WO2025096581A1

WO2025096581A1 - Methods of spatial analysis and single nuclei sequencing

Info

Publication number: WO2025096581A1
Application number: PCT/US2024/053617
Authority: WO
Inventors: Mohammadreza MIRZAZADEH; Zaneta ANDRUSIVOVA; Mengxiao HE; Leire ALONSO GALICIA; Marco Vicari; Joakim Lundeberg
Original assignee: 10X Genomics Inc
Current assignee: 10X Genomics Inc
Priority date: 2023-10-31
Filing date: 2024-10-30
Publication date: 2025-05-08
Anticipated expiration: 2026-04-30

Abstract

Provided herein are methods and compositions disclosing an approach that combines single nuclear RNA-seq (snRNA-seq) with spatial transcriptomics. The methods include processing and analyzing multiple analytes in a biological sample, and utilizes multiple substrates, one having a spatial array having a plurality of capture probes, each having a capture domain and a spatial barcode. The methods can be used to detect location of both nucleic acid and protein analytes. The methods also include isolating cells and/or nuclei from a sample and determining analyte expression in the isolated cells and/or nuclei.

Description

Attorney Docket No.: 47706-0398WO1 METHODS OF SPATIAL ANALYSIS AND SINGLE NUCLEI SEQUENCING CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No.63/594,760, filed October 31, 2023, which is herein incorporated by reference in its entirety. BACKGROUND Cells within a tissue of a subject 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. Spatial heterogeneity has been previously studied using techniques that only provide data for a small handful of analytes in the context of an intact tissue or a portion of a tissue, or provides substantial analyte data for dissociated tissue (i.e., single cells), but fail to provide information regarding the position of the single cell in a parent biological sample (e.g., tissue sample). Spatial transcriptomics has emerged as a powerful tool for studying cellular and molecular heterogeneity in tissues. Indeed, the field of spatial transcriptomics offers a range of approaches, varying in the number of transcripts that can be measured as well as resolution. Single cell RNA-seq (scRNA-seq) offers the most comprehensive high-resolution coverage of the transcriptome but does not retain the spatial context. This highlights the importance of developing strategies to allow for a more complete understanding of complex biological systems (Longo et al., Nature Reviews Genetics, 22, 627–644 (2021)). In addition, unlike scRNA-seq, single nuclear RNA-seq (snRNA-seq) allows transcriptomic analysis of tissues that have already been preserved while also being less prone to inducing changes in gene expression during cell extraction procedure as well as exhibiting lower bias towards isolation of specific cell types compared to scRNA-seq (Lacar et al. Nat Commun., 2016 Apr 19:7:11022) (Bakken et al., PLoS One, 2018 Dec 26;13(12):e0209648). Nevertheless, these benefits are accompanied by drawbacks, such as profiling fewer mRNA molecules resulting in a slight decrease in cluster separation between highly similar cell types. In order to combine spatial transcriptomics with single nucleus sequencing from the same tissue, current techniques require separate tissue sections, as well as larger quantities of Attorney Docket No.: 47706-0398WO1 input material for snRNA-seq. Such experimental setups can lead to considerable variations in cellular content and composition. This inconsistency is particularly significant in tumor samples, where cellular and interpatient heterogeneity is a defining characteristic of the disease, and not even neighboring sections may accurately represent the same cellular structures. Thus, there remains a need for methods to detect analytes for spatial transcriptomic, single-cell and/or single nuclear analyses, while avoiding such drawbacks. I. SUMMARY The present disclosure features methods of spatial analysis combined with single nuclei sequencing, all from the same biological sample. This is achieved using “SIMPlex- seq” (Single-section Integrated Multilayer Profiling in tissues, also referred to as combinatorial methods of spatial transcriptomics and single-nucleus sequence, or simply combinatorial methods), an approach that combines Single cell RNA-seq (scRNA-seq) with spatial transcriptomics. This method provides several advantages and opens new possibilities within the field. First, the methods disclosed herein require a reduced input material compared to previous methods. In particular, the methods demonstrate the feasibility of extracting sufficient amounts of intact nuclei from a single biological sample (e.g., a tissue section), which is typically only achievable by processing multiple or larger tissue samples. This enables a more efficient and streamlined workflow which minimizes required input material and thus preserving valuable samples. The ability to utilize only one or a few tissue samples (e.g., sections) would be particularly useful in instances where sample size is a limiting factor, such as precious clinical biopsy specimens. Second, the methods integrate single nuclei and spatial transcriptomics datasets and allow for enhanced accuracy in sample profiling. By utilizing a single tissue sample (e.g., section), the disclosed methods mitigate the discrepancies arising from variations in cellular content between adjacent sections. This ensures a more accurate representation of the cell types and states in the single nuclei dataset and therefore facilitates more comprehensive analysis of samples with highly dynamic environments such as tumor biopsies, allowing for deeper understanding of the complex interplay between various cell types and molecular pathways. Third, the methods provide flexibility and adaptability to additional methods. For example, the combinatorial methods provided herein can potentially incorporate other modalities such as proteomics (e.g., before the tissue is processed by the spatial transcriptomics protocol), making it a flexible platform that can evolve with the rapidly advancing field of -omics. As nuclei are extracted from the same tissue section (e.g., after spatial transcriptomics methods), the methods also facilitate Attorney Docket No.: 47706-0398WO1 the simultaneous assessment of multiple molecular modalities, such as single-nuclei Whole Genome Sequencing (snWGS). These methods facilitate the comprehensive exploration of tumor biology, revealing insights into gene expression, cellular interactions, and spatial organization. Thus, included herein is a method for processing multiple analytes in a biological sample mounted on a first substrate. In some instances, the method includes: (a) hybridizing a first probe and a second probe to a first analyte in the biological sample, wherein the first probe and the second probe each comprise a nucleic acid sequence that is substantially complementary to a nucleic acid sequence of the first analyte, and wherein the second probe comprises a capture probe binding domain; (b) coupling the first probe and the second probe, thereby generating a connected probe; (c) 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; (d) releasing the connected probe from the first analyte when at least a portion of the biological sample is aligned with at least a portion of the array; (e) hybridizing the connected probe to the capture domain of the capture probe in the array; (f) isolating one or more cells or nuclei from the biological sample on the first substrate, wherein the one or more cells or nuclei comprise a second analyte; and (g) hybridizing a nucleic acid barcode molecule to the second analyte, a complement thereof, or an intermediate agent of the second analyte. In another embodiment, disclosed herein is a method for processing multiple analytes in a biological sample mounted on a first substrate, the method comprising: (a) hybridizing a first probe and a second probe to a first analyte in the biological sample, wherein the first probe and the second probe each comprise a nucleic acid sequence that is substantially complementary to a nucleic acid sequence of the first analyte, and wherein the second probe comprises a capture probe binding domain; (b) coupling the first probe and the second probe, thereby generating a connected probe; (c) 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; (d) hybridizing the connected probe to the capture domain of the capture probe in the array; (e) isolating one or more cells or nuclei from the biological sample, wherein the one or more cells or nuclei comprise a second analyte; and (f) hybridizing a Attorney Docket No.: 47706-0398WO1 nucleic acid barcode molecule to the second analyte, a complement thereof, or an intermediate agent of the second analyte. In some instances, aligning the first substrate with a second substrate includes bringing a first surface of the first substrate including the biological sample and a second surface of the second substrate including the array within proximity of each other. Such proximity can be, but is not limited to, less than about 30 microns, less than about 25 microns, less than about 20 microns, less than about 15 microns, less than about 12 microns, less than about 10 microns, less than about 8 microns, less than about 5 microns, or less. In some embodiments, aligning the first substrate with a second substrate includes contacting a first surface of the first substrate including the biological sample with a second surface of the second substrate including the array. In some instances, the proximity or contact allows for migration of the connected probe to the array, e.g., for hybridization to the capture domain of the capture probe. In some instances, the methods also include separating the first substrate and the second substrate. In some instances, separating the first substrate and the second substrate occurs after hybridizing the connected probe to the capture domain of the capture probe in the array. In some instances, the methods also include determining (i) all or a part of a sequence of the connected probe corresponding to the first analyte, or a complement thereof, and (ii) the spatial barcode, or a complement thereof, and using the determined sequences of (i) and (ii) to determine a location of the first analyte in the biological sample. In some instances, determining (i) all or a part of a sequence of the connected probe corresponding to the first analyte, or a complement thereof, and (ii) the spatial barcode, or a complement thereof, comprises sequencing. In some instances, the methods also include determining presence and/or abundance of the second analyte from the one or more cells or nuclei isolated from the biological sample. In some instances, determining presence and/or abundance of the second analyte from the one or more cells or nuclei isolated from the biological sample comprises sequencing. In some instances, the first probe comprises a 5’ handle sequence, wherein the 5’ handle sequence comprises about 5 nucleotides to 50 nucleotides. In some instances, the second probe comprises a 3’ handle sequence, wherein the 3’ handle sequence comprises about 5 nucleotides to 50 nucleotides. In some instances, the 3’ handle sequence comprises a poly(A) sequence. In some instances, the poly(A) sequence is at a 3’ end of the second probe. In some instances, the first probe and the second probe hybridize to the nucleic acid sequence Attorney Docket No.: 47706-0398WO1 of the first analyte, wherein the nucleic acid sequence of the first analyte is about 25 to 100 nucleotides in length. In some instances, the first probe and/or the second probe comprises DNA. In some instances, the hybridizing the first probe and the second probe to the nucleic acid sequence of the first analyte comprises contacting the biological sample with 100 or more probe pairs comprising the first probe and the second probe. In some instances, the hybridizing the first probe and the second probe to the first analyte comprises contacting the biological sample with 5,000 or more probe pairs comprising the first probe and the second probe. In some instances, the first probe and the second probe hybridize to adjacent sequences of the first analyte. In some instances, the coupling the first probe to the second probe comprises use of a ligase selected from a PBCV-1 DNA ligase, a Chlorella virus DNA ligase, a single stranded DNA ligase, or a T4 DNA ligase. In some instances, the first probe and the second probe hybridize to sequences in the first analyte that are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides away from one another. In some instances, the methods include generating an extended first probe, wherein the extended first probe comprises a sequence complementary to a sequence between the sequence hybridized to the first probe and the sequence hybridized to the second probe. In some instances, the methods include ligating the extended first probe and the second probe using a ligase selected from a PBCV-1 DNA ligase, a Chlorella virus DNA ligase, a single stranded DNA ligase, or a T4 DNA ligase. In some instances, the methods include generating an extended second probe, wherein the extended second probe comprises a sequence complementary to a sequence between the sequence hybridized to the first probe and the sequence hybridized to the second probe. In some instances, the methods include ligating the first probe and the extended second probe using a ligase selected from a PBCV-1 DNA ligase, a Chlorella virus DNA ligase, a single stranded DNA ligase, or a T4 DNA ligase. In some instances, generating an extended first probe and/or generating an extended second probe includes use of a polymerase, e.g., a DNA polymerase. In some instances, releasing the connected probe from the first analyte comprises applying heat to the biological sample. In some instances, releasing the connected probe from the first analyte comprises applying an enzyme to the biological sample. In some instances, the enzyme is an endoribonuclease. In some instances, the endoribonuclease is one or more of RNase H, RNase A, RNase C, or RNase I. In some instances, the endoribonuclease is RNase H. Attorney Docket No.: 47706-0398WO1 In some instances, the RNase H comprises RNase H1, RNase H2, or both RNase H1 and RNase H2. In some instances, the methods also include contacting the biological sample with a reagent medium comprising a permeabilization agent. In some instances, the permeabilization agent comprises a protease. In some instances, the protease is selected from trypsin, pepsin, elastase, or proteinase K. In some instances, the protease is pepsin or proteinase K. In some instances, the reagent medium further comprises a detergent. In some instances, the detergent is selected from sodium dodecyl sulfate (SDS), sarkosyl, or saponin. In some instances, the reagent medium further comprises polyethylene glycol (PEG). In some instances, the methods include passively migrating the connected probe to the array (e.g., for hybridizing to the capture probe). In some instances, the methods include actively migrating the connected probe to the array (e.g., for hybridizing to the capture probe). In some instances, the capture probe further comprises one or more functional domains, a unique molecular identifier (UMI), a cleavage domain, or combinations thereof. In some instances, the one or more functional domains comprises a primer binding site. In some instances, the capture domain comprises a homopolymeric sequence. In some instances, the capture domain comprises a poly(T) sequence. In some instances, the methods include extending the capture probe using the connected probe as a template, thereby generating an extended capture probe; and/or extending the connected probe using the capture probe as a template, thereby generating an extended connected probe. In some instances, the methods include separating the extended capture probe from the connected probe. In some instances, the separating comprises use of potassium hydroxide. In some instances, the methods include amplifying all or part of the connected probe hybridized to the capture domain. In some instances, the first analyte and/or the second analyte comprises RNA. In some instances, the RNA is mRNA. In some instances, the first analyte and/or the second analyte comprises DNA. In some instances, the DNA is genomic DNA. In another embodiment, disclosed herein is a method for processing multiple analytes in a biological sample mounted on a first substrate, the method comprising: (a) 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 capture agent barcode domain comprises an analyte Attorney Docket No.: 47706-0398WO1 binding moiety barcode and a capture handle sequence, and wherein upon the contacting, the analyte binding moiety binds to a first analyte; (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, 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; (c) hybridizing the capture agent barcode domain from the analyte capture agent that is bound to the first analyte to the capture domain of the capture probe; (d) isolating one or more cells or nuclei from the biological sample on the first substrate, wherein the one or more cells or nuclei comprises a second analyte; and (e) hybridizing a nucleic acid barcode molecule to the second analyte a complement thereof or an intermediate agent. In some instances, when the biological sample is aligned with at least a portion of the array, the method includes releasing the capture agent barcode domain from the analyte capture agent that is bound to the first analyte. In some instances, aligning the first substrate with a second substrate comprising an array includes bringing a first surface of the first substrate including the biological sample and a second surface of the second substrate including the array within proximity of each other. Such proximity can be, but is not limited to, less than about 30 microns, less than about 25 microns, less than about 20 microns, less than about 15 microns, less than about 12 microns, less than about 10 microns, less than about 8 microns, less than about 5 microns, or less. In some embodiments, aligning the first substrate with a second substrate comprising an array includes contacting a first surface of the first substrate including the biological sample with a second surface of the second substrate including the array. In some instances, the proximity or contact allows for migration of the capture agent barcode domain to the array, e.g., for hybridization to the capture domain of the capture probe. In some instances, the methods include separating the first substrate and the second substrate. In some instances, separating the first substrate and the second substrate occurs after hybridizing the capture agent barcode domain to the capture domain of the array. In some instances, the methods also include determining (i) all or a part of a sequence of the capture agent barcode domain, or a complement thereof, and (ii) a sequence of the spatial barcode, or a complement thereof, and using the determined sequences of (i) and (ii) to determine a location of the first analyte in the biological sample. In some instances, determining (i) all or a part of a sequence of the capture agent barcode domain, or a complement thereof, and (ii) a sequence of the spatial barcode, or a complement thereof, comprises sequencing. Attorney Docket No.: 47706-0398WO1 In some instances, the methods also include determining presence and/or abundance of a second analyte from the one or more cells or nuclei isolated from the biological sample. In some instances, determining presence and/or abundance of a second analyte from the one or more cells or nuclei isolated from the biological sample comprises sequencing. In some instances, the methods further include extending the capture agent barcode domain using the capture probe as a template, thereby incorporating a complement of the spatial barcode to generate a spatially tagged capture agent barcode domain. In some instances, the capture handle sequence of the capture agent barcode domain is substantially complementary to the capture domain of the capture probe. In some instances, the analyte binding moiety barcode is associated with or identifies the analyte binding moiety. In some instances, the analyte binding moiety comprises an antibody or an antigen- binding fragment thereof. In some instances, the analyte capture agent comprises a linker that couples the capture agent barcode domain to the analyte binding moiety. In some instances, the linker is a cleavable linker. In some instances, the cleavable linker is a disulfide linker, a photo- cleavable linker, a UV-cleavable linker, or an enzyme cleavable linker. In some instances, the enzyme cleavable linker is an RNase cleavable linker. In some instances, the first analyte is a protein. In some instances, the protein is an intracellular or extracellular protein. In some instances, the aligning comprises: mounting the first substrate on a first member of a support device, the first member configured to retain the first substrate; and/or mounting the second substrate on a second member of the support device; and/or applying a reagent medium to the first substrate and/or the second substrate; and/or operating an alignment mechanism of the support device to move the first member and/or the second member such that at least a portion of the biological sample is aligned with at least a portion of the array, and such that the portion of the biological sample and the portion of the array contact the reagent medium. In some instances, the alignment mechanism is coupled to the first member, the second member, or both the first member and the second member. In some instances, the alignment mechanism comprises a linear actuator, optionally wherein: the linear actuator is configured to move the second member along an axis orthogonal to the first member and/or the second member, and/or the linear actuator is configured to move the first member along an axis orthogonal to a plane of the first member and/or the second member, and/or 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, and/or the linear Attorney Docket No.: 47706-0398WO1 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. In some instances, at least one of the first substrate and the second substrate further comprise a spacer disposed on the first substrate or the second substrate, wherein when at least the portion of the biological sample is aligned with at least a portion of the array such that the portion of the biological sample and the portion of the array contact the reagent medium, the spacer is disposed between the first substrate and the second substrate and is configured to maintain the reagent medium within a chamber formed by the first substrate, the second substrate, and the spacer, and to maintain a separation distance between the first substrate and the second substrate, wherein the spacer is positioned to surround an area on the first substrate on which the biological sample is disposed and/or the array disposed on the second substrate, wherein the area of the first substrate, the spacer, and the second substrate at least partially encloses a volume comprising the biological sample. In some instances, the methods include fixing the one or more cells or nuclei. In some instances, the one or more cells or nuclei are fixed in formaldehyde. In some instances, the one or more cells or nuclei are fixed in 4% formaldehyde. In some instances, the nucleic acid barcode molecule comprises one or more of a cell or nuclei barcode, a second unique molecule identifier, and a primer. In some instances, the intermediate agent is a second connected probe. In some instances, the intermediate agent is a second capture agent barcode domain from a second analyte capture agent comprising a second analyte binding moiety and the second capture agent barcode domain, wherein the second capture agent barcode domain comprises a second analyte binding moiety barcode and a second capture handle sequence. In some instances, the methods include generating a copy of the second analyte, a complement thereof, or the intermediate agent, or a complement thereof. In some instances, generating the copy of the second analyte, a complement thereof, or the intermediate agent, or a complement thereof, uses a polymerase or a reverse transcriptase. In some instances, the methods include hybridizing the nucleic acid barcode molecule to the complement of the second analyte, or the intermediate agent. In some instances, the nucleic acid barcode molecule comprises a hybridization region of a template switching oligonucleotide (TSO). In some instances, the hybridization region of the TSO comprises a poly(G) sequence and wherein the nucleic acid barcode molecule comprises a poly(C) sequence. In some instances, the methods include extending the complement of the second analyte or the intermediate agent using the nucleic acid barcode molecule as a template, thereby generating an extended nucleic acid barcode molecule. Attorney Docket No.: 47706-0398WO1 In some instances, the methods also include amplifying the extended nucleic acid barcode molecule. In some instances, determining the presence and/or abundance of the second analyte in the biological sample comprises determining (i) the sequence of the cell or nuclei barcode, or a complement thereof, and (ii) all or a portion of the sequence of the second analyte, or a complement thereof, or all or a portion of the sequence of the intermediate agent, or a complement thereof. In some instances, the nucleic acid barcode molecule is coupled to a particle. In some instances, the particle is a bead. In some instances, the nucleic acid barcode molecule is released from the particle upon application of a stimulus, optionally wherein the stimulus comprises a biological stimulus, a chemical stimulus, a thermal stimulus, an electrical stimulus, a magnetic stimulus, or a photo stimulus. In some instances, the nuclei are separated into a plurality of partitions, wherein a partition of the plurality of partitions comprises the nucleic acid barcode molecule and a nucleus of the nuclei, and wherein the method further comprises lysing the nucleus. In some instances, the partition is a droplet, microwell, or well. In some instances, the second analyte comprises RNA. In some instances, the RNA is mRNA. In some instances, the second analyte comprises DNA. In some instances, the DNA is genomic DNA. In some instances, the biological sample is a tissue sample. In some instances, the tissue sample is a tissue section. In some instances, the biological sample is a fresh tissue sample and/or a frozen tissue sample. In some instances, the biological sample is a fixed tissue sample. In some instances, the fixed tissue sample is a formalin fixed paraffin embedded (FFPE) tissue sample. In some instances, the FFPE tissue sample is deparaffinized and decrosslinked prior to step (a). In some instances, the biological sample is stained prior to step (a). In some instances, the biological sample is stained using immunofluorescence, immunohistochemistry, hematoxylin, and/or eosin. 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. 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 Attorney Docket No.: 47706-0398WO1 specific numerical values that fall within such ranges irrespective of whether a specific numerical value or specific sub-range is expressly stated. 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. 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. II. DESCRIPTION OF DRAWINGS 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 in the drawings indicate like elements. FIG.1A shows an exemplary sandwiching process where a first substrate (e.g., a slide), including a biological sample, and a second substrate (e.g., array slide) are brought into proximity with one another. FIG.1B shows a fully formed sandwich configuration creating a chamber formed from one or more spacers, the first substrate, and the second substrate. FIG.2A shows a perspective view of an exemplary sample handling apparatus in a closed position. FIG.2B shows a perspective view of an exemplary sample handling apparatus in an open position. FIG.3A shows the first substrate angled over (superior to) the second substrate. FIG.3B shows that as the first substrate lowers, and/or as the second substrate rises, the dropped side of the first substrate may contact a drop of reagent medium. FIG.3C shows a full closure of the sandwich between the first substrate and the second substrate with one or more spacers contacting both the first substrate and the second substrate. FIG.4A shows a side view of the angled closure workflow. Attorney Docket No.: 47706-0398WO1 FIG.4B shows a top view of the angled closure workflow. FIG.5 is a schematic diagram showing an example of a barcoded capture probe, as described herein. FIG.6 shows a schematic illustrating a cleavable capture probe. FIG.7 shows exemplary capture domains on capture probes. FIG.8 shows an exemplary arrangement of barcoded features within an array. FIG.9A shows an exemplary workflow for performing templated capture and producing a ligation product. FIG.9B shows an exemplary workflow for capturing a ligation product from FIG. 9A on a substrate. FIG.10 is a schematic diagram of an exemplary analyte capture agent. FIG.11 is a schematic diagram depicting an exemplary interaction between a feature- immobilized capture probe and an analyte capture agent. FIG.12 shows a schematic overview of the workflow combining spatial transcriptomics and single nuclei RNA sequencing. FIGs.13A-13F show quality control analysis and results of mouse brain samples showing representative image of H&E stained mouse brain sections placed in one 11x11mm capture area (FIG.13A), distributions of unique genes (left panel) and number of UMI counts (right panel) per spot visualized as violin (FIG.13B), distributions of unique genes per spot (FIG.13C), distributions of unique genes (left panel) and number of UMI counts (right panel) per nucleus visualized as violin plots (FIG.13D), number of genes and UMIs per nuclei for each sample visualized as a scatter plot (FIG.13E), gene-gene scatter plots (FIG.13F). MB1 and MB2: mouse brain samples processed by combinatorial methods of spatial transcriptomics and single nuclei sequencing. MFB_10x: dataset generated by single cell sequencing. FIG.14 shows UMAP visualization of mouse brain single nuclei data detected by unsupervised clustering (top right image) and heatmap representation of cluster marker genes for all 41 UMAP clusters. Each cluster is indicated by number as well as a color at the top of the heat map. FIG.15 shows dot plots representing expression of top marker genes per cluster in mouse brain datasets. Each dot was sized to represent the proportion of each cluster expressing the marker genes. Attorney Docket No.: 47706-0398WO1 FIGs.16A-16P shows representative images showing spatial distribution of selected factors obtained by non-negative matrix factorization of the spatial transcriptomics data . FIGs.17A-17C shows spatial distribution of neurons (left image), oligodendrocytes (middle image) and tranthyretin (right image) in mouse brain sample attained by cell-type deconvolution of the spatial transcriptomics data using the single-nuclei data as reference with broad cell type annotations.. Oligo: oligodendrocytes; Ttr: tranthyretin FIGs.18A-18H shows spatial distribution of various cell types in distinct morphological areas of the mouse brain, attained by cell-type deconvolution of the spatial transcriptomics data using the single-nuclei data as reference with cell type annotations of higher level of granularity. FIG.19 shows spatial distribution of cell types in the cortex and hippocampal area of the brain. L2/3 IT: Layer 2/3 intratelencephalic; L4: Layer 4; L5 IT: Layer 5 intratelencephalic; L5 PT: Layer 5 pyramidal tract; L6 CT: Layer 6 corticothalamic; L6 IT: Layer 6 intratelencephalic; L6b: subtype of neurons in Layer 6. Single-cell data annotated using neuron subclasses found in the cortex area of the brain was used for this deconvolution of the spatial transcriptomics data. FIGs.20A-20C shows UMAP visualization of single nuclei mouse brain data, colored by cluster and annotated through label transfer. FIGs.21A-21C shows quality control analysis of combinatorial methods in fresh- frozen (FF) and FFPE breast cancer (BC) samples. FIG.21A shows Patient 1 section 1 (left) and patient 1 section 2 (right) FF BC distributions of median number of unique genes, number of UMI counts per nucleus, percentage of mitochondrial transcripts visualized as violin plots with unique genes per nucleus also visualized as a histogram. FIG.21B shows Patient 2 section 1 (left) and patient 2 section 2 (right) FF BC distributions of median number of unique genes, number of UMI counts per nucleus and percentage of mitochondrial transcripts visualized as violin plots with unique genes per nucleus also visualized as a histogram. FIG.21C shows Patient 1 (left) and patient 2 (right) FFPE BC distributions of median number of unique genes, number of UMI counts per nucleus, percentage of mitochondrial transcripts visualized as violin plots with unique genes per nucleus also visualized as a histogram. FIGs.22A-22F show clusters from FF (FIGs.22A and 22B) and FFPE (FIG.22C and FIG.22D) breast cancer sections. FIG.22E shows FFPE breast cancer snRNA-seq data generated using publicly available single nuclei isolation and sequencing protocol with Attorney Docket No.: 47706-0398WO1 clusters manually annotated FIG.22F shows FF 4% formalin-fixed (24h) BC publicly available single cell RNA-seq dataset generated using standard an RNA Profiling Multiplexed protocol. III. DETAILED DESCRIPTION A. Spatial Analysis Methods Spatial analysis methodologies described herein can provide a vast amount of analyte and/or expression data for a variety of analytes within a biological sample at high spatial resolution, while retaining native spatial context. Spatial analysis methods 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 protein and/or 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. 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. Non-limiting aspects of spatial analysis methodologies and compositions are described in U.S. Patent Nos.11,447,807, 11,352,667, 11,168,350, 11,104,936, 11,008,608, 10,995,361, 10,913,975, 10,774,374, 10,724,078, 10,640,816, 10,494,662, 10,480,022, 10,364,457, 10,317,321, 10,059,990, 10,041,949, 10,030,261, 10,002,316, 9,879,313, 9,783,841, 9,727,810, 9,593,365, 8,951,726, 8,604,182, and 7,709,198; U.S. Patent Application Publication Nos.2020/0239946, 2020/0080136, 2020/0277663, 2019/0330617, 2020/0256867, 2020/0224244, 2019/0085383, and 2013/0171621; PCT Patent Application Publication Nos. WO2018/091676, WO2020/176788, WO2017/144338, and WO2016/057552; Non-patent literature references 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; and the Visium Spatial Gene Expression Reagent Kits User Guide (e.g., Rev F, dated January 2022); and/or the Visium Spatial Gene Expression Reagent Kits - Tissue Optimization User Guide (e.g., Rev E, dated February 2022), both of which are available at the 10x Genomics Support Attorney Docket No.: 47706-0398WO1 Documentation website, and can be used herein in any combination, and each of which is incorporated herein by reference in its entirety. Further non-limiting aspects of spatial analysis methodologies and compositions are described herein. Some general terminology that may be used in this disclosure can be found in Section (I)(b) of PCT Patent Application Publication No. WO2020/176788 and/or U.S. Patent Application Publication No.2020/0277663, which is herein incorporated by reference. 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. 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. 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 PCT Patent Application Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663, which is herein incorporated by reference. In some embodiments, an analyte can be detected indirectly, such as through detection of an intermediate agent, for example, a ligation product or an analyte capture agent (e.g., an oligonucleotide-conjugated antibody), such as those described herein. 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 Attorney Docket No.: 47706-0398WO1 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 may be paraffin blocks produced by extracting cylindrical tissue cores from different paraffin donor blocks and re-embedding these tissue cores into a single recipient (microarray) block at defined array coordinates. The biological sample as used herein can be any suitable biological sample described herein or known in the art. In some embodiments, the biological sample is a tissue sample. In some embodiments, the tissue sample is a solid tissue sample. In some embodiments, the biological sample is a tissue section (e.g., a fixed tissue section). In some embodiments, the tissue is flash-frozen and sectioned. Any suitable method described herein or known in the art can be used to flash-freeze and section the tissue sample. In some embodiments, the biological sample, e.g., the tissue, is flash-frozen using liquid nitrogen before sectioning. In some embodiments, the biological sample, e.g., a tissue sample, is flash-frozen using nitrogen (e.g., liquid nitrogen), isopentane, or hexane. 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 by cryosectioning, for example using a microtome. In some embodiments, the methods further comprise a thawing step, after the cryosectioning. 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 plant, an insect, an arachnid, a nematode (e.g., Caenorhabditis elegans), a fungus, an amphibian, or a fish (e.g., zebrafish)). A biological sample can be obtained from a prokaryote such as a bacterium, e.g., Escherichia coli, Staphylococci or Mycoplasma pneumoniae; an archaeon; a virus such as Hepatitis C virus or human immunodeficiency virus; or a viroid. A biological sample can be obtained from a eukaryote, such as a patient derived organoid (PDO) or patient derived xenograft (PDX). The biological sample can include organoids, a miniaturized and simplified version of an organ produced in vitro in three dimensions that shows realistic micro-anatomy. Organoids can be generated from one or more cells from a tissue, embryonic stem cells, and/or induced pluripotent stem cells, which can self-organize in three-dimensional culture Attorney Docket No.: 47706-0398WO1 owing to their self-renewal and differentiation capacities. In some embodiments, an organoid is a cerebral organoid, an intestinal organoid, a stomach organoid, a lingual organoid, a thyroid organoid, a thymic organoid, a testicular organoid, a hepatic organoid, a pancreatic organoid, an epithelial organoid, a lung organoid, a kidney organoid, a gastruloid, a cardiac organoid, or a retinal organoid. Subjects from which biological samples can be obtained can be healthy or asymptomatic individuals, individuals that have or are suspected of having a disease (e.g., cancer) or a pre-disposition to a disease, and/or individuals that are in need of therapy or suspected of needing therapy. Biological samples can be derived from a homogeneous culture or population of the subjects or organisms mentioned herein or alternatively from a collection of several different organisms, for example, in a community or ecosystem. 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. 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, when the biological sample is fixed using a fixative including an alcohol (e.g., methanol or acetone-methanol mixture), the biological sample is not decrosslinked afterward. In some preferred embodiments, the biological sample is fixed using a fixative including an alcohol (e.g., methanol or an acetone-methanol mixture) after freezing and/or sectioning. In some instances, the biological sample is flash-frozen, and then the biological sample is sectioned and fixed (e.g., using methanol, acetone, or an acetone-methanol mixture). In some instances when methanol, acetone, or an acetone- methanol mixture is used to fix the biological sample, the sample is not decrosslinked at a later step. In instances when the biological sample is frozen (e.g., flash frozen using liquid nitrogen and embedded in OCT) followed by sectioning and alcohol (e.g., methanol, acetone- methanol) fixation or acetone fixation, the biological sample is referred to as “fresh frozen”. In some embodiments, fixation of the biological sample e.g., using acetone and/or alcohol (e.g., methanol, acetone-methanol) is performed while the sample is mounted on a substrate (e.g., glass slide, such as a positively charged glass slide). Attorney Docket No.: 47706-0398WO1 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 (PFA) or formalin. In some embodiments, the fixative induces crosslinks within the biological sample. In some embodiments, after fixing, e.g., by formalin or PFA, 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, the sample can be rehydrated using 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. In such instances, the biological sample is referred to as “fixed frozen”. In preferred embodiments, a fixed frozen biological sample is not treated with methanol. In preferred embodiments, a fixed frozen biological sample is not paraffin-embedded. Thus, in preferred embodiments, a fixed frozen biological sample is not deparaffinized. In some embodiments, a fixed frozen biological sample is rehydrated in an ethanol gradient. In some instances, the biological sample (e.g., a fixed frozen tissue sample) is treated with a citrate buffer. Citrate buffer can be used to decrosslink antigens and fixation medium in the biological sample for antigen retrieval. Thus, any suitable decrosslinking agent can be used in addition to or alternatively to citrate buffer. In some embodiments, for example, the biological sample (e.g., a fixed frozen tissue sample) is decrosslinked using TE buffer. In any of the foregoing, the biological sample can further be stained, imaged, and/or destained. For example, in some embodiments, a fresh frozen tissue sample or fixed frozen tissue sample is stained (e.g., via eosin and/or hematoxylin), imaged, destained (e.g., via HCl), or a combination thereof. In some embodiments, when a fresh frozen tissue sample is fixed in methanol, the sample is treated with isopropanol prior to being stained (e.g., via eosin and/or hematoxylin), imaged, destained (e.g., via HCl), or a combination thereof. In some embodiments when a fixed frozen tissue sample is treated with a sucrose gradient, the sample can be rehydrated using an ethanol gradient before being stained, (e.g., via eosin and/or hematoxylin), imaged, destained (e.g., via HCl), decrosslinked (e.g., via TE buffer or citrate buffer), or a combination thereof. In some embodiments, the biological sample can undergo further fixation (e.g., while mounted on a substrate), stained, imaged, and/or destained. For example, a fixed frozen biological sample may be subject to an additional Attorney Docket No.: 47706-0398WO1 fixing step (e.g., using PFA) before optional ethanol rehydration, staining, imaging, and/or destaining. In any of the foregoing, 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, an acid, and a soluble organic compound that preserves morphology of biomolecules. PAXgene provides 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, e.g., Ergin B. et al., J Proteome Res.2010 Oct 1;9(10):5188-96; Kap M. et al., PLoS One.; 6(11):e27704 (2011); and Mathieson W. et al., Am J Clin Pathol.; 146(1):25-40 (2016), each of which is hereby incorporated by reference in its entirety, for a description and evaluation of PAXgene for tissue fixation. Thus, in some embodiments, when the biological sample, e.g., the tissue sample, is fixed in a fixative including alcohol, the fixative is PAXgene. In some embodiments, a fresh frozen tissue sample is fixed with PAXgene. In some embodiments, a fixed frozen tissue sample is fixed with PAXgene. In some embodiments, the biological sample, e.g., the tissue sample, is fixed, for example in methanol, acetone, acetone-methanol, PFA, and/or PAXgene or is formalin-fixed and paraffin-embedded (FFPE). In some embodiments, the biological sample includes intact cells. In some embodiments, the biological sample is a cell pellet, e.g., a fixed cell pellet, e.g., an FFPE cell pellet. FFPE samples are used in some instances in the RNA-templated ligation (RTL) methods disclosed herein. A limitation of direct RNA capture for fixed samples is that the RNA integrity of fixed (e.g., FFPE) samples can be lower than of a fresh sample, thereby capturing RNA directly from fixed samples, e.g., by capture of a common sequence such as a poly(A) tail of an mRNA molecule, can be more difficult. However, by utilizing RTL probes that hybridize to RNA target sequences in the transcriptome, RNA analytes can be captured without requiring that both a poly(A) tail and target sequences remain intact. Accordingly, RTL probes can be utilized to beneficially improve capture and spatial analysis of fixed samples. 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 destaining 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 Attorney Docket No.: 47706-0398WO1 (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. 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 mouse sample. In some instances, the sample is a human sample. In some embodiments, the 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 instances, the sample is a human or mouse breast tissue sample. In some instances, the sample is a human or mouse brain tissue sample. In some instances, the sample is a human or mouse lung tissue sample. In some instances, the sample is a human or mouse tonsil tissue sample. In some instances, the sample is a human or mouse liver tissue sample. In some instances, the sample is a human or mouse bone, skin, kidney, thymus, testes, or prostate tissue sample. In some embodiments, the tissue sample is derived from normal or diseased tissue. 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. Biological samples are also described in Section (I)(d) of PCT Patent Application Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663, which is herein incorporated by reference. The following embodiments can be used with any of the methods described herein. In some embodiments, the biological sample (e.g., a fixed and/or stained biological sample) is imaged. In some embodiments, the biological sample is visualized or imaged using bright field microscopy. In some embodiments, the biological sample is visualized or imaged using fluorescence microscopy. The biological sample can be visualized or imaged using additional methods of visualization and imaging known in the art. Non-limiting examples of visualization and imaging include expansion microscopy, bright field microscopy, dark field microscopy, phase contrast microscopy, electron microscopy, fluorescence microscopy, reflection microscopy, interference microscopy and confocal microscopy. In some embodiments, the sample is stained and imaged prior to adding reagents for analyzing captured analytes as disclosed herein to the biological sample. In some embodiments, the methods include staining the biological sample. In some embodiments, the staining includes the use of hematoxylin and/or eosin. 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 can be stained using any number of biological stains, including but not limited to, acridine Attorney Docket No.: 47706-0398WO1 orange, Bismarck brown, carmine, coomassie blue, cresyl violet, DAPI (4',6-diamidino-2- phenylindole), eosin, ethidium bromide, acid fuchsine, hematoxylin, Hoechst stains, iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red, osmium tetroxide, propidium iodide, rhodamine, or safranin. In some instances, the biological sample can be stained using known staining techniques, including Can-Grunwald, Giemsa, hematoxylin and eosin (H&E), 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 staining includes the use of a detectable label, such as a radioisotope, a fluorophore, a chemiluminescent compound, a bioluminescent compound, or a combination thereof. 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 PCT Patent Application Publication No. WO2020/176788 and/or U.S. Patent Application Publication No.2020/0277663, which is herein incorporated by reference. Briefly, in any of the methods described herein, the method includes a step of permeabilizing the biological sample. For example, the biological sample can be permeabilized to facilitate transfer of extension products to the capture probes on the array. In some embodiments, the permeabilizing includes the use of an organic solvent (e.g., acetone, ethanol, or methanol), a detergent (e.g., saponin, Triton X-100™, Tween-20™, or sodium dodecyl sulfate (SDS)), an enzyme (e.g., an endopeptidase, an exopeptidase, or a protease), or a combination thereof. In some embodiments, the permeabilizing includes the use of an endopeptidase, a protease, SDS, polyethylene glycol tert-octylphenyl ether, polysorbate 80, polysorbate 20, N-lauroylsarcosine sodium salt solution, saponin, Triton X-100™, Tween-20™, or a combination thereof. In some embodiments, the endopeptidase is pepsin. In some embodiments, the endopeptidase is Proteinase K. Additional methods for sample permeabilization are described, for example, in Jamur et al., Method Mol. Biol.588:63-66, 2010, which is herein incorporated herein by reference. Array-based spatial analysis methods can involve the transfer of one or more analytes or derivatives thereof 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 Attorney Docket No.: 47706-0398WO1 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. 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 instances, the capture probe includes a homopolymer sequence, such as a poly(T) sequence. 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)). See, e.g., Section (II)(b) (e.g., subsections (i)-(vi)) of PCT Patent Application Publication No. WO2020/176788 and/or U.S. Patent Application Publication No.2020/0277663, which is herein incorporated by reference. Generation of capture probes can be achieved by any appropriate method, including those described in Section (II)(d)(ii) of PCT Patent Application Publication No. WO2020/176788 and/or U.S. Patent Application Publication No.2020/0277663, which is herein incorporated by reference. In some instances, a capture probe and a nucleic acid analyte interaction (or any other nucleic acid to nucleic acid interaction) occurs because the sequences of the two nucleic acids are substantially complementary to one another. By “substantial,” “substantially,” and the like, two nucleic acid sequences can be complementary when at least 60% of the nucleotide residues of one nucleic acid sequence are complementary to nucleotide residues of the other nucleic acid sequence. The complementary residues within a particular complementary nucleic acid sequence need not always be contiguous with each other, but can be interrupted by one or more non-complementary residues within the complementary nucleic acid sequence. In some embodiments, at least 60%, but less than 100%, of the residues of one of the two complementary nucleic acid sequences are complementary to residues of the other nucleic acid sequence. In some embodiments, at least 70%, 80%, 90%, 95%, or 99% of the residues of one nucleic acid sequence are complementary to residues in the other nucleic acid sequence. Sequences are said to be “substantially complementary” when at least 60% (e.g., at least 70%, at least 80%, or at least 90%) of the residues of one nucleic acid sequence are complementary to residues of the other nucleic acid sequence. In some embodiments, the biological sample is mounted on a first substrate and the array of capture probes is on (e.g., affixed to) a second substrate. In this configuration, one or more analytes or analyte derivatives (e.g., intermediate agents, e.g., ligation products) are then released from the Attorney Docket No.: 47706-0398WO1 biological sample and migrate to the second substrate comprising an array of capture probes. In some embodiments, the release and migration of the analytes or analyte derivatives to the second substrate comprising the array of capture probes occurs in a manner that preserves the original spatial context of the analytes in the biological sample. This method can be referred to as a sandwiching process, which is described, e.g., in U.S. Patent Application Publication No.2021/0189475 and PCT Patent Application Publication Nos. WO2021/252747 A1, WO2022/061152 A2, and WO2022/140028 A1, each of which is herein incorporated by reference. FIG.1A shows an exemplary sandwiching process 100 where a first substrate (e.g., slide 103), including a biological sample 102, and a second substrate (e.g., array slide 104 including an array having spatially barcoded capture probes 106) are brought into proximity with one another. As shown in FIG.1A, a drop of liquid reagent (e.g., permeabilization solution 105) is introduced on the second substrate in proximity to the capture probes 106 and in between the biological sample 102 and the second substrate (e.g., slide 104 including an array having spatially barcoded capture probes 106). The permeabilization solution 105 may release analytes or analyte derivatives (e.g., intermediate agents, e.g., ligation products) that can be captured by the capture probes of the array 106. 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 capture probes (e.g., aligned in a sandwich configuration). As shown, the second substrate (e.g., array slide 104) is in an inferior position to the first substrate (e.g., slide 103). In some embodiments, the first substrate (e.g., slide 103) may be positioned superior to the second substrate (e.g., slide 104). A reagent medium 105 within a gap between the first substrate (e.g., slide 103) and the second substrate (e.g., slide 104) creates a liquid interface between the two substrates. The reagent medium may be a permeabilization solution which permeabilizes and/or digests the biological sample 102. In some embodiments wherein the biological sample 102 has been pre-permeabilized, the reagent medium is not a permeabilization solution. Herein, the reagent medium may also comprise one or more of a monovalent salt, a divalent salt, ethylene carbonate, and/or glycerol. In some embodiments, analytes (e.g., mRNA transcripts) and/or analyte derivatives (e.g., intermediate agents, e.g., ligation products) of the biological sample 102 may release from the biological sample, and actively or passively migrate (e.g., diffuse) across the gap toward the capture probes on the array 106. Alternatively, in certain embodiments, migration of the analyte or analyte derivative (e.g., intermediate agent, e.g., ligation product) from the biological sample is Attorney Docket No.: 47706-0398WO1 performed actively (e.g., electrophoretic, by applying an electric field to promote migration). Exemplary methods of electrophoretic migration are described in WO2020/176788, and U.S. Patent Application Publication No.2021/0189475, each of which is hereby incorporated by reference. As further shown, one or more spacers 110 may be positioned between the first substrate (e.g., slide 103) and the second substrate (e.g., array slide 104 including spatially barcoded capture probes 106). The one or more spacers 110 may be configured to maintain a separation distance between the first substrate and the second substrate. While the one or more spacers 110 is shown as disposed on the second substrate, the spacer may additionally or alternatively be disposed on the first substrate. In some embodiments, the one or more spacers 110 is configured to maintain a separation distance between first and second substrates that is between about 2 microns (µm) and about 1 millimeters (mm), e.g., between about 2 µm and about 800 µm, between about 2 µm and about 700 µm, between about 2 µm and about 600 µm, between about 2 µm and about 500 µm, between about 2 µm and about 400 µm, between about 2 µm and about 300 µm, between about 2 µm and about 200 µm, between about 2 µm and about 100 µm, between about 2 µm and about 25 µm, or between about 2 µm and about 10 µm, measured in a direction orthogonal to the surface of the first substrate that supports the biological sample and the surface of the second substrate including the capture probes. In some instances, the separation distance is about 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 µm. In some embodiments, the separation distance is less than 50 µm. In some embodiments, the separation distance is less than 25 µm. In some embodiments, the separation distance is less than 20 µm. The separation distance may include a distance of at least 2 µm. FIG.1B shows a fully formed sandwich configuration 125 creating a chamber 150 formed from the one or more spacers 110, the first substrate (e.g., the slide 103), and the second substrate (e.g., the slide 104 including an array 106 having spatially barcoded capture probes) in accordance with some example implementations. In the example of FIG.1B, the liquid reagent (e.g., the permeabilization solution 105) fills the volume of the chamber 150 and may create a permeabilization buffer that allows analytes (e.g., mRNA transcripts and/or other molecules) or analyte derivatives (e.g., intermediate agents, e.g., ligation products) to diffuse from the biological sample 102 toward the capture probes of the second substrate (e.g., slide 104). In some aspects, flow of the permeabilization buffer may deflect transcripts and/or molecules from the biological sample 102 and may affect diffusive transfer of analytes Attorney Docket No.: 47706-0398WO1 or analyte derivatives (e.g., intermediate agents, e.g., ligation products) for spatial analysis. A partially or fully sealed chamber 150 resulting from the one or more spacers 110, the first substrate (e.g., slide 103), and the second substrate (e.g., slide 104) may reduce or prevent flow from undesirable movement (e.g., convective movement) of transcripts and/or molecules during the diffusive transfer from the biological sample 102 to the capture probes. The sandwiching process methods described above can be implemented using a variety of hardware components. For example, the sandwiching process methods can be implemented using a sample holder (also referred to herein as a support device, a sample handling apparatus, and an array alignment device). Further details on support devices, sample holders, sample handling apparatuses, or systems for implementing a sandwiching process are described in, e.g., U.S. Patent Application Publication No.2021/0189475, and PCT Patent Application Publication No. WO2022/061152 A2, each of which is incorporated by reference in its entirety. In some embodiments, the sample holder can include a first member including a first retaining mechanism configured to retain a first substrate including a biological 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 adjustment 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. In some embodiments, the adjustment mechanism includes a linear actuator. 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. 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. 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. In some Attorney Docket No.: 47706-0398WO1 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. FIG.2A is a perspective view of an example sample handling apparatus 200 in a closed position in accordance with some example implementations. As shown, the sample handling apparatus 200 includes a first member 204, a second member 210, optionally an image capture device 220, a first substrate 206, optionally a hinge 215, and optionally a mirror 216. The hinge 215 may be configured to allow the first member 204 to be positioned in an open or closed configuration by opening and/or closing the first member 204 in a clamshell manner along the hinge 215. FIG.2B is a perspective view of the example sample handling apparatus 200 in an open position in accordance with some example implementations. As shown, the sample handling apparatus 200 includes one or more first retaining mechanisms 208 configured to retain one or more first substrates 206. In the example of FIG.2B, the first member 204 is configured to retain two first substrates 206, however the first member 204 may be configured to retain more or fewer first substrates 206. In some aspects, when the sample handling apparatus 200 is in an open position (e.g., in FIG.2B), the first substrate 206 and/or the second substrate 212 may be loaded and positioned within the sample handling apparatus 200 such as within the first member 204 and the second member 210, respectively. As noted, the hinge 215 may allow the first member 204 to close over the second member 210 and form a sandwich configuration. In some aspects, after the first member 204 closes over the second member 210, an adjustment mechanism of the sample handling apparatus 200 may actuate the first member 204 and/or the second member 210 to form the sandwich configuration for the permeabilization step (e.g., bringing the first substrate 206 and the second substrate 212 closer to each other and within a threshold distance for the sandwich configuration). The adjustment mechanism may be configured to control a speed, an angle, a force, or the like of the sandwich configuration. In some embodiments, the biological sample (e.g., sample 102 from FIG.1A) may be aligned within the first member 204 (e.g., via the first retaining mechanism 208) prior to closing the first member 204 such that a desired region of interest of the sample is aligned with the barcoded array of the second substrate (e.g., the slide 104 from FIG.1A), e.g., when the first and second substrates are aligned in the sandwich configuration. Such alignment may be accomplished manually (e.g., by a user) or automatically (e.g., via an automated alignment mechanism). After or before alignment, spacers may be applied to the first substrate 206 Attorney Docket No.: 47706-0398WO1 and/or the second substrate 212 to maintain a minimum spacing between the first substrate 206 and the second substrate 212 during sandwiching. In some aspects, the permeabilization solution (e.g., permeabilization solution 305) may be applied to the first substrate 206 and/or the second substrate 212. The first member 204 may then close over the second member 210 and form the sandwich configuration. Analytes or analyte derivatives (e.g., intermediate agents, e.g., ligation products) may be captured by the capture probes of the array and may be processed for spatial analysis. In some embodiments, during the permeabilization step, the image capture device 220 may capture images of the overlap area between the biological sample and the capture probes on the array 106. If more than one first substrates 206 and/or second substrates 212 are present within the sample handling apparatus 200, the image capture device 220 may be configured to capture one or more images of one or more overlap areas. 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. FIGs.3A-3C depict a side view and a top view of an exemplary angled closure workflow 300 for sandwiching a first substrate (e.g., slide 303) having a biological sample 302 and a second substrate (e.g., slide 304 having capture probes 306) in accordance with some exemplary implementations. FIG.3A depicts the first substrate (e.g., the slide 303 including a biological sample 302) angled over (superior to) the second substrate (e.g., slide 304). As shown, reagent medium (e.g., permeabilization solution) 305 is located on the spacer 310 toward the right- hand side of the side view in FIG.3A. While FIG.3A depicts the reagent medium on the right-hand side of side view, it should be understood that such depiction is not meant to be limiting as to the location of the reagent medium on the spacer. FIG.3B shows that as the first substrate lowers and/or as the second substrate rises, the dropped side of the first substrate (e.g., a side of the slide 303 angled toward the slide 304) may contact the reagent medium 305. The dropped side of the slide 303 may urge the reagent medium 305 toward the opposite direction (e.g., towards an opposite side of the spacer 310, towards an opposite side of the slide 303 relative to the dropped side). For example, in the side view of FIG.3B the reagent medium 305 may be urged from right to left as the sandwich is formed. In some embodiments, the first substrate and/or the second substrate are further moved to achieve an approximately parallel arrangement of the first substrate and the second substrate. Attorney Docket No.: 47706-0398WO1 FIG.3C depicts a full closure of the sandwich between the first substrate and the second substrate with the spacer 310 contacting both the first substrate and the second substrate and maintaining a separation distance and optionally the approximately parallel arrangement between the two substrates. As shown in the top view of FIG.3C, the spacer 310 fully encloses and surrounds the biological sample 302 and the capture probes 306, and the spacer 310 form the sides of chamber 350 which holds a volume of the reagent medium 305. While FIG.3C depicts the first substrate (e.g., the slide 303 including biological sample 302) angled over (superior to) the second substrate (e.g., slide 304) and the second substrate including the spacer 310, it should be understood that an exemplary angled closure workflow can include the second substrate angled over (superior to) the first substrate and the first substrate including the spacer 310. It may be desirable that the reagent medium be free from air bubbles between the substrates to facilitate transfer of target analytes with spatial information. Additionally, air bubbles present between the substrates 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 303 and slide 304) during a permeabilization step (e.g., step 104). In some aspects, it may be possible to reduce or eliminate bubble formation between the substrates using a variety of filling methods and/or closing methods. In some instances, the first substrate and the second substrate are arranged in an angled sandwich assembly as described herein. For example, during the sandwiching of the two substrates (e.g., the slide 303 and the slide 304), an angled closure workflow may be used to suppress or eliminate bubble formation. FIG.4A is a side view of the angled closure workflow 400 in accordance with some exemplary implementations. FIG.4B is a top view of the angled closure workflow 400 in accordance with some exemplary implementations. As shown at step 405, reagent medium 401 is positioned to the side of the substrate 402. At step 410, the dropped side of the angled substrate 406 contacts the reagent medium 401 first. The contact of the substrate 406 with the reagent medium 401 may form a linear or low curvature flow front that fills the gap between the two substrates 406 and 402 uniformly with the slides closed. At step 415, the substrate 406 is further lowered toward the substrate 402 (or the substrate 402 is raised up toward the substrate 406) and the dropped side of the substrate 406 may contact and may urge the reagent medium toward the side opposite the dropped side, Attorney Docket No.: 47706-0398WO1 thereby creating a linear or low curvature flow front that may prevent or reduce bubble trapping between the substrates. At step 420, the reagent medium 401 fills the gap between the substrate 406 and the substrate 402. The linear flow front of the liquid reagent may be formed by squeezing the reagent medium 401 volume along the contact side of the substrate 402 and/or the substrate 406. Additionally, capillary flow may also contribute to filling the gap area. In some embodiments, the reagent medium (e.g., 105 in FIG.1A) includes a permeabilization agent. In some embodiments, following initial contact between the biological sample and a permeabilization agent, the permeabilization agent can be removed from contact with the biological sample (e.g., by opening the sample holder). Suitable agents for this purpose include, but are not limited to, organic solvents (e.g., acetone, ethanol, or methanol), cross-linking agents (e.g., paraformaldehyde), detergents (e.g., saponin, Triton X- 100™, Tween-20™, or SDS), and enzymes (e.g., trypsin or other proteases (e.g., Proteinase K)). In some embodiments, the detergent is an anionic detergent (e.g., SDS or N- lauroylsarcosine sodium salt solution). In some embodiments, the reagent medium includes a lysis reagent. Lysis solutions can include ionic surfactants such as, for example, sarkosyl, and SDS. More generally, chemical lysis agents can include, without limitation, organic solvents, chelating agents, detergents, surfactants, and chaotropic agents. In some embodiments, the reagent medium includes a protease. Exemplary proteases include, e.g., pepsin, trypsin, elastase, and Proteinase K. In some embodiments, the reagent medium includes a nuclease. In some embodiments, the nuclease includes an RNase. In some embodiments, the RNase includes RNase A, RNase C, RNase H, and/or RNase I. In some embodiments, the reagent medium includes one or more of SDS or a sodium salt thereof, Proteinase K, pepsin, N- lauroylsarcosine, and RNase. In some embodiments, the reagent medium includes polyethylene glycol (PEG). In some embodiments, the molecular weight of the PEG is from about 2K to about 16K. In some embodiments, the molecular weight of the PEG is about 2K, about 3K, about 4K, about 5K, about 6K, about 7K, about 8K, about 9K, about 10K, about 11K, about 12K, about 13K, about 14K, about 15K, or about 16K. In some embodiments, the PEG is present at a concentration from about 2% to about 25%, from about 4% to about 23%, from about 6% to about 21%, or from about 8% to about 20% (v/v). In certain embodiments, a dried permeabilization reagent is applied or formed as a layer on the first substrate, the second substrate, or both prior to contacting the biological Attorney Docket No.: 47706-0398WO1 sample with the array. For example, a permeabilization reagent can be deposited in solution on the first substrate or the second substrate or both and then dried. In some instances, the aligned portions of the biological sample and the array are in contact with the reagent medium 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 for about 1-60 minutes. In some instances, the device is configured to control a temperature of the first and second substrates. In some embodiments, the temperature of the first and second members is lowered to a first temperature that is below room temperature. 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. 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 ligation product or an analyte capture agent), or a portion thereof), or derivatives thereof (see, e.g., Section (II)(b)(vii) of PCT Patent Application Publication No. WO2020/176788 and/or U.S. Patent Application Publication No.2020/0277663 regarding extended capture probes, which is herein incorporated by reference). In some cases, capture probes may be configured to form ligation products with a template (e.g., a DNA or RNA template, such as an analyte or an intermediate agent, or portion thereof), thereby creating ligation products that serve as proxies for the template. As used herein, an “extended capture probe” refers to a capture probe having additional nucleotides added to a terminus (e.g., a 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 Attorney Docket No.: 47706-0398WO1 (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 bound to the capture domain of the capture probe. In some embodiments, the capture probe is extended using a reverse transcriptase. In some embodiments, the capture probe is extended using one or more DNA polymerases. In some embodiments, the extended capture probes include the sequence of the capture domain, the sequence of the spatial barcode of the capture probe, and the complementary sequence of the template used for extension of the capture probe. 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., sequencing. In some embodiments, extended capture probes (e.g., DNA molecules) can act as templates for an amplification reaction (e.g., a polymerase chain reaction). Additional variants of spatial analysis methods, including in some embodiments, an imaging step, are described in Section (II)(a) of PCT Patent Application Publication No. WO2020/176788 and/or U.S. Patent Application Publication No.2020/0277663, which is herein incorporated by reference. Analysis of captured analytes (and/or intermediate agents or portions thereof), for example, including sample removal, extension of capture probes using the capture analyte as a template, 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 PCT Patent Application Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663, which is herein incorporated by reference. Some quality control measures are described in Section (II)(h) of PCT Patent Application Publication No. WO2020/176788 and/or U.S. Patent Application Publication No.2020/0277663, which is herein incorporated by reference. Spatial information can provide information of medical importance. For example, the methods 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 Attorney Docket No.: 47706-0398WO1 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. Exemplary methods for identifying spatial information of biological and/or medical importance can be found in U.S. Patent Application Publication Nos.2021/0140982, 2021/0198741, and 2021/0199660, each of which is herein incorporated by reference in its entirety. Spatial information can provide information of biological importance. For example, the methods 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 or proximity based analysis); determination of up-regulated 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 healthy and diseased 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). For spatial array-based methods, a substrate may function 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 PCT Patent Application Publication No. WO2020/176788 and/or U.S. Patent Application Publication No.2020/0277663, which is herein incorporated by reference. Exemplary features and geometric attributes of an array can be found in Sections (II)(d)(i), (II)(d)(iii), and (II)(d)(iv) of PCT Patent Application Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663, which is herein incorporated by reference. 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 or wells) including 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 Attorney Docket No.: 47706-0398WO1 capture is further described in Section (II)(e) of PCT Patent Application Publication No. WO2020/176788 and/or U.S. Patent Application Publication No.2020/0277663, which is herein incorporated by reference. FIG.5 is a schematic diagram showing an exemplary capture probe, as described herein. As shown, the capture probe 502 is optionally coupled to a feature 501 by a cleavage domain 503, such as a disulfide linker. The capture probe can include a functional sequence 504 that is useful for subsequent processing. The functional sequence 504 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 505. The capture probe can also include a unique molecular identifier (UMI) sequence 506. While FIG.5 shows the spatial barcode 505 as being located upstream (5’) of UMI sequence 506, it is to be understood that capture probes wherein UMI sequence 506 is located upstream (5’) of the spatial barcode 505 is also suitable for use in any of the methods described herein. The capture probe can also include a capture domain 507 to facilitate capture of a target analyte. 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 an analyte capture sequence present in an analyte capture agent. The capture domain can have a sequence complementary to a splint oligonucleotide. A splint oligonucleotide, in addition to having a sequence complementary to a capture domain of a capture probe, can have a sequence complementary to a sequence of a nucleic acid analyte, a portion of a connected probe described herein, a capture handle sequence described herein, and/or a methylated adaptor described herein. FIG.6 is a schematic illustrating a cleavable capture probe, wherein the cleaved capture probe can enter into a non-permeabilized cell and bind to analytes within the cell. The capture probe 601 can contain a cleavage domain 602, a cell penetrating peptide 603, a reporter molecule 604, and a disulfide bond (-S-S-).605 represents all other parts of a capture probe, for example, a spatial barcode and a capture domain. FIG.7 is a schematic diagram of an exemplary multiplexed spatially-barcoded feature. In FIG.7, the feature 701 can be coupled to spatially-barcoded capture probes, wherein 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 include four different Attorney Docket No.: 47706-0398WO1 types of spatially-barcoded capture probes, each type of spatially-barcoded capture probe possessing the spatial barcode 702. One type of capture probe associated with the feature can include the spatial barcode 702 in combination with a poly(T) capture domain 703, designed to capture mRNA target analytes. A second type of capture probe associated with the feature can include the spatial barcode 702 in combination with a random N-mer capture domain 704 for gDNA analysis. A third type of capture probe associated with the feature can include the spatial barcode 702 in combination with a capture domain complementary to the analyte capture agent of interest 705. A fourth type of capture probe associated with the feature can include the spatial barcode 702 in combination with a capture probe that can bind a nucleic acid molecule 706 that can function in a CRISPR assay (e.g., CRISPR/Cas9). While only four different capture probe-barcoded constructs are shown in FIG.7, 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.7 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/or metabolites, and gDNA; (b) mRNA, accessible chromatin (e.g., ATAC-seq, DNase-seq, and/or MNase-seq), cell surface or intracellular proteins and/or 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. 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. Attorney Docket No.: 47706-0398WO1 In some embodiments, the spatial barcode 505 and functional sequence 504 are common to all of the probes attached to a given feature. In some embodiments, the UMI sequence 506 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. FIG.8 depicts an exemplary arrangement of barcoded features within an array. From left to right, FIG.8 shows (left) a slide including six spatially-barcoded arrays, (center) an enlarged schematic of one of the six spatially-barcoded arrays, showing a grid of barcoded features in relation to a biological sample, and (right) an enlarged schematic of one section of an array, showing the specific identification of multiple features within the array (e.g., labelled as ID578, ID579, ID580, etc.). 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 PCT Patent Application Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663, which is herein incorporated by reference. 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 or cell nucleus 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 or cell nuclei 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, single cell nuclei, or cell groups for analysis. Some such methods of spatial analysis are described in Section (III) of PCT Patent Application Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663, which is herein incorporated by reference. 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 et al., Nucleic Acids Res.2017 Aug 21; 45(14):e128, which is herein incorporated by reference. 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 Attorney Docket No.: 47706-0398WO1 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 or a non-homopolymeric sequence). After hybridization to the analyte, a ligase (e.g., a T4 RNA ligase (Rnl2), a PBCV-1 DNA Ligase or Chlorella virus DNA Ligase, a single-stranded DNA ligase, or a T4 DNA ligase) ligates the two oligonucleotides together, creating 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 can create 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 ligation product is released from the analyte. In some instances, the ligation product is released using an endonuclease (e.g., RNase H). In some instances, the ligation product is removed using heat. In some instances, the ligation product is removed using KOH. The released 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. In some instances, one or both of the oligonucleotides may hybridize to genomic DNA (gDNA) which can lead to false positive sequencing data from ligation events on gDNA (off target) in addition to the desired (on target) ligation events on target nucleic acids, (e.g., mRNA). Thus, in some embodiments, the disclosed methods can include contacting the biological sample with a deoxyribonuclease (DNase). The DNase can be an endonuclease or exonuclease. In some embodiments, the DNase digests single-stranded and/or double- stranded DNA. Suitable DNases include, without limitation, a DNase I and a DNase II. Use of a DNase as described can mitigate false positive sequencing data from off target gDNA ligation events. A non-limiting example of templated ligation methods disclosed herein is depicted in FIG.9A. After a biological sample is contacted with a substrate including a plurality of capture probes and contacted with (a) a first probe 901 having a target-hybridization sequence 903 and a primer sequence 902 and (b) a second probe 904 having a target- hybridization sequence 905 and a capture domain (e.g., a poly(A) sequence) 906, the first probe 901 and the second probe 904 hybridize 910 to an analyte 907. A ligase 921 ligates 920 the first probe 901 to the second probe 904, thereby generating a ligation product 922. The ligation product 922 is then released 930 from the analyte 931 by digesting the analyte 907 Attorney Docket No.: 47706-0398WO1 using an endoribonuclease 932. The sample is permeabilized 940 and the ligation product 941 is able to hybridize to a capture probe on the substrate. Methods and compositions for spatial detection using templated ligation have been described in PCT Patent Application Publication No. WO 2021/133849 A1, U.S. Patent Nos.11,332,790 and 11,505,828, each of which is incorporated by reference in its entirety. In some embodiments, as shown in FIG.9B, the ligation product 9001 includes a capture probe capture domain 9002, which can bind to a capture probe 9003 (e.g., a capture probe immobilized, directly or indirectly, on a substrate 9004). In some embodiments, methods provided herein include contacting 9005 a biological sample with a substrate 9004, wherein the capture probe 9003 is affixed to the substrate (e.g., immobilized to the substrate, directly or indirectly). In some embodiments, the capture probe capture domain 9002 of the ligated product 9001 binds to the capture domain 9006. The capture probe can also include a unique molecular identifier (UMI) 9007, a spatial barcode 9008, a functional sequence 9009, and a cleavage domain 9010. In some embodiments, methods provided herein include permeabilization of the biological sample such that the capture probe can more easily bind to target analytes (i.e., compared to no permeabilization). In some embodiments, reverse transcription (RT) reagents can be added to permeabilize biological samples. Incubation with the RT reagents can be used to extend the capture probes 9011 to produce spatially-barcoded full-length cDNA 9012 and 9013 from the captured analytes (e.g., polyadenylated mRNA). Second strand reagents (e.g., second strand primers, enzymes, etc.) can be added to the biological sample to initiate second strand synthesis. In some embodiments, methods provided herein include permeabilization of the biological sample such that the capture probe can more easily capture the ligation products (i.e., compared to no permeabilization). In some embodiments, polymerization (e.g., reverse transcription (RT)) reagents can be added to permeabilized biological samples. Incubation with the RT reagents can be used to extend the capture probes 9011 to produce spatially- barcoded full-length cDNA 9012 and 9013 from the captured ligation products (e.g., polyadenylated ligation products). In some embodiments, the extended ligation products can be denatured 9014, released from the capture probe, and transferred (e.g., to a clean tube) for amplification, and/or library construction. The spatially-barcoded ligation products can be amplified 9015 via PCR prior to library construction. P59016 and P79019 sequences can be used for sequencing, while i5 9017 and i79018 sequences can be used as sample indexes. The amplicons can then be Attorney Docket No.: 47706-0398WO1 sequenced using paired-end sequencing using TruSeq Read 1 and TruSeq Read 2 as sequencing primer sites for Illumina sequencers. Other sequencing systems can be used, all that is required is the sequences specific to a particular instrument and workflow is incorporated into the sequencing libraries. In some embodiments, in addition to the detection of genetic variants in a biological sample, detection of one or more other analytes (e.g., protein analytes) can be performed, either sequentially or concurrently, 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) an analyte capture 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” 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 cases, an analyte binding moiety barcode (or portion thereof) may be able to be removed (e.g., cleaved) from the analyte capture agent. Additional description of analyte capture agents can be found in Section (II)(b)(ix) of PCT Patent Application Publication No. WO2020/176788 and/or Section (II)(b)(viii) U.S. Patent Application Publication No. 2020/0277663, which is herein incorporated by reference. FIG.10 is a schematic diagram of an exemplary analyte capture agent 1002 comprised of an analyte binding moiety 1004 and an analyte binding moiety barcode domain 1008. The analyte binding moiety 1004 is a molecule capable of binding to an analyte 1006 and the analyte capture agent 1002 is capable of interacting with a spatially-barcoded capture probe, e.g., on an array. The analyte binding moiety 1004 can bind to the analyte 1006 with high affinity and/or with high specificity. The analyte capture agent 1002 can include: (i) an analyte binding moiety barcode domain 1008, which serves to identify the analyte binding moiety, and (ii) a capture domain, which can hybridize to at least a portion or an entirety of a capture domain of a capture probe. The analyte binding moiety 1004 can include a polypeptide and/or an aptamer. The analyte binding moiety 1004 can include an antibody or antibody fragment (e.g., an antigen binding fragment). FIG.11 is a schematic diagram depicting an exemplary interaction between a feature-immobilized capture probe 1124 and an analyte capture agent 1126. The feature- Attorney Docket No.: 47706-0398WO1 immobilized capture probe 1124 can include a spatial barcode 1108 as well as functional sequence 1106 and a UMI 1110, as described elsewhere herein. The capture probe can be affixed 1104 to a feature such as a bead 1102. The capture probe 1124 can also include a capture domain 1112 that is capable of binding to an analyte capture agent 1126. The analyte binding moiety barcode domain of the analyte capture agent 1126 can include a functional sequence 1118, analyte binding moiety barcode 1116, and an analyte capture sequence 1114 that is capable of binding (e.g., hybridizing) to the capture domain 1112 of the capture probe 1124. The analyte capture agent 1126 can also include a linker 1120 that allows the analyte binding moiety barcode domain (e.g., including the functional sequence 1118, analyte binding moiety barcode 1116, and analyte capture sequence 1114) to couple to the analyte binding moiety 1122. In some embodiments, the linker 1120 is a cleavable linker. In some embodiments, the cleavable linker is a photo-cleavable linker, a UV-cleavable linker, chemical-cleavable linker, thermal-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). 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. 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 each spatial barcode is 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. 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 Attorney Docket No.: 47706-0398WO1 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 or a fiducial marker) of the array. Accordingly, each feature location has an “address” or location in the coordinate space of the array. Some exemplary spatial analysis workflows are described in the Exemplary Embodiments section of PCT Patent Application Publication No. WO2020/176788 and/or U.S. Patent Application Publication No.2020/0277663, which is herein incorporated by reference. See, e.g., the Exemplary embodiment starting with “In some non-limiting examples of the workflows described herein, the sample can be immersed…” of PCT Patent Application Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663, which is herein incorporated by reference. See also, e.g., the Visium Spatial Gene Expression Reagent Kits User Guide (e.g., Rev F, dated January 2022); and/or the Visium Spatial Gene Expression Reagent Kits - Tissue Optimization User Guide (e.g., Rev E, dated February 2022), each of which is herein incorporated by reference in its entirety. 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 PCT Patent Application Publication No. WO2020/176788 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 PCT Patent Application Publication No. WO2020/123320, which is herein incorporated by reference. Suitable systems for performing spatial analysis can include components such as a chamber (e.g., a flow cell or a 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. 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 Attorney Docket No.: 47706-0398WO1 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 or CMOS) used to capture images. The systems can also optionally include one or more light sources (e.g., LED-based, diode-based, or lasers) for illuminating a sample, a substrate with features, analytes from a biological sample captured on a substrate, and various control and calibration media. 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. 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 Patent Application Publication No. WO2021/102003 and/or U.S. Patent Application Publication No.2021/0150707, each of which is incorporated herein by reference in its entirety. 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-dimensional and/or three- dimensional map of the analyte presence and/or level are described in PCT Patent Application Publication No. WO2020/053655 and spatial analysis methods are generally described in PCT Patent Application Publication No. WO2021/102039 and/or U.S. Patent Application Publication No.2021/0155982, each of which is incorporated herein by reference in its entirety. 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 Attorney Docket No.: 47706-0398WO1 Attributes Section, Control Slide for Imaging Section of PCT Patent Application Publication Nos. WO2020/123320, WO2021/102005, and/or U.S. Patent Application Publication No. 2021/0158522, each of which is incorporated herein by reference in its entirety. 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, or 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. B. Methods of Spatial Analysis and Single Nuclei Sequencing To fully understand the spatial organization and function of tissue, it is important to obtain spatially resolved gene expression profiles. Despite their value, typical spatial transcriptomics methods targeting the entire transcriptome do not provide single-cell resolution. Combining single-cell data with spatial data allows finer interpretation of spatially resolved datasets. However, this relies on well matched single-cell and spatial data. In response to this need, disclosed herein are methods that generate both spatial transcriptomics and single cell or single nuclei RNA sequencing (snRNA-seq) data from a single (e.g., the same) biological sample, e.g., a tissue sample (e.g., a tissue section). This approach facilitates more accurate representation of cell types within the analyzed biological sample compared to unmatched datasets (e.g., from two different biological samples, such as serial tissue sections). The disclosed methods and working examples demonstrate the feasibility of extracting sufficient amounts of intact nuclei from a single tissue section (e.g., 5-18 µm thick), which is typically only achievable by processing larger tissue pieces. This facilitates a more efficient and streamlined workflow which minimizes required input material, thus preserving valuable samples. This ability to utilize only one tissue section is particularly useful in instances where sample size is a limiting factor, such as precious clinical biopsy specimens. The disclosed methods are advantageous for integration of single nuclei and spatial transcriptomics datasets and enhanced accuracy in sample profiling. For example, by utilizing a single tissue section, the disclosed methods mitigate the discrepancies that can arise from variations in cellular content between adjacent tissue sections. This ensures a more accurate representation of the cell types and states in the single nuclei dataset and therefore facilitates more comprehensive analysis of samples with highly dynamic environments such Attorney Docket No.: 47706-0398WO1 as tumor biopsies, allowing for deeper understanding of the complex interplay between various cell types and molecular pathways. In some embodiments, the methods disclosed herein utilize a single substrate (e.g., a first substrate, such as, a glass slide). For example, in some embodiments, a biological sample is contacted with an array comprising a plurality of capture probes where the array is disposed on a first substrate. In such embodiments, one or more analytes are processed from the biological sample disposed on the array on the first substrate in accordance with the spatial analysis methods disclosed herein. Thereafter, the biological sample can be harvested from the first substrate and one or more cells or nuclei are isolated therefrom to perform analysis of a second analyte in accordance with the methods disclosed herein. In some embodiments, the methods disclosed herein utilize multiple substrates (e.g., a first substrate and a second substrate). In some embodiments, the first substrate is a slide (e.g., a glass slide; e.g. a SuperFrost^TM Plus microscope slide as shown in FIG.12) and the second substrate is a slide (e.g., comprising a spatial array). In some embodiments, a biological sample is placed on the first substrate, and the biological sample is manipulated by the addition of templated ligation probes and/or analyte capture agents (e.g., only templated ligation probes, only analyte capture agents, or both). After, the substrates (e.g., slides) are “sandwiched” together as exemplified in FIGs.3A-3B and described in Section A above. In some instances, the first substrate and the second substrate are aligned. In some instances, aligning includes mounting the first substrate on a first member of a support device, the first member configured to retain the first substrate. In some instances, aligning includes mounting the second substrate on a second member of the support device. In some instances, aligning includes applying a reagent medium to the first substrate and/or the second substrate. In some instances, aligning includes operating an alignment mechanism of the support device to move the first member and/or the second member such that at least a portion of the biological sample is aligned with at least a portion of the array, and such that the portion of the biological sample and the portion of the array contact the reagent medium. In some instances, aligning includes arranging the first and second substrates such that a first side of the first substrate comprising the biological sample is positioned opposite or adjacent to a first side of the second substrate comprising the spatial array when the first substrate and second substrate are brought into proximity or contact. In some instances, the alignment mechanism is coupled to the first member, the second member, or both the first member and the second member. In some instances, the alignment mechanism comprises a linear actuator. In some instances, the linear actuator is Attorney Docket No.: 47706-0398WO1 configured to move the second member along an axis orthogonal to the first member and/or the second member. In some instances, the linear actuator is configured to move the first member along an axis orthogonal to a plane of the first member and/or the second member. In some instances, 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. In some instances, 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. In some instances, aligning the first substrate with the second substrate includes bringing a first surface of the first substrate including the biological sample and a second surface of the second substrate including the array within proximity of each other. Such proximity can be, but is not limited to, less than about 30 microns, less than about 25 microns, less than about 20 microns, less than about 15 microns, less than about 12 microns, less than about 10 microns, less than about 8 microns, less than about 5 microns, or less. In some embodiments, aligning the first substrate with the second substrate includes contacting a first surface of the first substrate including the biological sample with a second surface of the second substrate including the array. In some instances, the proximity or contact allows for migration of an analyte or analyte derivative (e.g., intermediate agent) to the array, e.g., for hybridization to the capture probe. In some instances, at least one of the first substrate and the second substrate further comprise a spacer disposed on the first substrate or the second substrate. In some instances, when at least the portion of the biological sample is aligned with at least a portion of the array such that the portion of the biological sample and the portion of the array contact the reagent medium, the spacer is disposed between the first substrate and the second substrate. In some instances, the spacer is configured to maintain the reagent medium within a chamber formed by the first substrate, the second substrate, and the spacer. In some instances, the spacer is configured to maintain a separation distance between the first substrate and the second substrate, wherein the spacer is positioned to surround an area on the first substrate on which the biological sample is disposed and/or the array disposed on the second substrate. In some instances, the area of the first substrate, the spacer, and the second substrate at least partially encloses a volume comprising the biological sample. In some instances, one or more dimensions of the spacer (e.g., height) comprises less than about 30 microns, less than about 25 microns, less than about 20 microns, less than Attorney Docket No.: 47706-0398WO1 about 15 microns, less than about 12 microns, less than about 10 microns, less than about 8 microns, less than about 5 microns, or less. Intermediate agents such as ligation products and/or the oligonucleotide of the analyte capture agent hybridize to capture probes on the array on the second substrate, where they can be further processed and analyzed (e.g., sequenced) After, referring to FIG.12, in some embodiments, the biological sample remaining on the first substrate is fixed (e.g., with formaldehyde) and transferred to a tube, where it is incubated with nucleic acid barcode molecules. In some embodiments, after a series of purification steps, one or more cells or nuclei are isolated from the biological sample. The cells or nuclei are sorted, counted, a library is prepared e.g., from the generated GEMs, and the nucleic acids are sequenced. In some embodiments, the biological sample (e.g., tissue section) has a thickness of about 2-20 µm. In some embodiments, the biological sample (e.g., tissue section) has a thickness of about 5-18 µm. An exemplary embodiment described herein includes a method for processing multiple analytes in a biological sample mounted on a first substrate, the method comprising: (a) hybridizing a first probe and a second probe to a first analyte in the biological sample, wherein the first probe and the second probe each comprise a nucleic acid sequence that is substantially complementary to a nucleic acid sequence of the first analyte, and wherein the second probe comprises a capture probe binding domain; (b) coupling the first probe and the second probe, thereby generating a connected probe; (c) 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; (d) releasing the connected probe from the first analyte when at least a portion of the biological sample is aligned with at least a portion of the array; (e) hybridizing the connected probe to the capture domain of the capture probe in the array; (f) isolating one or more cells or nuclei from the biological sample on the first substrate, wherein the one or more cells or nuclei comprise a second analyte; and (g) hybridizing a nucleic acid barcode molecule to the second analyte, a complement thereof, or an intermediate agent of the second analyte. Another exemplary embodiment described herein includes a method for processing multiple analytes in a biological sample mounted on a first substrate, the method comprising: (a) hybridizing a first probe and a second probe to a first analyte in the biological sample, Attorney Docket No.: 47706-0398WO1 wherein the first probe and the second probe each comprise a nucleic acid sequence that is substantially complementary to a nucleic acid sequence of the first analyte, and wherein the second probe comprises a capture probe binding domain; (b) coupling the first probe and the second probe, thereby generating a connected probe; (c) 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; (d) hybridizing the connected probe to the capture domain of the capture probe in the array; (e) isolating one or more cells or nuclei from the biological sample, wherein the one or more cells or nuclei comprise a second analyte; and (f) hybridizing a nucleic acid barcode molecule to the second analyte, a complement thereof, or an intermediate agent of the second analyte. A third exemplary embodiment described herein includes a method for processing multiple analytes in a biological sample mounted on a first substrate, the method comprising: (a) 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 capture agent barcode domain comprises an analyte binding moiety barcode and a capture handle sequence, and wherein upon the contacting, the analyte binding moiety binds to a first analyte; (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, 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; (c) hybridizing the capture agent barcode domain from the analyte capture agent that is bound to the first analyte to the capture domain of the capture probe; (d) isolating one or more cells or nuclei from the biological sample on the first substrate, wherein the one or more cells or nuclei comprises a second analyte; and (e) hybridizing a nucleic acid barcode molecule to the second analyte a complement thereof or an intermediate agent. Generally, the methods of the present disclosure can be used with any biological sample (e.g., any biological sample described herein) as described in Section A above. For instance, in some embodiments, the biological sample is a tissue section. In some embodiments, the biological sample is a tissue sample. In some embodiments, the biological sample is a fresh-frozen biological sample. In some embodiments, the biological sample is a fresh-frozen tissue section. In some embodiments, the biological sample is a fixed biological Attorney Docket No.: 47706-0398WO1 sample (e.g., a formalin-fixed sample (such as FFPE), paraformaldehyde-, acetone-, or methanol-fixed). In some embodiments, the biological sample is an FFPE sample. In some embodiments, the biological sample is an FFPE tissue section. In some instances, the FFPE tissue sample is deparaffinized and decrosslinked. In some instances, the biological sample is a healthy or non-diseased sample; in some instances, it is a diseased sample (e.g., a cancer sample). In some instances, the biological sample can be a cell culture sample. In some embodiments, the biological sample can be stained using immunofluorescence, immunohistochemistry, hematoxylin, and/or eosin. In some embodiments, the biological sample can be imaged, e.g., either after staining the biological sample or when no stain is used. In some embodiments, the biological sample is visualized or imaged using bright field microscopy, fluorescence microscopy, expansion microscopy, dark field microscopy, phase contrast microscopy, electron microscopy, fluorescence microscopy, reflection microscopy, interference microscopy and confocal microscopy. The methods of spatial transcriptomics disclosed in this process can detect both nucleic acids and/or proteins. As disclosed in more detail below, in some embodiments, nucleic acid capture uses templated ligation, and protein detection utilizes antibody-tagged oligonucleotides called analyte capture agents. i. Spatial Nucleic Acid Detection using Templated Ligation In some instances, it may be difficult to directly capture a nucleic acid. For instance, in samples that are fixed, the poly(A) tail of mRNA molecules may degrade, such that it could not be captured on an array. In such instances (and even in situations where the samples are not fixed), the methods disclosed herein include templated ligation of probe pairs that are designed to hybridize to adjacent or abutting sequences of a nucleic acid (e.g., DNA or RNA). In these embodiments, the RNA can include mRNA, and the DNA can include gDNA. Methods of performing templated ligation are depicted in FIGs.9A and 9B and are described in detail in Section A of this disclosure. In some instances, templated ligation occurs on the first substrate and constitutes one embodiment of the spatial transcriptomics aspect of the combinatorial methods in this disclosure. During templated ligation, the probes (e.g., a first probe and a second probe) are complementary to portions of the target nucleic acid. In some instances, the methods include hybridizing a first probe and a second probe to the analyte (e.g., a first analyte), wherein the first probe and the second probe each comprise a sequence that is substantially Attorney Docket No.: 47706-0398WO1 complementary to adjacent sequences of the analyte, and wherein the second probe comprises a capture probe binding domain; and coupling the first probe and the second probe, thereby generating the connected probe. The methods can also 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, and wherein the method further comprises using the determined sequence of (i) and (ii) to determine the location and/or abundance of the analyte in the biological sample. In some instances, the adjacent sequences abut one another. In some instances, when both probes hybridize to the target nucleic acid, a gap can be present between the two probes. In some embodiments, the probes hybridize to sequences that are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides away from one another. To fill in this gap, a polymerase can be used to generate an extended first probe (or an extended second probe). In some instances, the first probe comprises a 5’ handle sequence, wherein the 5’ handle sequence comprises about 5 nucleotides to 50 nucleotides. In some instances, the second probe comprises a 3’ handle sequence, wherein the 3’ handle sequence comprises about 5 nucleotides to 50 nucleotides. In some instances, the 3’ handle sequence comprises a poly(A) sequence. In some instances, the poly(A) sequence is at a 3’ end of the second probe. In some instances, the first probe and the second probe hybridize to the nucleic acid sequence of the first analyte, wherein the nucleic acid sequence of the first analyte is about 25 to 100 nucleotides in length. In some instances, the first probe and/or the second probe comprises DNA. In some instances, the first probe and the second probe hybridize to adjacent sequences of the first analyte. In some instances, a plurality of probe pairs are added to the biological sample. For instance, at least 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, or 20000 probe pairs are added to the biological sample. In some instances, at least about 100 probe pairs are added. In some instances, at least about 5000 probe pairs are added. In some embodiments, the probes, one of which may be extended, can be ligated, thereby generating a connected probe (e.g., a templated ligation product (e.g., DNA or RNA templated ligation product)). In some embodiments, one of the pair of probes includes a flanking sequence complementary to a capture domain of the capture probe in the array. In some embodiments, the sequence of the templated ligation product complementary to the capture domain hybridizes to the capture domain of the capture probe. In some instances, the Attorney Docket No.: 47706-0398WO1 ligase is selected from a PBCV-1 DNA ligase, Chlorella virus DNA ligase, a single stranded DNA ligase, or a T4 DNA ligase. The connected probe is then captured on the array via hybridization to the capture domain of a capture probe in the array on the second substrate. An extension step is performed using a polymerase or reverse transcriptase, generating a single nucleic acid molecule that has the sequences (or complements thereof) of the capture probe and the connected probe. After, in some instances, the extended capture probe is separated from the extended connected probe using e.g., potassium hydroxide (KOH). This sequence can be amplified, further processed, and sequenced using methods similar to those described in Section A and in Section B, subsection (b)(1). Additional methods of templated ligation have been described in PCT Publ. No. WO 2021/133849 A1, and U.S. Pat. Nos.11,332,790 and 11,505,828, each of which is incorporated by reference in its entirety. ii. Spatial Protein Detection In some embodiments, analyte capture agents, as depicted in FIGs.10 and 11, and described in Section A of this disclosure, can also be contacted with the biological sample. In some embodiments, the analyte capture agents are contacted with the biological sample before the biological sample is aligned (e.g., brought into proximity to or contacted with) to the spatial array. In some embodiments, the analyte capture agents are contacted with the biological sample after the biological sample is aligned (e.g., brought into proximity to or contacted with) to the array. In some embodiments, the analyte binding moiety of the analyte capture agent interacts (e.g., binds) to an analyte (e.g., protein) in a biological sample. In some instances, the analyte is a protein. In some instances, the analyte is an intracellular protein, or an extracellular protein. In some instances, the analyte is a cell membrane protein. In some embodiments, the analyte binding moiety is an antibody or antigen-binding fragment thereof. Analyte capture agents can also include a coupled oligonucleotide that can comprise one or more domains. For example, the oligonucleotide can include an analyte binding moiety barcode and an analyte capture sequence. In some embodiments, the analyte binding moiety barcode, or a complement thereof, refers to (e.g., identifies) a barcode that is associated with or otherwise identifies the analyte binding moiety. In some embodiments, the conjugated oligonucleotide can include an analyte capture sequence. In some embodiments, Attorney Docket No.: 47706-0398WO1 the analyte capture sequence is capable of interacting with (e.g., hybridizing) to a capture domain of a capture probe on a substrate. In some instances, the analyte capture sequence of the capture agent barcode domain is substantially complementary to the capture domain of the capture probe. In some instances, the analyte binding moiety barcode is associated with or identifies the analyte binding moiety. In some instances, the analyte binding moiety comprises an antibody or an antigen- binding fragment thereof. In some instances, the analyte capture agent comprises a linker that couples the oligonucleotide (e.g., capture agent barcode domain) to the analyte binding moiety. In some instances, the linker is a cleavable linker. In some instances, the cleavable linker is a disulfide linker, a photo-cleavable linker, a UV-cleavable linker, or an enzyme cleavable linker. In some instances, the enzyme cleavable linker is an RNase cleavable linker. The methods provided herein can also utilize blocking probes to block the non- specific binding (e.g., hybridization) of the analyte capture sequence and the capture domain of a capture probe on an array. In some embodiments, following contact between the biological sample and the array, the biological sample is contacted with a plurality of analyte capture agents, where an analyte capture agent includes an analyte capture sequence that is reversibly blocked with a blocking probe. In some embodiments, the analyte capture sequence is reversibly blocked with more than one blocking probe (e.g., 2, 3, 4, or more blocking probes). In some embodiments, the analyte capture agent is blocked prior to binding the protein. In some embodiments, methods of protein detection using analyte capture agents can use two substrates: one with the biological sample and a second with a spatial array. In some instances, the methods include contacting the biological sample on the first substrate with a plurality of analyte capture agents, wherein the analyte binding moiety of the analyte capture agent specifically binds to the protein upon said contacting, and wherein the capture agent barcode domain comprises an analyte binding moiety barcode and an analyte capture sequence. Then, when the biological sample is aligned with at least a portion of the spatial array on the second substrate, the capture agent barcode domain hybridizes to the capture domain of the capture probe (e.g., after being released from the analyte capture agent). After, all or a part of the sequence of the capture agent barcode domain, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof, are determined in order to determine the location and/or abundance of the protein in the biological sample. Attorney Docket No.: 47706-0398WO1 In some embodiments, the methods can combine both protein and nucleic acid detection using the spatial array (i.e., locations of both proteins and nucleic acids can be determined). In some embodiments, the templated ligation probes are allowed to bind the target nucleic acid before the analyte capture agents are delivered to the biological sample. In some embodiments, the templated ligation probes can be ligated together before, concurrently, or after the analyte capture agents are delivered to the biological sample. In some embodiments, the analyte capture agents are delivered to the biological sample and the analyte binding moiety is allowed to bind the target analyte (e.g., protein) before the templated ligation probes are delivered. In some embodiments, the analyte capture agents are delivered to the biological sample and the analyte capture sequence is blocked (e.g., blocked by any of the methods described herein). In some embodiments, the analyte capture sequence of the analyte capture agents is unblocked (e.g., unblocked by any of the methods described herein) before, concurrently, or after the templated ligation probes (e.g., RNA templated ligation probes) are delivered and/or before, concurrently, or after the templated ligation probes are ligated together. iii. Post-Capture Spatial Transcriptomics Methods After capture of the analyte or the intermediate agent, further processing of the captured analyte or the intermediate agent is performed to prepare the analyte or the intermediate agent for sequencing. For instance, in some embodiments, the methods include (a) extending the captured analyte or intermediate agent, such as a connected probe (e.g., ligation product), and/or the captured oligonucleotide of an analyte capture agent, wherein the extension product that is generated includes the spatial barcode or a complement thereof, (b) releasing the extension product, or a complement thereof, from the spatial array, (c) producing a library from the released extension products or complements thereof, and (d) sequencing the library. In some embodiments, extension is performed with a polymerase (e.g., any suitable polymerase, e.g., T4 polymerase). In some embodiments, the released extension products can be prepared for downstream applications, such as generation of a sequencing library and next-generation sequencing. Producing sequencing libraries are known in the art. For example, the released extension products can be purified and collected for downstream amplification steps. The released extension products can be amplified using PCR, where primer binding sites flank the spatial barcode and ligation product or analyte binding moiety barcode, or complements Attorney Docket No.: 47706-0398WO1 thereof, generating a library associated with a particular spatial barcode. In some embodiments, the library preparation can be quantitated and/or quality controlled to verify the success of the library preparation steps. The library amplicons are sequenced and analyzed to decode spatial information and the connected probe (e.g., ligation product) or analyte binding moiety barcode, or complements thereof. In some embodiments, the methods can include a pre-amplification step. For example, a complementary strand to the extension product can be generated and further include a pre-amplification step of the extension products or complements thereof (e.g., extended products) prior to library production (e.g., RTL library production; captured oligonucleotide of the analyte capture agent production). In some embodiments, the amplicons can then be enzymatically fragmented and/or size-selected in order to provide for desired amplicon size. In some embodiments, when utilizing an Illumina® library preparation methodology, for example, P5 and P7, sequences can be added to the amplicons thereby allowing for capture of the library preparation on a sequencing flowcell (e.g., on Illumina sequencing instruments). Additionally, i7 and i5 can index sequences be added as sample indexes if multiple libraries are to be pooled and sequenced together. Further, Read 1 and Read 2 sequences can be added to the library preparation for sequencing purposes. The aforementioned sequences can be added to a library preparation sample, for example, via End Repair, A-tailing, Adaptor Ligation, and/or PCR. The cDNA fragments can then be sequenced using, for example, paired-end sequencing using TruSeq Read 1 and TruSeq Read 2 as sequencing primer sites, although other methods are known in the art. iv. Single-Cell or Single-Nuclei Sequencing Subsequent to processing a first analyte (e.g., spatial analysis of a first analyte, connected probe or other intermediate agent), the biological sample remaining on the first substrate can be further processed to analyze a second analyte in one or more cells or nuclei from the biological sample. For example, after hybridizing the intermediate agent (e.g., a connected probe such as a ligation product, or a capture handle sequence) to the capture domain of the capture probe on the second substrate, the biological sample remaining on the first substrate is subject to further processing in order to detect a second analyte in one or more cells or nuclei from the biological sample. Attorney Docket No.: 47706-0398WO1 Reagents, composition, devices and methods for detecting and/or analyzing expression of an analyte (e.g., a second analyte) in one or more cells or nuclei from a biological sample are described in CG000477, a Chromium fixed RNA profiling user guide from 10x Genomics, and US Patent. No.11,639,928; US Pat. No.11,713,457; US Pat. No. 9,410,201; US Pat. No.11,078,522; and US Pat. No.11,591,637, each of which are hereby incorporated by reference in their entirety. In some instances, the second analyte comprises RNA. In some instances, the RNA is mRNA. In some instances, the RNA is nuclear RNA. In some instances, the RNA is pre- mRNA. In some instances, the second analyte comprises DNA. In some instances, the DNA is genomic DNA. In some instances, the methods include fixing the biological sample (e.g., the cells or nuclei of the biological sample). In some instances, the biological sample (e.g., cells and/or nuclei) are fixed in formaldehyde (e.g., about 1-10% formaldehyde, such as 4% formaldehyde). In some instances, after fixing the biological sample, the biological sample is removed from the first substrate. For example, the biological sample can be removed from the substrate e.g., via scraping, optionally added to a tube, and one or more cells and/or nucleic can be isolated from the biological sample. A plurality of nucleic acid barcode molecules is introduced to the one or more cells or nuclei from the biological sample. In some cases, the nucleic acid barcode molecule (e.g., each nucleic acid barcode molecule in the plurality of nucleic acid barcode molecules) can further comprise a unique molecular identifier (UMI). In some cases, the nucleic acid barcode molecule can comprise one or more functional sequences, for example, for attachment to a sequencing flow cell, such as, for example, a P5 sequence (or a portion thereof) for Illumina® sequencing. In some cases, the nucleic acid barcode molecule or derivative thereof (e.g., an oligonucleotide or polynucleotide generated from the nucleic acid molecule) can comprise another functional sequence, such as, for example, a P7 sequence (or a portion thereof) for attachment to a sequencing flow cell for Illumina sequencing. In some cases, the nucleic acid molecule can comprise an R1 primer sequence for Illumina sequencing. In some cases, the nucleic acid molecule can comprise an R2 primer sequence for Illumina sequencing. In some cases, a functional sequence can comprise a partial sequence, such as a partial barcode sequence, partial anchoring sequence, partial sequencing primer sequence (e.g., partial R1 sequence, partial R2 sequence, etc.), a partial sequence configured to attach to the flow cell of a sequencer (e.g., partial P5 sequence, partial P7 sequence, etc.), or a partial sequence of any Attorney Docket No.: 47706-0398WO1 other type of sequence described elsewhere herein. A partial sequence may contain a contiguous or continuous portion or segment, but not all, of a full sequence, for example. In some cases, a downstream procedure may extend the partial sequence, or derivative thereof, to achieve a full sequence of the partial sequence, or derivative thereof. Examples of such nucleic acid barcode molecules (e.g., oligonucleotides, polynucleotides, etc.) and uses thereof, as may be used with compositions, devices, methods and systems of the present disclosure, are provided in U.S. Patent Application Pub. Nos. 2014/0378345 and 2015/0376609, each of which is entirely incorporated herein by reference. In some instances, the methods include generating a copy of the second analyte, a complement thereof, or the intermediate agent, or a complement thereof. In some instances, generating the copy of the second analyte, a complement thereof, or the intermediate agent, or a complement thereof, uses a polymerase or a reverse transcriptase. In some instances, the methods include hybridizing the nucleic acid barcode molecule to the complement of the second analyte, or the intermediate agent. In some instances, the nucleic acid barcode molecule comprises a hybridization region, e.g., which can be complementary to non- templated nucleotides comprised in the complement of the second analyte. In some instances, the hybridization region comprises a poly(G) sequence and the non-templated nucleotides comprise a poly(C) sequence. In some instances, the complement of the second analyte is generated by a reverse transcription reaction, e.g., using a primer and the second analyte as a template. In some instances, the nucleic acid barcode molecule comprises a hybridization region of a template switching oligonucleotide (TSO). In some instances, the hybridization region of the TSO comprises a poly(G) sequence and wherein the nucleic acid barcode molecule comprises a poly(C) sequence. In some instances, the methods include extending the nucleic acid barcode molecule using the complement of the second analyte or the intermediate agent as a template, thereby generating an extended nucleic acid barcode molecule. In some instances, the methods also include amplifying the extended nucleic acid barcode molecule. In some instances, determining the presence and/or abundance of the second analyte in the biological sample comprises determining (i) the sequence of the cell or nuclei barcode, or a complement thereof, and (ii) all or a portion of the sequence of the second analyte, or a complement thereof, or all or a portion of the sequence of the intermediate agent, or a complement thereof. In some instances, sequences (i) and (ii) are determined from (e.g., by sequencing) the extended nucleic acid barcode molecule or a complement or derivative thereof. Attorney Docket No.: 47706-0398WO1 As used herein, the term “barcoded nucleic acid molecule” generally refers to a nucleic acid molecule that results from, for example, the processing of a nucleic acid barcode molecule with a nucleic acid sequence (e.g., nucleic acid sequence complementary to a nucleic acid primer sequence encompassed by the nucleic acid barcode molecule). The nucleic acid sequence may be a targeted sequence or a non-targeted sequence. For example, in the methods and systems described herein, hybridization and reverse transcription of an analyte such as a nucleic acid molecule (e.g., a messenger RNA (mRNA) molecule) of a cell or nucleus with a nucleic acid barcode molecule (e.g., a nucleic acid barcode molecule containing a barcode sequence and a nucleic acid primer sequence complementary to a nucleic acid sequence of the nucleic acid (e.g., mRNA) molecule) results in a barcoded nucleic acid molecule that has a sequence corresponding to the nucleic acid sequence of the mRNA and the barcode sequence (or a reverse complement thereof). A barcoded nucleic acid molecule may serve as a template, such as a template polynucleotide, that can be further processed (e.g., amplified) and sequenced to obtain the target nucleic acid (e.g., mRNA or other analyte) sequence. For example, in the methods and systems described herein, a barcoded nucleic acid molecule may be further processed (e.g., amplified) and sequenced to obtain the nucleic acid sequence of the mRNA. The term “bead,” as used herein, generally refers to a particle. The bead may be a solid or semi-solid particle. The bead may be a gel bead. The gel bead may include a polymer matrix (e.g., matrix formed by polymerization or cross-linking). The polymer matrix may include one or more polymers (e.g., polymers having different functional groups or repeat units). Polymers in the polymer matrix may be randomly arranged, such as in random copolymers, and/or have ordered structures, such as in block copolymers. Cross- linking can be via covalent, ionic, or inductive, interactions, or physical entanglement. The bead may be a macromolecule. The bead may be formed of nucleic acid molecules bound together. The bead may be formed via covalent or non-covalent assembly of molecules (e.g., macromolecules), such as monomers or polymers. Such polymers or monomers may be natural or synthetic. Such polymers or monomers may be or include, for example, nucleic acid molecules (e.g., DNA or RNA). The bead may be formed of a polymeric material. The bead may be magnetic or non-magnetic. The bead may be rigid. The bead may be flexible and/or compressible. The bead may be disruptable or dissolvable. The bead may be a solid particle (e.g., a metal-based particle including but not limited to iron oxide, gold or silver) covered with a coating comprising one or more polymers. Such coating may be disruptable or dissolvable. Attorney Docket No.: 47706-0398WO1 The term “partition,” as used herein, generally, refers to a space or volume that may be suitable to contain one or more species or conduct one or more reactions. A partition may be a physical compartment, such as a droplet or well. The partition may isolate space or volume from another space or volume. The droplet may be a first phase (e.g., aqueous phase) in a second phase (e.g., oil) immiscible with the first phase. The droplet may be a first phase in a second phase that does not phase separate from the first phase, such as, for example, a capsule or liposome in an aqueous phase. A partition may comprise one or more other (inner) partitions. In some cases, a partition may be a virtual compartment that can be defined and identified by an index (e.g., indexed libraries) across multiple and/or remote physical compartments. For example, a physical compartment may comprise a plurality of virtual compartments. Provided herein are methods for sample processing and/or analysis. A method of the present disclosure may comprise barcoding one or more types of biomolecules (e.g., a nucleic acid molecule, a protein, a lipid, a carbohydrate, or a combination thereof). The biomolecule may be, for instance, a nucleic acid molecule (e.g., a ribonucleic acid (RNA) molecule) or a protein. Such a method may involve attaching one or more probes (e.g., nucleic acid probes) to the biomolecules and subsequently attaching a nucleic acid barcode molecule comprising a barcode sequence to the one or more probes. For example, the nucleic acid barcode molecule may attach to an overhanging sequence of a probe or to the end of a probe. Extension from an end of the probe to an end of the nucleic acid barcode molecule may form an extended nucleic acid molecule comprising both a sequence complementary to the barcode sequence and a sequence complementary to a target region of the nucleic acid molecule. The extended nucleic acid molecule may then be denatured from the nucleic acid barcode molecule and the nucleic acid molecule may be duplicated. One or more processes of the method may be carried out within a partition such as a droplet or well. The present disclosure also provides a method of processing a sample (e.g., a cell sample or a tissue sample) that provides a barcoded nucleic acid molecule having linked probe molecules attached thereto. The method may comprise providing a sample (e.g., a nucleus or cell) comprising a nucleic acid molecule (e.g., an RNA or DNA molecule) having a first and second target region; a first probe having a (i) first probe sequence that is complementary to the first target region and (ii) an additional probe sequence; and a second probe having a second probe sequence that is complementary to the second target region. In some instances, the first target region and the second target region are adjacent. The first and second probe sequences may also comprise first and second reactive moieties, respectively. Attorney Docket No.: 47706-0398WO1 Upon hybridization of the first probe sequence of the first probe to the first target region of the nucleic acid molecule, and hybridization of the second probe sequence of the second probe to the second target region of the nucleic acid molecule, the reactive moieties may be adjacent to one another. Subsequent reaction between the adjacent reactive moieties under sufficient conditions may link the first and second probes to yield a probe-linked nucleic acid molecule. The probe-linked nucleic acid molecule may also be referred to as a probe-ligated nucleic acid molecule or a connected probe. In other instances, the first target region and the second target region are not adjacent, and a nucleic acid reaction (e.g., a nucleic acid extension reaction, a gap-filling reaction) may be performed to yield a probe-linked nucleic acid molecule. The probe-linked nucleic acid molecule (connected probe) may be barcoded with a barcode sequence of a nucleic acid barcode molecule to provide a barcoded probe-linked nucleic acid molecule (a barcoded connected probe). Barcoding may be achieved by hybridizing a binding sequence of the nucleic acid barcode molecule to the additional probe sequence of the first probe of the probe-linked nucleic acid molecule. The barcoded probe linked-nucleic acid molecule may be subjected to amplification reactions to yield an amplified product comprising the first and second target regions and the barcode sequence or sequences complementary to these sequences. Accordingly, the method may provide amplified products without the use of reverse transcription. One or more processes may be performed within a partition such as a droplet or well. The present disclosure also provides a method of generating barcoded, probe-linked nucleic acid molecules. The method may comprise providing a sample (e.g., a nucleus or cell) comprising a nucleic acid molecule (e.g., an RNA molecule) having a first target region and a second target region; a first probe having a first probe sequence that is complementary to the first target region and optionally an additional probe sequence; and a second probe having a second probe sequence that is complementary to the second target region. The additional probe sequence of the first probe may comprise a probe capture sequence. Alternatively, or in addition to, the second probe may comprise a probe capture sequence. The first probe sequence of the first probe may hybridize to the first target region of the nucleic acid molecule, generating a probe-associated nucleic acid molecule, and a nucleic acid reaction (e.g., a nucleic acid extension reaction using a polymerase or reverse transcriptase) may be performed to generate an extended nucleic acid molecule comprising a sequence complementary to the second target region. Prior to, during, or subsequent to the nucleic acid extension reaction, the second probe may hybridize to the nucleic acid molecule (or extended Attorney Docket No.: 47706-0398WO1 nucleic acid molecule, or complement thereof), and optionally, a nucleic acid extension reaction may be performed. The extended nucleic acid molecule may be barcoded, such as by (a) hybridization of a barcode binding sequence of the nucleic acid barcode molecule to the first probe (e.g., the additional probe sequence of the first probe) or the second probe (e.g., a probe capture sequence of the second probe), or (b) via a probe binding molecule (also referred to herein as a “splint molecule” or “splint oligonucleotide”), in which the probe binding molecule comprises (i) a probe binding sequence complementary to the additional probe sequence of the first probe (which may comprise the probe capture sequence) and/or a capture sequence of the second probe and a (ii) barcode binding sequence complementary to a sequence (e.g., a common sequence) of the barcode molecule. In some instances, the barcoding may be performed prior to hybridization of the second probe to the second target region. In such cases, the barcoded nucleic acid molecule may be subjected to conditions sufficient for hybridization of the second probe sequence of the second probe to the second target region of the nucleic acid molecule (or barcoded nucleic acid molecule). A nucleic acid reaction (e.g., nucleic acid extension) may be performed, thereby generating a barcoded, probe-linked nucleic acid molecule. The methods of the present disclosure may comprise methods for generating one or more partitions such as droplets. In some instances, the partition is a droplet, microwell, or well. In some instances, the partition is a droplet. Droplets can be formed by creating an emulsion by mixing and/or agitating immiscible phases. Mixing or agitation may comprise various agitation techniques, such as vortexing, pipetting, tube flicking, or other agitation techniques. In some cases, mixing or agitation may be performed without using a microfluidic device. In some examples, the droplets may be formed by exposing a mixture to ultrasound or sonication. Systems and methods for droplet and/or emulsion generation by agitation are described in International Application Publication No. WO2020167862, which is entirely incorporated herein by reference for all purposes. In another aspect, in addition to or as an alternative to droplet-based partitioning, biological particles (e.g., cells or nuclei) may be comprised within (e.g., encapsulated within) a particulate material to form a “cell bead”. A cell bead can contain a biological particle (e.g., a cell or nucleus) or macromolecular constituents (e.g., RNA, DNA, proteins, etc.) of a biological particle. A cell bead may include a single cell or multiple cells, or a derivative of the single cell or multiple cells. For example after lysing and washing the cells, inhibitory components from cell lysates can be washed away and the macromolecular constituents can be bound as cell beads. Attorney Docket No.: 47706-0398WO1 Systems and methods disclosed herein can be applicable to both cell beads (and/or droplets or other partitions) containing biological particles and cell beads (and/or droplets or other partitions) containing macromolecular constituents of biological particles. Cell beads may be or include a cell, cell derivative, cellular material and/or material derived from the cell in, within, or encased in a matrix, such as a polymeric matrix. In some cases, a cell bead may comprise a live cell. In some instances, the live cell may be capable of being cultured when enclosed in a gel or polymer matrix, or of being cultured when comprising a gel or polymer matrix. In some instances, the polymer or gel may be diffusively permeable to certain components and diffusively impermeable to other components (e.g., macromolecular constituents). In some cases, a plurality of nucleic acid barcode molecules may be attached to a bead. The nucleic acid barcode molecules may be attached directly or indirectly to the bead. In some cases, the nucleic acid barcode molecules may be covalently linked to the bead. In some cases, the nucleic acid barcode molecules are covalently linked to the bead via a linker. In some cases, the linker is a degradable linker. In some cases, the linker comprises a labile bond configured to release said nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules. In some cases, the labile bond comprises a disulfide linkage. A nucleic acid barcode molecule may contain one or more barcode sequences. A plurality of nucleic acid barcode molecules may be coupled to a bead. The one or more barcode sequences may include sequences that are the same for all nucleic acid molecules coupled to a given bead and/or sequences that are different across all nucleic acid molecules coupled to the given bead. The nucleic acid molecule may be incorporated into the bead. Nucleic acid barcode molecules can comprise one or more functional sequences for coupling to an analyte, analyte tag such as a reporter oligonucleotide, a derivative of an analyte (e.g., cDNA). Such functional sequences can include, e.g., a template switch oligonucleotide (TSO) sequence, a primer sequence (e.g., a poly T sequence, or a nucleic acid primer sequence complementary to a target nucleic acid sequence and/or for amplifying a target nucleic acid sequence, a random primer, and a primer sequence for messenger RNA). In addition, in the case of encapsulated biological particles (e.g., a cell or a nucleus in a polymer matrix), the biological particles may be exposed to an appropriate stimulus to release the biological particles or their contents from a bead. For example, in some cases, a chemical stimulus may be co-partitioned along with an encapsulated biological particle to allow for the degradation of the bead and release of the cell or its contents into the larger partition. In some cases, this stimulus may be the same as the stimulus described elsewhere Attorney Docket No.: 47706-0398WO1 herein for release of nucleic acid molecules (e.g., oligonucleotides) from their respective bead. In alternative examples, this may be a different and non-overlapping stimulus, in order to allow an encapsulated biological particle to be released into a partition at a different time from the release of nucleic acid molecules into the same partition. For a description of methods, compositions, and systems for encapsulating cells (also referred to as a “cell bead”), see, e.g., U.S. Pat.10,428,326 and U.S. Pat. Pub.20190100632, which are each incorporated by reference in their entirety. In some instances, the nucleic acid barcode molecule is released from the particle upon application of a stimulus, optionally wherein the stimulus comprises a biological stimulus, a chemical stimulus, a thermal stimulus, an electrical stimulus, a magnetic stimulus, or a photo stimulus. In some instances, the nuclei are separated into a plurality of partitions, wherein a partition of the plurality of partitions comprises the nucleic acid barcode molecule and a nucleus of the nuclei, and wherein the method further comprises lysing the nucleus. In an aspect, the present disclosure provides a method for barcoding nucleic acid molecules. The method may generally comprise contacting a nucleic acid molecule with a pair of probes and a barcode molecule to generate a barcoded molecule (e.g., a barcoded probe-linked molecule). The nucleic acid molecule may comprise a sequence corresponding to a target sequence or a template sequence. One or more nucleic acid reactions (e.g., a ligation, a nucleic acid extension reaction, amplification, etc.) may be performed to generate the barcoded molecule. In some aspects, the method comprises: contacting a nucleic acid molecule with a first probe to generate a probe-associated nucleic acid molecule, wherein the nucleic acid molecule comprises a first target region and a second target region, wherein the first probe comprises a first probe sequence complementary to the first target region; performing a nucleic acid reaction (e.g., a nucleic acid extension reaction, e.g., by using a polymerase or reverse transcriptase, etc.) to generate an extended probe molecule comprising a sequence complementary to the second target region; providing (i) a second probe comprising a second probe sequence corresponding to or complementary to the second target region and (ii) a nucleic acid barcode molecule; and subjecting the extended probe molecule or derivative thereof to conditions sufficient to generate a barcoded molecule. The first target region and the second target region may be disposed adjacent to one another or may be separate from one another (e.g., disposed on opposite ends of a gap region). In some instances, barcoding may be facilitated by providing a probe binding molecule (also referred to herein as a “splint molecule” or in some instances, a “splint oligonucleotide”). For example, the first probe and/or the second probe may comprise a probe capture sequence, and Attorney Docket No.: 47706-0398WO1 the probe-binding molecule may comprise a probe-binding sequence complementary to the probe capture sequence. In addition to or alternatively, the nucleic acid barcode molecule may comprise a barcode sequence and a barcode capture sequence, and the probe-binding molecule may comprise a barcode binding sequence complementary to the barcode capture sequence. In some instances, the probe-binding molecule may be pre-annealed to the nucleic acid barcode molecule. Barcoding may comprise hybridization of the probe binding molecule to the probe capture sequence (or complement thereof) of the first probe and/or second probe and to the barcode capture sequence of the nucleic acid barcode molecule. Accordingly, the barcoded molecule may comprise a sequence corresponding to the first target region, a sequence corresponding to the second target region, a sequence corresponding to the probe capture sequence, and a sequence corresponding to the barcode sequence. One or more operations may be performed within a partition (e.g., droplet or well). The methods described herein may facilitate gene expression profiling with single- cell, single-nucleus or single-cell bead resolution using, for example, nucleic acid extension reactions, probe hybridization, chemical or enzymatic ligation, barcoding, amplification, and sequencing. The methods described herein may allow for gene expression analysis while avoiding the use of specialized imaging equipment and, in certain instances, reverse transcription, which may be highly error prone and inefficient. In some instances, the methods may be used to analyze a pre-determined panel of target genes in a population of single cells, nuclei, or cell beads in a sensitive and accurate manner. The methods described herein may also be useful in detecting or characterizing genetic variants, for example, in instances where the sequence of a region disposed between the target regions (e.g., a gap region) is not known. In some cases, the methods described herein may be useful in analyzing a single nucleotide polymorphism (SNP), an alternative-spliced junction, an insertion, a mutation, a deletion, a gene rearrangement (e.g., V(D)J rearrangements), a transposon, or other genetic element or variants. In some cases, the nucleic acid molecule analyzed by the methods described herein may comprise a fusion gene (e.g., a hybrid gene generated via translocation, interstitial deletion, or chromosomal inversion). In some cases, the methods described herein may be useful in analyzing genomic, transcriptomic, exomic and/or proteomic elements in cells, nuclei, cell beads, tissue samples, spatial arrays of cells, nuclei or tissues, etc. The nucleic acid molecule analyzed by the methods described herein may be a single- stranded or a double-stranded nucleic acid molecule. A double-stranded nucleic acid molecule may be completely or partially denatured to provide access to a target region (e.g., a Attorney Docket No.: 47706-0398WO1 target sequence) of a strand of the nucleic acid molecule. Denaturation may be achieved by, for example, adjusting the temperature or pH of a solution comprising the nucleic acid molecule; using a chemical agent such as formamide, guanidine, sodium salicylate, dimethyl sulfoxide, propylene glycol, urea, or an alkaline agent (e.g., NaOH); or using mechanical agitation (e.g., centrifuging or vortexing a solution including the nucleic acid molecule). The nucleic acid molecule may be a target nucleic acid molecule. The target nucleic acid molecule may be an RNA molecule. The RNA molecule may be, for example, a transfer RNA (tRNA) molecule, ribosomal RNA (rRNA) molecule, mitochondrial RNA (mtRNA) molecule, messenger RNA (mRNA) molecule, non-coding RNA molecule, synthetic RNA molecule, or another type of RNA molecule. For example, the RNA molecule may be an mRNA molecule. In some cases, the nucleic acid molecule may be a viral or pathogenic RNA. In some cases, the nucleic acid molecule may be a synthetic nucleic acid molecule previously introduced into or onto a cell. For example, the nucleic acid molecule may comprise a plurality of barcode sequences, and two or more barcode sequences may be target regions of the nucleic acid molecule. In some instances, the nucleic acid molecule is a guide RNA (gRNA), which may be exogenously introduced in a cell or cell bead. In some instances, the nucleic acid molecule is an RNA molecule derived from an exogenously introduced nucleic acid molecule, e.g., an RNA derived from a plasmid, an integrated DNA sequence (e.g., using viral transduction in a cell), a gRNA from a CRISPR genetic element, etc. The nucleic acid molecule may comprise one or more target regions. In some cases, a target region may correspond to a gene or a portion thereof. Each region may have the same or different sequences. For example, the nucleic acid molecule may comprise two target regions having the same sequence located at different positions along a strand of the nucleic acid molecule. Alternatively, the nucleic acid molecule may comprise two or more target regions having different sequences. Different target regions may be interrogated by different probes. Target regions may be located adjacent to one another or may be spatially separated along a strand of the nucleic acid molecule. The target regions may be located on the same strand or different strands. As used herein with regard to two entities, “adjacent,” may mean that the entities directly next to one other (e.g., contiguous) or in proximity to one another. For example, a first target region may be directly next to a second target region (e.g., having no other entity disposed between the first and second target regions) or in proximity to a second target region (e.g., having an intervening sequence or molecule between the first and second target regions). In some cases, a double-stranded nucleic acid molecule may Attorney Docket No.: 47706-0398WO1 comprise a target region in each strand that may be the same or different. For a nucleic acid molecule comprising multiple target regions, the methods described herein may be performed for one or more target regions at a time. For example, a single target region of the multiple target regions may be analyzed (e.g., as described herein) or two or more target regions may be analyzed at the same time. Analyzing two or more target regions may involve providing two or more probes, where a first probe has a sequence that is complementary to the first target region, a second probe has a sequence that is complementary to the second target region, etc. Each probe (e.g., the first probe and the second probe) may further comprise one or more additional sequences (e.g., additional probe sequences, unique molecular identifiers (UMIs), a barcode sequence, a primer sequence, a capture sequence, or other functional sequence). For example, in some instances, the first probe and/or the second probe may comprise the same or different barcode sequences. In some examples, the first probe and the second probe may be configured to hybridize to one or more nucleic acid barcode molecules. For example, the first probe and/or the second probe may comprise a probe capture sequence, which may be configured to hybridize to a nucleic acid barcode molecule or to a probe binding molecule (e.g., a splint oligonucleotide) that is configured to hybridize to a nucleic acid barcode molecule (e.g., via a barcode binding sequence that is complementary to a capture sequence of the nucleic acid barcode molecule). The probe capture sequence may be any useful length; for example, the probe capture sequence may be about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100 or more nucleotides in length. The probe capture sequence may be at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100 or more nucleotides in length. The probe capture sequence may be at most 100, at most 90, at most 80, at most 70, at most 60, at most 50, at most 40, at most 30, at most 20, at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1 nucleotide in length. A range of lengths of the probe capture sequence, such as from about 8 to about 50 nucleotides in length, etc. In some instances, the probe capture sequence length may be varied based on any useful application and properties, e.g., melting temperature, annealing temperature, annealing strength (e.g., GC content), hybridization stringency, etc. Similarly, the probe binding molecule and nucleic acid barcode molecule may further comprise one or more additional sequences (e.g., unique molecular identifiers (UMIs), a Attorney Docket No.: 47706-0398WO1 barcode sequence, a primer sequence, a capture sequence, or other functional sequence). For example, in some instances, the probe binding molecule or barcode molecule may comprise a functional sequence, a primer sequence (e.g., sequencing primer sequence or partial sequencing primer sequence), a UMI, etc. The probe binding molecule and the nucleic acid barcode molecule may be any useful length; for example, either or both may be about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100 or more nucleotides in length. The probe binding molecule or the barcode molecule may be at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100 or more nucleotides in length. The probe capture binding molecule or the barcode molecule may be at most 100, at most 90, at most 80, at most 70, at most 60, at most 50, at most 40, at most 30, at most 20, at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1 nucleotide in length. A range of lengths of the probe binding molecule or barcode molecule may be used, such as from about 16 to about 100 nucleotides in length, etc. In some instances, the probe binding molecule or barcode molecule length may be varied based on any useful application and properties, e.g., melting temperature, annealing temperature, etc. In some instances, the first target region and the second target region of the nucleic acid molecule are not adjacent. For instance, the first target region and the second target region may be separated by one or more gap regions disposed between the first target region and the second target region. The gap region may comprise, for example, at least one nucleotide base, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, or more bases. The gap region may comprise at most about 1000, at most about 500, at most about 400, at most about 300, at most about 200, at most about 100, at most about 90, at most about 80, at most about 70, at most about 60, at most about 50, at most about 40, at most about 30, at most about 20, at most about 10, or at most about 5 bases. The gap region may comprise a range of number of bases, such as between about 1 and 30 bases. Access to a nucleic acid molecule included in a cell, nucleus, or cell bead may be provided by lysing or permeabilizing the cell or nucleus. Lysing the cell, nucleus or cell bead may release the nucleic acid molecule contained therein from the cell, nucleus, or cell bead. Attorney Docket No.: 47706-0398WO1 A cell or nucleus may be lysed using a lysis agent such as a bioactive agent. A bioactive agent useful for lysing a cell or nucleus may be, for example, an enzyme (e.g., as described herein). An enzyme used to lyse a cell or nucleus may or may not be capable of carrying out additional functions such as degrading, extending, reverse transcribing, or otherwise altering a nucleic acid molecule. Alternatively, an ionic or non-ionic surfactant such as TritonX-100, Tween 20, sarcosyl, or sodium dodecyl sulfate may be used to lyse a cell or nucleus. Cell/nucleus lysis may also be achieved using a cellular disruption method such as an electroporation or a thermal, acoustic, or mechanical disruption method. Alternatively, a cell or nucleus may be permeabilized to provide access to a nucleic acid molecule included therein. Permeabilization may involve partially or completely dissolving or disrupting a cell/nuclear membrane or a portion thereof. Permeabilization may be achieved by, for example, contacting a cell membrane with an organic solvent (e.g., methanol) or a detergent such as Triton X-100 or NP-40. The cell, nucleus or cell bead may be fixed, as described elsewhere herein. The probe-linked nucleic acid molecule may be barcoded to provide a barcoded probe-linked nucleic acid molecule, or barcoding may occur prior to generation of the probe- linked nucleic acid molecule. Barcoding may be performed using a variety of techniques. For example, the first probe or the second probe may comprise a probe capture sequence. The nucleic acid barcode molecule may comprise a barcode capture sequence capable of hybridizing to the probe capture sequence. Alternatively, barcoding may be mediated by a probe binding molecule (e.g., a splint oligonucleotide) comprising (i) a probe binding sequence, which may be complementary to the probe capture sequence of the first probe or the second probe, and (ii) a barcode binding sequence, which may be complementary to the barcode capture sequence of the nucleic acid barcode molecule. In some instances, the barcoding may be followed by ligation, e.g., chemically or enzyme-mediated, to covalently link the nucleic acid barcode molecule to the probe (or to the probe binding sequence, and the probe binding sequence may be ligated to the probe). Examples of chemical ligation of nucleic acid molecules may include “click chemistry” approaches, e.g., reaction of azide and alkyne moieties, as described in U.S. Pat. Pub. No.2020/0239874, which is incorporated by reference herein in its entirety. In some aspects, the first probe and/or the second probe; of each probe pair, may comprise a barcode that acts as a sample-specific barcode. A second barcode may be introduced during partitioning, as described herein, which acts as a partition specific barcode, such that the probe pairs, after ligation. In some aspects, multiple samples can be prepared at the same time, with each sample receiving probe pairs with barcodes Attorney Docket No.: 47706-0398WO1 specific to each sample. After hybridization of the probes to target regions of interest of the nucleic acid sequences of interest, and further after partitioning of the cells within partitions and the oligonucleotides comprising partition-specific barcodes, the output nucleic acid sequences comprise complements thereof or copies of the probe pair and further comprises the sample-specific barcode and the partition specific barcode, thus enabling users to multiplex multiple samples, pool together, process, and sequence, and demultiplex the determined sequences. By way of example, the first probe may comprise a first probe sequence and a probe capture sequence, and the first probe may be subjected to conditions sufficient to hybridize the first probe sequence to the first target region, thereby generating a probe-associated nucleic acid molecule. In some instances, the probe-associated nucleic acid molecule may be subjected to washing or other conditions to remove unannealed probes from a mixture. The probe-associated nucleic acid molecule may be extended from an end of the first probe towards an end of the nucleic acid molecule to which it is hybridized (towards the end which is proximal to the second target region) to provide an extended nucleic acid molecule. The extended nucleic acid barcode molecule may comprise the first probe sequence and a complement to the second target region. In some instances, the extended nucleic acid molecule may be barcoded, e.g., by hybridizing the barcode capture sequence of the nucleic acid barcode molecule to the probe capture sequence, or by hybridizing (i) a probe-binding molecule comprising a probe binding sequence and a barcode binding sequence to the probe capture sequence and (ii) the barcode capture sequence of the nucleic acid barcode molecule to the barcode binding sequence of the probe binding molecule. In some instances, the probe- binding molecule may be provided pre-annealed to the nucleic acid barcode molecule. Subsequently, a second probe comprising a second probe sequence may be provided. The barcoded, extended nucleic acid molecule may be subjected to conditions sufficient to hybridize the second probe sequence to the second target region or complement thereof. A nucleic acid extension reaction may be performed, thereby generating a barcoded molecule (e.g., barcoded probe-linked molecule) comprising a sequence corresponding to the first target region, a sequence corresponding to the second target region, a sequence corresponding to the probe capture sequence, and a sequence corresponding to the barcode sequence. v. Systems and methods for sample compartmentalization In an aspect, the systems and methods described herein provide for the compartmentalization, depositing, or partitioning of one or more particles (e.g., biological Attorney Docket No.: 47706-0398WO1 particles, macromolecular constituents of biological particles, beads, reagents, etc.) into discrete compartments or partitions (referred to interchangeably herein as partitions), where each partition maintains separation of its own contents from the contents of other partitions. The partition can be a droplet in an emulsion or a well. A partition may comprise one or more other partitions. A partition may include one or more particles. A partition may include one or more types of particles. For example, a partition of the present disclosure may comprise one or more biological particles and/or macromolecular constituents thereof. A partition may comprise one or more beads. A partition may comprise one or more gel beads. A partition may comprise one or more cell beads. A partition may include a single gel bead, a single cell bead, or both a single cell bead and single gel bead. A partition may include one or more reagents. Alternatively, a partition may be unoccupied. For example, a partition may not comprise a bead. A cell bead can be a biological particle and/or one or more of its macromolecular constituents encased inside of a gel or polymer matrix, such as via polymerization of a droplet containing the biological particle and precursors capable of being polymerized or gelled. Unique identifiers, such as barcodes, may be injected into the droplets previous to, subsequent to, or concurrently with droplet generation, such as via a support (e.g., bead), as described elsewhere herein. The methods and systems of the present disclosure may comprise methods and systems for generating one or more partitions such as droplets. The droplets may comprise a plurality of droplets in an emulsion. In some examples, the droplets may comprise droplets in a colloid. In some cases, the emulsion may comprise a microemulsion or a nanoemulsion. In some examples, the droplets may be generated with aid of a microfluidic device and/or by subjecting a mixture of immiscible phases to agitation (e.g., in a container). In some cases, a combination of the mentioned methods may be used for droplet and/or emulsion formation. Droplets can be formed by creating an emulsion by mixing and/or agitating immiscible phases. Mixing or agitation may comprise various agitation techniques, such as vortexing, pipetting, tube flicking, or other agitation techniques. In some cases, mixing or agitation may be performed without using a microfluidic device. In some examples, the droplets may be formed by exposing a mixture to ultrasound or sonication. Systems and methods for droplet and/or emulsion generation by agitation are described in International Application No. PCT/US20/17785, which is entirely incorporated herein by reference for all purposes. Attorney Docket No.: 47706-0398WO1 Microfluidic devices or platforms comprising microfluidic channel networks (e.g., on a chip) can be utilized to generate partitions such as droplets and/or emulsions as described herein. Methods and systems for generating partitions such as droplets, methods of encapsulating biological particle methods of increasing the throughput of droplet generation, and various geometries, architectures, and configurations of microfluidic devices and channels are described in U.S. Patent Publication Nos.2019/0367997 and 2019/0064173, each of which is entirely incorporated herein by reference for all purposes. In some examples, individual particles can be partitioned to discrete partitions by introducing a flowing stream of particles in an aqueous fluid into a flowing stream or reservoir of a non-aqueous fluid, such that droplets may be generated at the junction of the two streams/reservoir, such as at the junction of a microfluidic device provided elsewhere herein. The methods of the present disclosure may comprise generating partitions and/or encapsulating particles, such as biological particles, in some cases, individual biological particles such as single cells, nuclei or cell beads. In some examples, reagents may be encapsulated and/or partitioned (e.g., co-partitioned with biological particles) in the partitions. Various mechanisms may be employed in the partitioning of individual particles. An example may comprise porous membranes through which aqueous mixtures of cells may be extruded into fluids (e.g., non-aqueous fluids). Beads Nucleic acid barcode molecules may be delivered to a partition (e.g., a droplet or well) via a solid support or carrier (e.g., a bead). In some cases, nucleic acid barcode molecules are initially associated with the solid support and then released from the solid support upon application of a stimulus, which allows the nucleic acid barcode molecules to dissociate or to be released from the solid support. In specific examples, nucleic acid barcode molecules are initially associated with the solid support (e.g., bead) and then released from the solid support upon application of a biological stimulus, a chemical stimulus, a thermal stimulus, an electrical stimulus, a magnetic stimulus, and/or a photo stimulus. A nucleic acid barcode molecule may contain a barcode sequence and a functional sequence, such as a nucleic acid primer sequence or a template switch oligonucleotide (TSO) sequence. The solid support may be a bead. A solid support, e.g., a bead, may be porous, non- porous, hollow (e.g., a microcapsule), solid, semi-solid, and/or a combination thereof. Beads may be solid, semi-solid, semi-fluidic, fluidic, and/or a combination thereof. In some Attorney Docket No.: 47706-0398WO1 instances, a solid support, e.g., a bead, may be at least partially dissolvable, disruptable, and/or degradable. In some cases, a solid support, e.g., a bead, may not be degradable. In some cases, the solid support, e.g., a bead, may be a gel bead. A gel bead may be a hydrogel bead. A gel bead may be formed from molecular precursors, such as a polymeric or monomeric species. A semi-solid support, e.g., a bead, may be a liposomal bead. Solid supports, e.g., beads, may comprise metals including iron oxide, gold, and silver. In some cases, the solid support, e.g., the bead, may be a silica bead. In some cases, the solid support, e.g., a bead, can be rigid. In other cases, the solid support, e.g., a bead, may be flexible and/or compressible. A partition may comprise one or more unique identifiers, such as barcodes. Barcodes may be previously, subsequently or concurrently delivered to the partitions that hold the compartmentalized or partitioned biological particle. For example, barcodes may be injected into droplets or deposited in microwells previous to, subsequent to, or concurrently with droplet generation or providing of reagents in the microwells, respectively. The delivery of the barcodes to a particular partition allows for the later attribution of the characteristics of the individual biological particle to the partition. Barcodes may be delivered, for example on a nucleic acid molecule (e.g., an oligonucleotide), to a partition via any suitable mechanism. Barcoded nucleic acid molecules can be delivered to a partition via a support (e.g., a bead). A support, in some instances, can comprise a bead. Beads are described in further detail below. In some cases, barcoded nucleic acid molecules can be initially associated with the support (e.g., bead) and then released from the support. Release of the barcoded nucleic acid molecules can be passive (e.g., by diffusion from or out of the support). In addition, or alternatively, release from the support can be upon application of a stimulus which allows the barcoded nucleic acid nucleic acid molecules to dissociate or to be released from the support (e.g., bead). Such stimulus may disrupt the support, an interaction that couples the barcoded nucleic acid molecules to or within the support, or both. Such stimulus can include, for example, a thermal stimulus, photo-stimulus, chemical stimulus (e.g., change in pH or use of a reducing agent(s)), a mechanical stimulus, a radiation stimulus; a biological stimulus (e.g., enzyme), or any combination thereof. Methods and systems for partitioning barcode carrying beads into droplets are provided in US. Patent Publication Nos.2019/0367997 and 2019/0064173, and International Application No. PCT/US20/17785, each of which is herein entirely incorporated by reference for all purposes. In some examples, beads, biological particles, and droplets may flow along channels (e.g., the channels of a microfluidic device), in some cases at substantially regular flow Attorney Docket No.: 47706-0398WO1 profiles (e.g., at regular flow rates). Such regular flow profiles may permit a droplet to include a single bead and a single biological particle. Such regular flow profiles may permit the droplets to have an occupancy (e.g., droplets having beads and biological particles) greater than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. Such regular flow profiles and devices that may be used to provide such regular flow profiles are provided in, for example, U.S. Patent Publication No.2015/0292988, which is entirely incorporated herein by reference. A bead may be porous, non-porous, solid, semi-solid, semi-fluidic, fluidic, and/or a combination thereof. In some instances, a bead may be dissolvable, disruptable, and/or degradable. In some cases, a bead may not be degradable. In some cases, the bead may be a gel bead. A gel bead may be a hydrogel bead. A gel bead may be formed from molecular precursors, such as a polymeric or monomeric species. A semi-solid bead may be a liposomal bead. Solid beads may comprise metals including iron oxide, gold, and silver. In some cases, the bead may be a silica bead. In some cases, the bead can be rigid. In other cases, the bead may be flexible and/or compressible. A bead may be of any suitable shape. Examples of bead shapes include, but are not limited to, spherical, non-spherical, oval, oblong, amorphous, circular, cylindrical, and variations thereof. A bead may comprise natural and/or synthetic materials. For example, a bead can comprise a natural polymer, a synthetic polymer or both natural and synthetic polymers. In some aspects, a bead may be a gel bead, such as a hydrogel bead. Examples of natural polymers include proteins and sugars such as deoxyribonucleic acid, rubber, cellulose, starch (e.g., amylose, amylopectin), proteins, enzymes, polysaccharides, silks, polyhydroxyalkanoates, chitosan, dextran, collagen, carrageenan, ispaghula, acacia, agar, gelatin, shellac, sterculia gum, xanthan gum, Corn sugar gum, guar gum, gum karaya, agarose, alginic acid, alginate, or natural polymers thereof. Examples of synthetic polymers include acrylics, nylons, silicones, spandex, viscose rayon, polycarboxylic acids, polyvinyl acetate, polyacrylamide, polyacrylate, polyethylene glycol, polyurethanes, polylactic acid, silica, polystyrene, polyacrylonitrile, polybutadiene, polycarbonate, polyethylene, polyethylene terephthalate, poly(chlorotrifluoroethylene), poly(ethylene oxide), poly(ethylene terephthalate), polyethylene, polyisobutylene, poly(methyl methacrylate), poly(oxymethylene), polyformaldehyde, polypropylene, polystyrene, poly(tetrafluoroethylene), poly(vinyl acetate), poly(vinyl alcohol), poly(vinyl chloride), poly(vinylidene dichloride), poly(vinylidene difluoride), poly(vinyl fluoride) and/or Attorney Docket No.: 47706-0398WO1 combinations (e.g., co-polymers) thereof. Beads may also be formed from materials other than polymers, including lipids, micelles, ceramics, glass-ceramics, material composites, metals, other inorganic materials, and others. In some cases, the nucleic acid molecule can comprise a functional sequence, for example, for attachment to a sequencing flow cell, such as, for example, a P5 sequence (or a portion thereof) for Illumina® sequencing. In some cases, the nucleic acid molecule or derivative thereof (e.g., oligonucleotide or polynucleotide generated from the nucleic acid molecule) can comprise another functional sequence, such as, for example, a P7 sequence (or a portion thereof) for attachment to a sequencing flow cell for Illumina sequencing. In some cases, the nucleic acid molecule can comprise a barcode sequence. In some cases, the nucleic acid molecule can further comprise a unique molecular identifier (UMI). In some cases, the nucleic acid molecule can comprise an R1 primer sequence for Illumina sequencing. In some cases, the nucleic acid molecule can comprise an R2 primer sequence for Illumina sequencing. In some cases, a functional sequence can comprise a partial sequence, such as a partial barcode sequence, partial anchoring sequence, partial sequencing primer sequence (e.g., partial R1 sequence, partial R2 sequence, etc.), a partial sequence configured to attach to the flow cell of a sequencer (e.g., partial P5 sequence, partial P7 sequence, etc.), or a partial sequence of any other type of sequence described elsewhere herein. A partial sequence may contain a contiguous or continuous portion or segment, but not all, of a full sequence, for example. In some cases, a downstream procedure may extend the partial sequence, or derivative thereof, to achieve a full sequence of the partial sequence, or derivative thereof. Examples of such nucleic acid molecules (e.g., oligonucleotides, polynucleotides, etc.) and uses thereof, as may be used with compositions, devices, methods and systems of the present disclosure, are provided in U.S. Patent Pub. Nos.2014/0378345 and 2015/0376609, each of which is entirely incorporated herein by reference. IV. EXAMPLES 1. EXAMPLE 1 – General workflow of spatial analysis and nuclei sequencing A detailed workflow of the methods provided herein is depicted in FIG.12. Briefly, the procedure starts by performing spatial transcriptomics methods. Upon completion of the spatial transcriptomics procedure and connected probe release, libraries are prepared. The Attorney Docket No.: 47706-0398WO1 biological sample is fixed with 4% formaldehyde to enhance the resilience of the nuclei for downstream processing. More specifically, three different samples were used. First, a fresh-frozen mouse brain sample was sectioned at 12 µm thickness. Two consecutive sections were placed within a pre-marked 11x11 mm area on a glass slide. Slides with sections were stored at -80°C prior to processing with the combinatorial protocol (spatial transcriptomics and single-nucleus sequencing). Second, fresh-frozen breast cancer samples were sectioned at 18 µm thickness. Each breast cancer section was placed into a pre-marked 11x11mm area on a glass slide and stored at -80°C prior to processing with the combinatorial protocol. Third, FFPE breast cancer samples were sectioned at 5 µm thickness, placed on a glass slide (SuperFrost^TM Plus Gold), dewaxed, H&E stained, imaged, and hard-cover with a glass coverslip. The slides (SuperFrost^TM Plus slides) with fresh-frozen sections were incubated on a pre-heated thermal cycler at 37°C for 1 minute, followed by fixation with 4% paraformaldehyde for 10 minutes at room temperature. Upon completing hematoxylin and eosin staining and imaging, all the SuperFrost^TM slides, including slides with FFPE samples, were placed into cassettes and tissue sections were incubated with pre-hybridization mix for 15 minutes at room temperature. The pre-hybridization mix was replaced with probe hybridization mix and incubated at 50°C overnight. A post-hybridization wash was performed, followed by probe ligation and a post-ligation wash. The ligation probes were released and hybridized to capture domains of capture probes on a spatial array on a second substrate using the Visium CytAssist from 10x Genomics. After hybridization, the second substrate was incubated with a probe extension mix. Probes were eluted and each sample was collected into a tube for pre-amplification step. Next, samples were cleaned using SPRIselect beads and indexed via PCR reaction. After indexing, samples underwent a second cleanup using SPRIselect beads and the measurement of the concentration and length of the final libraries was performed prior to sequencing. The final libraries were sequenced on Nextseq2000 (Illumina) platform. Length of read 1 and read 2 were 28 base pairs and 50 base pairs, respectively. After migrating the ligation products to the capture probes, the remaining tissue sections left on the SuperFrost^TM slides were re-fixed with 4% paraformaldehyde for 10-15 minutes at room temperature, washed with 1xPBS and scraped using cell scraper with 50 µl of nuclease free water into a PCR tube pre-coated with 1xPBS + 2% BSA solution. Next, each sample was mixed with the probe hybridization mix according to the 10x Genomics protocol for Chromium Fixed single cell or single nuclear RNA profiling (CG000477), Attorney Docket No.: 47706-0398WO1 including 20 µl of DNA probes targeting the human or mouse whole transcriptome, and incubated overnight at 42°C. On the following day, each sample was combined with 175 µl of post-hybridization wash buffer and transferred to a 1.5 ml tube. The remaining post-hybridization solution volume was added to reach a total volume of 900 µl. Subsequently, the sample was incubated at 42°C for 10 minutes and centrifuged at 850 rcf for 10 minutes at room temperature. This step was repeated once, resulting in a total of two washes. Finally, the cell pellet was resuspended in 500 µl of Resuspension buffer. Next, nuclei were extracted. All tubes and pipette tips were coated with 1xPBS + 2% BSA solution. The isolated breast cancer samples were placed on ice and 300 µl of lysis buffer (10mM Tris-HCl, 10mM NaCl, 3mM MgCl₂, 0.1% Igepal, 1mM DTT, 1U/µl RNase Inhibitor) was added to the sample and pestle was used to homogenize the tissue. After homogenization with a pestle, 700 µl of lysis buffer was added to the sample followed by incubation on ice for 12 minutes for breast cancer samples. The sample was gently pipette-mixed a few times during the incubation period. The mouse brain sample pellet was directly homogenized using a pestle and pipette mixing without lysis. All the samples were then passed through a 70 µm strainer or 50 µm strainer to remove tissue debris. The strainer was washed with ~200 µl of lysis buffer to minimize sample loss. This procedure was repeated with a 20 µm strainer, including the strainer washing step with lysis buffer. Nuclei were centrifuged at 500 rcf for 10 minutes at 4°C. The pellet containing nuclei was resuspended in 200 µl of 1xPBS + 2% BSA solution, stained with DAPI and sorted using Fluorescence-activated Cell Sorting (FACS). Sorted nuclei were centrifuged at 500 rcf for 10 min at 4°C, resuspended in post-hybridization resuspension buffer, counted using Countess II automated cell counter using the DAPI channel. Aiming for a retrieval of 10,000 nuclei, nuclei were then diluted accordingly and mixed with enzymes. Partitioning oil and gel beads were used to form tens of thousands of partitions each containing a single cell or nucleus and a single gel bead using the Chromium X system from 10x Genomics. Recovered gel beads- in-emulsion were incubated in a thermocycler according to the protocol, followed by gel beads recovery and pre-amplification PCR. Then, the resulting sample was cleaned using SPRIselect beads, indexed via PCR reaction and cleaned again with SPRIselect beads. The protocol resulted in the production of high-quality single nuclei RNA-seq libraries. The final libraries were sequenced on Nextseq2000 (Illumina) platform. Length of read 1 and read 2 were 28 base pairs and 90 base pairs, respectively. Attorney Docket No.: 47706-0398WO1 2. EXAMPLE 2 – Quality control comparison of methods combining spatial analysis and single nuclei sequencing in fixed-frozen mouse brain tissue To assess the performance of the methods described in Example 1, experiments were performed using mouse brain tissue.12 µm-thick coronal sections of a fixed-frozen (FF) mouse brain sample were placed onto two SuperFrost^TM slides, each area spanning a size of roughly 11x11mm, as shown in FIG.13A. As described in Example 1, each section was fixed with 4% formaldehyde for 10 minutes prior to the templated ligation and hybridization to the capture probes on the spatial array. The templated ligation probes are capable of targeting and capturing 18,085 protein-coding genes. Following hybridization, capture, and probe release, the sections on the SuperFrost^TM Plus slides were re-fixed using 4% formaldehyde and scraped into a tube to generate snRNA-seq libraries. ~30,000-50,000 nuclei per sample were obtained after FACS and 10,000 nuclei per sample was targeted by loading 16,500 nuclei into the Chromium system (10x Genomics). It was observed that 8,000-12,000 reads/nucleus were sufficient to detect nuclear transcriptomes with 1,700-2,500 median genes/nucleus and 2,500-4,400 UMI counts/nucleus in each of the experiments respectively. In parallel, high-quality spatial transcriptomics data was generated from the same sections (FIGs.13B-13C). Over 5000 median gene/spot and 14,679-17,580 UMI counts/spot, respectively, were obtained. Additional data for these samples are provided in Table 1 below. Table 1. Spatial transcriptomics data from mouse brain technical replicates. Mouse Brain Replicate 1 Mouse Brain Replicate 2

Attorney Docket No.: 47706-0398WO1 Reads Mapped Confidently to 98.1% 98.0% Probe Set

e mouse brain was used. Standard single-cell protocols recommend isolation of more than 500,000 cells or nuclei for its execution. However, for this experiment, a single 12 µm-thick mouse brain section was enough to generate quality single nuclei data from 10,267 cells. Standard single nuclei RNA-seq analysis procedures were performed, and these methods included removing nuclei with few genes per nucleus, here less than 400, and correcting for doublets, among other steps. Overall, a high correlation in quality control metrics was observed between the combinatorial methods described herein and a standard single nuclei protocol, including distributions of median number of unique genes (FIG.13D, left panel) and number of UMI counts (FIG.13D, right panel) per nucleus visualized as violin plots; number of genes and UMIs per nuclei visualized as a scatter plot for each sample (FIG.13E); and as evaluated via gene-gene scatter plots between the combinatorial methods described herein and a standard single nuclei protocol (FIG. 13F). These results indicate that the present protocol effectively captures single nuclei gene expression even when using significantly less input material and performing spatial transcriptomics on the same tissue section prior to nuclei isolation, which is further supported by the comparable results obtained from both protocols. (FIGs.13D-13F) Following the initial quality checks, integration of the single nuclei data part and the public, single-cell data, as well as clustering was performed, resulting in identification of 41 clusters (Louvain algorithm, res 0.8). As shown in FIG.14 and FIG.15, shared marker genes for each cluster in both generated single nuclei data and control-group single-cell datasets were visualized through dot plots. These results suggest acceptable similarity between the two datasets, indicating comparable performance of these methods in terms of detecting gene expression differences across different cell types. Next, in order to get a quick rough cell-type annotation of the single-nuclei data. Already annotated publicly available mouse brain scRNAseq data (Zeisel et al., Cell, 2018 Aug 9;174(4):999-1014.e22) was utilized to automatically annotate the single-nuclei data generated using the combinatorial method by the function Label Transfer function (TranferData) in the widely used R package Seurat. As the original publicly available data set contained different Attorney Docket No.: 47706-0398WO1 granularity of cell-type annotations, label transfer on two selected resolutions (Subclass and Taxonomy level 4) was performed to cover both broader cell-type annotations as well as finer cell-type annotations. As shown in FIG. 20, a broad variety of neurons, microglia and astrocytes among others also were found, demonstrating the effectiveness and reliability of the combinatorial methods in dissecting the multilayered intricacies of brain function and pathology from tissue sections. Non-negative matrix factorization (NNMF or NMF) was performed as it can be a useful technique to decompose spatial transcriptomics data because of its ability to find gene expression signatures that are associated with different spatial patterns or cell types even when there is a mixture of cells in spots. This is particularly true when a reference single-cell RNA- seq data is not available to conduct cell type deconvolution. In particular, it is expected that factors map to clear morphological structures as well as distinct neuronal layers in the cortex that is a well-known characteristic of the mouse brain will be seen. As shown in FIGs. 16A- 16P, factors map to distinct morphological regions as well as different layers of the cortex. Next, to explore localization of cell types within the tissue, the single nuclei gene expression data obtained from the combinatorial methods described herein was utilized to deconvolve the spatial transcriptomics data in order to visualize spatial distribution of various cell types. By using a well-matched single cell or single nuclei dataset as reference, the cell- type proportions in each spot was able to be estimated. In this context, deconvolution allows a user to infer the proportions of previously identified cell types in each spot based on their unique gene expression signatures, providing a comprehensive map of cellular diversity that links identity and function with location. This approach has the potential to unlock new insights into how cellular composition, localization and spatial organization contribute to the function or disease in various tissue types. As shown in FIGs. 17A-17C, the broader cell-type annotations were used for deconvolution with the proportions of neurons, oligodendrocytes as well as Ttr (type of ependymal cells that express the secreted protein Transthyretin) shown. As expected, the neurons are mostly present in the CA1, CA3 and the dentate gyrus area of the hippocampus as well as the cortex while oligodendrocytes correctly map to the white matter of the brain. Ttr also correctly maps to the ventricles (Choroid plexus) where they are usually found. Further, as shown in FIGs.18A-18H, the finer cell-type annotations were used for deconvolution and different cell types as well as different neurons were mapped to distinct morphological areas of the brain. Attorney Docket No.: 47706-0398WO1 Annotations from another public scRNAseq dataset, Allen Brain Atlas Mouse Cortex, was also transferred using label transfer to the single nuclei data generated using the present method. The cortex area of the brain was evaluated here and as shown in FIG.19, the resulting cell-type proportions of neurons originating from different layers in the cortex mapped to their corresponding region in distinct layers. See e.g., Yao et al. Cell.2021 Jun 10; 184(12): 3222– 3241.e26, which is incorporated by reference in its entirety. 3. EXAMPLE 3 – Spatial analysis and single nuclei sequencing in fixed-frozen and FFPE breast cancer tissue samples In order to test the applicability of the combinatorial methods described in Example 1 in other tissue types, fresh-frozen (FF) and formalin-fixed paraffin embedded (FFPE) breast cancer (BC) samples were investigated. 18 µm commercial BC samples from two different patient blocks and 5 µm clinically available FFPE BC samples from two different patients were sectioned. Matched spatial transcriptomics data and single nuclei sequencing data were generated (as described in Example 1). A total of ~300,000 FF nuclei and ~450,000 FFPE nuclei were obtained across the 4 and 2 tissue sections respectively after FACS.40,000 FF nuclei and 20,000 FFPE nuclei were targeted, respectively, aiming for 10K cell recovery per section. FF BC samples averaged 5,000 reads per nucleus, and single nuclei sequencing generated nuclear transcriptomes with a median of 1,200 genes and 1,800 UMI counts per nucleus for each respective patient (FIGs.21A-21B). In parallel high quality spatial transcriptomics data was generated from the same sections averaging 4700 median gene/spot and 16,800 UMI counts/spot respectively. FFPE samples averaged 11,900 reads per nucleus, with a median of 884 genes and 1,141 UMI counts per nucleus for each respective patient (FIG.21C). In parallel high quality spatial transcriptomics data from the same sections was also generated, averaging 5,000 median gene/spot and 18,000 UMI counts/spot respectively. To assess the performance of the BC dataset using the combinatorial methods of spatial transcriptomics and single nuclei sequencing, a standard breast cancer dataset generated by single cell RNA-seq was used. Given potential batch effects, combinatorial analysis procedures, similar to those employed for the mouse brain data in Example 2, were executed. These procedures were fine-tuned for each section and included steps like excluding nuclei with fewer than 400 genes and rectifying doublet errors, among others. Attorney Docket No.: 47706-0398WO1 For the cluster annotation of the combinatorial BC dataset, a publicly available annotated BC dataset was leveraged, observing that the cells were successfully matched and annotated using this reference (FIGs. 22A-22F). This consistent annotation underscores the efficacy of the combinatorial approach, suggesting that the combinatorial dataset captures a comprehensive range of cellular profiles despite the low amount of the input material, consistent with previously characterized profiles in breast cancer research. The methods demonstrated in these examples offer an unparalleled view of gene activity across diverse tissues. Its adaptability to various tissue types, preservation methods, and thicknesses underlines its versatility in biomedical research. Reliable correlations between datasets and consistent annotations attest to its precision and reliability. Notably, its proficiency spans from elucidating mouse brain intricacies to decoding breast cancer complexities. The method's economic edge, using cost-effective nuclei isolation reagents and catering to low- input samples, enhances its appeal. In sum, the methods discussed herein stand out as a versatile and trustworthy tool, and are useful to decipher complex diseases like cancer. OTHER EMBODIMENTS It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Attorney Docket No.: 47706-0398WO1 WHAT IS CLAIMED IS: 1. A method for processing multiple analytes in a biological sample mounted on a first substrate, the method comprising: (a) hybridizing a first probe and a second probe to a first analyte in the biological sample, wherein the first probe and the second probe each comprise a nucleic acid sequence that is substantially complementary to a nucleic acid sequence of the first analyte, and wherein the second probe comprises a capture probe binding domain; (b) coupling the first probe and the second probe, thereby generating a connected probe; (c) 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; (d) releasing the connected probe from the first analyte when at least a portion of the biological sample is aligned with at least a portion of the array; (e) hybridizing the connected probe to the capture domain of the capture probe; (f) isolating one or more cells or nuclei from the biological sample on the first substrate, wherein the one or more cells or nuclei comprise a second analyte; and (g) hybridizing a nucleic acid barcode molecule to the second analyte, a complement thereof, or an intermediate agent of the second analyte. 2. A method for processing multiple analytes in a biological sample mounted on a first substrate, the method comprising: (a) hybridizing a first probe and a second probe to a first analyte in the biological sample, wherein the first probe and the second probe each comprise a nucleic acid sequence that is substantially complementary to a nucleic acid sequence of the first analyte, and wherein the second probe comprises a capture probe binding domain; (b) coupling the first probe and the second probe, thereby generating a connected probe; (c) 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; Attorney Docket No.: 47706-0398WO1 (d) hybridizing the connected probe to the capture domain of the capture probe; (e) isolating one or more cells or nuclei from the biological sample, wherein the one or more cells or nuclei comprise a second analyte; and (f) hybridizing a nucleic acid barcode molecule to the second analyte, a complement thereof, or an intermediate agent of the second analyte. 3. The method of claim 1 or 2, further comprising separating the first substrate and the second substrate. 4. The method of claim 3, wherein separating the first substrate and the second substrate occurs after hybridizing the connected probe to the capture domain of the capture probe. 5. The method of any one of claims 1-4, further comprising determining (i) all or a part of a sequence of the connected probe corresponding to the first analyte, or a complement thereof, and (ii) the spatial barcode, or a complement thereof, and using the determined sequences of (i) and (ii) to determine a location of the first analyte in the biological sample. 6. The method of claim 5, wherein determining (i) all or a part of a sequence of the connected probe corresponding to the first analyte, or a complement thereof, and (ii) the spatial barcode, or a complement thereof, comprises sequencing. 7 The method of any one of claims 1-6, further comprising determining presence and/or abundance of the second analyte from the one or more cells or nuclei isolated from the biological sample. 8. The method of claim 7, wherein determining presence and/or abundance of the second analyte from the one or more cells or nuclei isolated from the biological sample comprises sequencing. 9. The method of any one of claims 1-8, wherein the first probe comprises a 5’ handle sequence, wherein the 5’ handle sequence comprises about 5 nucleotides to 50 nucleotides. 10. The method of claim 9, wherein the second probe comprises a 3’ handle sequence, wherein the 3’ handle sequence comprises about 5 nucleotides to 50 nucleotides. Attorney Docket No.: 47706-0398WO1 11. The method of claim 10, wherein the 3’ handle sequence comprises the capture probe binding domain, optionally wherein the capture probe binding domain comprises a poly(A) sequence. 12. The method of claim 11, wherein the poly(A) sequence is at a 3’ end of the second probe. 13. The method of any one of claims 1-12, wherein the first probe and the second probe hybridize to the nucleic acid sequence of the first analyte, wherein the nucleic acid sequence of the first analyte is about 25 to 100 nucleotides in length. 14. The method of any one of claims 1-13, wherein the first probe and/or the second probe comprises DNA. 15. The method of any one of claims 1-14, wherein the hybridizing the first probe and the second probe to the first analyte comprises contacting the biological sample with 100 or more probe pairs comprising the first probe and the second probe. 16. The method of any one of claims 1-15, wherein the hybridizing the first probe and the second probe to the first analyte comprises contacting the biological sample with 5,000 or more probe pairs comprising the first probe and the second probe. 17. The method of any one of claims 1-16, wherein the first probe and the second probe hybridize to adjacent sequences of the first analyte. 18. The method of any one of claims 1-17, wherein the coupling the first probe and the second probe comprises use of a ligase selected from a PBCV-1 DNA ligase, a Chlorella virus DNA ligase, a single stranded DNA ligase, or a T4 DNA ligase. 19. The method of any one of claims 1-18, wherein the first probe and the second probe hybridize to sequences in the first analyte that are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides away from one another. Attorney Docket No.: 47706-0398WO1 20. The method of claim 19, further comprising generating an extended first probe, wherein the extended first probe comprises a sequence complementary to a sequence between the sequence hybridized to the first probe and the sequence hybridized to the second probe. 21. The method of claim 20, further comprising ligating the extended first probe and the second probe using a ligase selected from a PBCV-1 DNA ligase, a Chlorella virus DNA ligase, a single stranded DNA ligase, or a T4 DNA ligase. 22. The method of claim 19, further comprising generating an extended second probe, wherein the extended second probe comprises a sequence complementary to a sequence between the sequence hybridized to the first probe and the sequence hybridized to the second probe. 23. The method of claim 22, further comprising ligating the first probe and the extended second probe using a ligase selected from a PBCV-1 DNA ligase, a Chlorella virus DNA ligase, a single stranded DNA ligase, or a T4 DNA ligase. 24. The method of any one of claims 1 or 3-23, wherein releasing the connected probe from the first analyte comprises applying heat to the biological sample. 25. The method of any one of claims 1 or 3-23, wherein releasing the connected probe from the first analyte comprises contacting an enzyme to the biological sample. 26. The method of claim 25, wherein the enzyme is an endoribonuclease. 27. The method of claim 26, wherein the endoribonuclease is one or more of RNase H, RNase A, RNase C, or RNase I. 28. The method of claim 26 or 27, wherein the endoribonuclease is RNase H. 29. The method of claim 28, wherein the RNase H comprises RNase H1, RNase H2, or both RNase H1 and RNase H2. Attorney Docket No.: 47706-0398WO1 30. The method of any one of claims 1-29, further comprising contacting the biological sample with a reagent medium comprising a permeabilization agent. 31. The method of claim 30, wherein the permeabilization agent comprises a protease. 32. The method of claim 31, wherein the protease is selected from trypsin, pepsin, elastase, or proteinase K. 33. The method of claim 31 or 32, wherein the protease is pepsin or proteinase K. 34. The method of any one of claim 30-33, wherein the reagent medium further comprises a detergent. 35. The method of claim 34, wherein the detergent is selected from sodium dodecyl sulfate (SDS), sarkosyl, or saponin. 36. The method of any one of claims 30-35, wherein the reagent medium further comprises one or more crowding agents, preferably polyethylene glycol (PEG). 37. The method of any one of claims 1-36, further comprising passively migrating the connected probe to the array. 38. The method of any one of claims 1-36, further comprising actively migrating the connected probe to the array. 39. The method of any one of claims 1-38, wherein the capture probe further comprises one or more functional domains, a unique molecular identifier (UMI), a cleavage domain, or combinations thereof. 40. The method of claim 39, wherein the one or more functional domains comprises a primer binding site. 41. The method of any one of claims 1-40, wherein the capture domain comprises a homopolymeric sequence. Attorney Docket No.: 47706-0398WO1 42. The method of any one of claims 1-41, wherein the capture domain comprises a poly(T) sequence. 43. The method of any one of claims 1-42, further comprising extending the capture probe using the connected probe as a template, thereby generating an extended capture probe; and/or extending the connected probe using the capture probe as a template, thereby generating an extended connected probe. 44. The method of any one of claims 1-43, further comprising separating the extended capture probe from the connected probe and/or separating the extended connected probe from the capture probe. 45. The method of claim 44, wherein the separating comprises use of potassium hydroxide or heat. 46. The method of any one of claims 1-45, further comprising amplifying all or part of the connected probe hybridized to the capture domain, or amplifying the extended connected probe and/or extended capture probe. 47. The method of any one of claims 1-46, wherein the first analyte comprises RNA. 48. The method of claim 47, wherein the RNA is mRNA. 49. The method of any one of claims 1-46, wherein the first analyte comprises DNA or RNA, the second analyte comprises DNA or RNA, or a combination thereof. 50. The method of claim 49, wherein the DNA is genomic DNA. 51. A method for processing multiple analytes in a biological sample mounted on a first substrate, the method comprising: (a) 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 capture Attorney Docket No.: 47706-0398WO1 agent barcode domain comprises an analyte binding moiety barcode and a capture handle sequence, and wherein upon the contacting, the analyte binding moiety binds to a first analyte in the biological sample; (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, 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; (c) hybridizing the capture agent barcode domain from the analyte capture agent that is bound to the first analyte to the capture domain of the capture probe; (d) isolating one or more cells or nuclei from the biological sample on the first substrate, wherein the one or more cells or nuclei comprises a second analyte; and (e) hybridizing a nucleic acid barcode molecule to the second analyte, a complement thereof, or an intermediate agent. 52. The method of claim 51, further comprising, when the biological sample is aligned with at least a portion of the array, releasing the capture agent barcode domain from the analyte capture agent that is bound to the first analyte. 53. The method of claim 51 or 52, further comprising separating the first substrate and the second substrate. 54. The method of claim 53, wherein separating the first substrate and the second substrate occurs after hybridizing the capture agent barcode domain to the capture domain of the capture probe of the array. 55. The method of any one of claims 51-54, further comprising determining (i) all or a part of a sequence of the capture agent barcode domain, or a complement thereof, and (ii) a sequence of the spatial barcode, or a complement thereof, and using the determined sequences of (i) and (ii) to determine a location of the first analyte in the biological sample. 56. The method of claim 55, wherein determining (i) all or a part of a sequence of the capture agent barcode domain, or a complement thereof, and (ii) a sequence of the spatial barcode, or a complement thereof, comprises sequencing. Attorney Docket No.: 47706-0398WO1 57. The method of any one of claims 51-56, further comprising determining presence and/or abundance of the second analyte from the one or more cells or nuclei isolated from the biological sample. 58. The method of claim 57, wherein determining presence and/or abundance of the second analyte from the one or more cells or nuclei isolated from the biological sample comprises sequencing. 59. The method of any one of claims 51-58, further comprising extending the capture agent barcode domain using the capture probe as a template, thereby incorporating a complement of the spatial barcode to generate a spatially tagged capture agent barcode domain; and/or extending the capture probe using the capture agent barcode domain as a template, thereby generating an extended capture probe. 60. The method of any one of claims 51-59, wherein the capture handle sequence of the capture agent barcode domain is substantially complementary to the capture domain of the capture probe. 61. The method of any one of claims 51-60, wherein the analyte binding moiety barcode is associated with or identifies the analyte binding moiety. 62. The method of any one of claims 51-61, wherein the analyte binding moiety comprises an antibody or an antigen-binding fragment thereof. 63. The method of any one of claims 51-62, wherein the analyte capture agent comprises a linker that couples the capture agent barcode domain to the analyte binding moiety. 64. The method of claim 63, wherein the linker is a cleavable linker. 65. The method of claim 64, wherein the cleavable linker is a disulfide linker, a photo- cleavable linker, a UV-cleavable linker, or an enzyme cleavable linker. Attorney Docket No.: 47706-0398WO1 66. The method of claim 65, wherein the enzyme cleavable linker is an RNase cleavable linker. 67. The method of anyone of claims 51-66, wherein the first analyte is a protein. 68. The method of claim 67, wherein the protein is an intracellular or extracellular protein. 69. The method of any one of claims 1-68, wherein the aligning 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; applying a reagent medium to the first substrate and/or the second substrate; and operating an alignment mechanism of the support device to move the first member and/or the second member such that at least a portion of the biological sample is aligned with at least a portion of the array, and such that the portion of the biological sample and the portion of the array contact the reagent medium. 70. The method of claim 69, wherein the alignment mechanism is coupled to the first member, the second member, or both the first member and the second member. 71. The method of claim 69 or 70, wherein the alignment mechanism comprises a linear actuator, optionally wherein: the linear actuator is configured to move the second member along an axis orthogonal to the first member and/or the second member, and/or the linear actuator is configured to move the first member along an axis orthogonal to a plane of the first member and/or the second member, and/or 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, and/or 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. Attorney Docket No.: 47706-0398WO1 72. The method of any one of claims 69-71, wherein at least one of the first substrate and the second substrate further comprise a spacer disposed on the first substrate or the second substrate, wherein when at least the portion of the biological sample is aligned with at least a portion of the array such that the portion of the biological sample and the portion of the array contact the reagent medium, the spacer is disposed between the first substrate and the second substrate and is configured to maintain the reagent medium within a chamber formed by the first substrate, the second substrate, and the spacer, and to maintain a separation distance between the first substrate and the second substrate, wherein the spacer is positioned to surround an area on the first substrate on which the biological sample is disposed and/or the array disposed on the second substrate, wherein the area of the first substrate, the spacer, and the second substrate at least partially encloses a volume comprising the biological sample. 73. The method of any one of claims 1-72, further comprising fixing the one or more cells or nuclei, optionally wherein fixing the one or more cells or nuclei comprises fixing the biological sample before the isolating the one or more cells or nuclei from the biological sample. 74. The method of claim 73, wherein the one or more cells or nuclei are fixed in formaldehyde. 75. The method of claim 73 or 74, wherein the one or more cells or nuclei are fixed in 4% formaldehyde. 76. The method of any one of claims 1-75, wherein the nucleic acid barcode molecule comprises one or more of a cell or nuclei barcode, a second unique molecule identifier, and a primer. 77. The method of any one of claims 1-76, wherein the intermediate agent is a second connected probe. 78. The method of any one of claims 51-77, wherein the intermediate agent is a second capture agent barcode domain from a second analyte capture agent comprising a second analyte binding moiety and the second capture agent barcode domain, wherein the second Attorney Docket No.: 47706-0398WO1 capture agent barcode domain comprises a second analyte binding moiety barcode and a second capture handle sequence. 79. The method of any one of claims 1-78, further comprising generating a copy of the second analyte, a complement thereof, or the intermediate agent, or a complement thereof. 80. The method of claim 79, wherein generating the copy of the second analyte, a complement thereof, or the intermediate agent, or a complement thereof, comprises use of a polymerase or a reverse transcriptase. 81. The method of any one of claims 1-80, further comprising hybridizing the nucleic acid barcode molecule to the complement of the second analyte, or the intermediate agent of the second analyte. 82. The method of any one of claims 1-81, wherein the nucleic acid barcode molecule comprises a hybridization region of a template switching oligonucleotide (TSO). 83. The method of claim 82, wherein the hybridization region of the TSO comprises a poly(G) sequence and wherein the nucleic acid barcode molecule comprises a poly(C) sequence. 84. The method of any one of claims 1-83, further comprising extending the nucleic acid barcode molecule using the complement of the second analyte or the intermediate agent as a template, thereby generating an extended nucleic acid barcode molecule. 85. The method of claim 84, further comprising amplifying the extended nucleic acid barcode molecule. 86. The method of any one of claims 76-85, wherein determining the presence and/or abundance of the second analyte in the biological sample comprises determining (i) the sequence of the cell or nuclei barcode, or a complement thereof, and (ii) all or a portion of the sequence of the second analyte, or a complement thereof, or all or a portion of the sequence of the intermediate agent, or a complement thereof. Attorney Docket No.: 47706-0398WO1 87. The method of any one of claims 1-86, wherein the nucleic acid barcode molecule is coupled to a particle. 88. The method of claim 87, wherein the particle is a bead. 89. The method of claim 87 or 88, wherein the nucleic acid barcode molecule is released from the particle upon application of a stimulus, optionally wherein the stimulus comprises a biological stimulus, a chemical stimulus, a thermal stimulus, an electrical stimulus, a magnetic stimulus, or a photo stimulus. 90. The method of any one of claims 1-89, wherein the one or more cells or nuclei are separated into a plurality of partitions, wherein a partition of the plurality of partitions comprises the nucleic acid barcode molecule and a cell or nucleus of the one or more cells or nuclei, and wherein the method further comprises lysing the cell or nucleus. 91. The method of claim 90, wherein the partition is a droplet, microwell, or well. 92. The method of any one of claims 1-91, wherein the second analyte comprises RNA. 93. The method of claim 92, wherein the RNA is mRNA. 94. The method of any one of claims 1-93, wherein the second analyte comprises DNA. 95. The method of claim 94, wherein the DNA is genomic DNA. 96. The methods of any one of claims 1-95, wherein the biological sample is a tissue sample. 97. The method of claim 96, wherein the tissue sample is a tissue section. 98. The method of any one of claims 1-97, wherein the biological sample is a fresh tissue sample and/or a frozen tissue sample, preferably a fresh frozen tissue section. Attorney Docket No.: 47706-0398WO1 99. The method of any one of claims 1-97, wherein the biological sample is a fixed tissue sample, preferably a fixed tissue section. 100. The method of claim 99, wherein the fixed tissue sample is a formalin fixed paraffin embedded (FFPE) tissue sample, preferably a FFPE tissue section. 101. The method claim 100, wherein the FFPE tissue section is deparaffinized and decrosslinked prior to step (a). 102. The method of any one of claims 1-101, wherein the biological sample is stained prior to step (a). 103. The method of claim 102, wherein the biological sample is stained using immunofluorescence, immunohistochemistry, hematoxylin, and/or eosin. 104. The method of any one of claims 82-103, wherein the intermediate agent of the second analyte is a probe-linked nucleic acid molecule. 105. The method of claim 104, wherein the probe-linked nucleic acid molecule is generated by hybridizing a first probe comprising (i) a first probe sequence that is complementary to a first target region of the second analyte and (ii) an additional probe sequence; and a second probe comprising a second probe sequence that is complementary to a second target region of the second analyte; and linking the first probe and the second probe to generate the probe-linked nucleic acid molecule, optionally wherein the linking comprises ligating. 106. The method of claim 104 or 105, wherein the probe-linked nucleic acid molecule is generated before isolating the one or more cells or nucleic from the biological sample. 107. The method of any one of claims 104-106, further comprising hybridizing a binding sequence of the nucleic acid barcode molecule to the additional probe sequence of the first probe of the probe-linked nucleic acid molecule, and optionally performing a nucleic acid Attorney Docket No.: 47706-0398WO1 extension reaction, thereby generating a barcoded, probe-linked nucleic acid molecule or a complement thereof. 108. The method of claim 107 further comprising determining the sequence of the barcoded, probe-linked nucleic acid molecule or a complement thereof, optionally wherein the determining comprises sequencing. 109. The method of any one of claims 1-81, wherein the nucleic acid barcode molecule comprises a hybridization region, wherein the hybridization region is complementary to non- templated nucleotides comprised in the complement of the second analyte. 110. The method of claim 109, wherein the hybridization region comprises a poly(G) sequence and wherein the non-templated nucleotides comprise a poly(C) sequence; optionally wherein the complement of the second analyte is generated by a reverse transcription reaction using a primer and the second analyte as a template. 111. The method of any one of claims 1-110, wherein the first substrate and/or the second substrate is a glass slide. 112. The method of any one of claims 1-111, wherein the biological sample is a diseased sample, optionally wherein a subject from which the biological sample was derived has a disease or condition, optionally cancer. 113. The method of any one of claims 1-112, wherein the biological sample is a tissue section comprising a thickness of 2-20 µm, preferably 5-18 µm.