sequence.

[0440] In cases where the oligonucleotides on the existing array include a recognition sequence that can hybridize to a splint oligonucleotide, probes can also be generated by directly ligating other oligonucleotides to the existing oligonucleotides via the splint oligonucleotide. The recognition sequence can be located at the free 5′ end or the free 3′ end of the oligonucleotide on the existing array. Recognition sequences useful in the methods of the invention can not include restriction enzyme recognition sites or secondary structures (e.g., hairpins) and can include a high content of guanine and cytosine nucleotides and thus have high stability.

[0441] A bead array can be generated by attaching beads (e.g., barcoded beads) to a substrate in a regular pattern or an irregular arrangement. The beads can be attached to a selective area of the substrate by, for example, selectively activating areas on the substrate to enable bead attachment. Activating selective areas on the substrate can include activating a coating (e.g., a photocleavable coating) or a polymer applied to the substrate. Beads can be attached iteratively, e.g., a subset of beads can be attached at one time, and the same process can be repeated to attach the remaining beads. Alternatively, all of the beads can be attached to the substrate in one step.

[0442] Barcoded beads or beads containing multiple barcoded probes can be generated by first preparing multiple barcoded probes on a substrate, depositing multiple beads on the substrate, and using the probes on the substrate as templates to generate probes attached to the beads.

[0443] Large-scale commercial manufacturing methods enable millions of oligonucleotides to be attached to an array. Commercial arrays include arrays from Roche NimbleGen, Inc. (Wisconsin) and Affymetrix, Inc. (ThermoFisher Scientific).

[0444] In some embodiments, the arrays can be prepared according to the methods described in WO 2012 / 140224, WO 2014 / 060483, WO 2016 / 162309, WO 2017 / 019456, WO 2018 / 091676, and WO 2012 / 140224, as well as U.S. Patent Application No. 2018 / 0245142. The entire contents of the above documents are incorporated herein by reference.

[0445] In some embodiments, the features on the arrays include beads. In some embodiments, two or more beads are dispersed on a substrate to create an array, where each bead is a feature on the array. Optionally, the beads can be dispersed into the holes in the substrate such that, for example, each hole contains only a single bead.

[0446] A "bead" is a particle. The beads can be porous, non-porous, solid, semi-solid, and / or combinations thereof. In some embodiments, the beads can be soluble, breakable, and / or degradable, while in certain embodiments, the beads are non-degradable.

[0447] The beads can generally be any suitable shape. Examples of bead shapes include, but are not limited to, spherical, non-spherical, oval, rectangular, amorphous, circular, cylindrical, and variations thereof. A cross-section (e.g., a first cross-section) can correspond to the diameter or the maximum cross-sectional dimension of the bead. In some embodiments, the beads can be approximately spherical. In these embodiments, the first cross-section can correspond to the diameter of the bead. In some embodiments, the beads can be approximately cylindrical. In such embodiments, the first cross-section can correspond to the diameter, length, or width along the approximately cylindrical bead.

[0448] The beads can be of uniform size or non-uniform size. "Polydispersity" generally refers to the non-uniformity of the molecular or particle size. The polydispersity index (PDI) of the beads can be calculated using the equation PDI = Mw / Mn, where Mw is the weight-average molar mass and Mn is the number-average molar mass. In certain embodiments, the beads can be provided as a plurality of beads or groups of beads having a relatively monodisperse size distribution. Maintaining relatively consistent bead characteristics (e.g., size) helps with overall consistency when a relatively consistent dosage of the reagent is desired.

[0449] In some embodiments, the beads provided herein can have a size distribution with a coefficient of variation of the cross-sectional dimension of less than 50%, less than 40%, less than 30%, less than 20%, less than 15%, less than 10%, less than 5%, or lower. In some embodiments, the plurality of beads provided herein have a polydispersity index of less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or lower.

[0450] In some embodiments, the diameter or maximum dimension of the beads may be no greater than 100 μm (e.g., no greater than 95 μm, 90 μm, 85 μm, 80 μm, 75 μm, 70 μm, 65 μm, 60 μm, 55 μm, 50 μm, 45 μm, 40 μm, 35 μm, 30 μm, 25 μm, 20 μm, 15 μm, 14 μm, 13 μm, 12 μm, 11 μm, 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm, or 1 μm).

[0451] In some embodiments, the average diameter of the plurality of beads is no greater than 100 μm. In some embodiments, the average diameter or maximum dimension of the plurality of beads is no greater than 95 μm, 90 μm, 85 μm, 80 μm, 75 μm, 70 μm, 65 μm, 60 μm, 55 μm, 50 μm, 45 μm, 40 μm, 35 μm, 30 μm, 25 μm, 20 μm, 15 μm, 14 μm, 13 μm, 12 μm, 11 μm, 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm or 1 μm.

[0452] In some embodiments, the volume of the beads may be at least about 1 μm 3 , such as at least 1 μm 3 , 2 μm 3 , 3 μm 3 , 4 μm 3 , 5 μm 3 , 6 μm 3 , 7 μm 3 , 8 μm 3 , 9 μm 3 , 10 μm 3 , 12 μm 3 , 14 μm 3 , 16 μm 3 , 18 μm 3 , 20 μm 3 , 25 μm 3 , 30 μm 3 , 35 μm 3 , 40 μm 3 , 45 μm 3 , 50 μm 3 , 55 μm 3 , 60 μm 3 , 65 μm 3 , 70 μm 3 , 75 μm 3 , 80 μm 3 , 85 μm 3 , 90 μm 3 , 95 μm3 , 100 μm 3 , 125 μm 3 , 150 μm 3 , 175 μm 3 , 200 μm 3 , 250 μm 3 , 300 μm 3 , 350 μm 3 , 400 μm 3 , 450 μm 3 , μm 3 , 500 μm 3 , 550 μm 3 , 600 μm 3 , 650 μm 3 , 700 μm 3 , 750 μm 3 , 800 μm 3 , 850 μm 3 , 900 μm 3 , 950 μm 3 , 1000 μm 3 , 1200 μm 3 , 1400 μm 3 , 1600 μm 3 , 1800 μm 3 , 2000 μm 3 , 2200 μm 3 , 2400 μm 3 , 2600 μm 3 , 2800 μm 3 , 3000 μm 3 or greater.

[0453] In some embodiments, the beads can have a volume between about 1 μm 3 and 100 μm 3 , for example between about 1 μm 3 and 10 μm 3 , between about 10 μm 3 and 50 μm 3 , or between about 50 μm 3 and 100 μm 3 . In some embodiments, the beads can include a volume between about 100 μm 3 and 1000 μm 3 , for example between about 100 μm 3 and 500 μm 3 , or between about 500 μm 3 and 1000 μm 3 . In some embodiments, the beads can include about 1000 μm3 and a volume between 3000 μm 3 such as between about 1000 μm 3 and 2000 μm 3 or between about 2000 μm 3 and 3000 μm 3 In some embodiments, the beads can include a volume between about 1 μm 3 and 3000 μm 3 such as between about 1 μm 3 and 2000 μm 3 between, about 1 μm 3 and 1000 μm 3 between, about 1 μm 3 and 500 μm 3 or between about 1 μm 3 and 250 μm 3 In some embodiments, the beads can include a volume between about 1 μm

[0454] The bead may include one or more identical or different cross-sections. In some embodiments, the bead may have a first cross-section that is different from the second cross-section. The bead may have a first cross-section of at least about 0.0001 microns, 0.001 microns, 0.01 microns, 0.1 microns, or 1 micron. In some embodiments, the bead may include a cross-section (e.g., the first cross-section) of at least about 1 micron (μm), 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 100 μm, 120 μm, 140 μm, 160 μm, 180 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm, 1 millimeter (mm) or greater. In some embodiments, the bead may include a cross-section (e.g., the first cross-section) between about 1 μm and 500 μm, such as between about 1 μm and 100 μm, between about 100 μm and 200 μm, between about 200 μm and 300 μm, between about 300 μm and 400 μm, or between about 400 μm and 500 μm. For example, the bead may include a cross-section (e.g., the first cross-section) between about 1 μm and 100 μm. In some embodiments, the bead may have a second cross-section of at least about 1 μm. For example, the bead may include a second cross-section of at least about 1 micron (μm), 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 100 μm, 120 μm, 140 μm, 160 μm, 180 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm, 1 millimeter (mm) or greater.In some embodiments, the beads can include a second cross-section between about 1 μm and 500 μm, such as between about 1 μm and 100 μm, between about 100 μm and 200 μm, between about 200 μm and 300 μm, between about 300 μm and 400 μm, or between about 400 μm and 500 μm. For example, the beads can include a second cross-section between about 1 μm and 100 μm.

[0455] In some embodiments, the beads can be nanoscale (e.g., the beads can have a diameter or maximum cross-sectional dimension of about 100 nanometers (nm) to about 900 nanometers (nm)) (e.g., 850 nm or less, 800 nm or less, 750 nm or less, 700 nm or less, 650 nm or less, 600 nm or less, 550 nm or less, 500 nm or less, 450 nm or less, 400 nm or less, 350 nm or less, 300 nm or less, 250 nm or less, 200 nm or less, 150 nm or less). A plurality of beads can have an average diameter or average maximum cross-sectional dimension of about 100 nanometers (nm) to about 900 nanometers (nm) (e.g., 850 nm or less, 800 nm or less, 750 nm or less, 700 nm or less, 650 nm or less, 600 nm or less, 550 nm or less, 500 nm or less, 450 nm or less, 400 nm or less, 350 nm or less, 300 nm or less, 250 nm or less, 200 nm or less, 150 nm or less). In some embodiments, the diameter or size of the beads is about the size of a single cell (e.g., a single cell being evaluated).

[0456] In some embodiments, the beads can be gel beads. A "gel" is a semi-rigid material that is permeable to liquids and gases. Exemplary gels include, but are not limited to, gels having a colloidal structure, such as agarose; polymer networks, such as gelatin; hydrogels; and cross-linked polymer structures, such as polyacrylamide, SFA (e.g., see U.S. Patent Application Publication No. 2011 / 0059865, which is incorporated herein by reference in its entirety), and PAZAM (e.g., see U.S. Patent Application Publication No. 2014 / 0079923, which is incorporated herein by reference in its entirety).

[0457] Depending on the intended use, the gel can be formulated into various shapes and sizes. In some embodiments, the gel is prepared and formulated into gel beads (e.g., gel beads that include capture probes attached to or associated with the gel beads). The gel beads can be hydrogel beads. The hydrogel beads can be formed from molecular precursors, such as polymeric or monomeric substances.

[0458] In some embodiments, the hydrogel beads can comprise a polymer matrix (e.g., a matrix formed by polymerization or crosslinking). The polymer matrix can comprise one or more polymers (e.g., polymers having different functional groups or repeating units). Crosslinking can be achieved through covalent, ionic, and / or induced interactions and / or physical entanglements.

[0459] The semi-solid beads can be liposomal beads.

[0460] The solid beads can comprise metals, including but not limited to iron oxide, gold, and silver. In some embodiments, the beads can be silica beads. In some embodiments, the beads can be rigid. In some embodiments, the beads can be flexible and / or compressible.

[0461] The beads can be macromolecules. The beads can be formed from nucleic acid molecules bound together. The beads can be formed by covalent or non-covalent assembly of molecules (e.g., macromolecules), such as monomers or polymers. The polymer or monomer can be natural or synthetic. The polymer or monomer can be or comprise, for example, nucleic acid molecules (e.g., DNA or RNA).

[0462] The beads can be rigid, or can be flexible and / or compressible. The beads can comprise a coating comprising one or more polymers. Such a coating can be disrupted or dissolved. In some embodiments, the beads comprise a spectroscopic or optical marker (e.g., a dye) attached directly or indirectly (e.g., through a linker) to the bead. For example, the beads can be prepared as a colored formulation (e.g., beads that exhibit different colors within the visible spectrum), which can change color (e.g., colorimetric beads) upon application of a desired stimulus (e.g., heat and / or a chemical reaction) to form beads of a different color (e.g., opaque and / or transparent beads).

[0463] The beads can include natural and / or synthetic materials. For example, the beads can include natural polymers, synthetic polymers, or both natural and synthetic polymers. Examples of natural polymers include, but are not limited to, proteins, sugars such as deoxyribonucleic acid, rubber, cellulose, starch (e.g., amylose, amylopectin), enzymes, polysaccharides, silk, polyhydroxyalkanoates, chitosan, dextran, collagen, carrageenan, ibagu gum, gum arabic, agar, gelatin, shellac, sterculia gum, xanthan gum, corn syrup gum, guar gum, karaya gum, agarose, alginic acid, sodium alginate, or other natural polymers. Examples of synthetic polymers include, but are not limited to, acrylics, resins, nylon, silicone, spandex, viscose rayon, polycarboxylic acids, polyvinyl acetate, polyacrylamide, polyacrylates, polyethylene glycol, polyurethane, polylactic acid, silica, polystyrene, polyacrylonitrile, polybutadiene, polycarbonate, polyethylene, polyethylene terephthalate, polychlorotrifluoroethylene, polyethylene oxide, polyethylene terephthalate, polyethylene, polyisobutylene, polymethyl methacrylate, polyoxymethylene, polypropylene, polystyrene, polytetrafluoroethylene, polyvinyl acetate, polyvinyl alcohol, polyvinyl chloride, polyvinylidene chloride, polyvinylidene fluoride, polyvinyl fluoride, and / or combinations thereof (e.g., copolymers). The beads can also be formed from materials other than polymers, including, for example, lipids, micelles, ceramics, glass ceramics, material composites, metals, and / or other inorganic materials.

[0464] In some embodiments, the beads are degradable beads. The degradable beads can include one or more substances having labile bonds (e.g., disulfide linkers, primers, other oligonucleotides, etc.) such that when the bead / substance is exposed to an appropriate stimulant, the labile bonds are broken and the beads degrade. The labile bonds can be chemical bonds (e.g., covalent bonds, ionic bonds) or can be another type of physical interaction (e.g., van der Waals interactions, dipole-dipole interactions, etc.). In some embodiments, the crosslinking agent used to generate the beads can include labile bonds. When exposed to appropriate conditions, the labile bonds break and the beads degrade. For example, when polyacrylamide gel beads containing a cysteamine crosslinking agent are exposed to a reducing agent, the disulfide bonds of cystamine can be broken and the beads degrade.

[0465] Degradation can refer to the dissociation of bound or entrapped substances (e.g., disulfide linkers, primers, other oligonucleotides, etc.) from the beads, whether or not the physical beads themselves are structurally degraded. For example, due to changes in the chemical environment, the entrapped substances can be released from the beads by osmotic pressure differences. By way of example, changes in the pore size of the beads due to osmotic pressure differences can generally occur without structural degradation of the beads themselves. In some embodiments, an increase in pore size due to osmotic swelling of the beads can cause the release of entrapped substances within the beads. In some embodiments, osmotic shrinkage of the beads due to pore size contraction can result in better retention of the entrapped substances by the beads.

[0466] Any suitable reagent for degradable beads can be used. In some embodiments, changes in temperature or pH can be used to degrade thermosensitive or pH-sensitive bonds within the beads. In some embodiments, chemical degradants can be used to degrade chemical bonds within the beads through oxidation, reduction, or other chemical changes. For example, the chemical degradant can be a reducing agent such as DTT, where DTT can degrade the disulfide bonds formed between the crosslinker and the gel precursor, thereby degrading the beads. In some embodiments, a reducing agent can be added to degrade the beads, which can cause the beads to release their contents. Examples of reducing agents can include but are not limited to dithiothreitol (DTT), β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or DTBA), tris(2-carboxyethyl)phosphine (TCEP), or combinations thereof.

[0467] Any of a number of chemical reagents can be used to initiate the degradation of the beads. Examples of chemical reagents include but are not limited to pH-mediated alterations in the integrity of components within the beads, degradation of components of the beads through cleavage of crosslink bonds, and depolymerization of components of the beads.

[0468] In some embodiments, the beads can be formed from a material comprising a degradable chemical crosslinker such as N,N'-bis-(acryloyl)cystamine (BAC) or cystamine. Degradation of such a degradable crosslinker can be achieved through various mechanisms. In some instances, the beads can be contacted with a chemical degradant that can cause oxidation, reduction, or other chemical changes. For example, the chemical degradant can be a reducing agent such as dithiothreitol (DTT). Other examples of reducing agents can include β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or DTBA), tris(2-carboxyethyl)phosphine (TCEP), or combinations thereof.

[0469] In some embodiments, exposure to an aqueous solution (such as water) can trigger hydrolytic degradation, thereby initiating the degradation of the beads. When applying a heat stimulus, the beads can also be induced to release their contents. Changes in temperature can cause various changes in the beads. For example, heat can liquefy solid beads. Changes in heat can cause the beads to melt, causing partial degradation of the beads. In some embodiments, heat can increase the internal pressure of the bead components such that the beads rupture or explode. Heat can also act on thermosensitive polymers that are the materials for constructing the beads.

[0470] In the case of using degradable beads, it is beneficial to avoid exposing the beads to one or more stimuli that cause the degradation before a given time, in order to avoid, for example, premature degradation of the beads and problems caused by such degradation, including, for example, poor flow characteristics and aggregation. For example, in the case where the beads comprise reducible crosslinking groups (such as disulfide groups), it will be desirable to avoid contacting the beads with a reducing agent (such as DTT or other disulfide cleavage reagents). In these embodiments, in some embodiments, the treatment of the beads described herein will be free of reducing agents, such as DTT. Since reducing agents are typically provided in commercial enzyme preparations, it is desirable to provide enzyme preparations that are free of reducing agents (or free of DTT) when treating the beads described herein. Examples of such enzymes include, for example, polymerase preparations, reverse transcriptase preparations, ligase preparations, and many other enzyme preparations that can be used to treat the beads described herein. The term "reducing-agent-free" or "DTT-free" preparation refers to a preparation having less than about 1 / 10, about 1 / 50, or about 1 / 100 of the lower range of such materials used to degrade the beads. For example, for DTT, a reducing-agent-free preparation may have less than about 0.01 millimolar (mM), 0.005 mM, 0.001 mM DTT, 0.0005 mM DTT, or less than about 0.0001 mM DTT. In some embodiments, the amount of DTT may be undetectable.

[0471] Compared to non-degradable beads, when an appropriate stimulus is applied to the beads, the degradable beads can be used to release attached capture probes (such as nucleic acid molecules, spatial barcode sequences, and / or primers) from the beads more rapidly. For example, for a substance bound to the inner surface of a porous bead or in the case of an encapsulated substance, when the bead degrades, the substance may have greater mobility and accessibility to other substances in the solution. In some embodiments, the substance may also be attached to the degradable bead via a degradable linker (such as a disulfide linker). The degradable linker may respond to the same stimulus as the degradable bead, or the two degradable substances may respond to different stimuli. For example, a capture probe having one or more spatial barcodes can be attached to a polyacrylamide bead comprising cystamine via a disulfide bond. When the spatially barcoded bead is exposed to a reducing agent, the bead degrades and the capture probe having one or more spatial barcode sequences is released when the disulfide bond between the capture probe and the bead and the disulfide bond of cystamine within the bead are cleaved.

[0472] Adding multiple types of labile bonds to beads can produce beads that can respond to various stimuli. Each type of labile bond can be sensitive to a related stimulus (e.g., chemical stimulus, light, temperature, pH, enzyme, etc.), such that the release of a reagent attached to the bead via each labile bond can be controlled by applying an appropriate stimulus. Some non-limiting examples of labile bonds that can be coupled to a precursor or bead include ester bonds (e.g., cleavable with acid, base, or hydroxylamine), vicinal diol bonds (e.g., cleavable with sodium periodate), Diels-Alder bonds (e.g., cleavable with heat), sulfone bonds (e.g., cleavable with base), silyl ether bonds (e.g., cleavable with acid), glycosidic bonds (e.g., cleavable with amylase), peptide bonds (e.g., cleavable with protease), or phosphodiester bonds (e.g., cleavable with nuclease (e.g., DNase)). The bond can be cleaved by other nucleic acid molecule-targeting enzymes, such as restriction enzymes (e.g., restriction endonucleases). This functionality can be used to control the release of reagents from the beads. In some embodiments, another reagent including a labile bond can be attached to the bead via, for example, an activated functional group of the bead after gel bead formation. In some embodiments, the gel beads including labile bonds are reversible. In some embodiments, gel beads with reversible labile bonds are used to capture one or more regions of interest of a biological sample. For example, but not limited to, beads including heat-labile bonds can be heated by a light source (e.g., laser), and the changes in the gel beads contribute to the capture of the biological sample in contact with the gel beads. Capture probes having one or more spatially barcoded regions that are releasable, cleavable, or reversibly attached to the beads described herein include capture probes that are released or releasable upon cleavage of the linkage between the capture probe and the bead, or capture probes that are released upon degradation of the underlying bead itself, such that the capture probes having one or more spatially barcoded regions are made accessible or become accessible to other reagents, or both.

[0473] Beads can have different physical properties. The physical properties of the beads can be used to characterize the beads. Non-limiting examples of the physical properties of the beads include size, shape, roundness, density, symmetry, and hardness. For example, beads can have different sizes. By using a microfluidic channel network configured to provide beads of a specific size (e.g., based on channel size, flow rate, etc.), beads of different sizes can be obtained. In some embodiments, the beads have different hardness values, which can be obtained by changing the polymer concentration used to generate the beads. In some embodiments, the physical properties of the capture probes can be used to make the spatially barcoded regions attached to the beads optically detectable. For example, nucleic acid origami, such as deoxyribonucleic acid (DNA) origami, can be used to generate optically detectable spatial barcodes. To this end, one or more nucleic acid molecules can be folded to form two-dimensional and / or three-dimensional geometries. Different geometries can be detected optically.

[0474] In some embodiments, a special type of nanoparticle having more than one different physical property can be used to physically distinguish the beads. For example, Janus particles having hydrophilic and hydrophobic surfaces can be used to provide unique physical properties.

[0475] In some embodiments, the beads are capable of identifying multiple analytes (e.g., nucleic acids, proteins, chromatin, metabolites, drugs, gRNAs, and lipids) from a single cell. In some embodiments, the beads are capable of identifying a single analyte (e.g., mRNA) from a single cell.

[0476] The pore size of the beads can be adjusted. For example, the pore size can be selected to retain denatured nucleic acids. The pore size can be selected to maintain diffusion permeability to exogenous chemicals such as sodium hydroxide (NaOH) and / or endogenous chemicals such as inhibitors. The beads can be formed from biocompatible and / or biochemically compatible materials and / or materials that maintain or enhance cell viability. The beads can be formed from materials that are thermally, chemically, enzymatically, and / or optically depolymerizable.

[0477] In some embodiments, the beads can be non-covalently loaded with one or more reagents. The beads can be non-covalently loaded by (e.g.) subjecting the beads to conditions sufficient to swell the beads, allowing sufficient time for the reagent to diffuse into the interior of the beads, and subjecting the beads to conditions sufficient to deswell the beads. The swelling of the beads can be achieved by, for example, placing the beads in a thermodynamically favorable solvent, subjecting the beads to a higher or lower temperature, subjecting the beads to a higher or lower ionic concentration, and / or subjecting the beads to an electric field.

[0478] The swelling of the beads can be achieved by various swelling methods. In some embodiments, the swelling is reversible (e.g., by subjecting the beads to conditions that promote deswelling). In some embodiments, the deswelling of the beads is achieved, for example, by transferring the beads to a thermodynamically unfavorable solvent, subjecting the beads to a lower or higher temperature, subjecting the beads to a lower or higher ionic concentration, and / or adding or removing an electric field. The deswelling of the beads can be achieved by various deswelling methods. In some embodiments, the deswelling is reversible (e.g., by subjecting the beads to conditions that promote swelling). In some embodiments, the deswelling of the beads can include transferring the beads such that the pores in the beads contract. The contraction can impede the diffusion of the reagent out of the interior of the beads. The resulting impediment can be due to steric interactions between the reagent and the interior of the beads. Such transfer can be accomplished by microfluidics. For example, the transfer can be achieved by moving the beads from one co-flowing solvent stream to a different co-flowing solvent stream. The swellability and / or pore size of the beads can be adjusted by changing the polymer composition of the beads.

[0479] The beads may include a temperature-responsive polymer such that when the beads are heated or cooled, the properties or dimensions of the beads may change. For example, the polymer may include poly(N-isopropylacrylamide). The gel beads may include poly(N-isopropylacrylamide), and when heated, the gel beads may decrease in one or more dimensions (e.g., cross-sectional diameter, multiple cross-sectional diameters). The temperature sufficient to change one or more properties of the gel beads may be, for example, at least about 0 degrees Celsius (°C), 1 °C, 2 °C, 3 °C, 4 °C, 5 °C, 10 °C or higher. For example, the temperature may be about 4 °C. In some embodiments, the temperature sufficient to change one or more properties of the gel beads may be (e.g.) at least about 25 °C, 30 °C, 35 °C, 37 °C, 40 °C, 45 °C, 50 °C or higher. For example, the temperature may be about 37 °C.

[0480] Functionalization of the beads for attaching capture probes can be achieved by a variety of different methods, including but not limited to activating chemical groups within the polymer, introducing reactive or activatable functional groups in the polymer structure, or attachment at the prepolymer or monomer stage of bead production. The beads may be functionalized to bind to a target analyte, such as nucleic acid, protein, carbohydrate, lipid, metabolite, peptide, or other analyte.

[0481] In some embodiments, the beads may contain molecular precursors (e.g., monomers or polymers) that can form a polymer network through polymerization of the molecular precursors. In some embodiments, the precursor may be a polymerized substance that is capable of undergoing further polymerization via, for example, chemical crosslinking. In some embodiments, the precursor may include one or more of acrylamide or methacrylamide monomers, oligomers, or polymers. In some embodiments, the beads may include a prepolymer, which is an oligomer capable of further polymerization. For example, polyurethane beads can be prepared using a prepolymer. In some embodiments, the beads may contain individual polymers that can be further polymerized together (e.g., to form a copolymer). In some embodiments, beads can be generated by polymerization of different precursors such that they include a mixture of polymers, copolymers, and / or block copolymers. In some embodiments, the beads may include covalent or ionic bonds between polymer precursors (e.g., monomers, oligomers, and linear polymers), nucleic acid molecules (e.g., oligonucleotides), primers, and other entities. In some embodiments, the covalent bond may be a carbon-carbon bond or a thioether bond.

[0482] Crosslinking of the polymer can be either permanent or reversible, depending on the specific crosslinking agent used. Reversible crosslinking can linearize or dissociate the polymer under appropriate conditions. In some embodiments, reversible crosslinking can also allow for reversible attachment of materials bound to the bead surface. In some embodiments, the crosslinking agent can form a disulfide bond. In some embodiments, the chemical crosslinking agent that forms a disulfide bond can be cystamine or modified cystamine.

[0483] For example, in cases where the polymer precursor material includes a linear polymer material (such as linear polyacrylamide, PEG, or other linear polymer materials), the activator can include a crosslinking agent or a chemical that activates the crosslinking agent within the formed droplets. Similarly, for a polymer precursor that includes polymerizable monomers, the activator can include a polymerization initiator. For example, in certain embodiments, where the polymer precursor includes a mixture of acrylamide monomers and N,N'-bis-(acryloyl)cystamine (BAC) comonomers, a reagent such as N,N,N',N'-tetramethylethylenediamine (TEMED) can be provided, which can initiate the copolymerization of acrylamide and BAC into a crosslinked polymer network, or other conditions sufficient to polymerize or gel the precursor. Conditions sufficient to polymerize or gel the precursor can include exposure to heat, cooling, electromagnetic radiation, and / or light.

[0484] After polymerization or gelation, a polymer or gel can be formed. The polymer or gel can be diffusible and permeable to chemical or biochemical reagents. The polymer or gel can be non-diffusible and non-permeable to macromolecular components. The polymer or gel can include one or more of dithiothreitol-crosslinked polyacrylamide, agarose, alginate, polyvinyl alcohol, polyethylene glycol (PEG)-diacrylate, PEG acrylate, PEG thiol, PEG azide, PEG alkyne, other acrylates, chitosan, hyaluronic acid, collagen, fibrin, gelatin, or elastin. The polymer or gel can include any other polymer or gel.

[0485] In some embodiments, a disulfide bond can be formed between a molecular precursor unit (such as a monomer, oligomer, or linear polymer) or a precursor incorporated into a bead and a nucleic acid molecule (such as an oligonucleotide, capture probe). For example, cystamine (including modified cystamine) is an organic reagent that includes a disulfide bond and can be used as a crosslinking agent between individual monomers or polymerization precursors of a bead. Polyacrylamide can be polymerized in the presence of cystamine or a substance that includes cystamine (such as modified cystamine) to produce polyacrylamide gel beads that include disulfide bonds (such as chemically reducible beads that include a chemically reducible crosslinking agent). The disulfide bond can cause the bead to degrade (or dissolve) when exposed to a reducing agent.

[0486] In some embodiments, the linear polysaccharide polymer chitosan can be crosslinked with glutaraldehyde via hydrophilic chains to form beads. The crosslinking of the chitosan polymer can be achieved through chemical reactions initiated by heat, pressure, pH changes, and / or radiation.

[0487] In some embodiments, the bead may include an acrydite moiety, which in some aspects can be used to attach one or more capture probes to the bead. In some embodiments, the acrydite moiety may refer to an acrydite analogue generated by the reaction of acrydite with one or more substances (e.g., disulfide linkers, primers, other oligonucleotides, etc.), such as, but not limited to, the reaction of acrydite with other monomers and crosslinkers during a polymerization reaction. The acrydite moiety can be modified to form a chemical bond with the substance to be attached (e.g., a capture probe). The acrydite moiety can be modified with a thiol group capable of forming a disulfide bond, or can be modified with a group that already includes a disulfide bond. The thiol group or disulfide (through disulfide exchange) can be used as an anchor point for the substance to be attached, or another moiety of the acrydite moiety can be used for attachment. In some embodiments, the attachment can be reversible such that when the disulfide bond is broken (e.g., in the presence of a reducing agent), the attached substance is released from the bead. In some embodiments, the acrydite moiety may include a reactive hydroxyl group that can be used for substance...

Claims

1. A non-diagnostic method for determining the location of an analyte present in a biological sample, comprising: (a) providing a substrate comprising a plurality of spatially barcoded oligonucleotides, wherein the spatially barcoded oligonucleotides among the plurality of spatially barcoded oligonucleotides comprise a spatial barcode, a primer domain, and a first hybridization domain; (b) conjugating a cell label to the spatially barcoded oligonucleotide, wherein the conjugation comprises hybridizing a primer oligonucleotide to the primer domain, wherein the primer oligonucleotide is conjugated to the cell label, and wherein the primer oligonucleotide conjugated to the cell label is substantially complementary to the primer domain of the spatially barcoded oligonucleotide; (c) contacting the biological sample with the cell label such that cells in the biological sample are labeled with the spatially barcoded oligonucleotide; (d) dissociating the cells from the biological sample; (e) providing the cells comprising the spatially barcoded oligonucleotide to beads, wherein the beads comprise: (1) a first bead-binding oligonucleotide, wherein the first bead-binding oligonucleotide comprises a cell barcode and a second hybridization domain, and (2) a second bead-binding oligonucleotide, wherein the second bead-binding oligonucleotide comprises the cell barcode and a capture domain, and wherein the first hybridization domain hybridizes with the second hybridization domain; (f) allowing an analyte from the cell to interact with the capture domain of the second bead-binding oligonucleotide, wherein allowing the analyte from the cell to interact with the capture domain comprises permeabilizing the cell; (g) correlating the analyte bound to the capture domain of the second bead-binding oligonucleotide with the cell barcode; and (h) correlating the cell barcode with the spatial barcode, thereby determining the location of the analyte present in the biological sample.

2. The method according to claim 1, wherein the primer oligonucleotide conjugated to the cell label is substantially complementary to the primer domain, the spatial barcode, and the first hybridization domain of the spatially barcoded oligonucleotide.

3. The method according to claim 1, comprising determining the sequence of at least a portion of the spatially barcoded oligonucleotide.

4. The method according to claim 3, wherein determining the sequence comprises in situ sequencing.

5. The method according to claim 4, wherein in situ sequencing comprises one or more of sequencing by synthesis, sequencing by ligation, rolling circle amplification sequencing, fluorescence in situ sequencing (FISSEQ), and spatially resolved transcript amplicon read mapping (STARmap).

6. The method according to any one of claims 1-5, wherein providing the cells comprising the spatially barcoded oligonucleotide to the beads comprises hybridization.

7. The method according to any one of claims 1-5, wherein the spatially barcoded oligonucleotide comprises one or more of a unique molecular identifier, an attachment sequence, a cleavage domain, and a functional domain.

8. The method according to claim 7, wherein the attachment sequence comprises one or more of a flow cell attachment sequence and a substrate attachment sequence.

9. The method according to any one of claims 1-5, wherein correlating the analyte bound to the capture domain with the cell domain comprises identifying the analyte.

10. The method according to any one of claims 1-5, wherein correlating the cell barcode with the spatial barcode comprises extending the hybridized first bead-binding oligonucleotide and the spatially barcoded oligonucleotide.

11. The method according to claim 10, the method comprising determining the sequences of the extended first bead-binding oligonucleotide and the spatially barcoded oligonucleotide.

12. A non-diagnostic method for determining the location of an analyte present in a biological sample, comprising: (a) providing a substrate comprising a plurality of spatially barcoded oligonucleotides, wherein the spatially barcoded oligonucleotides in the plurality of spatially barcoded oligonucleotides comprise a spatial barcode, a first hybridization domain, and a cleavage domain; (b) conjugating a cell labeler to the spatially barcoded oligonucleotide, wherein the cell labeler comprises a first association domain that hybridizes to the spatially barcoded oligonucleotide, wherein the conjugation comprises hybridizing the first association domain to the spatially barcoded oligonucleotide; (c) contacting the biological sample with the cell labeler such that cells in the biological sample are labeled with the spatially barcoded oligonucleotide; (d) cleaving the spatially barcoded oligonucleotide from the substrate; (e) dissociating the cells from the biological sample; (f) providing the cells comprising the spatially barcoded oligonucleotide to beads, wherein the beads comprise: (1) a first bead-binding oligonucleotide, wherein the first bead-binding oligonucleotide comprises a cell barcode and a second hybridization domain, and (2) a second bead-binding oligonucleotide, wherein the second bead-binding oligonucleotide comprises the cell barcode and a capture domain, wherein the first hybridization domain hybridizes to the second hybridization domain; (g) allowing an analyte from the cells to interact with the capture domain of the second bead-binding oligonucleotide, wherein allowing the analyte from the cells to interact with the capture domain comprises permeabilizing the cells; (h) associating the analyte bound to the capture domain of the second bead-binding oligonucleotide with the cell barcode; (i) associating the cell barcode with the spatially barcoded oligonucleotide; and (j) determining the location of the analyte present in the biological sample.

13. The method according to claim 12, wherein the spatially barcoded oligonucleotide comprises a second binding domain, and the first association domain of the cell labeler hybridizes to the second association domain.

14. The method according to any one of claims 12-13, wherein the cell labeler is conjugated to the spatially barcoded oligonucleotide via a linker.

15. The method according to any one of claims 12-13, wherein the spatially barcoded oligonucleotide comprises one or more of a unique molecular identifier, an attachment sequence, a restriction endonuclease sequence, and a functional domain.

16. The method according to claim 15, wherein the attachment sequence comprises one or more of a flow cell attachment sequence and a substrate attachment sequence.

17. The method according to claim 1 or 12, wherein the biological sample is a tissue sample.

18. The method according to claim 17, wherein the tissue sample is a fresh frozen tissue sample.

19. The method according to claim 17, wherein the tissue sample is a formalin-fixed paraffin-embedded (FFPE) tissue sample.

20. The method according to claim 17, wherein the tissue sample comprises tumor cells.

21. The method according to claim 17, wherein the tissue sample comprises tissue sections.

22. The method according to claim 1 or 12, further comprising imaging the biological sample.

23. The method according to claim 1 or 12, wherein the analyte comprises at least one of RNA, DNA, protein, lipid, peptide, metabolite, small molecule, and cell labeling agent.

24. The method according to claim 1 or 12, wherein the analyte comprises RNA.

25. The method according to claim 1 or 12, wherein the cell labeling agent comprises an extracellular cell labeling agent.

26. The method according to claim 1 or 12, wherein the cell labeling agent comprises an intracellular cell labeling agent.

27. The method according to claim 1 or 12, wherein the cell labeling agent is localized to an internal component of the cell.

28. The method according to claim 27, wherein the internal component of the cell comprises one or more of mitochondria, Golgi apparatus, smooth endoplasmic reticulum, rough endoplasmic reticulum, nucleus, nucleolus, and lysosome.

29. The method according to claim 1 or 12, wherein the cell labeling agent comprises one or more of lipid, antibody, chitosan, lectin, streptavidin, click chemistry modified moiety, cell penetrating peptide, nanoparticle, TIVA tag, and liposome / polysome.

30. The method according to claim 1 or 12, wherein the cell labeling agent is amphiphilic.

31. The method according to claim 1 or 12, wherein the cell labeling agent is lipophilic.

32. The method according to claim 1 or 12, wherein the cell labeling agent is a cholesterol moiety.

33. The method according to claim 1 or 12, wherein the cell labeling agent is coupled to the initiating oligonucleotide via a linker.

34. The method according to any one of claims 14 or 33, wherein the linker comprises one or more of the following: N-hydroxysuccinimide (NHS) linker, bifunctional NHS linker, azide, alkyne, glycol chitosan, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG), and succinimidyl 3-(2-pyridyldithio)propionate (SPDP).

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