Spatial mapping of nucleic acid methylation

Abstract

The present disclosure is directed to an array comprising one or more capture sites, wherein the capture sites comprise one or more nucleic acid probes immobilized on a solid support, and wherein the nucleic acid probes comprise: (a) a first clustering adapter sequence and a second clustering adapter sequence; (b) a spatial barcode (SBC) sequence; (c) a sequencing adapter sequence; (d) a restriction enzyme recognition site (RS region); and (e) a random region (X region); and its use to spatially identifying nucleic acid methylation.

Description

SPATIAL MAPPING OF NUCLEIC ACID METHYLATIONCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority benefit of U.S. Provisional Application No. 63 / 616,248, filed December 29, 2023, which is hereby incorporated by reference in its entirety.BACKGROUND

[0002] The modified DNA cytosine 5-methylcytosine (5mC) is a well-studied epigenetic modification that plays fundamental roles in human development and disease. Cytosine methylation occurs throughout the human genome and is generally associated with transcriptional repression, though in some cases it may actually have the opposite effect. Methylation induced transcriptional regulation occurs in healthy cells in order for a specific cell type to establish and maintain their cellular identity. However improper maintenance of DNA methylation can cause transcriptional dysregulation which can result in disease phenotypes. Alterations of methylation levels at promoter and transcriptional regulator regions of the genome can result in altered expression profiles from healthy cells resulting in unchecked cellular division, a phenotype of cancerous cells.

[0003] Epigenetic analysis of tissues can reveal incidence of disease such as cancer through analyzing methylation levels or chromatin occupancy. Analysis of the bulk DNA methylation level of a tissue depends on the methylation level of each cell type present within that tissue and the proportion of the cells from each cell type. Single cell-type- specific analysis provides more information on if methylation levels of a specific cell type in a tissue align with methylation patterns from a disease state. However, spatial information is lost in bulk and single cell sequencing methods. Providing spatial context for epigenetic patterns would enable deeper understanding of how a cell type’s methylation profile shapes its development and control of cell states in the native context of complex tissues. Spatially resolved epigenomic information is informative for spatial and developmental biology with a wide range of applications in biological and biomedical research. However, DNA methylation signatures have not been characterized spatially with high resolution. The most common method for assessing DNA methylation signatures isbisulfite sequencing, but this conversion method intrinsically damages the DNA, making it difficult to use in high sensitivity or low input applications. Spatial mapping of DNA is inherently low input since there are typically only two copies of DNA per cell. Therefore, there is a need for non-destructive DNA methylation mapping to enable greater capture of spatial DNA methylation.BRIEF SUMMARY

[0004] The present disclosure is directed to an array comprising one or more capture sites, wherein the capture sites comprise one or more nucleic acid probes immobilized on a solid support, and wherein the nucleic acid probes comprise: (a) a first clustering adapter sequence and a second clustering adapter sequence; (b) a spatial barcode (SBC) sequence; (c) a sequencing adapter sequence; (d) a restriction enzyme recognition site (RS region); and (e) a random region (X region). In one aspect, the X region is located at the 3'-terminus of the SBC. In another aspect, the nucleic acid probe comprises: 5'-first clustering adapter sequence-SBC sequence-X region-sequencing adapter-RS region- second clustering adapter sequence sequence-3'. In another aspect, the X region comprises modified nucleoside triphosphates with different functional groups. In some aspects, the functional groups are azide, alykyne, DIBAC, tetrazine, alkene, or norbomene groups. In some aspects, the sequencing adaptor is the SBS12 sequencing adapter.

[0005] In another aspect, each of the nucleic acid probes on the solid support are amplified to form clusters , wherein each cluster comprises amplicons of a single nucleic acid probe on the solid support, and wherein the restriction enzyme recognition sequence and second clustering adapter sequence regions are cleaved to expose a capture region. In another aspect, each cluster has a unique SBC sequence. In another aspect, the first clustering adapter sequence is P7 and the second clustering adapter sequence is P5. In another aspect, the capture sites provide for spatial detection of nucleic acid methylation marks in a biological sample.

[0006] The present disclosure is also directed to a method for spatially identifying nucleic acid methylation marks in the DNA of a biological sample, comprising:

[0007] contacting a biological sample with a solid support that comprises a spatial bar code (SBC) sequence; permeabilizing the biological sample; fragmenting or tagmentingthe DNA within the biological sample with a transposome containing an adapter sequence and a capture sequence; capturing the fragments from step c by hybridization or other attachment method of the fragments to the nucleic acid probes of the array, preferably click attachment, whereby a SBC sequence is appended to the DNA fragments; contacting the DNA fragments with a non-strand displacing polymerase and a ligase; treating the spatially barcoded nucleic acids of step d with methylation conversion to enable methylcytosine discrimination, thereby spatially identifying nucleic acid methylation marks in a biological sample.

[0008] In one aspect, the transposome is a forked transposome. In one aspect, the transposome is a non-forked transposome. In one aspect, the capture sequence is a poly A sequence. In another aspect, the polymerase is T4 DNA polymerase and the ligase is Taq DNA ligase.

[0009] The present disclosure is also directed to a method for spatially identifying nucleic acid methylation marks in the DNA of a biological sample, comprising: contacting a biological sample with a solid support that comprises a spatial bar code (SBC) sequence; permeabilizing the biological sample; fragmenting or tagmenting the DNA within the biological sample using a non-forked transposome containing an adapter sequence and a poly U capture sequence; contacting the DNA fragments from step c with a DNA polymerase; denaturing the DNA fragments from step d using physical or chemical means; capturing the fragments from step c by hybridization of the fragments to the nucleic acid probes of the array whereby a SBC sequence is appended to the DNA fragments; treating the spatially barcoded nucleic acids of step d with methylation conversion to enable methylcytosine discrimination, thereby spatially identifying nucleic acid methylation marks in a biological sample.

[0010] In one aspect, the denaturing is performed by heating or treatment with sodium hydroxide. In another aspect, the denaturing is performed by treating the DNA sequences with USER or a combination of uracil DNA glycosylase (UDG) and an abasic site endonuclease. In another aspect, the abasic site endonuclease is Endonuclease VIII. In another aspect, the permeabilizing and fragmenting steps are performed prior to contacting the biological sample with the solid support. In another aspect, the methylation conversion is performed using a methylcytosine selective deaminase, bisulfite, or EM- seq. In another aspect, the DNA within the biological sample is targeted at a specific genomic locus of interest. In another aspect, the targeting is performed by a transposomecontaining a modified transposase that guides the targeting. In another aspect, the modified transposase comprises a catalytically dead Cas9 (dCas9) protein or a Protein A sequence. In another aspect, step (e) further comprises amplification of the sequences and sequencing-by-synthesis.

[0011] The present disclosure is also directed to a method for spatially identifying nucleic acid methylation marks in the DNA of a biological sample, comprising: providing the array as described herein; contacting a biological sample with a solid support that comprises the array; permeabilizing the biological sample thereby releasing nuclear DNA from the sample; fragmenting the nuclear DNA within the biological sample; contacting the nuclear DNA from step d with a hybridization oligonucleotide that comprises a 5'- terminal functional group and a hybridization region that corresponds to a target region on the nuclear DNA under conditions that allow for hybridization of the nuclear DNA and the hybridization oligonucleotide; capturing the hybridized DNA from step e onto the solid support by click reaction chemistry; extending the 3' end of the hybridization oligonucleotide strand of the hybridized DNA using the nuclear DNA strand as a template; digesting unoccupied nucleic acid probes, uncaptured hybridization oligonucleotides, and single-stranded regions of the hybridized DNA by treatment with a single-stranded exonuclease; extending the 3' end of the nuclear DNA strand using the hybridization oligonucleotide strand as a template; and treating the spatially barcoded DNA with methylation conversion to enable methylcytosine discrimination, thereby spatially identifying nucleic acid methylation marks in a biological sample.

[0012] In one aspect, the click reaction chemistry is copper-catalyzed azide-alkyne cycoaddition (CuAAC)-based, strain-promoted azide-alkyne cycloaddition (SPAAC)- based, Diels-Alder reaction with inverse electron demand (DARinv), or Staudinger ligation. In another aspect, during CuAAC -based chemistry, the nucleic acid probes on the solid support comprise azide or alkyne functional groups at their 3' terminus and the hybridization oligonucleotide comprises the corresponding functional group. In another aspect, during SPAAC -based chemistry, the nucleic acid probes on the solid support comprise azide or DIB AC functional groups at their 3' terminus and the hybridization oligonucleotide comprises the corresponding functional group. In another aspect, during Diels-Alder reaction chemistry, the nucleic acid probes on the solid support comprise tetrazine or norbornene functional groups at their 3' terminus and the hybridization oligonucleotide comprises the corresponding functional group. In another aspect, duringStaudinger ligation chemistry, the nucleic acid probes on the solid support comprise phosphie or azide functional groups at their 3' terminus and the hybridization oligonucleotide comprises the corresponding functional group. In another aspect, the method further comprises elution of the SBC-tagged DNA fragments and library generation.

[0013] The present disclosure is also directed to a method of preparing a solid support for tagmentation of a target DNA in a biological sample, comprising: seeding a template oligonucleotide having the general structure P7-SBC-Y-ME-RS-X-P5' onto a P7 / P5 grafted solid support, wherein X and Y are any defined sequence and RS is a restriction enzyme recognition site and wherein the template oligonucleotide is tethered to the solid support; amplifying the template oligonucleotide on the solid support to cluster the oligonucleotides such that each cluster have a unique SBC associated with it; cleaving the P5 strand off the surface; hybridizing an oligonucleotide with the general structure of X'- RS'-ME'-Y' onto the template oligonucleotide and sequencing the SBC of the template oligonucleotide by synthesis; cleaving the template oligonucleotide at the RS; denaturing and washing away the untethered nucleic acid strand; hybridizing a ME' oligonucleotide to the ME on the tethered strand to form a grafted oligonucleotide comprising a functional ME-ME' structure; and contacting the grafted oligonucleotide with a Tn5 transposase thereby generating a solid surface for tagmenting the target DNA.

[0014] The present disclosure is also directed to a method of preparing a solid surface for tagmentation of a target DNA in a biological sample, comprising: seeding a template oligonucleotide having the general structure P7-SBC-Y- RS-X-P5' onto a P7 / P5 grafted surface, wherein X and Y are any define sequence and RS is a restriction enzyme recognition site and wherein the template oligonucleotide is tethered to the solid support; amplifying the template oligonucleotide on the solid support to cluster the oligonucleotides such that each cluster have a unique SBC associated with it; cleaving the P5 strand of the surface; hybridizing an oligonucleotide with the general structure of X'- RS'-Y' onto the template oligonucleotide and sequencing the SBC of the template oligonucleotide by synthesis; cleaving the template oligonucleotide at the RS; denaturing and removing the untethered nucleic acid strand; hybridizing a ME'-RS'-Y' -blocker to the tethered strand and extending the ME sequence onto the tethered strand; denaturing and removing the ME'-RS'-Y' -blocker; hybridizing ME' sequence to ME on the tethered strand to form a grafted oligonucleotide comprising a functional ME-ME' structure; andcontacting the grafted oligonucleotide with a Tn5 transposase thereby generating a solid surface for tagmenting the target DNA.

[0015] The present disclosure is also directed to a method of preparing a solid surface for tagmentation of a target DNA in a biological sample, comprising: seeding a template oligonucleotide having the general structure P7-SBC-Y- RS-X-P5' onto a P7 / P5 grafted surface, wherein X and Y are any define sequence and RS is a restriction enzyme recognition site and wherein the template oligonucleotide is tethered to the solid support; amplifying the template oligonucleotide on the solid support to cluster the oligonucleotides such that each cluster have a unique SBC associated with it; cleaving the P5 strand of the surface; hybridizing an oligonucleotide with the general structure of X'- RS'-Y' onto the template oligonucleotide and sequencing the SBC of the template oligonucleotide by synthesis; cleaving the template oligonucleotide at the RS; denaturing and removing the untethered nucleic acid strand; hybridizing a ME / ME'-RS'-Y' -blocker to the tethered strand and ligating the ME sequence onto the tethered strand; denaturing and removing the ME'-RS'-Y' -blocker; hybridizing ME' sequence to ME on the tethered strand to form a grafted oligonucleotide comprising a functional ME-ME structure; and contacting the grafted oligonucleotide with a Tn5 transposase thereby generating a solid surface for tagmenting the target DNA.

[0016] In one aspect, the template oligonucleotide further comprises a sequencing index. In another aspect, the solid support is a flow cell. In another aspect, X and Y are A14 and B15 sequences. In another aspect, the RS is a BamHl recognition site. In another aspect, the amplification is performed by bridge amplification or ExAmp methods.

[0017] The present disclosure is also directed to a method for spatially identifying target DNA in a biological sample, comprising: preparing the solid support according to the disclosure herein; contacting the biological sample with the solid support; permeabilizing the biological sample to release the target DNA; contacting the target DNA with the Tn5 transposase on the solid support and a soluble Tn5 transposase comprising X-ME oligonucleotides in the presence of SBC sequence, thereby tagmenting the DNA; extending the untethered DNA to also contain the SBC sequence; and eluting the untethered DNA strand by incubation with a denaturing wash buffer. In one aspect, the method further comprises amplifying the untethered strand of by polymerase chain reaction (PCR) and generating a library of strands for sequencing.

[0018] The present disclosure is also directed to a method for spatially identifying nucleic acid methylation marks in the DNA of a biological sample, comprising: preparing the solid support as described herein; contacting the biological sample with the solid support; permeabilizing the biological sample to release the target DNA; contacting the target DNA with the Tn5 transposase on the solid support and a soluble Tn5 transposase comprising X-ME oligonucleotides in the presence of SBC sequence, thereby tagmenting the DNA; extending the untethered DNA to also contain the SBC sequence; eluting the untethered DNA strand by incubation with a denaturing wash buffer; and treating the DNA with methylation conversion chemistry to enable methylcytosine discrimination, thereby spatially identifying nucleic acid methylation marks in a biological sample.

[0019] The present disclosure is also directed to a method for spatially identifying nucleic acid methylation marks in the DNA of a biological sample, comprising: preparing the solid support as described herein; contacting the biological sample with the solid support; permeabilizing the biological sample to release the target DNA; contacting the target DNA with (i) the Tn5 transposase on the solid support, and (ii) an antibody target-specific histone modification region, and a solubilized protein A-conjugated soluble Tn5 transposase comprising X-ME oligonucleotides in the presence of SBC sequence, or a dead Cas9 (dCas9)- conjugated soluble Tn5 transposase comprising X-ME oligonucleotides in the presence of SBC sequence, thereby tagmenting the DNA; extending the untethered DNA to also contain both the X and SBC sequence; eluting the untethered DNA strand by incubation with a denaturing wash buffer; and generating a library of DNA comprising specific histone-modification regions and identifying target DNA of interest by sequencing.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure l is a schematic diagram illustrating a method of spatially capturing DNA in a tissue sample and preparing the DNA for sequencing to detect methylation marks.

[0021] Figure 2 is a schematic diagram illustrating a method of generating spatially barcoded DNA library fragments using a forked transposome and a poly A sequence.

[0022] Figure 3 is a schematic diagram illustrating a method of generating spatially barcoded DNA library fragments using a non-forked transposome and a poly U sequence.

[0023] Figure 4 is a schematic diagram illustrating a method for denaturation, methylation conversion, and indexing of libraries generated with the methods depicted in Figure 2 and Figure 3.

[0024] Figure 5 is a schematic diagram illustrating generating a spatially barcoded surface with a modified NTP containing a click-chemistry handle for downstream workflow steps.

[0025] Figure 6 is a schematic diagram illustrating a method for capturing DNA targets from a tissue sample using click-chemistry.

[0026] Figure 7 is a schematic diagram illustrating a method for generating a sequencing library from captured DNA targets.

[0027] Figure 8 is a schematic diagram illustrating a method for generating a Tn5-SBC flow cell surface.

[0028] Figure 9 is a schematic diagram illustrating a method for generating a Tn5-SBC flow cell surface.

[0029] Figure 10 is a schematic diagram illustrating a method for generating a Tn5-SBC flow cell surface.

[0030] Figure 11 is a schematic diagram illustrating a method for spatial ATAC or DNA methylation detection using a Tn5-SBC surface.

[0031] Figure 12 is a schematic diagram illustrating a method for detecting spatial histone modification using a Tn5-SBC surface.DETAILED DESCRIPTIONA. Definitions

[0032] The terminology used in the present disclosure is for the purpose of describing particular aspects only and is not intended to be limiting.

[0033] As used in this specification and the enumerated paragraphs herein, the singular forms "a," "an," and "the" include plural reference unless the context dictates otherwise.

[0034] As used herein, the terms “substantially,” "about," and "approximately" shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20-25 percent (%), for example, within 20 percent, 10 percent, 5 percent, 4 percent, 3 percent, 2 percent, or 1 percent of the stated value or range of values.

[0035] As used herein, the term “substrate” refers to a material used as a support for compositions described herein. In some aspects, the substrate can be a solid support. Any variety of solid supports can be used in a method, composition, or apparatus of the present disclosure.

[0036] As used herein, the term "solid support" refers to a rigid substrate that is insoluble in aqueous liquid. The substrate can be non-porous or porous. The substrate can optionally be capable of taking up a liquid (e.g., due to porosity) but will typically be sufficiently rigid that the substrate does not swell substantially when taking up the liquid and does not contract substantially when the liquid is removed by drying. A nonporous solid support is generally impermeable to liquids or gases. Exemplary solid supports include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene, and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon™, cyclic olefins, polyimides, etc.), nylon, ceramics, resins, Zeonor, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, optical fiber bundles, and polymers. In some aspects, the substrate is glass. Other suitable substrate materials may include polymeric materials, silicon, quartz (fused silica), boro float glass, silica, silica-based materials, carbon, metals including gold, optical fiber or optical fiber bundles, or sapphire. The particular material can be selected based on properties desired for a particular use. For example, materials that are transparent to a desired wavelength of radiation are useful for analytical techniques that will utilize radiation of the desired wavelength, such as one or more of the techniques set forth herein. Conversely, it may be desirable to select a material that does not pass radiation of a certain wavelength (e.g., being opaque, absorptive, or reflective). This can be useful for the formation of a mask to be used during the manufacture of the structured substrate, or to be used for a chemical reaction or analytical detection carried out using the structured substrate. Other properties of a material that can be exploited are inertness or reactivity to certain reagents used in a downstream process, ease of manipulation, or low cost during a manufacturing process manufacture. Further examples of materials that can be used in the structured substrates or methods of the present disclosure are described in US Pat. App. Pub. No. 2012 / 0316086 Al and 2013 / 0116153, the entire contents of each are incorporated by reference herein. In some aspects, the solid support is a flow cell as described herein below.

[0037] Other example substrate materials can include metal oxide, organo-silicate (e.g., polyhedral organic silsesquioxanes (POSS)), polyacrylates, tantalum oxide, complementary metal oxide semiconductor (CMOS), or combinations thereof. An example of POSS is described in Kehagias et al., Microelectronic Engineering 86 (2009), pp. 776- 778, which is incorporated by reference in its entirety. In some examples, substrates used in the present application include silica-based substrates, such as glass, fused silica, or other silica-containing material. In some examples, silica-based substrates can include silicon, silicon dioxide, silicon nitride, or silicone hydride. In some examples, substrates used in the present application include plastic materials or components such as polyethylene, polystyrene, poly(vinyl chloride), polypropylene, nylons, polyesters, polycarbonates, and poly(methyl methacrylate). Example plastic materials include poly(methyl methacrylate), polystyrene, cyclic olefin polymer substrates, COCs, and epoxies In some examples, the substrate is or includes a silica-based material or plastic material or a combination thereof. In particular examples, the substrate has at least one surface including glass or a silicon- based polymer. In some examples, the substrates can include a metal. In some such examples, the metal is gold. In some examples, the substrate has at least one surface including a metal oxide.

[0038] In some aspects, the surface includes a tantalum oxide or tin oxide. Acrylamides, enones, or acrylates may also be utilized as a substrate material or component. Other substrate materials can include, but are not limited to gallium arsenide, indium phosphide, aluminum, ceramics, polyimide, quartz, resins, polymers, and copolymers. In some examples, the substrate and / or the substrate surface can be, or include, quartz. In some other examples, the substrate and / or the substrate surface can be, or include, a semiconductor, such as GaAs or ITO. The foregoing lists are intended to illustrate, but not limit, the present application. Substrates can include a single material or a plurality of different materials. Substrates can be composites or laminates. In some examples, the substrate includes an organo-silicate material.

[0039] Substrates can be flat, round, spherical, rod-shaped, or any other suitable shape. Substrates may be rigid or flexible. In some examples, a substrate is a glass slide, an array, a bead, or a flow cell. Substrates can be non-patterned, textured, or patterned on one or more surfaces of the substrate. In some examples, the substrate is patterned. Such patterns may include posts, pads, wells, ridges, channels, or other three-dimensional concave or convex structures. Patterns may be regular or irregular across the surface of the substrate.Patterns can be formed, for example, by nanoimprint lithography or by the use of metal pads that form features on non-metallic surfaces, for example.

[0040] As used herein, the term “bead” refers to a small body made of a rigid or semi-rigid material. The body can have a shape characterized, for example, as a sphere, oval, microsphere, or other recognized particle shape whether having regular or irregular dimensions. Example materials that are useful for beads include, without limitation, glass; plastic such as acrylic, polystyrene or a copolymer of styrene and another material, polypropylene, polyethylene, polybutylene, polyurethane or polytetrafluoroethylene (TEFLON®, from Chemours); polysaccharides or cross-linked polysaccharides such as agarose or Sepharose; nylon; nitrocellulose; resin; silica or silica-based materials including silicon and modified silicon; carbon-fiber, metal; inorganic glass; optical fiber bundle, or a variety of other polymers. Example beads include, without limitation, controlled pore glass beads, paramagnetic beads, thoria sol, Sepharose beads, nanocrystals, and others known in the art as described, for example, in Microsphere Detection Guide from Bangs Laboratories, Fishers Ind. Beads may also be coated with a polymer that has a functional group that can attach to an oligonucleotide.

[0041] A solid support can include a collection of beads. The beads can be suspended in a solution or they can be located on the surface of a substrate. Examples of arrays having beads located on a surface include those wherein beads are located in wells such as a BeadChip array (Illumina Inc., San Diego Calif.), substrates used in sequencing platforms from 454 LifeSciences (a subsidiary of Roche, Basel Switzerland) or substrates used in sequencing platforms from Ion Torrent (a subsidiary of Life Technologies, Carlsbad Calif.). Other solid supports having beads located on a surface are described in U.S. Pat. Nos. 6,266,459; 6,355,431; 6,770,441; 6,859,570; 6,210,891; 6,258,568; or 6,274,320; US Pat. App. Publ. Nos. 2009 / 0026082 Al; 2009 / 0127589 Al; 2010 / 0137143 Al; or 2010 / 0282617 Al or PCT Publication No. WO 2000 / 63437, each of which is incorporated herein by reference. Several of the above references describe methods for attaching nucleic acids to beads before loading the beads in or on a solid support. It will, however, be understood that the oligonucleotides can be made first and then attached to the beads which can then be loaded onto an array and used in a method set forth herein. In some aspects, the oligonucleotides are released from the beads and attached to a solid support (for example, and without limitation, a flow cell).

[0042] In some aspects, a substrate described herein forms at least part of a flow cell or is located in or coupled to a flow cell. Flow cells may include a flow chamber that is divided into a plurality of lanes or a plurality of sectors. As used herein, the term “flow cell” is intended to mean a vessel having a chamber where a reaction can be carried out, an inlet for delivering reagents to the chamber, and an outlet for removing reagents from the chamber. In some aspects, the chamber is configured for detection of the reaction that occurs in the chamber. For example, the chamber can include one or more transparent surfaces allowing optical detection of tissue samples, optically labeled molecules, or the like in the chamber. Exemplary flow cells include, but are not limited to those used in a nucleic acid sequencing apparatus such as flow cells for the Genome Analyzer®, MiSeq®, NextSeq®, or HiSeq® platforms commercialized by Illumina, Inc. (San Diego, Calif.); or for the SOLiD™ or Ion Torrent™ sequencing platform commercialized by Life Technologies (Carlsbad, Calif.). Exemplary flow cells and methods for their manufacture and use are also described, for example, in WO 2014 / 142841 Al; U.S. Pat. App. Pub. No. 2010 / 0111768 Al and U.S. Pat. No. 8,951,781, each of which is incorporated herein by reference.

[0043] In some aspects, the solid supports typically used for bead arrays are used without beads. For example, nucleic acids, such as the oligonucleotides described herein, can be attached directly to the wells or gel material in wells. Thus, the above references are illustrative of materials, compositions, or apparatus that can be modified for use in the methods and compositions set forth herein.

[0044] A solid support used in a method set forth herein can include an array of beads, wherein different oligonucleotides are attached to different beads in the array. In various aspects, each bead can be attached to a different oligonucleotide and the beads can be randomly distributed on the solid support in order to effectively attach the different nucleic acid probes to the solid support.

[0045] Optionally, the solid support can include wells having dimensions that accommodate no more than a single bead. In such a configuration, the beads may be attached to the wells due to forces resulting from the fit of the beads in the wells. It is also possible to use attachment chemistries or adhesives to hold the beads in the wells.

[0046] As described herein, oligonucleotides that are attached to beads can comprise or consist of barcode sequences. According to methods provided herein, a population of beads can be configured such that each bead is attached to only one type of oligonucleotidecomprising a plurality of barcodes, and many different beads (each with a different oligonucleotide) are present in the population.

[0047] Optionally, the substrate can include a gel coating. Attachment of nucleic acids to a solid support via a gel is exemplified by flow cells available commercially from Illumina Inc. (San Diego, CA) or described in US Pat. App. Pub. Nos. 2011 / 0059865 Al, 2014 / 0079923 Al, or 2015 / 0005447 Al; or PCT Publ. No. WO 2008 / 093098, each of which is incorporated herein by reference. Exemplary gels that can be used in the methods and apparatus set forth herein include, but are not limited to, those having a colloidal structure, such as agarose; polymer mesh structure, such as gelatin; or cross-linked polymer structure, such as polyacrylamide, or SFA (see, for example, US Pat. App. Pub. No. 2011 / 0059865 Al, which is incorporated herein by reference).

[0048] In some aspects, the surface of the substrate can include PAZAM (see, for example, US Pat. App. Publ. Nos. 2014 / 0079923 Al, or 2015 / 0005447 Al, each of which is incorporated herein by reference). PAZAM can include azide moieties which may be reacted with moieties in molecules to couple the molecules to the surface. For example, molecules may include alkynes (such as alkyne, BCN, or DBCO) as moieties, PEG1 to PEG10 as linkers, and biotin as a moiety. In the non-limiting example in which PEG4 is the linker, molecules may be referred to as alkyne-PEG4-biotin molecules. Once attached to the surface via a reaction between moieties, biotin is available to be reacted. In some aspects, biotin is then reacted with an active site of a pre-incubated streptavidin-dual biotin- P5 / P7 complex, binding complexes to the surface. Each streptavidin-dual biotin-P5 / P7 complex can include a streptavidin central molecule and at least one oligonucleotide, e.g., one or more P5 and / or P7 oligonucleotides which are functionalized to dual biotin to which a respective active site of streptavidin binds. At this point, oligonucleotides can be used for appropriate clustering and sequencing processes (not specifically illustrated). To initiate regeneration and reuse of the flowcell, a reagent such as hot formamide or ethylene glycol is introduced to decouple the streptavidin central molecule from biotin, for example by denaturing the streptavidin. A nuclease digest is also performed such that nuclease digests polynucleotides in the flowcell, e.g., oligonucleotides and any polynucleotides coupled thereto, into nucleotides. At this point, or at a later time, another set of complexes can be introduced and the cycle repeated.

[0049] In some aspects, a solid support can be configured as an array of features to which nucleic acids can be attached. As used herein, the term "feature" means a location in anarray for a particular species of molecule. A feature can contain only a single molecule or it can contain a population of several molecules of the same species. Features of an array are typically discrete. The discrete features can be contiguous or they can have spaces between each other. The size of the features and / or spacing between the features can vary such that arrays can be high-density, medium-density, or lower-density. High-density arrays are characterized as having sites separated by less than about 15 pm. Mediumdensity arrays have sites separated by about 15 to 30 pm, while low-density arrays have sites separated by greater than 30 pm. An array can have, for example, sites that are separated by less than 100 pm, 50 pm, 10 pm, 5 pm, 1 pm, or 0.5 pm. An apparatus or method of the present disclosure can be used to detect an array at a resolution sufficient to distinguish sites at the above densities or density ranges. Exemplary features include without limitation, beads (or other particles) in or on a substrate, droplets, wells in a substrate, projections from a substrate, ridges on a substrate, or channels in a substrate.

[0050] Features may be present on a solid support before contacting the solid support with nucleic acid probes. For example, in aspects where probes are attached to a support via hybridization to primers, the primers can be attached at the features, whereas interstitial areas outside of the features substantially lack any of the primers. Nucleic acid probes can be captured at preformed features on a solid support, and optionally amplified on the solid support, using methods set forth in US Pat. No. 8,895,249, US Pat. No. 8,778,849, or US Pat App. Pub. No. 2014 / 0243224 Al, each of which is incorporated herein by reference. Alternatively, a solid support may have a lawn of primers or may otherwise lack features. In this case, a feature can be formed by virtue of the attachment of a nucleic acid probe to the solid support. Optionally, the captured nucleic acid probe can be amplified on the solid support such that the resulting cluster becomes a feature. Although the attachment is exemplified above as a capture between a primer and a complementary portion of a probe, it will be understood that capture moieties other than primers can be present at pre-formed features or as a lawn. Other exemplary capture moieties include, but are not limited to, chemical moieties capable of reacting with a nucleic acid probe to create a covalent bond or receptors capable of binding non- covalently to a ligand on a nucleic acid probe.

[0051] In some aspects, the step of attaching nucleic acid probes to a solid support can be carried out by providing a fluid that contains a mixture of different nucleic acid probes and contacting this fluidic mixture with the solid support. The contact can result in the fluidicmixture being in contact with a surface to which many different nucleic acid probes from the fluidic mixture will attach. Thus, the probes have random access to the surface (whether the surface has pre-formed features configured to attach the probes or a uniform surface configured for attachment). Accordingly, the probes can be randomly located on the solid support.

[0052] The total number and variety of different probes that end up attached to a surface can be selected for a particular application or use. For example, in aspects where a fluidic mixture of different nucleic acid probes is contacted with a solid support for purposes of attaching the probes to the support, the number of different probe species can exceed the occupancy of the solid support for probes. Thus, the number and variety of different probes that attach to the solid support can be equivalent to the probe occupancy of the solid support. Alternatively, the number and variety of different probe species on the solid support can be less than the occupancy (i.e. there will be redundancy of probe species such that the solid support may contain multiple features having the same probe species). Such redundancy can be achieved, for example, by contacting the solid support with a fluidic mixture that contains a number and variety of probe species that is substantially lower than the probe occupancy of the solid support.

[0053] Attachment of the nucleic acid probes can be mediated by hybridization of the nucleic acid probes to complementary primers that are attached to the solid support, chemical bond formation between a reactive moiety on the nucleic acid probe and the solid support (examples are set forth in US Pat. No. 8,895,249, US Pat. No. 8,778,849, or US Pat App. Pub. No. 2014 / 0243224 Al, each of which is incorporated herein by reference), affinity interactions of a moiety on the nucleic acid probe with a solid support-bound moiety (e.g. between known receptor-ligand pairs such as streptavidin-biotin, antibody-epitope, lectin-carbohydrate and the like), physical interactions of the nucleic acid probes with the solid support (e.g. hydrogen bonding, ionic forces, van der Waals forces and the like), or other interactions known in the art to attach nucleic acids to surfaces.

[0054] The features can be present in any of a variety of desired formats. For example, the features can be wells, pits, channels, ridges, raised regions, pegs, posts, or the like. In some aspects, the features can contain beads. However, in particular aspects, the features need not contain a bead or particle.

[0055] As used herein, the term “array” refers to a population of sites that can be differentiated from each other according to relative location. Different molecules that areat different sites of an array can be differentiated from each other according to the locations of the sites in the array. An individual site of an array can include one or more molecules of a particular type. For example, a site can include a single nucleic acid molecule having a particular sequence or a site can include several nucleic acid molecules having the same sequence (and / or complementary sequence, thereof). The sites of an array can be different features located on the same substrate. The sites of an array can be separate substrates each bearing a different molecule. Different molecules attached to separate substrates can be identified according to the locations of the substrates on a surface to which the substrates are associated or according to the locations of the substrates in a liquid or gel. Exemplary arrays in which separate substrates are located on a surface include, without limitation, those having beads in wells.

[0056] As used herein, the term "pitch," when used in reference to features of an array, is intended to refer to the center-to-center spacing for adjacent features. A pattern of features can be characterized in terms of average pitch. The pattern can be ordered such that the coefficient of variation around the average pitch is small or the pattern can be random in which case the coefficient of variation can be relatively large. In either case, the average pitch can be, for example, at least about 10 nm, 0.1 pm, 0.5 pm, 1 pm, 5 pm, 10 pm, 100 pm or more. Alternatively or additionally, the average pitch can be, for example, at most about 100 pm, 10 pm, 5 pm, 1 pm, 0.5 pm 0.1 pm or less. Of course, the average pitch for a particular pattern of features can be between one of the lower values and one of the upper values selected from the ranges above.

[0057] High-density arrays are characterized as having an average pitch of less than about 15 pm. Medium-density arrays have an average pitch of about 15 to 30 pm, while low- density arrays have an average pitch greater than 30 pm. An array useful in the invention can have an average pitch that is less than 100 pm, 50 pm, 10 pm, 5 pm, 1 pm or 0.5 pm. The average pitch values and ranges set forth above or elsewhere herein are intended to apply to ordered arrays or random arrays. In particular aspects, features on a solid support can each have an area that is larger than about 100 nm2, 250 nm2, 500 nm2, 1 pm2, 2.5 pm2, 5 pm210 pm2, 100 pm2or 500 pm2. Alternatively or additionally, features can each have an area that is smaller than about 1 mm2500 pm2100 pm225 pm2, 10 pm25 pm21 pm2500 nm2, or 100 nm2. The above ranges can describe the apparent area of a bead or other particle on a solid support when viewed or imaged from above.

[0058] A solid support can include or can be made by the methods set forth herein to attach, a plurality of different nucleic acid probes. For example, a solid support can include at least 10, 100, 1 x 103, 1 x 104, 1 x 105, 1 x 106, 1 x 107, 1 x 108, 1 x 109or more different probes. Alternatively or additionally, a solid support can include at most 1 x 109, 1 x 108, 1 x 107, 1 x 106, 1 x 105, l x 104, 1 x 103, 100, or fewer different probes. It will be understood that each of the different probes can be present in several copies, for example, when the probes have been amplified to form a cluster. Thus, the above ranges can describe the number of different nucleic acid clusters on a solid support. It will also be understood that the above ranges can describe the number of different barcodes, target capture sequences, or other sequence elements set forth herein as being unique to particular nucleic acid probes. Alternatively or additionally, the ranges can describe the number of extended probes or modified probes created on a solid support using a method set forth herein.

[0059] As used herein, terms such as “covalently coupled” or “covalently bonded” refer to the forming of a chemical bond that is characterized by the sharing of pairs of electrons between atoms. For example, a covalently coupled molecule refers to a molecule that forms a chemical bond, as opposed to a non-covalent bond such as electrostatic interaction.

[0060] As used herein, the term “linker” is intended to mean a portion of a molecule via which one element is attached to another element. For example, a linker may attach a first reactive moiety to a second reactive moiety. Linkers may be covalent.

[0061] As used herein, the term “different," when used in reference to nucleic acids, means that the nucleic acids have nucleotide sequences that are not the same as each other. Two or more nucleic acids can have nucleotide sequences that are different along their entire length. Alternatively, two or more nucleic acids can have nucleotide sequences that are different along a substantial portion of their length. For example, two or more nucleic acids can have target nucleotide sequence portions that are different for the two or more molecules while also having a universal sequence portion that is the same for the two or more molecules. The term can be similarly applied to proteins that are distinguishable as different from each other based on amino acid sequence differences.

[0062] As used herein, “complementary” means that an oligonucleotide comprises a sequence of nucleotides that can form a double-stranded structure by matching base pairs with another oligonucleotide or part thereof. As used herein “substantially complementary” means that the oligonucleotide has at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% overall sequence identity to the complementary sequence.

[0063] As used herein, the terms "nucleic acid" and "nucleotide" are intended to be consistent with their use in the art and to include naturally occurring species or functional analogs thereof. Particularly useful functional analogs of nucleic acids are capable of hybridizing to a nucleic acid in a sequence-specific fashion or capable of being used as a template for replication of a particular nucleotide sequence. Naturally occurring nucleic acids generally have a backbone containing phosphodiester bonds. An analog structure can have an alternate backbone linkage including any of a variety of those known in the art. Naturally occurring nucleic acids generally have a deoxyribose sugar (e.g. found in deoxyribonucleic acid (DNA)) or a ribose sugar (e.g. found in ribonucleic acid (RNA)). A nucleic acid can contain nucleotides having any of a variety of analogs of these sugar moieties that are known in the art. A nucleic acid can include native or non-native nucleotides. In this regard, a native deoxyribonucleic acid can have one or more bases selected from the group consisting of adenine, thymine, cytosine, or guanine and a ribonucleic acid can have one or more bases selected from the group consisting of uracil, adenine, cytosine or guanine. Useful non-native bases that can be included in a nucleic acid or nucleotide are known in the art. The terms "probe" or "target," when used in reference to a nucleic acid or sequence of a nucleic acid, are intended as semantic identifiers for the nucleic acid or sequence in the context of a method or composition set forth herein and do not necessarily limit the structure or function of the nucleic acid or sequence beyond what is otherwise explicitly indicated. The terms "probe" and "target" can be similarly applied to other analytes such as proteins, small molecules, cells, or the like.

[0064] As used herein, a “primer” is a nucleic acid molecule that can hybridize to a target sequence, such as an adapter attached to a library fragment. As one example, an amplification primer can serve as a starting point for template amplification and cluster generation. As another example, a synthesized nucleic acid (template) strand may include a site to which a primer e.g., a sequencing primer) can hybridize in order to prime the synthesis of a new strand that is complementary to the synthesized nucleic acid strand. Any primer can include any combination of nucleotides or analogs thereof. In some examples, the primer is a single-stranded oligonucleotide or polynucleotide. The primer length can be any number of bases long and can include a variety of non-natural nucleotides. In various aspects, the sequencing primer is a short strand, ranging from 5 to 60 bases, from 10 to 60 bases, from 10 to 20 bases, from 10 to 30 bases, from 10 to 40 bases, from 10 to 50 bases, or from 20 to 40 bases.

[0065] As used herein, the term “adapter” refers generally to any linear nucleic acid molecule that can be ligated to an oligonucleotide of the disclosure. In some aspects, adapters include two reverse complementary oligonucleotides forming a double-stranded structure. In some aspects, an adapter includes two oligonucleotides that are complementary at one portion and mismatched at another portion, forming a Y-shape or fork-shaped adapter that is double-stranded at the complementary portion and has two floppy overhangs at the mismatched portion.

[0066] As used herein, the terms “sample” or “tissue” or “tissue sample” are intended to mean an aggregation of cells, and, optionally, intercellular matter. In some aspects, the cells in a tissue are not free-floating in solution and instead are attached to each other to form a multicellular structure. Exemplary tissue types include muscle, nerve, epidermal, and connective tissues. In some aspects, the tissue sample is from a human.

[0067] In some aspects, the sample is one or more cells. The cell(s) can be individual and free from any tissue or multicellular structure at the time contact is made with the solid support. For example, the cell(s) can be present in a fluid (e.g. when a plurality of different cells are present the fluid can be a fluidic mixture of the different cells) and the fluid can be contacted with the solid support to which the different probes are attached. Any of a variety of cells can be used including, for example, those from a prokaryote, archaea, or eukaryote. One or more cells used in a method, composition, or apparatus of the present disclosure can be a single-celled organism or a multicellular organism. Exemplary organisms from which one or more cells can be obtained include, but are not limited to a mammal, plant, algae, nematode, insect, fish, reptile, amphibian, fungi, or Plasmodium falciparum. Exemplary species are set forth previously herein or known in the art.

[0068] Aspects of the present disclosure can also use one or more subcellular components as a tissue sample. For example, a fluidic mixture can include one or more nuclei, Golgi apparatus, mitochondria, chloroplasts, membrane fractions, vesicles, endoplasmic reticulum, or other components known in the art. Other useful types of tissue samples are one or more viruses or viroids. It will be understood that a tissue sample can be a homogeneous culture or population of the above cells, subcellular components, viruses, or viroids. Alternatively, the tissue sample can be a non-homogenous collection of cells, subcellular components, viruses, or viroids, for example, derived from several different organisms in a community or ecosystem. An exemplary community is the collection ofbacteria present in the digestive system, lung, or other organ of a multicellular organism such as a mammal.

[0069] One or more cells, subcellular components, viruses, or viroids that are contacted with a solid support in a method set forth herein can be attached to the solid support. Attachment can be achieved using methods known in the art such as those exemplified herein with respect to attachment of nucleic acids to a solid support. In some aspects, attachment is selective for specific types of cells, subcellular components, viruses, or viroids. For example, the solid support can include antibodies or other receptors that are selective for epitopes or ligands present on one or a subset of different cells, subcellular components, viruses, or viroids present in a fluidic mixture. In other aspects, the attachment of cells, subcellular components, viruses, or viroids can be mediated by non-selective moieties such as chemical moieties that are broadly reactive.

[0070] In some aspects, one or more cells, subcellular components, viruses, or viroids that have been contacted with a solid support can be lysed to release target nucleic acids. Lysis can be carried out using methods known in the art such as those that employ one or more chemical treatments, enzymatic treatments, electroporation, heat, hypotonic treatments, sonication, or the like. Exemplary lysis techniques are set forth in Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001) and in Ansubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1999).

[0071] In some aspects, the tissue sample may be fixed, sectioned, and mounted on a surface. The tissue can be derived from a multicellular organism such as those exemplified above in regard to cells. A tissue section can be contacted with a solid support, for example, by laying the tissue on the surface of the solid support. The tissue can be freshly excised from an organism or it may have been previously preserved for example by freezing, embedding in a material such as paraffin (e.g. formalin fixed paraffin embedded samples), formalin fixation, infiltration, dehydration, or the like. The methods disclosed herein may be performed before or after staining the tissue sample. For example, following hematoxylin and eosin staining, a tissue sample may be spatially analyzed in accordance with the methods provided herein. A method may include analyzing the histology of the sample (e.g., using hematoxylin and eosin staining) and then spatially analyzing the tissue.

[0072] In some aspects, the sample can be fixed by deep freezing the sample at a temperature suitable to maintain or preserve the integrity of the tissue structure. A fixed or embedded tissue sample can be sectioned, i.e., thinly sliced, using known methods.

[0073] Optionally, a tissue section can be attached to a solid support, for example, using techniques and compositions exemplified herein with regard to attaching nucleic acids, cells, viruses, beads, or the like to a solid support. As a further option, a tissue can be permeabilized and the cells of the tissue lysed when the tissue is in contact with a solid support. Any of a variety of treatments can be used such as those set forth above in regard to lysing cells. Target nucleic acids that are released from a tissue that is permeabilized can be captured by nucleic acid probes on the surface of the substrate.

[0074] As used herein, “hybridize” is intended to mean noncovalently associating a first oligonucleotide to a second oligonucleotide along the lengths of those polymers to form a double-stranded “duplex” or “complex.” For example, two DNA oligonucleotide strands may associate through complementary base pairing. The strength of the association between the first and second oligonucleotides increases with the complementarity between the sequences of nucleotides within those oligonucleotides. The strength of hybridization between oligonucleotides may be characterized by a temperature of melting (Tm) at which 50% of the duplexes have oligonucleotide strands that disassociate from one another. Oligonucleotides that are “partially” hybridized to one another means that they have sequences that are complementary to one another, but such sequences are hybridized with one another along only a portion of their lengths to form a partial duplex. Oligonucleotides with an “inability” to hybridize include those that are physically separated from one another such that an insufficient number of their bases may contact one another in a manner so as to hybridize with one another.

[0075] As used herein, the term “plurality” is intended to mean a population of two or more members, which may all be the same or two or more members may be different. Pluralities may range in size from small, medium, large, to very large. The size of a small plurality may range, for example, from a few members to tens of members. Medium-sized pluralities may range, for example, from tens of members to about 100 members or hundreds of members. Large pluralities may range, for example, from about hundreds of members to about 1,000 members, to thousands of members, and up to tens of thousands of members. Very large pluralities may range, for example, from tens of thousands of members to about hundreds of thousands, a million, millions, tens of millions, and up to or greater thanhundreds of millions of members. Therefore, a plurality may range in size from two to well over one hundred million members as well as all sizes, as measured by the number of members, in between and greater than the above example ranges. Accordingly, the definition of the term is intended to include all integer values greater than two. An upper limit of a plurality may be set, for example, by the theoretical diversity of bead types in an array.

[0076] As used herein, the term “attached” refers to the state of two things being joined, fastened, adhered, connected, or bound to each other. For example, an oligonucleotide can be attached to a material, such as a bead, by a covalent or non-covalent bond. A covalent bond is characterized by the sharing of pairs of electrons between atoms. A non-covalent bond is a chemical bond that does not involve the sharing of pairs of electrons and can include, for example, hydrogen bonds, ionic bonds, van der Waals forces, hydrophilic interactions, and hydrophobic interactions.

[0077] As used herein, the terms “semi-random” or “semi-randomer” refer to a nucleotide sequence that comprises or consists of a partially pre-determined nucleotide sequence combined with a random nucleotide sequence.

[0078] As used herein, the terms “target” or “target molecule” refer to any molecule found within a cell. For example, a target molecule can include, but is not limited to, a nucleic acid, a nucleic acid sequence, RNA, mRNA, siRNA, rRNA, tRNA, DNA, cDNA, genomic DNA, a protein, an oligonucleotide, or a polynucleotide. A target molecule may be a nucleic acid sequence to be analyzed. A target molecule may include nucleotide sequences additional to a target sequence to be analyzed. For example, a target molecule may include one or more adapters, including an amplification adapter that functions as a primer binding site, which flank(s) a target molecule sequence that is to be analyzed. In particular examples, target molecules may have different sequences than one another but may have first and second adapters that are the same as one another. The two adapters that may flank a particular target molecule sequence may have the same sequence as one another, or complementary sequences to one another, or the two adapters may have different sequences. Thus, species in a plurality of target molecules may include regions of known sequence that flank regions of unknown sequence that are to be evaluated by, for example, sequencing (e.g., SBS). In some examples, the target molecule carries an amplification adapter at a single end, and such adapter may be located at either the 3' end or the 5' end of the target polynucleotide. Target molecules may be used without any adapter, in which casea primer binding sequence may come directly from a sequence found in the target polynucleotide.

[0079] The terms “polynucleotide” and “oligonucleotide” are used interchangeably herein. The different terms are not intended to denote any particular difference in size, sequence, or other property unless specifically indicated otherwise. For clarity of description, the terms may be used to distinguish one species of polynucleotide from another when describing a particular method or composition that includes several polynucleotide species.

[0080] As used herein, a “polymerase” is intended to mean an enzyme having an active site that assembles polynucleotides by polymerizing nucleotides into polynucleotides. A polymerase can bind a primer and a single-stranded target polynucleotide, and can sequentially add nucleotides to the growing primer to form a “complementary copy” polynucleotide having a sequence that is complementary to that of the target polynucleotide. DNA polymerases may bind to the target polynucleotide and then move down the target polynucleotide sequentially adding nucleotides to the free hydroxyl group at the 3' end of a growing polynucleotide strand. DNA polymerases may synthesize complementary DNA molecules from DNA templates. RNA polymerases may synthesize RNA molecules from DNA templates (transcription). Other RNA polymerases, such as reverse transcriptases, may synthesize cDNA molecules from RNA templates. Still, other RNA polymerases may synthesize RNA molecules from RNA templates, such as RdRP. Polymerases may use a short RNA or DNA strand (primer), to begin strand growth. Some polymerases may displace the strand upstream of the site where they are adding bases to a chain. Such polymerases may be said to be strand displacing, meaning they have an activity that removes a complementary strand from a template strand being read by the polymerase.

[0081] Example DNA polymerases include Bst DNA polymerase, 9° Nm DNA polymerase, Phi29 DNA polymerase, DNA polymerase I (E. coli . DNA polymerase I (Large), (Klenow) fragment, Klenow fragment (3 '-5' exo-), T4 DNA polymerase, T7 DNA polymerase, Deep VentR™ (exo-) DNA polymerase, Deep VentR™ DNA polymerase, DyNAzyme™ EXT DNA, DyNAzyme™ II Hot Start DNA Polymerase, Phusion™ High- Fidelity DNA Polymerase, Therminator™ DNA Polymerase, Therminator™ II DNA Polymerase, VentR® DNA Polymerase, VentR® (exo-) DNA Polymerase, RepliPHI™ Phi29 DNA Polymerase, rBst DNA Polymerase, rBst DNA Polymerase (Large), Fragment (IsoTherm™ DNA Polymerase), Master Amp™ AmpliTherm™, DNA Polymerase, Taq DNA polymerase, Tth DNA polymerase, Tfl DNA polymerase, Tgo DNA polymerase, SP6DNA polymerase, Tbr DNA polymerase, DNA polymerase Beta, ThermoPhi DNA polymerase, and Isopol™ SD+ polymerase. In specific, nonlimiting examples, the polymerase is selected from a group consisting of Bst, Bsu, and Phi29. Some polymerases have an activity that degrades the strand behind them (3' exonuclease activity). Some useful polymerases have been modified, either by mutation or otherwise, to reduce or eliminate 3' and / or 5' exonuclease activity.

[0082] Example RNA polymerases include RdRps (RNA dependent, RNA polymerases) that catalyze the synthesis of the RNA strand complementary to a given RNA template. Example RdRps include polioviral 3Dpol, vesicular stomatitis virus L, and hepatitis C virus NS5B protein. Example RNA Reverse Transcriptases. A non-limiting example list to include are reverse transcriptases derived from Avian Myelomatosis Virus (AMV), Murine Moloney Leukemia Virus (MMLV) and / or the Human Immunodeficiency Virus (HIV), telomerase reverse transcriptases such as (hTERT), SuperScript™ III, SuperScript™ IV Reverse Transcriptase, ProtoScript® II Reverse Transcriptase.

[0083] As used herein, the term “primer” is defined as a polynucleotide to which nucleotides may be added via a free 3' OH group. A primer may include a 3' block inhibiting polymerization until the block is removed. A primer may include a modification at the 5' terminus to allow a coupling reaction or to couple the primer to another moiety. A primer may include one or more moieties, such as 8-oxo-G, which may be cleaved under suitable conditions, such as UV light, chemistry, enzyme, or the like. The primer length may be any suitable number of bases long and may include any suitable combination of natural and non-natural nucleotides. A target polynucleotide may include an “amplification adapter” or, more simply, an “adapter,” that hybridizes to (has a sequence that is complementary to) a primer and may be amplified so as to generate a complementary copy polynucleotide by adding nucleotides to the free 3' OH group of the primer.

[0084] As used herein, the term “double-stranded,” when used in reference to a polynucleotide, is intended to mean that all or substantially all of the nucleotides in the polynucleotide are hydrogen bonded to respective nucleotides in a complementary polynucleotide. A double-stranded polynucleotide also may be referred to as a “duplex.”

[0085] As used herein, the term “single-stranded,” when used in reference to a polynucleotide, means that essentially none of the nucleotides in the polynucleotide are hydrogen bonded to a respective nucleotide in a complementary polynucleotide.

[0086] As used herein, the term “cluster” refers to a population of nucleic acids that is attached to a solid support.

[0087] The method of present disclosure can include a step of performing a nucleic acid detection to determine the barcode sequence of the nucleic acid probes that are located on the solid support. In many aspects, the probes are randomly located on the solid support and the nucleic acid detection reaction provides information to locate each of the different probes. Exemplary nucleic acid detection methods include, but are not limited to nucleic acid sequencing of a probe, hybridization of nucleic acids to a probe, ligation of nucleic acids that are hybridized to a probe, extension of nucleic acids that are hybridized to a probe, extension of a first nucleic acid that is hybridized to a probe followed by ligation of the extended nucleic acid to a second nucleic acid that is hybridized to the probe, or other methods known in the art such as those set forth in US Pat. No. 8,288,103 or 8,486,625, each of which is incorporated herein by reference.

[0088] In some aspects, the methods of the present disclosure can include sequencing to spatially detect a target molecule. In some aspects, sequencing techniques, such as sequencing-by-synthesis (SBS) techniques, can be used in the disclosed methods. SBS can be carried out as follows. To initiate a first SBS cycle, one or more labeled nucleotides, DNA polymerase, SBS primers, etc., can be contacted with one or more features on a solid support (e.g. feature(s) where nucleic acid probes are attached to the solid support). Those features where SBS primer extension causes a labeled nucleotide to be incorporated can be detected. Optionally, the nucleotides can include a reversible termination moiety that terminates further primer extension once a nucleotide has been added to the SBS primer. For example, a nucleotide analog having a reversible terminator moiety can be added to a primer such that subsequent extension cannot occur until a deblocking agent is delivered to remove the moiety. Thus, for aspects that use reversible termination, a deblocking reagent can be delivered to the solid support (before or after detection occurs).

[0089] Washes can be carried out between the various delivery steps. The cycle can then be repeated n times to extend the primer by n nucleotides, thereby detecting a sequence of length n. Exemplary SBS procedures, fluidic systems, and detection platforms that can be readily adapted for use with a composition, apparatus, or method of the present disclosure are described, for example, in Bentley et al., Nature 456:53-59 (2008), PCT Publ. Nos. WO 91 / 06678, WO 04 / 018497 or WO 07 / 123744; US Pat. Nos. 7,057,026, 7,329,492,7,211,414, 7,315,019 or 7,405,281, and US Pat. App. Publ. No. 2008 / 0108082, each of which is incorporated herein by reference.

[0090] Other sequencing procedures that use cyclic reactions can be used, such as pyrosequencing. Pyrosequencing detects the release of inorganic pyrophosphate (PPi) as particular nucleotides are incorporated into a nascent nucleic acid strand (Ronaghi, et al., Analytical Biochemistry 242(1), 84-9 (1996); Ronaghi, Genome Res. 11(1), 3-11 (2001); Ronaghi et al. Science 281(5375), 363 (1998); or US Pat. Nos. 6,210,891, 6,258,568, or 6,274,320, each of which is incorporated herein by reference). In pyrosequencing, released PPi can be detected by being immediately converted to adenosine triphosphate (ATP) by ATP sulfurylase, and the level of ATP generated can be detected via luciferase-produced photons. Thus, the sequencing reaction can be monitored via a luminescence detection system. Excitation radiation sources used for fluorescence-based detection systems are not necessary for pyrosequencing procedures. Useful fluidic systems, detectors, and procedures that can be used for the application of pyrosequencing to apparatus, compositions, or methods of the present disclosure are described, for example, in PCT Pat. App. Publ. No. W02012 / 058096, US Pat. App. Publ. No. 2005 / 0191698 Al, or US Pat. Nos. 7,595,883 or 7,244,559, each of which is incorporated herein by reference.

[0091] In some aspects, sequencing-by-ligation reactions can also be used. Examples of sequencing-by-ligation reactions include, for example, those described in Shendure et al. Science 309: 1728-1732 (2005); or US Pat. Nos. 5,599,675 or 5,750,341, each of which is incorporated herein by reference. Some aspects can include sequencing-by-hybridization procedures as described, for example, in Bains et al., Journal of Theoretical Biology 135(3), 303-7 (1988); Drmanac et al., Nature Biotechnology 16, 54-58 (1998); Fodor et al., Science 251(4995), 767-773 (1995); or PCT Pat. App. Publ. No. WO 1989 / 10977, each of which is incorporated herein by reference. In both sequencing-by-ligation and sequencing-by- hybridization procedures, target nucleic acids (or amplicons thereof) that are present at sites of an array are subjected to repeated cycles of oligonucleotide delivery and detection. Compositions, apparatus, or methods set forth herein or in references cited herein can be readily adapted for sequencing-by- ligation or sequencing-by-hybridization procedures. Typically, the oligonucleotides are fluorescently labeled and can be detected using fluorescence detectors similar to those described with regard to SBS procedures herein or in references cited herein.

[0092] Some sequencing aspects can utilize methods involving the real-time monitoring of DNA polymerase activity. For example, nucleotide incorporations can be detected through fluorescence resonance energy transfer (FRET) interactions between a fluorophore-bearing polymerase and y-phosphate-labeled nucleotides, or with zeromode waveguides (ZMWs). Techniques and reagents for FRET -based sequencing are described, for example, in Levene et al. Science 299, 682-686 (2003); Lundquist et al. Opt. Lett. 33, 1026-1028 (2008); Korlach et al. Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008), each of which is incorporated herein by reference.

[0093] Some sequencing aspects include the detection of a proton released upon incorporation of a nucleotide into an extension product. For example, sequencing based on the detection of released protons can use an electrical detector and associated techniques that are commercially available from Ion Torrent (Guilford, CT, a Life Technologies and Thermo Fisher subsidiary) or sequencing methods and systems described in US Pat app. Publ. Nos. 2009 / 0026082 Al; 2009 / 0127589 Al; 2010 / 0137143 Al; or US 2010 / 0282617 Al, each of which is incorporated herein by reference

[0094] In some aspects, nucleic acid hybridization techniques are also useful methods for determining barcode sequences. In some cases, combinatorial hybridization methods can be used such as those used for decoding of multiplex bead arrays (see e.g. US Pat. No. 8,460,865, which is incorporated herein by reference). Such methods utilize labeled nucleic acid decoder probes that are complementary to at least a portion of a barcode sequence. A hybridization reaction can be carried out using decoder probes having known labels such that the location where the labels end up on the solid support identifies the nucleic acid probes according to the rules of nucleic acid complementarity. In some cases, pools of many different probes with distinguishable labels are used, thereby allowing a multiplex decoding operation. The number of different barcodes determined in a decoding operation can exceed the number of labels used for the decoding operation. For example, decoding can be carried out in several stages where each stage constitutes hybridization with a different pool of decoder probes. The same decoder probes can be present in different pools but the label that is present on each decoder probe can differ from pool to pool (i.e. each decoder probe is in a different "state" when in different pools). Various combinations of these states and stages can be used to expand the number of barcodes that can be decoded well beyond the number of distinct labels available for decoding. Such combinatorialmethods are set forth in further detail in US Pat. No. 8,460,865 or Gunderson et al., Genome Research 14:870-877 (2004), each of which is incorporated herein by reference.

[0095] The present disclosure is generally directed to compositions and methods for spatially identifying nucleic acid methylation marks. In some aspects, the composition can include at least one nucleic acid probe. In some aspects, the composition can include a plurality of nucleic acid probes. In some aspects, the nucleic acid probes can include, among other things, a spatial barcode sequence, a restriction enzyme recognition site, a random region, a mosaic end, and a capture sequence.B. Nucleic Acid Probes

[0096] As used herein, the terms “probe,” “nucleic acid probe,” and “capture probes,” refer to an oligonucleotide having a nucleotide sequence that is capable of specifically annealing to a single-stranded polynucleotide sequence to be analyzed or subjected to a nucleic acid interrogation under conditions encountered in a primer annealing step of, for example, an amplification or sequencing reaction.

[0097] In some aspects, the nucleic acid probe contains a capture sequence and a spatial barcode sequence. In some aspects, the nucleic acid probe contains additional nucleic acid sequences. For example, the nucleic acid probe can also include a sequencing by synthesis (SBS) sequence. In some aspects, the SBS sequence is SBS12 or a complement thereof (SBS12'). In some aspects, the SBS sequence is SBS3 or a complement thereof (SBS3').

[0098] In some aspects, the nucleic acid probe can also contain a flowcell clustering sequence. The flowcell clustering sequence can be selected from the group consisting of P5, P5', P7, P7'. As used herein, the terms “P5” and “P7” may be used when referring to examples of adapters. The terms “P5'” (P5 prime) and “P7'” (P7 prime) refer to the complement of P5 and P7, respectively. It will be understood that any suitable adapter can be used in the methods presented herein and that the use of P5 and P7 are exemplary aspects only. Uses of adapters such as P5 and P7 or their complements on flowcells are known in the art, as exemplified by the disclosures of WO 2007 / 010251, WO 2006 / 064199, WO 2005 / 065814, WO 2015 / 106941, WO 1998 / 044151, and WO 2000 / 018957, each of which is incorporated herein by reference in its entirety. For example, any suitable forward amplification primer, whether immobilized or in solution, can be useful in the methods presented herein for hybridization to a complementary sequence and amplification of a sequence. Similarly, any suitable reverse amplification primer, whether immobilized or insolution, can be useful in the methods presented herein for hybridization to a complementary sequence and amplification of a sequence. One of skill in the art will understand how to design and use primer sequences that are suitable for the capture and / or amplification of nucleic acids as presented herein.

[0099] In some aspects, the nucleic acid probes can include a unique molecular identifier (UMI) or a single molecule identifier (SMI). As used herein, the term “unique molecular identifier” or “UMI” refers to a molecular tag, either random, non-random, or semi-random, that may be attached to a nucleic acid. When incorporated into a nucleic acid, a UMI can be used to correct for subsequent amplification bias by directly counting unique molecular identifiers (UMIs) that are sequenced after amplification. A UMI can be attached to similar nucleic acids, e.g., adapters, making each nucleic acid unique.

[0100] In some aspects, the nucleic acid probes can also contain primer binding sites. In some aspects, a nucleic acid probe contains first and second universal primer binding sites, they will be located at the ends of the probe. In some aspects, it may be desirable to remove at least one of the primer binding sites from the nucleic acid probe or amplicons produced from the probe. Accordingly, the nucleic acid probes can optionally include a cleavage site between the target capture sequence and one of the universal primer binding sequences. In this case, a cleavage reaction can be performed to separate the universal primer binding site from the target capture sequence. Generally, the portion of the probe (or its amplicons) that contains the target capture sequence will be attached to the solid support resulting in removal of the primer binding site from the solid support and retention of the target capture sequence. Thus, the cleaved probe can be used for hybridizing target nucleic acids and the cleaved probe can be extended using the method set forth previously herein.

[0101] In some aspects, a nucleic acid probe can include one more cleavage sites. In some aspects, a nucleic acid probe can include two different cleavage sites. In aspects where the nucleic acid probe includes primer binding sites, a first cleavage site can be located between a first primer binding site and one or more other sequence elements of the probe. A second cleavage site can be located between a second primer binding site and one or more other sequence elements of the probe. The cleavage sites can be reactive to different cleavage reactions such that each one can be selectively cleaved without necessarily cleaving the other. Accordingly, the first cleavage site can be cleaved prior to modifying the probe (for example, prior to producing an extended probe), thereby separating the first primer binding site from one or more other sequence elements that remain attached to a solid support. Thesecond cleavage site can be cleaved after modifying the probe (for example, after producing the extended probe), thereby releasing the modified probe for subsequent detection.

[0102] Alternatively, a nucleic acid probe can include the first cleavage site and a primer that is used to capture or amplify the nucleic acid probe can include the second cleavage site. In this configuration, the first cleavage site can be located between a first primer binding site and one or more other sequence elements of the probe such that cleavage separates the first primer binding site from one or more other sequence elements of the probe that remain attached to a solid support. Again, this first cleavage step will typically be carried out prior to modifying the probe (for example, prior to producing an extended probe). A second cleavage step can be carried out to cleave the second cleavage site after modifying the probe (for example, after producing the extended probe), thereby releasing the modified probe for subsequent detection. Thus, this cleavage site is useful for the release of modified probes (e.g. extended probes) to detect the sequence information and determine what sequences are present in a biological specimen and where the sequences are present in the specimen.

[0103] In some aspects, one or more probes that are contacted with a solid support in a method set forth herein can include a sequencing primer binding site.

[0104] Accordingly, a modified probe (e.g. extended probe) can be detected in a sequencing technique that includes a step of hybridizing a sequencing primer to the sequencing primer binding site. The sequencing primer binding site can be located in the probe such that cleavage of a modified version of the probe (e.g. an extended probe) will yield a released probe that includes the sequencing primer binding site. The sequencing primer binding site can be a universal sequencing primer binding site such that a plurality of different probes (e.g. having different barcode and / or target sequences) will have the same sequencing primer binding site.

[0105] In some aspects, the nucleic acid probes can also contain a mosaic end (ME) that is a recognition site for a transposase.

[0106] In some aspects, the nucleic acid probes are arranged in specific locations on the substrate.C. Spatial Barcode

[0107] In some aspects, the nucleic acid probe comprises at least one spatial barcode. As used herein, the term “barcode,” “spatial barcode,” or “spatial tag,” is intended to mean aseries of nucleotides in an oligonucleotide that can be used to identify the oligonucleotide, a spatial address on a surface, a characteristic of the oligonucleotide, or a manipulation that has been carried out on the oligonucleotide. In some aspects, the spatial barcode correlates to a positional location on the substrate. The spatial barcode can be unique to a positional location on the substrate. In some aspects, the spatial barcode tags the nucleic acid probe.

[0108] The barcode can be a naturally occurring nucleotide sequence or a nucleotide sequence that does not occur naturally in the organism from which the barcoded nucleic acid was obtained. A barcode sequence can be unique to a single nucleic acid species in a population or a barcode sequence can be shared by several different nucleic acid species in a population. For example, each nucleic acid capture probe in a population on a substrate for spatial capture of nucleic acids in a biological sample, e.g., a permeabilized tissue sample, a cell suspension, can include different barcode sequences from all other nucleic acid capture probes in the population. Alternatively, each nucleic acid probe in a population can include different barcode sequences from some or most other nucleic acid capture probes in a population. For example, each capture probe in a population can have a barcode that is present for several different capture probes in the population even though the capture probes with the common barcode differ from each other at other sequence regions along their length. In various aspects, one or more barcode sequences that are used with a biological tissue are not present in the genome, transcriptome, or other nucleic acids of the tissue sample. For example, barcode sequences can have less than 80%, 70%, 60%, 50%, or 40% sequence identity to the nucleic acid sequences in a particular tissue.

[0109] Barcode sequences can be any of a variety of lengths. Longer sequences can generally accommodate a larger number and variety of barcodes for a population. Generally, all probes in a plurality will have the same length barcode (albeit with different sequences), but it is also possible to use different length barcodes for different probes. A barcode sequence can be at least 2, 4, 6, 8, 10, 12, 15, 20 or more nucleotides in length. Alternatively or additionally, the length of the barcode sequence can be at most 20, 15, 12, 10, 8, 6, 4, or fewer nucleotides. Examples of barcode sequences that can be used are set forth, for example in, US Pat. App. Publ. No. 2014 / 0342921 Al and US Pat. No. 8,460,865, each of which is incorporated herein by reference.D. Capture Sequence

[0110] In some aspects, the substrate can comprise a capture sequence. As used herein, a “capture sequence” refers to a nucleic acid sequence that is generally complementary to a target molecule. In some aspects, the capture sequence hybridizes to its complementary target molecule thereby capturing the target molecule. In multiplex aspects, a plurality of different nucleic acid probes can include different target capture sequences that hybridize to different target nucleic acid sequences from a tissue sample. Different target capture sequences can be used to selectively bind to one or more desired target molecules in a tissue sample.[OHl] In some aspects, the capture sequence is a gene-specific nucleic acid sequence. As used herein, the term "gene-specific" or "target specific" when used in reference to a capture probe or other nucleic acid is intended to mean a capture probe or other nucleic acid that includes a nucleotide sequence specific to a targeted nucleic acid, e.g., a nucleic acid from a tissue sample, namely a sequence of nucleotides capable of selectively annealing to an identifying region of a targeted nucleic acid. Gene-specific capture probes can have a single species of oligonucleotide, or can include two or more species with different sequences. Thus, the gene-specific capture probes can be two or more sequences, including 3, 4, 5, 6, 7, 8, 9 or 10 or more different sequences. The gene-specific capture probes can comprise a gene-specific capture primer sequence and a universal capture probe sequence. Other sequences such as sequencing primer sequences and the like also can be included in a genespecific capture primer.

[0112] In some aspects, the capture sequence can be a universal capture sequence. As used herein, the terms "universal capture sequence" or “universal sequence” refer to a series of nucleotides that is common to two or more nucleic acid molecules even if the molecules also have regions of sequence that differ from each other. A universal sequence that is present in different members of a collection of molecules can allow the capture of multiple different nucleic acids using a population of universal capture nucleic acids that are complementary to the universal sequence. Similarly, a universal sequence present in different members of a collection of molecules can allow the replication or amplification of multiple different nucleic acids using a population of universal primers that are complementary to the universal sequence. Thus, a universal capture nucleic acid or a universal primer includes a sequence that can hybridize specifically to a universal sequence. Target nucleic acid molecules may be modified to attach universal adapters, for example,at one or both ends of the different target sequences. In some aspects, the universal capture sequence can be a random nucleic acid sequence, or a semi-random nucleic acid sequence.

[0113] In some aspects, the capture sequence hybridizes to an adaptor region in a nucleic acid sequencing library. In some aspects, the capture sequence can contain a PolyA sequence. In some aspects, the capture sequence can contain a PolyT sequence As used herein, the terms “Poly T,” or “Poly A” refer to a nucleic acid sequence that comprises two or more thiamine (T) or adenine (A) bases, respectively. A Poly T or Poly A sequence can include at least about 2, 5, 8, 10, 12, 15, 18, 20, or more of the T or A bases.

[0114] Any of a variety of target nucleic acids can be captured and analyzed in a method set forth herein including, but not limited to, messenger RNA (mRNA), copy DNA (cDNA), genomic DNA (gDNA), ribosomal RNA (rRNA) or transfer RNA (tRNA). Particular target sequences can be selected from databases and appropriate capture sequences designed using techniques and databases known in the art.E. Methods of detecting methylation marks in a biological sample

[0115] In some aspects, a biological sample is permeabilized and the nuclear DNA is fragmented. In some aspects, the nuclear DNA is fragmented through treatment with a transposome structure. In some aspects, the transposome contains a transposase, a transposase recognition site Mosaic End (ME), and an adapter sequence. In some aspects, the transposome also contains a random sequence. In some aspects, the transposome is forked. In some aspects, the transposome is non-forked. In some aspects, the transposase is Tn5 transposase. In some aspects, the adapter is a poly-A sequence. In some aspects, the adapter is a poly-U sequence. In some aspects, the transposome tagments the DNA in the biological sample. As used herein, “tagmenf ’ or “tagmentation” refers to the simultaneous fragmentation of DNA and addition of a sequence to the DNA fragments. In some aspects, the tagmentation adds the adapter sequence to the 3' terminus of the DNA fragments in the biological sample. In some aspects, the transposase is fused to other proteins to enable guided tagmentation at specific genomic loci. In some aspects, the transposase is fused to a catalytically dead Cas9 (dCas9) protein. In some aspects, the transposase is fused to Protein A to enable antibody-guided targeting to specific histone markers.

[0116] In some aspects, the DNA fragments have a poly-U sequence added to the 3' terminus. In some aspects, the DNA fragments are treated with a polymerase to copy the poly-U sequence into a complementary poly-A sequence on the synthesized second DNAstrand. In some aspects, the DNA fragments are then denatured to release the strand with the poly-A sequence from the strand with the poly-U sequence to enable binding to a nucleic acid probe. In some aspects, the DNA fragments are denatured using physical methods. In some aspects, the physical method is heating the DNA fragments until the strands are denatured. In some aspects, the DNA fragments are denatured using chemical methods, for example dimethyl sulfoxide (DMSO), formamide, or other organic solvents. In some aspects, the chemical method is treating the DNA fragments with sodium hydroxide. In some aspects, the DNA fragments may contain uracil base(s) which enable treatment with USER to release the strand with the poly-A sequence for binding to a nucleic acid probe. In some aspects, the DNA fragments are treated with Uracil DNA Glycosylase (UDG) and an abasic site endonuclease to release the strand with the poly-A sequence for binding to a nucleic acid probe. In some aspects, the abasic site endonuclease is Endonuclease VIII.

[0117] In some aspects, a biological sample is permeabilized and the nuclear DNA is fragmented with dsDNA fragmentase and subsequently denatured to single-stranded DNA. In some aspects, the single-stranded fragmented DNA is hybridized to oligonucleotides designed to target specific genomic loci. In some aspects, the oligonucleotides further contain a functional group on the 5' terminus. In some aspects, the functional group is an azide, alkyne, DIBAC, tetrazine, norbornene, or phosphine group.

[0118] In some aspects, the tagmented DNA fragments can passively diffuse to a substrate containing nucleic acid probes containing a spatial barcode. In some aspects, the nucleic acid probes also contain a poly-T capture sequence on the 3' terminus. In some aspects, the DNA fragments contain a poly-A sequence on the 3' terminus. In some aspects, the DNA fragments bind to the nucleic acid probes through the complementary poly-A and poly-T sequences.

[0119] In some aspects, the nucleic acid probes also contain a functional group at the 3' terminus. In some aspects, the DNA fragments are hybridized to an oligonucleotide with a functional group at the 5' terminus. In some aspects, the functional group is an azide, alkyne, DIBAC, tetrazine, norbomene, or phosphine group. In some aspects, the nucleic acid probes and DNA fragments bind through a click reaction. In some aspects, the click reaction is a copper-catalyzed azide-alkyne cycloaddition (CuAAC), wherein copper is added to initialize the reaction. In some aspects, the click reaction is a strain-promoted azide-alkyne cycloaddition (SPAAC). In some aspects, the click reaction is a Diels- Alderreaction with inverse electron demand (DARinv). In some aspects, the click reaction is a Staudinger ligation.

[0120] In some aspects, the hybridized nucleic acid probe-DNA fragment complex is treated with a non-strand displacing polymerase to extend the DNA fragment using the nucleic acid probe as a template, thereby adding the spatial barcode sequence to the DNA fragment. In some aspects, the polymerase is T4 DNA polymerase. In some aspects, the DNA fragment is treated with a ligase to fill gaps remaining following tagmentation. In some aspects, the ligase is Taq DNA ligase. In some aspects, the sample is treated with single-stranded exonuclease to remove un-occupied nucleic acid probes and excess sequence on DNA fragments.

[0121] In some aspects, the nuclear DNA is tagmented with a transposase on a nucleic acid probe. In some aspects, a spatial barcode is added to the DNA during tagmentation. In some aspects, the nuclear DNA is treated with an antibody targeting a specific histone modification prior to tagmentation. In some aspects, the transposase is targeted to the antibody-bound regions.

[0122] In some aspects, the DNA fragment is denatured to untether the nucleic acid probe. In some aspects, the DNA fragment is treated under conditions that enable methycytosine (mC) discrimination. In some aspects, the discrimination condition is treatment with a mC- selective deaminase that converts mC to T, while producing less C to U conversion. In some aspects, the discrimination condition is treatment with a C-selective deaminase that converts C to U, while producing less mC to T conversion. In some aspects, the deaminase treated nucleotides have reverse selectivity. In some aspects, the discrimination condition is treatment with bisulfite conversion methods. In some aspects, the discrimination condition is treatment with EM-seq methods.

[0123] In some aspects, the DNA fragment is indexed and amplified to enable sequencing by synthesis. In some aspects, the amplification uses P5 and P7 primers.F. Methods of preparing a surface for spatially-barcoding a biological sample

[0124] In some aspects, oligonucleotides are grafted into a surface. In some aspects, the oligonucleotides are nucleic acid probes. In some aspects, the nucleic acid probes consist of a P7 adapter, a spatial barcode (SBC), a sequencing adapter (e.g., BSB12), a random sequence (X sequence), a restriction enzyme recognition site (RS region) and a P5 sequence. In some aspects, the oligonucleotides grafted onto the surface are P5 and P7adapters. In some aspects, a nucleic acid probe is hybridized onto the P5 or P7 adapters on the surface. In some aspects, the nucleic acid probe has the structure P7-i7-SBC-Y-ME- RS-X-P5’, wherein X and Y are any defined sequences. In some aspects, X and Y are A14 and B15 sequences.

[0125] In some aspects, the nucleic acid probes are amplified to form clusters. In some aspects, bridge amplification is used to form clusters. In some aspects, ExAmp is used to form clusters. In some aspects, each cluster has a unique spatial barcode sequence. In some aspects, the clusters do not overlap. In some aspects, the clusters overlap at the boundaries between clusters. In some aspects, the P5 strand on the surface is cleaved and washed away.

[0126] In some aspects, the spatial barcode is decoded by sequencing by synthesis (SBS).In some aspects, SBS proceeds after hybridizing an oligonucleotide to the nucleic acid probes. In some aspects, the oligonucleotide has the structure of X’ -RS’ -ME’- Y’. In some aspects, the oligonucleotide has the structure of X’-RS’-Y’.

[0127] In some aspects, a restriction enzyme is applied to the surface to cleave the RS region in the nucleic acid probe to expose a capture region. In some aspects, the restriction enzyme cuts at the end of a ME sequence. In some aspects, the nucleic acid probe is denatured to remove the untethered strand. In some aspects, an oligonucleotide is hybridized to the tethered strand and extended using DNA polymerase. In some aspects, the oligonucleotide has the structure ME’-RS’-Y’-blocker, wherein the blocker prevents extension of the hybridized oligonucleotide and wherein the RS' is complementary to the portion of the RS region left following digestion. In some aspects, the oligonucleotide has the structure ME / ME’ -RS’ -Y’ -blocker. In some aspects, the blocker is an inverted-T. In some aspects, ME' is added to form a functional ME-ME' structure to bind a transposase. In some aspects, a transposase is loaded onto the nucleic acid probe. In some aspects, the transposase is Tn5. In some aspects, terminal deoxynucleotidyl transferase is used to incorporate modified nucleotides at the 3 ' terminus of the nucleic acid probe, corresponding to the X region. In some aspects, the modified nucleotides are modified nucleoside triphosphate (NTP) with different functional groups. In some aspects, the functional groups can include azide, alkyne, DIBAC, tetrazine, alkene, and / or norbornene groups.EXAMPLESExample 1

[0128] A tissue sample is mounted to a spatially barcoded surface as shown in Figure 1, followed by permeabilization of the sample to enable access to the nucleic acids contained in each cell. The spatially barcoded surface contains nucleic acid probes attached and immobilized to the surface by their 5' end. The nucleic acid probes contain, in a 5' to 3' orientation, a P7 adapter, i7 sequencing index, spatial barcode, and a poly-T sequence. The sample is then tagmented using a forked transposome structure containing a transposase (e.g., Tn5 transposase) and a poly-A sequence, as shown in Figure 2. The transposome fragments the DNA in the tissue into smaller fragments, and simultaneously adds the poly- A sequence to the 3' end of each DNA fragment to be used as an adapter sequence in downstream steps. If methylation marks at particular genomic loci are of interest, the transposase may be fused to a catalytically dead Cas9 (dCas9) protein to enable targeting a specific genetic locus. Alternatively, the transposase may be fused to Protein A to enable antibody-guided targeting to histone markers of interest.

[0129] Following tagmentation, the DNA fragments can diffuse to the spatially barcoded surface where the poly-A sequence on each DNA fragment will bind to the complementary poly-T sequence on each nucleic acid probe. Subsequently, the DNA fragments will be treated with a non-strand displacing polymerase (e.g., T4 DNA polymerase exo-) and a ligase (e.g. Taq DNA ligase) to fill the 9-basepair gap remaining after tagmentation. The polymerase will further copy the adapter sequence and spatial barcode from the nucleic acid probes to the 3' end of the DNA fragments.Example 2

[0130] A tissue sample is mounted to a spatially barcoded surface, followed by permeabilization of the sample to enable access to the nucleic acids contained in each cell. The spatially barcoded surface contains nucleic acid probes attached and immobilized to the surface by their 5' end. The nucleic acid probes contain, in a 5' to 3' orientation, a P7 adapter, i7 sequencing index, spatial barcode, and a poly-T sequence. The sample is then tagmented using a non-forked transposome structure containing a transposase (e.g., Tn5 transposase) and a poly-U sequence, as shown in Figure 3. The transposome fragments the DNA in the tissue into smaller fragments, and simultaneously adds the poly-U sequence tothe 5' end of each DNA fragment. If methylation marks at particular genomic loci are of interest, the transposase may be fused to a catalytically dead Cas9 (dCas9) protein to enable targeting a specific genetic locus. Alternatively, the transposase may be fused to Protein A to enable antibody-guided targeting to histone markers of interest.

[0131] Following tagmentation, the sample is treated with a polymerase to extend each DNA fragment and to copy the poly-U sequence into a complementary poly-A sequence. The DNA fragments are then denatured using physical (e.g., heat) or chemical (e.g., sodium hydroxide) methods to release the poly-A sequence from the poly-U sequence to enable binding to the poly-T sequence on the nucleic acid probes. Alternatively, the DNA fragments are treated with USER or a cocktail of Uracil DNA Glycosylase (UDG) and an abasic site endonuclease (e.g. Endonuclease VIII) to digest away the poly-Us, thereby releasing the poly-A sequence for binding. Subsequently, the DNA fragments can diffuse to the spatially barcoded surface where the poly-A sequence on each DNA fragment will bind to the complementary poly-T sequence on each nucleic acid probe. The DNA fragments will then be treated with a non-strand displacing polymerase (e.g., T4 DNA polymerase exo-) to copy the adapter sequence and spatial barcode from the nucleic acid probes to the 3' end of the DNA fragments.Example 3

[0132] The spatially-barcoded DNA fragments as described in Example 1 or Example 2 are treated under conditions that enable methylcytosine discrimination. Figure 4A depicts the process using the DNA fragments generated in Example 1, and Figure 4B depicts using the DNA fragments generated in Example 2. The DNA fragments are denatured and then treated with an mC-selective deaminase to convert mC to T. Alternative methylation conversion methods such as bisulfite or EM-seq may be used, provided suitable adapter sequences are utilized. Following methylation conversion, the DNA fragment libraries are indexed and amplified using P5 and P7 primers to enable sequencing-by-synthesis.Example 4

[0133] A surface is prepared for appending spatial barcodes to DNA fragments using a click chemistry reaction. Nucleic acid probes as shown in Figure 5A, containing a P7 sequence, spatial barcode, SBS12 sequencing adapter, random (X) sequence, restriction enzyme recognition site (RS), and P5 sequence are grafted onto the surface. The nucleicacid probes are then amplified into clusters using bridge amplification or ExAmp amplification methods, as shown in Figure 5B. This results in each cluster of nucleic acid probes having a unique spatial barcode from each cluster’s original nucleic acid probe template. Following clustering, a restriction enzyme is applied to the surface to cleave the RS and P5 regions to expose the capture region. Terminal deoxynucleotidy transferase (TdT) is then used to incorporate modified nucleotides at the 3' end of the nucleic acid probe, in the X region. These modified nucleotides can include a range of modified nucleoside triphosphate (NTP) with different functional groups such as Azide, Alkyne, DIBAC, Tetrazine, Alkene, and Norbornene groups.Example 5

[0134] A tissue sample is mounted on a surface prepared according to Example 4, followed by addition of mild permeabilization buffer to lyse the cell membranes and expose the nuclear DNA. Nuclear DNA is then fragmented by the dsDNA fragmentase and denatured into single-stranded DNA. Then, hybridization oligonucleotides, containing a 5' functional group and a hybridization region, are introduced to bind their designated targets inside the tissue as shown in Figure 6A. During hybridization, DNA targets and hybridization oligonucleotides slowly diffuse from inside the tissue toward the surface under gravity, where click chemistry reactions may occur as shown in Figure 6B. A copper-catalyzed azide-alkyne cycloaddition (CuAAC) based click reaction may occur if copper is added into the reaction to initialize click reactions. For CuAAC to occur, the nucleic acid probes on the surface must have an Azide or Alkyne functional group at their 3' terminus, and the hybridization oligonucleotides must have the corresponding functional group on their 5' terminus. Alternatively, a strain-promoted azide-alkyne cycloaddition (SPAAC) based click reaction may occur if the surface nucleic acid probes have an Azide or DIBAC functional group at their 3' terminus, and the hybridization oligonucleotdies have the corresponding functional group on their 5' terminus. Alternatively, a Diels-Alder reaction with inverse electron demand (DARinv) click reaction may occur if the surface nucleic acid probes have a tetrazine or norbornene functional group at their 3' terminus, and the hybridization oligonucleotides have the corresponding functional group at their 5' terminus. Another alternative is a Staudinger ligation click reaction, requiring the surface nucleic acid probes to have a phosphine or azide functional group at their 3' terminus and the hybridization oligonucleotides to have the corresponding functional group at their 5'terminus. Hybridization oligonucleotides act to enrich for DNA regions of interest by localizing DNA fragments from those regions near the surface. DNA fragments that are not bound by a hybridization oligonucleotide will not be captured by the surface nucleic acid probes. Hybridization oligonucleotides also act as a primer to enable second-strand synthesis.Example 6

[0135] After the click chemistry reactions described in Example 5, the hybridization oligonucleotide is extended with the DNA target sequence as a template as shown in Figure 7, which increases the strength of the interaction between the DNA target sequence and the spatial barcode and protects the barcode from subsequent degradation. Single-stranded exonuclease is then used to digest and remove un-occupied surface nucleic acid probes, uncaptured hybridization oligonucleotides, and excess sequence on the DNA targets. Then, the DNA target sequence is extended toward the P7 region using the nucleic acid probe as a template. This extension requires copying over a scar in the DNA backbone created by the click chemistry reaction, therefore a polymerase that can replicate across that scar is essential. The full-length strand containing the DNA target is then eluted off of the nucleic acid probe on the surface and put into a mC-selective deaminase reaction, where 5mC is converted to T on the single-strand DNA. Using a P7 primer, the single-strand DNA is extended to generate a double-stranded DNA product. The P5 adapter and SB S3 regions are then ligated to the end of the double-stranded DNA fragment.Example 7

[0136] An alternative method for adding a spatial barcode onto DNA fragments is by directly linking the addition to the tagmentation step using a Tn5 transposase-spatial barcode flow cell surface wherein a ME sequence is linked with spatial barcodes on a surface for Tn5 to bind. To prepare such a surface, a template nucleic acid probe with the structure of P7-i7-SBC-Y-ME-RS-X-P5' is seeded onto P5 / P7 grafted flow cells, wherein X or Y are any defined sequences (e.g., A14 and B15 sequences), such that the P5' end of the template nucleic acid probe binds to the P5 molecules on the flow cell as shown in Figure 8. The template nucleic acid probe is then amplified into a cluster using bridge amplification or ExAmp methods such that each cluster has a unique spatial barcode corresponding to the original template oligonucleotide in the cluster. The P5 strand on thesurface is then cleaved and washed away, leaving only molecules attached to P7 following amplification. The spatial barcode is then decoded through sequencing by synthesis. This is accomplished by first hybridizing an oligonucleotide with the general structure of X'- RS'-ME'-Y' onto the surface-bound DNA, then performing the sequencing by synthesis reaction toward the surface of the flow cell. A restriction enzyme is then added onto the surface to digest at RS regions to cut precisely at the end of the ME region (e.g., BamHl). A denaturing wash then washes away the untethered strand, followed by the addition of an ME'oligonucleotide to hybridize with ME on the tethered strand to form a functional MEME' structure for Tn5 binding. Finally, Tn5 is loaded onto the grafted oligonucleotide.Example 8

[0137] An alternative strategy for preparing a Tn5 transposase-spatial barcode flow cell surface is shown in Figure 9, wherein a template nucleic acid probe with a general structure of P7-i7-SBC-Y-RS-X-P5’ is seeded onto flow cells grafted with P7 and P5 sequences. The template nucleic acid probe is then amplified into a cluster using bridge amplification or ExAmp methods such that each cluster has a unique spatial barcode corresponding to the original template nucleic acid probe in the cluster. The P5 strand on the surface is then cleaved and washed away, leaving only molecules attached to P7 following amplification. The spatial barcode is then decoded through sequencing by synthesis. This is accomplished by first hybridizing an oligonucleotide with the general structure of X'-RS'-Y' onto the surface-bound DNA, then performing the sequencing by synthesis reaction toward the surface of the flow cell. A restriction enzyme is then added onto the surface to digest at RS regions, followed by a denaturing wash to remove the untethered strand. An oligonucleotide with the structure ME'-RS'-Y'-blocker is added to hybridize with the tethered strand, followed by DNA polymerase and dNTPs to extend the tethered strand to copy the ME sequence. The blocker prevents extension of the hybridized oligonucleotide, and can be an inverted-T, for example. A denaturing wash is performed to remove the blocking oligonucleotide to leave only the tethered strand. A ME' sequence is added to hybridize with ME on the tethered strand to form a functional ME-ME' structure for Tn5 binding. Finally, Tn5 is loaded onto the grafted oligonucleotide.Example 9

[0138] Another alternative strategy for preparing a Tn5 transposase-spatial barcode flow cell surface is shown in Figure 10, wherein a template nucleic acid probe with a general structure of P7-i7-SBC-Y-RS-X-P5’ is seeded onto flow cells grafted with P7 and P5 sequences. The template nucleic acid probe is then amplified into a cluster using bridge amplification or ExAmp methods such that each cluster has a unique spatial barcode corresponding to the original template nucleic acid probe in the cluster. The P5 strand on the surface is then cleaved and washed away, leaving only molecules attached to P7 following amplification. The spatial barcode is then decoded through sequencing by synthesis. This is accomplished by first hybridizing an oligonucleotide with the general structure of X'-RS'-Y' onto the surface-bound DNA, then performing the sequencing by synthesis reaction toward the surface of the flow cell. A restriction enzyme is then added onto the surface to digest at RS regions, followed by a denaturing wash to remove the untethered strand. An oligonucleotide with the structure ME / ME'-RS'-Y'-blocker is added to hybridize with the tethered strand, followed by ligase to ligate ME with the tethered strand. A denaturing wash is then performed to remove the hybridized oligonucleotide to leave the tethered strand. A ME' sequence is added to hybridize with ME on the tethered strand to form a functional ME-ME' structure for Tn5 binding. Finally, Tn5 is loaded onto the grafted oligonucleotide.Example 10

[0139] A tissue sample is directly mounted onto a Tn5 transposase-spatial barcode flow cell prepared by any one of Examples 7-9, as shown in Figure 11. The tissue sample is then permeabilized to release DNA onto the surface. A soluble version of Tn5 loaded with X- ME oligonucleotides is added onto the tissue with Mg2+included buffer. The DNA region that is exposed or easily accessible is tagmented by both surface Tn5 and soluble Tn5. During the tagmentation process, a spatial barcode is added directly onto a DNA strand from the tissue. A DNA extension step follows to copy the spatial barcode from the nucleic acid probes onto the DNA strand from the tissue. A denaturing wash is performed to elute the untethered strand from the nucleic acid probe. The strand can be amplified directly with PCR sample index primers P7 and X-i5-P5 to make a sequencing library. Alternatively, the untethered DNA strand can be treated under conditions that enable methylcytosinediscrimination before the sample index PCR. This can be a mC-selective deaminase that converts mC to T. The methylation locations can then be detected through sequencing.Example 11

[0140] Detecting spatial histone modificationis performed as shown in Figure 12, by using the Tn5 transposase-spatial barcode flow cell prepared by any one of Examples 7-9 in combination with the CUT&Tag method. A tissue sample is mounted and permeabilized on the flow cell surface. An antibody to target a specific histone modification is added to the tissue, and excess antibodies are washed away. Then a solubilized protein A conjugated to X-ME-Tn5 is added onto the surface, subsequently being captured by an antibody. Tn5 would then cut near the histone modification region. After adding Mg2+to enable tagmentation, the X sequence from soluble proteinA-Tn5 is added near the histone modification region. The nucleic acid probes and bound DNA sequences are then extended to add the X and spatial barcode sequences onto the DNA near the histone modification region. The untethered strand is then eluted and amplified using PCR with P7 and X-i5-P5 primers. The library containing spatially-barcoded DNA at specific histone modification regions can then be sequenced. Alternatively, a catalytically dead Cas9 could be used to target the soluble Tn5 to regions of the genome of interest.

Claims

WHAT IS CLAIMED IS:

1. An array comprising one or more capture sites, wherein the capture sites comprise one or more nucleic acid probes immobilized on a solid support, and wherein the nucleic acid probes comprise: (a) a first clustering adapter sequence and a second clustering adapter sequence; (b) a spatial barcode (SBC) sequence; (c) a sequencing adapter sequence; (d) a restriction enzyme recognition site (RS region); and (e) a random region (X region).

2. The array of claim 1, wherein the X region is located at the 3’-terminus of the SBC.

3. The array of claim 1 or 2, wherein the nucleic acid probe comprises: 5’-first clustering adapter sequence-SBC sequence-X region-sequencing adapter-RS region-second clustering adapter sequence sequence-3 ’ .

4. The array of any one of claims 1-3, wherein the X region comprises modified nucleoside triphosphates with different functional groups.

5. The array of claim 4, wherein the functional groups are azide, alykyne, DIB AC, tetrazine, alkene, or norbornene groups.

6. The array of any one of claims 1-5, wherein the sequencing adaptor is the SBS12 sequencing adapter.

7. The array of any one of claims 1-6, wherein each of the nucleic acid probes on the solid support are amplified to form clusters , wherein each cluster comprises amplicons of a single nucleic acid probe on the solid support, and wherein the restriction enzyme recognition sequence and second clustering adapter sequence regions are cleaved to expose a capture region.

8. The array of claim 7, wherein each cluster has a unique SBC sequence.

9. The array of any one of claims 1-8, wherein the first clustering adapter sequence is P7 and the second clustering adapter sequence is P5.

10. The array of any one of claims 1-6, wherein the capture sites provide for spatial detection of nucleic acid methylation marks in a biological sample.

11. A method for spatially identifying nucleic acid methylation marks in the DNA of a biological sample, comprising: a. contacting a biological sample with a solid support that comprises a spatial bar code (SBC) sequence; b. permeabilizing the biological sample; c. fragmenting or tagmenting the DNA within the biological sample with a transposome containing an adapter sequence and a capture sequence; d. capturing the fragments from step c by hybridization or other attachment method of the fragments to the nucleic acid probes of the array, preferably click attachment, whereby a SBC sequence is appended to the DNA fragments; e. contacting the DNA fragments with a non-strand displacing polymerase and a ligase; treating the spatially barcoded nucleic acids of step d with methylation conversion to enable methylcytosine discrimination, thereby spatially identifying nucleic acid methylation marks in a biological sample.

12. The method of claim 9, wherein the transposome is a forked transposome.

13. The method of claim 9, wherein the transposome is a non-forked transposome.

14. The method of any one of claims 9-11, wherein the capture sequence is a poly A sequence.

15. The method of any one of claims 9-12, wherein the polymerase is T4 DNA polymerase and the ligase is Taq DNA ligase.

16. A method for spatially identifying nucleic acid methylation marks in the DNA of a biological sample, comprising: a. contacting a biological sample with a solid support that comprises a spatial bar code (SBC) sequence; b. permeabilizing the biological sample; c. fragmenting or tagmenting the DNA within the biological sample using a non-forked transposome containing an adapter sequence and a poly U capture sequence; d. contacting the DNA fragments from step c with a DNA polymerase; e. denaturing the DNA fragments from step d using physical or chemical means; f. capturing the fragments from step c by hybridization of the fragments to the nucleic acid probes of the array whereby a SBC sequence is appended to the DNA fragments; g. treating the spatially barcoded nucleic acids of step d with methylation conversion to enable methylcytosine discrimination, thereby spatially identifying nucleic acid methylation marks in a biological sample.

17. The method of claim 14, wherein the denaturing is performed by heating or treatment with sodium hydroxide.

18. The method of claim 15, wherein the denaturing is performed by treating the DNA sequences with USER or a combination of uracil DNA glycosylase (UDG) and an abasic site endonuclease.

19. The method of claim 16, wherein the abasic site endonuclease is Endonuclease VIII.

20. The method of claim 8 or 9, wherein the permeabilizing and fragmenting steps are performed prior to contacting the biological sample with the solid support.

21. The method of any one of claims 8-19, wherein the methylation conversion is performed using a methylcytosine selective deaminase, bisulfite, or EM-seq.

22. The method of any one of claims 8-20, wherein the DNA within the biological sample is targeted at a specific genomic locus of interest.

23. The method of claim 21, wherein the targeting is performed by a transposome containing a modified transposase that guides the targeting.

24. The method of claims 21 or 22, wherein the modified transposase comprises a catalytically dead Cas9 (dCas9) protein or a Protein A sequence.

25. The method of any one of claims 8-23, wherein step (e) further comprises amplification of the sequences and sequencing-by-synthesis.

26. A method for spatially identifying nucleic acid methylation marks in the DNA of a biological sample, comprising: a. providing the array of any one of claims 1-10 ; b. contacting a biological sample with a solid support that comprises the array of step a; c. permeabilizing the biological sample thereby releasing nuclear DNA from the sample; d. fragmenting the nuclear DNA within the biological sample; e. contacting the nuclear DNA from step d with a hybridization oligonucleotide that comprises a 5 ’-terminal functional group and a hybridization region that corresponds to a target region on the nuclear DNA under conditions that allow for hybridization of the nuclear DNA and the hybridization oligonucleotide; f. capturing the hybridized DNA from step e onto the solid support by click reaction chemistry; g. extending the 3' end of the hybridization oligonucleotide strand of the hybridized DNA using the nuclear DNA strand as a template; h. digesting unoccupied nucleic acid probes, uncaptured hybridization oligonucleotides, and single-stranded regions of the hybridized DNA by treatment with a single-stranded exonuclease;i. extending the 3’ end of the nuclear DNA strand using the hybridization oligonucleotide strand as a template; j . treating the spatially barcoded DNA with methylation conversion to enable methylcytosine discrimination, thereby spatially identifying nucleic acid methylation marks in a biological sample.

27. The method of claim 25, wherein the click reaction chemistry is copper-catalyzed azidealkyne cycoaddition (CuAAC)-based, strain-promoted azide-alkyne cycloaddition (SPAAC)-based, Diels-Alder reaction with inverse electron demand (DARinv), or Staudinger ligation.

28. The method of claim 26, wherein during CuAAC -based chemistry, the nucleic acid probes on the solid support comprise azide or alkyne functional groups at their 3’ terminus and the hybridization oligonucleotide comprises the corresponding functional group.

29. The method of claim 26, wherein during SPAAC -based chemistry, the nucleic acid probes on the solid support comprise azide or DIB AC functional groups at their 3’ terminus and the hybridization oligonucleotide comprises the corresponding functional group.

30. The method of claim 26, wherein during Diels-Alder reaction chemistry, the nucleic acid probes on the solid support comprise tetrazine or norbornene functional groups at their 3’ terminus and the hybridization oligonucleotide comprises the corresponding functional group.

31. The method of claim 26, wherein during Staudinger ligation chemistry, the nucleic acid probes on the solid support comprise phosphie or azide functional groups at their 3’ terminus and the hybridization oligonucleotide comprises the corresponding functional group.

32. The method of any one of claims 25-30, further comprising elution of the SBC-tagged DNA fragments and library generation.

33. A method of preparing a solid support for tagmentation of a target DNA in a biological sample, comprising: a. seeding a template oligonucleotide having the general structure P7-SBC- Y-ME-RS-X-P5’ onto a P7 / P5 grafted solid support, wherein X and Y are any defined sequence and RS is a restriction enzyme recognition site and wherein the template oligonucleotide is tethered to the solid support; b. amplifying the template oligonucleotide on the solid support to cluster the oligonucleotides such that each cluster have a unique SBC associated with it; c. cleaving the P5 strand off the surface; d. hybridizing an oligonucleotide with the general structure of X’-RS’-ME’- Y’ onto the template oligonucleotide and sequencing the SBC of the template oligonucleotide by synthesis; e. cleaving the template oligonucleotide at the RS; f. denaturing and washing away the untethered nucleic acid strand; g. hybridizing a ME’ oligonucleotide to the ME on the tethered strand to form a grafted oligonucleotide comprising a functional ME-ME’ structure; and h. contacting the grafted oligonucleotide with a Tn5 transposase thereby generating a solid surface for tagmenting the target DNA.

34. A method of preparing a solid surface for tagmentation of a target DNA in a biological sample, comprising: a. seeding a template oligonucleotide having the general structure P7-SBC- Y- RS-X-P5’ onto a P7 / P5 grafted surface, wherein X and Y are any define sequence and RS is a restriction enzyme recognition site and wherein the template oligonucleotide is tethered to the solid support; b. amplifying the template oligonucleotide on the solid support to cluster the oligonucleotides such that each cluster have a unique SBC associated with it; c. cleaving the P5 strand of the surface;d. hybridizing an oligonucleotide with the general structure of X’-RS’-Y’ onto the template oligonucleotide and sequencing the SBC of the template oligonucleotide by synthesis; e. cleaving the template oligonucleotide at the RS; f. denaturing and removing the untethered nucleic acid strand; g. hybridizing a ME’ -RS ’-Y’ -blocker to the tethered strand and extending the ME sequence onto the tethered strand; h. denaturing and removing the ME’-RS’-Y’-blocker; i. hybridizing ME’ sequence to ME on the tethered strand to form a grafted oligonucleotide comprising a functional ME-ME’ structure; and j . contacting the grafted oligonucleotide with a Tn5 transposase thereby generating a solid surface for tagmenting the target DNA.

35. A method of preparing a solid surface for tagmentation of a target DNA in a biological sample, comprising: a. seeding a template oligonucleotide having the general structure P7-SBC- Y- RS-X-P5’ onto a P7 / P5 grafted surface, wherein X and Y are any define sequence and RS is a restriction enzyme recognition site and wherein the template oligonucleotide is tethered to the solid support; b. amplifying the template oligonucleotide on the solid support to cluster the oligonucleotides such that each cluster have a unique SBC associated with it; c. cleaving the P5 strand of the surface; d. hybridizing an oligonucleotide with the general structure of X’-RS’-Y’ onto the template oligonucleotide and sequencing the SBC of the template oligonucleotide by synthesis; e. cleaving the template oligonucleotide at the RS; f. denaturing and removing the untethered nucleic acid strand; g. hybridizing a ME / ME’-RS’-Y’-blocker to the tethered strand and ligating the ME sequence onto the tethered strand; h. denaturing and removing the ME’-RS’-Y’-blocker; i. hybridizing ME’ sequence to ME on the tethered strand to form a grafted oligonucleotide comprising a functional ME-ME’ structure; andj . contacting the grafted oligonucleotide with a Tn5 transposase thereby generating a solid surface for tagmenting the target DNA.

36. The method of any one of claims 33-35, wherein the template oligonucleotide further comprises a sequencing index.

37. The method of any one of claims 32-34, wherein the solid support is a flow cell.

38. The method of any one of claims 32-35, wherein X and Y are Al 4 and B 15 sequences.

39. The method of any one of claims 32-36, wherein the RS is a BamHl recognition site.

40. The method of any one of claims 32-37, wherein step (b) is performed by bridge amplification or ExAmp methods.

41. A method for spatially identifying target DNA in a biological sample, comprising: a. preparing the solid support of any one of claims 32-38; b. contacting the biological sample with the solid support of step (a); c. permeabilizing the biological sample to release the target DNA; d. contacting the target DNA with the Tn5 transposase on the solid support and a soluble Tn5 transposase comprising X-ME oligonucleotides in the presence of SBC sequence, thereby tagmenting the DNA; e. extending the untethered DNA to also contain the SBC sequence; and f. eluting the untethered DNA strand by incubation with a denaturing wash buffer.

42. The method of claim 39, further comprising amplifying the untethered strand of step (e) by polymerase chain reaction (PCR) and generating a library of strands for sequencing.

43. A method for spatially identifying nucleic acid methylation marks in the DNA of a biological sample, comprising: a. preparing the solid support of any one of claims 32-38;b. contacting the biological sample with the solid support of step (a); c. permeabilizing the biological sample to release the target DNA; d. contacting the target DNA with the Tn5 transposase on the solid support and a soluble Tn5 transposase comprising X-ME oligonucleotides in the presence of SBC sequence, thereby tagmenting the DNA; e. extending the untethered DNA to also contain the SBC sequence; f. eluting the untethered DNA strand by incubation with a denaturing wash buffer; and g. treating the DNA with methylation conversion chemistry to enable methylcytosine discrimination, thereby spatially identifying nucleic acid methylation marks in a biological sample.

44. A method for spatially identifying nucleic acid methylation marks in the DNA of a biological sample, comprising: a. preparing the solid support of any one of claims 32-38; b. contacting the biological sample with the solid support of step (a); c. permeabilizing the biological sample to release the target DNA; d. contacting the target DNA with:(i) the Tn5 transposase on the solid support, and(ii) an antibody target-specific histone modification region, and a solubilized protein A-conjugated soluble Tn5 transposase comprising X-ME oligonucleotides in the presence of SBC sequence, or a dead Cas9 (dCas9)- conjugated soluble Tn5 transposase comprising X-ME oligonucleotides in the presence of SBC sequence, thereby tagmenting the DNA; e. extending the untethered DNA to also contain both the X and SBC sequence; f. eluting the untethered DNA strand by incubation with a denaturing wash buffer; and g. generating a library of DNA comprising specific histone-modification regions and identifying target DNA of interest by sequencing.

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