EP4500178A1

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Publication number: EP4500178A1
Application number: EP24740635.8A
Authority: EP
Inventors: Govinda M. KAMATH; Stephen R. Williams
Original assignee: 10X Genomics Inc
Current assignee: 10X Genomics Inc
Priority date: 2023-05-15
Filing date: 2024-05-15
Publication date: 2025-02-05
Also published as: WO2024238625A1

Abstract

Systems and methods for quantifying spatial analyte data are provided. A biological sample is exposed to analyte capture agents. A first subset of the analyte capture agents specifically binds to a first subset of analytes in the biological sample. A second subset of the analyte capture agents non-specifically associates with the biological sample. The biological sample is aligned with a substrate comprising an array that includes spatially barcoded features. While aligned, oligonucleotides of analyte capture agents in the first and second subsets are hybridized to respective capture domain sequences of capture probes in the array. For each respective spatially barcoded feature in the array, for each respective analyte capture agent in the first subset, a normalized count is obtained by normalizing a respective raw count of the respective oligonucleotides in the first and second subsets that hybridized in the respective barcoded feature by the raw count of one or more analyte capture agents in the second subset.

Description

SPATIAL ANTIBODY DATA NORMALIZATION

CROSS REFERENCE TO RELATED PATENT APPLICATION

[0001] The present application claims priority to United States Provisional Patent Application No. 63/502,352, entitled “SPATIAL ANTIBODY DATA NORMALIZATION,” filed May 15, 2023, which is hereby incorporated by reference.

TECHNICAL FIELD

[0002] This specification describes technologies relating to analyte data normalization, particularly for use in analyzing spatial analyte data.

BACKGROUND

[0003] Cells within a tissue have differences in cell morphology and/or function due to varied analyte levels (e.g., gene and/or protein expression) within the different cells. The specific position of a cell within a tissue (e.g., the cell’s position relative to neighboring cells or the cell’s position relative to the tissue microenvironment) can affect, e.g., the cell’s morphology, differentiation, fate, viability, proliferation, behavior, signaling, and cross-talk with other cells in the tissue.

[0004] Spatial heterogeneity has been previously studied using techniques that typically provide data for a handful of analytes in the context of intact tissue or a portion of a tissue (e.g., tissue section), or provide significant analyte data from individual, single cells, but fail to provide information regarding the position of the single cells from the originating biological sample (e.g., tissue).

[0005] Spatial analysis of an analyte within a biological sample may require determining the sequence of the analyte sequence or a complement thereof and the sequence of the spatial barcode or a complement thereof to identify the location of the analyte. The biological sample may be placed on a solid support to improve specificity and efficiency when being analyzed for identification or characterization of an analyte, such as protein, DNA, RNA or other genetic material, within the sample.

[0006] Thus, there is a need in the art for systems and methods that provide improved ways of using spatial analyte data and detailed tissue images to analyze biological samples, particularly in a spatial context. SUMMARY

[0007] Some embodiments of the present disclosure are directed to methods, compositions, devices, and systems for determining the location and/or abundance of an analyte in a biological sample. Determining the spatial location and/or abundance of analytes (e.g., proteins, DNA, or RNA) within a biological sample leads to better understanding of spatial heterogeneity in various contexts, such as disease models. Described herein are methods for capturing probes and/or barcodes to a capture domain. In some instances, the techniques disclosed herein facilitate downstream processing, such as sequencing of the probes and/or barcodes bound to a capture domain.

[0008] In some examples, the methods, compositions, devices, and systems disclosed herein utilize RNA-templated ligation (RTL) for analyzing an analyte (e.g., RNA) in a biological sample. In some examples, RTL is used in combination with a “sandwich process,” wherein the analyte (e.g., ligation product) is transferred from a first substrate to a second substrate for further downstream processing. In some examples, analyte capture agents are used for analyzing an analyte (e.g., protein) in a biological sample. In some examples, the methods disclosed herein allow spatial analysis of two or more different types of analytes, e.g., protein and nucleic acids concurrently.

[0009] In some instances, disclosed herein is a method for analyzing an analyte in a biological sample mounted on a first substrate comprising: hybridizing a first probe oligonucleotide and a second probe oligonucleotide to the analyte, wherein the first probe oligonucleotide and the second probe oligonucleotide each comprise a sequence that is substantially complementary to adjacent sequences of the analyte, and wherein the second probe oligonucleotide comprises a capture probe binding domain; coupling the first probe oligonucleotide and the second probe oligonucleotide, thereby generating a connected probe; aligning the first substrate with a second substrate comprising an array, such that at least a portion of the biological sample is aligned with at least a portion of the array, wherein the array comprises a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises: (i) a spatial barcode and (ii) a capture domain; when the biological sample is aligned with at least a portion of the array, (i) releasing the connected probe from the analyte and (ii) migrating the connected probe from the biological sample to the array; and hybridizing the connected probe to the capture domain.

[0010] In some instances, the first probe oligonucleotide and the second probe oligonucleotide are on a contiguous nucleic acid sequence. In some instances, the first probe oligonucleotide is on the 3’ end of the contiguous nucleic acid sequence. In some instances, the second probe oligonucleotide is on the 5’ end of the contiguous nucleic acid sequence. In some instances, the adjacent sequences abut one another. In some instances, the adjacent sequences are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides away from one another.

[0011] In some instances, the methods further include generating an extended first probe oligonucleotide, wherein the extended first probe oligonucleotide comprises a sequence complementary to a sequence between the sequence hybridized to the first probe oligonucleotide and the sequence hybridized to the second probe oligonucleotide.

[0012] In some instances, the methods further include generating an extended second probe oligonucleotide using a polymerase, wherein the extended second probe oligonucleotide comprises a sequence complementary to a sequence between the sequence hybridized to the first probe oligonucleotide and the sequence hybridized to the second probe oligonucleotide.

[0014] In some instances, the reagent medium further comprises polyethylene glycol (PEG). In some instances, the methods further include contacting the biological sample with a DNase, e.g., a DNasel or DNAse II.

[0015] In some instances, the biological sample and the array are contacted with the reagent medium for about 1 to about 60 minutes. In some instances, the biological sample and the array are contacted with the reagent medium for about 30 minutes. In some instances, the biological sample and the array are contacted with the reagent medium for less than 30 minutes. [0016] In some instances, the methods further include determining (i) all or a part of the sequence of the connected probe, or a complement thereof, and (ii) the sequence of the spatial barcode, or a complement thereof, optionally wherein the method further comprises using the determined sequence of (i) and (ii) to determine the location and abundance of the analyte in the biological sample. In some instances, the determining comprises sequencing (i) all or a part of the sequence of the connected probe, or a complement thereof, and (ii) the sequence of the spatial barcode, or a complement thereof.

[0017] In some instances, the capture probe comprises a poly(T) sequence. In some instances, the capture probe comprises a sequence specific to the analyte. In some instances, the capture probe further comprises one or more functional domains, a unique molecular identifier (UMI), a cleavage domain, and combinations thereof.

[0018] In some instances, the analyte is RNA. In some instances, the analyte is mRNA. In some instances, the analyte is DNA. In some instances, the analyte is genomic DNA.

[0019] In some instances, the methods further include analyzing a different analyte in the biological sample. In some instances, the analyzing of the different analyte comprises: contacting the biological sample with a plurality of analyte capture agents, wherein an analyte capture agent of the plurality of analyte capture agents comprises an analyte binding moiety and a capture agent barcode domain, wherein the analyte binding moiety specifically binds to the different analyte, and wherein the capture agent barcode domain comprises an analyte binding moiety barcode and a capture handle sequence; and hybridizing the capture handle sequence to the capture domain. In some instances, the methods further include determining (i) all or part of the sequence of the capture agent barcode domain; and (ii) the sequence of the spatial barcode, or a complement thereof. In some instances, the methods further include using the determined sequence of (i), and (ii) to analyze the different analyte in the biological sample.

[0024] In some instances, the biological sample and the array are contacted with the reagent medium for about 1 to about 60 minutes.

[0026] In some instances, the tissue sample is fixed and stained prior to step (a).

[0027] Some embodiments of the present disclosure are directed to methods, compositions, devices, and systems for concurrently detecting gene expression and proteins in a biological sample. A way to detect proteins is by using antibodies that bind to the complementary antigens. One of the main challenges of using antibodies for protein detection and/or quantification is that sometimes, the antibody data can include artifacts and/or show localized regions with disproportionately high or low protein expressions due to random background binding. Accordingly, there is a need for improved methods and systems that reduce and/or eliminate such spurious effects.

[0030] In some embodiments, the first subset of analytes are proteins and the plurality of analytes further comprises a second subset of analytes that are nucleic acids. The method further comprises contacting a first probe and a second probe to the biological sample under conditions suitable for hybridization of the first probe and the second probe to an analyte in the second subset of analytes. The first probe comprises a sequence that is substantially complementary to a first portion of a sequence of the analyte. The second probe comprises a sequence that is substantially complementary to a second portion of a sequence of the analyte. The second or the first probe comprises a sequence that is complementary to the capture domain sequence of the capture probe in a respective capture probe plurality. The method further comprises coupling the first probe and the second probe, thereby generating a connected probe, and hybridizing the connected probe to the capture domain sequence of one or more of the capture probes in one or more capture probe pluralities in the array.

[0031] Another aspect of the present disclosure provides a computer system comprising one or more processors, and memory. One or more programs are stored in the memory and are configured to be executed by the one or more processors. It will be appreciated that this memory can be on a single computer, a network of computers, one or more virtual machines, or in a cloud computing architecture. The one or more programs perform any of the disclosed methods.

[0032] Still another aspect of the present disclosure provides a computer readable storage medium storing one or more programs. The one or more programs comprise instructions, which when executed by an electronic device with one or more processors and a memory, cause the electronic device to perform any of the disclosed methods.

[0033] Other embodiments are directed to systems, portable consumer devices, and computer readable media associated with methods described herein.

[0034] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, patent application, or item of information was specifically and individually indicated to be incorporated by reference. To the extent publications, patents, patent applications, and items of information incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

[0035] Where values are described in terms of ranges, it should be understood that the description includes the disclosure of all possible sub-ranges within such ranges, as well as specific numerical values that fall within such ranges irrespective of whether a specific numerical value or specific sub-range is expressly stated.

[0036] The term “each,” when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection, unless expressly stated otherwise, or unless the context of the usage clearly indicates otherwise.

[0037] Various embodiments of the features of this disclosure are described herein. However, it should be understood that such embodiments are provided merely by way of example, and numerous variations, changes, and substitutions can occur to 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.

[0038] As disclosed herein, any embodiment disclosed herein, when applicable, can be applied to any aspect.

[0039] Various embodiments of systems, methods, and devices within the scope of the appended claims each have several aspects, no single one of which is solely responsible for the desirable attributes described herein. Without limiting the scope of the appended claims, some prominent features are described herein. After considering this discussion, and particularly after reading the section entitled “Detailed Description” one will understand how the features of various embodiments are used.

INCORPORATION BY REFERENCE

[0040] This Application incorporates each of the following applications by reference herein in its entirety (i) PCT Patent Publication No. W02022/140028 Al, entitled “METHODS, COMPOSITIONS, AND SYSTEMS FOR CAPTURING PROBES AND/OR BARCODES” published June 30, 2022; and (ii) PCT Patent Publication No. W02023/044071 Al entitled “Systems and Methods for Image Registration or Alignment” published March 23, 2023.

[0041] All publications, patents, patent applications, and information available on the Internet and mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, patent application, or item of information was specifically and individually indicated to be incorporated by reference. To the extent publications, patents, patent applications, or item of information available on the Internet incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

DESCRIPTION OF DRAWINGS

[0042] The following drawings illustrate certain embodiments of the features and advantages of this disclosure. These embodiments are not intended to limit the scope of the appended claims in any manner. Like reference symbols throughout the several views of the patent application indicate like elements.

[0043] FIG. 1 is a schematic diagram showing an example of a barcoded capture probe, as described herein.

[0044] FIG. 2 is a schematic illustrating a cleavable capture probe, where the cleaved capture probe can enter into a biological sample (e.g., non-permeabilized cell) and hybridize to target analytes within the cell.

[0045] FIG. 3 is a schematic diagram of an exemplary multiplexed spatially-barcoded feature. [0046] FIG. 4 is a schematic diagram of an exemplary analyte capture agent.

[0047] FIG. 5 is a schematic diagram depicting an exemplary interaction between a feature- immobilized capture probe 524 and an analyte capture agent 526.

[0048] FIG. 6A shows an exemplary workflow for using connected (e.g., ligated) probes to capture nucleic acid analytes.

[0049] FIG. 6B shows an exemplary schematic illustrating the tissue sample sandwiched between a substrate and a spatially-barcoded capture probe array, where the connected (e.g., ligated probes) are transferred to the spatially-barcoded capture probe array.

[0050] FIG. 7A shows an exemplary workflow for using ligated probes and analyte capture agents to capture different analytes.

[0051] FIG. 7B shows an exemplary schematic illustrating the tissue sample sandwiched between a substrate and a spatially-barcoded capture probe array, where the ligated probes and capture agent barcode domains are transferred to the spatially-barcoded capture probe array.

[0052] FIG. 8A shows an exemplary schematic illustrating the use of an RNase cleavable linker to release a capture agent barcode domain from an analyte binding moiety of an analyte capture agent.

[0053] FIG. 8B shows an exemplary schematic illustrating the use of a UV cleavable linker to release a capture agent barcode domain from an analyte binding moiety of an analyte capture agent.

[0054] FIG. 9 shows an exemplary schematic diagram depicting a sandwiching process.

[0055] FIG. 10 illustrates an example block diagram illustrating a computing device in accordance with some embodiments of the present disclosure.

[0056] FIGS. 11 A, 11B, 11C, 11D, HE, HF, 11G, HH, HI, HJ, 11K, and HL collectively illustrate a method for quantifying spatial analyte data for a plurality of analytes of a first species, in accordance with some embodiments.

[0057] FIGS. 12A and 12B collectively illustrate a list of antibodies that are used in accordance with some embodiments of the present disclosure.

[0058] FIG. 13 illustrates a table of analyte capture agents in accordance with some embodiments of the present disclosure. [0059] FIG. 14 illustrates an image where antibody data of a biological sample is spatially overlaid on a histological image 1404 of the same biological sample.

[0060] FIGS. 15A and 15B are exemplary data showing the spatial distribution of antibodies on the same tissue section before and after normalization in accordance with some embodiments of the present disclosure.

[0061] FIG. 16 illustrates an exemplary normalization pathway that utilizes the unique molecular identifier (UMI) of four control antibodies to control for non-specific biological expression in the data, in accordance with some embodiments of the present disclosure.

[0062] FIGS. 17A and 17B illustrate cross-correlation data of analyte capture agents (e.g., oligonucleotide-conjugated antibodies) before and after normalization, respectively, in accordance with some embodiments of the present disclosure.

[0063] FIG. 18 illustrates that the normalization process in the present disclosure aids the detection of biological structures and spatial analysis of analytes in accordance with some embodiments of the present disclosure.

[0064] FIGS. 19A, 19B, and 19C collectively illustrate an application quantifying spatial analyte data for a plurality of analytes of a first species, in accordance with some embodiments of the present disclosure.

DETAILED DESCRIPTION

[0065] I. Introduction

[0066] Spatial analysis methodologies, systems, and compositions described herein can provide a vast amount of analyte and/or expression data for a variety of analytes within a biological sample at high resolution, while retaining native spatial context. Spatial analysis methods and compositions can include, e.g., the use of a capture probe including a spatial barcode (e.g., a nucleic acid sequence that provides information as to the location or position of an analyte within a cell or a tissue sample (e.g., mammalian cell or a mammalian tissue sample) and a capture domain that is capable of binding to an analyte (e.g., a nucleic acid) produced by and/or present in a cell. Spatial analysis methods and compositions can also include the use of a capture probe having a capture domain that captures an intermediate agent for indirect detection of an analyte (e.g., protein). For example, the intermediate agent can include a nucleic acid sequence (e.g., a barcode) associated with the intermediate agent. Detection of the intermediate agent is therefore indicative of the analyte in the cell or tissue sample. [0067] Non-limiting aspects of spatial analysis methodologies and compositions are described in U.S. Patent Nos. 10,774,374, 10,724,078, 10,480,022, 10,059,990, 10,041,949, 10,002,316, 9,879,313, 9,783,841, 9,727,810, 9,593,365, 8,951,726, 8,604,182, 7,709,198, U.S. Patent Application Publication Nos. 2020/239946, 2020/080136, 2020/0277663, 2020/024641, 2019/330617, 2019/264268, 2020/256867, 2020/224244, 2019/194709, 2019/161796, 2019/085383, 2019/055594, 2018/216161, 2018/051322, 2018/0245142, 2017/241911, 2017/089811, 2017/067096, 2017/029875, 2017/0016053, 2016/108458, 2015/000854, 2013/171621, WO 2018/091676, WO 2020/176788, Rodriques et al., Science 363(6434): 1463- 1467, 2019; Lee et al., Nat. Protoc. 10(3):442-458, 2015; Trejo et al., PLoS ONE

14(2) :e0212031, 2019; Chen et al., Science 348(6233):aaa6090, 2015; Gao et al., BMC Biol.

15:50, 2017; and Gupta et al., Nature Biotechnol. 36: 1197-1202, 2018; the Visium Spatial Gene Expression Reagent Kits User Guide (e.g., Rev C, dated June 2020), and/or the Visium Spatial Tissue Optimization Reagent Kits User Guide (e.g., Rev C, dated July 2020), both of which are available at the lOx Genomics Support Documentation website, and can be used herein in any combination. Further non-limiting aspects of spatial analysis methodologies and compositions are described herein.

[0068] Some general terminology that may be used in this disclosure can be found in Section (I)(b) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.

Typically, a “barcode” is a label, or identifier, that conveys or is capable of conveying information (e.g., information about an analyte in a sample, a bead, and/or a capture probe). A barcode can be part of an analyte, or independent of an analyte. A barcode can be attached to an analyte. A particular barcode can be unique relative to other barcodes. For the purpose of this disclosure, an “analyte” can include any biological substance, structure, moiety, or component to be analyzed. The term “target” can similarly refer to an analyte of interest.

[0069] Analytes can be broadly classified into one of two groups: 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, amidation variants of proteins, hydroxylation variants of proteins, methylation variants of proteins, ubiquitylation variants of proteins, sulfation variants of proteins, viral proteins (e.g., viral capsid, viral envelope, viral coat, viral accessory, viral glycoproteins, viral spike, etc.), extracellular and intracellular proteins, antibodies, and antigen binding fragments. Examples of nucleic acid analytes include, but are not limited to, DNA (e.g., genomic DNA, cDNA) and RNA, including coding and noncoding RNA (e.g., mRNA, rRNA, tRNA, ncRNA).

[0070] In some embodiments, the analyte(s) can be localized to subcellular location(s), including, for example, organelles, e.g., mitochondria, Golgi apparatus, endoplasmic reticulum, chloroplasts, endocytic vesicles, exocytic vesicles, vacuoles, lysosomes, etc. In some embodiments, analyte(s) can be peptides or proteins, including without limitation antibodies and enzymes. Additional examples of analytes can be found in Section (I)(c) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. In some embodiments, an analyte can be detected indirectly, such as through detection of an intermediate agent, for example, a connected probe (e.g., a ligation product) or an analyte capture agent (e.g., an oligonucleotide- conjugated antibody), such as those described herein.

[0071] A “biological sample” is typically obtained from the subject for analysis using any of a variety of techniques including, but not limited to, biopsy, surgery, and laser capture microscopy (LCM), and generally includes cells and/or other biological material from the subject. In some embodiments, the biological sample is a tissue sample. In some embodiments, the biological sample (e.g., tissue sample) is a tissue microarray (TMA). A tissue microarray contains multiple representative tissue samples - which can be from different tissues or organisms - assembled on a single histologic slide. The TMA can therefore allow for high throughput analysis of multiple specimens at the same time. Tissue microarrays are paraffin blocks produced by extracting cylindrical tissue cores from different paraffin donor blocks and re-embedding these into a single recipient (microarray) block at defined array coordinates.

[0072] The biological sample can be any suitable biological sample described herein or known in the art. In some embodiments, the biological sample is a tissue. In some embodiments, the tissue sample is a solid tissue sample. In some embodiments, a biological sample can be a tissue section. The tissue sample can be obtained from any suitable location in a tissue or organ of a subject, e.g., a human subject. In some instances, the tissue sample is a mouse sample. In some instances, the tissue sample is a human sample. In some embodiments, the tissue sample can be derived from skin, brain, breast, lung, liver, kidney, prostate, tonsil, thymus, testes, bone, lymph node, ovary, eye, heart, or spleen. In some embodiments, the tissue sample is flash-frozen and sectioned. In some embodiments, the biological sample, e.g., the tissue, is flash-frozen using nitrogen (e.g., liquid nitrogen), isopentane, or hexane before sectioning.

[0073] In some embodiments, the biological sample, e.g., the tissue, is embedded in a matrix e.g., optimal cutting temperature (OCT) compound to facilitate sectioning. OCT compound is a formulation of clear, water-soluble glycols and resins, providing a solid matrix to encapsulate biological (e.g., tissue) specimens. In some embodiments, the sectioning is performed using cryosectioning. In some embodiments, the methods further comprise a thawing step, after the cryosectioning.

[0074] In some embodiments, the biological sample, e.g., the tissue sample, is fixed in a fixative including alcohol, for example methanol. In some embodiments, instead of methanol, acetone, or an acetone-methanol mixture can be used. In some embodiments, the fixation is performed after sectioning. In some instances, the biological sample is not fixed with paraformaldehyde (PF A). In some instances, when the biological sample is fixed with a fixative including an alcohol (e.g., methanol or acetone-methanol mixture), it is not decrosslinked afterward.

[0075] In some embodiments, the biological sample, e.g., the tissue sample, is fixed e.g., immediately after being harvested from a subject. In such embodiments, the fixative is preferably an aldehyde fixative, such as paraformaldehyde (PF A) or formalin. In some embodiments, the fixative induces crosslinks within the biological sample. In some embodiments, after fixing e.g., by formalin or PF A, the biological sample is dehydrated via sucrose gradient. In some instances, the fixed biological sample is treated with a sucrose gradient and then embedded in a matrix e.g., OCT compound. In some instances, the fixed biological sample is not treated with a sucrose gradient, but rather is embedded in a matrix e.g., OCT compound after fixation. In some embodiments when a fixed frozen tissue sample is treated with a sucrose gradient, it can be rehydrated with an ethanol gradient. In some embodiments, the PFA or formalin fixed biological sample, which can be optionally dehydrated via sucrose gradient and/or embedded in OCT compound, is then frozen e.g., for storage or shipment.

[0076] In some embodiments, the biological sample can be fixed using PAXgene. For example, the biological sample can be fixed using PAXgene in addition, or alternatively to, a fixative disclosed herein or known in the art (e.g., alcohol, acetone, acetone-alcohol, formalin, paraformaldehyde). PAXgene is a non-cross-linking mixture of different alcohols, acid and a soluble organic compound that preserves morphology and bio-molecules. It is a two-reagent fixative system in which tissue is firstly fixed in a solution containing methanol and acetic acid then stabilized in a solution containing ethanol. See, Ergin B. et cd.. J Proteome Res. 2010 Oct l;9(10):5188-96; Kap M. et al., PLoS One.; 6(l l):e27704 (2011); and Mathieson W. et al., Am J Clin Pathol.; 146(1) :25-40 (2016), each of which are hereby incorporated by reference in their entirety, for a description and evaluation of PAXgene for tissue fixation. [0077] The biological sample can be from a mammal. In some instances, the biological sample is from a human, mouse, or rat. In addition to the subjects described above, the biological sample can be obtained from non-mammalian organisms (e.g., a plants, an insect, an arachnid, a nematode (e.g., Caenorhabditis elegans . a fungi, an amphibian, or a fish (e.g., zebrafish)). Biological samples can include one or more diseased cells. A diseased cell can have altered metabolic properties, gene expression, protein expression, and/or morphologic features.

Examples of diseases include inflammatory disorders, metabolic disorders, nervous system disorders, and cancer. Cancer cells can be derived from solid tumors, hematological malignancies, cell lines, or obtained as circulating tumor cells.

[0078] In some embodiments, a biological sample can be a fixed and/or stained biological sample (e.g., a fixed and/or stained tissue section). Non-limiting examples of stains include histological stains (e.g., hematoxylin and/or eosin) and immunological stains (e.g., fluorescent stains). In some embodiments, a biological sample (e.g., a fixed and/or stained biological sample) can be imaged. Biological samples are also described in Section (I)(d) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.

[0079] In some embodiments, a biological sample is permeabilized with one or more permeabilization reagents. For example, permeabilization of a biological sample can facilitate analyte capture. Exemplary permeabilization agents and conditions are described in Section (I)(d)(ii)(13) or the Exemplary Embodiments Section of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.

[0080] Array-based spatial analysis methods involve the transfer of one or more analytes from a biological sample to an array of features on a substrate, where each feature is associated with a unique spatial location on the array. Subsequent analysis of the transferred analytes includes determining the identity of the analytes and the spatial location of the analytes within the biological sample. The spatial location of an analyte within the biological sample is determined based on the feature to which the analyte is bound (e.g., directly or indirectly) on the array, and the feature’s relative spatial location within the array.

[0081] A “capture probe” refers to any molecule capable of capturing (directly or indirectly) and/or labelling an analyte (e.g., an analyte of interest) in a biological sample. In some embodiments, the capture probe is a nucleic acid or a polypeptide. In some embodiments, the capture probe includes a barcode (e.g., a spatial barcode and/or a unique molecular identifier (UMI)) and a capture domain). In some embodiments, a capture probe can include a cleavage domain and/or a functional domain (e.g., a primer-binding site, such as for next-generation sequencing (NGS)).

[0082] In some instances, the capture domain is designed to detect one or more specific analytes of interest. For example, a capture domain can be designed so that it comprises a sequence that is complementary or substantially complementary to one analyte of interest. Thus, the presence of a single analyte can be detected. Alternatively, the capture domain can be designed so that it comprises a sequence that is complementary or substantially complementary to a conserved region of multiple related analytes. In some instances, the multiple related analytes are analytes that function in the same or similar cellular pathways or that have conserved homology and/or function. The design of the capture probe can be determined based on the intent of the user and can be any sequence that can be used to detect an analyte of interest. In some embodiments, the capture domain sequence can therefore be random, semi-random, defined or combinations thereof, depending on the target analyte(s) of interest.

[0083] FIG. 1 is a schematic diagram showing an exemplary capture probe, as described herein. As shown, the capture probe 102 is optionally coupled to a feature 101 by a cleavage domain 103, such as a disulfide linker. The capture probe can include a functional sequence 104 that are useful for subsequent processing. The functional sequence 104 can include all or a part of sequencer specific flow cell attachment sequence (e.g., a P5 or P7 sequence), all or a part of a sequencing primer sequence, e.g., a R1 primer binding site, a R2 primer binding site), or combinations thereof. The capture probe can also include a spatial barcode 105. The capture probe can also include a unique molecular identifier (UMI) sequence 106. While FIG. 1 shows the spatial barcode 105 as being located upstream (5’) of UMI sequence 106, it is to be understood that capture probes where UMI sequence 106 is located upstream (5’) of the spatial barcode 105 is also suitable for use in any of the methods described herein. The capture probe can also include a capture domain 107 to facilitate capture of a target analyte. In some embodiments, the capture probe comprises one or more additional functional sequences that can be located, for example between the spatial barcode 105 and the UMI sequence 106, between the UMI sequence 106 and the capture domain 107, or following the capture domain 107. The capture domain can have a sequence complementary to a sequence of a nucleic acid analyte. The capture domain can have a sequence complementary to a connected probe described herein. The capture domain can have a sequence complementary to a capture handle sequence present in an analyte capture agent. The capture domain can have a sequence complementary to a splint oligonucleotide. Such splint oligonucleotide, in addition to having a sequence complementary to a capture domain of a capture probe, can have a sequence of a nucleic acid analyte, a sequence complementary to a portion of a connected probe described herein, and/or a capture handle sequence described herein.

[0084] The functional sequences can generally be selected for compatibility with any of a variety of different sequencing systems, e.g., Ion Torrent Proton or PGM, Illumina sequencing instruments, PacBio, Oxford Nanopore, etc., and the requirements thereof. In some embodiments, functional sequences can be selected for compatibility with non-commercialized sequencing systems. Examples of such sequencing systems and techniques, for which suitable functional sequences can be used, include (but are not limited to) Ion Torrent Proton or PGM sequencing, Illumina sequencing, PacBio SMRT sequencing, and Oxford Nanopore sequencing. Further, in some embodiments, functional sequences can be selected for compatibility with other sequencing systems, including non-commercialized sequencing systems.

[0085] In some embodiments, the spatial barcode 105 and functional sequences 104 is common to all of the probes attached to a given feature. In some embodiments, the UMI sequence 106 of a capture probe attached to a given feature is different from the UMI sequence of a different capture probe attached to the given feature.

[0086] FIG. 2 is a schematic illustrating a cleavable capture probe, where the cleaved capture probe can enter into a non-permeabilized cell and bind to analytes within the sample (e.g., cell). The capture probe 201 contains a cleavage domain 202, a cell penetrating peptide 203, a reporter molecule 204, and a disulfide bond (-S-S-). 205 represents all other parts of a capture probe, for example a spatial barcode and a capture domain.

[0087] FIG. 3 is a schematic diagram of an exemplary multiplexed spatially-barcoded feature. In FIG. 3, the feature 301 can be coupled to spatially-barcoded capture probes, where the spatially-barcoded probes of a particular feature can possess the same spatial barcode, but have different capture domains designed to associate the spatial barcode of the feature with more than one target analyte. For example, a feature may be coupled to four different types of spatially- barcoded capture probes, each type of spatially-barcoded capture probe possessing the spatial barcode 302. One type of capture probe associated with the feature includes the spatial barcode 302 in combination with a poly(T) capture domain 303, designed to capture mRNA target analytes. A second type of capture probe associated with the feature includes the spatial barcode 302 in combination with a random N-mer capture domain 304 for gDNA analysis. A third type of capture probe associated with the feature includes the spatial barcode 302 in combination with a capture domain complementary to a capture handle sequence of an analyte capture agent of interest 305. A fourth type of capture probe associated with the feature includes the spatial barcode 302 in combination with a capture domain that can specifically bind a nucleic acid molecule 306 that can function in a CRISPR assay (e.g., CRISPR/Cas9). While only four different capture probe-barcoded constructs are shown in FIG. 3, capture-probe barcoded constructs can be tailored for analyses of any given analyte associated with a nucleic acid and capable of binding with such a construct. For example, the schemes shown in FIG. 3 can also be used for concurrent analysis of other analytes disclosed herein, including, but not limited to: (a) mRNA, a lineage tracing construct, cell surface or intracellular proteins and metabolites, and gDNA; (b) mRNA, accessible chromatin (e.g., ATAC-seq, DNase-seq, and/or MNase-seq) cell surface or intracellular proteins and metabolites, and a perturbation agent (e.g., a CRISPR crRNA/sgRNA, TALEN, zinc finger nuclease, and/or antisense oligonucleotide as described herein); (c) mRNA, cell surface or intracellular proteins and/or metabolites, a barcoded labelling agent (e.g., the MHC multimers described herein), and a V(D)J sequence of an immune cell receptor (e.g., T-cell receptor). In some embodiments, a perturbation agent can be a small molecule, an antibody, a drug, an aptamer, a miRNA, a physical environmental e.g., temperature change), or any other known perturbation agents. See, e.g., Section (II)(b) e.g., subsections (i)- (vi)) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Generation of capture probes can be achieved by any appropriate method, including those described in Section (II)(d)(ii) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.

[0088] In some embodiments, more than one analyte type e.g., nucleic acids and proteins) from a biological sample can be detected e.g., simultaneously or sequentially) using any appropriate multiplexing technique, such as those described in Section (IV) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.

[0089] In some embodiments, detection of one or more analytes e.g., protein analytes) can be performed using one or more analyte capture agents. As used herein, an “analyte capture agent” refers to an agent that interacts with an analyte e.g., an analyte in a biological sample) and with a capture probe e.g., a capture probe attached to a substrate or a feature) to identify the analyte. In some embodiments, the analyte capture agent includes: (i) an analyte binding moiety e.g., that binds to an analyte), for example, an antibody or antigen-binding fragment thereof; (ii) analyte binding moiety barcode; and (iii) a capture handle sequence. As used herein, the term “analyte binding moiety barcode” refers to a barcode that is associated with or otherwise identifies the analyte binding moiety. As used herein, the term “analyte capture sequence” or “capture handle sequence” refers to a region or moiety configured to hybridize to, bind to, couple to, or otherwise interact with a capture domain of a capture probe. In some embodiments, a capture handle sequence is complementary to a capture domain of a capture probe. In some cases, an analyte binding moiety barcode (or portion thereof) may be able to be removed (e.g., cleaved) from the analyte capture agent.

[0090] The term “antibody,” as used herein, refers to an immunoglobulin molecule which specifically binds with an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Unless specifically noted otherwise, use of the term “antibody” or “antibodies” may specifically include “antibody fragment” and “antibody fragments” which include any form of an antibody other than the full-length form. Thus, a disclosed antibody may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, single chain Fv (scFv), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, combinations of CDRs, variable regions, framework regions, constant regions, heavy chains, light chains, alternative scaffold non-antibody molecules, and bispecific antibodies, and humanized antibodies.

[0091] FIG. 4 is a schematic diagram of an exemplary analyte capture agent 402 comprised of an analyte-binding moiety 404 and an oligonucleotide (also referred to herein as analytebinding moiety barcode domain) 408. The exemplary analyte-binding moiety 404 is a molecule capable of binding to an analyte 406 and the oligonucleotide is capable of interacting with a spatially-barcoded capture probe. The analyte binding moiety can bind to the analyte 406 with high affinity and/or with high specificity. The analyte capture agent can include an oligonucleotide 408, which can include a nucleotide sequence which can hybridize to at least a portion or an entirety of a capture domain of a capture probe. The oligonucleotide 408 can comprise an analyte binding moiety barcode and a capture handle sequence described herein. The analyte binding moiety 404 can include a polypeptide and/or an aptamer. The analyte-binding moiety 404 can include an antibody or antibody fragment (e.g., an antigen-binding fragment).

[0092] FIG. 5 is a schematic diagram depicting an exemplary interaction between a feature- immobilized capture probe 524 and an analyte capture agent 526. The feature-immobilized capture probe 524 can include a spatial barcode 508 as well as functional sequences 506 and UMI 510, as described elsewhere herein. The capture probe can also include a capture domain 512 that is capable of binding to an analyte capture agent 526. The analyte capture agent 526 can include a functional sequence 518, analyte binding moiety barcode 516, and a capture handle sequence 514 that is capable of binding to the capture domain 512 of the capture probe 524. The analyte capture agent can also include a linker 520 that allows the capture agent barcode domain 516 to couple to the analyte binding moiety 522.

[0093] In some embodiments, streptavidin cell tags can be utilized in an array -based system to produce a spatially-barcoded cell or cellular contents. In some embodiments, peptide-bound major histocompatibility complex (MHC) can be individually associated with biotin (P2m) and bound to a streptavidin moiety such that the streptavidin moiety comprises multiple pMHC moieties. Each of these moieties can bind to a TCR such that the streptavidin binds to a target T- cell via multiple MCH/TCR binding interactions. Multiple interactions synergize and can substantially improve binding affinity. Such improved affinity can improve labelling of T-cells and also reduce the likelihood that labels will dissociate from T-cell surfaces. In some embodiments, a capture agent barcode domain can be modified with streptavidin and contacted with multiple molecules of biotinylated MHC such that the biotinylated MHC molecules are coupled with the streptavidin conjugated capture agent barcode domain. The result is a barcoded MHC multimer complex. In some embodiments, the capture agent barcode domain sequence can identify the MHC as its associated label and also includes optional functional sequences such as sequences for hybridization with other oligonucleotides. In some embodiments, one example oligonucleotide is a capture probe that comprises a complementary sequence (e.g., rGrGrG corresponding to C C C), a barcode sequence and other functional sequences, such as, for example, a UMI, an adapter sequence (e.g., comprising a sequencing primer sequence (e.g., R1 or a partial R1 (“pRl”), R2), a flow cell attachment sequence (e.g., P5 or P7 or partial sequences thereof)), etc. In some cases, capture probe may at first be associated with a feature (e.g., a gel bead) and released from the feature. In other embodiments, capture probe can hybridize with a capture agent barcode domain of the MHC-oligonucleotide complex. The hybridized oligonucleotides (Spacer C C C and Spacer rGrGrG) can then be extended in primer extension reactions such that constructs comprising sequences that correspond to each of the two spatial barcode sequences (the spatial barcode associated with the capture probe, and the barcode associated with the MHC-oligonucleotide complex) are generated. In some cases, one or both of these corresponding sequences may be a complement of the original sequence in capture probe or capture agent barcode domain. In other embodiments, the capture probe and the capture agent barcode domain are ligated together. The resulting constructs can be optionally further processed (e.g., to add any additional sequences and/or for clean-up) and subjected to sequencing. As described elsewhere herein, a sequence derived from the capture probe spatial barcode sequence may be used to identify a feature and the sequence derived from spatial barcode sequence on the capture agent barcode domain may be used to identify the particular peptide MHC complex bound on the surface of the cell (e.g., when using MHC -peptide libraries for screening immune cells or immune cell populations).

[0094] Additional description of analyte capture agents can be found in Section (II)(b)(ix) of WO 2020/176788 and/or Section (II)(b)(viii) U.S. Patent Application Publication No. 2020/0277663.

[0095] There are at least two methods to associate a spatial barcode with one or more neighboring cells, such that the spatial barcode identifies the one or more cells, and/or contents of the one or more cells, as associated with a particular spatial location. One method is to promote analytes or analyte proxies (e.g., intermediate agents) out of a cell and towards a spatially- barcoded array (e.g., including spatially-barcoded capture probes). Another method is to cleave spatially-barcoded capture probes from an array and promote the spatially-barcoded capture probes towards and/or into or onto the biological sample.

[0096] In some cases, capture probes may be configured to prime, replicate, and consequently yield optionally barcoded extension products from a template (e.g., a DNA or RNA template, such as an analyte or an intermediate agent (e.g., a connected probe (e.g., a ligation product) or an analyte capture agent), or a portion thereof), or derivatives thereof (see, e.g., Section (II)(b)(vii) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663 regarding extended capture probes). In some cases, capture probes may be configured to form a connected probe (e.g., a ligation product) with a template (e.g., a DNA or RNA template, such as an analyte or an intermediate agent, or portion thereof), thereby creating ligations products that serve as proxies for a template.

[0097] As used herein, an “extended capture probe” refers to a capture probe having additional nucleotides added to the terminus (e.g., 3’ or 5’ end) of the capture probe thereby extending the overall length of the capture probe. For example, an “extended 3’ end” indicates additional nucleotides were added to the most 3’ nucleotide of the capture probe to extend the length of the capture probe, for example, by polymerization reactions used to extend nucleic acid molecules including templated polymerization catalyzed by a polymerase (e.g., a DNA polymerase or a reverse transcriptase). In some embodiments, extending the capture probe includes adding to a 3’ end of a capture probe a nucleic acid sequence that is complementary to a nucleic acid sequence of an analyte or intermediate agent specifically bound to the capture domain of the capture probe. In some embodiments, the capture probe is extended using reverse transcription. In some embodiments, the capture probe is extended using one or more DNA polymerases. The extended capture probes include the sequence of the capture probe and the sequence of the spatial barcode of the capture probe.

[0098] In some embodiments, extended capture probes are amplified (e.g., in bulk solution or on the array) to yield quantities that are sufficient for downstream analysis, e.g., via DNA sequencing. In some embodiments, extended capture probes (e.g., DNA molecules) act as templates for an amplification reaction (e.g., a polymerase chain reaction).

[0099] Additional variants of spatial analysis methods, including in some embodiments, an imaging step, are described in Section (II)(a) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Analysis of captured analytes (and/or intermediate agents or portions thereof), for example, including sample removal, extension of capture probes, sequencing (e.g., of a cleaved extended capture probe and/or a cDNA molecule complementary to an extended capture probe), sequencing on the array (e.g., using, for example, in situ hybridization or in situ ligation approaches), temporal analysis, and/or proximity capture, is described in Section (II)(g) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Some quality control measures are described in Section (II)(h) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.

[00100] Spatial information can provide information of biological and/or medical importance. For example, the methods and compositions described herein can allow for: identification of one or more biomarkers (e.g., diagnostic, prognostic, and/or for determination of efficacy of a treatment) of a disease or disorder; identification of a candidate drug target for treatment of a disease or disorder; identification (e.g., diagnosis) of a subject as having a disease or disorder; identification of stage and/or prognosis of a disease or disorder in a subject; identification of a subject as having an increased likelihood of developing a disease or disorder; monitoring of progression of a disease or disorder in a subject; determination of efficacy of a treatment of a disease or disorder in a subject; identification of a patient subpopulation for which a treatment is effective for a disease or disorder; modification of a treatment of a subject with a disease or disorder; selection of a subject for participation in a clinical trial; and/or selection of a treatment for a subject with a disease or disorder.

[00101] Spatial information can provide information of biological importance. For example, the methods and compositions described herein can allow for: identification of transcriptome and/or proteome expression profiles (e.g., in healthy and/or diseased tissue); identification of multiple analyte types in close proximity (e.g., nearest neighbor analysis); determination of up- and/or down-regulated genes and/or proteins in diseased tissue; characterization of tumor microenvironments; characterization of tumor immune responses; characterization of cells types and their co-localization in tissue; and identification of genetic variants within tissues (e.g., based on gene and/or protein expression profiles associated with specific disease or disorder biomarkers).

[00102] Typically, for spatial array -based methods, a substrate functions as a support for direct or indirect attachment of capture probes to features of the array. A “feature” is an entity that acts as a support or repository for various molecular entities used in spatial analysis. In some embodiments, some or all of the features in an array are functionalized for analyte capture. Exemplary substrates are described in Section (II)(c) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Exemplary features and geometric attributes of an array can be found in Sections (II)(d)(i), (II)(d)(iii), and (II)(d)(iv) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.

[00103] Generally, analytes and/or intermediate agents (or portions thereof) can be captured when contacting a biological sample with a substrate including capture probes (e.g., a substrate with capture probes embedded, spotted, printed, fabricated on the substrate, or a substrate with features (e.g., beads, wells) comprising capture probes). As used herein, “contact,” “contacted,” and/or “contacting,” a biological sample with a substrate refers to any contact (e.g., direct or indirect) such that capture probes can interact (e.g., bind covalently or non-covalently (e.g., hybridize)) with analytes from the biological sample. Capture can be achieved actively (e.g., using electrophoresis) or passively (e.g., using diffusion). Analyte capture is further described in Section (II)(e) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.

[00104] In some cases, spatial analysis can be performed by attaching and/or introducing a molecule (e.g., a peptide, a lipid, or a nucleic acid molecule) having a barcode (e.g., a spatial barcode) to a biological sample (e.g., to a cell in a biological sample). In some embodiments, a plurality of molecules (e.g., a plurality of nucleic acid molecules) having a plurality of barcodes (e.g., a plurality of spatial barcodes) are introduced to a biological sample (e.g., to a plurality of cells in a biological sample) for use in spatial analysis. In some embodiments, after attaching and/or introducing a molecule having a barcode to a biological sample, the biological sample can be physically separated (e.g., dissociated) into single cells or cell groups for analysis. Some such methods of spatial analysis are described in Section (III) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. [00105] In some cases, spatial analysis can be performed by detecting multiple oligonucleotides that hybridize to an analyte. In some instances, for example, spatial analysis can be performed using RNA-templated ligation (RTL). Methods of RTL have been described previously. See, e.g., Credle etal., Nucleic Acids Res. 2017 Aug 21;45(14):el28. Typically, RTL includes hybridization of two oligonucleotides to adjacent sequences on an analyte (e.g., an RNA molecule, such as an mRNA molecule). In some instances, the oligonucleotides are DNA molecules. In some instances, one of the oligonucleotides includes at least two ribonucleic acid bases at the 3’ end and/or the other oligonucleotide includes a phosphorylated nucleotide at the 5’ end. In some instances, one of the two oligonucleotides includes a capture domain (e.g., a poly(A) sequence, a non-homopolymeric sequence). After hybridization to the analyte, a ligase (e.g., SplintR ligase) ligates the two oligonucleotides together, creating a connected probe (e.g., a ligation product). In some instances, the two oligonucleotides hybridize to sequences that are not adjacent to one another. For example, hybridization of the two oligonucleotides creates a gap between the hybridized oligonucleotides. In some instances, a polymerase (e.g., a DNA polymerase) can extend one of the oligonucleotides prior to ligation. After ligation, the connected probe (e.g., a ligation product) is released from the analyte. In some instances, the connected probe (e.g., a ligation product) is released using an endonuclease (e.g., RNase A, RNase C, RNase H, or RNase I). The released connected probe (e.g., a ligation product) can then be captured by capture probes (e.g., instead of direct capture of an analyte) on an array, optionally amplified, and sequenced, thus determining the location and optionally the abundance of the analyte in the biological sample.

[00106] During analysis of spatial information, sequence information for a spatial barcode associated with an analyte is obtained, and the sequence information can be used to provide information about the spatial distribution of the analyte in the biological sample. Various methods can be used to obtain the spatial information. In some embodiments, specific capture probes and the analytes they capture are associated with specific locations in an array of features on a substrate. For example, specific spatial barcodes can be associated with specific array locations prior to array fabrication, and the sequences of the spatial barcodes can be stored (e.g., in a database) along with specific array location information, so that each spatial barcode uniquely maps to a particular array location.

[00107] Alternatively, specific spatial barcodes can be deposited at predetermined locations in an array of features during fabrication such that at each location, only one type of spatial barcode is present so that spatial barcodes are uniquely associated with a single feature of the array. Where necessary, the arrays can be decoded using any of the methods described herein so that spatial barcodes are uniquely associated with array feature locations, and this mapping can be stored as described above.

[00108] When sequence information is obtained for capture probes and/or analytes during analysis of spatial information, the locations of the capture probes and/or analytes can be determined by referring to the stored information that uniquely associates each spatial barcode with an array feature location. In this manner, specific capture probes and captured analytes are associated with specific locations in the array of features. Each array feature location represents a position relative to a coordinate reference point (e.g., an array location, a fiducial marker) for the array. Accordingly, each feature location has an “address” or location in the coordinate space of the array.

[00109] Some exemplary spatial analysis workflows are described in the Exemplary Embodiments section of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. See, for example, the Exemplary embodiment starting with “In some non-limiting examples of the workflows described herein, the sample can be immersed. . .” of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. See also, e.g., the Visium Spatial Gene Expression Reagent Kits User Guide (e.g., Rev C, dated June 2020), and/or the Visium Spatial Tissue Optimization Reagent Kits User Guide (e.g., Rev C, dated July 2020).

[00110] In some embodiments, spatial analysis can be performed using dedicated hardware and/or software, such as any of the systems described in Sections (II)(e)(ii) and/or (V) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663, or any of one or more of the devices or methods described in Sections Control Slide for Imaging, Methods of Using Control Slides and Substrates for, Systems of Using Control Slides and Substrates for Imaging, and/or Sample and Array Alignment Devices and Methods, Informational labels of WO 2020/123320.

[00111] Suitable systems for performing spatial analysis can include components such as a chamber (e.g., a flow cell or sealable, fluid-tight chamber) for containing a biological sample. The biological sample can be mounted for example, in a biological sample holder. One or more fluid chambers can be connected to the chamber and/or the sample holder via fluid conduits, and fluids can be delivered into the chamber and/or sample holder via fluidic pumps, vacuum sources, or other devices coupled to the fluid conduits that create a pressure gradient to drive fluid flow. One or more valves can also be connected to fluid conduits to regulate the flow of reagents from reservoirs to the chamber and/or sample holder. [00112] The systems can optionally include a control unit that includes one or more electronic processors, an input interface, an output interface (such as a display), and a storage unit (e.g., a solid state storage medium such as, but not limited to, a magnetic, optical, or other solid state, persistent, writeable and/or re-writeable storage medium). The control unit can optionally be connected to one or more remote devices via a network. The control unit (and components thereof) can generally perform any of the steps and functions described herein. Where the system is connected to a remote device, the remote device (or devices) can perform any of the steps or features described herein. The systems can optionally include one or more detectors (e.g., CCD, CMOS) used to capture images. The systems can also optionally include one or more light sources (e.g., LED-based, diode-based, lasers) for illuminating a sample, a substrate with features, analytes from a biological sample captured on a substrate, and various control and calibration media.

[00113] The systems can optionally include software instructions encoded and/or implemented in one or more of tangible storage media and hardware components such as application specific integrated circuits. The software instructions, when executed by a control unit (and in particular, an electronic processor) or an integrated circuit, can cause the control unit, integrated circuit, or other component executing the software instructions to perform any of the method steps or functions described herein.

[00114] In some cases, the systems described herein can detect (e.g., register an image) the biological sample on the array. Exemplary methods to detect the biological sample on an array are described in PCT Application No. 2020/061064 and/or U.S. Patent Publication No. US2021/0155982.

[00115] Prior to transferring analytes from the biological sample to the array of features on the substrate, the biological sample can be aligned with the array. Alignment of a biological sample and an array of features including capture probes can facilitate spatial analysis, which can be used to detect differences in analyte presence and/or level within different positions in the biological sample, for example, to generate a three-dimensional map of the analyte presence and/or level. Exemplary methods to generate a two- and/or three-dimensional map of the analyte presence and/or level are described in PCT Application No. 2020/053655 and spatial analysis methods are generally described in WO 2020/061108 and/or U.S. Patent Publication No. US2021/0155982.

[00116] In some cases, a map of analyte presence and/or level can be aligned to an image of a biological sample using one or more fiducial markers, e.g., objects placed in the field of view of an imaging system which appear in the image produced, as described in the Substrate Attributes Section, Control Slide for Imaging Section of WO 2020/123320, PCT Application No. 2020/061066, and/or U.S. Patent Application Publication No. 2021/0158522. Fiducial markers can be used as a point of reference or measurement scale for alignment (e.g., to align a sample and an array, to align two substrates, to determine a location of a sample or array on a substrate relative to a fiducial marker) and/or for quantitative measurements of sizes and/or distances.

[00117] The sandwich process is described in PCT Patent Application Publication No. WO 2020/123320, which is incorporated by reference in its entirety.

II. Methods, Compositions, Devices, and Systems for Capturing Analytes and Derivatives Thereof

[00118] (a) Introduction

[00119] Provided herein are methods, devices, compositions, and systems for analyzing the location and abundance of a nucleic acid or protein analyte in a biological sample. In some instances, the methods include aligning (i.e., sandwiching) a first substrate having the biological sample with a second substrate that includes a plurality of capture probes, thereby “sandwiching” the biological sample between the two substrates. Upon interaction of the biological sample with the substrate having a plurality of probes (in either instance), the location and abundance of a nucleic acid or protein analyte in a biological sample can be determined, as provided herein. These methods include an advantage in that steps provided herein prior to analyte or analyte- derived molecule by the capture probe, most — if not all — steps can be performed on a substrate that does not have capture probes, thereby providing a method that is cost effective.

[00120] The methods and systems provided herein can be applied to an analyte or an analyte- derived molecule(s). As used herein, an analyte derived molecule includes, without limitation, a connected probe (e.g., a ligation product) from an RNA-templated ligation (RTL) assay, a product of reverse transcription (e.g., an extended capture probe), and an analyte binding moiety barcode (e.g., a binding moiety barcode that identifies that analyte binding moiety (e.g., an antibody)). In some embodiments, the analyte or analyte derived molecules comprise RNA and/or DNA. In some embodiments, the analyte or analyte derived molecules comprise one or more proteins.

[00121] In some instances, the methods, devices, compositions, and systems disclosed herein provide efficient release of an analyte or analyte derived molecule from a biological sample so that it can be easily captured or detected using methods disclosed herein. [00122] In some instances, the methods, devices, compositions, and systems disclosed herein allow for detection of analytes or analyte derived molecules from different biological samples using a single array comprising a plurality of capture probes. As such, in some instances, the methods, devices, compositions, and systems allow for serial capture of analytes or analyte derived molecules from multiple samples. The analytes or analyte derived molecules can then be demultiplexed using biological-sample-specific index sequences to identify it biological sample origin.

[00123] Embodiments of the methods, devices, compositions, and systems disclosed herein are provided below.

[00124] (I) Exemplary Biological Samples

Accordingly, RTL probes can be utilized to beneficially improve capture and spatial analysis of fixed samples.

[00126] The biological sample, e.g., tissue sample, can be stained, and imaged prior, during, and/or after each step of the methods described herein. Any of the methods described herein or known in the art can be used to stain and/or image the biological sample. In some embodiments, the imaging occurs prior to deaminating the sample. In some embodiments, the biological sample is stained using an H&E staining method. In some embodiments, the tissue sample is stained and imaged for about 10 minutes to about 2 hours (or any of the subranges of this range described herein). Additional time may be needed for staining and imaging of different types of biological samples.

[00127] The tissue sample can be obtained from any suitable location in a tissue or organ of a subject, e.g., a human subject. In some instances, the sample is a human sample. In some instances, the sample is a human breast tissue sample. In some instances, the sample is a human brain tissue sample. In some embodiments, the sample is an embryo sample. The embryo sample can be a non-human embryo sample. In some instances, the sample is a mouse embryo sample.

[00128] (II) Exemplary First and Second Substrates

[00129] In some instances, the biological sample is placed (e.g., mounted or otherwise immobilized) on a first substrate. The first substrate can be any solid or semi-solid support upon which a biological sample can be mounted. In some instances, the first substrate is a slide. In some instances, the slide is a glass slide. In some embodiments, the substrate is made of glass, silicon, paper, hydrogel, polymer monoliths, or other material known in the art. In some embodiments, the first substrate is comprised of an inert material or matrix (e.g., glass slides) that has been functionalized by, for example, treating the substrate with a material comprising reactive groups which facilitate mounting of the biological sample.

[00130] In some embodiments, the first substrate does not comprise a plurality (e.g., array) of capture probes, each comprising a spatial barcode.

[00131] A substrate, e.g., a first substrate and/or a second substrate, can generally have any suitable form or format. For example, a substrate can be flat, curved, e.g., convexly or concavely curved. For example, a first substrate can be curved towards the area where the interaction between a biological sample, e.g., tissue sample, and a first substrate takes place. In some embodiments, a substrate is flat, e.g., planar, chip, or slide. A substrate can contain one or more patterned surfaces within the first substrate (e.g., channels, wells, projections, ridges, divots, etc.).

[00132] A substrate, e.g., a first substrate and/or second substrate, can be of any desired shape. For example, a substrate can be typically a thin, flat shape (e.g., a square or a rectangle). In some embodiments, a substrate structure has rounded comers (e.g., for increased safety or robustness). In some embodiments, a substrate structure has one or more cut-off corners (e.g., for use with a slide clamp or cross-table). In some embodiments where a substrate structure is flat, the substrate structure can be any appropriate type of support having a flat surface (e.g., a chip or a slide such as a microscope slide).

[00133] First and/or second substrates can optionally include various structures such as, but not limited to, projections, ridges, and channels. A substrate can be micropattemed to limit lateral diffusion of analytes (e.g., to improve resolution of the spatial analysis). A substrate modified with such structures can be modified to allow association of analytes, features (e.g., beads), or probes at individual sites. For example, the sites where a substrate is modified with various structures can be contiguous or non-contiguous with other sites.

[00134] In some embodiments, the surface of a first and/or second substrate is modified to contain one or more wells, using techniques such as (but not limited to) stamping, microetching, or molding techniques. In some embodiments in which a first and/or second substrate includes one or more wells, the first substrate can be a concavity slide or cavity slide. For example, wells can be formed by one or more shallow depressions on the surface of the first and/or second substrate. In some embodiments, where a first and/or second substrate includes one or more wells, the wells can be formed by attaching a cassette (e.g., a cassette containing one or more chambers) to a surface of the first substrate structure.

[00135] In some embodiments where the first and/or second substrate is modified to contain one or more structures, including but not limited to, wells, projections, ridges, features, or markings, the structures can include physically altered sites. For example, a first and/or second substrate modified with various structures can 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. In some embodiments where the first substrate is modified to contain various structures, including but not limited to wells, projections, ridges, features, or markings, the structures are applied in a pattern. Alternatively, the structures can be randomly distributed.

[00136] In some embodiments, a first substrate includes one or more markings on its surface, e.g., to provide guidance for aligning at least a portion of the biological sample with a plurality of capture probes on the second substrate during a sandwich process disclosed herein. For example, the first substrate can include a sample area indicator identifying the sample area. In some embodiments, during a sandwiching process described herein the sample area indicator on the first substrate is aligned with an area of the second substrate comprising a plurality of capture probes. In some embodiments, the first and/or second substrate can include a fiducial mark. In some embodiments, the first and/or second substrate does not comprise a fiducial mark. In some embodiments, the first substrate does not comprise a fiducial mark and the second substrate comprises a fiducial mark. Such markings can be made using techniques including, but not limited to, printing, sand-blasting, and depositing on the surface.

[00137] In some embodiments, imaging can be performed using one or more fiducial markers, i.e., objects placed in the field of view of an imaging system which appear in the image produced. Fiducial markers are typically used as a point of reference or measurement scale. Fiducial markers can include, but are not limited to, detectable labels such as fluorescent, radioactive, chemiluminescent, and colorimetric labels. The use of fiducial markers to stabilize and orient biological samples is described, for example, in Carter etal., Applied Optics 46:421- 427, 2007), the entire contents of which are incorporated herein by reference. In some embodiments, a fiducial marker can be a physical particle (e.g., a nanoparticle, a microsphere, a nanosphere, a bead, a post, or any of the other exemplary physical particles described herein or known in the art).

[00138] In some embodiments, a fiducial marker can be present on a first substrate to provide orientation of the biological sample. In some embodiments, a microsphere can be coupled to a first substrate to aid in orientation of the biological sample. In some examples, a microsphere coupled to a first substrate can produce an optical signal (e.g., fluorescence). In some embodiments, a quantum dot can be coupled to the first substrate to aid in the orientation of the biological sample. In some examples, a quantum dot coupled to a first substrate can produce an optical signal.

[00139] In some embodiments, a fiducial marker can be an immobilized molecule with which a detectable signal molecule can interact to generate a signal. For example, a marker nucleic acid can be linked or coupled to a chemical moiety capable of fluorescing when subjected to light of a specific wavelength (or range of wavelengths). Although not required, it can be advantageous to use a marker that can be detected using the same conditions (e.g., imaging conditions) used to detect a labelled cDNA.

[00140] In some embodiments, a fiducial marker can be randomly placed in the field of view. For example, an oligonucleotide containing a fluorophore can be randomly printed, stamped, synthesized, or attached to a first substrate (e.g., a glass slide) at a random position on the first substrate. A tissue section can be contacted with the first substrate such that the oligonucleotide containing the fluorophore contacts, or is in proximity to, a cell from the tissue section or a component of the cell (e.g., an mRNA or DNA molecule). An image of the first substrate and the tissue section can be obtained, and the position of the fluorophore within the tissue section image can be determined (e.g., by reviewing an optical image of the tissue section overlaid with the fluorophore detection). In some embodiments, fiducial markers can be precisely placed in the field of view (e.g., at known locations on a first substrate). In this instance, a fiducial marker can be stamped, attached, or synthesized on the first substrate and contacted with a biological sample. Typically, an image of the sample and the fiducial marker is taken, and the position of the fiducial marker on the first substrate can be confirmed by viewing the image.

[00141] In some embodiments, a fiducial marker can be an immobilized molecule (e.g., a physical particle) attached to the first substrate. For example, a fiducial marker can be a nanoparticle, e.g., a nanorod, a nanowire, a nanocube, a nanopyramid, or a spherical nanoparticle. In some examples, the nanoparticle can be made of a heavy metal (e.g., gold). In some embodiments, the nanoparticle can be made from diamond. In some embodiments, the fiducial marker can be visible by eye.

[00142] A wide variety of different first substrates can be used for the foregoing purposes. In general, a first substrate can be any suitable support material. Exemplary first substrates include, but are not limited to, glass, modified and/or functionalized glass, hydrogels, films, membranes, plastics (including e.g., acrylics, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, TeflonTM, cyclic olefins, polyimides etc.), nylon, ceramics, resins, Zeonor, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, optical fiber bundles, and polymers, such as polystyrene, cyclic olefin copolymers (COCs), cyclic olefin polymers (COPs), polypropylene, polyethylene polycarbonate, or combinations thereof.

[00143] Among the examples of first substrate materials discussed above, polystyrene is a hydrophobic material suitable for binding negatively charged macromolecules because it normally contains few hydrophilic groups. For nucleic acids immobilized on glass slides, by increasing the hydrophobicity of the glass surface the nucleic acid immobilization can be increased. Such an enhancement can permit a relatively more densely packed formation (e.g., provide improved specificity and resolution).

[00144] In another example, a first substrate can be a flow cell. Flow cells can be formed of any of the foregoing materials, and can include channels that permit reagents, solvents, features, and analytes to pass through the flow cell. In some embodiments, a hydrogel embedded biological sample is assembled in a flow cell (e.g., the flow cell is utilized to introduce the hydrogel to the biological sample). In some embodiments, a hydrogel embedded biological sample is not assembled in a flow cell. In some embodiments, the hydrogel embedded biological sample can then be prepared and/or isometrically expanded as described herein.

[00145] Exemplary substrates similar to the first substrate (e.g., a substrate having no capture probes) and/or the second substrate are described in Section (I) above and in WO 2020/123320, which is hereby incorporated by reference in its entirety.

[00146] (b) Capturing Nucleic Acid Analytes using RNA-Templated Ligation

[00147] In some embodiments, the methods, devices, compositions, and systems described herein utilize RNA-templated ligation to detect the analyte. As used herein, spatial “RNA- templated ligation,” or “RTL” or simply “templated ligation” is a process where individual probe oligonucleotides (e.g., a first probe oligonucleotide, a second probe oligonucleotide) in a probe pair hybridize to adjacent sequences of an analyte (e.g., an RNA molecule) in a biological sample (e.g., a tissue sample). The RTL probe oligonucleotides are then coupled (e.g., ligated) together, thereby creating a connected probe (e.g., a ligation product). RNA-templated ligation is disclosed in PCT Publ. No. WO 2021/133849 Al and US Publ. No. US 2021/0285046 Al, each of which is incorporated by reference in its entirety.

[00151] Also provided herein are methods for analyzing an analyte in a biological sample mounted on a first substrate including (a) hybridizing a first probe oligonucleotide and a second probe oligonucleotide to the analyte, where the first probe oligonucleotide and the second probe oligonucleotide each include a sequence that is substantially complementary to adjacent sequences of the analyte, and where the second probe oligonucleotide includes a capture probe binding domain; (b) coupling (e.g., ligating) the first probe oligonucleotide and the second probe oligonucleotide, thereby generating a connected probe (e.g., a ligation product) including the capture probe binding domain; (c) aligning the first substrate with a second substrate including an array, such that at least a portion of the biological sample is aligned with at least a portion of the array, where the array includes a plurality of capture probes, where a capture probe of the plurality of capture probes includes: (i) a spatial barcode and (ii) a capture domain; (d) when the biological sample is aligned with at least a portion of the array, (i) releasing the connected probe (e.g., a ligation product) from the analyte and (ii) passively or actively migrating the connected probe (e.g., a ligation product) from the biological sample to the array; and (e) hybridizing the capture probe binding domain of the connected probe (e.g., a ligation product) to the capture domain.

[00152] In some embodiments, the process of transferring the connected probe (e.g., a ligation product) from the first substrate to the second substrate is referred to as a “sandwich” process. The sandwich process is described in PCT Patent Application Publication No. WO 2020/123320, which is incorporated by reference in its entirety. Described herein are methods in which an array with capture probes located on a substrate and a biological sample located on a different substrate, are contacted such that the array is in contact with the biological sample (e.g., the substrates are sandwiched together). In some embodiments, the array and the biological sample can be contacted (e.g., sandwiched), without the aid of a substrate holder. In some embodiments, the array and biological sample substrates can be placed in a substrate holder (e.g., an array alignment device) designed to align the biological sample and the array. For example, the substrate holder can have placeholders for two substrates. In some embodiments, an array including capture probes can be positioned on one side of the substrate holder (e.g., in a first substrate placeholder). In some embodiments, a biological sample can be placed on the adjacent side of the substrate holder in a second placeholder. In some embodiments, a hinge can be located between the two substrate placeholders that allows the substrate holder to close, e.g., make a sandwich between the two substrate placeholders. In some embodiments, when the substrate holder is closed the biological sample and the array with capture probes are contacted with one another under conditions sufficient to allow analytes present in the biological sample to interact with the capture probes of the array. For example, dried permeabilization reagents can be placed on the biological sample and rehydrated. A permeabilization solution can be flowed through the substrate holder to permeabilize the biological sample and allow analytes in the biological sample to interact with the capture probes. Additionally, the temperature of the substrates or permeabilization solution can be used to initiate or control the rate of permeabilization. For example, the substrate including the array, the substrate including the biological sample, or both substrates can be held at a low temperature to slow diffusion and permeabilization efficiency. Once sandwiched, in some embodiments, the substrates can be heated to initiate permeabilization and/or increase diffusion efficiency. Transcripts that are released from the permeabilized tissue can diffuse to the array and be captured by the capture probes. The sandwich can be opened, and cDNA synthesis can be performed on the array.

[00153] In some embodiments, the methods as disclosed herein include hybridizing of one or more probe oligonucleotide probe pairs (e.g., RTL probes) to adjacent or nearby sequences of a target analyte (e.g., RNA; e.g., mRNA) of interest. In some embodiments, the probe oligonucleotide pairs include sequences that are complementary or substantially complementary to an analyte. For example, in some embodiments, each probe oligonucleotide includes a sequence that is complementary or substantially complementary to an mRNA of interest (e.g., to a portion of the sequence of an mRNA of interest). In some embodiments, each target analyte includes a first target region and a second target region. In some embodiments, the methods include providing a plurality of first probe oligonucleotides and a plurality of second probe oligonucleotides, where a pair of probe oligonucleotides for a target analyte comprises both a first and second probe oligonucleotide. In some embodiments, a first probe oligonucleotide hybridizes to a first target region of the analyte, and the second probe oligonucleotide hybridizes to a second, adjacent or nearly adjacent target region of the analyte.

[00154] In some instances, the probe oligonucleotides are DNA molecules. In some instances, the first probe oligonucleotide is a DNA molecule. In some instances, the second probe oligonucleotide is a DNA molecule. In some instances, the first probe oligonucleotide comprises at least two ribonucleic acid bases at the 3’ end. In some instances, the second probe oligonucleotide comprises a phosphorylated nucleotide at the 5’ end.

[00155] RTL probes can be designed using methods known in the art. In some instances, probe pairs are designed to cover an entire transcriptome of a species (e.g., a mouse or a human). In some instances, RTL probes are designed to cover a subset of a transcriptome e.g., a mouse or a human). In some instances, the methods disclosed herein utilize about 500, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000, about 10,000, about 15,000, about 20,000, or more probe pairs.

[00157] In some instances, the first and second target regions of an analyte are directly adjacent to one another. In some embodiments, the complementary sequences to which the first probe oligonucleotide and the second probe oligonucleotide hybridize are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 125, or about 150 nucleotides away from each other. Gaps between the probe oligonucleotides may first be filled prior to coupling (e.g., ligation), using, for example, dNTPs in combination with a polymerase such as polymerase mu, DNA polymerase, RNA polymerase, reverse transcriptase, VENT polymerase, Taq polymerase, and/or any combinations, derivatives, and variants (e.g., engineered mutants) thereof. In some embodiments, when the first and second probe oligonucleotides are separated from each other by one or more nucleotides, deoxyribonucleotides are used to extend and couple (e.g., ligate) the first and second probe oligonucleotides.

[00159] In some embodiments, the first and/or second probe as disclosed herein includes at least two ribonucleic acid bases at the 3’ end; a functional sequence; a phosphorylated nucleotide at the 5’ end; and/or a capture probe binding domain. In some embodiments, the functional sequence is a primer sequence.

[00162] In some embodiments, methods disclosed herein include a wash step after hybridizing the first and the second probe oligonucleotides. The wash step removes any unbound oligonucleotides and can be performed using any technique known in the art. In some embodiments, a pre-hybridization buffer is used to wash the sample. In some embodiments, a phosphate buffer is used. In some embodiments, multiple wash steps are performed to remove unbound oligonucleotides. For example, it is advantageous to decrease the amount of unhybridized probes present in a biological sample as they may interfere with downstream applications and methods.

[00163] In some embodiments, after hybridization of probe oligonucleotides (e.g., first and the second probe oligonucleotides) to the target analyte, the probe oligonucleotides (e.g., the first probe oligonucleotide and the second probe oligonucleotide) are coupled (e.g., ligated) together, creating a single connected probe (e.g., a ligation product) that is complementary to the target analyte. Ligation can be performed enzymatically or chemically, as described herein. For example, the first and second probe oligonucleotides are hybridized to the first and second target regions of the analyte, and the probe oligonucleotides are subjected to a nucleic acid reaction to ligate them together. For example, the probes may be subjected to an enzymatic ligation reaction using a ligase (e.g., T4 RNA ligase (Rnl2), a SplintR ligase, or a T4 DNA ligase). See, e.g., Zhang L., et al.,- Archaeal RNA ligase from thermoccocus kodakarensis for template dependent ligation RNA Biol. 2017; 14(1): 36-44 for a description of KOD ligase. A skilled artisan will understand that various reagents, buffers, cofactors, etc. may be included in a ligation reaction depending on the ligase being used.

[00164] In some embodiments, the first probe oligonucleotide and the second probe oligonucleotides are on a contiguous nucleic acid sequence. In some embodiments, the first probe oligonucleotide is on the 3’ end of the contiguous nucleic acid sequence. In some embodiments, the first probe oligonucleotide is on the 5’ end of the contiguous nucleic acid sequence. In some embodiments, the second probe oligonucleotide is on the 3’ end of the contiguous nucleic acid sequence. In some embodiments, the second probe oligonucleotide is on the 5’ end of the contiguous nucleic acid sequence.

[00167] In some embodiments, the method further includes amplifying the connected probe (e.g., a ligation product) prior to the releasing step. In some embodiments, the entire connected probe (e.g., a ligation product) is amplified. In some embodiments, only part of the connected probe (e.g., a ligation product) is amplified. In some embodiments, amplification is isothermal. In some embodiments, amplification is not isothermal. Amplification can be performed using any of the methods described herein such as, but not limited to, a strand-displacement amplification reaction, a rolling circle amplification reaction, a ligase chain reaction, a transcription-mediated amplification reaction, an isothermal amplification reaction, and/or a 10 loop-mediated amplification reaction. In some embodiments, amplifying the connected probe (e.g., a ligation product) creates an amplified connected probe (e.g., a ligation product) that includes (i) all or part of sequence of the connected probe (e.g., a ligation product) specifically bound to the capture domain, or a complement thereof, and (ii) the sequence of the spatial barcode, or a complement thereof.

[00168] In some embodiments, the method further includes determining (i) all or a part of the sequence of the connected probe (e.g., a ligation product), or a complement thereof, and (ii) the sequence of the spatial barcode, or a complement thereof. In some embodiments, the method further includes using the determined sequence of (i) and (ii) to determine the location and abundance of the analyte in the biological sample. [00169] In some embodiments, after coupling (e.g., ligation) of the first and second probe oligonucleotides to create a ligation product, the connected probe (e.g., a ligation product) is released from the analyte. To release the connected probe (e.g., a ligation product), an endoribonuclease (e.g., RNase A, RNase C, RNase H, or RNase I) is used. An endoribonuclease such as RNase H specifically cleaves RNA in RNA:DNA hybrids. In some embodiments, the connected probe (e.g., a ligation product) is released enzymatically. In some embodiments, an endoribonuclease is used to release the probe from the analyte. In some embodiments, the endoribonuclease is one or more of RNase H. In some embodiments, the RNase H is RNase Hl or RNase H2.

[00170] In some embodiments, the releasing of the connected probe (e.g., a ligation product) includes contacting the biological sample with a reagent medium comprising a permeabilization agent and an agent for releasing the connected probe (e.g., a ligation product), thereby permeabilizing the biological sample and releasing the connected probe (e.g., a ligation product) from the analyte. In some embodiments, the agent for releasing the connected probe (e.g., a ligation product) comprises a nuclease. In some embodiments, the nuclease is an endonuclease. In some embodiments, the nuclease is an exonuclease. In some embodiments, the nuclease includes an RNase. In some embodiments, the RNase is selected from RNase A, RNase C, RNase H, or RNase I.

[00171] In some embodiments, the reagent medium comprises polyethylene glycol (PEG). In some embodiments, the PEG is from about PEG 2K to about PEG 16K. In some embodiments, the PEG is PEG 2K, 3K, 4K, 5K, 6K, 7K, 8K, 9K, 10K, 1 IK, 12K, 13K, 14K, 15K, or 16K. In some embodiments, the PEG is present at a concentration from about 2% to 25%, from about 4% to about 23%, from about 6% to about 21%, or from about 8% to about 20% (v/v).

[00172] In some embodiments, the reagent medium includes a wetting agent.

[00173] In some instances, after creation of the connected probe (e.g., a ligation product), the methods disclosed herein include simultaneous treatment of the biological sample with a permeabilization agent such as proteinase K (to permeabilize the biological sample) and a releasing agent such as an endonuclease such as RNase H (to release the connected probe (e.g., a ligation product) from the analyte). In some instances, the permeabilization step and releasing step occur at the same time. In some instances, the permeabilization step occurs before the releasing step. In some embodiments, the permeabilization agent comprises a protease. In some embodiments, the protease is selected from trypsin, pepsin, elastase, or Proteinase K. In some embodiments, the protease is an endopeptidase. Endopeptidases that can be used include but are not limited to trypsin, chymotrypsin, elastase, thermolysin, pepsin, clostripan, glutamyl endopeptidase (GluC), ArgC, peptidyl-asp endopeptidase (ApsN), endopeptidase LysC and endopeptidase LysN. In some embodiments, the endopeptidase is pepsin.

[00174] In some embodiments, the reagent medium further includes a detergent. In some embodiments, the detergent is selected from sodium dodecyl sulfate (SDS), sarkosyl, saponin, Triton X-100™, or Tween-20™ In some embodiments, the reagent medium includes less than 5 w/v% of a detergent selected from sodium dodecyl sulfate (SDS) and sarkosyl. In some embodiments, the reagent medium includes as least 5% w/v% of a detergent selected from SDS and sarkosyl. In some embodiments, the reagent medium does not include SDS or sarkosyl.

[00176] In some embodiments, the connected probe (e.g., a ligation product) includes a capture probe binding domain, which can hybridize to a capture probe (e.g., a capture probe immobilized, directly or indirectly, on a substrate). In some embodiments, the capture probe includes a spatial barcode and the capture domain. In some embodiments, the capture probe binding domain of the connected probe (e.g., a ligation product) specifically binds to the capture domain of the capture probe.

[00177] In some embodiments, methods provided herein include mounting a biological sample on a first substrate, then aligning the first substrate with a second substrate including an array, such that at least a portion of the biological sample is aligned with at least a portion of the array, where the array includes a plurality of capture probes. After hybridization of the connected probe (e.g., a ligation product) to the capture probe, downstream methods as disclosed herein can be performed.

[00178] In some embodiments, at least 50% of connected probes (e.g., a ligation products) released from the portion of the biological sample aligned with the portion of the array are captured by capture probes of the portion of the array. In some embodiments, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of connected probe (e.g., a ligation products) are detected in spots directly under the biological sample.

[00179] In some embodiments, the capture probe includes a poly(T) sequence. In some embodiments, capture probe includes a sequence specific to the analyte. In some embodiments, the capture probe includes a functional domain. In some embodiments, the capture probe further includes one or more functional domains, a unique molecular identifier (UMI), a cleavage domain, and combinations thereof. In some embodiments, the capture probe binding domain includes a poly(A) sequence. In some embodiments, the capture probe binding domain includes a sequence complementary to a capture domain of a capture probe that detects a target analyte of interest. In some embodiments, the analyte is RNA. In some embodiments, the analyte is mRNA.

[00180] In some embodiments, the connected probe (e.g, a ligation product) (e.g, the analyte derived molecule) includes a capture probe binding domain, which can hybridize to a capture probe (e.g., a capture probe immobilized, directly or indirectly, on a substrate). Methods provided herein include contacting a biological sample with a substrate, where the capture probe is affixed to the substrate (e.g., immobilized to the substrate, directly or indirectly). After hybridization of the connected probe (e.g., a ligation product) to the capture probe, downstream methods as disclosed herein (e.g., sequencing, in situ analysis such as RCA) can be performed.

[00181] In some embodiments, the method further includes analyzing a different analyte in the biological sample. In some embodiments, the analysis of the different analyte includes (a) further contacting the biological sample with a plurality of analyte capture agents, where an analyte capture agent of the plurality of analyte capture agents includes an analyte binding moiety and a capture agent barcode domain, where the analyte binding moiety specifically binds to the different analyte, and where the capture agent barcode domain includes an analyte binding moiety barcode and an capture handle sequence that is complementary to a capture domain of a capture probe; and (b) hybridizing the analyte capture sequence to the capture domain.

[00182] An exemplary embodiment of a workflow for analysis of protein and RNA analytes is shown in FIG. 7A. As shown in FIG. 7A, a fixed tissue sample mounted on a first substrate (e.g., a slide-mounted tissue sample) is decrosslinked, followed by hybridization of probe pairs to nucleic acid analyte analytes. Also as shown in FIG. 7A, a first and second probe of a probe pair is connected, e.g., ligated. The sample is optionally washed (e.g., with a buffer), prior to incubation with an analyte capture agent (e.g., an antibody) that specifically binds a different analyte, e.g., a protein analyte. The analyte capture agent comprises a capture agent barcode domain. In some embodiments, the analyte capture agent is an antibody with an oligonucleotide tag, the oligonucleotide tag comprising a capture agent barcode domain. In some embodiments, the connected probes (e.g., the ligation products) and antibody oligonucleotide tags are released from the tissue under sandwich conditions as described herein. For the sandwich conditions, the tissue-mounted slide can be aligned with an array and permeabilized with a reagent medium in the sandwich configuration as described herein (see, e.g., FIG. 7B). In some embodiments, the reagent medium comprises RNase and a permeabilization agent (e.g., Proteinase K).

Permeabilization releases the connected probe (e.g., a ligation product) and capture agent barcode domain, for capture onto a second substrate comprising an array with a plurality of capture probes (see, e.g., FIG. 7B). After capture of the connected probe and capture agent barcode domain, the tissue slide can be removed (e.g., the sandwich can be “opened” or “broken”). [00183] In some embodiments, following opening of the sandwich, the capture probes can be extended, sequencing libraries can be prepared and sequenced, and the results can be analyzed computationally.

[00184] In some embodiments, the method further includes determining (i) all or part of the sequence of the capture agent barcode domain; and (ii) the sequence of the spatial barcode, or a complement thereof. In some embodiments, the method further includes using the determined sequence of (i), and (ii) to analyze the different analyte in the biological sample. In some embodiments, the releasing step further releases the capture agent barcode domain from the different analyte. In some embodiments, the different analyte is a protein analyte. In some embodiments, the protein analyte is an extracellular protein. In some embodiments, the protein analyte is an intracellular protein.

[00185] (c) Capturing Analytes for Spatial Detection using Analyte Capture Agents

[00186] In some embodiments, the methods, compositions, devices, and systems provided herein utilize analyte capture agents for spatial detection. An “analyte capture agent” refers to a molecule that interacts with a target analyte (e.g., a protein) and with a capture probe. Such analyte capture agents can be used to identify the analyte. In some embodiments, the analyte capture agent can include an analyte binding moiety and a capture agent barcode domain. In some embodiments, the analyte capture agent includes a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the cleavable linker is a photo-cleavable linker, a UV-cleavable linker, or an enzyme cleavable linker. In some instances, the cleavable linker is a disulfide linker. A disulfide linker can be cleaved by use of a reducing agent, such as dithiothreitol (DTT), Beta-mercaptoethanol (BME), or Tris (2-carboxyethyl) phosphine (TCEP).

[00187] An analyte binding moiety is a molecule capable of binding to a specific analyte. In some embodiments, the analyte binding moiety comprises an antibody or antibody fragment. In some embodiments, the analyte binding moiety comprises a polypeptide and/or an aptamer. In some embodiments, the analyte is a protein (e.g., a protein on a surface of a cell or an intracellular protein).

[00188] A capture agent barcode domain can include a capture handle sequence which can hybridize to at least a portion or an entirety of a capture domain of a capture probe. In some embodiments, the capture handle sequence is complementary to a portion or entirety of a capture domain of a capture probe. In some embodiments, the capture handle sequence includes a poly (A) tail. In some embodiments, the capture handle sequence includes a sequence capable of binding a poly (T) domain. In some embodiments, the capture agent barcode domain comprises an analyte binding moiety barcode and a capture handle sequence. The analyte binding moiety barcode refers to a barcode that is associated with or otherwise identifies the analyte binding moiety, and the capture handle sequence can hybridize to a capture probe. In some embodiments, the capture handle sequence specifically binds to the capture domain of the capture probe. Other embodiments of an analyte capture agent useful in spatial analyte detection are described herein.

[00189] Provided herein are methods for analyzing an analyte in a biological sample including (a) contacting the biological sample with a plurality of analyte capture agents, where an analyte capture agent of the plurality of analyte capture agents includes an analyte binding moiety and a capture agent barcode domain, where the analyte binding moiety specifically binds to the analyte, and where the capture agent barcode domain includes an analyte binding moiety barcode and an capture handle sequence; (b) contacting the biological sample with a reagent medium including an agent for releasing the capture agent barcode domain from the analyte binding moiety, thereby releasing the capture agent barcode domain from the analyte binding moiety; and (c) hybridizing the capture handle sequence to a capture domain of a capture probe, where the capture probe includes (i) a spatial barcode and (ii) a capture domain.

[00190] Also provided herein are methods for analyzing an analyte in a biological sample mounted on a first substrate including (a) contacting the biological sample with a plurality of analyte capture agents, where an analyte capture agent of the plurality of analyte capture agents includes an analyte binding moiety and a capture agent barcode domain, where the analyte binding moiety specifically binds to the analyte, and where the capture agent barcode domain includes an analyte binding moiety barcode and an capture handle sequence; (b) aligning the first substrate with a second substrate comprising an array, such that at least a portion of the biological sample is aligned with at least a portion of the array, where the array includes a plurality of capture probes, where a capture probe of the plurality of capture probes includes (i) a spatial barcode and (ii) a capture domain; (c) when the biological sample is aligned with at least a portion of the array, (i) releasing the capture agent barcode domain from the analyte and (ii) passively or actively migrating the capture agent barcode domain from the biological sample to the array; and (d) coupling the capture handle sequence to the capture domain.

[00191] Also provided herein are methods for analyzing an analyte in a biological sample mounted on a first substrate including (a) contacting the biological sample with a plurality of analyte capture agents, where an analyte capture agent of the plurality of analyte capture agents includes an analyte binding moiety and a capture agent barcode domain, where the analyte binding moiety specifically binds to the analyte, and where the capture agent barcode domain includes an analyte binding moiety barcode and an capture handle sequence; (b) aligning the first substrate with a second substrate comprising an array, such that at least a portion of the biological sample is aligned with at least a portion of the array, where the array includes a plurality of capture probes, where a capture probe of the plurality of capture probes includes (i) a spatial barcode and (ii) a capture domain; (c) when the biological sample is aligned with at least a portion of the array, (i) releasing the capture agent barcode domain from the analyte and (ii) passively or actively migrating the capture agent barcode domain from the biological sample to the array; and (d) hybridizing the capture handle sequence to the capture domain.

[00192] In some embodiments, the process of transferring the connected probe (e.g., a ligation product) from the first substrate to the second substrate is referred to as a “sandwich process”. The sandwich process is described above and in PCT Patent Application Publication No. WO 2020/123320, which is incorporated by reference in its entirety.

[00193] In some embodiments, the method further includes determining (i) all or a part of the capture agent barcode domain, or a complement thereof; and (ii) the sequence of the spatial barcode, or a complement thereof. In some embodiments, the method further includes using the determined sequence of (i) and (ii) to determine the location and abundance of the analyte in the biological sample.

[00194] In some embodiments, an analyte capture agent is introduced to a biological sample, where the analyte binding moiety specifically binds to a target analyte, and then the biological sample can be treated to release the capture agent barcode domain from the biological sample. In some embodiments, the capture agent barcode domain can then migrate and bind to a capture domain of a capture probe, and the capture agent barcode domain can be extended to generate a spatial barcode complement at the end of the capture agent barcode domain. In some embodiments, the spatially-tagged capture agent barcode domain can be denatured from the capture probe, and analyzed using methods described herein.

[00197] In some embodiments, the reagent medium further includes a detergent. In some embodiments, the detergent is selected from sodium dodecyl sulfate (SDS), sarkosyl, saponin, Triton X-100™, or Tween-20™ In some embodiments, the reagent medium includes less than 5 w/v% of a detergent selected from sodium dodecyl sulfate (SDS) and sarkosyl. In some embodiments, the reagent medium includes as least 5% w/v% of a detergent selected from SDS and sarkosyl. In some embodiments, the reagent medium does not include SDS or sarkosyl.

[00199] Also provided herein are methods further including analyzing a different analyte in the biological sample. In some embodiments, the analysis of the different analyte includes (a) hybridizing a first probe oligonucleotide and a second probe oligonucleotide to the different analyte, where the first probe oligonucleotide and the second probe oligonucleotide each comprise a sequence that is substantially complementary to adjacent sequences of the different analyte, and where the second probe oligonucleotide comprises a capture probe binding domain; (b) ligating the first probe oligonucleotide and the second probe oligonucleotide, thereby generating a connected probe (e.g., a ligation product) comprising the capture probe binding domain; and (c) hybridizing the capture probe binding domain of the connected probe (e.g., a ligation product) to the capture domain.

[00200] In some embodiments, the method further includes determining (i) all or part of the sequence of the connected probe (e.g., a ligation product), or a complement thereof, and (ii) the sequence of the spatial barcode, or a complement thereof. In some embodiments, the method further includes using the determined sequence of (i), and (ii) to analyze the different analyte in the biological sample. In some embodiments, the releasing step further releases the connected probe (e.g., a ligation product) from the different analyte. In some embodiments, the different analyte is RNA. In some embodiments, the different analyte is mRNA.

[00201] In some embodiments, the capture probe comprises a poly(T) sequence. In some embodiments, the capture probe comprises a sequence complementary to the capture handle sequence. In some embodiments, the capture probe comprises a functional domain. In some embodiments, the capture probe further comprises one or more functional domains, a unique molecular identifier (UMI), a cleavage domain, and combinations thereof.

[00203] In some instances, RTL is performed between two oligonucleotides that each are affixed to an analyte binding moiety (i.e., a protein-binding moiety). Generally, the methods of RTL in this setting is as follows. In some embodiments, provided herein is a method of determining a location of at least one analyte in a biological sample including: (a) hybridizing a first analyte-binding moiety to a first analyte in the biological sample, where the first analytebinding moiety is bound to a first oligonucleotide, where the first oligonucleotide comprises: (i) a functional sequence; (ii) a first barcode; and (iii) a first bridge sequence; (b) hybridizing a second analyte-binding moiety to a second analyte in the biological sample, where the second analytebinding moiety is bound to a second oligonucleotide; where the second oligonucleotide comprises: (i) capture probe binding domain sequence, (ii) a second barcode; and (ii) a second bridge sequence; (c) contacting the biological sample with a third oligonucleotide; (d) hybridizing the third oligonucleotide to the first bridge sequence of the first oligonucleotide and second bridge sequence of the second oligonucleotide; (e) ligating the first oligonucleotide and the second oligonucleotide, creating a connected probe (e.g., a ligation product); (f) contacting the biological sample with a substrate, where a capture probe is affixed to the substrate, where the capture probe comprises a spatial barcode and the capture domain; and (g) allowing the capture probe binding domain sequence of the second oligonucleotide to specifically bind to the capture domain. In some instances, the connected probe (e.g., a ligation product) is cleaved from the analyte biding moieties.

[00204] In some instances, two analytes (e.g., two different proteins) in close proximity in a biological sample are detected by a first analyte-binding moiety and a second analyte-binding moiety, respectively. In some embodiments, a first analyte-binding moiety and/or the second analyte-binding moiety is an analyte capture agent (e.g., any of the exemplary analyte capture agents described herein). In some embodiments, the first analyte-binding moiety and/or the second analyte-binding moiety is a first protein. In some embodiments, the first analyte-binding moiety and/or the second analyte-binding moiety is an antibody. For example, the antibody can include, without limitation, a monoclonal antibody, recombinant antibody, synthetic antibody, a single domain antibody, a single-chain variable fragment (scFv), and or an antigen-binding fragment (Fab). In some embodiments, the first analyte-binding moiety binds to a cell surface analyte (e.g., any of the exemplary cell surface analytes described herein). In some embodiments, binding of the analyte is performed metabolically. In some embodiments, binding of the analyte is performed enzymatically. In some embodiments, the methods include a secondary antibody that binds to a primary antibody, enhancing its detection.

[00205] In some embodiments, the first analyte-binding moiety and the second analytebinding moiety each bind to the same analyte. In some embodiments, the first analyte-binding moiety and/or second analyte-binding moiety each bind to a different analyte. For example, in some embodiments, the first analyte-binding moiety binds to a first polypeptide and the second analyte-binding moiety binds to a second polypeptide.

[00206] In some embodiments of any of the methods of determining a location of at least one analyte in a biological sample, a first and/or a second oligonucleotide are bound (e.g., conjugated or otherwise attached using any of the methods described herein) to a first analyte-binding moiety and/or a second analyte-binding moiety, respectively.

[00207] In some embodiments of any of the methods of determining a location of at least one analyte in a biological sample as described herein, a second oligonucleotide is bound (e.g., conjugated or otherwise attached using any of the methods described herein) to a second analytebinding moiety. For example, the second oligonucleotide can be covalently linked to the second analyte-binding moiety. In some embodiments, the second oligonucleotide is bound to the second analyte-binding moiety via its 5’ end. In some embodiments, the second oligonucleotide includes a free 3’ end. In some embodiments the second oligonucleotide is bound to the second analytebinding moiety via its 3’ end. In some embodiments, the second oligonucleotide includes a free 5’ end.

[00208] In some embodiments, the oligonucleotides are bound to the first and/or second analyte-binding moi eties via a linker (e.g., any of the exemplary linkers described herein). In some embodiments, the linker is a cleavable linker. In some embodiment, the linker is a linker with photo-sensitive chemical bonds (e.g., photo-cleavable linkers). In some embodiments, the linker is a cleavable linker that can undergo induced dissociation.

[00209] FIGs. 8A-8B show exemplary schematics illustrating methods to release the capture agent barcode domain from the analyte binding moiety. FIG. 8A shows the use of an RNase cleavable linker to release a capture agent barcode domain from an analyte binding moiety of an analyte capture agent. In particular, a target (indicated by the circle in FIG. 8A interacts with an antibody. After an interaction is established, an enzyme (e.g., an RNAse) cleaves a linker, freeing a barcode from the antibody. The barcode, which is specific to the antibody, is captured on a slide comprising a plurality of probes. Similarly, FIG. 8B shows the use of a UV cleavable linker to release a capture agent barcode domain from an analyte binding moiety of an analyte capture agent.

[00210] In some embodiments, the oligonucleotides are bound (e.g., attached via any of the methods described herein) to an analyte-binding domain via a 5’ end.

[00211] In some embodiments, a barcode is used to identify the analyte-binding moiety to which it is bound. The barcode can be any of the exemplary barcodes described herein. In some embodiments, the first and/or second oligonucleotide include a capture probe binding domain sequence. For example, a capture probe binding domain sequence can be a poly(A) sequence when the capture domain sequence is a poly(T) sequence.

[00212] In some embodiments, a third oligonucleotide (e.g., a splint oligonucleotide) hybridizes to both the first and second oligonucleotides and enables ligation of the first oligonucleotide and the second oligonucleotide. In some embodiments, a ligase is used. In some aspects, the ligase includes a DNA ligase. In some aspects, the ligase includes an RNA ligase. In some aspects, the ligase includes T4 DNA ligase. In some embodiments, the ligase is a SplintR ligase.

[00213] (d) Sandwich Processes

[00214] In some embodiments, one or more analytes from the biological sample are released from the biological sample and migrate to a substrate comprising an array of capture probes for attachment to the capture probes of the array. In some embodiments, the release and migration of the analytes to the substrate comprising the array of capture probes occurs in a manner that preserves the original spatial context of the analytes in the biological sample. In some embodiments, the biological sample is mounted on a first substrate and the substrate comprising the array of capture probes is a second substrate. In some embodiments, the alignment of the first substrate and the second substrate is facilitated by a sandwiching process. Accordingly, described herein are methods, compositions, devices, and systems for sandwiching together the first substrate as described herein with a second substrate having an array with capture probes.

[00215] FIG. 9 is a schematic diagram depicting an exemplary sandwiching process between a first substrate comprising a biological sample (e.g., a tissue section 902 on a slide 903) and a second substrate comprising a spatially barcoded array, e.g., a slide 904 that is populated with spatially-barcoded capture probes 906. During the exemplary sandwiching process, the first substrate is aligned with the second substrate, such that at least a portion of the biological sample is aligned with at least a portion of the array (e.g., aligned in a sandwich configuration). As shown, the second substrate (e.g., slide 904) is in a superior position to the first substrate (e.g., slide 903). In some embodiments, the first substrate (e.g., slide 903) may be positioned superior to the second substrate (e.g., slide 904). In some embodiments, the first and second substrates are aligned to maintain a gap or separation distance 907 between the two substrates. When the first and second substrates are aligned, one or more analytes are released from the biological sample and actively or passively migrate to the array for capture. In some embodiments, the migration occurs while the aligned portions of the biological sample and the array are contacted with a reagent medium 905. The released one or more analytes may actively or passively migrate across the gap 907 via the reagent medium 905 toward the capture probes 906, and be captured by the capture probes 906.

[00217] In some embodiments, the sandwiching process may be facilitated by a device, sample holder, sample handling apparatus, or system described in, e.g., US. Patent Application Pub. No. 20210189475, PCT/US2021/036788, or PCT/US2021/050931.

[00218] In some embodiments, the first and second substrates are placed in a substrate holder (e.g., an array alignment device) configured to align the biological sample and the array. In some embodiments, the device comprises a sample holder. In some embodiments, the sample holder includes a first member and a second member that receive a first substrate and a second substrate, respectively. The device can include an alignment mechanism that is connected to at least one of the members and aligns the first and second members. Thus, the devices of the disclosure can advantageously align the first substrate and the second substrate and any samples, barcoded probes, or permeabilization reagents that may be on the surface of the first and second substrates.

[00219] In some embodiments, the sandwiching process comprises: mounting the first substrate on a first member of a support device, the first member configured to retain the first substrate; mounting the second substrate on a second member of the support device, the second member configured to retain the second substrate, applying a reagent medium to the first substrate and/or the second substrate, the reagent medium comprising a permeabilization agent, operating an alignment mechanism of the support device to move the first member and/or the second member such that a portion of the biological sample is aligned (e.g., vertically aligned) with a portion of the array of capture probes and within a threshold distance of the array of capture probes, and such that the portion of the biological sample and the capture probe contact the reagent medium, where the permeabilization agent releases the analyte from the biological sample.

[00220] In some embodiments of a sample holder, the sample holder can include a first member including a first retaining mechanism configured to retain a first substrate comprising a sample. The first retaining mechanism can be configured to retain the first substrate disposed in a first plane. The sample holder can further include a second member including a second retaining mechanism configured to retain a second substrate disposed in a second plane. The sample holder can further include an alignment mechanism connected to one or both of the first member and the second member. The alignment mechanism can be configured to align the first and second members along the first plane and/or the second plane such that the sample contacts at least a portion of the reagent medium when the first and second members are aligned and within a threshold distance along an axis orthogonal to the second plane. The alignment mechanism may be configured to move the second member along the axis orthogonal to the second plane and/or move the first member along an axis orthogonal to the first plane.

[00221] In some embodiments, the alignment mechanism includes a linear actuator. In some embodiments, the alignment mechanism includes one or more of a moving plate, a bushing, a shoulder screw, a motor bracket, and a linear actuator. The moving plate may be coupled to the first member or the second member. The alignment mechanism may, in some cases, include a first moving plate coupled to the first member and a second moving plate coupled to the second member. In some embodiments, the linear actuator is configured to move the second member along an axis orthogonal to the plane of the first member and/or the second member. For example, the moving plate may be coupled to the second member and adjust the separation distance along a z axis (e.g., orthogonal to the second substrate) by moving the moving plate up in a superior direction toward the first substrate. In some embodiments, the linear actuator is configured to move the first member along an axis orthogonal to the plane of the first member and/or the second member. The movement of the moving plate may be accomplished by the linear actuator configured to move the first member and/or the second member at a velocity. The velocity may be controlled by a controller communicatively coupled to the linear actuator. In some embodiments, the linear actuator is configured to move the first member, the second member, or both the first member and the second member at a velocity of at least 0.1 mm/sec (e.g., at least 0.1 mm/sec to 2 mm/sec). In some aspects, the velocity may be selected to reduce or minimize bubble generation or trapping within the reagent medium. In some embodiments, the linear actuator is configured to move the first member, the second member, or both the first member and the second member with an amount of force of at least 0.1 lbs (e.g., between 0.1-4.0 pounds of force).

[00224] Provided herein are methods for delivering a fluid to a biological sample disposed on an area of a first substrate and an array disposed on a second substrate.

[00225] In some embodiments and with reference to FIG. 9, the sandwich configuration described herein between a first substrate comprising a biological sample (e.g., slide 903) and a second substrate comprising a spatially barcoded array (e.g., slide 904 with barcoded capture probes 906) may include a reagent medium (e.g., a liquid reagent medium, e.g., a permeabilization solution 905 or other target molecule release and capture solution) to fill a gap (e.g., gap 907). It may be desirable that the reagent medium be free from air bubbles between the slides to facilitate transfer of target molecules with spatial information. Additionally, air bubbles present between the slides may obscure at least a portion of an image capture of a desired region of interest. Accordingly, it may be desirable to ensure or encourage suppression and/or elimination of air bubbles between the two substrates (e.g., slide 903 and slide 904) during a permeabilization step.

[00226] In some aspects, it may be possible to reduce or eliminate bubble formation between the slides using a variety of filling methods and/or closing methods.

[00227] Workflows described herein may include contacting a drop of the reagent medium (e.g., liquid reagent medium, e.g., a permeabilization solution 905) disposed on a first substrate or a second substrate with at least a portion of the second substrate or first substrate, respectively. In some embodiments, the contacting comprises bringing the two substrates into proximity such that the sample on the first substrate is aligned with the barcode array of capture probes on the second substrate.

[00228] In some embodiments, the drop includes permeabilization reagents (e.g., any of the permeabilization reagents described herein). In some embodiments, the rate of permeabilization of the biological sample is modulated by delivering the permeabilization reagents (e.g., a fluid containing permeabilization reagents) at various temperatures. [00229] In the example sandwich maker workflows described herein, the reagent medium (e.g., liquid reagent medium, permeabilization solution 905) may fill a gap (e.g., the gap 907) between a first substrate (e.g., slide 903) and a second substrate (e.g., slide 904 with barcoded capture probes 906) to warrant or enable transfer of target molecules with spatial information. Described herein are examples of filling methods that may suppress bubble formation and suppress undesirable flow of transcripts and/or target molecules or analytes. Robust fluidics in the sandwich making described herein may preserve spatial information by reducing or preventing deflection of molecules as they move from the tissue slide to the capture slide.

[00230] Further details on angled closure workflows, and devices and systems for implementing an angled closure workflow, that can be used in conjunction with the present disclosure are described in PCT/US2021/036788 and PCT/US2021/050931, which are hereby incorporated by reference in their entirety.

[00231] Additional configurations for reducing or eliminating bubble formation, and/or for reducing unwanted fluid flow, are described in PCT/US2021/036788, which is hereby incorporated by reference in its entirety.

[00232] In some embodiments, the reagent medium comprises a permeabilization agent. Suitable agents for this purpose include, but are not limited to, organic solvents (e.g., acetone, ethanol, and methanol), cross-linking agents (e.g, paraformaldehyde), detergents (e.g, saponin, Triton X-100™, Tween-20™, or sodium dodecyl sulfate (SDS)), and enzymes (e.g., trypsin, proteases (e.g., proteinase K). In some embodiments, the detergent is an anionic detergent (e.g., SDS or N-lauroylsarcosine sodium salt solution). Exemplary permeabilization reagents are described in in PCT Patent Application Publication No. WO 2020/123320, which is incorporated by reference in its entirety.

[00233] In some embodiments, the reagent medium comprises a lysis reagent. Lysis solutions can include ionic surfactants such as, for example, sarkosyl and sodium dodecyl sulfate (SDS). More generally, chemical lysis agents can include, without limitation, organic solvents, chelating agents, detergents, surfactants, and chaotropic agents. Exemplary lysis reagents are described in PCT Patent Application Publication No. WO 2020/123320, which is incorporated by reference in its entirety.

[00234] In some embodiments, the reagent medium comprises a protease. Exemplary proteases include, e.g., pepsin, trypsin, pepsin, elastase, and proteinase K. Exemplary proteases are described in PCT Patent Application Publication No. WO 2020/123320, which is incorporated by reference in its entirety. [00235] In some embodiments, the reagent medium comprises a detergent. Exemplary detergents include sodium dodecyl sulfate (SDS), sarkosyl, saponin, Triton X-100TM, and Tween-20TM. Exemplary detergents are described in PCT Patent Application Publication No. WO 2020/123320, which is incorporated by reference in its entirety.

[00236] In some embodiments, the reagent medium comprises a nuclease. In some embodiments, the nuclease comprises am RNase. In some embodiments, the RNase is selected from RNase A, RNase C, RNase H, and RNase I. In some embodiments, the reagent medium comprises one or more of sodium dodecyl sulfate (SDS), proteinase K, pepsin, N- lauroylsarcosine, RNAse, and a sodium salt thereof.

[00237] The sample holder is compatible with a variety of different schemes for contacting the aligned portions of the biological sample and array with the reagent medium to promote analyte capture. In some embodiments, the reagent medium is deposited directly on the second substrate (e.g., forming a reagent medium that includes the permeabilization reagent and the feature array), and/or directly on the first substrate. In some embodiments, the reagent medium is deposited on the first and/or second substrate, and then the first and second substrates aligned in the sandwich configuration such that the reagent medium contacts the aligned portions of the biological sample and array. In some embodiments, the reagent medium is introduced into the gap 907 while the first and second substrates are aligned in the sandwich configuration.

[00238] In certain embodiments a dried permeabilization reagent is applied or formed as a layer on the first substrate or the second substrate or both prior to contacting the sample and the feature array. For example, a reagent can be deposited in solution on the first substrate or the second substrate or both and then dried. Drying methods include, but are not limited to spin coating a thin solution of the reagent and then evaporating a solvent included in the reagent or the reagent itself. Alternatively, in other embodiments, the reagent can be applied in dried form directly onto the first substrate or the second substrate or both. In some embodiments, the coating process can be done in advance of the analytical workflow and the first substrate and the second substrate can be stored pre-coated. Alternatively, the coating process can be done as part of the analytical workflow. In some embodiments, the reagent is a permeabilization reagent. In some embodiments, the reagent is a permeabilization enzyme, a buffer, a detergent, or any combination thereof. In some embodiments, the permeabilization enzyme is pepsin. In some embodiments, the reagent is a dried reagent (e.g., a reagent free from moisture or liquid). In some instances, the substrate that includes the sample (e.g., a histological tissue section) is hydrated. The sample can be hydrated by contacting the sample with a reagent medium, e.g., a buffer that does not include a permeabilization reagent. In some embodiments, the hydration is performed while the first and second substrates are aligned in a sandwich configuration.

[00239] In some instances, the aligned portions of the biological sample and the array are in contact with the reagent medium 905 for about 1 minute. In some instances, the aligned portions of the biological sample and the array are in contact with the reagent medium 905 for about 5 minutes. In some instances, the aligned portions of the biological sample and the array are in contact with the reagent medium 905 in the gap 907 for about 1 minute, about 5 minutes, about 10 minutes, about 12 minutes, about 15 minutes, about 18 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 36 minutes, about 45 minutes, or about an hour. In some instances, the aligned portions of the biological sample and the array are in contact with the reagent medium 905 for about 1-60 minutes. In some instances, the aligned portions of the biological sample and the array are in contact with the reagent medium 905 for about 30 minutes.

[00240] In some embodiments, following initial contact between sample and a permeabilization agent, the permeabilization agent can be removed from contact with sample (e.g., by opening sample holder) before complete permeabilization of sample. For example, in some embodiments, only a portion of sample is permeabilized, and only a portion of the analytes in sample may be captured by feature array. In some instances, the reduced amount of analyte captured and available for detection can be offset by the reduction in lateral diffusion that results from incomplete permeabilization of sample. In general, the spatial resolution of the assay is determined by the extent of analyte diffusion in the transverse direction (i.e., orthogonal to the normal direction to the surface of sample). The larger the distance between the sample on the first substrate and the feature array on the second substrate, the greater the extent of diffusion in the transverse direction, and the concomitant loss of resolution. Analytes liberated from a portion of the sample closest to the feature array have a shorter diffusion path, and therefore do not diffuse as far laterally as analytes from portions of the sample farthest from the feature array. As a result, in some instances, incomplete permeabilization of the sample (by reducing the contact interval between the permeabilization agent and the sample) can be used to maintain adequate spatial resolution in the assay.

[00242] In an exemplary embodiment, the second substrate is contacted with the permeabilization reagent. In some embodiments, the permeabilization reagent is dried. In some embodiments, the permeabilization reagent is a gel or a liquid. Also in the exemplary embodiment, the biological sample is contacted with buffer. Both the first and second substrates are placed at lower temperature to slow down diffusion and permeabilization efficiency. Alternatively, in some embodiments, the sample can be contacted directly with a liquid permeabilization reagent without inducing an unwanted initiation of permeabilization due to the substrates being at the second temperature. In some embodiments, the low temperature slows down or prevents the initiation of permeabilization. In a second step, keeping the sample holder and substrates at a cold temperature (e.g., at the first or second temperatures) continues to slow down or prevent the permeabilization of the sample. In a third step, the sample holder (and consequently the first and second substrates) is heated up to initiate permeabilization. In some embodiments, the sample holder is heated up to a third temperature. In some embodiments, the third temperature is above room temperature (e.g., 25 degrees Celsius) (e.g., 30 degrees Celsius or higher, 35 degrees Celsius or higher, 40 degrees Celsius or higher, 50 degrees Celsius or higher, 60 degrees Celsius or higher). In some embodiments, analytes that are released from the permeabilized tissue of the sample diffuse to the surface of the second substrate and are captured on the array (e.g., barcoded probes) of the second substrate. In a fourth step, the first substrate and the second substrate are separated (e.g., pulled apart) and temperature control is stopped.

[00243] In some embodiments, where either the first substrate or substrate second (or both) includes wells, a permeabilization solution can be introduced into some or all of the wells, and then the sample and the feature array can be contacted by closing the sample holder to permeabilize the sample. In certain embodiments, a permeabilization solution can be soaked into a hydrogel film that is applied directly to the sample, and/or soaked into features (e.g., beads) of the array. When the first and second substrates are aligned in the sandwich configuration, the permeabilization solution promotes migration of analytes from the sample to the array.

[00244] In certain embodiments, different permeabilization agents or different concentrations of permeabilization agents can be infused into array features (e.g., beads) or into a hydrogel layer as described above. By locally varying the nature of the permeabilization reagent(s), the process of analyte capture from the sample can be spatially adjusted.

[00245] In some instances, migration of the analyte from the biological sample to the second substrate is passive (e.g., via diffusion). Alternatively, in certain embodiments, migration of the analyte from the biological sample is performed actively (e.g., electrophoretic, by applying an electric field to promote migration). In some instances, first and second substrates can include a conductive epoxy. Electrical wires from a power supply can connect to the conductive epoxy, thereby allowing a user to apply a current and generate an electric field between the first and second substrates. In some embodiments, electrophoretic migration results in higher analyte capture efficiency and better spatial fidelity of captured analytes (e.g., on a feature array) than random diffusion onto matched substrates without the application of an electric field (e.g., via manual alignment of the two substrates). Exemplary methods of electrophoretic migration are described in WO 2020/176788, including at FIGs. 13-15, 24A-24B, and 25A-25C, which is hereby incorporated by reference in its entirety.

[00246] Loss of spatial resolution can occur when analytes migrate from the sample to the feature array and a component of diffusive migration occurs in the transverse (e.g., lateral) direction, approximately parallel to the surface of the first substrate on which the sample is mounted. To address this loss of resolution, in some embodiments, a permeabilization agent deposited on or infused into a material with anisotropic diffusion can be applied to the sample or to the feature array. The first and second substrates are aligned by the sample holder and brought into contact. A permeabilization layer that includes a permeabilization solution infused into an anisotropic material is positioned on the second substrate. [00247] In some embodiments, the feature array can be constructed atop a hydrogel layer infused with a permeabilization agent. The hydrogel layer can be mounted on the second substrate, or alternatively, the hydrogel layer itself may function as the second substrate. When the first and second substrates are aligned, the permeabilization agent diffuses out of the hydrogel layer and through or around the feature array to reach the sample. Analytes from the sample migrate to the feature array. Direct contact between the feature array and the sample helps to reduce lateral diffusion of the analytes, mitigating spatial resolution loss that would occur if the diffusive path of the analytes was longer.

[00248] Spatial analysis workflows can include a sandwiching process described herein, e.g., a process as described in FIG. 9. In some embodiments, the workflow includes provision of the first substrate comprising the biological sample. In some embodiments, the workflow includes, mounting the biological sample onto the first substrate. In some embodiments where the biological sample is a tissue sample, the workflow includes sectioning of the tissue sample (e.g., cryostat sectioning). In some embodiments, the workflow includes a fixation step. In some instances, the fixation step can include fixation with methanol. In some instances, the fixation step includes formalin (e.g., 2% formalin).

[00249] In some embodiments, the biological sample on the first substrate is stained using any of the methods described herein. In some instances, the biological sample is imaged, capturing the stain pattern created during the stain step. In some instances, the biological sample then is destained prior to the sandwiching process.

[00250] The biological sample can be stained using known staining techniques, including, without limitation, Can-Grunwald, Giemsa, hematoxylin and eosin (H&E), hematoxylin, Jenner’s, Leishman, Masson’s trichrome, Papanicolaou, Romanowsky, silver, Sudan, Wright’s, and/or Periodic Acid Schiff (PAS) staining techniques. PAS staining is typically performed after formalin or acetone fixation. In some embodiments, the biological sample can be stained using a detectable label (e.g., radioisotopes, fluorophores, chemiluminescent compounds, bioluminescent compounds, and dyes) as described elsewhere herein. In some embodiments, a biological sample is stained using only one type of stain or one technique. In some embodiments, staining includes biological staining techniques such as H&E staining. In some embodiments, staining includes biological staining using hematoxylin. In some embodiments, staining includes identifying analytes using fluorescently-conjugated antibodies, e.g., by immunofluorescence. In some embodiments, a biological sample is stained using two or more different types of stains, or two or more different staining techniques. For example, a biological sample can be prepared by staining and imaging using one technique (e.g., H&E staining and brightfield imaging), followed by staining and imaging using another technique (e.g., IHC/IF staining and fluorescence microscopy) for the same biological sample. In some instances, a biological sample on the first substrate is stained.

[00251] In some instances, methods for immunofluorescence include a blocking step. The blocking step can include the use of blocking probes to decrease unspecific binding of the antibodies. The blocking step can optionally further include contacting the biological sample with a detergent. In some instances, the detergent can include Triton X-100™. The method can further include an antibody incubation step. In some embodiments, the antibody incubation step effects selective binding of the antibody to antigens of interest in the biological sample. In some embodiments, the antibody is conjugated to an oligonucleotide (e.g., an oligonucleotide-antibody conjugate as described herein). In some embodiments, the antibody is not conjugated to an oligonucleotide. In some embodiments, the method further comprises an antibody staining step. The antibody staining step can include a direct method of immunostaining in which a labelled antibody binds directly to the analyte being stained for. Alternatively, the antibody staining step can include an indirect method of immunostaining in which a first antibody binds to the analyte being stained for, and a second, labelled antibody binds to the first antibody. In some embodiments, the antibody staining step is performed prior to sandwich assembly. In some embodiments where an oligonucleotide-antibody conjugate is used in the antibody incubation step, the method does not comprise an antibody staining step.

[00252] In some instances, the methods include imaging the biological sample. In some instances, imaging occurs prior to sandwich assembly. In some instances, imaging occurs while the sandwich configuration is assembled. In some instances, imaging occurs during permeabilization of the biological sample. In some instances, images are captured using high resolution techniques (e.g., having 300 dots per square inch (dpi) or greater). For example, images can be captured using brightfield imaging (e.g., in the setting of hematoxylin or H&E stain), or using fluorescence microscopy to detect adhered labels. In some instances, high resolution images are captured temporally using e.g., confocal microscopy. In some instances, a low resolution image is captured. A low resolution image (e.g., images that are about 72dpi and normally have an RGB color setting) can be captured at any point of the workflow, including but not limited to staining, destaining, permeabilization, sandwich assembly, and migration of the analytes. In some instances, a low resolution image is taken during permeabilization of the biological sample. [00253] In some embodiments, the location of the one or more analytes in a biological sample are determined by immunofluorescence. In some embodiments, one or more detectable labels (e.g., fluorophore-labeled antibodies) bind to the one or more analytes that are captured (hybridized to) by a probe on the first slide and the location of the one or more analytes is determined by detecting the labels under suitable conditions. In some embodiments, one or more fluorophore-labeled antibodies are used to conjugate to a moiety that associates with a probe on the first slide or the analyte that is hybridized to the probe on the first slide. In some instances, the location(s) of the one or more analytes is determined by imaging the fluorophore-labeled antibodies when the fluorophores are excited by a light of a suitable wavelength. In some embodiments, the location of the one or more analytes in the biological sample is determined by correlating the immunofluorescence data to an image of the biological sample. In some instances, the tissue is imaged throughout the permeabilization step.

[00254] In some instances, the biological samples can be destained. In some instances, destaining occurs prior to permeabilization of the biological sample. By way of example only, H&E staining can be destained by washing the sample in HC1. In some instances, the hematoxylin of the H&E stain is destained by washing the sample in HC1. In some embodiments, destaining can include 1, 2, 3, or more washes in HC1. In some embodiments, destaining can include adding HC1 to a downstream solution (e.g., permeabilization solution).

[00255] Between any of the methods disclosed herein, the methods can include a wash step (e.g., with SSC (e.g., O. lx SSC)). Wash steps can be performed once or multiple times (e.g., lx, 2x, 3x, between steps disclosed herein). In some instances, wash steps are performed for about 10 seconds, about 15 seconds, about 20 seconds, about 30 seconds, or about a minute. In some instances, three washes occur for 20 seconds each. In some instances, the wash step occurs before staining the sample, after destaining the sample, before permeabilization the sample, after permeabilization the sample, or any combination thereof.

[00256] In some instances, after the sandwiching process the first substrate and the second substrate are separated (e.g., such that they are no longer aligned in a sandwich configuration, also referred to herein as opening the sandwich). In some embodiments, subsequent analysis (e.g., cDNA synthesis, library preparation, and sequences) can be performed on the captured analytes after the first substrate and the second substrate are separated.

[00257] In some embodiments, the process of transferring the ligation product or methylated- adaptor-containing nucleic acid from the first substrate to the second substrate is referred to interchangeably herein as a “sandwich process,” “sandwiching process,” or “sandwiching”. The sandwich process is further described in PCT Patent Application Publication No. WO 2020/123320, PCT/US2021/036788, and PCT/US2021/050931, which are incorporated by reference in its entirety.

[00258] (e) Use of Multiplexed Sandwich Makers

[00259] This disclosure also provides methods, compositions, devices, and systems for using a single capture probe-containing to detect analytes from different biological samples (e.g., tissues) on different slides using serial sandwich processes. Thus, only one capture probe-containing array is necessary for the methods disclosed herein. In this way, as described herein, analytes from different samples or tissues can be captured serially and demultiplexed by sample-specific index sequences.

[00260] The methods include generating a connected probe (e.g., a ligation product) in multiple biological samples (i.e.., a first sample, a second sample, a third sample, etc.). Generation of a connected probe (e.g., a ligation product) has been described above, and the same methods are used herein to generate a connected probe (e.g., a ligation product) from analytes that are either protein analytes or nucleic acid (i.e., mRNA) analytes. That is, in some instances, the multiplexed sandwich maker methods disclosed herein can be used to detect protein analytes. In other instances, the multiplexed sandwich maker methods disclosed herein can be used to detect nucleic acid (i.e., mRNA) analytes.

[00261] The methods, compositions, devices, and systems include utilizing an analyte capture agent in multiple biological samples (e.g., a first sample, a second sample, a third sample, etc.). Using analyte capture agents for spatial detection has been described above, and the same methods are used herein to use an analyte capture agent to identify analytes in a biological sample. In some embodiments, the multiplexed sandwich maker methods disclosed herein can be used to detect protein analytes.

[00262] As discussed below, each connected probe (e.g., a ligation product) that is generated or analyte capture agent includes a sample index sequence, which is a nucleotide sequence that is associated with a particular sample of origin in the multiplex sandwich methods. After generation of each connected probe (e.g., a ligation product) or analyte capture agent, each sample is serially sandwiched to an array or slide having a plurality of capture probes that can detect and hybridize to a capture probe binding domain from the connected probe (e.g., a ligation product) or analyte capture agent. During the sandwiching process, the indexed connected probe or analyte capture agent actively or passively migrates from the sample to the array for capture by a capture probe.

Then the sandwich is opened, and the next sample is sandwiched with the array. In some embodiments, the array is washed prior to sandwiching with the next sample. Additional samples or tissues (e.g., 2 or more) can then be sandwiched with the array or slide having a plurality of capture probes, where connected probes (e.g., ligation products) or analyte capture agents from the additional samples or tissues can be transferred to the array in a similar manner. Because each sample includes a unique sample index, the sample of origin for each connected probe (e.g., a ligation product) or analyte capture agent that is captured on the array can be identified. In addition, the location of the connected probe (e.g., a ligation product) can be identified. In some embodiments, the location of the analyte capture agent can be identified. In some instances, the location is identified using fiducial markers on the gene expression slide (e.g., array) so that location of the ligation probe on the array mirrors the location of the sample on the sample slide. Exemplary fiducial markers are described in PCT Patent Application Publication No. WO 2020/123320, which is incorporated by reference in its entirety.

[00263] Such methods, compositions, devices, and systems allow for detection of analytes in multiple samples using only one gene expression slide and can be performed on any slide sample described herein. For example, in some instances, the biological sample is a formalin-fixed, paraffin-embedded (FFPE) sample, a frozen sample, or a fresh sample. In some instances, the biological sample is contacted with one or more stains. In some instances, the one or more stains include hematoxylin and eosin. In some instances, cell markers are detected using methods known in the art (e.g., using one or more optical labels such as fluorescent labels, radioactive labels, chemiluminescent labels, calorimetric labels, or colorimetric labels. In some instances, the biological sample is imaged before generating a connected probe (e.g., a ligation product) and before transferring the connected probe (e.g., a ligation product) to the gene expression slide.

[00264] The multiplex sandwich methods, compositions, devices, and systems described herein allow for detection of different types of samples and different analytes. For example in some instances, the samples used in the multiplex sandwich methods are from different species. In some instances, the samples used in the multiplex sandwich methods are from the same species but different individuals in the same species. In some instances, the samples used in the multiplex sandwich methods are from the same individual organism. In some instances, the samples are from different tissues or cell types. In some instances, the samples are from the same tissues or cell types. In some instances, the samples are from the same subject taken at different time points (e.g., before and after treatment). It is appreciated that the samples can be from any source so long as ligated products having sample index sequences unique to each sample are generated. [00265] Multiple samples can be used in the methods described herein. For example, in some instances, at least two samples are used. In some instances, more than two samples (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, or more) samples are used in the methods disclosed herein. It is appreciated that each sample can be from different sources (e.g., different species, different organisms). In some embodiments, from each sample, the same gene is detected and identified. In some embodiments, for each sample, different genes are detected and identified.

[00266] In order to differentiate one sample from another, probe oligonucleotide for each sample in a multiplexed setting can include one or more unique sequences to identify the origin of the connected probe (e.g., a ligation product). In some instances, the unique sequence is a sample index sequence. In some instances, probe oligonucleotides for each sample include one or more (e.g., at least 1, 2, 3, 4, 5, or more) unique sample index sequence to identify the origin of the connected probe (e.g., a ligation product).

[00267] In some instances, the sample index is about 5 nucleotides to about 50 nucleotides long (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides long. In some embodiments, the sample index is about 5-15 nucleotides long. In some embodiments, the sample index is about 10-12 nucleotides long. Both synthetic and/or naturally-occurring nucleotides can be used to generate a sample index sequence. It is appreciated that any sequence can be designed so long as it is unique among other sample index sequences and optionally that it can be distinguished from any sequence in the genome of the sample.

[00268] A sample index sequence can be located anywhere on the connected probe (e.g., a ligation product) so long as it does not affect (1) hybridization of the probe oligonucleotides to the analyte, (2) ligation of the probe oligonucleotides to generate the connected probe (e.g, a ligation product), and (3) hybridization of the capture probe binding domain to the capture probe on an array. For example, in some instances, the sample index sequence can be located on the first probe oligonucleotide (e.g, the left hand probe; i.e., as shown in FIG. 6A-B). In some instances, the sample index is located on the flap of the first probe oligonucleotide that does not hybridize to the analyte. In some instances, the sample index sequence can be located on the second probe oligonucleotide (e.g., the right hand probe; i.e., as shown in FIG. 6A-B). In some instances, the sample index is located on the flap of the second probe oligonucleotide that does not hybridize to the analyte.

[00269] (f) Computer Systems for Normalization [00270] FIG. 10 illustrates a block diagram illustrating an exemplary, non-limiting system 1000 for quantifying spatial analyte data for a plurality of analytes of a first species in accordance with some embodiments. In some embodiments, the system 1000 includes one or more processing units CPU(s) 1002 (also referred to as processors), one or more network interfaces 1004, a user interface 1006, a memory 1012, and one or more communication buses 1014 for interconnecting these components. The communication buses 1014 optionally include circuitry (sometimes called a chipset) that interconnects and controls communications between system components. The memory 1012 typically includes high-speed random access memory, such as DRAM, SRAM, DDR RAM, ROM, EEPROM, flash memory, CD-ROM, digital versatile disks (DVD) or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, other random access solid state memory devices, or any other medium which can be used to store desired information; and optionally includes non-volatile memory, such as one or more magnetic disk storage devices, optical disk storage devices, flash memory devices, or other non-volatile solid state storage devices. The memory 1012 optionally includes one or more storage devices remotely located from the CPU(s) 1002. The memory 1012, or alternatively the non-volatile memory device(s) within the memory 1012, comprises a non- transitory computer readable storage medium. It will be appreciated that this memory 1012 can be distributed across one or more computers. In some implementations, the memory 1012 or alternatively the non-transitory computer readable storage medium stores the following programs, modules and data structures, or a subset thereof:

• an optional operating system 1016 that includes procedures for handling various basic system services and for performing hardware dependent tasks;

• an optional network communication module (or instructions) 1018 for connecting the system 1000 with other devices, or a communication network;

• a normalization module 1020 for obtaining normalized counts for respective analyte capture agents;

• a feature data construct 1021 comprising electronic representation of one or more features 1022 (e.g., 1022-1, 1022-2, ...1022-M) (e.g., spatially barcoded features) on a substrate, a respective electronic representation of a feature 1022 including: o respective raw counts 1024 of one or more respective analyte capture agents whose oligonucleotide hybridized to the respective feature 1022. For example, feature 1 includes a raw count 1024-1- 1 of analyte capture agent 1-1, . . ., and a raw count 1024-1-Q of analyte capture agent 1-Q, where Q is a positive integer of 2 or greater. Feature 2 includes a raw count 1024-2- 1 of analyte capture agent 2- 1, and a raw count 1024-2-Q of analyte capture agent 2-Q, where Q is a positive integer of 2 or greater; o respective normalized counts 1026 of the one or more respective analyte capture agents whose oligonucleotide hybridized to the respective feature 1022. For example, feature 1 includes a normalized count 1026-1-1 of analyte capture agent 1-1, . . ., a normalized count 1026 -1-Q of analyte capture agent 1-Q, where Q is a positive integer of 2 or greater. Feature 2 includes a normalized count 1026-1- 1 of analyte capture agent 2-1, . . ., a normalized count 1026-2-Q of analyte capture agent 2-Q, where Q is a positive integer of 2 or greater; and o a respective spatial barcode 1028 (e.g., 1028-1, 1028-2, . . . 1028-M), where M is a positive integer of 2 or greater, that spatially identifies the spatially barcoded feature on the substrate.

[00271] In some implementations, the user interface 1006 includes an input device (e.g., a keyboard, a mouse, a touchpad, a track pad, and/or a touch screen) 1010 for a user to interact with the system 1000 and a display 1008.

[00272] In some implementations, one or more of the above identified elements are stored in one or more of the previously mentioned memory devices and correspond to a set of instructions for performing a function described above. The above identified modules or programs (e.g., sets of instructions) need not be implemented as separate software programs, procedures or modules, and thus various subsets of these modules may be combined or otherwise re-arranged in various implementations. In some implementations, the memory 1012 optionally stores a subset of the modules and data structures identified above. Furthermore, in some embodiments, the memory stores additional modules and data structures not described above. In some embodiments, one or more of the above identified elements is stored in a computer system, other than that of system 1000, that is addressable by system 1000 so that system 1000 may retrieve all or a portion of such data when needed.

[00273] Although FIG. 10 shows an exemplary system 1000, the figure is intended more as functional description of the various features that may be present in computer systems than as a structural schematic of the implementations described herein. In practice, and as recognized by those of ordinary skill in the art, items shown separately could be combined and some items could be separated. [00274] (g) Methods for Normalization

[00275] FIGS. 11 A, 11B, 11C, 11D, HE, HF, 11G, HH, HI, HJ, 11K, and HL collectively illustrate a method 1100 for quantifying spatial analyte data for a plurality of analytes, in accordance with some embodiments. In some embodiment the plurality of analytes is in a biological sample. In some embodiments the biological sample is of a first species (e.g., human, dog, cat, mouse, rat, etc.). In some embodiments, the biological sample is of mammal, a bird, a reptile, an amphibian, a fish, an insect, an arachnid, a mollusk, a crustacean, or a plant.

[00276] In some embodiments, the plurality of analytes comprises five or more analytes, ten or more analytes, fifty or more analytes, one hundred or more analytes, five hundred or more analytes, 1000 or more analytes, 2000 or more analytes, or between 2000 and 100,000 analytes.

[00277] In some embodiments, the plurality of analytes comprises DNA, RNA, proteins, or a combination thereof. For instance, in some embodiments, each respective analyte in the plurality of analytes is the same type of analyte. In some embodiments, the plurality of analytes includes at least an analyte of a first type (e.g., nucleic acids, such as DNA or RNA ) and an analyte of a second type (e.g., protein). In some embodiments, the plurality of analytes comprises a plurality of analyte types (e.g., RNA and protein, RNA and DNA, DNA and protein, or a combination of RNA, DNA, and protein).

[00278] In some embodiments, the plurality of analytes comprises at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 2000, at least 3000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 20,000, at least 30,000, at least 40,000, at least 50,000, at least 100,000, at least 200,000, or at least 300,000 analytes. In some embodiments, the plurality of analytes comprises no more than 500,000, no more than 200,000, no more than 100,000, no more than 80,000, no more than 50,000, no more than 30,000, no more than 20,000, no more than 10,000, no more than 5000, no more than 3000, no more than 2000, no more than 1000, no more than 500, no more than 100, or no more than 50 analytes. In some embodiments, the plurality of analytes comprises between 5 and 2000, between 1000 and 100,000, between 2000 and 10,000, between 5000 and 50,000, between 50 and 5000, or between 100 and 10,000 analytes. In some embodiments, the plurality of analytes falls within another range starting no lower than 5 analytes and ending no higher than 500,000 analytes.

[00279] Referring to block 1102, in some embodiments, the method 1100 includes exposing a plurality of analyte capture agents to a biological sample from a subject that is a member of the first species. In some embodiments the first species is human. In some embodiments the first species is a mammalian species. In some embodiments the first species is a plant species. In some embodiments, the biological sample is of mammal, a bird, a reptile, an amphibian, a fish, an insect, an arachnid, a mollusk, a crustacean, or a plant.

[00281] Each respective analyte capture agent in the first and second subsets of the plurality of analyte capture agents comprises a corresponding analyte binding moiety and a corresponding oligonucleotide comprising an analyte binding moiety barcode (e.g., a barcode that identifies that analyte binding moiety (e.g., an antibody)) and a capture handle sequence as disclosed herein.

[00282] In some embodiments, the biological sample is a sectioned tissue sample having a depth of 500 microns or less. In some embodiments, the biological sample is a sectioned tissue sample having a depth of 100 microns or less. In some embodiments, the sectioned tissue sample has a depth of 80 microns or less, 70 microns or less, 60 microns or less, 50 microns or less, 40 microns or less, 25 microns or less, 20 microns or less, 15 microns or less, 12 microns or less, 10 microns or less, 5 microns or less, 2 microns or less, or 1 micron or less. In some embodiments, the biological sample is a sectioned tissue sample having a depth of at least 0.1 microns, at least 1 micron, at least 5 microns, at least 10 microns, at least 12 microns, at least 15 microns, at least 20 microns, at least 30 microns, at least 50 microns, or at least 80 microns. In some embodiments, the sectioned tissue sample has a depth of between 10 microns and 20 microns, between 10 microns and 15 microns, between 1 and 10 microns, between 0.1 and 5 microns, between 20 and 100 microns, between 1 and 50 microns, or between 0.5 and 10 microns. In some embodiments, the sectioned tissue sample falls within a range starting no lower than 0.1 microns and ending no higher than 500 microns.

[00283] Referring to block 1104, in some embodiments, the biological sample is a fixed tissue sample. Example details of suitable fixed tissue samples in accordance with some embodiments of the present disclosure are disclosed in section (c) “Capturing Analytes for Spatial Detection using Analyte Capture Agents.”

[00284] Referring to block 1106, in some embodiments, the fixed tissue sample is a formalin fixed paraffin embedded (FFPE) tissue sample. Example details of suitable formalin fixed tissue samples in accordance with some embodiments of the present disclosure are disclosed in section (c) “Capturing Analytes for Spatial Detection using Analyte Capture Agents.”

[00285] Referring to block 1108, in some embodiments, the biological sample is a fresh frozen tissue sample.

[00286] Referring to block 1110, in some embodiments, each analyte in the first subset of analytes comprises a protein. Referring to block 1112, in some such embodiments, the protein is an intracellular or extracellular protein, optionally a cell surface protein. Suitable cell surface proteins include transmembrane proteins.

[00287] Referring to block 1114, in some embodiments, the first subset of the plurality of analyte capture agents comprises two, three, four, five, six, seven, eight, or nine or more antibodies (including antigen binding fragments thereof) that each bind to a different antigen selected from the group consisting of alphaSMA, PanCK, EPCAM, CD31, PDL1, PD1, PCNA, BCL2, Vimentin (VIM), CD3 (e.g., CD3E), CD4, CD8A, CXCR5, CD45RA, CD45RO, CD40, CD19, CD20, PAX5, CD27, CCR7, CD21, CDl lc, CD68, CD163, CD14, CDl lb, HLA-DR, CD 16, CD66B, and CD 138, optionally where the antigen is a human antigen.

[00288] In some embodiments, the first subset of the plurality of analyte capture agents comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40 or more analyte capture agents each containing an antibody (including antigen binding fragments thereof) that each bind an antigen that is not selected from the group consisting of alphaSMA, PanCK, EPCAM, CD31, PDL1, PD1, PCNA, BCL2, Vimentin (VIM), CD3 (e g, CD3E), CD4, CD8A, CXCR5, CD45RA, CD45RO, CD40, CD19, CD20, PAX5, CD27, CCR7, CD21, CDl lc, CD68, CD163, CD14, CDl lb, HLA-DR, CD 16, CD66B, and CD 138. In some embodiments, none of the analyte capture agents in the first subset of the plurality of analyte capture agents bind an antigen in the group consisting of alphaSMA, PanCK, EPCAM, CD31, PDL1, PD1, PCNA, BCL2, Vimentin, CD3, CD4, CD8A, CXCR5, CD45RA, CD45RO, CD40, CD19, CD20, PAX5, CD27, CCR7, CD21, CDl lc, CD68, CD163, CD14, CDllb, HLA-DR, CD16, CD66B, and CD138.

[00289] Continuing to FIG. 11B, referring to block 1116, in some embodiments, the first subset of the plurality of analyte capture agents comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 antibodies that each bind to a different antigen selected from the group consisting of alphaSMA, PanCK, EPCAM, CD31, PDL1, PD1, PCNA, BCL2, Vimentin (VIM), CD3 (e.g., CD3E), CD4, CD8A, CXCR5, CD45RA, CD45RO, CD40, CD19, CD20, PAX5, CD27, CCR7, CD21, CDl lc, CD68, CD163, CD14, CDl lb, HLA-DR, CD 16, CD66B, and CD 138, optionally where the antigen is a human antigen.

[00290] FIG. 12A illustrates exemplary analyte binding moieties of the second subset of the plurality of analyte capture agents in accordance with one embodiment of the present disclosure (subset 1250). FIG. 12A and FIG. 12B collectively illustrate exemplary analyte binding moieties of the first subset of the plurality of analyte capture agents and the respective analytes (e.g., proteins) bound thereby in accordance with one embodiment of the present disclosure (subset 1252). In some embodiments, the second subset of the plurality of analyte capture agents includes any 1, 2, 3, or all 4 of the analyte binding moieties of subset 1250. In some embodiments, the first subset of the plurality of analyte capture agents includes any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or all 31 of the analyte binding moieties of subset 1252.

[00293] Referring to block 1120, in some embodiments, the method includes aligning the biological sample with a first substrate comprising an array, such that at least a portion of the biological sample is aligned with the array. Suitable alignment methods are disclosed herein as well as in PCT Patent Publication No. W02022/140028 Al entitled “METHODS, COMPOSITIONS, AND SYSTEMS FOR CAPTURING PROBES AND/OR BARCODES”, published June 30, 2022 start here ; and (ii) PCT Patent publication No. W02023/044071 Al, entitled “Systems and Methods for Image Registration or Alignment” published March 23, 2023.

[00294] In some embodiments, the array comprises at least 100, 200, 500, 1000, 2000, 3000, 4000, or 5000 spatially barcoded features. Further disclosure of example arrays is disclosed in the section entitled “Introduction” above. In some embodiments an “array” refers to a specific arrangement of a plurality of spatially barcoded features, where the arrangement is either irregular or forms a regular pattern. Individual features in the array differ from one another based on their relative spatial locations. In general, at least two of the features in the array include a distinct capture probe (e.g., any of the examples of capture probes described herein). More disclosure on example arrays used in accordance with some embodiments of the present disclosure are disclosed in PCT Patent Publication No. W02023/044071, entitled “Systems and Methods for Image Registration or Alignment” published March 23, 2023.

[00295] Each spatially barcoded feature comprises a respective capture probe plurality. Each capture probe in the respective capture probe plurality comprises: (i) a respective spatial barcode, in a plurality of spatial barcodes, that spatially identifies the spatially barcoded feature on the first substrate and (ii) a capture domain sequence. More disclosure on capture probes, and their spatial barcodes and capture domain sequences is provided in the section above entitled “Introduction.”

[00296] In FIG. 11C, referring to block 1122, in some embodiments, for each respective analyte capture agent in the first subset of the plurality of analyte capture agents, the analyte binding moiety of the respective analyte capture agent comprises an antibody that reacts with a respective analyte of the first subset of the plurality of analytes. [00297] In some embodiments, the corresponding antibody is conjugated to the corresponding oligonucleotide of the respective analyte capture agent. The oligonucleotide of the respective analyte capture agent is also referred to herein as an analyte-binding moiety barcode domain 408. FIG. 4 illustrates an analyte capture agent 402 comprised of an analyte-binding moiety 404 conjugated to a corresponding oligonucleotide (analyte-binding moiety barcode domain 408). As discussed in the section entitled “Introduction” in conjunction with FIG. 4, the analyte-binding moiety barcode domain (oligonucleotide) 408 can comprise an analyte binding moiety barcode and a capture handle sequence. In more detail, FIG. 5 illustrates how the analyte binding moiety 522 is conjugated by linker 520 to an analyte binding moiety barcode 516 and capture handle sequence 514. In FIG. 5, the oligonucleotide conjugated to the analyte binding moiety 522 of the analyte capture agent 526 comprises the linker 520, an optional functional sequence 518, analyte binding moiety barcode 516, and capture handle sequence 514.

[00298] Referring to FIG. 5, in some embodiments, the corresponding capture handle sequence 514 of the corresponding oligonucleotide comprises a nucleic acid sequence that is substantially complementary to the capture domain sequence (in particular the capture domain sequence of capture domain 512) of a capture probe 524 in a respective capture probe plurality.

[00299] For a second analyte capture agent in the second subset of the plurality of analyte capture agents, the analyte binding moiety of the second analyte capture agent comprises a control antibody reactive to an antigen of a second species. The second species is other than the first species. In some embodiments, the control antibody is conjugated to the oligonucleotide of the second analyte capture agent. The capture handle sequence of the oligonucleotide of the second analyte capture agent comprises a nucleic acid sequence that is substantially complementary to the capture domain sequence of a capture probe in a respective capture probe plurality.

[00300] Referring to block 1124, in some embodiments, each analyte binding moiety of each respective analyte capture agent in the first subset of the plurality of analyte capture agents comprises an IgA, IgD, IgE, IgG, or IgM antibody (or a fragment thereof).

[00302] Referring to block 1128, in some embodiments, the first species is human and the second subset of the plurality of analyte capture agents collectively comprises analyte capture agents comprising control antibodies reactive to a non-human species, for example, an immunoglobulin from mouse, an immunoglobulin from rat, an immunoglobulin from rabbit, an immunoglobulin from dog, or a combination thereof. In some embodiments, the first species is human and the second subset of the plurality of analyte capture agents comprises a first analyte capture agent comprising an analyte binding moiety that was raised on an antigen from a second species, and a second analyte capture agent that was raised on an antigen from a third species, where the second species and the third species are not human and are different from each other. In some such embodiments, the second species is a mammalian. In some such embodiments, both the second species and the third species are mammalian.

[00303] In some embodiments, the first species is not human. For example, in some embodiments, the first species is selected from mouse, rat, hamster, rabbit, dog, cat, sheep, goat, or horse, while the second species is selected from mouse, rat, hamster, rabbit, dog, cat, sheep, goat, or horse, but is mutually exclusive to the first species.

[00304] Referring to block 1130, in some embodiments, the first species is human and the second subset of the plurality of analyte capture agents comprises a first analyte capture agent comprising a first control antibody reactive to mouse IgG2a, a second analyte capture agent comprising a second control antibody reactive to mouse IgGlk, a third analyte capture agent comprising a third control antibody reactive to mouse IgG2bk, and a fourth analyte capture agent comprising a fourth control antibody reactive to rat IgG2a.

[00305] In FIG. HE, referring to block 1132, in some embodiments, the capture domain sequence of one or more capture probes in one or more capture probe pluralities comprises a poly(T) sequence. In some embodiments the poly(T) sequence consists of between two and one hundred contiguous thymine residues. In some embodiments the poly(T) sequence comprises between two and one hundred contiguous thymine residues. In some embodiments the poly(T) sequence consists of between three and fifty contiguous thymine residues. In some embodiments the poly(T) sequence comprises between three and fifty contiguous thymine residues. In some embodiments the poly(T) sequence consists of between three and twenty -five contiguous thymine residues. In some embodiments the poly(T) sequence comprises between three and twenty-five contiguous thymine residues.

[00306] Referring to block 1134, and with reference to FIG. 5, in some embodiments, the capture probe 524 in one or more capture probe pluralities further comprises one or more functional domains (e.g., functional sequence 506 of FIG. 5), a unique molecular identifier (UMI), a cleavage domain 504, or a combination thereof.

[00307] Referring to block 1136, in some embodiments, a respective capture probe plurality includes 10 or more capture probes, 100 or more capture probes, 500 or more capture probes, 1000 or more capture probes, 2000 or more capture probes, 10,000 or more capture probes, 100,000 or more capture probes, 1 x 10⁶ or more capture probes, 2 x 10⁶ or more capture probes, or 5 x 10⁶ or more capture probes.

[00308] Referring to block 1138, in some embodiments, each capture probe in a respective capture probe plurality includes the same spatial barcode from the plurality of spatial barcodes. In other words, in some embodiments there is only one unique spatial barcode for a particular feature, each capture probe in the feature has this spatial barcode, and no capture probe in any other feature in the array has this spatial barcode.

[00309] In some embodiments, each capture probe in a respective capture probe plurality collectively includes two spatial barcodes from the plurality of spatial barcodes. In other words, in some embodiments there are two unique spatial barcodes for a particular feature, each capture probe in the feature has one of these two spatial barcodes, and no capture probe in any other feature in the array has either of these spatial barcodes.

[00310] In some embodiments, each capture probe in a respective capture probe plurality collectively includes M spatial barcodes from the plurality of spatial barcodes, where M is 3, 4, 5, 6, 7, 8, 9, 10 or some other positive integer. In other words, in some embodiments there are M unique spatial barcodes for a particular feature, each capture probe in the feature has one of these M spatial barcodes, and no capture probe in any other feature in the array has any of the M spatial barcodes. [00311] Referring to block 1140, in some embodiments, each respective spatially barcoded feature in the array is contained within a 100 micron by 100 micron square on the first substrate. Referring to block 1142, in some embodiments, a distance between a center of each respective spatially barcoded feature to a neighboring spatially barcoded feature in the array on the first substrate is between 10 microns and 100 microns. With continued reference to FIG. 11F, referring to block 1144, in some embodiments, a shape of each spatially barcoded feature in the array on the first substrate is a closed-form shape. Referring to block 1146, in some embodiments, the closed-form shape is circular, elliptical, or an N-gon, where N is a value between 1 and 20. Referring to block 1148 in some embodiments, the closed-form shape is circular and each spatially barcoded feature in the array has a diameter of 80 microns or less. Referring to block 1150, in some embodiments, the closed-form shape is circular and each spatially barcoded feature in the array has a diameter of between 8 microns and 40 microns. Referring to block 1152, in some embodiments, a distance between a center of each respective spatially barcoded feature to a neighboring spatially barcoded feature in the array on the first substrate is between 5 microns and 50 microns.

[00312] In some embodiments, each respective spatially barcoded feature in the array is contained within a 10 micron by 10 micron square on a substrate. In some embodiments, the distance between a center of each respective spatially barcoded feature to a neighboring spatially barcoded feature in the array is between 2 microns and 10 microns. In some embodiments, the distance between a center of each respective spatially barcoded feature to a neighboring spatially barcoded feature in the array is between 4 microns and 8 microns. In some embodiments, the shape of each feature in the array is a closed-form shape. In some embodiments, the closed-form shape is circular and each feature in the array has a width of between 3 microns and 7 microns. In some embodiments, the closed-form shape is square and each feature in the array has a width of between 6 microns and 10 microns. In some embodiments, a feature is not visible by a human without magnification.

[00313] Further non-limiting embodiments relating to features (interchangeably referred to as capture spots), including but not limited to beads, bead arrays, bead properties (e.g., structure, materials, construction, cross-linking, degradation, reagents, and/or optical properties), synthesis and conjugation of capture probes to beads, and for covalently and non-covalently bonding beads to substrates are described in U.S. Patent Publication No. US20210062282, U.S. Patent Publication No. 20110059865A1, U.S. Patent No. 9,012,022, PCT Publication No.

202020176788 Al; U.S. Patent No. 11,501,440; U.S. Patent Publication No. US 2021-0150707; U.S. Patent No. 11,514,575; U.S. Patent Publication No. 2021-0123040; U.S. Patent Publication No. 20220389409; and U.S. Patent Publication No. US 2021-0155982 each of which is hereby incorporated herein by reference in its entirety. In a specific embodiment, a plurality of barcoded features as described herein includes a plurality of barcoded beads. The plurality of barcoded beads can be disposed on a substrate in a form of a monolayer, where each barcoded bead in the plurality of barcoded beads occupies a distinct position on the substrate.

[00314] Referring to block 1154, in some embodiments, the method includes hybridizing, while the at least a portion of the biological sample is aligned with the array, (i) the oligonucleotide of each analyte capture agent in the first subset of the plurality of analyte capture agents, which specifically bound the first subset of analytes, to the capture domain sequence of a capture probe in a respective capture probe plurality; and (ii) the oligonucleotide of each analyte capture agent in the second subset of the plurality of analyte capture agents, which non- specifically associated with the biological sample, to the capture domain sequence of a capture probe in a respective capture probe plurality.

[00315] In FIG. 11G, referring to block 1156, in some embodiments, the biological sample is mounted on a second substrate. Optionally, during the aligning (block 1120) and/or the hybridizing (block 1154), a separation distance is maintained between the first substrate and the second substrate. Referring to block 1158, in some embodiments, the separation distance is less than 50 microns as measured in a direction orthogonal to the surface of the second substrate. In some embodiments, the separation distance is between 1 micron and 25 microns or between 2 microns and 10 microns as measured in a direction orthogonal to the surface of the second substrate. In some embodiments, there is no separation distance between the biological sample and the array. FIG. 9 illustrates this separation distance with the annotation gap 905.

[00316] Referring to block 1160, in some embodiments, during the hybridizing (block 1154), the first substate and the second substrate are coplanar.

[00319] Referring to block 1164, in some embodiments, the respective raw count of the respective analyte capture agent comprises a UMI count. The UMI provides one way for block 1162 to reliably determine raw counts (e.g., which capture probe has bound to an oligonucleotide from a respective analyte capture agent 526). Referring to FIG. 1 and FIG. 5 as a guide, in some such embodiments, the disclosed methodology gives rise to a unique sequenced oligonucleotide (not shown in FIG. 1 or FIG. 5) each time an analyte capture agent 526 binds to a capture probe 102 / 524 on the array. In some embodiments, this unique sequenced oligonucleotide includes the analyte binding moiety barcode 516 from the analyte capture agent 526, as well as the UMI sequence 106 / 510 and the spatial barcode 105 / 508 from the coupled capture probe 524. Detection of the unique sequenced oligonucleotide having this combination of a particular analyte binding moiety barcode 516, a particular UMI 106 / 510 and a particular spatial barcode 105 / 508 means that a particular analyte capture agent (one that has the particular analyte binding moiety barcode 516) has been captured by the capture domain sequence of a particular capture probe in a capture probe plurality (identified by the spatial barcode 105 /508 that uniquely identifies that capture probe plurality and the UMI 510 which uniquely identifies a particular capture probe in that capture probe plurality) for a particular spatially barcoded feature. In this instance, each capture probe 524 in a particular capture probe plurality has the same spatial barcode 105 / 508. In this way, the same capture probe is not counted twice (for example in instances where two oligonucleotides are sequenced that have the same binding moiety barcode 516, same UMI 106 / 510 and a particular spatial barcode 105 / 508, only one count is attributed since the two oligonucleotides both represent the same capture probe / analyte capture agent binding event.

[00320] In response to detecting this sequence, block 1164 increments the raw count for the corresponding capture probe for the corresponding spatially barcoded feature by a single count. If 37 oligonucleotides having this unique combination of UMI 510 and binding moiety barcode 516 are sequenced, the raw count for the analyte capture agent 526 associated with the binding moiety barcode 516 is assigned a raw count of 37 for the spatially barcoded feature associated with UMI 510.

[00321] Blocks 1222 and 1224 further illustrate how polynucleotides are generated and sequences that contain both an analyte binding moiety barcode of a first analyte capture agent and a sequence of a first spatial barcode in the plurality of spatial barcodes.

[00324] In some embodiments, a constant factor is used, as discussed above. In some embodiments, the constant factor is a value greater than 1. In some embodiments, the constant factor is a value between 5 and 100,000. In some embodiments, the constant factor is a value between 10 and 10,000. In some embodiments, the constant factor is a value between 100 and 2,000.

[00326] Referring to block 1170, in some embodiments, the measure of central tendency is a mean, median, mode, a weighted mean, weighted median, or weighted mode of the raw count of the one or more (e.g., two or more) analyte capture agents in the second subset of the plurality of analyte capture agents in the respective spatially barcoded feature.

[00327] Referring to block 1172, in some embodiments, the raw count of one or more analyte capture agents in the second subset of the plurality of analyte capture agents consists of a raw count of between 2 capture agents and 20 capture agents in the second subset of the plurality of analyte capture agents. In some embodiments, the raw count of one or more analyte capture agents in the second subset of the plurality of analyte capture agents consists of a raw count of two or more, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 capture agents in the second subset of the plurality of analyte capture agents.

[00328] In FIG. Ill, referring to block 1174, in some embodiments, the first subset of analytes are proteins and the plurality of analytes further comprises a second subset of analytes that are nucleic acids. Some such embodiments support interrogating a biological sample using both antibodies that bind to particular antigens (see, for example, section (c) entitled “Capturing Analytes for Spatial Detection using Analyte Capture Agents”, above), and probes that detect transcription products (e.g., in the form or mRNA) (see section (b) “Capturing Nucleic Acid Analytes using RNA-Templated Ligation”, above) .

[00329] Referring to block 1176, in some embodiments, the method further comprises contacting a first probe and a second probe to the biological sample under conditions suitable for hybridization of the first probe and the second probe to an analyte in the second subset of analytes. The first probe comprises a sequence that is substantially complementary to a first portion of a sequence of the analyte. The second probe comprises a sequence that is substantially complementary to a second portion of a sequence of the analyte. The second or the first probe comprises a sequence that is complementary to the capture domain sequence of the capture probe in a respective capture probe plurality. See, for example, section (b) “Capturing Nucleic Acid Analytes using RNA-Templated Ligation”, above.

[00330] Referring to block 1178, in some embodiments, the first portion of the sequence of the analyte and the second portion of the sequence of the analyte are a contiguous nucleic acid sequence of the analyte. Referring to block 1180, in some embodiments, the first portion of the sequence of the analyte and the second portion of the sequence of the analyte are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides away from one another in the analyte. See, for example, section (b) “Capturing Nucleic Acid Analytes using RNA-Templated Ligation” above, for more disclosure on such embodiments.

[00331] Referring to block 1182, in some embodiments, the biological sample is contacted with a set of about 5-100,000 probes, where the set comprises the first probe and the second probe.

[00332] With continued reference to FIG. 11J, referring to block 1184, the method further comprises coupling the first probe and the second probe, thereby generating a connected probe, and hybridizing the connected probe to the capture domain sequence of one or more of the capture probes in one or more capture probe pluralities in the array. See, for example, section (b) “Capturing Nucleic Acid Analytes using RNA-Templated Ligation” above, for more disclosure on such embodiments.

[00333] Referring to block 1186, in some embodiments, the coupling the first probe and the second probe comprises ligating, via a ligase, the first probe and the second probe. Referring to block 1188, in some such embodiments, the ligase is selected from the group consisting of splintR ligase, a single stranded DNA ligase, and a T4 DNA ligase. See, for example, section (b) “Capturing Nucleic Acid Analytes using RNA-Templated Ligation” above, for more disclosure on such embodiments.

[00334] Referring to block 1190, in some embodiments, the method further comprises treating the biological sample with a reagent medium comprising a permeabilization agent, thereby permeabilizing the biological sample. Referring to block 1198, in some such embodiments, the permeabilization agent comprises a protease. Referring to block 1200, in some such embodiments, the protease is selected from trypsin, pepsin, elastase, or proteinase K. See, for example, section (b) “Capturing Nucleic Acid Analytes using RNA-Templated Ligation” above, for more disclosure on such embodiments. [00335] Referring to block 1192, in some embodiments, the method further comprises treating the biological sample with a releasing agent, thereby releasing the connected probe from the biological sample. Referring to block 1194, in some such embodiments, the releasing agent comprises a nuclease. Referring to block 1196, in some such embodiments, the nuclease comprises an RNase, optionally where the RNase is selected from RNase A, RNase C, RNase H, or RNase I. See, for example, section (b) “Capturing Nucleic Acid Analytes using RNA- Templated Ligation” above, for more disclosure on such embodiments.

[00336] Referring to block 1202, in some embodiments, the reagent medium further comprises a detergent. See, for example, section (b) “Capturing Nucleic Acid Analytes using RNA- Templated Ligation”, above for more disclosure on such embodiments.

[00337] In some instances of nucleic acid analysis, the first and second probe oligonucleotides hybridize to genomic DNA (gDNA) which can lead to Unique Molecular Identifier (UMI) counts from probe ligation events on gDNA (off target) in addition to the desired (on target) probe ligation events on a target nucleic acid e.g., mRNA. Thus, in some embodiments, the method further comprises treating the biological sample with a deoxyribonuclease (DNase). The DNase can be an endonuclease or exonuclease. In some embodiments, the DNase digests single- and/or double-stranded DNA. Suitable DNases include, without limitation, a DNase I and a DNase II.

[00338] Referring to block 1208, in some embodiments, the method further comprises overlaying the histological image onto a second image comprising a normalized count for at least one analyte capture agent in the first subset of the plurality of analyte capture agents for each respective spatially barcoded feature in the array. Example 1 illustrates the benefits of the disclosed normalized process. Methods for taking histological images are disclosed in PCT Publication No. W02023/044071, which is hereby incorporated by reference. Thus, in such embodiments, the histological image and the second image are put in the same frame of reference with respect to the biological sample. This allows for the observer to notice any spatial correlations between features in the histological image and features in the second image. In the context of an image, a feature is some region of high or low intensity. Correlations in such spatial features signifies some association between features observable in histological images and the spatial abundance of the antigen or antigens associated with the analyte capture agents whose normalized counts were used to create the second image.

[00339] In terms of the second image, consider the case where there are 1000 spatially barcoded features and the normalized count of analyte capture agent “A” in each of these spatially barcoded features is used to form the second image. In this instance, the second image comprises an array of values, where each element in the array of values has (i) the coordinates of the location of the corresponding spatially barcoded feature in the array of spatially barcoded features and (ii) the normalized count of the analyte capture agent “A” in the corresponding spatially barcoded feature.

[00340] In some embodiments, for the second image, the normalized count of each analyte capture agent in the first subset of analyte capture agents is summed together to provide a total count for each of the spatially barcoded features and these total count values are used to form the second image. In this instance, the second image comprises an array of values, where each element in the array of values has (i) the coordinates of the location of the corresponding spatially barcoded feature in the array of spatially barcoded features and (ii) the summed normalized count of each of the analyte capture agent in the first subset of analyte capture agents for the corresponding spatially barcoded feature. In some such embodiments the histological image is overlayed onto this second image. In other embodiments the histological image is optional. In other embodiments the histological image is not generated.

[00342] In some embodiments, an image based on a normalized count of one analyte capture agent in the first subset of analyte capture agents or a raw count of one analyte capture agent in the second subset of analyte capture agents is generated and observed without determining a histological image of the biological sample.

[00343] Referring to block 1210, in some embodiments where a histological image is generated, the histological image comprises 10,000 or more pixel values. In some embodiments where the second image is generated, the second image comprises 10,000 or more pixel values.

In some embodiments, the overlaying between the histological image and the second image takes into consideration an alignment of at least one percent of the pixels in the histological image with corresponding pixels in the second image.

[00344] Referring to block 1212, in some embodiments, the histological image comprises 100,000 or more pixel values while the second image comprises 100,000 or more pixel values. In some such embodiments, the overlaying takes into consideration an alignment of at least one percent of the pixels in the histological image with corresponding pixels in the second image.

[00345] Referring to block 1214, in some embodiments, the histological image comprises 500,000 or more pixel values and the second image comprises 500,000 or more pixel values. In some such embodiments, the overlaying takes into consideration an alignment of at least one percent of the pixels in the histological image with corresponding pixels in the second image.

[00346] In some embodiments, the histological image comprises 250,000, 750,000, 1 x 10⁶, 2 x 10⁶, 3 x 10⁶, 4 x 10⁶ or more pixel values and the second image comprises 250,000, 750,000, 1 x 10⁶, 2 x 10⁶, 3 x 10⁶, 4 x 10⁶ or more pixel values. In some such embodiments, the overlaying takes into consideration an alignment of at least one percent, two percent, three percent, four percent, five percent, six percent, seven percent, eight percent, nine percent, or ten percent of the pixels in the histological image with corresponding pixels in the second image.

[00347] In some embodiments, the histological image comprises 250,000, 750,000, 1 x 10⁶, 2 x 10⁶, 3 x 10⁶, 4 x 10⁶ or more pixel values and the second image comprises 250,000, 750,000, 1 x 10⁶, 2 x 10⁶, 3 x 10⁶, 4 x 10⁶ or more pixel values. In some such embodiments, the overlaying takes into consideration an alignment of at least 5 percent, 10 percent, 15 percent, 20 percent, 25 percent, 30 percent, 35 percent, or 40 percent of the pixels in the histological image with corresponding pixels in the second image.

[00348] Referring to block 1206, in some embodiments where a histological image of the biological sample is taken, any of the methods for taking histological images disclosed in PCT Publication No. W02023/044071, which is hereby incorporated by reference, is used. [00349] In some embodiments, a histological image generally refers to any image that contains structural information for a biological sample and/or a biological tissue. In some embodiments, a histological image is obtained using any detectable marker such as an antibody, a fluorescent label (e.g., a fluorophore), a radioactive label, a chemiluminescent label, a calorimetric label, a colorimetric label, a stain and/or a combination thereof. For instance, in some embodiments, the biological sample is prepared for taking a histological image using a stain selected from the group consisting of: live/dead stain, trypan blue, periodic acid-Schiff reaction stain, Masson’s trichrome, Alcian blue, van Gieson, reticulin, Azan, Giemsa, Toluidine blue, isamin blue, Sudan black and osmium, acridine orange, Bismarck brown, carmine, Coomassie blue, cresyl violet, DAPI, eosin, ethidium bromide, acid fuchsine, hematoxylin, Hoechst stains, iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red, osmium tetroxide, propidium iodide, rhodamine, safranin, or a combination thereof.

[00350] For instance, in some embodiments, a histological image is acquired using transmission light microscopy (e.g., bright field transmission light microscopy, dark field transmission light microscopy, oblique illumination transmission light microscopy, dispersion staining transmission light microscopy, phase contrast transmission light microscopy, differential interference contrast transmission light microscopy, emission imaging, etc.). See, for example, Methods in Molecular Biology, 2018, Light Microscopy Method and Protocols, Markaki and Harz eds., Humana Press, New York, New York, ISBN-13: 978-1493983056, which is hereby incorporated by reference.

[00351] In some embodiments, a histological image is a bright-field microscopy image in which the imaged biological sample appears dark on a bright background. In some such embodiments, the biological sample has been stained. For instance, in some embodiments, the biological sample has been stained with Hematoxylin and Eosin and the histological image is a bright-field microscopy image. In some embodiments the biological sample has been stained with a Periodic acid-Schiff reaction stain (stains carbohydrates and carbohydrate rich macromolecules a deep red color) and the histological image is a bright-field microscopy image. In some embodiments the biological sample has been stained with a Masson’s tri chrome stain (nuclei and other basophilic structures are stained blue, cytoplasm, muscle, erythrocytes and keratin are stained bright-red, collagen is stained green or blue, depending on which variant of the technique is used) and the histological image is a bright-field microscopy image. In some embodiments, the biological sample has been stained with an Alcian blue stain (a mucin stain that stains certain types of mucin blue, and stains cartilage blue and can be used with H&E, and with van Gieson stains) and the histological image is a bright-field microscopy image. In some embodiments the biological sample has been stained with a van Gieson stain (stains collagen red, nuclei blue, and erythrocytes and cytoplasm yellow, and can be combined with an elastin stain that stains elastin blue/black) and the histological image is a bright-field microscopy image. In some embodiments the biological sample has been stained with a reticulin stain, an Azan stain, a Giemsa stain, a Toluidine blue stain, an isamin blue/eosin stain, a Nissl and methylene blue stain, and/or a Sudan black and osmium stain and the histological image is a bright-field microscopy image.

[00352] In some embodiments, rather than being a bright-field microscopy image of a sample, the histological image is an immunohistochemistry (IHC) image. IHC imaging may utilize a staining technique using antibody labels. One form of immunohistochemistry (IHC) imaging is immunofluorescence (IF) imaging. In an example of IF imaging, primary antibodies are used that specifically label a protein in the biological sample, and then a fluorescently labelled secondary antibody or other form of probe is used to bind to the primary antibody, to show up where the first (primary) antibody has bound. A light microscope, equipped with fluorescence, is used to visualize the staining. The fluorescent label is excited at one wavelength of light and emits light at a different wavelength. Using the right combination of filters, the staining pattern produced by the emitted fluorescent light is observed. In some embodiments, a biological sample is exposed to several different primary antibodies (or other forms of probes) in order to quantify several different proteins in a biological sample. In some such embodiments, each such respective different primary antibody (or probe) is then visualized with a different fluorescence label (different channel) that fluoresces at a unique wavelength or wavelength range (relative to the other fluorescence labels used). In this way, several different proteins in the biological sample can be visualized.

[00353] More generally, in some embodiments of the present disclosure, in addition to bright- field imaging or instead of bright-field imaging, fluorescence imaging is used to acquire a respective image of the biological sample. As used herein the term “fluorescence imaging” refers to imaging that relies on the excitation and re-emission of light by fluorophores, regardless of whether they are added experimentally to the sample and bound to antibodies (or other compounds) or naturally occurring features of the sample. The above-described IHC imaging, and in particular IF imaging, is just one form of fluorescence imaging.

[00354] In some such embodiments, a histological image is acquired using confocal microscopy, two-photon imaging, wide-field multiphoton microscopy, single plane illumination microscopy or light sheet fluorescence microscopy. See, for example, Adaptive Optics for Biological Imaging, 2013, Kubby ed., CRC Press, Boca Raton, Florida; and Confocal and Two-Photon Microscopy: Foundations, Applications and Advances, 2002, Diaspro ed., Wiley Liss, New York, New York; and Handbook of Biological Confocal Microscopy, 2002, Pawley ed., Springer Science+Business Media, LLC, New York, New York each of which is hereby incorporated by reference.

[00355] In some embodiments, a histological image is obtained using various immunohistochemistry (IHC) probes that excite at various different wavelengths. See, for example, Day and Davidson, 2014, “The Fluorescent Protein Revolution (In Cellular and Clinical Imaging),” CRC Press, Taylor & Francis Group, Boca Raton, Florida; “Quantitative Imaging in Cell Biology” Methods in Cell Biology 123, 2014, Wilson and Tran, eds.; Advanced Fluorescence Reporters in Chemistry and Biology IL Molecular Constructions, Polymers and Nanoparticles (Springer Series on Fluorescence), 2010, Demchenko, ed., Springer-Verlag, Berlin, Germany; Fluorescence Spectroscopy and Microscopy: Methods and Protocols (Methods in Molecular Biology) 2014th Edition, 2014, Engelborghs and Visser, eds., HumanPress, each of which is hereby incorporated by reference for their disclosure on fluorescence imaging.

[00356] An image can be obtained in any electronic image file format, including but not limited to JPEG/JFIF, TIFF, Exif, PDF, EPS, GIF, BMP, PNG, PPM, PGM, PBM, PNM, WebP, HDR raster formats, HEIF, BAT, BPG, DEEP, DRW, ECW, FITS, FLIF, ICO, ILBM, IMG, PAM, PCX, PGF, JPEG XR, Layered Image File Format, PLBM, SGI, SID, CD5, CPT, PSD, PSP, XCF, PDN, CGM, SVG, PostScript, PCT, WMF, EMF, SWF, XAML, and/or RAW.

[00357] In some embodiments, a histological image is obtained in any electronic color mode, including but not limited to grayscale, bitmap, indexed, RGB, CMYK, HSV, lab color, duotone, and/or multichannel. In some embodiments, the image is manipulated (e.g., stitched, compressed and/or flattened). In some embodiments, the histological image is a color image (e.g., 3 x 8 bit, 2424 x 2424 pixel resolution). In some embodiments, the histological is a monochrome image (e.g., 14 bit, 2424 x 2424 pixel resolution).

[00358] In some embodiments, a histological image comprises a plurality of pixels. In some embodiments, the plurality of pixels comprises at least 100, at least 500, at least 1000, at least 5000, at least 10,000, at least 50,000, at least 100,000, at least 500,000, at least 1 x 10⁶, at least 2 x 10⁶, at least 3 x 10⁶, at least 5 x 10⁶, at least 8 x 10⁶, at least 1 x 10⁷, at least 1 x 10⁸, at least 1 x 10⁹, at least 1 x 10¹⁰, or at least 1 x 10¹¹ pixels. In some embodiments, the plurality of pixels comprises no more than 1 x 10¹², no more than 1 x 10¹¹, no more than 1 x 10¹⁰, no more than 1 x 10⁹, no more than 1 x 10⁸, no more than 1 x 10⁷, no more than 1 x 10⁶ , no more than 100,000, no more than 10,000, or no more than 1000 pixels. In some embodiments, the plurality of pixels comprises from 1000 to 100,000, from 10,000 to 500,000, from 100,000 to 1 x 10⁶, from 500,000 to 1 x 10⁹, or from 1 x 10⁶ to 1 x 10⁸ pixels. In some embodiments, the plurality of pixels falls within another range starting no lower than 100 pixels and ending no higher than 1 x 10¹² pixels.

[00359] In some embodiments, each pixel in the plurality of pixels of the histological image has a pixel size (resolution) between 0.8pm and 4.0pm. In some embodiments this pixel size is derived by dividing the camera pixel size (resolution) by the magnification of the objective lens of the camera used to capture values for the plurality of pixels. In some embodiments, each pixel in the plurality of pixels has a pixel size between 0.4pm and 5.0pm. In some embodiments, each pixel in the plurality of pixels of the first image has a pixel size (resolution) between 0.8pm and 4.0pm or between 0.4pm and 5.0pm.

[00360] In some embodiments, a histological image comprises a plurality of pixels, such that the location of each respective pixel in the plurality of pixels in the array (e.g., matrix) corresponds to its original location in the image. In some embodiments, a histological image is represented as a vector comprising a plurality of pixels, such that each respective pixel in the plurality of pixels in the vector comprises spatial information corresponding to its original location in the image.

[00362] In some embodiments, a histological image has an image size between 1 KB and 1 MB, between 1 MB and 0.5 GB, between 0.5 GB and 5 GB, between 5 GB and 10 GB, or greater than 10 GB. [00363] In some embodiments, a histological image is obtained using an image capture device, such as a microscope.

[00364] Referring to block 1216 in FIG. 11L, in some embodiments, the method further comprises using the normalized count for each respective analyte capture agent in the first subset of the plurality of analyte capture agents and the count for each respective analyte capture agent in the second subset of the plurality of analyte capture agents for each respective spatially barcoded feature in the array to determine whether or not the subject has a condition.

[00365] For instance, in some embodiments, any of the spatial analyte data acquired using the disclosed methods (e.g., normalized count of each respective analyte capture agent in the first subset of the plurality of analyte capture agents, images created using these counts, or alignment of images created using these counts to histological images) is used to determine whether or not the subject has a disease or a stage of a disease. In some embodiments, the spatial analyte data is used to determine a probability or likelihood that the subject has a disease or a stage of a disease. In some embodiments, it is known that the subject has a disease and the spatial analyte data is used to determine a prognosis for the subject regarding the disease. In some embodiments, the disease is cancer or diabetes. In some embodiments, the disease is a cancer, hematologic disorder, autoimmune disease, inflammatory disease, immunological disorder, metabolic disorder, neurological disorder, genetic disorder, psychiatric disorder, gastroenterological disorder, renal disorder, cardiovascular disorder, dermatological disorder, respiratory disorder, or a viral infection.

[00366] Referring to block 1218, in some embodiments, the method further comprises providing a treatment to the subject when it is determined that the subject has the condition. Referring to block 1220, in some such embodiments, the treatment comprises a composition comprising a small molecule compound and one or more excipient and/or one or more pharmaceutically acceptable carrier and/or one or more diluent. These include all conventional solvents, dispersion media, fillers, solid carriers, coatings, antifungal and antibacterial agents, dermal penetration agents, surfactants, isotonic and absorption agents and the like.

[00367] An exemplary carrier is pharmaceutically “acceptable” in the sense of being compatible with the other ingredients of the composition (e.g., the composition comprising the modified polymer) and not injurious to the patient. The compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier that constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product.

[00368] Exemplary compounds, compositions, or combinations of the present disclosure (e.g., the composition comprising the modified polymer) formulated for intravenous, intramuscular, or intraperitoneal administration, and a compound of the invention or a pharmaceutically acceptable salt, solvate or prodrug thereof may be administered by injection or infusion.

[00369] Referring to block 1222, in some embodiments, the method further comprises sequencing a first polynucleotide comprising (i) a sequence of an analyte binding moiety barcode of a first analyte capture agent in the plurality of analyte capture agents or a complement thereof, and (ii) a sequence of a first spatial barcode in the plurality of spatial barcodes, or a complement thereof.

[00370] Referring to block 1224, in some embodiments, the method further comprises generating the first polynucleotide. The generating comprises extending the capture probe in the respective capture probe plurality having the first spatial barcode using the oligonucleotide of the first analyte capture agent as a template.

[00371] Examples

[00372] EXAMPLE 1 - NORMALIZATION

[00373] Example 1 illustrates the benefits of the disclosed normalization process.

[00374] FIG. 14 illustrates an unnormalized image. In FIG. 14 a second image 1402 is overlaid on a histological image 1404 of the same biological sample. Each circle in the second image represents a respective spatially barcoded feature in the array. The color of each respective circle in the array represents a total raw count of a particular analyte capture agent whose oligonucleotide hybridized to a capture probe of the respective spatially barcoded feature in accordance with the color code to the right of the image. The data shows presence of a few regions that have disproportionately high or low expression of the protein bound by the analyte capture agent.

[00375] FIGS. 15A and 15B are exemplary data showing the spatial distribution of antibodies on the same tissue section before and after normalization in accordance with the present disclosure, respectively. FIG. 15A shows that before normalization in accordance with the present disclosure, the tissue section appears to include a region 1502 of relatively high protein expression, as depicted by the high-intensity (e.g., yellow- and green-colored) spots. FIG. 15B shows that after normalization, the region 1502 that were observed in FIG. 15A vanishes and another region 1504 of relatively high protein expression is observed on an edge of the tissue section. Absent the normalization process of the present disclosure, the region 1504 would have been masked, and an artifact (e.g., 1502) would have been assessed as genuine biological observation.

[00376] FIG. 16 illustrates an exemplary normalization pathway that utilizes the unique molecular identifier (UMI) counts of four control antibodies to reduce artifacts in the data, in accordance with some embodiments of the present disclosure.

[00377] FIGS. 17A and 17B illustrate cross-correlation data of analyte capture agents (e.g., oligonucleotide-conjugated antibodies) before and after normalization, respectively. FIG. 17A shows spurious correlations before normalization. FIG. 17B shows an improvement in the crosscorrelation data after normalization.

[00378] FIG. 18 illustrates that the normalization process described in the present disclosure aids the detection of biological structures.

[00379] FIGS. 19A, 19B, and 19C collectively illustrate screenshots of a user interface that provides interactive visualization functionality to analyze data in accordance with some embodiments of the present disclosure. FIG. 19A illustrates the user interface 1900. The user interface 1900 displays an initial screen 1902 that allows a user to see clustering identified by the application. Such clustering techniques are disclosed in United States Patent Publication No. US 2021-0062272 Al. FIG. 19B illustrates a control antibody normalization screen 1904. In some embodiments, panel 1904 represents the default when analyzing data that includes antibodies. FIG. 19C illustrates user interaction with a slider bar 1906, which allows a user to “gate” their data and identify area(s) of interest in the biological sample.

REFERENCES CITED AND ALTERNATIVE EMBODIMENTS

[00380] All publications, patents, patent applications, and information available on the internet and mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, patent application, or item of information was specifically and individually indicated to be incorporated by reference. To the extent publications, patents, patent applications, and items of information incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material. [00381] The present invention can be implemented as a computer program product that comprises a computer program mechanism embedded in a non-transitory computer readable storage medium. For instance, the computer program product could contain the program modules shown in FIG. 10, and/or described in FIGS. 11 A, 11B, 11C, 11D, HE, HF, 11G, 11H, HI, 11 J, 11K, and HL. These program modules can be stored on a CD-ROM, DVD, magnetic disk storage product, USB key, or any other non-transitory computer readable data or program storage product.

[00382] As used herein, the terms “about” or “approximately” refer to an acceptable error range for a particular value as determined by one of ordinary skill in the art, which can depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. “About” can mean a range of ±20%, ±10%, ±5%, or ±1% of a given value. The term “about” or “approximately” can mean within an order of magnitude, within 5-fold, or within 2- fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed. The term “about” can have the meaning as commonly understood by one of ordinary skill in the art. The term “about” can refer to ±10%. The term “about” can refer to ±5%.

[00383] Where values are described in terms of ranges, it should be understood that the description includes the disclosure of all possible sub-ranges within such ranges, as well as specific numerical values that fall within such ranges irrespective of whether a specific numerical value or specific sub-range is expressly stated. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. As used herein, the term “between” used in a range is intended to include the recited endpoints. For example, a number “between X and Y” can be X, Y, or any value from X to Y.

[00384] The term “each,” when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection, unless expressly stated otherwise, or unless the context of the usage clearly indicates otherwise. [00385] As used herein, the singular forms “a,” “an,” and “the” include the plural forms as well, unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only,” and the like in connection with the recitation of claim elements or use of a “negative” limitation. Furthermore, to the extent that the terms “including,” “includes,” “having,” “has,” “with,” or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[00386] Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only. The embodiments were chosen and described in order to best explain the principles of the invention and its practical applications, to thereby enable others skilled in the art to best utilize the invention and various embodiments with various modifications as are suited to the particular use contemplated. The invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

Claims

What is claimed:

1. A method for quantifying spatial analyte data for a plurality of analytes of a first species, the method comprising:

(a) exposing a plurality of analyte capture agents to a biological sample from a subject that is a member of the first species under conditions that (i) cause at least a first subset of the plurality of analyte capture agents to specifically bind to a first subset of analytes in the plurality of analytes in the biological sample, and (ii) cause at least a second subset of the plurality of analyte capture agents to non-specifically associate with the biological sample, wherein each respective analyte capture agent in the first and second subsets of the plurality of analyte capture agents comprises a corresponding analyte binding moiety and a corresponding oligonucleotide comprising an analyte binding moiety barcode and a capture handle sequence;

(b) aligning the biological sample with a first substrate comprising an array, such that at least a portion of the biological sample is aligned with the array, wherein the array comprises at least 1000 spatially barcoded features, and wherein each spatially barcoded feature comprises a respective capture probe plurality, wherein each capture probe in the respective capture probe plurality comprises: (i) a respective spatial barcode, in a plurality of spatial barcodes, that spatially identifies the spatially barcoded feature on the first substrate and (ii) a capture domain sequence;

(c) hybridizing, while the at least a portion of the biological sample is aligned with the array, (i) the oligonucleotide of each analyte capture agent in the first subset of the plurality of analyte capture agents, which specifically bound the first subset of analytes, to the capture domain sequence of a capture probe in a respective capture probe plurality; and (ii) the oligonucleotide of each analyte capture agent in the second subset of the plurality of analyte capture agents, which non-specifically associated with the biological sample, to the capture domain sequence of a capture probe in a respective capture probe plurality; and

3. The method of claim 1, wherein for each respective analyte capture agent in the first subset of the plurality of analyte capture agents, the respective analyte binding moiety of the respective analyte capture agent comprises a corresponding antibody that reacts with a respective analyte of the first subset of the plurality of analytes, wherein the corresponding antibody is conjugated to the corresponding oligonucleotide of the respective analyte capture agent, and wherein the corresponding capture handle sequence of the corresponding oligonucleotide comprises a nucleic acid sequence that is substantially complementary to the capture domain sequence of a capture probe in a respective capture probe plurality, and for each respective analyte capture agent in the second subset of the plurality of analyte capture agents, the respective analyte binding moiety of the respective analyte capture agent comprises a corresponding control antibody reactive to an antigen of a species other than the first species, and wherein the corresponding control antibody is conjugated to the oligonucleotide of the respective analyte capture agent, and wherein the corresponding capture handle sequence of the corresponding oligonucleotide comprises a nucleic acid sequence that is complementary to a capture domain sequence of the capture probe in a respective capture probe plurality.

4. The method of claim 2 or 3, wherein each analyte binding moiety of each respective analyte capture agent in the first subset of the plurality of analyte capture agents comprises an IgA, IgD, IgE, IgG, or IgM antibody.

5. The method of claim 3, wherein the first species is human and the second subset of the plurality of analyte capture agents collectively comprises analyte capture agents comprising control antibodies reactive to an immunoglobulin from mouse, an immunoglobulin from rat, an immunoglobulin from rabbit, an immunoglobulin from dog, or a combination thereof.

6. The method of claim 5, wherein the first species is human and the second subset of the plurality of analyte capture agents comprises a first analyte capture agent comprising a first control antibody reactive to mouse IgG2a, a second analyte capture agent comprising a second control antibody reactive to mouse IgGlk, a third analyte capture agent comprising a third control antibody reactive to mouse IgG2bk, and a fourth analyte capture agent comprising a fourth control antibody reactive to rat IgG2a.

7. The method of any one of claims 1-6, wherein the first subset of the plurality of analyte capture agents comprises five or more antibodies that each react with a different antigen selected from the group consisting of alphaSMA, PanCK, EPCAM, CD31, PDL1, PD1, PCNA, BCL2, Vimentin, CD3, CD4, CD8A, CXCR5, CD45RA, CD45RO, CD40, CD19, CD20, PAX5, CD27, CCR7, CD21, CDl lc, CD68, CD163, CD14, CDl lb, HLA-DR, CD16, CD66B, and CD138, optionally wherein the antigen is a human antigen.

8. The method of any one of claims 1-6, wherein the first subset of the plurality of analyte capture agents comprises ten or more antibodies that each react with a different antigen selected from the group consisting of alphaSMA, PanCK, EPCAM, CD31, PDL1, PD1, PCNA, BCL2, Vimentin, CD3, CD4, CD8A, CXCR5, CD45RA, CD45RO, CD40, CD19, CD20, PAX5, CD27, CCR7, CD21, CDl lc, CD68, CD163, CD14, CDl lb, HLA-DR, CD16, CD66B, and CD138, optionally wherein the antigen is a human antigen.

9. The method of any one of claims 1-8, wherein the first subset of analyte capture agents consists of between 10 and 500 analyte capture agents, and the second subset of analyte capture agents consists of between 2 and 25 analyte capture agents.

13. The method of claim 12, wherein the measure of central tendency is a mean, median, mode, a weighted mean, weighted median, or weighted mode of the raw count of the two or more analyte capture agents in the second subset of the plurality of analyte capture agents in the respective spatially barcoded feature.

14. The method of any one of claims 11-13, wherein the raw count of two or more analyte capture agents in the second subset of the plurality of analyte capture agents consists of a raw count of between 2 capture agents and 20 capture agents in the second subset of the plurality of analyte capture agents.

15. The method of any one of claims 1-13, wherein the first subset of analytes are proteins and the plurality of analytes further comprises a second subset of analytes that are nucleic acids, and wherein the method further comprises contacting a first probe and a second probe to the biological sample under conditions suitable for hybridization of the first probe and the second probe to an analyte in the second subset of analytes, wherein the first probe comprises a sequence that is substantially complementary to a first portion of a sequence of the analyte, the second probe comprises a sequence that is substantially complementary to a second portion of a sequence of the analyte, and the second or the first probe comprises a sequence that is complementary to the capture domain sequence of the capture probe in a respective capture probe plurality, and wherein the method further comprises: coupling the first probe and the second probe, thereby generating a connected probe, and hybridizing the connected probe to the capture domain sequence of one or more of the capture probes in one or more capture probe pluralities in the array.

16. The method of claim 15, wherein the first portion of the sequence of the analyte and the second portion of the sequence of the analyte are a contiguous nucleic acid sequence of the analyte.

17. The method of claim 15, wherein the first portion of the sequence of the analyte and the second portion of the sequence of the analyte are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides away from one another in the analyte.

18. The method of claim 15, wherein the coupling the first probe and the second probe comprises ligating, via a ligase, the first probe and the second probe.

19. The method of claim 18, wherein the ligase is selected from the group consisting of splintR ligase, a single stranded DNA ligase, and a T4 DNA ligase.

20. The method of any one of claims 15-19, further comprising treating the biological sample with a reagent medium comprising a permeabilization agent, thereby permeabilizing the biological sample.

21. The method of claim 20, wherein the method further comprises treating the biological sample with a releasing agent, thereby releasing the connected probe from the biological sample.

22. The method of claim 21, wherein the releasing agent comprises a nuclease.

23. The method of claim 22, wherein the nuclease comprises an RNase, optionally wherein the RNase is selected from RNase A, RNase C, RNase H, or RNase I.

24. The method of any one of claims 20-23, wherein the permeabilization agent comprises a protease.

25. The method of claim 24, wherein the protease is selected from the group consisting of trypsin, pepsin, elastase, and proteinase K.

26. The method of any one of claim 20-25, wherein the reagent medium further comprises a detergent.

27. The method of any one of claims 1-26, wherein the biological sample is mounted on a second substrate, optionally wherein during the aligning (b) and/or the hybridizing (c), a separation distance is maintained between the first substrate and the second substrate.

28. The method of claim 27, wherein the separation distance is less than 50 microns as measured in a direction orthogonal to the surface of the second substrate.

29. The method of claim 27 or 28, wherein during the hybridizing (c), the first substate and the second substrate are coplanar.

30. The method of any one of claims 1-29, wherein the capture domain sequence of one or more capture probes in one or more capture probe pluralities comprises a poly(T) sequence.

31. The method of any one of claims 1-30, wherein the capture probe in one or more capture probe pluralities further comprises one or more functional domains, a unique molecular identifier (UMI), a cleavage domain, or a combination thereof.

32. The method of any one of claims 1-31, wherein the biological sample is a fixed tissue sample.

33. The method of claim 32, wherein the fixed tissue sample is a formalin fixed paraffin embedded (FFPE) tissue sample.

34. The method of any one of claims 1-31, wherein the biological sample is a fresh frozen tissue sample.

35. The method of any one of claims 1-34, wherein a respective capture probe plurality includes 1000 or more capture probes, 2000 or more capture probes, 10,000 or more capture probes, 100,000 or more capture probes, 1 x 10⁶ or more capture probes, 2 x 10⁶ or more capture probes, or 5 x 10⁶ or more capture probes.

36. The method of claim 35, wherein each capture probe in a respective capture probe plurality includes the same spatial barcode from the plurality of spatial barcodes.

37. The method of any one of claims 1-36, the method further comprising taking a histological image of the biological sample.

38. The method of claim 37, the method further comprising overlaying the histological image onto a second image comprising a normalized count for at least one analyte capture agent in the first subset of the plurality of analyte capture agents for each respective spatially barcoded feature in the array.

39. The method of any one of claims 15-29, the method further comprising: taking a histological image of the biological sample; and overlaying the histological image onto a second image comprising a normalized count for at least one analyte capture agent in the second subset of the plurality of analyte capture agents for each respective spatially barcoded feature in the array.

40. The method of claim 38 or 39, wherein the histological image comprises 10,000 or more pixel values, the second image comprises 10,000 or more pixel values, and the overlaying takes into consideration an alignment of at least one percent of the pixels in the histological image with corresponding pixels in the second image.

41. The method of claim 38 or 39, wherein the histological image comprises 100,000 or more pixel values, the second image comprises 100,000 or more pixel values, and the overlaying takes into consideration an alignment of at least one percent of the pixels in the histological image with corresponding pixels in the second image.

42. The method of claim 38 or 39, wherein the histological image comprises 500,000 or more pixel values, the second image comprises 500,000 or more pixel values, and the overlaying takes into consideration an alignment of at least one percent of the pixels in the histological image with corresponding pixels in the second image.

43. The method of any one of claims 1-42, wherein each respective spatially barcoded feature in the array is contained within a 100 micron by 100 micron square on the first substrate.

44. The method of any one of claims 1-43, wherein a distance between a center of each respective spatially barcoded feature to a neighboring spatially barcoded feature in the array on the first substrate is between 10 microns and 100 microns.

45. The method of any one of claims 1-44, wherein a shape of each spatially barcoded feature in the array on the first substrate is a closed-form shape.

46. The method of claim 45, wherein the closed-form shape is circular, elliptical, or an N- gon, where N is a value between 1 and 20.

47. The method of claim 45, wherein the closed-form shape is circular and each spatially barcoded feature in the array has a diameter of 80 microns or less.

48. The method of claim 45, wherein the closed-form shape is circular and each spatially barcoded feature in the array has a diameter of between 8 microns and 40 microns.

49. The method of claim 48, wherein a distance between a center of each respective spatially barcoded feature to a neighboring spatially barcoded feature in the array on the first substrate is between 5 microns and 50 microns.

50. The method of any one of claims 1-49, the method further comprising using the normalized count for each respective analyte capture agent in the first subset of the plurality of analyte capture agents and the count for each respective analyte capture agent in the second subset of the plurality of analyte capture agents for each respective spatially barcoded feature in the array to determine whether or not the subject has a condition.

51. The method of any one of claims 1-50, wherein each analyte in the first subset of analytes comprises a protein.

52. The method of claim 51, wherein the protein is an intracellular or extracellular protein, optionally a cell surface protein.

53. The method of any one of claims 1-52, wherein the respective raw count of the respective analyte capture agent comprises a UMI count.

54. The method of any one of claims 15-53, wherein the method further comprises contacting the biological sample with a set of about 5-100,000 probes, wherein the set comprises the first probe and the second probe.

55. The method of any one of claims 1-54, wherein the method further comprises sequencing a first polynucleotide comprising (i) a sequence of an analyte binding moiety barcode of a first analyte capture agent in the plurality of analyte capture agents or a complement thereof, and (ii) a sequence of a first spatial barcode in the plurality of spatial barcodes, or a complement thereof.

56. The method of claim 55, the method further comprising generating the first polynucleotide, wherein the generating comprises extending the capture probe in the respective capture probe plurality having the first spatial barcode using the oligonucleotide of the first analyte capture agent as a template.

57. The method of claim 50, wherein the method further comprises providing a treatment of the subject when it is determined that the subject has the condition.

58. The method of claim 57, wherein the treatment comprises a composition comprising a small molecule compound and one or more excipient and/or one or more pharmaceutically acceptable carrier and/or one or more diluent.