Increasing spatial array resolution

Abstract

The invention relates to increasing spatial array resolution. The present invention relates to methods of making spatial arrays and determining the location of an analyte present in a biological sample (e.g., by using a cell labeling agent).

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201980071233.1.

[0002] Cross-references to related applications

[0003] This application claims U.S. Provisional Patent Application No. 62 / 724,561, filed August 29, 2018; U.S. Provisional Patent Application No. 62 / 723,950, filed August 28, 2018; U.S. Provisional Patent Application No. 62 / 723,957, filed August 28, 2018; U.S. Provisional Patent Application No. 62 / 723,960, filed August 28, 2018; U.S. Provisional Patent Application No. 62 / 723,964, filed August 28, 2018; U.S. Provisional Patent Application No. 62 / 723,970, filed August 28, 2018; and U.S. Provisional Patent Application No. 62 / 723,964, filed August 28, 2018. U.S. Provisional Patent Application No. 972, filed August 29, 2018; U.S. Provisional Patent Application No. 62 / 724,483, filed August 29, 2018; U.S. Provisional Patent Application No. 62 / 724,487, filed August 29, 2018; U.S. Provisional Patent Application No. 62 / 724,489, filed August 29, 2018; U.S. Provisional Patent Application No. 62 / 788,905, filed January 6, 2019; U.S. Provisional Patent Application No. 62 / 788,867, filed January 6, 2019; U.S. Provisional Patent Application No. 62 / 788,871, filed January 6, 2019; and U.S. Provisional Patent Application No. 62 / 788,89, filed January 6, 2019. 7. U.S. Provisional Patent Application No. 62 / 788,885, filed January 6, 2019; U.S. Provisional Patent Application No. 62 / 779,342, filed December 13, 2018; U.S. Provisional Patent Application No. 62 / 822,565, filed March 22, 2019; U.S. Provisional Patent Application No. 62 / 819,496, filed March 15, 2019; U.S. Provisional Patent Application No. 62 / 819,486, filed March 15, 2019; U.S. Provisional Patent Application No. 62 / 819,467, filed March 22, 2019; and U.S. Provisional Patent Application No. 62 / 822,6... U.S. Provisional Patent Application No. 62 / 822,618, filed March 22, 2019; U.S. Provisional Patent Application No. 62 / 822,592, filed March 22, 2019; U.S. Provisional Patent Application No. 62 / 819,468, filed March 15, 2019; U.S. Provisional Patent Application No. 62 / 822,627, filed March 22, 2019; U.S. Provisional Patent Application No. 62 / 819,448, filed March 15, 2019; U.S. Provisional Patent Application No. 62 / 822,649, filed March 22, 2019; and U.S. Provisional Patent Application No. 62 / 819, filed March 15, 2019.U.S. Provisional Patent Application No. 456, filed March 15, 2019; U.S. Provisional Patent Application No. 62 / 819,478, filed March 15, 2019; U.S. Provisional Patent Application No. 62 / 819,449, filed March 15, 2019; U.S. Provisional Patent Application No. 62 / 822,554, filed March 22, 2019; U.S. Provisional Patent Application No. 62 / 822,575, filed March 22, 2019; U.S. Provisional Patent Application No. 62 / 822,605, filed March 22, 2019; U.S. Provisional Patent Application No. 62 / 812,219, filed February 28, 2019; and U.S. Provisional Patent Application No. 62 / 819,458, filed March 15, 2019. Priority claims are made to U.S. Provisional Patent Application No. 62 / 839,223, filed April 26, 2019; U.S. Provisional Patent Application No. 62 / 839,320, filed April 26, 2019; U.S. Provisional Patent Application No. 62 / 839,346, filed April 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 June 13, 2019; U.S. Provisional Patent Application No. 62 / 839,526, filed April 26, 2019; and U.S. Provisional Patent Application No. 62 / 858,331, filed June 7, 2019. The contents of each of these applications are incorporated herein by reference in their entirety. Background Technology

[0004] Cells within a tissue exhibit differences in morphology and / or function due to varying levels of analytes (e.g., gene and / or protein expression) within different cells. A cell's specific location within the tissue (e.g., its position relative to neighboring cells or its position relative to the tissue microenvironment) can influence, for example, cell morphology, differentiation, fate, viability, proliferation, behavior, and signal transduction and crosstalk with other cells in the tissue.

[0005] Spatial heterogeneity has been previously studied, but the techniques used in those studies only provide data on a small amount of analyte in intact tissue or partial tissue contact, or a large amount of analyte data for single cells, but cannot provide information on the location of single cells in the parent biological sample (e.g., tissue sample).

[0006] The spatial architecture of gene expression can be observed in single cells, tissues, or organisms. Genetic material, along with the expression of associated genes and proteins, influences cell fate and behavior. Spatial heterogeneity in developmental systems is typically investigated through RNA hybridization, immunohistochemistry, fluorescent reporters, purification or induction of predefined subsets, and subsequent genome profiling (e.g., RNA-seq). However, such methods rely on a small subset of predefined markers, thus introducing selection bias that limits discovery and making localization across the entire RNA transcriptome costly and laborious. Summary of the Invention

[0007] This article provides methods for fabricating spatial arrays and determining the location of analytes present in biological samples (e.g., by using cell-tagging agents).

[0008] In one aspect, a method for determining the location of an analyte present in a biological sample includes (a) providing a substrate comprising an array of a plurality of spatially barcoded oligonucleotides, wherein the spatially barcoded oligonucleotides in the plurality of spatially barcoded oligonucleotides contain spatial barcodes, priming domains, etc. (a) conjugating a cell marker to a spatially barcoded oligonucleotide; (b) contacting a biological sample with a cell marker such that cells in the biological sample are labeled with the spatially barcoded oligonucleotide; (c) providing cells containing the spatially barcoded oligonucleotide to a bead, wherein the bead contains (1) a first bead-binding oligonucleotide, wherein the first bead-binding oligonucleotide contains a cell barcode and a second hybridization domain, and (2) a second bead-binding oligonucleotide, wherein the second bead-binding oligonucleotide contains the cell barcode and a capture domain; (e) allowing an analyte from the cell 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 to determine the location of the analyte present in the biological sample.

[0009] In some implementations, the method for determining the location of an analyte present in a biological sample includes dissociating cells from the biological sample.

[0010] In some embodiments, providing a cell marker includes hybridizing a priming oligonucleotide to a priming domain, wherein the priming oligonucleotide is coupled to the cell marker. In some embodiments, the priming oligonucleotide coupled to the cell marker is substantially complementary to the priming domain of the spatially barcoded oligonucleotide. In some embodiments, the priming oligonucleotide coupled to the cell marker is substantially complementary to the priming domain, spatial barcode, and first hybridization domain of the spatially barcoded oligonucleotide.

[0011] In some embodiments, methods for determining the location of an analyte present in a biological sample include determining at least a portion of the sequence 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).

[0012] In some embodiments, providing the beads with cells containing spatially barcoded oligonucleotides includes hybridization. In some embodiments, hybridization includes a first hybridization domain hybridizing with a second hybridization domain.

[0013] In some embodiments, the spatially barcoded oligonucleotide includes 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.

[0014] In some embodiments, allowing an analyte from the cell to interact with the capture domain includes releasing the analyte from the cell. In some embodiments, release includes permeation of the cell. In some embodiments, associating an analyte bound to the capture domain with a cellular structural domain includes recognizing the analyte. In some embodiments, associating cellular barcodes with spatial barcodes includes extending the first bead-binding oligonucleotide and the spatially barcoded oligonucleotide through hybridization. In some embodiments, associating cellular barcodes with spatial barcodes includes determining the sequences of the extended bead-binding oligonucleotide and the spatially barcoded oligonucleotide.

[0015] In another aspect, a method for determining the location of an analyte present in a biological sample includes (a) providing a substrate comprising an array of a plurality of spatially barcoded oligonucleotides, wherein the spatially barcoded oligonucleotides comprise spatial barcodes, a first hybridization domain, and a cleavage domain; (b) conjugating a cell marker to the spatially barcoded oligonucleotides; 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 conjugation includes hybridizing the first association domain with the spatially barcoded oligonucleotide. In some embodiments, the spatially barcoded oligonucleotide includes a second association domain, wherein the first association domain of the cell marker hybridizes with the second association domain.

[0016] In some embodiments, the cell marker is coupled to a spatially barcoded oligonucleotide via a linker. In some embodiments, the spatially barcoded oligonucleotide includes 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 includes one or more of a flow cell attachment sequence and a substrate attachment sequence.

[0017] In some embodiments, a method for determining the location of an analyte present in a biological sample includes (a) contacting the biological sample with a cell marker 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 cells containing the spatially barcoded oligonucleotide to a bead, wherein the bead contains (1) a first bead-binding oligonucleotide, wherein the first bead-binding oligonucleotide contains a cell barcode and a second hybridization domain, and (2) a second bead-binding oligonucleotide, wherein the second bead-binding oligonucleotide contains the cell barcode and a capture domain; (d) allowing an analyte from the cell 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.

[0018] 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 contains tumor cells. In some embodiments, the tissue sample comprises tissue sections. In some embodiments, the method for determining the location of an analyte present in the biological sample includes 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.

[0019] In another aspect, a method for generating an array includes (a) providing a plurality of spatially barcoded oligonucleotides to a substrate, wherein two or more of the plurality of spatially barcoded oligonucleotides include a first attachment sequence and a second attachment sequence, and wherein the substrate includes 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 to generate a spatially barcoded oligonucleotide array; and (b) amplifying the spatially barcoded oligonucleotides on the substrate, thereby generating the array.

[0020] In some embodiments, amplifying two or more spatially barcoded oligonucleotides on a substrate includes bridging amplification. In some embodiments, the method of generating an array includes determining the identity of two or more spatially barcoded oligonucleotides in the array. In some embodiments, determining the identity of two or more spatially barcoded oligonucleotides includes sequencing the spatially barcoded oligonucleotides. In some embodiments, sequencing includes in situ sequencing. In some embodiments, in situ sequencing includes hybridizing a trigger oligonucleotide with two or more spatially barcoded oligonucleotides. In some embodiments, the trigger oligonucleotide is conjugated to a cell marker. In some embodiments, in situ sequencing includes one or more of sequencing by synthesis, sequencing by ligation, and rolling circle amplification sequencing. In some embodiments, in situ sequencing includes sequencing by synthesis. In some embodiments, sequencing by synthesis includes hybridizing a trigger oligonucleotide with two or more spatially barcoded oligonucleotides. In some embodiments, the trigger oligonucleotide is conjugated to a cell marker.

[0021] In some embodiments, the two or more spatially barcoded oligonucleotides include one or more of a spatial barcode, an initiation 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.

[0022] In some embodiments, the cell marker comprises an extracellular cell marker. In some embodiments, the cell marker comprises an intracellular cell marker. In some embodiments, the cell marker is localized to an internal component of the cell. In some embodiments, the internal component of the cell includes one or more of mitochondria, Golgi apparatus, smooth endoplasmic reticulum, rough endoplasmic reticulum, nucleus, nucleolus, and lysosomes. In some embodiments, the cell marker comprises one or more of lipids, antibodies, chitosan, lectins, streptavidin, click chemically modified moieties, cell-penetrating peptides, nanoparticles, TIVA tags, and liposomes / polyribosomes. In some embodiments, the cell marker is amphiphilic. In some embodiments, the cell marker is lipophilic. In some embodiments, the cell marker is a cholesterol moiety. In some embodiments, the cell marker is coupled to an initiating oligonucleotide via a linker. In some embodiments, the connector comprises one or more of the following: N-hydroxysuccinimide (NHS) connector, bifunctional NHS connector, azide, alkyne, ethylene glycol chitosan, 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-poly(ethylene glycol) (DSPE-PEG), and succinimide-3-(2-pyridyl dithio)propionate (SPDP).

[0023] All publications, patents, and patent applications available on the Internet mentioned in this specification are incorporated herein by reference as if each individual publication, patent, patent application, and information item were incorporated herein by reference separately and individually. In the event of any conflict between the publications, patents, patent applications, and information items incorporated by reference and the disclosure contained in this specification, this specification shall prevail and / or take precedence over any conflicting material.

[0024] When a value is described as a range, it should be understood that the description includes the disclosure of all possible subranges within that range, as well as the disclosure of a specific numerical value within that range, regardless of whether the specific numerical value or specific subrange is explicitly stated.

[0025] When the term “each” refers to a group of items, it is intended to identify a single item in that group, but not necessarily every item in that group, unless otherwise explicitly stated, or unless the context of the usage clearly indicates otherwise.

[0026] This document describes various embodiments of the features of the present invention. However, it should be understood that these embodiments are provided by way of example only, and many variations, modifications, and substitutions can be made by those skilled in the art without departing from the scope of this disclosure. It should also be understood that various alternatives to the specific embodiments described herein are also within the scope of this disclosure. Attached Figure Description

[0027] The following figures illustrate certain embodiments of the features and advantages of the invention. These embodiments are not intended to limit the scope of the appended claims in any way. Like reference numerals in the figures denote like elements.

[0028] Figure 1 An exemplary spatial analysis workflow is shown.

[0029] Figure 2 An exemplary spatial analysis workflow is shown.

[0030] Figure 3 An exemplary spatial analysis workflow is shown.

[0031] Figure 4 An exemplary spatial analysis workflow is shown.

[0032] Figure 5 An exemplary spatial analysis workflow is shown.

[0033] Figure 6 This is a schematic diagram illustrating an example of a barcode-based capture probe as described in this article.

[0034] Figure 7This is a schematic diagram illustrating a lysable capture probe, in which the lysed capture probe can enter non-permeable cells and bind to the target analyte within the sample.

[0035] Figure 8 This is a schematic diagram illustrating an exemplary multi-spatial label feature.

[0036] Figure 9 This is a schematic diagram of an exemplary analyte trapping agent.

[0037] Figure 10 This is a schematic diagram depicting an exemplary interaction between the feature-immobilized capture probe 1024 and the analyte capture agent 1026.

[0038] Figure 11A , 11B Figures 1 and 11C are schematic diagrams illustrating how streptavidin cell tags can be used to generate spatially barcoded cells or cell contents in an array-based system.

[0039] Figure 12 This is a schematic diagram showing the arrangement of barcode features within the array.

[0040] Figure 13 This is a schematic diagram showing a side view of an anti-diffusion medium (e.g., a cap).

[0041] Figure 14A and 14B This is a diagram showing the unfolding of the electrophoretic transfer system. Figure 14A and side view Figure 14B The diagram illustrates that the electrophoretic transfer system is configured to guide transcript analytes to a spatially barcode-capture probe array.

[0042] Figure 15 This is a schematic diagram illustrating an exemplary workflow scheme utilizing an electrophoretic transfer system.

[0043] Figure 16 An example of a microfluidic channel structure 1600 for dividing a dissociated sample (e.g., biological particles or individual cells from the sample) is shown.

[0044] Figure 17A An example of a microfluidic channel structure 1700 is shown, which is used to deliver a bead carrying a spatial barcode to a droplet.

[0045] Figure 17B A cross-sectional view of another example of a microfluidic channel structure 1750 with geometric features for controlled partitioning is shown.

[0046] Figure 17C A workflow diagram is shown.

[0047] Figure 18 It is a schematic diagram of cell labeling using the covalent coupling of the analyte binding portion to the cell surface or the non-covalent interaction with cell membrane elements.

[0048] Figure 19 This is a schematic diagram depicting cell labeling using cell-penetrating peptides or delivery systems.

[0049] Figure 20A This is a schematic diagram illustrating an exemplary, non-limiting, and non-exhaustive process for “pixelating” a sample, in which a small portion of the sample is moved into an individual partition or well by cutting, imprinting, microdissecting, or transferring the sample using a hollow needle or microneedle.

[0050] Figure 20B This is a schematic diagram depicting a multi-needle pixelation, where a set of needles passes through a sample on a scaffold and into nanopores containing gel beads and reagents below. Once the needles are inside the nanopores, the cells pop out.

[0051] Figure 21 A schematic diagram of an exemplary, non-limiting, non-exhaustive workflow for dissociating spatially barcoded samples for analysis by droplet or flow cell analysis methods is shown.

[0052] Figure 22 This is a schematic diagram (depicted as a gray rectangle) illustrating an exemplary spatially barcoded oligonucleotide on a substrate. Exemplary components of the spatially barcoded oligonucleotide include: an attachment sequence (depicted as P5 / P7), a priming domain (depicted as R1 / R2), a hybridization domain (depicted as Cap), and a spatial barcode (depicted as SpBC).

[0053] Figure 23 This is a schematic diagram illustrating an exemplary spatially barcoded oligonucleotide attached to a cell marker. Figure 23 In the illustrated embodiment, the cell marker is a lipid. For example... Figure 23 As shown, the spatially barcoded oligonucleotides attached to the cell marker are complements of the spatially barcoded oligonucleotides attached to the substrate. Exemplary components of the spatially barcoded oligonucleotides are as follows: attachment sequence (described as P5 / P7), initiation domain (described as R1 / R2), hybridization domain (described as Cap), and spatial barcode (described as SpBC).

[0054] Figure 24 This is a schematic diagram illustrating the contact between an exemplary biological sample (depicted as a light gray rectangle above a substrate) and spatially barcoded oligonucleotides attached to a cell marker. Figure 24 In the illustrated embodiment, the cell marker is a lipid. For example... Figure 24As shown, the spatially barcoded oligonucleotides attached to the cell marker are complements of the spatially barcoded oligonucleotides attached to the substrate. Exemplary components of the spatially barcoded oligonucleotides are as follows: attachment sequence (described as P5 / P7), initiation domain (described as R1 / R2), hybridization domain (described as Cap), and spatial barcode (described as SpBC).

[0055] Figure 25 This is a schematic diagram illustrating an exemplary gel emulsion (GEM) droplet, in which cells labeled with spatially barcoded oligonucleotides hybridize with beads via complementary hybridization domains. Figure 25 In the embodiment shown, the cell marker is a lipid, and the beads are gel beads.

[0056] Figure 26 This is a schematic diagram (depicted as a gray rectangle) illustrating an exemplary spatially barcoded oligonucleotide on a substrate. Exemplary oligonucleotides partially complementary to the spatially barcoded oligonucleotide are depicted as means for determining at least a portion of the sequence of the spatially barcoded oligonucleotide. Exemplary components of the spatially barcoded oligonucleotide include: an attachment sequence (depicted as P5 / P7), a priming domain (depicted as R1 / R2), an association domain (depicted as hyb), a hybridization domain (depicted as Cap), and a spatial barcode (depicted as SpBC).

[0057] Figure 27 This is a schematic diagram illustrating an exemplary spatially barcoded oligonucleotide attached to a cell marker. Figure 27 In the illustrated embodiment, the cell marker is a lipid. Exemplary components of the spatially barcoded oligonucleotide are as follows: an attachment sequence (described as P7), a priming domain (described as R2), an association domain (described as hyb), a hybridization domain (described as Cap), and a spatial barcode (described as SpBC).

[0058] Figure 28A This is a schematic diagram illustrating an exemplary sample processing apparatus that can be used to implement the various steps and methods described herein.

[0059] Figure 28B This is a schematic diagram illustrating an example imaging device that can be used to obtain images of biological samples, analytes, and feature arrays.

[0060] Figure 28C yes Figure 28A and Figure 28B A schematic diagram of an example of the control unit of the device. Detailed Implementation

[0061] I. Introduction

[0062] This invention describes apparatus, systems, methods, and compositions for spatial analysis of biological samples. Certain general terms, analytes, sample types, and preparation steps are specifically described in this section, and will be referred to later in the invention.

[0063] (a) Spatial Analysis

[0064] Tissues and cells can be obtained from any source. For example, tissues and cells can be obtained from single-celled or multicellular organisms (e.g., mammals). Tissues and cells obtained from mammals (e.g., humans) often have different levels of analytes (e.g., gene and / or protein expression), which can lead to differences in cell morphology and / or function. The location of cells within a tissue can influence cell fate, behavior, morphology, and signaling and crosstalk with other cells in the tissue. Information about differences in intracellular analyte levels (gene and / or protein expression) in mammalian tissues can also help physicians select or administer treatments effective against single-celled or multicellular organisms (e.g., mammals) based on detected differences in intracellular analyte levels in tissues. Differences in intracellular analyte levels in mammalian tissues can also provide information on how tissues (e.g., healthy and diseased tissues) function and / or develop. Differences in intracellular analyte levels in mammalian tissues can also provide information on different disease pathogenesis mechanisms within tissues and on the mechanisms of action of treatments within tissues. Differences in intracellular analyte levels in mammalian tissues can also provide information on drug resistance mechanisms and the development of drug resistance mechanisms in mammalian tissues. Differences in the presence or absence of analytes in different cells within tissues of multicellular organisms (e.g., mammals) can provide information about the mechanisms and development of drug resistance in multicellular organism tissues.

[0065] 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 single cells of mammals. For example, spatial analysis methods can be used to detect differences in analyte levels (e.g., gene and / or protein expression) within different cells of a tissue section sample, from which data can be reconstructed to generate a three-dimensional map of analyte levels (e.g., gene and / or protein expression) in a tissue sample obtained from a mammal, for example at a degree of spatial resolution (e.g., single-cell resolution).

[0066] Spatial heterogeneity in developmental systems is typically investigated through RNA hybridization, immunohistochemistry, fluorescent reporters, purification or induction of predefined subsets, and subsequent genome profiling (e.g., RNA-seq). However, these methods rely on a relatively small set of predefined markers, thus introducing selection bias that limits discovery. These previous methods also depend on prior knowledge. Traditionally, spatial RNA analysis has relied on staining a limited amount of RNA material. In contrast, single-cell RNA sequencing allows for in-depth profiling of cellular gene expression, including non-coding RNA, but established methods isolate cells from their native spatial context.

[0067] Current spatial analysis methods provide a wealth of analyte level and / or expression data for multiple analytes in a sample with high spatial resolution, while preserving the natural spatial environment. Spatial analysis methods include, for example, the use of capture probes comprising a spatial barcode (e.g., a nucleic acid sequence providing information about the location of the capture probe within a cellular or tissue sample (e.g., mammalian cell or mammalian tissue sample)) and a capture domain capable of binding to analytes (e.g., proteins and / or nucleic acids) produced by or present in cells. As described herein, the spatial barcode can be a nucleic acid having a unique sequence, a unique fluorophore or a unique combination of fluorophores, a unique amino acid sequence, a unique heavy metal or a unique combination of heavy metals, or any other unique detectable reagent. The capture domain can be any reagent capable of binding to analytes produced by and / or present in cells (e.g., a nucleic acid capable of hybridizing with nucleic acids from cells (e.g., mRNA, genomic DNA, mitochondrial DNA, or miRNA), the substrate or binding chaperone of the analyte, or an antibody capable of specifically binding to the analyte). The capture probe may also include a nucleic acid sequence complementary to a universal forward and / or universal reverse primer sequence. The capture probe may also include cleavage sites (e.g., cleavage recognition sites of restriction endonucleases), light-labile bonds, thermosensitive bonds, or chemically sensitive bonds.

[0068] A variety of different methods can be used to detect the binding of analytes to capture probes, such as nucleic acid sequencing, fluorophore detection, nucleic acid amplification, nucleic acid ligation detection, and / or nucleic acid lysis product detection. In some examples, detection is used to associate a specific spatial barcode with a specific analyte produced by and / or present in cells (e.g., mammalian cells).

[0069] The capture probe can be attached to a surface, such as a solid array, beads, or coverslip. In some examples, the capture probe is not attached to a surface. In some instances, the capture probe may be encapsulated within, embedded in, or layered on the surface of a permeable composition (e.g., any of the substrates described herein). For example, the capture probe may be encapsulated or disposed within a permeable bead (e.g., a gel bead). In some instances, the capture probe may be encapsulated within, embedded in, or layered on the surface of a substrate (e.g., any of the exemplary substrates described herein, such as a hydrogel or porous membrane).

[0070] In some embodiments, cells or tissue samples comprising cells are contacted with a capture probe attached to a substrate (e.g., the surface of the substrate), and the cells or tissue sample are permeated to release analytes from the cells and bind to the capture probe attached to the substrate. In some examples, a variety of methods (e.g., electrophoresis, chemical gradients, pressure gradients, fluid flow, or magnetic fields) can be used to actively guide the analytes released from the cells to the capture probe attached to the substrate.

[0071] In other examples, a variety of methods can be used to guide the capture probe to interact with the cell or tissue sample, including lipid anchors in the capture probe, reagents that can specifically bind to or form covalent bonds with membrane proteins in the capture probe, fluid flow, pressure gradient, chemical gradient, or magnetic field.

[0072] The non-restrictive aspects of spatial analysis methods are discussed in WO 2011 / 127099, WO 2014 / 210233, WO 2014 / 210225, WO 2016 / 162309, WO 2018 / 091676, WO 2012 / 140224, WO 2014 / 060483, US Patent No. 10,002,316, US Patent No. 9,727,810, US 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, US 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 2011 / 094669, US Patent No. 7,709,198, US Patent No. 8,604,182, US Patent No. 8,951,726, US Patent No. 9,783,841, US Patent No. 10,041,949, WO 2016 / 057552, WO2017 / 147483, WO 2018 / 022809, WO 2016 / 166128, WO 2017 / 027367, WO 2017 / 027368, WO2018 / 136856, WO 2019 / 075091, US Patent No. 10,059,990, WO 2018 / 057999, WO 2015 / 161173, and Gupta et al., Nature The methods described in Biotechnol. 36:1197-1202, 2018 can be used herein in any combination. Further non-limiting aspects of the spatial analysis methods are described herein.

[0073] (b) General Terminology

[0074] Specific terminology is used in this invention to explain various aspects of the described apparatuses, systems, methods, and compositions. This section includes explanations of certain terms that appear in later sections of this invention. If the descriptions in this section conflict with their usage in other sections of this invention, the definitions in this section shall prevail.

[0075] (i) Barcode

[0076] A barcode is a label or identifier that conveys or is capable of conveying information (e.g., information about analytes, beads, and / or capture probes in a sample). A barcode can be part of the analyte or independent of it. A barcode can be attached to the analyte. A particular barcode may be unique relative to other barcodes.

[0077] Barcodes can come in a variety of different formats. For example, barcodes can include polynucleotide barcodes, random nucleic acid and / or amino acid sequences, and synthetic nucleic acid and / or amino acid sequences. Barcodes can be attached to an analyte or another part or structure in a reversible or irreversible manner. For example, barcodes can be added to fragments of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) samples before or during sequencing. Barcodes can allow for the identification and / or quantification of individual sequencing reads (e.g., barcodes can be or can include a unique molecular identifier or "UMI").

[0078] Barcodes can spatially resolve molecular components found in biological samples, for example, at single-cell resolution (e.g., a barcode may be or may include a "spatial barcode"). In some embodiments, a barcode simultaneously 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 may include two or more polynucleotide sequences separated by one or more non-barcode sequences (e.g., sub-barcodes).

[0079] (ii) Nucleic acids and nucleotides

[0080] The terms “nucleic acid” and “nucleotide” are intended to be consistent with their use in the art and include naturally occurring substances or their functional analogs. Particularly useful nucleic acid functional analogs are capable of hybridizing with nucleic acids in a sequence-specific manner (e.g., capable of hybridizing with two nucleic acids such that a link can occur between the two hybridized nucleic acids) or can be used as templates for replicating specific nucleotide sequences. Naturally occurring nucleic acids typically have a backbone containing phosphodiester bonds. Analog structures may 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)).

[0081] Nucleic acids may comprise any of a nucleotide having any of the various analogs of these sugar moieties known in the art. Nucleic acids may include natural or non-natural nucleotides. In this regard, natural deoxyribonucleic acid may have one or more bases selected from adenine (A), thymine (T), cytosine (C), or guanine (G), and ribonucleic acid may have one or more bases selected from uracil (U), adenine (A), cytosine (C), or guanine (G). Useful non-natural bases that may be included in nucleic acids or nucleotides are known in the art.

[0082] (iii) Probes and targets

[0083] "Probe" or "target", when used in relation to nucleic acids or nucleic acid sequences, means as 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 explicitly indicated.

[0084] (iv) Oligonucleotides and Polynucleotides

[0085] The terms "oligonucleotide" and "polynucleotide" are used interchangeably to refer to single-stranded polymers of nucleotides with a length of about 2 to about 500 nucleotides. Oligonucleotides can be synthesized, prepared enzymatically (e.g., by polymerization), or prepared using a "split-assemble" method. Oligonucleotides may include ribonucleotide monomers (i.e., may be oligoribonucleotides) and / or deoxyribonucleotide monomers (i.e., oligodeoxyribonucleotides). In some examples, oligonucleotides may include combinations of deoxyribonucleotide monomers and ribonucleotide monomers (e.g., random or ordered combinations 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. An oligonucleotide may include (e.g., covalently or non-covalently) one or more functional portions attached to a multimeric structure. For example, an oligonucleotide may include one or more detectable labels (e.g., radioisotopes or fluorophores).

[0086] (v) object

[0087] "Object" is an animal, such as a mammal (like a human or ape), or a bird (like a bird), or other organism, such as a plant. Instances of objects include, but are not limited to, mammals such as rodents, mice, rats, rabbits, guinea pigs, ungulates, horses, sheep, pigs, goats, cattle, cats, dogs, and primates (i.e., humans or non-human primates); plants such as Arabidopsis thaliana, maize, sorghum, oats, wheat, rice, rapeseed, or soybeans; 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; and fungi such as Pneumocystis carinii. The species *Carinii*, *Takifugu rubripes*, yeast, *Saccharomyces cerevisiae* or *Schizosaccharomyces pombe*; or *Plasmodium falciparum*.

[0088] (vi) Genome

[0089] "Genome" generally refers to genomic information from an object, such as at least part or all of the genetic information encoded by the object's genes. A genome can include coding regions (e.g., regions that encode proteins) and non-coding regions. A genome can include sequences of some or all of the object's chromosomes. For example, the human genome typically has 46 chromosomes in total. Some or all of these sequences can constitute the genome.

[0090] (vii) Adapter, Connector, and Label

[0091] "Adaptor," "conjugate," and "tag" are terms that may be used interchangeably in this disclosure and refer to a substance that can be coupled to a polynucleotide sequence using any of a number of different techniques, including (but not limited to) ligation, hybridization, and tagging (in a process referred to as "tagging"). An adaptor may also be a functionally enhanced nucleic acid sequence, such as a spacer sequence, primer sequence / site, barcode sequence, or unique molecular identifier sequence.

[0092] (viii) Heterozygium, hybridization, annealing, and chain recombination.

[0093] The terms “hybridization,” “hybridization,” “annealing,” and “chain recombination” are used interchangeably in this disclosure and refer to the pairing of essentially complementary or complementary nucleic acid sequences within two different molecules. Pairing can be achieved through any process in which nucleic acid sequences are linked to essentially complementary or fully complementary sequences by base pairing to form a hybridization complex. For the purpose of hybridization, two nucleic acid sequences are “essentially complementary” if at least 60% (e.g., at least 70%, at least 80%, or at least 90%) of the individual bases in each other are complementary.

[0094] (ix) primers

[0095] A primer is a single-stranded nucleic acid sequence with a 3' end that can be used as a substrate for nucleic acid polymerases in nucleic acid extension reactions. 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 may also include RNA nucleotides and DNA nucleotides (e.g., in a random or designed pattern). Primers may also include other natural or synthetic nucleotides with other functionalities as described herein. In some examples, DNA primers can be used to initiate RNA synthesis and vice versa (e.g., RNA primers can be used to initiate DNA synthesis). Primer lengths can vary. For example, primers can be from about 6 bases to about 120 bases. For example, primers may include up to about 25 bases.

[0096] (x) Primer extension

[0097] "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 overlapping their respective complementary terminal nucleic acid sequences (e.g., 3′ ends). Following this ligation, another nucleic acid sequence can be used as an extension template to perform one or both-end extension (e.g., enzyme extension). Enzyme extension can be performed by enzymes including, but not limited to, polymerases and / or reverse transcriptases.

[0098] (xi) Neighborhood connection

[0099] "Proximity joining" is a method of joining two (or more) nucleic acid sequences that are adjacent to each other by enzymatic means (e.g., ligases). In some embodiments, proximity joining may include a "gap filling" step, which involves incorporating one or more nucleic acids across a distance spanning two nucleic acid molecules of interest using a polymerase based on the nucleic acid sequence of a template nucleic acid molecule (see, for example, U.S. Patent No. 7,264,929, the entire contents of which are incorporated herein by reference).

[0100] Various methods can be used for proximity ligation of nucleic acid molecules, including (but not limited to) "sticky-end" and "blunt-end" ligation. Furthermore, single-stranded ligation can be used for proximity ligation of single-stranded nucleic acid molecules. Sticky-end proximity ligation involves hybridizing the complementary single-stranded sequences between the two nucleic acid molecules to be ligated before the ligation event occurs. Blunt-end proximity ligation typically does not include hybridization from the complementary regions of each nucleic acid molecule because both nucleic acid molecules lack single-stranded overhangs at the ligation site.

[0101] (xii) Nucleic acid extension

[0102] Nucleic acid extension typically 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 nucleic acid sequences) in a template-dependent manner, such that consecutive nucleic acids are incorporated by an enzyme (e.g., polymerase or reverse transcriptase) to generate a newly synthesized nucleic acid molecule. For example, by using a complementary nucleic acid sequence as a template for nucleic acid synthesis, a new nucleic acid molecule can be synthesized using primers that hybridize with the complementary nucleic acid sequence. Similarly, the 3' polyadenylated tail of an mRNA transcript that hybridizes with a poly(dT) sequence (e.g., a capture domain) can be used as a template for the single-stranded synthesis of the corresponding cDNA molecule.

[0103] (xiii) PCR amplification

[0104] “PCR amplification” refers to the generation of copies of genetic material, including DNA and RNA sequences, using polymerase chain reaction (PCR). Suitable reagents and conditions for performing PCR are described, for example, 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 typical PCR amplification, the reaction mixture includes the genetic material to be amplified, an enzyme, one or more primers for primer extension, and reagents for the reaction. Oligonucleotide primers are of sufficient length to hybridize with complementary genetic material under annealing conditions. The length of primers typically depends on the length of the amplified domain, but is usually at least 4, 5, 6, 8, 9, 10 base pairs (bp), at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or 35 bp, and can be as long as 40 bp or more, with primer length generally between 18 and 50 bp. Genetic material can be contacted with a single primer or a pair of primers (forward and reverse primers), depending on whether primer extension, linear, or exponential amplification of the genetic material is required.

[0105] 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 some embodiments, the DNA polymerase is derived from bacteria; for example, the DNA polymerase is a bacterial DNA polymerase. For instance, the DNA polymerase can be derived from bacteria of the genera *Escherichia coli*, *Bacillus*, *Thermophilus*, or *Thermococcus*.

[0106] Suitable examples of usable DNA polymerases include, but are not limited to: Escherichia coli DNA polymerase I, Bsu DNA polymerase, Bst DNA polymerase, Taq DNA polymerase, and 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, including 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.

[0107] The term "DNA polymerase" includes not only naturally occurring enzymes but also all modified derivatives thereof, as well as derivatives of naturally occurring DNA polymerases. For example, in some embodiments, DNA polymerases may 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 partial function, such as DNA polymerase activity with wild-type sequences. Under different reaction conditions, such as temperature, template concentration, primer concentration, etc., mutations can affect the enzyme's activity profile, such as increasing or decreasing the polymerization rate. Mutations or sequence modifications may also affect the enzyme's exonuclease activity and / or thermal stability.

[0108] In some implementations, PCR amplification may include reactions such as, but not limited to, strand displacement amplification, rolling circle amplification, ligase chain reaction, transcription-mediated amplification, isothermal amplification, and / or loop-mediated amplification.

[0109] In some embodiments, PCR amplification uses a single primer complementary to the 3' tag of the target DNA fragment. In some embodiments, PCR amplification uses first and second primers, wherein at least the 3' terminal portion of the first primer is complementary to at least a portion of the 3' tag of the target nucleic acid fragment, and wherein at least the 3' terminal portion of the second primer displays at least a portion of the sequence 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 display at least a portion of the sequence 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.

[0110] In some implementations (e.g., when PCR amplification amplifies captured DNA), DNA ligases can be used to ligate PCR amplification products to other sequences. DNA ligase activity can be provided by one or more different DNA ligases. In some implementations, the DNA ligase is derived from bacteria; for example, the DNA ligase is a bacterial DNA ligase. In some implementations, the DNA ligase is derived from viruses (e.g., bacteriophages). 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, Thermococcus spp. (strain 9oN) DNA ligase (9oN™ DNA ligase, available from New England Biolabs in Ipswich, Massachusetts), and Ampligase. TM (Available from Epicentre Biotechnologies, Madison, Wisconsin). Derivatives, such as sequence-modified derivatives and / or their mutants, may also be used.

[0111] In some implementations, genetic material is amplified via 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, and ArrayScript. TM MultiScribe TM ThermoScript TM ,and Reverse transcriptases include enzymes I, II, III, and IV. The term "reverse transcriptase" encompasses not only naturally occurring enzymes but also all their modified derivatives, as well as derivatives of naturally occurring reverse transcriptases.

[0112] Furthermore, reverse transcription can be performed using sequence-modified derivatives or mutants of M-MLV, MuLV, AMV, and HIV reverse transcriptases, including mutants that retain at least some functional (e.g., reverse transcriptase) activity while retaining the wild-type sequence. The reverse transcriptase can be provided as part of a composition comprising other components, such as stabilizing components that enhance or improve the activity of the reverse transcriptase, such as RNase inhibitors or inhibitors of DNA-dependent DNA synthesis, for example, actinomycin D. Sequence-modified derivatives or mutants of many reverse transcriptases, such as M-MLV, and compositions comprising unmodified and modified enzymes, such as ArrayScript, are also available. TM MultiScribe TM ThermoScript TM ,and Enzymes I, II, III, and IV are commercially available.

[0113] Some reverse transcriptases (such as avian myeloblastosis virus (AMV) reverse transcriptase and Moroni murine leukemia virus (M-MuLV, MMLV) reverse transcriptase) can use both RNA (cDNA synthesis) and single-stranded DNA (ssDNA) as templates to synthesize complementary DNA strands. Therefore, in some embodiments, the reverse transcription reaction can use enzymes (reverse transcriptases) capable of using both RNA and ssDNA as templates for the extension reaction, such as AMV or MMLV reverse transcriptases.

[0114] In some implementations, RNA and / or DNA quantification 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 the 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 analyzed gene may correspond to expression (mRNA) and quantity (DNA) compared with a reference nucleic acid extract (DNA and RNA) to compare the expression level of the target nucleic acid.

[0115] (xiv) antibody

[0116] An antibody is a polypeptide molecule that recognizes and binds to a complementary target antigen. Antibodies typically have a Y-shaped molecular structure. Naturally occurring antibodies, known as immunoglobulins, belong to the immunoglobulin class, including IgG, IgM, IgA, IgD, and IgE. Antibodies can also be synthesized artificially. For example, recombinant antibodies, as monoclonal antibodies, can be synthesized using synthetic genes by recovering the antibody gene from a source cell, amplifying it into a suitable vector, and introducing the vector into a host to induce the host to express the recombinant antibody. Generally, recombinant antibodies can be cloned from any antibody-producing animal species using suitable oligonucleotide primers and / or hybridization probes. Recombinant technology can be used to generate antibodies and antibody fragments, including non-endogenous substances.

[0117] Synthetic antibodies can be obtained from non-immunoglobulin sources. For example, antibodies can be produced from nucleic acids (e.g., aptamers) and non-immunoglobulin scaffolds (e.g., peptide aptamers) with hypervariable loops inserted to form antigen-binding sites. Synthetic antibodies based on nucleic acid or peptide structures can be smaller than immunoglobulin-derived antibodies, resulting in greater tissue permeability.

[0118] Antibodies may also include affinity proteins, which are affinity agents typically having a molecular weight of about 12-14 kDa. Affinity proteins typically bind to targets (e.g., target proteins) with high affinity and specificity. Examples of such targets include, but are not limited to, ubiquitin chains, immunoglobulins, and C-reactive proteins. In some embodiments, the affinity protein is derived from a cysteine ​​protease inhibitor and includes a peptide ring and a variable N-terminal sequence that provides a binding site.

[0119] Antibodies can also include single-domain antibodies (V) H H domain and VNAR domain), scFv and Fab fragments.

[0120] (xv) affinity group

[0121] An “affinity group” is a molecule or part of a molecule that has a high affinity or preference for association or binding with another particular or specific molecule or part. Association or binding with another particular or specific molecule or part can be achieved 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 association or binding with proteavidin or streptavidin. For example, an affinity group can also refer to avidin or streptavidin, which has an affinity for biotin. Other examples of affinity groups and the particular or specific molecules or parts they bind to or associate with include, but are not limited to, antibodies or antibody fragments and their respective antigens, such as digoxigenin and anti-digoxigenin antibodies, lectins and carbohydrates (e.g., sugars, monosaccharides, disaccharides, or polysaccharides), and receptors and receptor ligands.

[0122] Any pair of affinity groups and the specific or particular molecule or part thereof that binds to or associates with them can reverse their effects, for example, between a first molecule and a second molecule, in a first instance the first molecule is characterized as the affinity group of the second molecule, and in a second instance the second molecule is characterized as the affinity group of the first molecule.

[0123] (xvi) Markers, detectable markers and optical markers.

[0124] The terms “detectable label,” “optical label,” and “label” are used interchangeably herein to refer to the directly or indirectly detectable portion associated (e.g., coupled) with a molecule to be detected (e.g., a capture probe or analyte). A detectable label may be directly detectable by itself (e.g., a radioisotope label or a fluorescent label), or, in the case of an enzyme label, may be indirectly detectable, for example, by catalyzing a chemical change in a substrate compound or composition that makes it directly detectable. Detectable labels may be suitable for small-scale detection and / or for high-throughput screening. Therefore, suitable detectable labels include, but are not limited to, radioisotopes, fluorophores, chemiluminescent compounds, bioluminescent compounds, and dyes.

[0125] Detectable markers can be detected qualitatively (e.g., optically or spectroscopically) or quantitatively. Qualitative detection typically involves detection methods that confirm the presence or occurrence of the detectable marker, while quantitative detection typically involves detection methods that have quantifiable (e.g., digitally reportable) values, such as intensity, duration, polarization, and / or other properties. In some embodiments, the detectable marker is incorporated into a feature or a capture probe associated with the feature. For example, a detectably tagged feature may include a fluorescent, colorimetric, or chemiluminescent marker attached to a bead (see, for example, Rajeswali 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).

[0126] In some implementations, multiple detectable markers can be attached to the feature to be detected, the capture probe, or the composition. For example, the detectable markers can be incorporated during nucleic acid polymerization or amplification (e.g., -Tag nucleotides, for example -dCTP). Any suitable detectable marker can be used. In some embodiments, the detectable marker is a fluorophore. For example, the fluorophore may be from the following group: 7-AAD (7-aminoactinomycin D), acridine orange (+DNA), acridine orange (+RNA), 350 430 488、 532, 546、 555、 568、 594、 633、 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, O-Feulgen (Auramine), 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, Tetrazolium Chloride Blue, Calcein, Calcein Blue, Calcium Crimson TM Calcium Green-1 TM Calcium Orange TM , White, 5-Carboxyfluorescein (5-FAM), 5-Carboxynaphthalenefluorescein, 6-Carboxyrhodamine 6G, 5-Carboxytetramethylrhodamine (5-TAMRA), Carboxy-X-rhodamine (5-ROX), Cascade Cascade Yellow TM CCF2 (GeneBlaze) TM CFP (cyan fluorescent protein), CFP / YFPFRET, chromopycin A3, Cl-NERF (low pH), CPM, 6-CR 6G, CTC-methyl Cychrome (PE-Cy5), dansylamide, dansyl cadaverine, 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-4 ANEPPS, Di-8 ANEPPS, DM-NERF (4.5-6.5pH), 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 phosphoramide, 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, Fura Red TM (High calcium), Fura Red TM / Fluo-3, GeneBLAzer TM (CCF2), GFP redshift (rsGFP), GFP wild-type, GFP / BFP-FRET, GFP / DsRed-FRET, Hoechst 33342&33258, 7-hydroxy-4-methylcoumarin (pH 9), 1,5-indoleacetic acid, Indo-1 (high calcium), Indo-1 (low calcium), indole dicarboxycyanine, indole tricarboxycyanine, 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 (pH 5), LysoSensor TM Yellow / Blue (pH 4.2) green, red, Yellow, Mag-Fura-2, Mag-Indo-1, Magnesium Green TM Marina 4-Methylumbelliferone, Miteradine green, orange, 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 (polydinophyll-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, Pyrrolidone Y, Quantum Red (PE-Cy5), Quinacrine Mustard, R670 (PE-Cy5), Red 613 (PE-Texas Red), Red Fluorescent Protein (DsRed), Halogen, RH414, 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), thiazolinone, thiazolinone orange, -1 / TO- -1、 -3 / TO- -3、TO- -5. Tricolor (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, ATTO488, ATTO 532, ATTO 550, ATTO 565, ATTO Rho101, ATTO 590, ATTO 633, ATTO 647N, TYE563, 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).

[0127] As described above, in some embodiments, the detectable marker 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 a chemiluminescent reaction given a suitable substrate (e.g., an oxidizing agent plus a chemiluminescent compound). Many families of compounds are known to provide chemiluminescence under a variety of 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 are luminescent in the presence of alkaline hydrogen peroxide or calcium hypochlorite and a base. Other examples of families of chemiluminescent compounds include, for example, 2,4,5-triphenylimidazolium, p-dimethylamino and -methoxy substituents, oxalates (e.g., oxaloyl active esters), p-nitrophenyl, N-alkyl acridine esters, luciferin, lusterin, or acridine esters.

[0128] (xvii) Template-converting oligonucleotides

[0129] A template-transfer oligonucleotide is an oligonucleotide that hybridizes with an untemplated nucleotide added by a reverse transcriptase (e.g., an enzyme with terminal transferase activity) during reverse transcription. In some embodiments, the template-transfer oligonucleotide hybridizes with an untemplated poly(C) nucleotide added by a reverse transcriptase. In some embodiments, the template-transfer oligonucleotide adds a common 5' sequence to full-length cDNA used for cDNA amplification.

[0130] In some embodiments, template-converting oligonucleotides add a common sequence to the 5' end of the RNA being reverse transcribed. For example, the template-converting oligonucleotide can hybridize with an untemplated poly(C)nucleotide added to the end of a cDNA molecule and provide a template for reverse transcriptase to continue replicating to the 5' end of the template-converting oligonucleotide, thereby generating full-length cDNA capable of further amplification. In some embodiments, once the full-length cDNA molecule is generated, the template-converting oligonucleotide can be used as a primer in the cDNA amplification reaction.

[0131] In some embodiments, template-converting oligonucleotides are added before, during, or after reverse transcription or other terminal transferase-based reactions. In some embodiments, the capture probe includes template-converting oligonucleotides. In some embodiments, sample analysis methods using template-converting oligonucleotides may involve generating nucleic acid products from analytes in tissue samples, followed by further processing of the nucleic acid products using template-converting oligonucleotides.

[0132] The template-converting oligonucleotide may include a hybridization region and a template region. The hybridization region may include any sequence capable of hybridizing with a target. In some embodiments, the hybridization region may, for example, include consecutive G bases complementary to a C base overhang at the 3' end of the cDNA molecule. Consecutive G bases may 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 may include any sequence of the cDNA to be incorporated. In other embodiments, the hybridization region may include at least one base in addition to at least one G base. In other embodiments, hybridization may include non-G bases. In some embodiments, the template region includes at least one (e.g., at least 2, 3, 4, 5, or more) tag sequence and / or functional sequence. In some embodiments, the template region and the hybridization region are separated by a spacer.

[0133] In some embodiments, the template region includes a barcode sequence. The barcode sequence may function as a spatial barcode and / or as a unique molecular identifier. The template-transforming oligonucleotide may include deoxyribonucleic acid; ribonucleic acid; modified nucleic acid, including 2-aminopurine, 2,6-diaminopurine (2-amino-dA), reverse dT, 5-methyl dC, 2′-deoxyinosine, superT (5-hydroxybutyl-2'-deoxyuridine), superG (8-aza-7-dezoguanosine), 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., fluorine C, fluorine U, fluorine A, and fluorine G), or any combination thereof.

[0134] In some embodiments, the template-converting oligonucleotide may be at least about 1, 2, 10, 20, 50, 75, 100, 150, 200, or 250 nucleotides long or longer. In some embodiments, the template-converting oligonucleotide may be at most about 2, 10, 20, 50, 100, 150, 200, or 250 nucleotides long or longer.

[0135] (xviii) Clamping oligonucleotides

[0136] A "sandwich oligonucleotide" is an oligonucleotide that acts as a "sandwich" when hybridizing with other polynucleotides, keeping the polynucleotides adjacent to each other so they can be linked together. In some embodiments, the sandwich oligonucleotide is DNA or RNA. A sandwich oligonucleotide may include a nucleotide sequence that is partially complementary to the nucleotide sequence of two or more different oligonucleotides. In some embodiments, the sandwich oligonucleotide assists in linking a "donor" oligonucleotide and a "recipient" oligonucleotide. Generally, RNA ligase, DNA ligase, or other types of ligase are used to link two nucleotide sequences together.

[0137] In some embodiments, the length of the splice oligonucleotide is between 10 and 50 oligonucleotides, for example, 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 splice 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.

[0138] (c) Analytes

[0139] The apparatus, systems, methods, and compositions described in this disclosure can be used to detect and analyze a wide variety of analytes. For the purposes of this invention, "analyte" can include any biological substance, structure, part, or component to be analyzed. The term "target" can similarly refer to the analyte of interest.

[0140] 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 capsid proteins, extracellular and intracellular proteins, antibodies, and antigen-binding fragments. In some embodiments, the analyte can be an organelle (e.g., the cell nucleus or mitochondria).

[0141] Cell surface features corresponding to the analyte may include, but are not limited to, receptors, antigens, surface proteins, transmembrane proteins, differentiation protein clusters, protein channels, protein pumps, carrier proteins, phospholipids, glycoproteins, glycolipids, cell-cell interaction protein complexes, antigen-presenting complexes, major histocompatibility complexes, engineered T cell receptors, T cell receptors, B cell receptors, chimeric antigen receptors, extracellular matrix proteins, post-translational modifications (e.g., phosphorylation, glycosylation, ubiquitination, nitrosation, methylation, acetylation, or lipidation) of cell surface proteins, gap junctions, and adhesion junctions.

[0142] Analytes can originate from specific cell types and / or specific subcellular regions. For example, analytes can originate from the cytoplasm, nucleus, mitochondria, microsomes, and more generally, any other compartment, organelle, or part of the cell. Permeabilizers specifically targeting certain cell compartments and organelles can be used to selectively release analytes from the cell for analysis.

[0143] 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.

[0144] Examples of nucleic acid analytes also include RNA analytes, such as various types of coding and non-coding RNA. 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 tissue sections). RNA can be small (e.g., less than 200 nucleic acid bases in length) or large (e.g., RNA longer 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 nucleolar RNA (snRNA), Piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA), and small rDNA-derived RNA (srRNA). RNA can be double-stranded or single-stranded. RNA can be circular RNA. RNA can be bacterial rRNA (e.g., 16S rRNA or 23S rRNA).

[0145] Other examples of analytes include mRNA and cell surface features (e.g., using markers described herein), mRNA and intracellular proteins (e.g., transcription factors), mRNA and cellular methylation status, mRNA and available chromatin (e.g., ATAC-seq, DNase-seq, and / or micrococcal enzyme-seq), mRNA and metabolites (e.g., using markers described herein), barcoded markers (e.g., oligonucleotide-labeled antibodies described herein), and V(D)J sequences of immune cell receptors (e.g., T cell receptors), mRNA and perturbators (e.g., CRISPR crRNA / sgRNA, TALEN, zinc finger nucleases, and / or antisense oligonucleotides as described herein).

[0146] The analyte may 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 first generated from the reverse transcription of the corresponding mRNA using a poly(T)-containing primer. The generated cDNA can then be barcoded using a capture probe having a barcoded sequence (and optionally a UMI sequence) that hybridizes to at least a portion of the generated cDNA. In some embodiments, a template-converting oligonucleotide hybridizes to a poly(C) tail added to the 3′ end of the cDNA via reverse transcriptase. The original mRNA template and template-converting oligonucleotide can be denatured from the cDNA, and then the barcoded capture probe can hybridize with the cDNA to generate a complement to 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, are described in PCT patent application PCT / US2017 / 057269 filed October 18, 2017, and U.S. Patent Application Serial No. 15 / 825,740 filed November 29, 2017, both of which are incorporated herein by reference in their entirety. V(D)J analysis can also be performed using one or more markers that bind to specific surface features of immune cells and are associated with barcode sequences. One or more markers may include MHC or MHC multimers.

[0147] As described above, the analyte may include nucleic acids capable of functioning as components of a gene editing reaction, such as gene editing based on regularly clustered, spaced short palindromic repeats (CRISPR). Therefore, the capture probe may include a nucleic acid sequence complementary to the analyte (e.g., a sequence that can hybridize to CRISPR RNA (crRNA), single-guide RNA (sgRNA), or an adaptor sequence engineered into crRNA or sgRNA).

[0148] In some implementations, analytes can be extracted from live cells. Processing conditions can be adjusted to ensure that the biological sample remains active during analysis and that the analyte is extracted (or released) from the live cells of the sample. Live cell-derived analytes can be obtained only once from the sample, or they can be obtained periodically from a sample that continues to remain viable.

[0149] Generally, the systems, apparatus, methods, and compositions described herein can be used to analyze any number of analytes. For example, the number of analytes analyzed may 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 10000, or at least about 100000 or more different analytes present in the sample region or within the individual characteristics of the substrate. Methods for performing multiple analyses to analyze two or more different analytes will be discussed in later sections of this invention.

[0150] (d) Biological samples

[0151] (i) Types of biological samples

[0152] "Biological samples" are 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 typically include cells and / or other biological material from the subject. In addition to the subjects mentioned above, biological samples can be obtained from prokaryotes, such as bacteria like *Escherichia coli*, *Staphylococcus*, 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). 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), individuals undergoing pre-treatment for a disease, and / or individuals requiring or suspected of requiring treatment.

[0153] Biological samples can include any number of macromolecules, such as cellular macromolecules and organelles (e.g., mitochondria and the nucleus). 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 aspiration, or fine-needle aspiration. 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.

[0154] Cell-free biological samples may include extracellular polynucleotides. Extracellular polynucleotides can be isolated from bodily samples such as blood, plasma, serum, urine, saliva, mucosal excretions, sputum, feces, and tears.

[0155] Biological samples may be derived from homogeneous cultures or populations of the objects or organisms described herein, or alternatively from a collection of several different organisms in, for example, a community or ecosystem.

[0156] Biological samples may include one or more diseased cells. Diseased cells may exhibit altered metabolic properties, gene expression, protein expression, and / or morphological characteristics. Examples of diseases include inflammatory disorders, metabolic disorders, nervous system disorders, and cancer. Cancer cells may originate from solid tumors, hematologic malignancies, cell lines, or be obtained as circulating tumor cells.

[0157] 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 many genetic disorders, including, for example, aneuploidy, such as Down syndrome, Edwards syndrome, and Patau syndrome. Furthermore, cell surface features of fetal cells can be used to identify any of a variety of diseases or conditions.

[0158] Biological samples can also include immune cells. Sequence analysis of the immune function of these cells, including genomics, proteomics, and cell surface characteristics, can provide a wealth of information to help understand the state and function of the immune system. For example, determining the status of minimal residual disease (MRD) (e.g., negative or positive) in patients with multiple myeloma (MM) after autologous stem cell transplantation is considered a predictor of MRD in MM patients (see, for example, U.S. Patent Application Publication No. 2018 / 0156784, which is incorporated herein by reference in its entirety).

[0159] 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 / multi-segmented neutrophils), monocytes / macrophages, mast cells, platelets / megakaryocytes, and dendritic cells.

[0160] As described above, a biological sample may include a single analyte of interest or multiple analytes of interest. Methods for performing multiplex analyses to analyze two or more different analytes in a single biological sample will be discussed in later sections of this invention.

[0161] (ii) Preparation of biological samples

[0162] Various steps can be performed to prepare biological samples for analysis. Unless otherwise stated, the preparation steps described below can generally be combined in any way to appropriately prepare a specific sample for analysis.

[0163] (1) Tissue section

[0164] Biological samples can be obtained from a subject (e.g., via surgical biopsy, whole-subject section) or grown in vitro as cell populations on a growth substrate or culture dish, and prepared as tissue sections or tissue slides for analysis. The grown samples can be thin enough to be analyzed without further processing. Alternatively, the grown samples and samples obtained via biopsy or sectioning can be prepared as thin tissue slides using mechanical cutting equipment (e.g., a vibrating blade microtome). As another alternative, in some embodiments, thin tissue slides can be prepared by applying a biological sample tough imprint to a suitable substrate material.

[0165] The thickness of a tissue section can be a fraction of the largest cross-sectional size of the cells (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 thicker than the largest cross-sectional cell size can also be used. For example, cryogenic sections, such as 10–20 micrometers thick, can be used.

[0166] More generally, the thickness of tissue sections typically depends on the method used to prepare the sections and the physical properties of the tissue; therefore, sections with a variety of thicknesses can be prepared and used. For example, tissue section thicknesses 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 micrometers. Thicker sections, such as at least 70, 80, 90, or 100 micrometers or greater, can also be used if desired or convenient. Typically, tissue section thicknesses are between 1-100 micrometers, 1-50 micrometers, 1-30 micrometers, 1-25 micrometers, 1-20 micrometers, 1-15 micrometers, 1-10 micrometers, 2-8 micrometers, 3-7 micrometers, or 4-6 micrometers; however, as mentioned above, sections with thicknesses greater than or less than these ranges can also be analyzed.

[0167] 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 sequentially slicing the biopsy sample using a slicing blade. In this way, spatial information between consecutive sections can be preserved, and sections can be analyzed sequentially to obtain three-dimensional information about the biological sample.

[0168] (2) Freezing

[0169] In some embodiments, biological samples (e.g., tissue sections as described above) can be prepared by deep freezing at temperatures suitable for maintaining or preserving the integrity (e.g., physical properties) of the tissue structure. For example, this temperature may 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. Frozen tissue samples can be sliced ​​(e.g., thinly sliced) onto a substrate surface using any number of suitable methods. For example, tissue samples can be prepared using a cryostat (e.g., a cryostat) 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, this temperature may be below -15°C, below -20°C, or below -25°C.

[0170] (3) Formaldehyde fixation and paraffin embedding

[0171] 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 and embedding the sample in paraffin or resin blocks, the sample can be sectioned as described above. Prior to analysis, the paraffin embedding material can be removed from the tissue sections by incubating them in a suitable solvent (e.g., xylene) followed by rinsing (e.g., 99.5% ethanol for 2 minutes, 96% ethanol for 2 minutes, and 70% ethanol for 2 minutes) (e.g., deparaffining).

[0172] (4) Fixed

[0173] As an alternative to formaldehyde fixation, biological samples can be fixed in any of a variety of other fixatives to preserve the biological structure of the sample prior to analysis. For example, samples can be fixed by immersing them in ethanol, methanol, acetone, paraformaldehyde-triton, or combinations thereof.

[0174] In some embodiments, acetone fixation is used in conjunction with freshly frozen samples, which may include, but are not limited to, cortical tissue, mouse olfactory bulbs, human brain tumors, post-mortem human brains, and breast cancer samples. When performing acetone fixation, a pre-permeabilization step (described below) may be omitted. Alternatively, acetone fixation may be performed in conjunction with a permeabilization step.

[0175] (5) Embedding

[0176] As an alternative to 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 before analyzing tissue sections obtained from the sample. Suitable embedding materials include, but are not limited to, waxes, resins (e.g., methacrylates), epoxy resins, and agar.

[0177] (6) Staining

[0178] To facilitate visualization, biological samples can be stained using a variety of staining agents and techniques. In some implementations, for example, any number of staining agents can be used to stain the samples, including but not limited to acridine orange, Bismarck brown, carmine, Coomassie blue, cresol purple, DAPI, eosin, ethidium bromide, acid fuchsin, hematoxylin, Höller's stain, iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red, osmium tetroxide, propidium iodide, rhodamine, or saffron.

[0179] Samples can be stained using hematoxylin and eosin (H&E), Papanicolaou staining, Masson's trichrome staining, silver staining, and / or periodic acid Schiff (PAS) staining. PAS staining is typically performed after fixation with formaldehyde or acetone. In some embodiments, samples can be stained using Romanovsky staining methods, including Wright's staining, Jenner's staining, Canglenwald staining, Leishman's staining, and Giemsa staining.

[0180] In some embodiments, biological samples can be destained. Methods for decontaminating or destaining biological samples are known in the art and generally depend on the nature of the staining agent applied to the sample. For example, in some embodiments, one or more immunofluorescent staining agents are applied to the sample via antibody coupling. These staining agents can be removed using techniques such as disulfide bond cleavage by washing with reducing agents and detergents, hygroscopic salt treatment, antigen recovery solution treatment, and acidic glycine buffer treatment. For example, methods for multiple staining and destaining are described in, for example, 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.

[0181] (7) Hydrogel embedding

[0182] In some embodiments, the biological sample may be embedded in a hydrogel substrate. Embedding the sample in this manner typically 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 polymer material and activating the polymer material to form a hydrogel. In some embodiments, the hydrogel is formed such that it is internalized within the biological sample.

[0183] In some embodiments, biological samples are immobilized in a hydrogel by crosslinking 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.

[0184] The composition and application of hydrogel substrates for biological samples typically depend on the nature and preparation of the biological sample (e.g., sectioned, unsectioned, fixed type). As an example, where the biological sample is a tissue section, the hydrogel substrate may comprise a monomer solution and an ammonium persulfate (APS) initiator / tetramethylethylenediamine (TEMED) promoter solution. As another example, when the biological sample consists of cells (e.g., cultured cells or cells isolated from a tissue sample), the cells may be incubated with both the monomer solution and the APS / TEMED solution. For cells, the hydrogel substrate gel forms within compartments, including but not limited to devices for culturing, maintaining, or transporting cells. For example, the hydrogel substrate may be formed using a monomer solution plus APS / TEMED, added to the compartments to a depth between approximately 0.1 μm and approximately 2 mm.

[0185] 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 contents of which are incorporated herein by reference.

[0186] (8) Isometric Expansion

[0187] In some embodiments, the biological sample embedded in the hydrogel can be expanded at equal intervals. Available methods of equal expansion include hydration, a preparatory step in the expansion microscope, as described by Chen et al., Science 347(6221): 543–548 (2015).

[0188] Isometric expansion can be achieved by anchoring one or more components of a biological sample to a gel, followed by gel formation, protein hydrolysis, and swelling. Isometric expansion of the biological sample can occur before 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 contact with the substrate by a capture probe, which will be discussed in more detail later.

[0189] Generally, the steps used for isometric expansion of biological samples can depend on the characteristics of the sample (e.g., the thickness of the tissue section, fixation, cross-linking) and / or the analyte of interest (e.g., different conditions for anchoring RNA, DNA, and proteins to the gel).

[0190] In some embodiments, proteins in a biological sample are anchored to a swellable gel (e.g., a polyelectrolyte gel). Antibodies can be directed to proteins before, after, or during anchoring to the swellable gel. DNA and / or RNA in a biological sample can also be anchored to the swellable gel via suitable adapters. Examples of such adapters include, but are not limited to, 6-((acryloyl)amino)hexanoic acid (acryloyl-X-SE) (available from Thermo Fisher Scientific, Waltham, MA), LabelIT amine (available from MirusBio, Madison, Wisconsin), and Label X (described, for example, in Chen et al., Nat. Methods 13:679-684, 2016, the entire contents of which are incorporated herein by reference).

[0191] Isometric scaling of a sample can improve the spatial resolution of subsequent analyses. The increased resolution in a spatial profile can be determined by comparing isometrically scaled samples with non-isometrically scaled samples.

[0192] In some embodiments, the biological sample is equidistantly extended 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-extended size. In some embodiments, the sample is equidistantly extended to at least twice and less than 20 times its non-extended size.

[0193] (9) Substrate attachment

[0194] In some embodiments, biological samples may be attached to a hydrogel substrate. Examples of substrates suitable for this purpose are described in detail below. The attachment of biological samples can be irreversible or reversible, depending on the nature of the sample and subsequent steps in the analytical method.

[0195] In some 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 an example of polymers suitable for this purpose.

[0196] More generally, in some embodiments, the substrate can be coated or functionalized with one or more substances to facilitate sample adhesion to the substrate. Suitable substances that can be used to coat or functionalize the substrate include, but are not limited to, lectins, polylysine, antibodies, and polysaccharides.

[0197] (10) Cell depolymerization

[0198] In some implementations, the biological sample corresponds to cells (e.g., from cell cultures or tissue samples). In a cell sample containing multiple cells, the multiple individual cells may naturally not aggregate. For example, the cells may be derived from cell suspensions and / or isolated or deaggregated cells from tissues or tissue sections.

[0199] Alternatively, the cells in the sample may aggregate and can be deaggregated into multiple individual cells using techniques such as enzymes or mechanical methods. Examples of enzymes used for enzymatic deaggregation include, but are not limited to, dispersases, collagenases, trypsin, and combinations thereof. For example, a tissue homogenizer can be used for mechanical deaggregation.

[0200] (11) Suspension and adherent cells

[0201] In some embodiments, the biological sample may be obtained from an in vitro grown cell culture. The sample from the cell culture may include one or more suspension cells that are anchored independently within the cell culture. Examples of such cells include, but are not limited to, hematopoietic cells derived from 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.

[0202] Samples from cell cultures may include one or more adherent cells grown on the surface of a container containing culture medium. Non-limiting examples of adherent cells include DU145 (prostate cancer) cells, H295R (adrenocortical carcinoma) cells, HeLa (cervical cancer) cells, KBM-7 (chronic myeloid 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, and SH-SY5Y (neuroblastoma, cloned from myeloma) cells. Cells, including T-47D (breast cancer) cells, THP-1 (acute myeloid leukemia) cells, U87 (glioblastoma) cells, National Cancer Institute 60 Cancer Cell Line Group (NCI60), Vero (African green monkey kidney epithelial cell line) cells, MC3T3 (embryonic skull) cells, GH3 (pituitary adenoma) cells, PC12 (pheochromocytoma) cells, canine MDCK kidney epithelial cells, Xenopus A6 kidney epithelial cells, zebrafish AB9 cells, and Sf9 insect epithelial cells.

[0203] Other examples of adherent cells are shown in Table 1 and are categorized in, for example, “A Catalog of in Vitro Cell Lines, Transplantable Animal and Human Tumors and Yeast”, Division of Diagnostic and Therapeutic Use of Cancer, National Cancer Institute (DCTD) (2013) and “The exomes of the NCI-60 panel: a genomic resource for cancer biology and systems pharmacology”, Cancer Research 73(14):4372-82, 2013, the full contents of which are included in this paper by reference.

[0204] Table 1: Examples of adherent cells

[0205]

[0206]

[0207]

[0208] 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, canine MDCK renal epithelium, Xenopus A6 renal epithelium, zebrafish AB9, and Sf9 insect epithelial cell lines.

[0209] (12) Tissue permeability

[0210] In some implementations, biological samples may be permeabilized to facilitate the transfer of analytes from the sample and / or to facilitate the transfer of substances (e.g., capture probes) into the sample. If the sample is not permeabilized sufficiently, the amount of analyte captured from the sample may be too low for adequate analysis. Conversely, if the tissue sample is too permeabilized, the relative spatial relationships of analytes within the tissue sample may be lost. Therefore, a balance is ideal between adequately permeating the tissue sample to obtain good signal intensity while still maintaining the spatial resolution of the analyte distribution within the sample.

[0211] Generally, biological samples can be permeated by exposing them to one or more permeabilizing agents. Suitable reagents for this purpose include, but are not limited to, organic solvents (e.g., acetone, ethanol, and methanol), crosslinking agents (e.g., paraformaldehyde), and detergents (e.g., saponins, Triton X-100). TM Or Tween-20 TMAnd enzymes (such as trypsin, proteases). In some embodiments, biological samples may be incubated with cell permeabilizing agents to promote sample permeation. For example, other methods of sample permeation are described in Jamur et al., Method Mol. Biol. 588:63-66, 2010, the entire contents of which are incorporated herein by reference. Any suitable method for sample permeation may generally be used in conjunction with the samples described herein.

[0212] In some embodiments, where an antidiffusion medium is used to restrict the migration of analytes or other substances during the analytical procedure, the antidiffusion medium may include at least one permeation reagent. For example, the antidiffusion medium may include pores (e.g., micropores, nanopores, or lenticels) containing a permeation buffer or reagent. In some embodiments, the antidiffusion medium is a hydrogel, which may include a permeation buffer. In some embodiments, the hydrogel is soaked in a permeation buffer before contacting the sample. In some embodiments, when the antidiffusion medium is applied to a biological sample, the hydrogel or other antidiffusion medium may contain a drying reagent or monomer to deliver the permeation reagent. In some embodiments, the antidiffusion medium (i.e., the hydrogel) is covalently attached to a solid substrate (i.e., an acrylic glass slide). In some embodiments, the hydrogel may be modified to both contain a capture probe and deliver a permeation reagent. For example, the hydrogel membrane may be modified to include a spatially barcoded capture probe. The spatially barcoded hydrogel membrane is then soaked in a permeation buffer before contacting the sample. Therefore, spatially barcoded hydrogel membranes deliver permeation reagents 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 amount of permeation solution. In some embodiments, a hydrogel membrane soaked in permeation reagent is sandwiched between the sample and the spatially barcoded array. In some embodiments, the target analyte is able to diffuse through the permeation reagent-soaked hydrogel and hybridize or bind to the capture probe on the other side of the hydrogel. In some embodiments, the thickness of the hydrogel is proportional to the resolution loss. In some embodiments, the pores (e.g., micropores, nanopores, or picometer pores) may contain spatially barcoded capture probes and permeation reagents and / or buffers. In some embodiments, the spatially barcoded capture probes and permeation reagents are held between spacers. In some embodiments, the sample is perforated, cut, or transferred into the pores, where the target analyte diffuses through the permeation reagent / buffer to the spatially barcoded capture probe. In some embodiments, the resolution loss may be proportional to the gap thickness (e.g., the amount of permeation buffer between the sample and the capture probe). In some embodiments, the thickness of the anti-diffusion medium (e.g., hydrogel) is between about 50 and 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.

[0213] In some embodiments, the permeation solution can be delivered to the sample through a porous membrane. In some embodiments, the porous membrane is used to limit the loss of diffused analytes while allowing the permeation reagent to reach the sample. 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 permeation reagent flows through microfluidic chambers or channels on the porous membrane. In some embodiments, flow control measures the sample's accessibility to the permeation reagent. In some embodiments, the porous membrane is sandwiched between a spatial barcode array and the sample, wherein the permeation solution is applied to the porous membrane. The permeation reagent diffuses into the tissue through the membrane pores.

[0214] In some implementations, biological samples can be permeated by adding one or more lysing agents to the sample. Examples of suitable lysing agents include, but are not limited to, bioactive reagents such as lysins for lysing different cell types, such as Gram-positive or Gram-negative bacteria, plants, yeast, and mammals, such as lysozyme, colorless peptidase, lysostaphin, Labiase, kitalase, cytolysins, and a variety of other commercially available lysins.

[0215] Other lysis agents may be added to biological samples, either additionally or alternatively, to facilitate permeation. For example, surfactant-based lysis solutions can be used to lyse sample cells. Lysis solutions may include ionic surfactants such as creatine amide and sodium dodecyl sulfate (SDS). More generally, chemical lysis agents may include, but are not limited to, organic solvents, chelating agents, detergents, surfactants, and dissociation agents.

[0216] In some embodiments, biological samples can be permeated using non-chemical permeation methods. Non-chemical permeation methods are known in the art. For example, usable non-chemical permeation methods include, but are not limited to, physical dissolution techniques such as electroporation, mechanical permeation methods (e.g., bead milling to mechanically disrupt the tissue structure of the sample using a homogenizer and grinding balls), acoustic permeation (e.g., ultrasound), and thermal decomposition techniques, such as heating to induce thermal permeation of the sample.

[0217] (13) Selective enrichment of RNA substances

[0218] In some embodiments where 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 the sample. In some embodiments, the other oligonucleotides are sequences used to initiate a reaction via a polymerase. For example, one or more primer sequences having sequence complementarity with 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 having sequence complementarity with 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 the various methods known in the art (e.g., streptavidin beads) utilizing biotinylation-streptavidin affinity.

[0219] Alternatively, any of a variety of methods can be used to downselect (e.g., remove) one or more RNAs. For example, a probe can be given to the sample that selectively hybridizes with ribosomal RNA (rRNA), thereby reducing the pooling and concentration of rRNA in the sample. Subsequent application of the capture probe to the sample can result in improved capture of other types of RNA due to the reduction of nonspecific RNA present in the sample. Alternatively, double-stranded specific nuclease (DSN) treatment can remove rRNA (see, for example, Archer et al., Selective and flexible removal of problematic sequences from RNA-seq libraries at the cDNA stage, BMC Genomics, 15 401, (2014), the entire contents of which are incorporated herein by reference). In addition, hydroxyapatite chromatography can remove abundant substances (e.g., rRNA) (see, for example, Vandernoot, VA, cDNA normalization by hydroxyapatite chromatography to enrich transcriptome diversity in RNA-seq applications, Biotechniques, 53(6) 373-80, (2012), the entire contents of which are incorporated herein by reference).

[0220] (14) Other reagents

[0221] Before analyzing a sample, other reagents can be added to the biological sample for various functions. In some embodiments, DNase and RNase inactivators or inhibitors (e.g., proteinase K) and / or chelating agents (e.g., EDTA) can be added to the sample.

[0222] In some implementations, 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.

[0223] In some implementations, reverse transcriptase may be added to the sample, including enzymes with terminal transferase activity, primers, and conversion oligonucleotides. Template conversion can be used to increase the length of cDNA, for example, by appending a predefined nucleic acid sequence to the cDNA.

[0224] (15) Preprocessing for capturing probe interactions

[0225] In some embodiments, the analyte in the biological sample may be pretreated before interacting with the capture probe. For example, a polymerization reaction catalyzed by a polymerase (e.g., DNA polymerase or reverse transcriptase) may be performed in the biological sample before interaction with the capture probe. In some embodiments, the primers used for the polymerization reaction include functional groups that enhance hybridization with the capture probe. The capture probe may include appropriate capture domains to capture the bioanalyte of interest (e.g., a poly(dT) sequence to capture poly(A) mRNA).

[0226] In some embodiments, the bioanalyte is pretreated using next-generation sequencing to generate a library. For example, the analyte can be pretreated by adding a modifier (e.g., ligating a sequence that interacts with a capture probe). In some embodiments, the analyte (e.g., DNA or RNA) is fragmented using fragmentation techniques (e.g., using transposases and / or fragmentation buffers).

[0227] After fragmentation, the analyte can be modified. For example, modification can be added by ligating an adaptor sequence that hybridizes with the capture probe. 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 promotes hybridization with a capture probe containing a capture domain with a functional amount of poly(dT) sequence.

[0228] In some embodiments, a ligation reaction catalyzed by a ligase is performed in the biological sample prior to interaction with the capture probe. In some embodiments, ligation can be performed by chemical ligation. In some embodiments, click chemistry can be used for ligation, as described below. In some embodiments, the capture domain includes a DNA sequence 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 detected directly.

[0229] In some embodiments, a target-specific reaction is performed in the biological sample prior to interaction with the 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 to one or more analytes; and target-specific detection using in situ hybridization, DNA microscopy, and / or antibody detection. In some embodiments, the capture probe includes a capture domain (e.g., amplified or ligated) that is targeted to a target-specific product.

[0230] II. General Spatial Array-Based Analysis Methods

[0231] This part of the invention describes methods, apparatus, systems, and compositions for spatial array-based analysis of biological samples.

[0232] (a) Spatial analysis methods

[0233] Array-based spatial analysis methods involve transferring one or more analytes from a biological sample onto a feature array on a substrate, each associated with a unique spatial location within the array. Subsequent analysis of the transferred analytes includes determining the analyte identity and the spatial location of each analyte within the sample. The spatial location of each analyte within the sample is determined based on the features bound to each analyte in the array and the relative spatial location of those features within the array.

[0234] There are at least two general methods for associating spatial barcodes with one or more neighboring cells, such that the spatial barcode identifies one or more cells and / or the contents of one or more cells as associated with a specific spatial location. One general method is to remove the target analyte from the cell and direct it toward a spatially barcoded array. Figure 1 Exemplary implementations of this general method are described. Figure 1In this process, a spatially barcoded array containing capture probes (as further described herein) is brought into contact with sample 101, and the sample is permeated, allowing 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.

[0235] Another common approach is to capture probes from array fragmentation spatial barcodes and drive the spatial barcode capture probes toward the sample and / or into or onto the sample. Figure 2 An exemplary implementation of this general method is depicted, enabling a spatially barcoded array of capture probes (as further described herein) to contact sample 201. The spatially barcoded capture probes are cleaved and then interact with 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 optionally be removed for analysis. The sample can be selectively segregated prior to analysis. Once the labeled cells are associated with the spatially barcoded capture probes, the capture probes can be analyzed to obtain spatial resolution information about the labeled cells 203.

[0236] Figure 3 An exemplary workflow including sample preparation on a spatially barcoded array 301 is shown. Sample preparation may include placing the sample on a glass slide, fixing the sample, and / or staining the sample for imaging. The stained sample is then imaged on array 302 using bright-field (imaging for hematoxylin and eosin staining of the sample) and fluorescence (imaging for features) modes. In some embodiments, a target analyte is then released from the sample, and a capture probe forming the spatially barcoded array hybridizes to or binds to the released target analyte 303. The sample is then removed from array 304, and the capture probe is lysed from array 305. A second imaging of the sample and array is then optionally performed in two modes 305B, while the analyte is reverse transcribed into cDNA, and an amplicon library is prepared 306 and sequenced 307. The two sets of images are then spatially overlaid to correlate spatially identifiable sample information 308. When the sample and array are not subjected to a second imaging 305B, a point coordinate file is provided by the manufacturer. The point coordinate file replaces the second imaging step 305B. In addition, a unique PCR adaptor and sequencing can be used for amplicon library preparation.

[0237] Figure 4Another exemplary workflow is shown, which utilizes a spatially labeled array on a substrate, where capture probes labeled with spatial barcodes are clustered in regions called features. The spatially labeled capture probes may include cleavage domains, one or more functional sequences, spatial barcodes, unique molecular identifiers, and capture domains. The spatially labeled capture probes may also include 5' end modifications for reversible attachment to the substrate. The spatially barcode array contacts sample 401 and permeates the sample by applying a permeation reagent 402. The permeation reagent can be administered by placing the array / sample assembly in a bulk solution. Alternatively, the permeation reagent can be administered to the sample via an antidiffusion medium and / or a physical barrier (e.g., a cap), wherein the sample is sandwiched between the antidiffusion medium and / or barrier and the substrate containing the array. Analytes are migrated to the spatially barcode-labeled capture array using any number of techniques disclosed herein. For example, analyte migration can be performed using an antidiffusion medium cap and passive migration. As another example, analyte migration can be active migration, for example, using an electrophoretic transfer system. Once the analyte is near the spatially barcode-labeled capture probe, the capture probe may hybridize or otherwise bind to the target analyte 403. The sample can be optionally removed from array 404.

[0238] The capture probe can optionally be cleaved from 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 template-converting oligonucleotides. For example, the template-converting oligonucleotides can hybridize with a poly(C) tail added to the 3′ end of the cDNA via reverse transcriptase. The original mRNA template and template-converting oligonucleotides can be denatured from the cDNA, and then the barcoded capture probe can hybridize with the cDNA to generate a cDNA complement. 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 are side-attached to the spatial barcode of interest and the analyte region, producing a library associated with a specific spatial barcode. In some embodiments, the cDNA contains sequencing-by-synthesis (SBS) primer sequences. The library amplicon is sequenced and analyzed to decode the spatial information 407.

[0239] Figure 5An exemplary workflow is described, in which a sample is removed from a spatially barcoded array, and a spatially barcoded capture probe is removed from the array for barcoded analyte amplification and library preparation. Another embodiment includes first-strand synthesis using template-converting oligonucleotides on the spatially barcoded array without cleaving the capture probe. In this embodiment, sample preparation 501 and permeabilization 502 are performed as described elsewhere herein. Once the capture probe captures the target analyte, the first-strand cDNA generated via template conversion and reverse transcriptase 503 is subsequently denatured, and the second strand is subsequently extended 504. The second-strand cDNA is then denatured, neutralized, and transferred from the first-strand cDNA to tube 505. cDNA quantification and amplification can be performed using the standard techniques discussed herein. The cDNA can then undergo library preparation 506 and indexing 507, including fragmentation, end repair and A-tailing, and indexing PCR steps.

[0240] In some non-limiting examples of the above workflow, the sample can be immersed in 100% frozen methanol and incubated at -20°C for 30 minutes. After 20 minutes, the sample can be removed and rinsed with 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 the laboratory wipe and air dry). The sample can be evenly covered in 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 the laboratory wipe). The slide containing the sample can be immersed in water, and excess liquid can be removed. Then, the sample is 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 methods disclosed herein can be used to image the sample.

[0241] The following are non-limiting exemplary steps for sample permeabilization and cDNA generation. The sample can be exposed to the permeabilizing enzyme and incubated at 37°C for 6 minutes. Other permeabilization methods are described herein. The permeabilizing enzyme can be removed by adding SSC buffer, and a sample can be prepared for analyte capture. The sample is then subjected to a pre-equilibrated thermal cycling protocol, and the SSC buffer is removed. A master mixture containing nuclease-free water, reverse transcriptase reagent, template-converting oligonucleotide, reducing agent, and reverse transcriptase can be added, and the sample with the master mixture 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. Sodium hydroxide can be removed, and elution buffer can be added and removed from the sample. A second-strand mixture, 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, elution buffer is added, the sample is removed, sodium hydroxide is added to the sample, and the sample is incubated at room temperature for 10 minutes. Tris-HCl can be added and the reagents mixed.

[0242] The following steps are non-limiting exemplary procedures for cDNA amplification and quality control. A qPCR mixture, comprising nuclease-free water, a qPCR master mixture, and cDNA primers, can be prepared, and a NaOH / Tris-HCl mixture can be mixed with the qPCR mixture and the sample, and thermally cycled according to a predetermined thermal cycling protocol. After thermal cycling is complete, a cDNA amplification mixture can be prepared and combined with the sample. The sample is then incubated and thermally cycled. The sample can then be resuspended in SPRIselect reagent and pipetted to ensure proper mixing. The sample can then be incubated at room temperature for 5 minutes and cleaned by placing it on a magnet (e.g., with the magnet at a high position). The supernatant is removed, 80% ethanol is added to the precipitate, and incubation is performed for 30 seconds. The ethanol can be removed, and the precipitate can be washed again. The sample is then centrifuged and placed on a magnet (e.g., with the magnet at a low position). Any residual ethanol can be removed, and the sample can be air-dried. The magnet can be removed, 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., at a high position) until the solution becomes clear. Partial samples can be run on the Agilent Bioanalyst High Sensitivity Chip, where a region can be selected and cDNA concentration measured to calculate total cDNA yield. Alternatively, quantification can be performed using an Agilent Bioanalyst or Agilent TapeStation.

[0243] The following steps are non-limiting exemplary steps for constructing a spatial gene expression library. A fragmentation mixture, including fragmentation buffer and fragmentation enzyme, can be prepared on ice. Elution buffer and the fragmentation mixture can be added to each sample, mixed, and centrifuged. The sample mixture can then 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., at a 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., at a high position) until the solution is clear. The supernatant is removed, 80% ethanol is added to the precipitate, and the precipitate is incubated for 30 seconds to remove the ethanol. The ethanol wash can be repeated, with the sample placed on a magnet (e.g., at a low position) until the solution is clear. Residual 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., at a high position) until the solution becomes clear, and a portion of the sample can be transferred to a new tubing. A ligation mixture including ligation buffer, DNA ligase, and adaptor oligonucleotides can be prepared and centrifuged. The adaptor ligation mixture can be added to the sample, pipette-mixed, and briefly centrifuged. The sample can then be thermally cycled according to a predetermined protocol. SPRIsleect reagent can be added to the sample, incubated at room temperature for 5 minutes, and placed on a magnet (e.g., at a high position) until the solution becomes clear. The supernatant is removed, and the precipitate is washed with 80% ethanol and incubated for 30 seconds to remove the ethanol. The ethanol wash can be repeated, and the sample can be briefly centrifuged before placing it on a magnet (e.g., at a low position). Any residual ethanol can be removed, and the sample can be air-dried. Elution buffer can be added to the sample, the sample removed from the magnet, and then pipette-mixed, incubated at room temperature for 2 minutes, and placed on a magnet (e.g., at a low position) until the solution becomes clear. A portion of the sample can be transferred to a new tubing. Sample index PCR mixtures, including amplification mixtures and SI primers, can be prepared and combined with samples. Sample / sample index PCR mixtures can be loaded into individual Chromium i7 sample index wells and can be used with a thermal cycling protocol. SPRIselect reagent can be added to each sample, mixed, and incubated at room temperature for 5 minutes. Samples can be placed on a magnet (e.g., at a high position) until the solution is clear, and the supernatant can be transferred to a new tube. SPRIselect reagent can be added to each sample, pipette mixed, and incubated at room temperature for 5 minutes. Samples can then be placed on a magnet (e.g., at a high position) until the solution is clear. Remove the supernatant, wash the precipitate with 80% ethanol, incubate for 30 seconds, and then remove the ethanol. Ethanol washing can be repeated by centrifuging the sample and placing it on a magnet (e.g., at a low position) to remove any remaining ethanol.The sample can be removed from the magnet, elution buffer can be added to the sample, pipette mixing is performed, and incubation at room temperature for 2 minutes. The sample can be placed on the magnet (e.g., at a high position) until the solution becomes clear, and a portion of the sample can be transferred to a new strip. The average fragment size can be determined using a bioanalyzer tracer or an Agilent TapeStation.

[0244] In some implementations, correlation analysis of the data generated by this workflow and other workflows described herein can produce a correlation of more than 95% (e.g., 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher) across genes expressed in two capture regions. When the workflow is performed using single-cell RNA sequencing of the cell nucleus, in some implementations, correlation analysis of the data can produce a correlation of more than 90% (e.g., more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) across genes expressed in two capture regions.

[0245] (b) Capture probe

[0246] A “capture probe” is any molecule capable of capturing (directly or indirectly) and / or labeling an analyte of interest in a biological sample. In some embodiments, the capture probe is a nucleic acid or peptide. In some embodiments, the capture probe is a conjugate (e.g., an oligonucleotide-antibody conjugate). In some embodiments, the capture probe includes a barcode (e.g., a spatial barcode and / or a unique molecular identifier (UMI)) and a capture domain.

[0247] Figure 6This is a schematic diagram illustrating an example of a capture probe as described herein. As shown, capture probe 602 is optionally coupled to feature 601 via a cleavage domain 603, such as a disulfide linker. The capture probe may include functional sequences useful for subsequent processing, such as functional sequence 604, which may include sequencer-specific flow cell attachment sequences, such as the P5 sequence, and functional sequence 606, which may include sequencing primer sequences, such as the R1 primer binding site. In some embodiments, sequence 604 is the P7 sequence and sequence 606 is the R2 primer binding site. Spatial barcode 605 may be included within the capture probe for barcoding of the target analyte. Functional sequences are typically selected for compatibility 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, functional sequences may be selected for compatibility with non-commercial sequencing systems. Examples of such sequencing systems and techniques that can use appropriate functional sequences include (but are not limited to) Roche 454 sequencing, ion-flux proton or PGM sequencing, Illumina X10 sequencing, PacBio SMRT sequencing, and Oxford nanopore sequencing. Furthermore, in some implementations, functional sequences can be selected for compatibility with other sequencing systems, including non-commercial sequencing systems.

[0248] In some implementations, the spatial barcode 605, functional sequence 604 (e.g., flow cell attachment sequence), and 606 (e.g., sequencing primer sequence) may be shared by all probes attached to a given feature. The spatial barcode may also include a capture field 607 to facilitate the capture of the target analyte.

[0249] Capture Domain

[0250] As described above, each capture probe includes at least one capture domain. A “capture domain” is an oligonucleotide, peptide, small molecule, or any combination thereof that specifically binds to the desired analyte. In some embodiments, the capture domain can be used to capture or detect the desired analyte.

[0251] 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) configured to interact with multiple DNA molecules. In some embodiments, the functional sequence may include a poly(T) sequence configured to interact with messenger RNA (mRNA) molecules via a poly(A) tail of an 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.

[0252] The capture probe may include ribonucleotides and / or deoxyribonucleotides, as well as 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, along with an RNA or DNA sequence (e.g., degenerate RNA), may be used to ligate single-stranded DNA or RNA to the capture domain. In some embodiments, ligases with RNA template ligase activity (e.g., SplintR ligase, T4 RNA ligase 2, or KOD ligase) may be used to ligate single-stranded DNA or RNA to the capture domain. In some embodiments, the capture domain comprises a splint oligonucleotide. In some embodiments, the capture domain captures the splint oligonucleotide.

[0253] 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 with nucleic acids (e.g., RNA or other analytes) present in cells of a tissue sample in contact with the array. In some embodiments, the capture domain may be selected or designed to bind selectively or specifically to a target nucleic acid. For example, the capture domain may be selected or designed to capture mRNA by hybridizing with the poly(A) tail of mRNA. Thus, in some embodiments, the capture domain includes a poly(T) DNA oligonucleotide, which is a series of consecutive deoxythymidine residues linked by phosphodiester bonds that are capable of hybridizing with the poly(a) tail of mRNA. In some embodiments, the capture domain may include a nucleotide that is functionally or structurally similar to a poly(T) tail. For example, a poly(U) oligonucleotide or an oligonucleotide containing a deoxythymidine analogue. In some embodiments, the capture domain comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides. In some embodiments, the capture domain comprises at least 25, 30, or 35 nucleotides.

[0254] In some implementations, a random sequence (e.g., a random hexamer or similar sequence) may be used to form all or part of the capture domain. For example, the random sequence may be used in conjunction with a poly(T) (or poly(T) analog) sequence. Therefore, in cases where the capture domain comprises a poly(T) (or “poly(T)-like”) oligonucleotide, it may also comprise a random oligonucleotide sequence (e.g., a “poly(T)-random sequence” probe). This may be located, for example, at the 5' or 3' of the poly(T) sequence, such as at the 3' end of the capture domain. The poly(T)-random sequence probe facilitates the capture of the poly(A) tail of the mRNA. In some implementations, the capture domain may be a completely random sequence. In some implementations, a degenerate capture domain may be used.

[0255] In some embodiments, sets of two or more capture probes form 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, sets of two or more capture probes form 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, sets of two or more capture probes form 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, a probe having a degenerate capture domain may be added to any of the foregoing combinations listed herein. In some embodiments, a probe having a degenerate capture domain may replace one of the probes in each pair described herein.

[0256] The capture domain may be based on a specific gene sequence or motif sequence or common / conserved sequence that it is designed to capture (i.e., a sequence-specific capture domain). Therefore, in some embodiments, the capture domain is capable of selectively binding to a desired nucleic acid subtype or subset, such as a specific type of RNA, such as mRNA, rRNA, tRNA, SRP RNA, tRNA, snRNA, snRNA, SmY RNA, sARNA, gRNA, RNase P, RNase 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 acid, such as microbiome RNA, such as 16S rRNA.

[0257] In some embodiments, the capture domain includes an "anchor" or "anchor sequence," which is a nucleotide sequence designed to ensure hybridization of the capture domain with a desired bioanalyte. In some embodiments, the anchor sequence includes 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 including a poly(T) sequence can be designed to capture mRNA. In such embodiments, the anchor sequence may include a random 3-mer (e.g., GGG) that helps ensure hybridization of the poly(T) capture domain with mRNA. In some embodiments, the anchor sequence may 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.

[0258] In some embodiments, the capture domain of the capture probe is blocked before the biological sample comes into contact with the array, and blocking probes are used when nucleic acids in the biological sample are modified before they are captured onto the array. In some embodiments, blocking probes are used to block or modify the free 3' end of the capture domain. In some embodiments, blocking probes may hybridize with capture probes to mask the free 3' end of the capture domain, such as hairpin probes or partially double-stranded probes. In some embodiments, the free 3' end of the capture domain can be blocked by chemical modification, for example, by adding an azidomethyl group as a chemically reversible capping moiety, such that the capture probe does not include a free 3' end. Blocking or modifying the capture probe, particularly the free 3' end of the capture domain, before the biological sample comes into contact with the array prevents modification of the capture probe, for example, preventing the addition of a poly(A) tail to the free 3' end of the capture probe.

[0259] Non-limiting examples of 3′ modifications include dideoxyC-3′ (3′-ddC), 3′ inverted dT, 3′C3 spacers, 3′ amino groups, and 3′ phosphorylation. In some embodiments, nucleic acids in a biological sample can be modified such that they can be captured by a capture domain. For example, an adaptor sequence (including a binding domain capable of binding to the capture domain of a capture probe) can be added to the end of a nucleic acid (e.g., fragmented genomic DNA). In some embodiments, this is achieved by ligating the adaptor sequence or by nucleic acid extension. In some embodiments, an enzyme is used to incorporate other nucleotides, such as poly(A) tails, at the ends of the nucleic acid sequence. 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 rendered inaccessible such that the capture domain is less susceptible to the effects of processes (e.g., ligation or extension) used to modify the nucleic acid in the biological sample.

[0260] In some embodiments, the capture domain of the capture probe is modified to remove any modifications to the capture probe that occur during the modification of nucleic acid molecules in the biological sample. In some embodiments, the capture probe may include other sequences downstream of the capture domain, i.e., up to the 3′ of the capture domain, i.e., a blocking domain.

[0261] In some implementations, the capture domain of the capture probe may be a non-nucleic acid domain. Examples of suitable capture domains that are not entirely based on nucleic acids include, but are not limited to, proteins, peptides, aptamers, antigens, antibodies, and molecular analogs that mimic the function of any capture domain described herein.

[0262] Fracturing domain

[0263] Each capture probe may optionally include at least one cleavage domain. A cleavage domain represents a portion of the probe used to reversibly attach the probe to the array feature, as described below. Furthermore, 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, spatial barcodes and / or universal molecular identifiers (UMIs) may be released by cleaving the cleavage domain.

[0264] Figure 7 This is a schematic diagram illustrating a cleavable capture probe, where the cleaved capture probe can enter non-permeable cells and bind to the target analyte within the sample. Capture probe 701 includes a cleavage domain 702, a cell-penetrating peptide 703, a reporter molecule 704, and a disulfide bond (-SS-). 705 represents all other parts of the capture probe, such as the spatial barcode and the capture domain.

[0265] In some embodiments, the cleavage domain connecting 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 specific locations. 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).

[0266] Other examples of cleavage domains include unstable chemical bonds, such as, but not limited to, ester bonds (e.g., cleavable by acids, bases, or hydroxylamine), vicinal diol bonds (e.g., cleavable by sodium periodate), Diels-Alder bonds (e.g., cleavable by heat), sulfone bonds (e.g., cleavable by bases), silyl ether bonds (e.g., cleavable by acids), glycosidic bonds (e.g., cleavable by amylases), peptide bonds (e.g., cleavable by proteases), or phosphodiester bonds (e.g., cleavable by nucleases (e.g., DNases)).

[0267] In some embodiments, the cleavage domain includes a sequence recognized by one or more enzymes capable of cleaving nucleic acid molecules, such as those capable of breaking phosphodiester bonds between two or more nucleotides. These bonds can be cleaved by other nucleic acid molecule-targeting enzymes, such as restriction enzymes (e.g., restriction endonucleases). For example, the cleavage domain may include a restriction endonuclease (restriction enzyme) recognition sequence. Restriction endonucleases cleave double-stranded or single-stranded DNA at a specific recognition nucleotide sequence known as a restriction site. In some embodiments, rare-cut restriction enzymes, i.e., enzymes with long recognition sites (at least 8 base pairs in length), are used to reduce the likelihood of cleavage at other locations within the capture probe.

[0268] In some embodiments, the cleavage domain includes a poly(U) sequence, which may be generated by uracil DNA glycosylase (UDG) and DNA glycosylase-lyase endonuclease VIII (commercially known as USER). TM The mixture of enzymes cleaves the target. Once released, the releasable capture probe can be used for the reaction. Therefore, for example, the activatable capture probe can be activated by releasing the capture probe from the target.

[0269] In some embodiments, when the capture probe is indirectly attached to the substrate, for example via a surface probe, the cleavage domain comprises 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 may be one, two, or three base pairs). Such mismatches are recognized, for example, by MutY and T7 endonuclease I enzymes, leading to the cleavage of the nucleic acid molecule at the mismatch site.

[0270] In some embodiments, when the capture probe is indirectly attached to the feature, for example via a surface probe, the cleavage domain includes a nickase recognition site or sequence. A nickase is a nuclease that can only cleave single strands of a DNA double-strand. Therefore, the cleavage domain may 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 double-strand between the surface probe and the capture probe, thereby releasing the capture probe from the feature.

[0271] Cleavage enzymes can also be used in some embodiments where the capture probe is directly attached to the feature. For example, the substrate can be contacted with a nucleic acid molecule hybridized to the cleavage domain of the capture probe to provide or reconstitute a cleavage enzyme recognition site, such as a cleavage helper probe. Therefore, contact with the cleavage enzyme will result in cleavage of the cleavage domain, thereby releasing the capture probe from the feature. Such cleavage helper probes can also be used to provide or reconstitute cleavage recognition sites for other cleavage enzymes, such as restriction endonucleases.

[0272] Some nickases introduce single-strand cleavage only at specific sites on the DNA molecule by binding to and recognizing specific nucleotide recognition sequences. Many natural nickases have been discovered, and the sequence recognition characteristics of at least four nickases have been identified. 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 a complementary nickase recognition site on the cleavage domain. After use, the nickase can be removed from the analysis or inactivated after the release of the capture probe to prevent unnecessary cleavage of the capture probe.

[0273] This includes not only examples of suitable capture domains based on nucleic acids, but also, but not limited to, proteins, peptides, aptamers, antigens, antibodies, and molecular analogs that mimic the function of any capture domain described herein.

[0274] In some implementations, the capture probe does not contain a cleavage domain. For example, an example of a substrate with an attachment capture probe lacking a cleavage domain is described in Macosko et al., (2015) Cell 161, 1202–1214, the entire contents of which are incorporated herein by reference.

[0275] 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 may be included at the location where the cleavage domain would normally be located. In such embodiments, the region may complement the functional domain or even exist as another functional domain. In some embodiments, a cleavage domain is present, but its use is optional.

[0276] Functional domain

[0277] Each capture probe may optionally include at least one functional domain. Each functional domain typically includes a functional nucleotide sequence for downstream analytical steps throughout the analytical procedure.

[0278] In some implementations, the capture probe may include a functional domain for attaching to the sequencing flow cell, for example, for... The sequenced P5 sequence. In some embodiments, the capture probe or a derivative thereof may include another functional domain, for example, for attachment to... The P7 sequence of the sequencing flow cell. Selectable functional domains allow for compatibility with any of the various sequencing systems, such as 454 sequencing, ion-fluid proton or PGM, Illumina X10, and their requirements.

[0279] In some implementations, the functional domain includes a primer. The primer may include components for use with... The R1 primer sequence for sequencing, and in some embodiments, including those for... The R2 primer sequence for sequencing. Examples of such capture probes and their uses are described in U.S. Patent Publications 2014 / 0378345 and 2015 / 0376609, the entire contents of which are incorporated herein by reference.

[0280] Spatial Barcode

[0281] As described above, the capture probe may include one or more spatial barcodes (e.g., two or more, three or more, four or more, five or more). A “spatial barcode” is a continuous nucleic acid segment or two or more non-contiguous nucleic acid segments used as a tag or identifier that conveys or is capable of conveying spatial information. In some embodiments, the capture probe includes spatial barcodes with spatial aspects, wherein the barcodes are associated with a specific location within an array or a specific location on a substrate.

[0282] Spatial barcodes can be part of the analyte or independent of the analyte (i.e., part of the capture probe). A spatial barcode can be a tag attached to the analyte (e.g., a nucleic acid molecule) or a combination of tags other than endogenous characteristics of the analyte (e.g., the size or terminal sequence of the analyte). Spatial barcodes can be unique. In some implementations where the spatial barcode is unique, it functions both as a spatial barcode and as a unique molecular identifier (UMI) associated with a specific capture probe.

[0283] Spatial barcodes can have a variety of different formats. For example, spatial barcodes may include polynucleotide spatial barcodes, random nucleic acid and / or amino acid sequences, and synthetic nucleic acid and / or amino acid sequences. In some embodiments, spatial barcodes are attached to analytes in a reversible or irreversible manner. In some embodiments, spatial barcodes are added to fragments of, for example, DNA or RNA samples before, during, and / or after sample sequencing. In some embodiments, spatial barcodes enable the identification and / or quantification of individual sequence readouts. In some embodiments, spatial barcodes are used as fluorescent barcodes, wherein fluorescently labeled oligonucleotide probes hybridize with the spatial barcode.

[0284] In some embodiments, the spatial barcode is a nucleic acid sequence that substantially does not hybridize with the analyte nucleic acid molecule in the biological sample. In some embodiments, the spatial barcode has less than 80% sequence identity to the nucleic acid sequence on a substantial portion (e.g., 80% or more) of the nucleic acid molecule in the biological sample (e.g., less than 70%, 60%, 50%, or less than 40% sequence identity).

[0285] The spatial barcode sequence may include about 6 to about 20 or more nucleotides within the capture probe sequence. In some embodiments, the length of the spatial barcode sequence may 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 may 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 may be at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or shorter.

[0286] These nucleotides can be completely continuous, i.e., in a single stretch of adjacent nucleotides, or they can be divided into two or more separate subsequences separated by one or more nucleotides. The length of the separated spatial barcode subsequences is approximately 4 to 16 nucleotides. In some embodiments, the spatial barcode subsequence 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 subsequence 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 subsequence can be at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or shorter.

[0287] For multiple capture probes attached to a common array feature, one or more spatial barcode sequences of the multiple capture probes may include the same sequence for all capture probes coupled to the feature and / or different sequences among all capture probes coupled to the feature.

[0288] Figure 8 This is a schematic diagram illustrating an exemplary multi-spatial labeling feature. Figure 8 In this design, feature 801 can be coupled to a spatially barcoded capture probe, wherein the spatially barcoded probe for a specific feature may 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 having a spatial barcode 802. One type of capture probe associated with this feature includes a combination of spatial barcode 802 and a poly(T) capture domain 803, designed to capture mRNA target analytes. A second type of capture probe associated with this feature includes a 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 a combination of spatial barcode 802 and a capture domain complementary to a capture domain on the analyte capture agent capture barcode structural domain 805. A fourth type of capture probe associated with this feature includes a combination of a spatial barcode 802 and a capture probe that specifically binds to nucleic acid molecule 806, which can function in CRISPR analysis (e.g., CRISPR / Cas9). Although Figure 8 Only four different capture probe barcoded constructs are shown, but capture probe barcoded constructs can be customized for the analysis of any given analyte related to nucleic acids and can be bound to such constructs. For example, Figure 8The protocols 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, available chromatin (e.g., ATAC-seq, DNase-seq, and / or MNAase-seq), cell surface or intracellular proteins and metabolites, and perturbators (e.g., CRISPR-crRNA / sgRNA, TALEN, zinc finger nucleases, and / or antisense oligonucleotides as described herein); and (c) V(D)J sequences of mRNA, cell surface or intracellular proteins and / or metabolites, barcode markers (e.g., MHC multimers as described herein), and immune cell receptors (e.g., T cell receptors).

[0289] Capture probes attached to a single array feature may include the same (or common) spatial barcode sequence, different spatial barcode sequences, or a combination of both. Capture probes attached to a feature may include multiple sets of capture probes. A given set of capture probes may include the same spatial barcode sequence. The same spatial barcode sequence may differ from the spatial barcode sequence of another set of capture probes.

[0290] Multiple capture probes may include spatial barcode sequences (e.g., nucleic acid barcode sequences) associated with specific locations on a spatial array. For example, a first plurality of capture probes may be associated with a first region based on a spatial barcode sequence common to capture probes within a first region, and a second plurality of capture probes may be associated with a second region based on a spatial barcode sequence common to capture probes within a second region. The second region may or may not be associated with the first region. Other plurality of capture probes may be associated with spatial barcode sequences common to capture probes within other regions. In some embodiments, the spatial barcode sequence may be identical across the plurality of capture probe molecules.

[0291] In some implementations, multiple distinct spatial barcodes are combined into a single array capture probe. For example, a mixed but known set of spatial barcode sequences can provide stronger addressing or attributes of the spatial barcode to a given point or location by offering repeated or independent verification of location characteristics. In some implementations, multiple spatial barcodes represent an increased specificity of the location of a particular array point.

[0292] Unique molecular identifier

[0293] Capture probes 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 continuous nucleic acid segment or two or more non-continuous nucleic acid segments that serve as a tag or identifier for a particular analyte or a capture probe bound to a particular analyte (e.g., by a capture domain).

[0294] UMIs can be unique. A 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.

[0295] In some embodiments, the UMI is a nucleic acid sequence that substantially does not hybridize with the analyte nucleic acid molecule in the biological sample. In some embodiments, the UMI has less than 80% sequence identity to the nucleic acid sequence in a substantial portion (e.g., 80% or more) of the nucleic acid molecule in the biological sample (e.g., less than 70%, 60%, 50%, or less than 40% sequence identity).

[0296] The UMI may include about 6 to about 20 or more nucleotides within the capture probe sequence. In some embodiments, the length of the UMI may 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 may 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 may be at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or shorter.

[0297] These nucleotides can be completely continuous, i.e., within a single sequence segment of adjacent nucleotides, or they can be divided into two or more separate subsequences separated by one or more nucleotides. The length of the separated UMI subsequence is approximately 4 to 16 nucleotides. In some embodiments, the UMI subsequence can be about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some embodiments, the UMI subsequence 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 subsequence can be at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or shorter.

[0298] 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, DNA or RNA samples before, during, and / or after sequencing the analyte. In some embodiments, the UMI enables the identification and / or quantification of individual sequence reads. In some embodiments, the UMI is used as a fluorescent barcode, wherein a fluorescently labeled oligonucleotide probe hybridizes with the UMI.

[0299] Other aspects of the capture probe

[0300] For capture probes attached to an array feature, an individual array feature may include one or more capture probes. In some embodiments, an individual array feature includes hundreds or thousands of capture probes. In some embodiments, capture probes are associated with a specific individual feature, wherein the individual feature contains capture probes, which include spatial barcodes unique to a defined area or location on the array.

[0301] In some implementations, a particular feature may include capture probes comprising more than one spatial barcode (e.g., one capture probe at a particular feature may include a spatial barcode different from the spatial barcode included in another capture probe at the same particular feature, while both capture probes include a second common spatial barcode), wherein each spatial barcode corresponds to a specific defined region or location on the array. For example, multiple spatial barcode sequences associated with a particular feature on the array can provide a stronger address or attribute to a given location by providing repeating or independent confirmation of the location. In some implementations, multiple spatial barcodes represent an increased specificity of the location of a particular array point. In a non-limiting example, a particular array point may be encoded using two different spatial barcodes, wherein each spatial barcode identifies a specific defined region within the array, and an array point having both spatial barcodes identifies a sub-region where the two defined regions overlap, such as, for example, the overlapping portion of a Venn diagram.

[0302] In another non-limiting example, a particular array point can be encoded with three different spatial barcodes, wherein a first spatial barcode identifies a first region within the array, a second spatial barcode identifies a second region, wherein the second region is a sub-region entirely within the first region, and a third spatial barcode identifies a third region, wherein the third region is a sub-region entirely within the first and second sub-regions.

[0303] In some embodiments, the capture probe attached to the array feature is released from the array feature for sequencing. Alternatively, in some embodiments, the capture probe remains attached to the array feature and is sequenced while remaining attached to the array feature (e.g., by in situ sequencing). Other aspects of sequencing of the capture probe are described in subsequent sections of the invention.

[0304] In some implementations, an array feature may include different types of capture probes attached to the feature. For example, an array feature may 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 class of analytes, and the second type of capture probe having a capture domain designed to bind to a second type of analytes. Typically, an array feature may 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.

[0305] In some embodiments, the capture probe is a nucleic acid. In some embodiments, the capture probe is attached to an array feature via its 5' end. In some embodiments, the capture probe from its 5' end to its 3' 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 its 5' end to its 3' end includes: a barcode (e.g., a spatial barcode or UMI) and a capture domain. In some embodiments, the capture probe from its 5' end to its 3' 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 its 5' end to its 3' end includes: 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 from its 5' end to its 3' end includes: a cleavage domain, a functional domain, a spatial barcode, a UMI, and a capture domain. In some embodiments, the capture probe does not include a spatial barcode. In some embodiments, the capture probe does not include a UMI. In some implementations, the capture probe includes a sequence used to initiate the sequencing reaction.

[0306] In some embodiments, the capture probe is fixed to the feature via its 3' end. In some embodiments, the capture probe from its 3' end to its 5' end includes: one or more barcodes (e.g., spatial barcodes and / or UMIs) and one or more capture fields. In some embodiments, the capture probe from its 3' end to its 5' end includes: a barcode (e.g., a spatial barcode or a UMI) and a capture field. In some embodiments, the capture probe from its 3' end to its 5' end includes: a fragmentation field, a functional field, one or more barcodes (e.g., spatial barcodes and / or UMIs) and a capture field. In some embodiments, the capture probe from its 3' end to its 5' end includes: a fragmentation field, a functional field, a spatial barcode, a UMI, and a capture field.

[0307] 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, one or more of which are used as initiation 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, such as a variable barcode sequence. In some embodiments, the in-situ synthesized oligonucleotide is attached to features of the array.

[0308] In some embodiments, the capture probe is the product of two or more oligonucleotide sequences, for example, two or more oligonucleotide sequences linked together. In some embodiments, one of the oligonucleotide sequences is an oligonucleotide synthesized in situ.

[0309] In some embodiments, the capture probe comprises a splint oligonucleotide. Two or more oligonucleotides can be ligated together using the splint oligonucleotide and any type of ligase known in the art or described herein (e.g., splint ligase).

[0310] In some embodiments, one of the oligonucleotides includes: a constant sequence (e.g., a sequence partially complementary to a splint oligonucleotide), a degenerate sequence, and a capture domain (e.g., as described herein). In some embodiments, the capture probe is generated by causing an enzyme to add a polynucleotide to the end of the oligonucleotide sequence. The capture probe may include a degenerate sequence that can be used as a unique molecular identifier.

[0311] The capture probe may include a degenerate sequence, which is a sequence of nucleotides containing a number of possible bases at certain positions. The degenerate sequence may be a degenerate nucleotide sequence comprising 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 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 0, 10, 15, 20, 25, or more degenerate positions within the nucleotide sequence. In some embodiments, the degenerate sequence is used as a UMI.

[0312] In some embodiments, the capture probe comprises a restriction endonuclease recognition sequence or a nucleotide sequence that can be cleaved by a specific enzyme activity. For example, a uracil sequence can be cleaved by a specific enzyme activity. As another example, other modified bases (e.g., methylated) can be recognized and cleaved by specific endonucleases. 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 modification. Removing the blocking domain will reveal and / or restore the free 3' end of the capture domain of the capture probe. In some embodiments, other nucleotides may be removed to reveal and / or restore the 3' end of the capture domain of the capture probe.

[0313] In some embodiments, the blocking domain may 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., a 3′-O-blocking reversible terminator and a 3′-unblocking reversible terminator) and may be included in the capture probe during or after probe synthesis.

[0314] Extended capture probe

[0315] "Extended capture probe" is a capture probe with an expanded nucleic acid sequence. For example, in cases where the capture probe comprises nucleic acids, "extended 3' end" means that additional nucleotides are added to the terminal 3' nucleotide of the capture probe to extend its length, for example, 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).

[0316] In some implementations, extending the capture probe includes generating cDNA from the captured (hybridized) RNA. This process involves synthesizing a complementary strand of the hybridized nucleic acid, for example, generating cDNA based on a 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 a template for extension (e.g., a reverse transcription step).

[0317] In some embodiments, the capture probe uses reverse transcription extension. For example, reverse transcription includes the synthesis of cDNA (complementary or copy DNA) from RNA (e.g., messenger RNA) using a reverse transcriptase. In some embodiments, reverse transcription is performed while the tissue is still in situ, producing an analyte library, wherein the analyte library includes spatial barcodes from adjacent capture probes. In some embodiments, the capture probe uses one or more DNA polymerases for extension.

[0318] In some embodiments, the capture domain of the capture probe includes primers for generating a complementary strand of a nucleic acid that hybridizes to the capture probe, such as primers for DNA polymerase and / or reverse transcription. The nucleic acid (e.g., DNA and / or cDNA) molecule produced by the extension reaction contains the sequence of the capture probe. The extension of the capture probe, such as by DNA polymerase and / or reverse transcription, can be performed using a variety of suitable enzymes and protocols.

[0319] In some embodiments, a full-length DNA molecule (e.g., cDNA) is 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 differ in length from the initial RNA in the tissue sample. In some embodiments, the 3' end of the extended probe (e.g., a first-strand cDNA molecule) is modified. For example, a adapter or adaptor can be attached to the 3' end of the extended probe. This can be achieved by using a single-stranded ligase such as T4 RNA ligase or CircleGase. TM (Available from Epicentre Biotechnologies, Madison, Wisconsin). In some embodiments, template-converting oligonucleotides are used to extend cDNA to produce full-length cDNA (or cDNA as close to full-length as possible). In some embodiments, a second-strand synthetic helper probe (a partial 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, such as the first-stranded cDNA molecule, using a double-stranded ligase (e.g., 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, and Thermococcus spp. (strain 9). o N) DNA ligase (9 o N TM DNA ligase, New England Biolabs, Ampligase TM(Available from Epicentre Biotechnologies, Madison, Wisconsin) and SplintR (available from New England Biolabs, Ipswich, Massachusetts). In some embodiments, a polynucleotide tail (e.g., a poly(A) tail) is incorporated into the 3' end of the extended probe molecule. In some embodiments, a terminal transferase-active enzyme is used to incorporate the polynucleotide tail.

[0320] In some implementations, the double-stranded extended capture probe is treated to remove any unextended capture probes prior to amplification and / or analysis (e.g., sequence analysis). This can be achieved through various methods, such as enzymatic degradation of unextended probes, like exonucleases, or the use of purification columns.

[0321] In some embodiments, the extended capture probe is amplified to produce a sufficient quantity for analysis, such as by DNA sequencing. In some embodiments, the first strand of the extended capture probe (e.g., DNA and / or cDNA molecules) is used as a template for an amplification reaction (e.g., polymerase chain reaction).

[0322] In some embodiments, the amplification reaction uses primers comprising an affinity group to incorporate the affinity group onto an extended capture probe (e.g., RNA-cDNA hybridization). In some embodiments, the primers comprise an affinity group, and the extended capture probe comprises an affinity group. The affinity group may correspond to any of the affinity groups described above.

[0323] In some embodiments, the extended capture probe, including an affinity group, can be coupled to an affinity group-specific array feature. In some embodiments, the substrate may include an antibody or 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 a maltose-binding protein. In some embodiments, the array feature includes a maltose-binding protein, and the affinity group includes maltose. In some embodiments, the amplified extended capture probe can serve to release the amplified probe from the array feature, provided that a copy of the extended probe is not attached to the array feature.

[0324] In some embodiments, a capture probe or its complement or amplicon extended from the array feature is released. The step of releasing the capture probe or its complement or amplicon extended from the array feature can be implemented in a variety of ways. In some embodiments, the capture probe or its complement is released from the feature by nucleic acid lysis and / or denaturation (e.g., by heating to denature double-stranded molecules).

[0325] In some embodiments, extended capture probes or their complements or amplicones are released from an array feature 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 using a solution that interferes with the hydrogen bonds of the double-stranded molecules. In some embodiments, extended capture probes are released by applying heated water (e.g., water at least 85°C or a buffer solution, such as 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, solutions including salts, surfactants, etc., are added to further destabilize the interactions between nucleic acid molecules to release extended capture probes from the array feature. In some embodiments, formamide solutions can be used to destabilize the interactions between nucleic acid molecules to release extended capture probes from the array feature.

[0326] Analyte trap

[0327] This invention also provides methods and materials for spatial profiling of bioanalytes (e.g., mRNA, genomic DNA, proteins and / or metabolites accessible to chromatin and cell surface or intracellular) using analyte trapping agents. As used herein, an analyte trapping agent (formerly sometimes also referred to as a "cell marker") is a substance that interacts with an analyte (e.g., an analyte in a sample) and a trapping probe (e.g., a trapping probe attached to a substrate) to identify the analyte. In some embodiments, the analyte trapping agent includes an analyte-binding portion and a trapping agent barcode domain.

[0328] Figure 9 This is a schematic diagram of an exemplary analyte trapping agent 902, comprising an analyte binding portion 904 and a trapping agent barcode domain 908. The analyte binding portion 904 is a molecule capable of binding to an analyte 906 and interacting with a spatially barcode-coded trapping probe. The analyte binding portion can bind to the analyte 906 with high affinity and / or high specificity. The analyte trapping agent may include the trapping agent barcode domain 908, a nucleotide sequence (e.g., an oligonucleotide), which can hybridize to at least part or all of the trapping domain of the trapping probe. The analyte binding portion 904 may include a polypeptide and / or aptamer (e.g., an oligonucleotide or peptide molecule bound to a specific target analyte). The analyte binding portion 904 may include an antibody or antibody fragment (e.g., an antigen-binding fragment).

[0329] As used herein, the term "analyte-binding moiety" refers to a molecule or portion capable of binding to a macromolecular component (e.g., an analyte, such as a bioanalyte). In some embodiments of any spatial profile analysis method described herein, the analyte-binding moiety of an analyte trap bound to a bioanalyte may include, but is not limited to, antibodies or epitope-binding fragments thereof, cell surface receptor-binding molecules, receptor ligands, small molecules, bispecific antibodies, bispecific T-cell conjugates, T-cell receptor conjugates, B-cell receptor conjugates, precursors, aptamers, monomers, adhesins, DARPin, and protein scaffolds, or any combination thereof. The analyte-binding moiety may bind to a macromolecular component (e.g., an analyte) with high affinity and / or high specificity. The analyte-binding moiety may include a nucleotide sequence (e.g., an oligonucleotide) that may correspond to at least part or all of the analyte-binding moiety. The analyte-binding moiety may include peptides and / or aptamers (e.g., peptides and / or aptamers bound to a specific target molecule, such as an analyte). The analyte-binding moiety may include an antibody or antibody fragment (e.g., an antigen-binding fragment) bound to a specific analyte (e.g., a peptide).

[0330] In some embodiments, the analyte trapping agent is capable of binding analytes present within the cell. In some embodiments, the analyte trapping agent is capable of binding cell surface analytes, which may include, but are not limited to, receptors, antigens, surface proteins, transmembrane proteins, differentiation protein clusters, protein channels, protein pumps, carrier proteins, phospholipids, glycoproteins, glycolipids, cell-cell interaction protein complexes, antigen-presenting complexes, major histocompatibility complexes, engineered T cell receptors, T cell receptors, B cell receptors, chimeric antigen receptors, extracellular matrix proteins, and post-translational modifications (e.g., phosphorylation, glycosylation, ubiquitination, nitrosation, methylation, acetylation, or lipidation) states, gap junctions, and adhesion junctions of cell surface proteins. In some embodiments, the analyte trapping agent is capable of binding to post-translational modified cell surface analytes. In such embodiments, the analyte trapping agent may be specific to cell surface analytes based on a given state of post-translational modification (e.g., phosphorylation, glycosylation, ubiquitination, nitrosation, methylation, acetylation, or lipidation), such that the cell surface analyte profile may include post-translational modification information of one or more analytes.

[0331] In some embodiments, the analyte capture agent includes a capture agent barcode domain coupled to or otherwise attached to the analyte binding moiety. In some embodiments, the capture agent barcode domain is covalently linked to the analyte binding moiety. 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 moiety barcode and an analyte capture sequence.

[0332] As used herein, the term "analyte binding moiety barcode" refers to a barcode associated with or otherwise identifying an analyte binding moiety. In some embodiments, the analyte bound to the analyte binding moiety can also be identified by recognizing the analyte binding moiety (by recognizing its associated analyte binding moiety barcode). The analyte binding moiety barcode can be a nucleic acid sequence of a given length and / or a sequence associated with the analyte binding moiety. The analyte binding moiety barcode can generally include any of the various aspects of barcodes described herein. For example, an analyte-specific analyte capture agent of one type may have a first capture agent barcode domain coupled thereto (e.g., which includes a first analyte binding moiety barcode), while analyte capture agents of different analyte specificities may have different capture agent barcode domains coupled thereto (e.g., which include a second barcode analyte binding moiety barcode). In some aspects, such a capture agent barcode domain may include an analyte binding portion barcode that couples the capture agent barcode domain to an analyte binding portion. The selection of the capture agent barcode domain allows for significant sequence diversity while also facilitating attachment to most analyte binding portions (e.g., antibodies) and easy detection (e.g., using sequencing or array technologies). In some embodiments, the analyte capture agent may include an analyte binding portion having a capture agent barcode domain attached thereto. For example, the analyte capture agent may include a first analyte binding portion (e.g., an antibody bound to an analyte, such as a first cell surface feature) having a capture agent barcode domain associated thereto, which includes the first analyte binding portion barcode.

[0333] In some embodiments, the capture agent barcode domain of the analyte capture agent includes an analyte capture sequence. As used herein, the term "analyte capture sequence" refers to a region or portion configured to hybridize to, bind to, couple to, or otherwise interact with the capture domain of a capture probe. In some embodiments, the analyte capture sequence includes a nucleic acid sequence complementary or substantially complementary to the capture domain of a capture probe, such that the analyte capture sequence hybridizes with the capture domain of the capture probe. In some embodiments, the analyte capture sequence comprises a poly(A) nucleic acid sequence that hybridizes with a capture domain containing a poly(T) nucleic acid sequence. In some embodiments, the analyte capture sequence comprises a poly(T) nucleic acid sequence that hybridizes with a capture domain containing a poly(A) nucleic acid sequence. In some embodiments, the analyte capture sequence comprises a non-homogeneous nucleic acid sequence that hybridizes with a capture domain containing a non-homogeneous nucleic acid sequence complementary (or substantially complementary) to a non-homogeneous nucleic acid sequence in the analyte capture region.

[0334] In some embodiments of any spatial analysis method described herein using analyte trapping agents, trapping agent barcode domains may be directly coupled to the analyte binding portion, or they may be attached to beads, molecular lattices, such as linear, spherical, cross-linked, or other polymers, or other frameworks attached to or otherwise associated with the analyte binding portion, enabling the attachment of multiple trapping agent barcode domains to a single analyte binding portion. The attachment (coupling) of trapping agent barcode domains to the analyte binding portion can be achieved through any of a variety of direct or indirect, covalent or non-covalent binding or attachment. For example, in the case where trapping agent barcode domains are coupled to an analyte binding portion comprising an antibody or antigen-binding fragment, chemical conjugation techniques (e.g., Lightning, available from Innova Biosciences) can be used. Antibody labeling kits) covalently attach these capture agent barcode domains to portions of antibody or antigen-binding fragments. In some embodiments, the capture agent barcode domains may be coupled to antibody or antigen-binding fragments using non-covalent attachment mechanisms (e.g., using biotinylated antibodies and oligonucleotides or beads comprising one or more biotinylated linkers, coupled to the oligonucleotides with avidin or streptavidin linkers). Antibody and oligonucleotide biotinylation techniques may be used, and are described, for example, in Fang et al., Nucleic Acids Res. (2003), 31(2):708-715, the entire contents of which are incorporated herein by reference. Similarly, protein and peptide biotinylation techniques have been developed and may be used, and are described, for example, in U.S. Patent No. 6,265,552, the entire contents of which are incorporated herein by reference. Furthermore, click reaction chemistry (e.g., methyltetraazine-PEG5-NHS ester reaction, TCO-PEG4-NHS ester reaction, etc.) may be used to couple capture agent barcode domains to analyte binding portions. The reactive portion on the analyte-binding moiety may also include an amine for targeting aldehydes, an amine for targeting maleimides (e.g., free thiol groups), an azide for targeting click chemistry compounds (e.g., alkynes), biotin for targeting streptavidin, or a phosphate for targeting EDCs, which in turn target active esters (e.g., NH2). The reactive portion on the analyte-binding moiety may be a compound or group bound to the reactive portion on the analyte-binding moiety. Exemplary strategies for coupling the analyte-binding moiety to the capture agent barcode domain include using commercial kits (e.g., Solulink, Thunderlink), mild reduction of the hinge region and maleimide-labeled binding, click chemistry reactions facilitated by staining of labeled amides (e.g., copper-free), coupling via periodate oxidation of sugar chains, and coupling with amines. In the case where the analyte-binding moiety is an antibody, the antibody may be modified prior to or concurrently with oligonucleotide coupling. For example, the antibody can be glycosylated using a β-1,4-galactosyltransferase-based mutant, GalT (Y289L), and an azide-containing uridine diphosphate-N-acetylgalactosamine analog, uridine diphosphate-GalNAz. The modified antibody can then be conjugated to an oligonucleotide having a dibenzocyclooctylene-PEG4-NHS group. In some embodiments, certain steps (e.g., COOH activation (e.g., EDC) and bifunctional crosslinking agents) can be avoided to prevent the analyte-binding moiety from conjugating to itself.In some embodiments of any spatial profiling method described herein, analyte traps (e.g., analyte-binding moieties coupled to oligonucleotides) may be delivered intracellularly via transfection (e.g., using transfected amines, cationic polymers, calcium phosphate, or electroporation), transduction (e.g., using phages or recombinant viral vectors), mechanical delivery (e.g., magnetic beads), lipid delivery (e.g., 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC)), or transport proteins. Analyte traps may be delivered intracellularly via exosomes. For example, a first cell may be generated that releases an exosome containing the analyte trap. The analyte trap may attach to the exosome membrane. The analyte trap may be contained in the cytoplasm of the exosome. The released exosome may be harvested and provided to a second cell, thereby delivering the analyte trap into the second cell. The analyte trap may be released from the exosome membrane before, during, or after delivery to the cell. In some embodiments, the cell is permeated to couple the analyte trap to intracellular cellular components, such as, but not limited to, intracellular proteins, metabolites, and nuclear membrane proteins. Following intracellular delivery, the analyte trap can be used to analyze the intracellular components as described herein.

[0335] In some embodiments of any spatial profiling analysis method described herein, the capture agent barcode domain coupled to the analyte capture agent may include modifications that prevent it from being amplified by polymerase. In some embodiments, the capture agent barcode domain may serve as a template instead of a primer when bound to the capture domain of a nucleic acid or capture probe in a sample used for primer extension reactions. When the capture agent barcode domain also includes a barcode (e.g., an analyte-binding partial barcode), such a design can improve the efficiency of molecular barcoding by increasing the affinity between the capture agent barcode domain and uncoded sample nucleic acids and eliminating the potential formation of adaptor artifacts. In some embodiments, the capture agent barcode domain may include a random N-mer sequence capped by modifications that prevent it from being extended by polymerase. In some cases, the composition of the random N-mer sequence may be designed to maximize binding efficiency with free, uncoded ssDNA molecules. Designs may include random sequence compositions with high GC content, partially random sequences with fixed G or C at specific positions, the use of guanosine, the use of locked nucleic acids, or any combination thereof.

[0336] Modifications that block primer extension via polymerase can be carbon spacer groups of varying lengths or dideoxynucleotides. In some embodiments, the modification can be a baseless site with a purine- or pyrimidine-free structure, a base analog, or an analog of a phosphate backbone, such as an N-(2-aminoethyl)-glycine backbone linked by an amide bond, tetrahydrofuran, or 1',2'-dideoxyribose. Modifications can also be uracil bases, 2'OMe-modified RNA, C3-18 spacers (e.g., structures with 3-18 consecutive carbon atoms, such as C3 spacers), ethylene glycol polymeric spacers (e.g., spacer 18 (hexaethylene glycol spacer)), biotin, dideoxynucleotide triphosphates, ethylene glycol, amines, or phosphates.

[0337] In some embodiments of any spatial profiling analysis method described herein, the trapping agent barcode domain coupled to the analyte-binding portion includes a cleavable domain. For example, after the analyte trapping agent binds to the analyte (e.g., a cell surface analyte), the trapping 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 trapping agent barcode domain includes a U-removal element that releases the substance from the bead. In some embodiments, the U-removal element may include a single-stranded DNA (ssDNA) sequence containing at least one uracil. The substance can be attached to the bead via the ssDNA sequence. The substance can be released via a combination of a uracil-DNA glycosylase (e.g., removing uracil) and a nuclease (e.g., inducing ssDNA breakage). If the nuclease produces a 5' phosphate group from the cleavage, further enzymatic treatment to remove the phosphate group can be included in downstream processing, for example, before ligating other sequencing handle elements (e.g., Illumina full P5 sequence, partial P5 sequence, full R1 sequence, and / or partial R1 sequence).

[0338] In some embodiments, the analyte-binding portion of an analyte capture agent comprises 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 comprising multiple analyte-binding portions bind multiple analytes present in a biological sample. In some embodiments, the multiple analytes comprise a single class of analytes (e.g., a single class of peptides). In some embodiments where the multiple analytes comprise a single class of analytes, the analyte-binding portions of the multiple analyte capture agents are identical. In some embodiments where the multiple analytes comprise a single class of analytes, the analyte-binding portions of the multiple analyte capture agents are different (e.g., a member of the multiple analyte capture agents may have two or more analyte-binding portions, each of which binds a single class of analyte (e.g., at a different binding site)). In some embodiments, the multiple analytes comprise multiple different classes of analytes (e.g., multiple different classes of peptides).

[0339] In some implementations, multiple different types of analytes (e.g., peptides) from a biological sample can subsequently be associated with one or more physical properties of the biological sample. For example, multiple different types of analytes can be associated with the location of the analytes within the biological sample. Such information (e.g., proteomics information when the analyte-binding moiety recognizes the peptide) can be combined with other spatial information (e.g., genetic information from the biological sample, such as DNA sequence information, transcriptome information (i.e., transcript sequences), or both). For example, cell surface proteins can be associated with one or more physical properties of the cell (e.g., cell shape, size, activity, or type). One or more physical properties can be characterized by cell imaging. Cells can be bound by an analyte trapping agent comprising an analyte-binding moiety that binds to cell surface proteins and an analyte-binding moiety barcode that identifies the analyte-binding moiety, and cells can be spatially analyzed (e.g., any of the various spatial analysis methods described herein). For example, an analyte trapping agent bound to a cell surface protein can bind to a trapping probe (e.g., a trapping probe on an array) comprising a trapping domain that interacts with an analyte trapping sequence present on a trapping barcode domain of the analyte trapping agent. All or part of the trapping barcode domain (including the analyte-binding portion barcode) can be replicated using a polymerase, using the 3' end of the trapping domain as a priming site, to generate an extended trapping probe comprising all or part of the trapping probe (including the spatial barcode present on the trapping probe) and a copy of the analyte-binding portion barcode. In some embodiments, a spatial array having extended trapping probes can contact a sample, wherein the analyte trapping agent associated with the spatial array traps the target analyte. The analyte trapping agent containing the extended trapping probe (which includes the spatial barcode and the analyte-binding portion barcode of the trapping probe) can then be denatured from the trapping 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 using the single-cell / droplet method 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 barcode of the analyte-binding portion of the analyte trap. Therefore, the nucleic acid sequence of the extended capture probe can be associated with an analyte (e.g., a 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 an intracellular analyte in a nearby cell, wherein the intracellular analyte is released using any of the cell permeation or analyte migration techniques described herein.

[0340] In some embodiments of any of the spatial profiling methods described herein, the capture agent barcode domains released from the analyte capture agent can then be sequenced to identify which analyte capture agents bind to the analyte. Based on the presence of the capture agent barcode domain and analyte binding portion barcode sequences associated with features on the spatial array (e.g., features at a specific location), analyte files can be created for biological samples. Files for individual cells or cell populations can be compared with files for other cells (e.g., “normal” cells) to identify variations in the analyte, thereby providing diagnostic information. In some embodiments, these files can be used to diagnose various diseases characterized by changes in cell surface receptors, such as cancer and other conditions.

[0341] Figure 10 This 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 may include a spatial barcode 1008 and one or more functional sequences 1006 and 1010, as described elsewhere herein. The capture probe may also include a capture domain 1012 capable of binding to the analyte capture agent 1026. The analyte capture agent 1026 may include a functional sequence 1018, a capture agent barcode structure 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 may also include a connector 1020 that couples the capture agent barcode structure domain 1016 to an analyte binding portion 1022.

[0342] In some embodiments of any spatial profile analysis method described herein, the method is used to identify immune cell files. Immune cells express various adaptive immune receptors associated with immune function, such as T cell receptors (TCRs) and B cell receptors (BCRs). T cell receptors and B cell receptors play a role in the immune response by specifically recognizing and binding antigens and assisting in their destruction.

[0343] T cell receptors (TCRs) are molecules present on the surface of T cells that are typically responsible for recognizing antigen fragments as peptides that bind to the major histocompatibility complex (MHC) molecule. TCRs are usually heterodimers of two chains, each a member of the immunoglobulin superfamily, possessing an N-terminal variable (V) domain and a C-terminal constant domain. In humans, 95% of T cells have TCRs composed of α and β chains, while in 5% of T cells, the TCR consists of γ and δ (γ / δ) chains. This ratio varies during individual development, disease states, and between different species. When a TCR binds to an antigenic peptide and MHC (peptide / MHC or pMHC), T lymphocytes are activated through signal transduction.

[0344] Each of the two strands of the TCR contains multiple copies of gene segments—variable “V” gene segments, diverse “D” gene segments, and linked “J” gene segments. The TCRα chain (TCRa) is generated by recombination of the V and J segments, while the β chain (TCRb) is generated by recombination of the V, D, and J segments. Similarly, the generation of the TCRγ chain involves recombination of the V and J gene segments, and the generation of the TCRδ chain is achieved through recombination of the V, D, and J gene segments. The intersections of these specific regions (V and J in the α or γ chain, and V, D, and J in the β or δ chain) correspond to the CDR3 region, which is important for antigen MHC recognition. Complementarity-determining regions (e.g., CDR1, CDR2, and CDR3), or hypervariable regions, are sequences in the variable domain of antigen receptors (e.g., T cell receptors and immunoglobulins) that are complementary to the antigen. Much of the diversity of CDRs resides in CDR3, and this diversity arises from somatic recombination events during T lymphocyte development. The unique nucleotide sequence produced during gene arrangement can be called a clone.

[0345] B cell receptors (BCRs) are molecules present on the surface of B cells. The antigen-binding portion of the BCR consists of membrane-bound antibodies, which, like most antibodies (such as immunoglobulins), have unique and randomly determined antigen-binding sites. The antigen-binding portion of the BCR includes a membrane-bound immunoglobulin molecule of an isotype (e.g., IgD, IgM, IgA, IgG, or IgE). When a B cell is activated by its first encounter with a homologous antigen, the cell proliferates and differentiates into a population of antibody-secreting plasma B cells and memory B cells. Different immunoglobulin isotypes differ in biological characteristics, structure, target specificity, and distribution. Multiple molecular mechanisms exist to generate this initial diversity, including gene recombination at multiple sites.

[0346] The BCR consists of two genes encoding the antibody heavy and light chains: IgH and IgK (or IgL). Immunoglobulins are formed through recombination between gene segments, sequence diversification at the junctions of these segments, and point mutations throughout the gene. Each heavy chain gene contains multiple copies of three distinct gene segments—a variable "V" gene segment, a diverse "D" gene segment, and a connecting "J" gene segment. Each light chain gene contains multiple copies of two distinct gene segments within the protein's variable region—a variable "V" gene segment and a connecting "J" gene segment.

[0347] Recombination can produce a molecule with one of each of the V, D, and J segments. Furthermore, several bases can be deleted and other bases (called N and P nucleotides) added at each of the two linkages, resulting in further diversity. Upon B cell activation, affinity maturation occurs through somatic hypermutation. During this process, progeny cells of activated B cells accumulate different somatic mutations throughout the genome, with a higher concentration of mutations in the CDR region, resulting in antibodies with higher affinity for the antigen.

[0348] Besides somatic hypermutation, activated B cells also undergo allotype conversion. Antibodies with the same variable regions can have different forms (isotypes) depending on the constant regions. All primitive B cells express IgM (or IgD), while most activated B cells express IgG, but also IgM, IgA, and IgE. This conversion from IgM (and / or IgD) to IgG, IgA, or IgE occurs through recombination events, which cause a cell to specifically produce a particular allotype. The unique nucleotide sequences produced during gene arrangement can be similarly referred to as clonal types.

[0349] Some of the methods described herein are used to analyze various sequences of TCRs and BCRs from immune cells, such as various clonal types. In some embodiments, the methods are used to analyze the sequences of the TCRα chain, TCRβ chain, TCRδ chain, TCRγ chain, or any fragment thereof (e.g., including variable regions, constant regions, transmembrane regions, fragments thereof, combinations thereof, and combinations thereof of V(D)J or VJ regions). In some embodiments, the methods described herein can be used to analyze the sequences of the B cell receptor heavy chain, B cell receptor light chain, or any fragment thereof (e.g., including variable regions, constant regions, transmembrane regions, fragments thereof, combinations thereof, and combinations thereof of V(D)J or VJ regions).

[0350] In cases where immune cells are to be analyzed, primer sequences used for any of the various operations involving attaching barcode sequences and / or amplification reactions may include gene-specific sequences targeting genes or gene regions of immune cell proteins (e.g., immune receptors). These gene sequences include, but are not limited to, sequences of various T-cell receptor α variable genes (TRAV genes), T-cell receptor α linker genes (TRAJ genes), T-cell receptor α constant genes (TRAC genes), T-cell receptor β variable genes (TRBV genes), T-cell receptor β diversity genes (TRBD genes), T-cell receptor β linker genes (TRBJ genes), T-cell receptor β constant genes (TRBC genes), T-cell receptor γ variable genes (TRGV genes), T-cell receptor γ linker genes (TRGJ genes), T-cell receptor γ constant genes (TRGC genes), T-cell receptor δ variable genes (TRDV genes), T-cell receptor δ diversity genes (TRDD genes), T-cell receptor δ linker genes (TRDJ genes), and T-cell receptor δ constant genes (TRDC genes).

[0351] In some embodiments, the analyte-binding moiety is based on a class I or class II major histocompatibility complex (MHC). In some embodiments, the analyte-binding moiety is an MHC multimer, including but not limited to the dextrorotatory MHC, MHC tetramer, and MHC pentamer (e.g., see U.S. Patent Application Publications US2018 / 0180601 and US2017 / 0343545, the entire contents of which are incorporated herein by reference). MHCs comprising all or part of an MHC peptide (e.g., soluble MHC monomer molecules) can be used as the analyte-binding moiety of an analyte trapping agent coupled to a trapping agent barcode domain comprising an analyte-binding moiety barcode that identifies its associated MHC (thus, for example, a TCR binding chaperone of MHC). In some embodiments, MHC is used to analyze one or more cell surface features of T cells, such as the TCR. In some cases, multiple MHCs are bound together in a larger complex (MHC multimer) to enhance the binding affinity of MHC to the TCR through multi-ligand binding synergy.

[0352] Figure 11A , 11BFigures 11 and 11C illustrate how streptavidin cellular tags can be used to generate spatially barcoded cells or cell contents in an array-based system. For example, as shown in Figure 11, the peptide-bound major histocompatibility complex (pMHC) can bind biotin alone to and to the streptavidin moiety, such that the streptavidin moiety contains multiple pMHC moieties. Each of these moieties can bind to the TCR, allowing streptavidin to bind to target T cells via multiple MCH / TCR binding interactions. These multiple interactions work synergistically to significantly enhance binding affinity. This improved affinity can improve T cell labeling and reduce the likelihood of the label dissociating from the T cell surface. Figure 11B As shown, the capture agent barcode domain 1101 can be modified by streptavidin 1102 and contacted with multiple molecules (e.g., pMHC) of biotinylated MHC 1103, such that the biotinylated MHC 1103 molecule is coupled to the streptavidin-coupled capture agent barcode domain 1101. The result is a barcode-coded MHC multimer complex 1105. Figure 11B As shown, the trapper barcode domain sequence 1101 can identify MHC as its associated tag, and also includes optional functional sequences, such as sequences for hybridization with other oligonucleotides. Figure 11CAs shown, an example oligonucleotide is capture probe 1106, which includes a complementary sequence (e.g., rGrGrG corresponding to CCC), a barcode sequence, and other functional sequences, such as UMI, adaptor sequences (e.g., including sequencing primer sequences (e.g., R1 or a portion of R1 (“pR1”)), flow cell attachment sequences (e.g., P5 or P7 or a portion thereof), etc. In some cases, capture probe 1106 may first be associated with and released from a feature (e.g., a gel bead). In other embodiments, capture probe 1106 may hybridize with the capture 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 to generate a sequence containing two spatial barcode sequences corresponding to the two spatial barcode sequences (the spatial barcode associated with the capture probe and the spatial barcode associated with the MHC-oligonucleotide complex). The construct is a construct of the sequence of each of the associated barcodes. In some cases, one or both of these corresponding sequences may be complements to the original sequence in the capture probe 1106 or the 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 the capture probe 1106 can be used to identify features, and sequences derived from the spatial barcode sequence on the 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 immune cell populations).

[0353] (c) Substrate

[0354] For the spatial array-based analytical methods described in this section, the substrate serves to support the direct or indirect attachment of capture probes to array features. Furthermore, in some embodiments, the substrate (e.g., the same substrate or different substrates) can be used to support biological samples, particularly, for example, thin tissue sections. Therefore, "substrate" refers to a support insoluble in aqueous solutions that positions biological samples, analytes, features, and / or capture probes on the substrate.

[0355] A variety of different substrates can be used for the above purposes. Typically, the substrate can be any suitable support material. Exemplary substrates include, but are not limited to, glass, modified and / or functionalized glass, hydrogels, membranes, films, plastics (including, for example, copolymers of acrylic, polystyrene, styrene and other materials, polypropylene, polyethylene, polybutene, polyurethane, Teflon, etc.). TMMaterials include cycloolefins, polyimides, nylon, ceramics, resins, zeonor, silica or silica-based materials, including silicon and modified silicon, carbon, metals, inorganic glasses, fiber bundles and polymers such as polystyrene, cycloolefin copolymers (COC), cycloolefin polymers (COP), polypropylene, polyethylene and polycarbonate.

[0356] The substrate can also correspond to the flow cell. The flow cell can be formed from any of the materials described above and can include channels for passing reagents, solvents, features, and molecules through the flow cell.

[0357] In the examples of substrate materials discussed above, polystyrene is a suitable hydrophobic material for binding negatively charged macromolecules because it typically contains almost no hydrophilic groups. For nucleic acids immobilized on glass slides, increasing the hydrophobicity of the glass surface can enhance nucleic acid immobilization. This enhancement allows for the formation of relatively denser stacks (e.g., providing improved specificity and resolution).

[0358] In some embodiments, the substrate is coated with a surface treatment agent (e.g., poly(L)-lysine). Alternatively or additionally, the substrate may be treated by silanization (e.g., using epoxy silane, amino silane) and / or by polyacrylamide treatment.

[0359] The substrate can typically have any suitable form or shape. For example, the substrate can be flat, curved, such as convex or concave, towards the region where the biological sample (e.g., tissue sample) and the substrate interact. In some embodiments, the substrate is flat, such as a plane, chip, or glass slide. The substrate may include one or more patterned surfaces (e.g., channels, holes, protrusions, ridges, pits, etc.) within the substrate.

[0360] The substrate can be any desired shape. For example, the substrate can typically be a thin, flat shape (e.g., a square or rectangle). 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 cutoff angles (e.g., for sliding clamps or cross-shaped platforms). In some embodiments, when the substrate structure is flat, the substrate structure can be any suitable type of support with a flat surface (e.g., a chip or slide, such as a microscope slide).

[0361] The substrate may optionally include various structures, such as, but not limited to, protrusions, ridges, and channels. The substrate may be micropatterned to limit lateral diffusion (e.g., to prevent overlap of spatial barcodes). Substrates modified with such structures may be modified to allow the binding of analytes, features (e.g., beads), or probes at various locations. For example, sites on the substrate modified with various structures may be continuous or discontinuous with other sites.

[0362] 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 feature adheres to random sites.

[0363] In some embodiments, techniques such as (but not limited to) stamping, micro-etching, and molding 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 may be a concave slide or a cavity slide. For example, the holes may be formed by one or more shallow recesses on the surface of the substrate. In some embodiments where the substrate includes one or more holes, the holes can be formed by attaching a box (e.g., a box containing one or more chambers) to the surface of the substrate structure.

[0364] In some embodiments, the structure of the substrate (e.g., holes) may each carry different capture probes. Different capture probes attached to each structure can be identified based on the location of the structure in or on the substrate surface. Exemplary substrates include arrays of separate structures located on the substrate, including, for example, arrays of holes with receiving features.

[0365] In some embodiments, the substrate includes one or more markings on its surface, for example, to provide guidance for associating spatial information with the characterization of the object of interest. For example, the substrate may be marked with a grid of lines (e.g., to facilitate estimation of the size of an object seen at magnification and / or to provide a reference area for counting objects). In some embodiments, reference markings may be included on the substrate. Such markings may be produced using techniques including, but not limited to, printing, sandblasting, and surface deposition.

[0366] In some embodiments, the substrate is modified to include one or more structures, including but not limited to holes, protrusions, ridges, or markings, which may include physically altered sites. For example, substrates modified with various structures may include physical properties, including but not limited to physical configurations, magnetic or compressive forces, chemically functionalized sites, chemically altered sites, and / or electrostatically altered sites.

[0367] In some embodiments, the substrate is modified to include various structures, including but not limited to holes, protrusions, ridges, or markings, said structures being applied in a pattern. Alternatively, the structures may be randomly distributed.

[0368] In some embodiments, the substrate is treated to minimize or reduce nonspecific analyte hybridization within or between features. For example, the treatment may include coating the substrate with a hydrogel, membrane, and / or thin film that forms a physical barrier against nonspecific 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 Publications Nos. US2017 / 0253918 and US2018 / 0052081 can be used. The entire contents of the foregoing documents are incorporated herein by reference.

[0369] The treatment may include adding a reactive or activatable functional group, making it reactive upon receiving a stimulus (e.g., photoactivity). The treatment may include treatment with a polymer having one or more physical properties (e.g., mechanical, electrical, magnetic, and / or thermal) to minimize nonspecific binding (e.g., activating the substrate at certain sites to enable analyte hybridization at those sites).

[0370] The substrate (e.g., features on beads or arrays) may 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, 1000,000, 1000,000, 1000,000, 1000,000, 1000,000,000, 1000,000,000, or 100,000,000,000).

[0371] In some embodiments, the surface of the substrate is coated with a cell-permissive coating to enable the adhesion of living cells. A “cell-permissive coating” is a coating that enables or helps cells to maintain cell viability (e.g., retain viability) on a substrate. For example, a cell-permissive coating can enhance cell adhesion, cell growth, and / or cell differentiation; for instance, it can provide nutrients to living cells. Cell-permissive coatings can include biomaterials and / or synthetic materials. Non-limiting examples of cell-permissive coatings include those characterized by having one or more extracellular matrix (ECM) components (e.g., proteoglycans and fibrous proteins such as collagen, elastin, fibronectin, and laminin), polylysine, poly(L)-ornithine, and / or biocompatible silicones (e.g., [missing information]). The coating may include, for example, one or more extracellular substrate components, such as type I collagen, type II collagen, type IV collagen, elastin, fibronectin, laminin, and / or hyalin. In some embodiments, the cell acceptor coating comprises components derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma (e.g., A soluble base membrane formulation extracted from [the sample / structure]. In some embodiments, the cell acceptor coating includes collagen. The cell acceptor coating can be used to culture adherent cells on a spatially barcoded array, or to maintain cell viability of tissue samples or sections when in contact with a spatially barcoded array.

[0372] When the substrate includes a gel (e.g., a hydrogel or gel substrate), oligonucleotides within the gel can attach to the substrate. The terms "hydrogel" and "hydrogel substrate" are used interchangeably herein to refer to macromolecular polymer gels comprising a network. Within the network, some polymer chains may be selectively crosslinked, although crosslinking does not always occur.

[0373] In some embodiments, the hydrogel may include hydrogel subunits. A “hydrogel subunit” is a hydrophilic monomer, molecular precursor, or polymerizable (e.g., crosslinkable) to form a three-dimensional (3D) hydrogel network. Hydrogel subunits may 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 polyurethane, polyester polyurethane, polyethylene copolymers, polyamides, polyvinyl alcohol, polypropylene glycol, polyoxybutane, polyvinylpyrrolidone, polyacrylamide, hydroxyethyl polyacrylate and polyhydroxyethyl methacrylate, collagen, hyaluronic acid, chitosan, dextran, agarose, gelatin, alginate, protein polymers, methylcellulose, and combinations thereof.

[0374] In some embodiments, the hydrogel comprises a hybrid material, for example, the hydrogel material comprising elements of synthetic polymers and natural polymers. Examples of suitable hydrogels are described, for example, in U.S. Patent Nos. 6,391,937, 9,512,422, and 9,889,422 and U.S. Patent Application Publications Nos. 2017 / 0253918, 2018 / 0052081, and 2010 / 0055733, the entire contents of which are incorporated herein by reference.

[0375] In some embodiments, a crosslinking agent and / or initiator is added to the hydrogel subunit. Examples of crosslinking agents include, but are not limited to, bisacrylamide and bisacrylamide. Examples of initiators include, but are not limited to, azobisisobutyronitrile (AIBN), riboflavin, and L-arginine. The inclusion of a crosslinking agent and / or initiator can lead to increased covalent bonding between biomacromolecules interacting in later polymerization steps.

[0376] In some embodiments, the hydrogel may have a colloidal structure (e.g., agarose) or a polymer network structure (e.g., gelatin).

[0377] In some implementations, some hydrogel subunits are covalently or physically crosslinked and polymerized (e.g., undergoing "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, photocrosslinking, irradiation crosslinking (e.g., X-rays, electron beams), and combinations thereof. Techniques such as photolithography polymerization can also be used to form hydrogels.

[0378] Polymerization methods for hydrogel subunits can be selected to form hydrogels with different properties (e.g., pore size, swelling properties, biodegradability, conductivity, transparency, and / or permeability). For example, the hydrogel may include pores of sufficient size to allow macromolecules (e.g., nucleic acids, proteins, chromatin, metabolites, gRNA, antibodies, carbohydrates, peptides, metabolites, and / or small molecules) to pass through a sample (e.g., a tissue section). It is well known that pore size generally decreases with increasing hydrogel subunit concentration and generally increases with increasing ratio of hydrogel subunit to crosslinking agent. Therefore, fixative / hydrogel compositions containing a certain concentration of hydrogel subunits that allow such biomacromolecules to pass through can be prepared.

[0379] 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 one or more second materials. For example, the hydrogel can be pre-formed and then placed on top, bottom, or in any other configuration with one or more second materials. In some embodiments, hydrogel formation occurs after contact with one or more second materials during substrate formation. Hydrogel formation can also occur within structures located on the substrate (e.g., holes, ridges, protrusions, and / or markings).

[0380] In some embodiments, hydrogel formation on the substrate occurs before, simultaneously with, or after the feature (e.g., bead) is attached to the substrate. For example, hydrogel formation can be performed on a substrate that already contains a capture probe when the capture probe is attached (e.g., directly or indirectly) to the substrate.

[0381] In some embodiments, hydrogel formation occurs within a biological sample. In some embodiments, the biological sample (e.g., a tissue section) is embedded in the hydrogel. In some embodiments, hydrogel subunits are injected into the biological sample, and the polymerization of the hydrogel is initiated by external or internal stimuli.

[0382] In embodiments where a hydrogel is formed within a biological sample, functionalizing chemistry may be used. In some embodiments, functionalizing chemistry includes hydrogel histochemistry (HTC). Any hydrogel tissue framework suitable for HTC (e.g., synthetic or natural) can be used to anchor biomolecules and modulate functionalization. Non-limiting examples of methods using HTC framework variants include CLARITY, PACT, ExM, SWITCH, and ePACT. In some embodiments, hydrogel formation within the biological sample is permanent. For example, biomolecules can permanently adhere to the hydrogel, thereby enabling multi-round interrogation. In some embodiments, hydrogel formation within the biological sample is reversible.

[0383] In some embodiments, other reagents are added to the hydrogel subunits before, during, and / or after polymerization. These other reagents may include, but are not limited to, oligonucleotides (e.g., capture probes), endonucleases for DNA fragmentation, DNA fragmentation buffers, DNA polymerases, and dNTPs for amplifying nucleic acids and attaching barcodes to the amplified fragments. Other enzymes may be used, including but not limited to RNA polymerases, transposases, ligases, proteinase K, and DNases. Other reagents may also include reverse transcriptases, including enzymes with terminal transferase activity, primers, and converting oligonucleotides. In some embodiments, optical labels are added to the hydrogel subunits before, during, and / or after polymerization.

[0384] In some embodiments, an HTC reagent is added to the hydrogel before, during, and / or after polymerization. In some embodiments, a cell marker is added to the hydrogel before, during, and / or after polymerization. In some embodiments, a cell permeabilizer is added to the hydrogel before, during, and / or after polymerization.

[0385] Hydrogels embedded in biological samples can be removed by any suitable method. For example, electrophoretic tissue removal methods can be used to remove biomacromolecules from hydrogel-embedded samples. In some embodiments, the hydrogel-embedded sample is stored in a medium (e.g., a fixation medium, methylcellulose, or other semi-solid medium) before or after hydrogel removal.

[0386] "Conditionally removable coating" refers to a coating that can be removed from a substrate surface after the application of a release agent. In some embodiments, conditionally removable coatings include hydrogels as described herein, for example, hydrogels comprising peptide-based materials. Non-limiting examples of hydrogels having peptide-based materials include transmembrane segments having spider silk and human muscle L-type calcium channels (e.g., Materials based on synthetic peptides containing a combination of arginine-alanine-aspartic acid-alanine sequences (RADARADARADARADA) (e.g.) EAK16 (AEAEAKAKAEAEAKAK), KLD12 (KLDLKLDLKLDL), and PGMATRIX TM An amphiphilic 16-residue peptide.

[0387] In some embodiments, the hydrogel in the condition-removable coating is a stimulus-responsive hydrogel. A stimulus-responsive hydrogel may undergo a gel-to-solution and / or gel-to-solid transition upon application of one or more external triggers (e.g., a release agent). See, for example, Willner, Acc. Chem. Res. 50:657-658, 2017, the entire contents of which are incorporated herein by reference. Non-limiting examples of stimulus-responsive hydrogels include thermoresponsive hydrogels, pH-responsive hydrogels, photoresponsive hydrogels, redox-responsive hydrogels, analyte-responsive hydrogels, or combinations thereof. In some embodiments, the stimulus-responsive hydrogel may be a multi-stimuli-responsive hydrogel.

[0388] A “releasing agent” or “external trigger” is an agent that causes the conditionally removable coating to be removed from the substrate when the releasing agent is applied to it. External triggers or releasing agents may include physical triggers, such as thermal, magnetic, ultrasonic, electrochemical, and / or photostimulation, and chemical triggers, such as pH, redox reactions, supramolecular complexes, and / or biocatalytically driven reactions. See, for example, Echeverria et al., Gels (2018), 4, 54; doi:10.3390 / gels4020054, the full text of which is incorporated herein by reference. The type of “releasing agent” or “external trigger” may depend on the type of conditionally removable coating. For example, a conditionally removable coating characterized by a redox-responsive hydrogel may be removable upon application of a releasing agent comprising a reducing agent (e.g., dithiothreitol (DTT)). As another example, a pH-responsive hydrogel may be removed upon application of a releasing agent that changes the pH.

[0389] (d) Array

[0390] In many of the methods described herein, features (as further described below) are collectively positioned on a substrate. An “array” is a specific arrangement of multiple features that are irregular or form a regular pattern. The individual features in an array are distinct from each other 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).

[0391] Arrays can be used to simultaneously measure a large number of analytes. In some embodiments, oligonucleotides are used at least in part to create the array. For example, one or more copies of a single type of oligonucleotide (e.g., a capture probe) may correspond to or be directly or indirectly attached to a given feature in the array. In some embodiments, a given feature in the array comprises two or more oligonucleotides (e.g., capture probes). In some embodiments, two or more oligonucleotides (e.g., capture probes) directly or indirectly attached to a given feature on the array comprise a common (e.g., identical) spatial barcode.

[0392] 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), holes, and hydrogel pads. In some embodiments, features are attached or fixed directly or indirectly to a substrate. In some embodiments, the features are not attached or fixed directly or indirectly to a substrate, but are arranged, for example, within a closed or partially closed three-dimensional space (e.g., a hole or pit).

[0393] In addition to the features described above, a variety of other features may be used to form the arrays described herein. For example, in some embodiments, features formed by polymers and / or biopolymers printed, screen-printed, or electrostatically deposited on a substrate may be used to form the arrays. For example, inkjet printing of biopolymers is described in PCT patent application publication number WO 2014 / 085725. For example, inkjet printing of polymers is described in de Gans et al., Adv Mater. 16(3):203-213 (2004). Electrostatic deposition methods for polymers and biopolymers are described, for example, Hoyer et al., Anal. Chem. 68(21):3840-3844 (1996). The entire contents of the foregoing references are incorporated herein by reference.

[0394] As another example, in some implementations, the features are formed from metallic microparticles or nanoparticles. For instance, Lee et al., Beilstein J. Nanotechnol. 8:1049-1055 (2017) describe a suitable method for depositing such particles to form an array, the entire contents of which are incorporated herein by reference.

[0395] As another example, in some implementations, the features are formed by magnetic particles assembled on a substrate. Examples of 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.

[0396] As another example, in some implementations, the feature corresponds to a substrate area in which one or more optical markers have been incorporated and / or altered by a process such as permanent photobleaching. Suitable substrates for implementing features in this way include, for example, a variety of polymers. For example, a method for forming such a feature is described in Moshrefzadeh et al., Appl. Phys. Lett. 62:16 (1993), the entire contents of which are incorporated herein by reference.

[0397] As yet another example, in some implementations, the features may correspond to colloidal particles 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 contents of which are incorporated herein by reference.

[0398] As another example, in some implementations, features can be formed by dot array photopolymerization of monomer solutions on a substrate. In particular, two-photon and three-photon polymerization can be used to fabricate features of relatively small (e.g., submicron) dimensions. A suitable method for fabricating features on a substrate in this manner is described in Nguyen et al., Materials Today 20(6):314-322 (2017), the entire contents of which are incorporated herein by reference.

[0399] In some embodiments, the features are directly or indirectly attached to or fixed to a liquid-permeable substrate. In some embodiments, the features are directly or indirectly attached to or fixed to a biocompatible substrate. In some embodiments, the features are directly or indirectly attached to or fixed to a hydrogel substrate.

[0400] Figure 12 An exemplary arrangement of barcode-like features in an array is described. From left to right, Figure 12 The diagram shows (L) a slide comprising six spatially barcoded arrays, (C) an enlarged schematic diagram of one of the six spatially barcoded arrays showing a grid of barcoded features associated with the biological sample, and (R) an enlarged schematic diagram of a portion of the array showing specific identifiers (labeled ID578, ID579, ID560, etc.) for multiple features in the array.

[0401] As used herein, the term "bead array" refers to an array comprising a plurality of beads as features in an array. In some embodiments, the beads are attached to a substrate. For example, the beads may optionally be attached to a substrate such as a microscope slide and close to a biological sample (e.g., a tissue section comprising cells). The beads may 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)).

[0402] Examples of on- or in-substrate bead arrays include beads located in pores, such as BeadChip arrays (available from Illumina Inc., San Diego, California), arrays used in the 454LifeSciences sequencing platform (a subsidiary of Roche AG, Basel, Switzerland), and arrays used in ion-fluid sequencing platforms (a subsidiary of Carlsbad Life Sciences, California). Examples of bead arrays are described, for example, 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.

[0403] In some embodiments, the bead array comprises a plurality of beads. For example, the bead array may 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 comprises beads of a single type (e.g., substantially uniform in size, shape, and other physical properties, such as translucent). In some embodiments, the plurality of beads comprises two or more different types of beads.

[0404] In some embodiments, a bead array is formed when beads are embedded in a hydrogel layer, wherein the hydrogel polymerizes and fixes the relative positions of the beads. The bead array may be pre-equilibrated and combined with a reaction buffer and an enzyme (e.g., a reverse transcription mixture). In some embodiments, the bead array is frozen.

[0405] A “flexible array” comprises multiple spatially barcoded features attached to or embedded in a flexible substrate (e.g., a membrane or strip) placed on a biological sample. In some embodiments, the flexible array comprises multiple spatially barcoded features embedded in a hydrogel substrate. To form such an array, features of a microarray are copied into a 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 may include copying multiple features from a microarray into a first hydrogel, wherein the first hydrogel is in contact with the microarray; reducing the size of the first hydrogel including the copied features by removing water to form a first shrinking hydrogel including the copied features; copying the features in the first shrinking hydrogel into a second hydrogel, wherein the second hydrogel is in contact with the first hydrogel; and reducing the size of the second hydrogel including the copied features by removing water to form a second shrinking hydrogel including the copied features, thereby generating a high-density spatially barcoded array. The result is a high-density flexible array comprising spatially barcoded features.

[0406] In some implementations, spatially barcode-coded beads can be loaded onto a substrate (e.g., a hydrogel) to produce a high-density self-assembled bead array.

[0407] The flexible array can be pre-equilibrated, incorporating reaction buffers and functional concentrations of enzymes (e.g., reverse transcription mixtures). In some embodiments, the flexible bead array can be stored for extended periods (e.g., several days) or frozen until ready for use. In some embodiments, permeabilization of the biological sample (e.g., tissue sections) can be performed by adding an enzyme / washing agent before contact with the flexible array. The flexible array can be placed directly on the sample or indirectly in contact with the biological sample (e.g., with 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 targeted capture of the analyte can be performed on solid microspheres or first-size and second-size spherical beads.

[0408] A “microtube array” is a series of array features divided by microtubes. A “microtube channel” is a separate partition created by microtubes. For example, a microtube channel can be fluid-isolated from other microtube channels, such that the fluid or other contents in one microtube channel in the array are separated from the fluid or other contents in adjacent microtube channels in the array. The density and arrangement order of the microtubes can be any suitable density or arrangement order of discrete sites.

[0409] In some implementations, the microtube array is treated to create conditions conducive to loading. One example is the use of a corona bar (BD-20AC, Electro Technic Products) to create a hydrophilic surface. In some implementations, features (e.g., beads to which capture probes are attached) are loaded onto the microtube array such that the exact location of the features within the array is known. For example, capture probes containing spatial barcodes can be placed into microtube channels, allowing the spatial barcodes to identify the location from which a barcode sequence of barcoded nucleic acid molecules is derived.

[0410] In some implementations, when using a random distribution to distribute features, empirical testing can be performed to generate loading / distribution conditions that favor the individual features of each microtube. In some implementations, it may be desirable to achieve distribution conditions that promote only a single feature (e.g., a bead) in each microtube channel. In some implementations, it may be desirable to achieve distribution conditions that promote more than one feature (e.g., a bead) in each microtube channel by allowing features to flow through the microtube channel.

[0411] In some embodiments, the microtube array is positioned in contact with the sample (e.g., on top or below) such that the microtubes containing features (e.g., beads that may include capture probes) come into 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 a fluid-isolated reaction chamber containing the biological sample.

[0412] In some embodiments, a microchannel comprising a portion of beads and biological sample is created by contacting a microtube array with the biological sample to divide the biological sample. In some embodiments, the portion of the biological sample contained in the microtube channel is one or more cells. In some embodiments, after one or more cells are added to the microtube channel, a feature is introduced into the microtube array via a flow.

[0413] In some embodiments, reagents are added to the microcapillary array. The added reagents may include enzyme reagents and mixtures of reagents for nucleic acid amplification. In some embodiments, the reagents include reverse transcriptase, ligase, one or more nucleotides, and any combination thereof. After the reagents are added to the microcapillary channels, one or more microcapillary channels can be sealed, for example, using silicone oil, mineral oil, a non-porous material, or a cap.

[0414] In some implementations, after incubation for a specified time and at a specified temperature or temperature range (e.g., after hybridization or amplification reactions), the reagent solution is removed from each microcapillary channel. The reagent solutions can be processed individually for sequencing or combined for sequencing analysis.

[0415] In some embodiments, some or all of the features in the array include capture probes. In some embodiments, the array may include capture probes that are directly or indirectly attached to the substrate.

[0416] Capture probes include capture domains (e.g., nucleotide sequences) that can specifically bind (e.g., hybridize) to target analytes (e.g., mRNA, DNA, or proteins) 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 incorporated into the target (e.g., a fluorophore, a heavy metal (e.g., silver ions), or a chemiluminescent label). In some embodiments, the intensity of the visual signal is correlated with the relative abundance of each analyte in the biological sample. Since an array can contain thousands or millions (or more) of capture probes, a feature array with capture probes can query many analytes in parallel.

[0417] 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 include one or more capture probes designed to capture mRNA from one organism (e.g., human) and one or more capture probes designed to capture DNA from a second organism (e.g., bacteria).

[0418] Capture probes can be attached to substrates or features using various techniques. In some embodiments, capture probes are directly attached to features fixed on an array. In some embodiments, capture probes are chemically fixed to the substrate. For example, chemical fixation can occur between functional groups on the substrate and corresponding functional elements on the capture probe. Exemplary corresponding functional elements in the capture probe can be intrinsic chemical groups of the capture probe, such as hydroxyl groups, or functional elements 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 fixed comprises functional amine groups or is chemically modified to include functional amine groups. Means and methods for such chemical modification are well known in the art.

[0419] In some embodiments, the capture probe is a nucleic acid. In some embodiments, the capture probe is attached to a feature or substrate via its 5' end. In some embodiments, the capture probe is attached to a feature or substrate via its 5' end and includes, 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 attached to a feature via its 5' end and includes, from the 5' end to the 3' end: a barcode (e.g., a spatial barcode or UMI) and a capture domain. In some embodiments, the capture probe is attached to a feature or substrate via its 5' end and includes, 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.

[0420] In some embodiments, the capture probe is anchored to a feature or substrate via its 5' end and includes, from the 5' end to the 3' end: a fragmentation 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 anchored to a feature or substrate via its 5' end and includes, from the 5' end to the 3' end: a fragmentation domain, a functional domain, a spatial barcode, a UMI, and a capture domain. In some embodiments, the capture probe is anchored to a feature or substrate via its 5' end and does not include a spatial barcode. In some embodiments, the capture probe is anchored to a feature or substrate via its 5' end and does not include a UMI. In some embodiments, the capture probe includes a sequence for initiating a sequencing reaction.

[0421] In some embodiments, the capture probe is fixed to the feature or substrate via its 3' end. In some embodiments, the capture probe is fixed to the feature or substrate via its 3' end and includes, from the 3' end to the 5' end: one or more barcodes (e.g., spatial barcodes and / or UMIs) and one or more capture fields. In some embodiments, the capture probe is fixed to the feature or substrate via its 3' end and includes, from the 3' end to the 5' end: a barcode (e.g., a spatial barcode or a UMI) and a capture field. In some embodiments, the capture probe is fixed to the feature or substrate via its 3' end and includes, from the 3' end to the 5' end: a fragmentation field, a functional field, one or more barcodes (e.g., spatial barcodes and / or UMIs) and a capture field. In some embodiments, the capture probe is fixed to the feature or substrate via its 3' end and includes, from the 3' end to the 5' end: a fragmentation field, a functional field, a spatial barcode, a UMI, and a capture field.

[0422] The positioning of functional groups within the capture probe to be immobilized can be used to control and shape the binding behavior and / or orientation of the capture probe. For example, functional groups may 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 may 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 of the capture probe to be immobilized include portions capable of binding to the capture probe (e.g., binding to amine-functionalized nucleic acids). Examples of such substrates are carboxyl, aldehyde, or epoxy-based supports.

[0423] In some embodiments, the substrate to which the capture probe can be anchored may be chemically activated, for example by activating functional groups available on the substrate. The term "activated (substrate)" refers to a material in which interacting or reactive chemical functional groups are established or realized through a chemical modification process. For example, a substrate including carboxyl groups may be activated prior to use. Furthermore, some substrates contain functional groups capable of reacting with specific portions already present in the capture probe.

[0424] 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 by a linker (i.e., a chemical linker) separating the "first" nucleotide of the capture probe from the substrate. In some embodiments, the capture probe does not bind directly to the array but interacts indirectly, for example, by binding to molecules that are themselves directly or indirectly bound to the array. In some embodiments, the capture probe is indirectly attached to the substrate (e.g., via a solution comprising a polymer).

[0425] In some embodiments where the capture probe is indirectly immobilized on an array feature, the capture probe may also include an upstream sequence capable of hybridizing with the 5′ end of the surface probe (in the 5′ direction of a sequence hybridizing to a nucleic acid, such as RNA from a tissue sample), for example, by hybridizing with a surface probe capable of binding to the capture probe. Separately, the capture domain of the capture probe may be considered as a capture domain oligonucleotide, which may be used in the synthesis of the capture probe in embodiments where the capture probe is indirectly immobilized on an array.

[0426] In some embodiments, the substrate comprises an inert material or matrix (e.g., a glass slide) that has been functionalized, for example, by treatment with a material containing reactive groups capable of immobilizing the probe. See, for example, WO 2017 / 019456, the entire contents of which are incorporated herein by reference. Non-limiting examples include polyacrylamide hydrogels supported on an inert substrate (e.g., glass slides; see WO 2005 / 065814 and U.S. Patent Application No. 2008 / 0280773, the entire contents of which are incorporated herein by reference).

[0427] In some embodiments, covalent methods are used to immobilize functionalized biomolecules (e.g., capture probes) onto functionalized substrates. Covalent attachment methods include, for example, the condensation of amines with activated carboxylic esters (e.g., N-hydroxysuccinimide esters); the condensation of amines with aldehydes under reductive amination conditions; and cycloaddition reactions, such as the Diels–Alder [4+2] reaction, 1,3-dipolar cycloaddition, and [2+2] cycloaddition. Covalent attachment methods also include, for example, click chemistry reactions, including [3+2] cycloaddition reactions (e.g., the Huisgen 1,3-dipolar cycloaddition and copper(I)-catalyzed azidoyne cycloaddition (CuAAC)); mercapto-alkene reactions; Diels–Alder reactions and electron-demanding Diels–Alder reactions; cycloaddition of [4+1] isonitriles and tetrazides; and nucleophilic ring-opening of small carbocyclic rings (e.g., epoxide ring-opening of amino oligonucleotides). Covalent attachment methods also include, for example, maleimide and thiol; and p-nitrobenzene ester-functionalized oligonucleotides and polylysine-functionalized substrates. Covalent attachment methods also include, for example, disulfide bond reactions; radical reactions (see, for example, U.S. Patent No. 5,919,626, the entire contents of which are incorporated herein by reference); and hydrazide-functionalized substrates (e.g., wherein the hydrazide functional group is directly or indirectly attached to the substrate) and aldehyde-functionalized oligonucleotides (see, for example, Yershov et al., (1996) Proc. Natl. Acad. Sci. USA 93, 4913–4918, the entire contents of which are incorporated herein by reference).

[0428] In some embodiments, photochemical covalent methods are used to immobilize functionalized biomolecules (e.g., capture probes) onto a functionalized substrate. Photochemical covalent linking methods include, for example, the immobilization of amtraone-coupled oligonucleotides (see, for example, Koch et al., (2000) Bioconjugate Chem. 11, 474–483, the entire contents of which are incorporated herein by reference).

[0429] In some embodiments, non-covalent methods are used to immobilize functionalized biomolecules (e.g., capture probes) onto a functionalized substrate. Methods for non-covalent attachment include, for example, biotin-functionalized oligonucleotides and streptavidin-treated substrates (see, for example...). (Et., (1993) Analytical Biochemistry 209, 278–283 and Gilles et al., (1999) Nature Biotechnology 17, 365–370, the entire contents of which are incorporated herein by reference).

[0430] In some embodiments, oligonucleotides (e.g., capture probes) may be attached to a substrate or feature according to the methods described below: 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 Research24,3031–3039; 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 full contents of all the above documents are incorporated herein by reference.

[0431] Arrays can be prepared by a variety of methods. In some embodiments, arrays are prepared by synthesizing oligonucleotides on the array (e.g., in situ synthesis) 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, synthetic adapters modified with photochemical protecting groups can be attached to a substrate, and photolithographic masks (applied to specific areas of the substrate) and light can be used to modify the photochemical protecting groups, thereby producing arrays with localized 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, all of which are incorporated herein by reference.

[0432] In some embodiments, the array is "spotted" or "printed" with oligonucleotides, and then these oligonucleotides (e.g., capture probes) are attached to the substrate. Oligonucleotides can be applied by non-contact or contact printing. Non-contact printers can use methods similar to computer printers (e.g., bubble jet or inkjet) to spray small droplets of probe solution onto the substrate. Such specialized inkjet printers can spray nanoliter to picoliter volumes of oligonucleotide solution droplets (instead of ink) onto the substrate. In contact printing, each printing needle directly applies the oligonucleotide solution to a specific location on the surface. Oligonucleotides can be attached to the substrate surface via electrostatic interactions between the negative charge of the DNA phosphate backbone and a positively charged coating on the substrate surface, or via UV-crosslinking covalent bonds between thymine bases in the DNA and amino groups on the treated substrate surface. In some embodiments, the substrate is a glass slide. In some embodiments, oligonucleotides (e.g., capture probes) are covalently attached to a chemical substrate, such as epoxysilanes, aminosilanes, lysine, polyacrylamide, etc.

[0433] These arrays can also be prepared using in-situ synthesis methods. In some embodiments, these arrays can be prepared using photolithography. This method typically relies on a combination of UV masking and photodirected combinatorial chemistry on a substrate to selectively synthesize probes directly on the array surface, one nucleotide at a time, for multiple sites simultaneously. In some embodiments, the substrate contains covalent linker molecules with photoremovable protecting groups at their free ends. UV light is guided through a photolithographic mask to deprotect and activate selected sites with hydroxyl groups, initiating coupling with the incoming protected nucleotide attached to the activated site. The mask is designed to selectively expose sites, thus specifying the coordinates on the array where each nucleotide can be attached. This process can be repeated, with a new mask 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 techniques can be used. It uses a programmable micromirror array to create a digital mask that reflects the desired UV pattern to remove the protection of the features.

[0434] In some implementations, inkjet printing can also be used for in-situ oligonucleotide synthesis. Different nucleotide precursors plus catalysts can be printed onto a substrate, followed by coupling and deprotection steps. This method relies on printing picoliter volumes of nucleotides onto an array surface in repeating rounds of base-by-base printing, which extends the length of oligonucleotide probes on the array.

[0435] Arrays can also be prepared via active hybridization using an electric field to control nucleic acid transport. 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 may contain binding molecules, such as streptavidin, which enable the formation of bonds (e.g., streptavidin-biotin bonds) when an electronically addressed biotinylated probe reaches its target site. The positive current is then removed from the activated feature, and new test sites can be activated by targeted application of the positive current. This process is repeated until all sites on the array are covered.

[0436] Arrays for spatial analysis can be generated using various methods as described herein. In some embodiments, the array has multiple capture probes containing spatial barcodes. These spatial barcodes and their relationship to their positions on the array can be determined. In some cases, such information is readily available because oligonucleotides are spotted, printed, or synthesized on the array in a predetermined pattern. In some cases, the spatial barcodes can be decoded using 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 subarrays. Thus, spatial barcodes can be transferred to subarrays with known patterns.

[0437] In some implementations, an array of barcoded probes can be generated by ligating multiple oligonucleotides. In some instances, the multiple oligonucleotides contain portions of a barcode, and a complete barcode is generated when the multiple oligonucleotides are ligated. For example, a first oligonucleotide containing a first portion of the barcode can be attached to a substrate (e.g., using any method described herein for attaching oligonucleotides to a substrate), and then a second oligonucleotide containing 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 before ligation, the first and / or second oligonucleotides can be attached to the substrate via a surface adapter containing a cleavage site. After ligation, the ligated oligonucleotides are linearized by cleaving at the cleavage site.

[0438] To increase barcode diversity, multiple second oligonucleotides containing two or more different barcode sequences can be linked to multiple first oligonucleotides containing the same barcode sequence, thereby generating two or more different types of barcodes. For selective linking, the first oligonucleotide attached to the substrate containing the first portion of the barcode can initially be protected with a protecting group (e.g., a photolytically cleavable protecting group), and the protecting group can be removed before linking the first and second oligonucleotides. In the case where barcoded probes on an array are generated by linking two or more oligonucleotides, an oligonucleotide concentration gradient can be applied to the substrate, allowing different combinations of oligonucleotides to be incorporated into the barcoded probe according to their position on the substrate.

[0439] Barcode probes on an array can also be generated by adding individual nucleotides to existing oligonucleotides on the array, for example, using a polymerase that operates in a template-independent manner. Individual nucleotides can be added to existing oligonucleotides in a concentration gradient, resulting in probes of different lengths depending on their position on the array.

[0440] Arrays can also be prepared by modifying existing arrays, for example, by modifying the oligonucleotides attached to the array. For example, probes can be generated on an array containing oligonucleotides, with the oligonucleotides attached to the array at the 3' end and having a free 5' end. The oligonucleotides can be in-situ synthesized oligonucleotides 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, primers partially complementary to the oligonucleotides (e.g., a constant sequence common to the oligonucleotides) can be hybridized with the oligonucleotides 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., via splint-type oligonucleotide-mediated ligation), wherein the added oligonucleotides may include a sequence of the capture domain or a portion of that sequence.

[0441] In cases where the oligonucleotides on the existing array include recognition sequences capable of hybridizing with the splice oligonucleotide, probes can also be generated by directly linking other oligonucleotides to the existing oligonucleotides via the splice oligonucleotide. The recognition sequence can be located at the free 5' or free 3' end of the oligonucleotide on the existing array. Recognition sequences useful to the method of the present invention may not contain restriction enzyme recognition sites or secondary structures (e.g., hairpins) and may include high levels of guanine and cytosine nucleotides, thus exhibiting high stability.

[0442] A bead array can be generated by attaching beads (e.g., barcode-coded beads) to a substrate in a regular pattern or irregular arrangement. Bead attachment can be achieved by selectively activating regions on the substrate, for example, by selectively activating areas on the substrate. Activating selective regions on the substrate may include activating a coating (e.g., a photolytically degradable coating) or polymer applied to the substrate. Beads can be attached iteratively; for example, a subset of beads can be attached at a time, and the same process can be repeated to attach the remaining beads. Alternatively, all beads can be attached to the substrate in one step.

[0443] 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.

[0444] Large-scale commercial manufacturing methods enable the attachment of millions of oligonucleotides to an array. Commercial arrays include those from Roche NimbleGen (Wisconsin) and Affymetrix (Thermo Fisher Scientific).

[0445] In some embodiments, the array may be fabricated 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 foregoing documents are incorporated herein by reference.

[0446] In some embodiments, the features on the array include beads. In some embodiments, two or more beads are dispersed on a substrate to create an array, wherein each bead is a feature on the array. Optionally, the beads can be dispersed into holes in the substrate, for example, such that each hole accommodates only a single bead.

[0447] "Bead" refers to a particle. Beads can be porous, non-porous, solid, semi-solid, and / or a combination thereof. In some embodiments, beads are soluble, ruptureable, and / or degradable, while in other embodiments, beads are non-degradable.

[0448] Beads can typically be any suitable shape. Examples of bead shapes include, but are not limited to, spherical, non-spherical, elliptical, rectangular, amorphous, circular, cylindrical, and variations thereof. A cross-section (e.g., a first cross-section) may correspond to the diameter or maximum cross-sectional dimension of the bead. In some embodiments, the bead may be approximately spherical. In these embodiments, the first cross-section may correspond to the diameter of the bead. In some embodiments, the bead may be approximately cylindrical. In such embodiments, the first cross-section may correspond to the diameter, length, or width along the approximately cylindrical bead.

[0449] Beads can be of uniform or non-uniform size. "Polydispersibility" generally refers to the non-uniformity of molecular or particle size. The polydispersity index (PDI) of 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 some embodiments, beads can be provided as multiple beads or groups of beads with a relatively monodisperse size distribution. Maintaining relatively consistent bead characteristics (e.g., size) contributes to overall consistency when a relatively consistent amount of reagent is required.

[0450] In some embodiments, the beads provided herein may have a size distribution with a coefficient of variation of cross-sectional dimensions 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.

[0451] In some embodiments, the diameter or maximum size of the bead may not exceed 100 μm (e.g., not 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 average diameter of the plurality of beads is not greater than 100 μm. In some embodiments, the average diameter or maximum size of the plurality of beads is not 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.

[0453] In some embodiments, the volume of the bead may be at least about 1 μm. 3 For example, at least 1μm 3 2μm 3 3μm 3 4μm 3 5μm 3 6μm 3 7μm 3 8μm 3 9μm 3 10μm 3 12μm 3 14μm 3 16μm 3 18μm 3 20μm 3 25μm 3 30μm 3 35μm 3 40μm 3 45μm 3 50μm 3 55μm 3 60μm 3 65μm 3 70μm 3 75μm 3 80μm 3 85μm 3 90μm 3 95μm3 100μm 3 125μm 3 150μm 3 175μm 3 200μm 3 250μm 3 300μm 3 350μm 3 400μm 3 450μm 3 μm 3 500μm 3 550μm 3 600μm 3 650μm 3 700μm 3 750μm 3 800μm 3 850μm 3 900μm 3 950μm 3 1000μm 3 1200μm 3 1400μm 3 1600μm 3 1800μm 3 2000μm 3 2200μm 3 2400μm 3 2600μm 3 2800μm 3 3000μm 3 Or larger.

[0454] In some embodiments, the beads may have a diameter of about 1 μm. 3 With 100μm 3 The volume between, for example, about 1 μm 3 With 10μm 3 Between, approximately 10μm 3 With 50μm 3 Between or approximately 50 μm 3 With 100μm 3 The volume between [the two sizes]. In some embodiments, the beads may include approximately 100 μm. 3 With 1000μm 3 The volume between, for example, about 100 μm 3 With 500μm 3 Between or approximately 500 μm 3 With 1000μm 3 The volume between [specific dimensions]. In some embodiments, the beads may include approximately 1000 μm.3 With 3000μm 3 The volume between, for example, approximately 1000 μm 3 With 2000μm 3 Between or approximately 2000μm 3 With 3000μm 3 The volume between [a certain value]. In some embodiments, the beads may include approximately 1 μm. 3 With 3000μm 3 The volume between, for example, about 1 μm 3 With 2000μm 3 Between, approximately 1μm 3 With 1000μm 3 Between, approximately 1μm 3 With 500μm 3 Between or approximately 1 μm 3 With 250μm 3 The volume between.

[0455] The bead may include one or more identical or different cross-sections. In some embodiments, the bead may have a first cross-section different from the second cross-section. The bead may have a first cross-section of at least about 0.0001 micrometers, 0.001 micrometers, 0.01 micrometers, 0.1 micrometers, or 1 micrometer. In some embodiments, the bead may include at least about 1 micrometer (μm), 2μm, 3μm, 4μm, 5μm, 6μm, 7μm, 8μm, 9μm, 10μm, 11μm, 12μm, 13μm, 14μm, 15μm, 16μm, 17μm, 18μm, 19μm, 20μm, 25μm, 30μm, 35μm, 40μm, 45μm, 50μm, 55μm, 60μm, 65μm, 70μm, 75μm. Cross-sections of 80μm, 85μm, 90μm, 100μm, 120μm, 140μm, 160μm, 180μm, 200μm, 250μm, 300μm, 350μm, 400μm, 450μm, 500μm, 550μm, 600μm, 650μm, 700μm, 750μm, 800μm, 850μm, 900μm, 950μm, 1 mm or larger (e.g., the first cross-section). In some embodiments, the bead may include a cross-section between about 1 μm and 500 μm (e.g., a first cross-section), such as between about 1 μm and 100 μm, about 100 μm and 200 μm, about 200 μm and 300 μm, about 300 μm and 400 μm, or about 400 μm and 500 μm. For example, the bead may include a cross-section between about 1 μm and 100 μm (e.g., a first cross-section). In some embodiments, the bead may have a second cross-section of at least about 1 μm. For example, beads may include 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 , 16μm, 17μm, 18μm, 19μm, 20μm, 25μm, 30μm, 35μm, 40μm, 45μm, 50μm, 55μm, 60μm, 65μm, 70μm, 75μm , 80μm, 85μm, 90μm, 100μm, 120μm, 140μm, 160μm, 180μm, 200μm, 250μm, 300μm, 350μm, 400μm, 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 larger second cross-section.In some embodiments, the bead may 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 bead may include a second cross-section between about 1 μm and 100 μm.

[0456] In some embodiments, the beads can be nanoscale (e.g., the beads can have a diameter or maximum cross-sectional size 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). Multiple beads may have an average diameter or average maximum cross-sectional size 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 approximately the size of a single cell (e.g., the single cell being evaluated).

[0457] In some embodiments, the beads may 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; polymeric network structures, such as gelatin; hydrogels; and cross-linked polymeric 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).

[0458] Depending on the intended use, gels can be formulated into various shapes and sizes. In some embodiments, the gel is prepared and formulated into gel beads (e.g., gel beads comprising a capture probe attached to or associated with the gel beads). Gel beads can be hydrogel beads. Hydrogel beads can be formed from molecular precursors, such as polymers or monomeric substances.

[0459] In some embodiments, the hydrogel beads may comprise a polymer matrix (e.g., a matrix formed by polymerization or crosslinking). The polymer matrix may comprise one or more polymers (e.g., polymers with different functional groups or repeating units). Crosslinking may be achieved through covalent, ionic, and / or induced interactions and / or physical entanglement.

[0460] Semi-solid beads can be liposome beads.

[0461] Solid beads may include metals, including but not limited to iron oxide, gold, and silver. In some embodiments, the beads may be silica beads. In some embodiments, the beads may be rigid. In some embodiments, the beads may be flexible and / or compressible.

[0462] Beads can be macromolecules. Beads can be formed from nucleic acid molecules linked together. Beads can be formed through the covalent or non-covalent assembly of molecules (e.g., macromolecules), such as monomers or polymers. Polymers or monomers can be natural or synthetic. Polymers or monomers can be or include, for example, nucleic acid molecules (e.g., DNA or RNA).

[0463] Beads can be rigid, or they can be flexible and / or compressible. Beads may include a coating comprising one or more polymers. This coating can be broken down or dissolved. In some embodiments, beads include spectral or optical markers (e.g., dyes) directly or indirectly (e.g., via a connector) attached to the beads. For example, beads can be prepared as colored formulations (e.g., beads exhibiting different colors within the visible spectrum) that can change color upon application of a desired stimulus (e.g., heat and / or a chemical reaction) to form beads of different colors (e.g., opaque and / or transparent beads).

[0464] Beads may include natural and / or synthetic materials. For example, beads may 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, ibaguar gum, gum arabic, agar, gelatin, shellac, tung oil gum, xanthan gum, corn gum, guar gum, carrageenan, agarose, alginate, sodium alginate, or natural polymers thereof. Examples of synthetic polymers include, but are not limited to, acrylic acid, resins, nylon, silicone, spandex, viscose rayon, polycarboxylic acid, polyvinyl acetate, polyacrylamide, polyacrylate, 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). 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.

[0465] In some embodiments, the beads are biodegradable beads. Biodegradable beads may include one or more substances having unstable bonds (e.g., disulfide linkers, primers, other oligonucleotides, etc.) such that when the bead / substance is exposed to a suitable stimuli, the unstable bonds are broken and the bead degrades. The unstable bonds may be chemical bonds (e.g., covalent bonds, ionic bonds) or may 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 may include unstable bonds. When exposed to suitable conditions, the unstable bonds break, and the bead degrades. For example, when polyacrylamide gel beads containing a cysteine ​​crosslinking agent are exposed to a reducing agent, the disulfide bonds of cysteine ​​can be broken and the bead degrades.

[0466] Degradation can refer to the dissociation of bound or entrained substances (e.g., disulfide linkers, primers, other oligonucleotides, etc.) from the bead, regardless of whether the physical bead itself is structurally degraded. For example, due to changes in the chemical environment, entrained substances can be released from the bead through a permeation pressure difference. For instance, changes in bead pore size caused by a permeation pressure difference can typically occur without structural degradation of the bead itself. In some embodiments, the increase in pore size due to permeation swelling of the bead can release entrained substances within the bead. In some embodiments, permeation shrinkage of the bead due to pore size contraction can result in the bead better retaining entrained substances.

[0467] Any suitable reagent for biodegradable 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 degrading agents can be used to degrade chemical bonds within the beads through oxidation, reduction, or other chemical changes. For example, a chemical degrading agent can be a reducing agent, such as DTT, which can degrade disulfide bonds formed between the crosslinking agent 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 include, but are not limited to, dithiothreitol (DTT), β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane (dithiobutane or DTBA), tris(2-carboxyethyl)phosphine (TCEP), or combinations thereof.

[0468] Any chemical reagent can be used to initiate the degradation of beads. Examples of chemical reagents include, but are not limited to, pH-mediated alteration of the integrity of the bead's internal components, degradation of bead components through the cleavage of cross-links, and depolymerization of bead components.

[0469] In some embodiments, the beads may be formed from materials comprising a biodegradable chemical crosslinking agent (e.g., N,N'-bis-(acryloyl)cysteine ​​(BAC) or cystamine). Degradation of this biodegradable crosslinking agent can be achieved through various mechanisms. In some instances, the beads may be contacted with a chemical degrading agent that can cause oxidation, reduction, or other chemical changes. For example, the chemical degrading agent may be a reducing agent, such as dithiothreitol (DTT). Other examples of reducing agents may include β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane (dithiobutane or DTBA), tris(2-carboxyethyl)phosphine (TCEP), or combinations thereof.

[0470] In some embodiments, exposure to an aqueous solution (e.g., water) can trigger hydrolytic degradation, thereby initiating the degradation of the beads. Applying heat can also induce the beads 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, leading to partial degradation. In some embodiments, heat can increase the internal pressure of the bead components, causing the beads to rupture or explode. Heat can also act on heat-sensitive polymers used as building blocks of the beads.

[0471] When using biodegradable 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 arising from such degradation, including, for example, poor flow properties and aggregation. For example, when the beads comprise reducible crosslinking groups (e.g., disulfide groups), it would be desirable to avoid contact between the beads and reducing agents (e.g., DTT or other disulfide cleavage agents). In some of these embodiments, the treatment of the beads described herein will be free of reducing agents, such as DTT. Since reducing agents are commonly provided in commercial enzyme formulations, it is desirable to provide enzyme formulations that are free of reducing agents (or DTT) when treating the beads described herein. Examples of such enzymes include, for example, polymerase formulations, reverse transcriptase formulations, ligase formulations, and many other enzyme formulations that can be used to treat the beads described herein. The terms "reducing agent-free" or "DTT-free" formulations refer to formulations having a fraction of about 1 / 10, about 1 / 50, or about 1 / 100 of such materials used for degrading beads. For example, for DTT, the reducing agent-free formulation may have less than about 0.01 mmol (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.

[0472] Compared to non-degradable beads, degradable beads can be used to release attached capture probes (e.g., nucleic acid molecules, spatial barcode sequences, and / or primers) more rapidly when appropriate stimulation is applied. For example, in the case of substances bound to the inner surface of porous beads or encapsulated substances, the substance may have greater mobility and accessibility to other substances in solution upon bead degradation. In some embodiments, substances may also be attached to degradable beads via degradable connectors (e.g., disulfide connectors). The degradable connectors may respond to the same stimulation as the degradable beads, or the two degradable substances may respond to different stimuli. For example, capture probes having one or more spatial barcodes can be attached to polyacrylamide beads comprising cystamine via disulfide bonds. When the spatially barcoded beads are exposed to a reducing agent, the beads degrade and release capture probes having one or more spatial barcode sequences upon the breaking of the disulfide bonds between the capture probe and the bead and the disulfide bonds of the cystamine within the bead.

[0473] Adding multiple types of unstable bonds to beads can create beads capable of responding to a variety of stimuli. Each type of unstable bond can be sensitive to the relevant stimulus (e.g., chemical stimuli, light, temperature, pH, enzymes, etc.), such that the release of reagents attached to the beads via each unstable bond can be controlled by applying an appropriate stimulus. Some non-limiting examples of unstable bonds that can couple to precursors or beads include ester bonds (e.g., cleavable with acids, bases, or hydroxylamine), vicinal diol bonds (e.g., cleavable with sodium periodate), Diels-Alder bonds (e.g., cleavable by heat), sulfone bonds (e.g., cleavable with bases), silyl ether bonds (e.g., cleavable with acids), glycosidic bonds (e.g., cleavable with amylases), peptide bonds (e.g., cleavable with proteases), or phosphodiester bonds (e.g., cleavable with nucleases, such as DNases). Bonds can be cleaved by enzymes targeting other nucleic acid molecules, 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 the unstable bond can be attached to the beads after gel bead formation via, for example, the activated functional groups of the beads described above. In some embodiments, the gel beads including unstable bonds are reversible. In some embodiments, gel beads with reversible unstable bonds are used to capture one or more regions of interest in a biological sample. For example, but not limited to, beads including heat-sensitive bonds can be heated by a light source (e.g., a laser) that causes changes in the gel beads to facilitate the capture of biological samples in contact with the gel beads. Capture probes having one or more spatial barcodes releasable, cleavable, or reversibly attached to the beads described herein include capture probes that are released or releasable through the cleavage of the connection between the capture probe and the bead, or capture probes released through the degradation of the underlying bead itself, such that the capture probes having one or more spatial barcodes are to be touched by other reagents or become accessible to other reagents, or both.

[0474] Beads can have different physical properties. These physical properties can be used to characterize the beads. Non-limiting examples of physical properties of beads include size, shape, roundness, density, symmetry, and hardness. For example, beads can have different sizes. Beads of different sizes can be obtained by using a microfluidic channel network configured to provide beads of a specific size (e.g., based on channel size, flow rate, etc.). In some embodiments, beads have different hardness values, which can be obtained by varying the polymer concentration used to generate the beads. In some embodiments, the physical properties of the capture probe can be used to make the spatial barcode attached to the bead optically detectable. For example, nucleic acid origami, such as deoxyribonucleic acid (DNA) origami, can be used to generate optically detectable spatial barcodes. For this purpose, one or more nucleic acid molecules can be folded to form two-dimensional and / or three-dimensional geometries. Different geometries can be detected optically.

[0475] In some embodiments, special types of nanoparticles having more than one different physical property can be used to make the beads physically distinguishable. For example, Janus particles with hydrophilic and hydrophobic surfaces can be used to provide unique physical properties.

[0476] In some embodiments, the beads are capable of recognizing multiple analytes (e.g., nucleic acids, proteins, chromatin, metabolites, drugs, gRNA, and lipids) from a single cell. In some embodiments, the beads are capable of recognizing a single analyte (e.g., mRNA) from a single cell.

[0477] 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 depolymerizable, chemically depolymerizable, enzymatically depolymerizable, and / or optically depolymerizable.

[0478] In some embodiments, the beads may be non-covalently loaded with one or more reagents. Non-covalent loading of the beads can be achieved, for example, by subjecting the beads to conditions sufficient to cause swelling, allowing sufficient time for the reagent to diffuse into the interior of the beads, and subjecting the beads to conditions sufficient to cause deswelling. Swelling of the beads can be achieved, for example, by placing the beads in a thermodynamically favorable solvent, subjecting the beads to higher or lower temperatures, subjecting the beads to higher or lower ion concentrations, and / or subjecting the beads to an electric field.

[0479] Bead swelling can be achieved through various swelling methods. In some embodiments, the swelling is reversible (e.g., by subjecting the beads to conditions that promote deswelling). In some embodiments, deswelling of the beads is achieved, for example, by transferring the beads in a thermodynamically unfavorable solvent, subjecting the beads to lower or higher temperatures, subjecting the beads to lower or higher ion concentrations, and / or adding or removing an electric field. Bead deswelling can be achieved through various deswelling methods. In some embodiments, deswelling is reversible (e.g., by subjecting the beads to conditions that promote swelling). In some embodiments, bead deswelling may include transferring the beads to cause the pores in the beads to shrink. The shrinkage will impede the diffusion of reagents from the interior of the beads. The resulting impediment may be due to spatial interactions between the reagent and the interior of the beads. This transfer can be accomplished using microfluidics. For example, the transfer can be achieved by moving the beads from one co-current solvent stream to a different co-current solvent stream. The swelling capacity and / or pore size of the beads can be tuned by changing the polymer composition of the beads.

[0480] The beads may include a temperature-responsive polymer so that the properties or size of the beads can be changed when the beads are heated or cooled. For example, the polymer may include poly(N-isopropylacrylamide). 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). Temperatures sufficient to change one or more properties of the gel beads may be, for example, at least about 0 degrees Celsius (°C), 1 degree Celsius, 2 degrees Celsius, 3 degrees Celsius, 4 degrees Celsius, 5 degrees Celsius, 10 degrees Celsius, or higher. For example, the temperature may be about 4 degrees Celsius. In some embodiments, temperatures sufficient to change one or more properties of the gel beads may be, for example, at least about 25 degrees Celsius, 30 degrees Celsius, 35 degrees Celsius, 37 degrees Celsius, 40 degrees Celsius, 45 degrees Celsius, 50 degrees Celsius, or higher. For example, the temperature may be about 37 degrees Celsius.

[0481] Functionalization of beads used to attach capture probes can be achieved through a variety of methods, including but not limited to activating chemical groups within the polymer, introducing active or activatable functional groups into the polymer structure, or attachment during the prepolymer or monomer stage of bead production. Beads can be functionalized to bind to target analytes, such as nucleic acids, proteins, carbohydrates, lipids, metabolites, peptides, or other analytes.

[0482] In some embodiments, the beads may comprise a molecular precursor (e.g., a monomer or polymer) that can form a polymer network through polymerization of the molecular precursor. In some embodiments, the precursor may be a polymerized material capable of further polymerization via, for example, chemical crosslinking. In some embodiments, the precursor may comprise one or more of acrylamide or methacrylamide monomers, oligomers, or polymers. In some embodiments, the beads may comprise a prepolymer that is an oligomer capable of further polymerization. For example, prepolymers can be used to prepare polyurethane beads. In some embodiments, the beads may comprise a single polymer that can be further polymerized together (e.g., to form a copolymer). In some embodiments, the beads may be generated by polymerization of different precursors, such that they comprise a mixture of polymers, copolymers, and / or block copolymers. In some embodiments, the beads may comprise 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 bonds may be carbon-carbon bonds or thioether bonds.

[0483] The crosslinking of polymers can be permanent or reversible, depending on the specific crosslinking agent used. Reversible crosslinking allows the polymer to linearize or dissociate under suitable conditions. In some embodiments, reversible crosslinking also allows for the reversible attachment of material to the bead surface. In some embodiments, the crosslinking agent can form disulfide bonds. In some embodiments, the chemical crosslinking agent forming disulfide bonds can be cystamine or modified cystamine.

[0484] For example, in cases where the polymer precursor material includes a linear polymer material (e.g., linear polyacrylamide, PEG, or other linear polymer materials), the activator may include a crosslinking agent or a chemical of the crosslinking agent within the activated droplets. Similarly, for polymer precursors comprising polymerizable monomers, the activator may include a polymerization initiator. For example, in some embodiments where the polymer precursor comprises a mixture of acrylamide monomer and N,N'-bis-(acryloyl)cysteine ​​(BAC) comonomer, reagents such as tetraethylmethylenediamine (TEMED) may 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 may include exposure to heating, cooling, electromagnetic radiation, and / or light.

[0485] Following polymerization or gelation, a polymer or gel may be formed. The polymer or gel may be diffusely permeable to chemical or biochemical reagents. Alternatively, the polymer or gel may be non-diffusionally permeable to macromolecular components. The polymer or gel may include one or more of the following: cross-linked polyacrylamide disulfide, agarose, alginate, polyvinyl alcohol, polyethylene glycol (PEG) diacrylate, PEG acrylate, PEG thiol, PEG azide, PEG alkyne, other acrylates, chitosan, hyaluronic acid, collagen, fibroin, gelatin, or elastin. The polymer or gel may include any other polymer or gel.

[0486] In some embodiments, disulfide bonds can be formed between molecular precursor units (e.g., monomers, oligomers, or linear polymers) or precursors incorporated into the beads and nucleic acid molecules (e.g., oligonucleotides, capture probes). For example, cystamine (including modified cystamine) is an organic agent comprising disulfide bonds and can be used as a crosslinking agent between individual monomers or polymerization precursors of the beads. Polyacrylamide can be polymerized in the presence of cystamine or substances including cystamine (e.g., modified cystamine) to produce polyacrylamide gel beads comprising disulfide bonds (e.g., chemically degradable beads comprising chemically reducible crosslinking agents). The disulfide bonds allow the beads to degrade (or dissolve) upon exposure to a reducing agent.

[0487] In some embodiments, the linear polysaccharide polymer chitosan can be crosslinked with glutaraldehyde via hydrophilic chains to form beads. Crosslinking of the chitosan polymer can be achieved through chemical reactions induced by heat, pressure, pH changes, and / or radiation.

[0488] In some embodiments, the beads may include an acrydite moiety, which in some respects can be used to attach one or more capture probes to the beads. 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 crosslinking agents during polymerization. The acrydite moiety may be modified to form a chemical bond with the substance to be attached (e.g., a capture probe). The acrydite moiety may be modified with a thiol group capable of forming a disulfide bond, or with a group that already includes a disulfide bond. A thiol group or disulfide (via disulfide exchange) may be used as an anchoring site for the substance to be attached, or another portion of the acrydite moiety may be used for attachment. In some embodiments, the attachment may be reversible, such that when the disulfide bond breaks (e.g., in the presence of a reducing agent), the attached substance is released from the beads. In some embodiments, the acrydite moiety may include reactive hydroxyl groups that can be used for substance attachment.

[0489] In some embodiments, the precursor (e.g., monomer or cross-linking agent) polymerized to form the bead may include a phosphorus acrylate moiety, such that the bead also includes the phosphorus acrylate moiety when it is produced. The phosphorus acrylate moiety may be attached to a nucleic acid molecule (e.g., an oligonucleotide), which may include a priming sequence (e.g., a primer for amplifying a target nucleic acid, a random primer, a primer sequence for messenger RNA) and / or one or more capture probes. One or more capture probes may include the same sequence for all capture probes coupled to a given bead and / or different sequences for all capture probes coupled to a given bead. The capture probe may be incorporated into the bead. In some embodiments, the capture probe may be incorporated into or attached to the bead such that the capture probe retains a free 3' end. In some embodiments, the capture probe may be incorporated into or attached to the bead such that the capture probe retains a free 5' end. In some embodiments, the beads may be functionalized such that each bead contains multiple different capture probes. For example, a bead may include multiple capture probes, such as capture probe 1, capture probe 2, and capture probe 3, and each of capture probe 1, capture probe 2, and capture probe 3 contains a different capture domain (e.g., the capture domain of capture probe 1 includes a poly(dT) capture domain, the capture domain of capture probe 2 includes a gene-specific capture domain, and the capture domain of capture probe 3 includes a CRISPR-specific capture domain). By functionalizing the beads so that each bead contains multiple different capture domains, the level of reusability for analyte detection can be improved.

[0490] In some embodiments, the precursors (e.g., monomers or crosslinking agents) polymerized to form beads may include reactive or activatable functional groups, such that when they become reactive, they can polymerize with other precursors to produce beads including said activated or activatable functional groups. Other substances (e.g., disulfide linkers, primers, other oligonucleotides, etc.) can then be attached to the beads using these functional groups. For example, some precursors including carboxylic acid (COOH) groups can copolymerize with other precursors to form beads that also include COOH functional groups. In some embodiments, acrylic acid (a substance including free COOH groups), acrylamide, and bis(acryloyl)cysteine ​​can copolymerize together to produce beads including...

Claims

1. A method for determining the location of an analyte in a biological sample, the method comprising: Multiple capture probes are introduced into the biological sample, wherein the capture probes include a spatial barcode and a capture domain, wherein the capture domain is specifically capable of binding to an analyte, and wherein the capture probe is capable of passing through the cell membrane of the biological sample; and Determine (i) all or part of the sequence corresponding to the spatial barcode or its complement, and (ii) all or part of the sequence corresponding to the analyte or its complement, and use the determined sequences of (i) and (ii) to determine the location of the analyte in the biological sample.

2. The method of claim 1, wherein the incorporation includes injection, particle bombardment, photoperforation, magnetic transfer and / or water perforation.

3. The method of claim 2, wherein the injection comprises microinjection.

4. The method of claim 1, wherein the capture probe further comprises a lysis domain, and the method includes contacting the biological sample with a substrate containing a plurality of capture probes and lysing the plurality of capture probes from the substrate.

5. The method of any one of claims 1-4, wherein the capture probe is directly or indirectly coupled to a lipophilic molecule.

6. The method of claim 5, wherein the lipophilic molecule is selected from the group consisting of cholesterol, tocopherol, sterol palmitate, lignans, squalene, fatty acids and their derivatives.

7. The method of any one of claims 1-4, wherein the capture probe is directly or indirectly coupled to a cell-penetrating agent.

8. The method of claim 7, wherein the cell-penetrating agent is a cell-penetrating peptide.

9. A method for determining the location of an analyte in a biological sample, the method comprising: (a) Bring the biological sample into contact with the substrate; (b) Dividing the biological sample into multiple slices, wherein slices of the multiple slices are translocated into the wells of a plurality of wells, wherein the slices contain one or more analytes; (c) Providing a plurality of capture probes, wherein one of the plurality of capture probes comprises a spatial barcode and a capture field, wherein the capture field is specifically capable of binding to an analyte in one or more analytes in the slice; and (d) Determine (i) all or part of the sequence corresponding to the spatial barcode or its complement, and (ii) all or part of the sequence corresponding to the analyte or its complement, and use the determined sequences of (i) and (ii) to determine the location of the analyte in the biological sample.

10. The method of claim 9, wherein the capture probe is provided on a substrate.

11. The method of claim 9, wherein the capture probe is provided in the orifice.

12. The method of any one of claims 9-11, wherein the pore is a nanopore.

13. The method of any one of claims 9-12, wherein the partitioning includes cutting and / or imprinting the biological sample.

14. The method of any one of claims 9-12, wherein the partitioning comprises laser-captured micro-cutting of the biological sample.

15. The method of any one of claims 9-12, wherein the division includes using a hollow needle or a microneedle.

16. The method of any one of claims 9-15, wherein the method further comprises identifying the region of interest in the biological sample prior to partitioning.

17. A method for generating an array, comprising: (a) Providing a plurality of spatially barcoded oligonucleotides to a substrate, wherein two or more of the plurality of spatially barcoded oligonucleotides include a first attachment sequence and a second attachment sequence, and wherein the substrate includes 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 to generate a spatially barcoded oligonucleotide array; and (b) Amplifying spatially barcoded oligonucleotides on the substrate. This generates an array.

18. The method of claim 17, wherein the two or more spatially barcoded oligonucleotides on the amplification substrate comprise bridging amplification.

19. The method of claim 17 or 18, comprising determining the identity of two or more spatially barcoded oligonucleotides in the array.

20. The method of claim 19, wherein determining the similarity of two or more spatially barcoded oligonucleotides comprises sequencing the spatially barcoded oligonucleotides.

21. The method of claim 20, wherein the sequencing comprises in situ sequencing.

22. The method of claim 21, wherein the in situ sequencing comprises hybridizing a priming oligonucleotide with two or more spatially barcoded oligonucleotides.

23. The method of claim 22, wherein the oligonucleotide is initiated to couple with the cell marker.

24. The method of any one of claims 21-23, wherein the in situ sequencing comprises one or more of sequencing by synthesis, sequencing by ligation, and sequencing by rolling circle amplification.

25. The method of any one of claims 21-23, wherein the in situ sequencing comprises sequencing by synthesis.

26. The method of claim 24 or 25, wherein sequencing by synthesis comprises hybridizing the priming oligonucleotide with two or more spatially barcoded oligonucleotides.

27. The method of claim 26, wherein the oligonucleotide is initiated to couple with the cell marker.

28. The method of any one of claims 17-27, wherein the two or more spatially barcoded oligonucleotides comprise one or more of a spatial barcode, an initiation domain, a hybridization domain, a unique molecular identifier, a functional domain, and a cleavage domain.

29. The method of any one of claims 17-27, wherein two or more spatially barcoded oligonucleotides comprise spatial barcodes.

30. The method of any one of claims 17-27, wherein two or more spatially barcoded oligonucleotides comprise a cleavage domain.

31. The method of any one of claims 23-30, wherein the cell marker comprises an extracellular cell marker.

32. The method of any one of claims 23-30, wherein the cell marker comprises an intracellular cell marker.

33. The method of any one of claims 23-30, wherein the cell marker is located on an internal component of the cell.

34. The method of claim 33, wherein the internal components of the cell include one or more of mitochondria, Golgi apparatus, smooth endoplasmic reticulum, rough endoplasmic reticulum, nucleus, nucleolus, and lysosomes.

35. The method of any one of claims 23-30, wherein the cell marker comprises one or more of lipids, antibodies, chitosan, lectins, streptavidin, click chemically modified moieties, cell-penetrating peptides, nanoparticles, TIVA tags, and liposomes / polyribosomes.

36. The method of any one of claims 23-30, wherein the cell marker is amphiphilic.

37. The method of any one of claims 23-30, wherein the cell marker is lipophilic.

38. The method of any one of claims 23-30, wherein the cell marker is a cholesterol moiety.

39. The method of any one of claims 23-30, wherein the cell marker is coupled to the initiating oligonucleotide via a linker.

40. The method of claim 39, wherein the connector comprises one or more of the following: N-hydroxysuccinimide (NHS) connector, bifunctional NHS connector, azide, alkyne, ethylene glycol chitosan, 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-poly(ethylene glycol) (DSPE-PEG), and succinimide-3-(2-pyridyl dithio)propionate (SPDP).

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