Single-cell and spatial DNA methylation measurements

Abstract

Methods for single-cell DNA methylation profiling are disclosed herein, with embodiments featuring use of droplet-based cell barcoding and conversion reagents to generate high-quality methylomes and / or methylome-plus-transcriptomes, for single cell genomic analysis and correlation of methylation state with transcript expression levels at single-cell resolution. The present disclosure is based, at least in part, upon discovery of methods and compositions capable of leveraging cell barcoding and conversion reagents to generate high-quality single-cell methylomes capable of being assessed rapidly in parallel across populations of cells, as well as coordinated single-cell methylome-plus-transcriptomes formatted for parallel, high-throughput assessment of cellular populations with precise single-cell resolution.

Description

SINGLE-CELL AND SPATIAL DNA METHYLATION MEASUREMENTSCROSS REFERENCE TO RELATED APPLICATION[0001 I This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 709,891, filed October 21, 2024, the entire content of which is incorporated herein by reference.STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0001] This invention was made with government support under Grant No. NS132135 awarded by the National Institutes of Health. The government has certain rights in the invention.TECHNICAL FIELD

[0002] The subject matter disclosed herein is generally directed to methods for DNA methylation profiling.BACKGROUND

[0003] DNA methylation is a fundamental component of epigenetic regulation, yet methods to profile DNA methylation at single-cell resolution are challenging to implement and have not been widely adopted. Droplet-based methods have greatly extended the accessibility and scale of single-cell sequencing studies, but there are limited options available for single-cell profiling of DNA methylation.

[0004] DNA methylation is a core layer of epigenetic regulation in mammalian cells that plays a crucial role in the development and maintenance of cellular identity. Existing methods to profile DNA methylation at single-cell resolution suffer from high reagent costs, complex protocols, and low throughput without extensive robotics infrastructure, which has limited the number and scope of single-cell DNA methylation studies to date. A need therefore exists for improved methods of single-cell DNA methylation profiling.

[0005] Citation or identification of any document in this application is not an admission that such a document is available as prior art to the present disclosure.SUMMARY

[0006] The disclosure is based, at least in part, upon discovery of methods for single-cell DNA methylation profiling that are compatible with existing approaches for isolating and assessing tagged nuclei of single cells (such extant single-cell approaches include, e.g., the "Slide-tags" approach previously presented in, e.g., WO 2024 / 138050). Aspects of the current disclosure provide methods for stabilizing single-cell barcode and / or spatial barcode sequences against conversion from unmethylated cytosine to uracil reactions subsequently performed upon genomic DNA fragments designed to discriminate naturally-occurring unmethylated cytosine residues from naturally-occurring methylated cytosine residues in a manner compatible with high- throughput assessment (e.g., bead-based and droplet-based manipulation, treatment, sequencing, and analysis approaches). In certain embodiments, a polymerase-mediated gap-fill reaction across a cell barcode-containing template strand is performed in the presence of methylated dCTP, thereby generating a conversion-protected cell barcode copy that can be selectively amplified and sequenced. Certain embodiments accordingly feature improved (single cell DNA methylation assessment-directed) use of droplet-based cell barcoding approaches and conversion reagents to generate high-quality methylomes and / or methylome-plus-transcriptomes, for single cell genomic analysis and correlation of methylation state with transcript expression levels and / or other phenotypes, selections, etc., at single-cell resolution.

[0007] In one aspect, the present disclosure provides a method for assessing DNA methylation in single nuclei, the method involving: fragmenting DNA to generate a population of DNA fragments, wherein for a plurality of the DNA fragments of the population, an adapter sequence is ligated to the DNA fragment, thereby generating a population of adapted DNA fragments, each comprising a top strand comprising a 5'-phosphate group at the 5'-end and a bottom strand; coencapsulating solid supports bound to ligation-based cell barcode oligonucleotides (L-CBOs) with the adapted DNA fragments in individual discrete volumes; ligating the L-CBOs to the 5’- ends- of the top strands of the adapted DNA fragments in the individual discrete volumes, thereby generating a population of barcoded DNA fragments, each including adapter and L-CBO sequences in the 5’-end region of the top strand; pooling the population of barcoded DNA fragments; and generating single-nucleus DNA methylation profiles from the population of barcoded DNA fragments, thereby assessing DNA methylation in single nuclei.

[0008] In one embodiment, the step of generating single-nucleus DNA methylation profiles from the population of barcoded DNA fragments includes: extending the bottom strands using a DNA polymerase enzyme and dNTPs with 5-methyl-dCTP substituted for dCTP to generate extended bottom strands where 5-methylcytosines are incorporated into the extended bottom strands at all positions complementary to guanine residues of the L-CBO sequences of the top strands; converting cytosine residues into uracil residues in the extended bottom strands, where unmethylated cytosine residues are converted to uracil residues, and 5'-methylcytosine residues (technically 5-methyl-cytadylyl moieties) are not converted into uracil residues, thereby generating converted extended bottom strands; amplifying and sequencing the amplified converted extended bottom strands; and detecting methylation sites in the converted extended bottom strand sequences and associating the methylation sites with individual L-CBO sequences of the converted extended bottom strands.

[0009] In a related embodiment, the converting step includes bisulfite-seq (BS-seq), enzymatic methyl-sequencing (EM-seq), or single-enzyme 5-methylcytosine sequencing (SEM-seq).

[0010] In an embodiment, the converting step uses a cytidine deaminase. Optionally, the cytidine deaminase is APOBEC1 or MsddA.

[0011] In another embodiment, the converting step uses a chemical reagent to deaminate unmethylated cytosines. Optionally, the chemical reagent is sodium bisulfite or ammonium bisulfite.

[0012] In one embodiment, the DNA is genomic DNA (gDNA).

[0013] In certain embodiments, the DNA is isolated from single cells or fragments thereof, nuclei, mitochondria, or chromosomes.

[0014] In some embodiments, the method further includes isolating nuclei containing the DNA and treating the nuclei with a nucleosome depletion agent prior to the fragmenting step. Optionally, the nucleosome depletion agent is sodium dodecylsulfate (SDS) or lithium diiodosalicylate (LIS).

[0015] In certain embodiments, the fragmenting step includes tagmentation, chemical fragmentation, or restriction enzyme digestion.

[0016] In one embodiment, the ligating step is performed using a lOx Single Cell Multi ome kit.

[0017] In some embodiments, fragmenting includes tagmentation using a transposase enzyme. Optionally, the transposase enzyme is hyperactive Tn5.

[0018] In certain embodiments, the solid support is a bead.

[0019] In some embodiments, the individual discrete volume is a droplet. Optionally, the nucleosome depletion agent is a detergent or a chaotropic agent.

[0020] In a related embodiment, the detergent is sodium dodecyl sulfate (SDS).

[0021] In another embodiment, the chaotropic agent is lithium diiodosalicylate (LIS).

[0022] In some embodiments, the method further includes treating the nuclei with a fixative agent prior to treating the nuclei with a nucleosome depletion agent. Optionally, the fixative agent is formaldehyde or dithio-bis(succinimidyl propionate).

[0023] In certain embodiments, the fragmenting step is performed using a mixture of: (1) a transposase loaded with a first adapter sequence having a 5 ’-phosphorylated single- stranded 5’-overhang, and (2) a transposase loaded with a second adapter sequence to generate a population of adapted DNA fragments, each having a first adapter sequence attached to the 5 ’-end of the top strand and a second adapter sequence attached to the 5 ’-end of the bottom strand; the ligating step includes ligating the L-CBOs to the 5 ’-overhang of the first adapter sequence; and the amplifying step includes using a primer specific to the L-CBO 5 ’-end and a primer specific to a derivative sequence including the second adapter sequence where the cytosines are replaced with uracils. Optionally, the ligating step is performed via a splint oligonucleotide having (i) a region complementary to the 5’-end of the first adapter sequence and (ii) a region complementary to the 3 ’-end of the L-CBO sequence.

[0024] In certain embodiments, the fragmenting step is performed using a transposase loaded with a first adapter sequence that includes a 5 ’-phosphorylated single-stranded 5 ’-overhang, thereby generating a population of adapted DNA fragments having the first adapter sequence attached to the original 5 ’-end of the top strand, where for any adapted DNA fragment, the strand with the 5 ’-overhang is the top strand and the strand complementary to the top strand is the bottom strand; the ligating step includes ligating the L-CBO to the 5 ’-overhang of the first adapter sequence; the step of generating single-nucleus DNA methylation profiles from the population of barcoded DNA fragments includes: extending the bottom strand using a DNA polymerase enzyme and dNTPs with 5-methyl-dCTP substituted for dCTP, thereby generating an extendedbottom strand where 5-methylcytosines are incorporated into the extended bottom strand at all positions complementary to guanine residues of the L-CBO sequence of the top strand; converting cytosine residues into uracil residues in the extended bottom strand, where unmethylated cytosine residues are converted to uracil residues, and 5'-methylcytosine residues are not converted into uracil residues, thereby generating a converted extended bottom strand; linearly amplifying the converted extended bottom strand using a primer specific to the L-CBO sequence at the 5’ end, thereby generating a double-stranded product comprising the converted extended bottom strand and its complement; adding a second adapter sequence to the double-stranded product comprising the converted extended bottom strand and its complement via (a) random priming or (b) adaptase chemistry, thereby generating a second adapter-comprising product; amplifying the second adapter-comprising product using a primer specific to the L-CBO sequence and a primer specific to the second adapter; sequencing the amplified second adapter-comprising product; and detecting methylation sites associated with each L-CBO sequence. Optionally, the ligating step is performed via a splint oligonucleotide having a first region complementary to the 5 ’-end of the first adapter sequence and a second region complementary to the 3 ’-end of the L-CBO.

[0025] In another aspect, the present disclosure provides a method for assessing DNA methylation and gene expression in single nuclei, the method involving: treating nuclei containing gDNA and mRNA with a nucleosome depletion agent (e.g., SDS, LIS); fragmenting the gDNA of the nuclei to generate a population of gDNA fragments, where for a plurality of the gDNA fragments of the population, an adapter sequence is ligated to the gDNA fragment, thereby generating a population of adapted gDNA fragments, each including a top strand including a 5'-phosphate group at the 5'-end and a bottom strand; co-encapsulating in individual discrete volumes solid supports bound to ligation-based cell barcode oligonucleotides (L-CBOs) and reverse transcription-based cell barcode oligonucleotides (RT-CBOs) with individual nuclei including the adapted gDNA fragments; cell-barcoding gDNA fragments and mRNA by: ligating L-CBOs to the 5’-ends- of the top strands of the adapted gDNA fragments in the individual discrete volumes, thereby generating a population of barcoded gDNA fragments, each with adapter and L-CBO sequences at the 5 ’-end of the top strand; and reverse-transcribing the mRNA using RT-CBOs as primers, thereby generating a population of cell-barcoded first-strand cDNA molecules; pooling the population of barcoded gDNA fragments and the population of cell-barcoded first-strand cDNA molecules; generating single-nucleus DNA methylation profiles by: extending the bottom strand of the gDNA fragments using a DNA polymerase enzyme and dNTPs with 5-methyl-dCTP substituted for dCTP to generate extended bottom strands wherein 5-methylcytosines are incorporated into the extended bottom strands at all positions complementary to guanine residues of the L-CBO sequence of the top strand; converting cytosine residues into uracil residues in the extended bottom strands, wherein unmethylated cytosine residues are converted to uracil residues, and 5-methylcytosine residues are not converted into uracil residues, thereby generating converted extended bottom strands; amplifying the converted extended bottom strands; sequencing the amplified converted extended bottom strands; detecting methylation sites associated with individual L-CBO sequences of the converted extended bottom strands; generating single-nucleus gene expression profiles by: amplifying the cell-barcoded first strand cDNA molecules; and sequencing the amplified cell-barcoded first strand cDNA molecules and associating sequenced cDNAs with individual RT-CBO sequences; thereby assessing DNA methylation and gene expression in single nuclei.

[0026] In one embodiment, the method includes, prior to the step of amplifying the cell-barcoded first strand cDNA molecules, performing tempi ate- switching reactions using Template-Switching Oligonucleotides (TSOs) within the individual discrete volumes, thereby generating a population of cell-barcoded first-strand cDNA molecules having 3 ’-terminal TSO sequences.

[0027] In a related embodiment, the TSO includes a 5 ’-biotin; the cell-barcoded first-strand cDNA molecules include 3’-terminal TSO sequences and biotin; and / or the method further includes, after the pooling step, separating the cell-barcoded first-strand cDNA molecules having 3’-terminal TSO sequences and biotin from the population of barcoded gDNA fragments via binding of the biotin to streptavidin beads.

[0028] In another aspect, the present disclosure provides a method for assessing DNA methylation, gene expression, and spatial location in single nuclei, the method involving: contacting a spatial array with a tissue section, where the spatial array includes spatial barcode oligonucleotides (SBOs) including spatial barcode sequences coupled to individual solid supports of the array via cleavable linkers, where the spatial barcode sequences are the same for an individual solid support of the array but differ between individual solid supports of the array, and where the spatial location and spatial barcode sequence of each individual solid support is known;cleaving the linkers, thereby delivering the SBOs to cells of the tissue section and generating tagged nuclei containing gDNA molecules and RNA molecules; isolating the tagged nuclei containing the gDNA molecules and RNA molecules from the tissue section; treating nuclei with a nucleosome depletion agent (e.g., SDS, LIS); fragmenting the nuclei to generate a population of gDNA fragments, where for a plurality of the gDNA fragments of the population, an adapter sequence is ligated to the gDNA fragment, thereby generating a population of adapted gDNA fragments, each including a top strand including a 5'-phosphate group at the 5'-end and a bottom strand; co-encapsulating in individual discrete volumes solid supports bound to ligation-based cell barcode oligonucleotides (L-CBOs) and reverse transcription-based cell barcode oligonucleotides (RT-CBOs) with the adapted gDNA fragments; cell-barcoding gDNA fragments, RNA molecules, and SBOs by: ligating L-CBOs to the 5’-ends of the top strands of the adapted gDNA fragments in the individual discrete volumes, thereby generating a population of barcoded gDNA fragments, each with adapter and L-CBO sequences at the 5’-end of the top strand; reverse-transcribing the RNA molecules using released RT-CBOs as primers, thereby generating a population of cell-barcoded first-strand cDNA molecules; extending SBOs using RT-CBOs as primers, thereby generating a population of cell-barcoded SBOs; and pooling the population of cell-barcoded gDNA fragments, the population of cell-barcoded first-strand cDNA molecules, and the population of cell-barcoded SBOs; generating single-nucleus DNA methylation profiles by: extending the bottom strand of the gDNA fragments using a DNA polymerase enzyme and dNTPs with 5-methyl-dCTP substituted for dCTP to generate extended bottom strands wherein 5-methylcytosines are incorporated into the extended bottom strands at all positions complementary to guanine residues of the L-CBO sequence of the top strand; converting cytosine residues into uracil residues in the extended bottom strands, wherein unmethylated cytosine residues are converted to uracil residues, and 5-methylcytosine residues are not converted into uracil residues, thereby generating converted extended bottom strands; amplifying and sequencing the amplified converted extended bottom strands; and detecting methylation sites in the converted extended bottom strand sequences and associating said methylation sites with individual L-CBO sequences of the converted extended bottom strands; generating single-nucleus gene expression profiles by: amplifying and sequencing the amplified cell-barcoded first strand cDNA molecules and associating sequenced cDNAs with individualRT-CBO sequences; generating single-nucleus spatial positions by: amplifying and sequencing the cell-barcoded SBO; counting the number of SBOs from each solid support in the spatial array associated with each nucleus to generate a distribution of SBOs; and determining the spatial locations of each nucleus within its original tissue section as the spatial centroid of the distribution for the single-nucleus DNA methylation profile and the single-nucleus gene expression profile for the nucleus, thereby assessing DNA methylation, gene expression, and spatial location of single nuclei.

[0029] In one embodiment, prior to the step of contacting the spatial array with the tissue section, the spatial array is sequence-verified by a process that includes in situ sequencing of nucleic acid sequences having spatial barcode sequences, thereby generating an index of the spatial barcode sequences.BRIEF DESCRIPTION OF THE DRAWINGS

[0030] An understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure may be utilized, and the accompanying drawings of which:

[0031] FIG. 1A shows a schematic for an exemplary method for single-cell DNA methylation sequencing (scDNAme-seq) that employs droplet-based cell barcoding ("dropMe"). Genomic DNA (gDNA) from a nucleosome-depleted nucleus is tagmented using a cocktail of transposases loaded with (i) 5 ’-phosphorylated Nextera Read 1 (pRln) or (ii) Nextera Read 2 (R2n), generating gDNA fragments flanked by pRln and R2n adapters, where the strand flanked by 5’-pRln is designated the top strand and the strand flanked by 5’-R2n is designated the bottom strand. Ligation-based cell-barcoded oligos (L-CBOs) released from a bead within a co-encapsulating droplet are ligated to top strand pRln adapters via a splint oligo. An extension step in which dCTP has been substituted with 5-methyl-dCTP generates an extended bottom strand with a conversion- protected copy of the cell barcode sequence. The resulting barcoded native gDNA fragments are used as input for DNA methylation profiling methods that label unmethylated cytosine residues through conversion to uracil. Following conversion, the bottom strand is selectively amplified and sequenced.

[0032] FIG. IB shows a schematic for another exemplary method for single-cell DNA methylation sequencing (scDNAme-seq) that employs droplet-based cell barcoding ("dropMe"). Genomic DNA (gDNA) from a nucleosome-depleted nucleus is tagmented transposase loaded with 5 ’-phosphorylated Nextera Read 1 (pRln), generating gDNA fragments flanked by pRln at both ends, where at each end the strand with the 5’-pRln is designated as the top strand and the strand with the 3’-end is designated as the bottom strand. Ligation-based cell-barcoded oligos (L-CBOs) released from a bead within a co-encapsulating droplet are ligated to top strand pRln adapters via a splint oligo. An extension step in which dCTP has been substituted with 5-methyl-dCTP generates an extended bottom strand with a conversion-protected copy of the cell barcode sequence. The resulting barcoded native gDNA fragments are used as input for DNA methylation profiling methods that label unmethylated cytosine residues through conversion to uracil. Following conversion, the bottom strand is linearly amplified using a primer specific to the 5’ end of the L-CBO. A R2n adapter sequence is added to these molecules by random priming. The resulting molecules are further amplified and sequenced.

[0033] FIG. 1C shows a schematic for an exemplary method for single-cell RNA sequencing (scRNA-seq) that employs droplet-based cell barcoding ("dropMe"). mRNA is reverse transcribed into cDNA using a primer containing an oligo-dT sequence and a unique molecular identifier (UMI) combined with a cell barcode (CB). A biotinylated Template Switching Oligo (TSO) binds to the cDNA. The TSO has a sequence that is complementary to the newly synthesized cDNA and includes a biotin tag. The reverse transcriptase extends the cDNA, incorporating the TSO sequence at the 3’ end of the cDNA. This adds a second sequencing handle to the cDNA. The cDNA fragments are purified using streptavidin (SA) beads.

[0034] FIG. 2 demonstrates a proof-of-principle dropMe experiment. About 3,000 nuclei isolated from a mouse brain sample were profiled. Nucleosome depletion was performed by crosslinking with SDS (xSDS), single-cell barcoding was performed using the lOx Single Cell Multiome kit, and conversion was performed using the NEB enzymatic methyl-sequencing (EM-seq) kit. Single-cell clustering and dimensionality reduction results are shown. Clustering was performed on mean %mCpG across 100-kb genomic bins. Cell types were annotated with reference to a mouse brain DNA methylation atlas.

[0035] FIG. 3 shows a schematic for "Slide-tags", a platform for spatially resolved singlenucleus RNA- and / or ATAC-seq, as set forth in PCT / US2023 / 085534 (published as WO 2024 / 138050). Slide-tags employs a dense array of oligo- conjugated 10-pm- diameter beads. In the lOx Multiome workflow, these bead-conjugated “spatial barcode oligos” (SBOs) comprise (from 5’ to 3’): (1) a photocleavable linker; (2) a bead-specific “spatial barcode” (SB) associated with the bead’s position in the array through SoLID sequencing; and (3) a poly-A tail, enabling their capture by cell-barcoded reverse transcription oligos released from a GEM bead within a coencapsulating droplet. A punch biopsy (up to 7 mm2in size) is taken from a 20-pm fresh frozen tissue section, melted onto a Slide-tags Multiome array, and exposed to UV light, whereupon photocleaved SBOs diffuse into the affixed tissue and become stably associated with nuclei. These nuclei are then extracted from the section and processed using the lOx Multiome kit. The original position of nuclei in the tissue section is later computationally inferred from the positions of beads whose SBOs became associated with a given nucleus, which can be done with approximately 3-pm spatial resolution and 99% placement accuracy.

[0036] FIGs. 4A-4C demonstrates a proof-of-principle Slide-tags dropMe experiment. About 2,000 nuclei isolated from a mouse hippocampus sample were profiled. Nucleosome depletion was performed by crosslinking with SDS (xSDS), cell barcoding was performed using the lOx Multiome kit, and conversion was performed using the EM-seq kit. Spatial barcodes were amplified from unconverted lOx Multiome output. FIG. 4A shows a Uniform Manifold Approximation and Projection (UMAP) of single-cell DNAme profiles, shaded by DNA methylation (“DNAme”) cluster. FIG. 4B shows a spatial reconstruction of the profiled sample, shaded by DNAme cluster. FIG. 4C shows a spatial reconstruction of the profiled sample, split and shaded by DNAme cluster.

[0037] FIGs. 5A-5C demonstrates a proof-of-principle Slide-tags dropMe+RNA experiment. About 4,000 nuclei isolated from a human glioblastoma sample were profiled. Nucleosome depletion was performed by crosslinking with SDS (xSDS), cell barcoding was performed using the lOx Multiome kit, and conversion was performed using the EM-seq kit. Spatial barcodes and cDNA were amplified from unconverted lOx Multiome output. FIG. 54 shows a Uniform Manifold Approximation and Projection (UMAP) of single-cell RNA profiles, shaded by RNA cluster, DNAme cluster, and fraction of CpG sites methylated (“%mCG”). FIG. 5B shows aUniform Manifold Approximation and Projection (UMAP) of single-cell gene DNAme profiles, shaded by RNA cluster, DNAme cluster, and fraction of CpG sites methylated. FIG. 5C shows a spatial reconstruction of the profiled sample, shaded by RNA cluster, DNAme cluster, and fraction of CpG sites methylated (“%mCG”).

[0038] FIG. 6A-6E demonstrate methylome mapping in the developing mammalian brain.FIG. 6A and FIG. 6B show dimensionality reduction and clustering on either RNA or mCG yielded spatially structured clusters corresponding to expected cell types and structures of the E16.5 forebrain. FIG. 6C shows identification of genes that are potentially regulated by large CG methylation domains in the cortical region by correlating each gene’s expression with Mean Squared Residue (MSR) scores of all VMRs within 50 kb of its transcription start site (TSS), merging significant like-signed cis-VMRs into aggregated methylation domains. This analysis identified 39 genes with >4.9 kb of negatively correlated cis-VMRs and 39 genes with >3.9 kb of positively correlated cis-VMRs. FIG. 6D shows GO terms relating to neurodevelopment. FIG.6E shows the relationship between CH methylation and gene expression across diencephalic, pallial, and subpallial regions.

[0039] The figures herein are for illustrative purposes only and are not necessarily drawn to scale.

[0040] These and other aspects, objects, features, and advantages of the example embodiments will become apparent to those having ordinary skill in the art upon consideration of the following detailed description of example embodiments.DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTSGeneral Definitions

[0041] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Definitions of common terms and techniques in molecular biology may be found in Molecular Cloning: A Laboratory Manual, 2ndedition (1989) (Sambrook, Fritsch, and Maniatis); Molecular Cloning: A Laboratory Manual, 4thedition (2012) (Green and Sambrook); Current Protocols in Molecular Biology (1987) (F.M. Ausubel et al. eds.); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (1995) (MJ. MacPherson,B.D. Hames, and G.R. Taylor eds.): Antibodies, A Laboratory Manual (1988) (Harlow and Lane, eds.): Antibodies A Laboratory Manual, 2ndedition 2013 (E.A. Greenfield ed.); Animal Cell Culture (1987) (R.I. Freshney, ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008 (ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 9780471185710); Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y.1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2ndedition (2011).

[0042] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.

[0043] The term “optional” or “optionally” means that the subsequent described event, circumstance or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0044] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

[0045] The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of + / - 10% or less, +7-5% or less, + / -1% or less, and + / -0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the current disclosure. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.

[0046] As used herein, a “biological sample” may contain whole cells and / or live cells and / or cell debris. The biological sample may contain (or be derived from) a “bodily fluid”. The present disclosure encompasses embodiments wherein the bodily fluid is selected from amniotic fluid, aqueous humour, vitreous humour, bile, blood serum, breast milk, cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudates, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritonealfluid, pleural fluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretion, vomit and mixtures of one or more thereof. Biological samples include cell cultures, bodily fluids, and cell cultures from bodily fluids. Bodily fluids may be obtained from a mammal organism, for example by puncture, or other collecting or sampling procedures.

[0047] As used herein, a "Ligation-Cell Barcode Oligonucleotide" (L-CBO) refers to an oligonucleotide designed for the purpose of tagging and identifying individual cells. Such oligonucleotides, for example, may be ligated to genomic DNA fragments, enabling the unique barcoding of each cell’s DNA.

[0048] As used herein, a "Reverse Transcription-Cell Barcode Oligonucleotide" (RT-CBO) refers to an oligonucleotide designed to barcode RNA molecules during the reverse transcription process. Such oligonucleotides may, for example, prime the reverse transcription of polyadenylated transcripts and / or spatial barcode oligonucleotides, incorporating unique barcodes into the complementary DNA (cDNA).

[0049] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.

[0050] Various embodiments are described hereinafter. It should be noted that the specific embodiments are not intended as an exhaustive description or as a limitation to the broader aspects discussed herein. One aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiment(s). Reference throughout this specification to “one embodiment”, “an embodiment,” “an example embodiment,” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” or “an example embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments describedherein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the disclosure. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

[0051] Reference is made to U.S. Patent Application publication number US 2021 / 0123040A1, now U.S. Patent 12,385,033. Reference is also made to "Slide-tags" International Patent Application PCT / US2023 / 085534 (published as WO 2024 / 138050). Reference is also made to “Slide-tags: scalable, single-nucleus barcoding for multi-modal spatial genomics,” Andrew J. C. Russell, Jackson A. Weir, Naeem M. Nadaf, Matthew Shabet, Vipin Kumar, Sandeep Kambhampati, Ruth Raichur, Giovanni J. Marrero, Sophia Liu, Karol S. Balderrama, Charles R. Vanderburg, Vignesh Shanmugam, Luyi Tian, Catherine J. Wu, Charles H. Yoon, Evan Z. Macosko, Fei Chen, bioRxiv 2023.04.01.535228. Reference is also made to Russell, A.J.C., Weir, J. A., Nadaf, N.M. et al. Slide-tags enables single-nucleus barcoding for multimodal spatial genomics. Nature (2023). All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as though each individual publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference.Overview

[0052] The present disclosure is based, at least in part, upon discovery of methods and compositions capable of leveraging cell barcoding and conversion reagents to generate high- quality single-cell methylomes capable of being assessed rapidly in parallel across populations of cells, as well as coordinated single-cell methylome-plus-transcriptomes formatted for parallel, high-throughput assessment of cellular populations with precise single-cell resolution (allowing for tracking of methylation state and associated transcript expression levels at single-cell resolution across a large population of cells, e.g., across a tissue section).

[0053] The methods disclosed herein dramatically reduce the barrier to conducting near-state- of-the-art single-cell DNA methylation studies and can be implemented by laboratories with diverse specialties. Spatial location and gene expression information is also obtained for the single-cell DNA methylation data of the currently disclosed methods. Application of the current methods to profding of new tissues and organisms, and / or using DNA methylation assessment asareadout for pooled single-cell perturbation screens is expressly contemplated, among other uses. Furthermore, the methods disclosed herein can be readily adapted to additional single-cell measurements, such as detection of chromatin accessibility and / or chromatin conformation.

[0054] Certain methods provided by the present disclosure expand upon the chemistry employed to generate single-cell AT AC libraries in a lOx Single Cell Multi ome kit, in which accessible DNA fragments from a tagmented nucleus are ligated to ATAC fragment-directed ligation cell barcode oligos (L-CBOs) released from a GEM bead (Gel Bead-in-emulsion) within a co-encapsulating droplet. The resulting pool of cell-barcoded unamplified genomic DNA (gDNA) fragments may be used as input for DNA methylation profiling methods that label unmethylated cytosine residues through conversion to uracil. To prevent C-to-U conversion within cell barcode sequences, which would cause significant overlap between previously distinct barcode sequences, a gap-filling step has been introduced herein that employs methylated dCTPs to generate a conversion-protected cell barcode sequence copy. In embodiments of the disclosure, the strand with the protected cell barcode sequence copy can then be selectively amplified following conversion. To generate genome-wide DNA methylation profiles, a nucleosome depletion step has been further introduced in the currently disclosed methods, with nucleosome depletion performed prior to tagmentation.

[0055] In embodiments, the present disclosure provides an accessible method for single-cell DNA methylation profiling that leverages the lOx Single Cell Multi ome kit. Genomic DNA fragments from nucleosome-depleted tagmented nuclei are ligated to cell barcodes using the ATAC component of lOx Multi ome kit. Unmethylated cytosines within these native fragments are converted to uracils through chemical or enzymatic treatment. Prior to conversion, a gap-fill reaction with methylated dCTP generates a conversion-protected cell barcode copy that can be selectively amplified and sequenced.

[0056] The present disclosure further provides methods and compositions for spatially resolved single-cell DNA methylation profiling involving combining methods and compositions capable of leveraging cell barcoding and conversion reagents to generate high-quality methylomes or methylom e-plus-transcriptomes with "slide-tags".

[0057] Certain methods described herein involve sequencing nucleic acid molecules from individual cells and nuclei. In eukaryotic genomes, chromosomal DNA wraps around histoneproteins to form structures called nucleosomes, resulting in a complex known as chromatin. The degree of chromatin packaging influences gene expression: tightly packed chromatin (closed chromatin) generally inhibits gene expression, while loosely packed, accessible chromatin (open chromatin) is associated with active gene transcription. Techniques for assessing genome-wide DNA accessibility have been highly effective in identifying regulatory elements across various cell types and in measuring changes that lead to gene activation or repression. One such technique is the Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq). ATAC-seq uses an artificial transposon to insert specific sequences into accessible chromatin regions. Since the transposase can only insert sequences into chromatin regions not bound by transcription factors or nucleosomes, sequencing reads can reveal areas of increased chromatin accessibility. Single-cell ATAC-seq (scATAC-seq) methods have also been developed, as detailed in PCT Patent Publications Nos. WO2018 / 218226, U.S. Pat. Nos.10,844,372 and 10,725,027, and U.S. Pat. App. Pub. 2020 / 0291454, all of which are incorporated by reference in their entirety.

[0058] The methods, compositions, systems, and kits described herein can be used to prepare samples for downstream analysis using Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) and RNA sequencing (RNA-seq) assays, either individually or together. This analysis can uncover regulatory factors that influence cis-element accessibility and / or trans-factor occupancy, and in certain of the current embodiments for assessing methylation of genomic DNA, associations of DNA methylation sites / patterns with corresponding gene regulatory events can be examined at single-cell resolution. In some instances, these methods, systems, and kits can reveal nucleosome positioning and regulatory nodes of coordinated activity within a cell type, such as coordinated trans-factor activities and the synergistic actions of co-binding transcription factors (TFs) on cis-elements. These methods can be performed in a high-throughput manner to obtain data from single biological particles (e.g., single cells or single nuclei), including epigenomic variability.

[0059] In one embodiment, the conditions used for nucleosome-depletion maintain the integrity of the isolated nuclei. Typically, nucleosome-depletion methods are used on a pellet or suspension of single cells, thus in those embodiments where an adherent cell culture or tissue is used as a source of the cells, the source is treated to obtain a pellet or suspension of single cells.

[0060] In one embodiment, the conditions for nucleosome depletion preserve the integrity of isolated nuclei. Typically, nucleosome depletion methods are applied to a pellet or suspension of single cells. Therefore, when using an adherent cell culture or tissue as the cell source, the source is processed to obtain a pellet or suspension of single cells.

[0061] Nucleosome depletion methods are well-established and include enzymatic and chemical treatments. In one embodiment, the conditions for nucleosome depletion involve a chemical treatment with a chaotropic agent that disrupts nucleic acid-protein interactions. An example of such a chaotropic agent is 3,5-lithium diiodosalicylic acid. The conditions for using 3,5-lithium diiodosalicylic acid involve adding it to a cell pellet and incubating on ice.

[0062] In certain embodiments, the conditions involve a chemical treatment with a detergent that can disrupt nucleic acid-protein interactions. One example of such a detergent is sodium dodecyl sulfate (SDS). The protocol for using SDS includes adding it to a cell pellet and incubating at an elevated temperature, such as 42°C. After SDS treatment, a nonionic detergent like Triton X-100 can be added to quench the SDS prior to further processing steps.

[0063] In some cases, when using a detergent like SDS, the nuclei are treated with a crosslinking agent before nucleosome depletion (WO 2018 / 018008). The cross-linking agent can be applied to nuclei either within cells or to isolated nuclei. Formaldehyde is an example of a crosslinking agent (Hoffman et al., 2015, J. Biol. Chem., 290:26404-26411). Cells can be treated with formaldehyde by adding it to a cell suspension and incubating at room temperature. After formaldehyde treatment, a chemical with primary amine groups such as glycine or tris can be added to quench the formaldehyde prior to further processing.

[0064] Throughout the nucleosome depletion process, the integrity of the isolated nuclei is preserved. The intactness of the nuclei after nucleosome depletion can be assessed using routine methods such as phase-contrast imaging. The number of intact nuclei post-depletion can range from 1 to 100 million.

[0065] Certain embodiments of the present disclosure provide sample preparation techniques for processing DNA and / or RNA nucleic acid molecules from a cell or cell nucleus. The method involves contacting a cell or cell nucleus with a transposase-nucleic acid complex, which includes a transposase molecule and one or more transposon end oligonucleotide molecules. This contact occurs in bulk solution, leading to a “tagmentation” reaction that generates template nucleic acidfragments (tagmented fragments) corresponding to target nucleic acid molecules (e.g., DNA) within the cell or nucleus.

[0066] Simultaneously, the cell or nucleus may be contacted with a primer molecule (e.g., one with a poly-T sequence) designed to interact with additional target nucleic acid molecules (e.g., RNA, such as mRNA). This interaction can occur in bulk solution or within a partition, producing additional template nucleic acid fragments (e.g., RNA fragments). The primer molecule, complementary to the target nucleic acid molecules, hybridizes to their sequences.

[0067] The cell or nucleus may be partitioned into droplets or wells, along with reagents like nucleic acid barcode molecules. The cell or nucleus is then lysed, permeabilized, fixed, crosslinked, or otherwise manipulated to access the template nucleic acid fragments. These fragments undergo various processing steps within the partition, such as barcoding, ligation, reverse transcription, template switching, linear amplification, and / or gap filling.

[0068] The processed template nucleic acid fragments, now including barcode sequences, are released from the partition and may undergo further processing in bulk. This includes gap filling, dA tailing, terminal-transferase, phosphorylation, ligation, and nucleic acid amplification processes. The fragments may also be subjected to polymerase chain reactions (PCR) to generate amplification products, which can be sequenced to identify the original target nucleic acid molecules (e.g., DNA and RNA) from the cell or nucleus. Additional methods, compositions, systems, and kits for processing DNA and / or RNA nucleic acid molecules from a cell or nucleus are detailed in U.S. Pat. Pub. No. 2020 / 0291454A1, incorporated by reference in its entirety.

[0069] Such processing may include, for example, cytosine to uracil conversion. As a result of cytosine conversion, unmethylated cytosines in the template nucleic acid are converted to uracil residues, while methylated cytosines remain unchanged. In some embodiments, the cytosine-converted construct may be amplified before hybridization to increase the material available for cluster amplification, converting uracil residues to thymine residues via amplification in the presence of free dNTPs. When adapter oligonucleotides include sequences for later steps (e.g., capture on a support or binding of a sequencing primer), the adapter can be synthesized using a bisulfite-resistant cytosine analog like 5-methyl dCTP (Me-C, or 5mC) at critical positions. Alternatively, a hairpin adapter can be ligated to one end of a linear template, serving as a primer to fill in the second strand of the template with dNTPs, including Me-C. After cytosineconversion, the second strand remains unconverted due to the incorporated Me-C bases and can serve as a reference for the original converted template strand.

[0070] The methods of the present disclosure may involve the following steps: (1) synthesizing a complementary strand containing methylated cytosine instead of unmethylated cytosine. This modification makes the strand resistant to subsequent deamination and allows for amplification with primers. After ligating the CBO to the template strand, generate a copy of the template strand using methylated-dCTP along with standard dATP, dGTP, and dTTP nucleotides. This ensures that the copy of the template strand is always methylated at cytosine positions, making it resistant to deamination. (2) converting unmethylated cytosines in the original template strand to uracils via deamination. This deamination can be performed using various methods, such as standard bisulfite deamination, enzymatic deamination with the EM-seq technique (utilizing TET2 and APOBEC2 enzymes), or the TET Assisted Pic-borane Sequencing (TAPS) method. (3) After deamination, amplifying the copy of the template strand using primers and proceeding with sequencing. This protocol ensures that the copy of the template strand’ s cytosine positions remain methylated and resistant to deamination, allowing for accurate sequencing results.

[0071] Certain embodiments disclosed herein provide for spatially tagged nuclei that are compatible with any genomic or multiomic single cell / nuclei assay to allow generation of a spatially resolved single cell sequencing library with single cell resolution. Previous methods were limited to detecting mRNA expression and were also limited by the amount of mRNA diffused from the cell and captured (see, e g., Stickels RR, Murray E, Kumar P, et al. Highly sensitive spatial transcriptomics at near-cellular resolution with Slide-seqV2. Nat Biotechnol.2021;39(3):313-319). The present disclosure overcomes these limitations by tagging nuclei with spatial barcodes, such that capturing analytes is not dependent upon diffusion from a cell. The nuclei can be completely lysed in a reaction volume to release all analytes (e.g., RNA and / or genomic DNA), such that all RNA and / or genomic DNA can be captured. Further, in order to perform multiomic spatial studies using a slide-seq type of method, each omic measurement is performed separately. The present disclosure does not require performing every spatial experiment twice or multiple times to obtain multiomic spatial results. In addition, the structure of the spatial barcodes allows either: plate-based, microfluidic-based, or nanowell-based captureof macromolecules and spatial barcodes from single-nuclei, in contrast to XYZeq and sci-Space, which require split-pool index based profding.

[0072] The current method requires only that one or more spatial barcode tags are delivered to permeabilized nuclei. The tagged nuclei can then be stored or directly used in any single cell genomics assay. In other words, the present disclosure unifies spatial profiling and single cell sequencing methods with DNA methylation state assessment. Further, the present disclosure provides for assessment of single-cell methylation state (and optionally associated single-cell transcript expression levels) across an array such that the distance between different spatial barcodes is less than the size of a cell, allowing single cell resolution. Further, the use of fluorescently labeled spatial barcode tags can allow for only tagged nuclei to be used in subsequent single cell genomics assays. Thus, because single tagged nuclei are used as the input for single nuclei / cell genomic assays, the analyte capture efficiency approaches the detection efficiency of the non-spatially resolved single-cell genomic sequencing techniques. For example, the genomic DNA and RNA capture rate approaches -100% of single nuclei genomic DNA and RNA sequencing data.Tagging nuclei in a tissue sample with spatial barcodes

[0073] In example embodiments, a tissue sample is placed on an array comprising spatial barcode sequences (spatial barcodes) and the spatial barcodes are released from the array to tag the nuclei in the tissue. The tissue sample is permeabilized to allow the spatial barcodes to tag the nuclei. Tagged single nuclei are then isolated from the tissue sample.Spatial Arrays

[0074] In example embodiments, nuclei in a tissue sample are tagged with spatial barcode nucleic acids. The spatial barcode nucleic acids are nucleic acids linked or attached to an array at specific positions and that include barcode sequences. Thus, the spatial barcode can identify the position in the array. As used herein, the term “array” refers to a population of features or sites that can be differentiated from each other according to relative location. Different molecules that are at 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 target nucleic acid molecule having aparticular sequence or a site can include several nucleic acid molecules having the same sequence (and / or a complementary sequence, thereof). The sites of an array can be different features located on the same substrate. Exemplary features include without limitation, wells in a substrate, beads (or other particles) in or on a substrate, projections from a substrate, ridges on a substrate and / or channels in a 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, beads arranged upon a flat surface (e.g., a slide), optionally beads captured upon a flat surface (e.g., a layer of beads adhered to or otherwise stably associated with a slide (e.g., a layer of beads adsorbed to a slide-attached elastomeric surface)), etc. In example embodiments, the array of the present disclosure includes greater than 10,000 individual locations, each location having a different spatial barcode. In example embodiments, the array of the present disclosure includes 10,000 to more than 1 million individual locations, each location having a different spatial barcode.

[0075] As used herein, the term “feature” means a location in an array 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. Medium density arrays have sites separated by about 15 to 30 pm, while low density arrays have sites separated by greater than 30 pm. An array useful herein 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.

[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 analyte, such as a nucleic acid, can be attached to a material, such as a gel or solid support, 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.Spatial barcode nucleic acids

[0077] In example embodiments, a spatial barcode nucleic acid is a nucleic acid sequence that includes a barcode sequence. In example embodiments, a spatial barcode nucleic acid is a nucleic acid sequence that includes from 5’ to 3’: a cleavable linker, a barcode sequence, and a capture sequence. In example embodiments, the spatial barcode nucleic acids are 3' end blocked to prevent extension. Thus, upon capture by a cell barcode, the cell barcode sequence primes extension into the spatial barcode nucleic acid to add the spatial barcode sequence to the cell barcode sequence and the spatial barcode nucleic acid is not extended. In example embodiments, the spatial barcode nucleic acids include a barcode sequence that identifies the location on the array. The term “barcode” as used herein refers to a short sequence of nucleotides (for example, DNA or RNA) or a series of nucleotides in a nucleic acid that can be used to identify the nucleic acid, a characteristic of the nucleic acid (e.g., the identity and optionally the location of the nucleic acid), or a manipulation that has been carried out on the nucleic acid (e.g., a perturbation) that is used as an identifier for an associated molecule, such as a target molecule and / or target nucleic acid, or as an identifier of the source of an associated molecule, such as a cell-of-origin. As used herein, the term “spatial barcode” or “spatial tag” is intended to mean a series of nucleotides in a nucleic acid that can be used to identify the location on an array to which a nucleic acid is fixed. As used herein, the term “spatial barcode” or “spatial tag” is also intended to mean a nucleic acid having a sequence that is indicative of a location. Typically, the nucleic acid is a synthetic molecule having a sequence that is not found in one or more biological specimen that will be used with the nucleic acid. However, in some embodiments the nucleic acid molecule can be naturally derived, or the sequence of the nucleic acid can be naturally occurring, for example, in a biological specimen that is used with the nucleic acid. The location indicated by a spatial tag can be a location in or on a biological specimen, in or on a solid support, or a combination thereof. A barcode sequence can function as a spatial tag. In example embodiments, the identification of the location of the tag that serves as a spatial tag is only determined after a population of beads (each possessing a distinct barcode sequence) has been arrayed upon a solid support (optionallyrandomly arrayed upon a solid support) and sequencing of such a bead-associated barcode sequence has been determined in situ upon the solid support.

[0078] The barcode sequence can be a naturally occurring sequence or a sequence that does not occur naturally in the organism from which the barcoded nucleic acid was obtained or from which the sample that is tagged 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 (e.g., all nucleic acid species attached to an array at a defined location or a single bead might possess the same barcode sequence, while different defined locations or beads present a different shared barcode sequence that serves to identify each such different location or bead). By way of further example, each nucleic acid probe in a population can include different barcode sequences from all other nucleic acid probes in the population. Alternatively, each nucleic acid in a population can include different barcode sequences from some or most other nucleic acids in a population. In particular embodiments, one or more barcode sequences that are used with a biological specimen (e.g., a tissue sample) are not present in the genome, transcriptome or other nucleic acids of the biological specimen. For example, barcode sequences can have less than 80%, 70%, 60%, 50% or 40% sequence identity to the nucleic acid sequences in a particular biological specimen. 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 on the two or more molecules.

[0079] As used herein, the term “biological specimen” is intended to mean one or more cell, tissue, organism or portion thereof. A biological specimen can be obtained from any of a variety of organisms. Exemplary organisms include, but are not limited to, a mammal such as a rodent, mouse, rat, rabbit, guinea pig, ungulate, horse, sheep, pig, goat, cow, cat, dog, or primate (i.e., human or non-human primate); a plant such as Arabidopsis thaliana, corn, sorghum, oat, wheat, rice, canola, or soybean; an algae such as Chlamydomonas reinhardtir, a nematode such as Caenorhabditis elegans, an insect such as Drosophila melanogaster, mosquito, fruit fly, honeybee or spider; a fish such as zebrafish; a reptile; an amphibian such as a frog or Xenopus laevis, a Dictyostelium discoideum, a fungi such as Pneumocystis carinii, Takifugu rubripes, yeast, Saccharamoyces cerevisiae or Schizosaccharomyces pom be, or a Plasmodium falciparum. Target nucleic acids can also be derived from a prokaryote such as a bacterium, Escherichia coll. Staphylococci or Mycoplasma pneumoniae, an archae; a virus such as Hepatitis C virus or human immunodeficiency virus; or a viroid. Specimens can be derived from a homogeneous culture or population of the above organisms or alternatively from a collection of several different organisms, for example, in a community or ecosystem.

[0080] In example embodiments, the spatial barcode nucleic acid includes a universal sequence used to capture the spatial barcode nucleic acid onto another nucleic acid sequence (e.g., a cell of origin identifying barcode nucleic acid). In some embodiments, the universal sequence is also referred to as a capture sequence, or handle sequence, such as a ligation, PCR, or indexing handle sequence. In preferred embodiments, the capture sequence is at the 3’ end of the spatial barcode nucleic acids. In example embodiments, the spatial barcode nucleic acid also includes a universal sequence used as a primer binding sequence. As used herein, the term “universal sequence” refers 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 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. Non-limiting examples of 3’ universal sequences used for capture of the spatial barcode nucleic acids include poly-A sequences for capture by poly-T sequences, or sequences complementary to commercially available capture sequences, such as tagmentation adapter sequences (e.g., Read 1 (Read IN) sequence on the beads in the Chromium Next GEM Single Cell ATAC Reagent Kit v 1.1 (10 x Genomics, Pleasanton, CA, USA)).

[0081] As used herein, the term “poly-T or poly-A,” when used in reference to a nucleic acid sequence, is intended to mean a series of two or more thiamine (T) or adenine (A) bases, respectively. A poly-T or poly-A can include at least about 2, 5, 8, 10, 12, 15, 18, 20, 25, 30 or more of the T or A bases, respectively. Alternatively or additionally, a poly-T or poly-A can include at most about 30, 25, 20, 18, 15, 12, 10, 8, 5 or 2 of the T or A bases, respectively.

[0082] In example embodiments, the spatial barcode nucleic acids do not require a unique molecular identifier (UMI) sequence because a UMI sequence is present on the single cell / nuclei genomics assay capture sequence, which also includes a cell of origin barcode sequence. Thus, when the spatial barcode is captured in a single cell assay, a UMI specific to each spatial barcode capture event will be present on each sequencing read.

[0083] In example embodiments, the spatial barcode nucleic acids are about 50 to 250 nucleotides in length. In one example, the spatial barcode nucleic acids include a linker, a primer binding sequence that is the same for all spatial barcode nucleic acids, a spatial barcode that is about 6 to 50 nucleotides, preferably 6 to 20 nucleotides, and a capture sequence of about 6 to 50 nucleotides. The spatial barcode nucleic acids may also include additional sequences, for example, to change the length of the sequence. In example embodiments, the spatial barcode nucleic acids are single stranded, preferably, ssDNA. In some embodiments, single stranded oligonucleotides diffuse into nuclei better than double stranded nucleotides. In example embodiments, single-stranded DNA (ssDNA) specifically stains the nuclei of permeabilized cells but not intact cells.

[0084] In example embodiments, the spatial barcode nucleic acids on the spatial array have the same spatial barcode for each location, but have different lengths for each location. Spatial barcode nucleic acids having different lengths allow for identifying the spatial location of a single cell in 3 dimensions because shorter nucleic acids will diffuse farther into a tissue section than a longer nucleic acid. In example embodiments, computational methods can be used to determine the location of single cells in a tissue section with multiple layers of cells, such as by quantitating the number of spatial barcodes having different lengths in the single nuclei.

[0085] 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 alsopossible 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, U.S. Patent Application Publication No. 2014 / 0342921 Al, now U.S. Patent 9,200,274 and U.S. Pat. No. 8,460,865, each of which is incorporated herein by reference.Linkers

[0086] In example embodiments, the spatial barcode nucleic acids include a linker sequence for attachment to the array or a solid support (e.g., a bead described further herein). In preferred embodiments, the linker is cleavable, such that the spatial barcode nucleic acids can be released when in contact with or in proximity to a tissue specimen. In example embodiments, the cleavable linker is chemically cleavable, photocleavable, or enzymatically cleavable.

[0087] In preferred embodiments, the linker is photocleavable. Photocleavable linkers are available that can be released by UV irradiation. A PC (Photo-Cleavable) spacer can be placed between DNA bases or between the oligo and a 5'-modifier group. The spacer arm can be cleaved with exposure to UV light in the 300-350 nm spectral range. Cleavage releases the oligo with a 5'-phosphate group. An exemplary photo-cleavable linker is commercially available (Integrated DNA Technologies, Inc., Coralville, Iowa) and shown:

[0088] In other example embodiments, the spatial barcode nucleic acids may contain one or more cleavable linkers, e.g., that can be cleaved upon application of a suitable stimulus. For example, the cleavable sequence may be a photocleavable linker that can be cleaved by applying light, a chemical cleavable linker that can be cleaved by applying a suitable chemical, or an enzymatically cleavable linker that can be cleaved by applying an enzyme.

[0089] Oligonucleotides with photo-sensitive chemical bonds (e.g., photo-cleavable linkers) have various advantages. They can be cleaved efficiently and rapidly (e.g., in nanoseconds and milliseconds). In some cases, photo-masks can be used such that only specific regions of the array are exposed to cleavable stimuli (e.g., exposure to UV light, exposure to light, exposure to heat induced by laser). When a photo-cleavable linker is used, the cleavable reaction is triggered by light, and can be highly selective to the linker and consequently biorthogonal. Non-limiting examples of a photo-sensitive chemical bond that can be used in a cleavage domain include those described inLeriche et al. Bioorg Med Chem. 2012 Jan 15;20(2): 571 -82; U.S. Patent Application Publication No. 2017 / 0275669, now U.S. Patent 10,266,874; and WO 2020 / 190509A9.Spatial barcode nucleic acid modifications

[0090] In example embodiments, the spatial barcode nucleic acids include a nucleotide modification to enhance diffusion into nuclei. In example embodiments, the spatial barcode nucleic acids are coupled to a lipophilic or amphiphilic moiety. Lipophilic molecules can associate with and / or insert into lipid membranes such as cell membranes and nuclear membranes. Non-limiting examples of lipophilic molecules that can be used in the methods provided herein include sterol lipids such as cholesterol, tocopherol, and derivatives thereof, lignoceric acid, and palmitic acid. Other lipophilic molecules that may be used in the methods provided herein comprise amphiphilic molecules wherein the headgroup (e.g., charge, aliphatic content, and / or aromatic content) and / or fatty acid chain length (e.g., C12, C14, C16, or C18) can be varied. For instance, fatty acid side chains (e.g., Cl 2, Cl 4, Cl 6, or Cl 8) can be coupled to glycerol or glycerol derivatives (e.g., 3-t-butyldiphenylsilylglycerol), which can also comprise, e.g., a cationic head group. The spatial barcode nucleic acids disclosed herein can be coupled (either directly or indirectly) to these amphiphilic molecules. An amphiphilic molecule may associate with and / or insert into a membrane (e.g., a nuclear membrane).

[0091] A spatial barcode nucleic acid may be attached to a lipophilic moiety (e.g., a cholesterol molecule). A spatial barcode nucleic acid may be attached to the lipophilic moiety via a linker, such as a tetra-ethylene glycol (TEG) linker. Other exemplary linkers include, but are not limited to, Amino Linker C6, Amino Linker Cl 2, Spacer C3, Spacer C6, Spacer Cl 2, Spacer 9, and Spacer 18. A spatial barcode nucleic acid may be attached to the lipophilic moiety or the linker on the 5' end of the spatial barcode nucleic acid. The linker may be a glycol or derivative thereof.For example, the linker may be tetra-ethylene glycol (TEG) or polyethylene glycol (PEG). A spatial barcode nucleic acid may be releasably attached to the linker or lipophilic moiety (e.g., as described elsewhere herein for releasable attachment of nucleic acid molecules) such that the spatial barcode nucleic acid or a portion thereof can be released from the lipophilic molecule. In example embodiments, a lipophilic moiety (e.g., a cholesterol) is indirectly (e.g., via hybridization or ligand-ligand interactions, such as biotin-streptavidin) coupled to an oligonucleotide.

[0092] In example embodiments, the spatial barcode nucleic acids include fluorescent labels such that tagged nuclei can be identified and sorted out from non-tagged nuclei. The efficiency of nuclei tagging can also be determined by quantitating the percentage of tagged nuclei. In example embodiments, a lipophilic molecule may comprise a fluorescent moiety. Many different fluorophores can be readily attached to oligonucleotides, such as, fluorescein and its tetra- and hexachlorinated derivatives TET and HEX.Gap filling, ligation, conversion and DNA methylation profiling

[0093] Certain aspects of the present disclosure relate to methods that involve gap filling and ligation of nucleic acid sequences. Such methods may involve annealing of a primer to a singlestranded overhang, extension of the primer and / or 3'-ends of nucleic acid strands by a DNA polymerase to achieve gap filling, and ligation of the extended product(s) to adjacent oligonucleotide strands. Such methods may join disruptions between adjacent 3'-end and 5'-end locations positioned in close / adjoining proximity to one another.Primer annealing

[0094] In certain embodiments, an oligonucleotide primer may be designed to anneal to a location downstream of a single-stranded 5'-overhang that includes a barcode sequence, such that polymerase-mediated extension of the oligonucleotide primer will occur in an orientation and location that captures the complement of the barcode sequence in the extended region. The primer is therefore complementary to a sequence within the single-stranded overhang and is introduced under conditions that promote hybridization. The complementary sequence is located 3' of the barcode sequence of the single-stranded 5'-overhang.

[0095] Subsequent steps may involve denaturation and annealing. Denaturation involves heating the nucleic acid to separate the strands. The annealing step involves cooling the mixture to allow the primer to hybridize with the single-stranded overhang.Polymerase extension reaction

[0096] Once the primer sequence has annealed to a complementary sequence, a polymerase extension reaction can be initiated via use of a DNA polymerase. A DNA polymerase enzyme extends the 3'-terminus of an oligonucleotide primer by adding nucleotides complementary to the template strand to which the primer has annealed. This process results in the synthesis of a new extension of a DNA strand, creating a double-stranded region downstream of the original doublestranded region.Gap Filling

[0097] Following the polymerase extension reaction, a gap-filling step may be performed to ensure the continuity of the DNA strand. This involves joining any nucleotide sequence gaps between adjacent oligonucleotides that form an otherwise continuous strand across a region of double-stranded nucleic acid. A DNA polymerase enzyme can add nucleotides to fill gaps, using the complementary DNA strand as template. This step ensures that the entire length of the DNA is double-stranded, and ready for performance of a final ligation step to form continuous strands.Ligation

[0098] Certain aspects of the disclosure feature ligation steps. Presence of a 5'-phosphate modification can be important for efficient ligation of oligonucleotide ends, with the 5'-phosphate modification for the formation of the phosphodiester bond during ligation. Ligation methods generally involve adding a DNA ligase enzyme to a reaction mixture. The ligase catalyzes the formation of a phosphodiester bond between the 3’-hydroxyl group of an upstream nucleic acid and the 5’-phosphate group of the downstream nucleic acid (e.g., oligonucleotide). This reaction effectively joins the two DNA fragments, resulting in a continuous DNA strand.

[0099] Several DNA ligases are known to be effective in joining oligonucleotide ends, including but not limited to T4 DNA Ligase, E. coli DNA Ligase, and Taq DNA Ligase.Methyl-Cytosine to Uracil Conversion

[0100] DNA methylation analysis most commonly involves the conversion of cytosine to uracil. This conversion process involves the deamination of cytosine, which results in the replacement of the amino group with a carbonyl group, thereby transforming cytosine into uracil (see image below). This process may be used to distinguish between methylated and unmethylated cytosines, particularly in mammalian genomic DNA.Conversion

[0101] The chemical conversion of cytosine to uracil may be represented, for example, as follows:Cytosine S-ul.fonat.ed Sulfonated Uracil Cytosine Uracil

[0102] Cytosine (C) + Bisulfite (HSO3 ) + Hydroxide ion (OH ) Uracil (U) + Sulfite ion (SO32) + Water (H2O)

[0103] This reaction involves the deamination of cytosine, facilitated by bisulfite treatment, which converts cytosine residues to uracil while leaving 5-methylcytosine residues unchanged.

[0104] Several agents are known to facilitate the conversion of cytosine to uracil, including but not limited to sodium bisulfite, hydroxylamine, and nitrous acid.Methylated cytosine in DNA methylation profiling

[0105] Methyl-cytosine (5-methylcytosine, Me-C) is a modified form of cytosine where a methyl group is added to the 5th carbon of the cytosine ring.

[0106] Methylated cytosine (5-methylcytosine, Me-C) may be involved in methods of profiling DNA methylation. DNA methylation is an epigenetic modification that involves the addition of a methyl group to the cytosine base, typically at CpG dinucleotides.

[0107] DNA methylation profiling may involve the detection and quantification of the presence of methylated cytosine. For example, (i) DNA is treated with sodium bisulfite, which converts unmethylated cytosine to uracil while leaving methylated cytosine unchanged, (ii) the bisulfite-treated DNA is then amplified using polymerase chain reaction (PCR). During thisprocess, uracil is replaced with thymine, allowing for the differentiation between methylated and unmethylated cytosines, and (iii) the amplified DNA is sequenced to determine the methylation status of individual cytosines. Methylated cytosines remain as cytosines, while unmethylated cytosines appear as thymines in the sequence data.Nucleosome Disruption

[0108] Preparing nucleosome-free genomic DNA may involve nucleosome disruption. Nucleosome disruption involves the use of specific enzymes and chemical agents to remove nucleosomes, thereby exposing the underlying DNA.

[0109] Several enzymes are capable of digesting nucleosomes by targeting the linker DNA between nucleosomes or the histone proteins themselves. The enzymes include, but are not limited to, Micrococcal Nuclease (MNase), Deoxyribonuclease I (DNase I), and Restriction Enzymes.

[0110] In addition to enzymatic digestion, several chemical agents can be used to achieve nucleosome-free genomic DNA. These agents disrupt the interactions between DNA and histone proteins, facilitating the removal of nucleosomes. The agents include, but are not limited to, Sodium Dodecyl Sulfate (SDS), Lithium Diiodosalicylate (LIS), Dimethyl Sulfate (DMS), Potassium Permanganate (KMnCL), and Ultraviolet (UV) Irradiation.Overhang sequences

[0111] In certain embodiments, the instant disclosure features methods involving ligation of tag and / or capture sequences to double-stranded nucleic acids (e.g., genomic DNA fragments, which can be blunt-ended or comprise 3'-overhang(s) and / or 5'-overhang(s) when initially prepared in a fragmented state). Linkage of tag and / or capture sequences to a double-stranded nucleic acid can produce terminal overhangs (terminal regions of single-stranded sequence, adjacent to a double-stranded region), where in one example of a 5'-overhang, (i) the doublestranded nucleic acid includes a first oligonucleotide strand and a second oligonucleotide strand; (ii) the tag and / or capture agent can be linked (e.g., ligated) to the 5' end of the first oligonucleotide strand; and (iii) the 3' end of the second oligonucleotide strand is recessed relative to the 5' end of the first oligonucleotide strand (when extended by the ligated tag and / or capture sequence), thereby producing a 5 '-overhang on the first oligonucleotide strand (post-ligation ofthe 5'-terminal tag and / or capture sequence to the first oligonucleotide strand) as compared to the 3 '-terminus of the second oligonucleotide strand. Where parallel ligation events occur upon both 5'-ends of a double-stranded nucleic acid molecule, 5'-overhangs formed by the ligated strands at each end of the double-stranded nucleic acid would be generated. Similarly, 3'-overhangs can be generated by extension of one or both 3 '-ends of a double-stranded nucleic acid beyond the length of the corresponding 5'-end of the strand complementary to such 3'-extended strand in forming single-stranded ends upon a double-stranded nucleic acid.Sequence verified array

[0112] In example embodiments, the spatial array is sequence verified. As used herein “sequence verified” refers to knowing the sequence of the spatial barcodes at each location in the array, or at essentially all locations, preferably greater than 50, 60, 70, 80, or 90% of locations. In an example embodiment, the array is sequence verified because the spatial barcodes attached to the array at each location were specifically placed at each location. In an example embodiment, the sequences of the spatial barcodes at each location are determined by in situ sequencing. In an example embodiment, the array is sequence verified by in situ sequencing before placing a tissue sample on the array. In example embodiments, in situ sequencing is performed by sequencing by ligation or sequencing by synthesis directly on the array and captured by microscopy.

[0113] In certain aspects of the disclosure, in situ sequencing is performed upon an array affixed to a surface, which can be performed by any art-recognized mode of parallel (optionally massively parallel) in situ sequencing, examples of which particularly include the previously described SOLiD™ method, which is a sequencing-by-ligation technique that can be performed in situ upon a solid support (refer, e.g., to Voelkerding et al, Clinical Chem., 55-641-658, 2009; U.S. Pat. Nos. 5,912,148; and 6,130,073, which are incorporated herein by reference in their entireties). In certain embodiments of the instant disclosure, such sequencing can be performed upon an array present on a standard microscope slide, optionally using a standard microscope fitted with sufficient computing power to track and associate individual sequences during progressive rounds of detection, with their spatial position(s). The instant disclosure also employs fluidics, incubation times, enzymatic mixes and imaging setup in performing in situ sequencing (see, e.g., US 2021 / 0123040A1, now U.S. Patent 12,385,033).

[0114] Sequencing techniques, such as sequencing-by-synthesis (SB S) techniques, are a useful method for determining barcode sequences. 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 bead or other solid support (e.g. feature(s) where nucleic acid probes are attached to the bead or other 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 embodiments that use reversible termination, a deblocking reagent can be delivered to the bead or other solid support (before or after detection occurs). 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; U.S. Pat. Nos. 7,057,026, 7,329,492, 7,211,414, 7,315,019 or 7,405,281, and U.S. Patent Application Publication No. 2008 / 0108082, now U.S. Patent 8,343,746, each of which is incorporated herein by reference.

[0115] Sequencing by ligation is a DNA sequencing method that uses the enzyme DNA ligase to identify the nucleotide present at a given position in a DNA sequence. Unlike most currently popular DNA sequencing methods, this method does not use a DNA polymerase to create a second strand. Instead, the mismatch sensitivity of a DNA ligase enzyme is used to determine the underlying sequence of the target DNA molecule. Sequencing-by-ligation reactions are also useful including, for example, those described in Shendure et al. Science 309:1728-1732 (2005); or U.S. Pat. No. 5,599,675 or 5,750,341, each of which is incorporated herein by reference. Some embodiments 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 Publication No. WO 1989 / 10977, each of which is incorporated herein by reference. In bothsequencing-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.

[0116] A method of the present disclosure can include a step of performing a nucleic acid detection reaction on a bead or other solid support to determine barcode sequences of nucleic acid probes that are located on the bead or other solid support. In many embodiments the probes are randomly located on the bead or other 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 U.S. Pat. No.8,288,103 or 8,486,625, each of which is incorporated herein by reference.Solid supports

[0117] In example embodiments, spatial barcode nucleic acids are attached via cleavable linkers to a solid support. In one embodiment, the spatial barcode nucleic acids are attached directly to a solid support. In one embodiment, the spatial barcode nucleic acids are attached to a plurality of solid supports (e.g., beads), each of the plurality of solid supports having a unique spatial barcode sequence, which are further attached to a solid support (e.g., a slide). 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 functionalizedglass, 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. Particularly useful solid supports for some embodiments are slides and beads capable of assorting / packing upon the surface of a slide (e.g., beads to which a large number of oligonucleotides are attached).

[0118] Any of a variety of solid supports can be used in a method, composition or apparatus of the present disclosure. Particularly useful solid supports are those used for nucleic acid arrays. Examples include glass, modified glass, functionalized glass, inorganic glasses, microspheres (e.g., inert and / or magnetic particles), plastics, polysaccharides, nylon, nitrocellulose, ceramics, resins, silica, silica-based materials, carbon, metals, an optical fiber or optical fiber bundles, polymers and multiwell (e.g., microtiter) plates. Exemplary plastics include acrylics, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes and Teflon™. Exemplary silica-based materials include silicon and various forms of modified silicon.

[0119] In particular embodiments, a solid support can be within or part of a vessel such as a well, tube, channel, cuvette, Petri plate, bottle or the like. Optionally, the vessel is a flow-cell, for example, as described in WO 2014 / 142841 Al; U.S. Pat. App. Pub. No. 2010 / 0111768 Al, now U.S. Pat. 8,241,573, and U.S. Pat. No. 8,951,781 or Bentley et al., Nature 456:53-59 (2008), each of which is incorporated herein by reference. Exemplary flow-cells are those that are commercially available from Illumina, Inc. (San Diego, Calif.) for use with a sequencing platform such as a Genome Analyzer®, MiSeq®, NextSeq® or HiSeq® platform. Optionally, the vessel is a well in a multiwell plate or microtiter plate.

[0120] In example embodiments, a solid support can include a gel coating. Attachment, e.g., of nucleic acids to a solid support via a gel is exemplified by flow cells available commercially from Illumina Inc. (San Diego, Calif.) or described in U.S. 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, SFA (see, for example, US Pat. App. Pub. No. 2011 / 0059865 Al, which is incorporated herein by reference) or PAZAM (see, for example, US Pat. App. Publ. Nos.2014 / 0079923 Al, or 2015 / 0005447 Al, each of which is incorporated herein by reference).

[0121] In some embodiments, a solid support can be configured as an array of features to which beads can be attached. 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. Exemplary features include wells that are present in substrates used for commercial sequencing platforms sold by 454 LifeSciences (a subsidiary of Roche, Basel Switzerland) or Ion Torrent (a subsidiary of Life Technologies, Carlsbad Calif.). Other substrates having wells include, for example, etched fiber optics and other substrates described in U.S. Pat. Nos. 6,266,459; 6,355,431; 6,770,441; 6,859,570; 6,210,891; 6,258,568; 6,274,320; U.S. Pat. App. Publ. Nos.2009 / 0026082 Al; 2009 / 0127589 Al; 2010 / 0137143 Al; 2010 / 0282617 Al or PCT Publication No. WO 00 / 63437, each of which is incorporated herein by reference. In some embodiments, wells of a substrate can include gel material (with or without beads) as set forth in U.S. Pat. App. Publ. No. 2014 / 0243224 Al, which is incorporated herein by reference.

[0122] Features can appear on a solid support as a grid of spots or patches. The features can be located in a repeating pattern or in an irregular, non-repeating pattern. Optionally, repeating patterns can include hexagonal patterns, rectilinear patterns, grid patterns, patterns having reflective symmetry, patterns having rotational symmetry, or the like. Asymmetric patterns can also be useful. The pitch of an array can be the same between different pairs of nearest neighbor features or the pitch can vary between different pairs of nearest neighbor features.

[0123] In particular embodiments, 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 pm2, 10 pm2or 50 pm2. Alternatively or additionally, features can each have an area that is smaller than about 50 pm2, 25 pm2, 10 pm2, 5 pm2, 1 pm2, 500 nm2, or 100 nm2. The preceding ranges can describe the apparent area of a bead or other particle on a solid support when viewed or imaged from above.

[0124] In example embodiments, the present disclosure provides a method for generating and using a spatially tagged array of microbeads to perform tagging of nuclei with spatial barcode nucleic acids upon cryosectioned tissue samples, with high image resolution and with detectionof genomic DNA methylation status and optionally transcript expression levels, at the single-cell level. The method can include the steps of (a) attaching different nucleic acid probes (spatial barcode nucleic acids) to beads that are then captured upon a solid support to produce randomly located probe-possessing beads on the solid support, wherein the different nucleic acid probes each includes a barcode sequence (that is shared by all such nucleic acid probes of a single bead), and wherein each of the randomly located beads includes a different barcode sequence(s) from other randomly located beads on the solid support; (b) performing a nucleic acid detection reaction on the solid support to determine the barcode sequences of the randomly located beads on the solid support; (c) contacting a biological specimen with the solid support that has the randomly located beads; (d) releasing the probes presented by the randomly located beads to tag the biological specimen that are proximal to the randomly located beads; and (e) isolating tagged nuclei, thereby spatially tagging the nuclei of the biological specimen.

[0125] As used herein, “beads”, “microbeads”, “microspheres” or “particles” or grammatical equivalents can include small discrete particles. The composition of the beads can vary, depending upon the class of capture probe, the method of synthesis, and other factors. In example embodiments of the instant disclosure, the sizes of the beads of the instant disclosure tend to range from 1 pm to 100 pm in diameter (with all subranges within this range expressly contemplated), e.g., depending upon the extent of image resolution desired, nature of the solid support to be used for spatial bead array construction, sequencing processes (e g., flow cell sequencing) to be employed, as well as other factors. In example embodiments, the smaller the bead used the tighter space each spatial barcode is located and the better the resolution. Using smaller beads, in particular when the distance between different spatial barcodes is less than the size of a single cell, may result in the same nuclei being tagged with more than one spatial barcode. This case would not affect the resolution because the single cell could be located at an overlapping site on the array. In the case where the bead was larger than a single cell the resolution would be decreased due to more than one nuclei being tagged with the same spatial barcode. In this case, a single cell could only be localized to a location on the array larger than a single cell.

[0126] Example embodiments of the instant disclosure employ a collection of beads or other particles, to which oligonucleotides are attached. The bead sizes can range from nanometers, for example, 100 nm, to millimeters, for example, 1 mm, with beads from about 0.2 pm to about 200pm commonly employed, and from about 5 to about 20 pm being within the range currently exemplified, although in some embodiments smaller or larger beads may be used. For example, beads less than 50 pm, such as 1 pm, 3 pm, 10 pm, 15 pm, 20 pm, in particular about 10 pm.

[0127] Suitable bead compositions include those used in peptide, nucleic acid and organic moiety synthesis, including, but not limited to, plastics, ceramics, glass, polystyrene, methylstyrene, acrylic polymers, paramagnetic materials, thoriasol, carbon graphite, titanium dioxide, latex or cross-linked dextrans such as Sepharose, cellulose, nylon, cross-linked micelles and Teflon may all be used. “Microsphere Detection Guide” from Bangs Laboratories, Fishers Ind. is a helpful guide, which is incorporated herein by reference in its entirety. The beads need not be spherical; irregular particles may be used. In addition, the beads may be porous, thus increasing the surface area of the bead available for either capture probe attachment or tag attachment.

[0128] In example embodiments, beads include any bead used for single cell genomics methods as described further herein. Non-limiting examples of beads include hydrogel particles (polyacrylamide, agarose, etc.), colloidal particles (polystyrene, magnetic or polymer particle, etc ), any bead which can leverage phosphoramidate chemistry such as those used in oligonucleotide synthesis known to those skilled in the art (e.g., methylacrylates, polysterenes, polyacrylamides, polyethylene glycols), paramagnetic beads, and magnetic beads.

[0129] In example embodiments, the bead may be a hydrogel particle (see, e.g., Int. Pat. Apl. Pub. No. WO 2008 / 109176 for examples of hydrogel particles, including hydrogel particles containing DNA). Examples of hydrogels include, but are not limited to, agarose or acrylamide -based gels, such as polyacrylamide, poly-N-isopropylacrylamide, or poly N-isopropylpolyacrylamide. For example, an aqueous solution of a monomer may be dispersed in a droplet, and then polymerized, e.g., to form a gel.

[0130] In example embodiments, the beads may comprise one or more polymers. Exemplary polymers include, but are not limited to, polystyrene (PS), polycaprolactone (PCL), polyisoprene (PIP), poly(lactic acid), polyethylene, polypropylene, polyacrylonitrile, polyimide, polyamide, and / or mixtures and / or co-polymers of these and / or other polymers. In addition, in some cases, the particles may be magnetic, which could allow for the magnetic manipulation of the particles. For example, the particles may comprise iron or other magnetic materials. The particles couldalso be functionalized so that they could have other molecules attached, such as proteins, nucleic acids or small molecules. In some embodiments, the particle may be fluorescent.

[0131] Beads comprising the spatial barcode nucleic acids of the present disclosure can be obtained by any previously described method. For example, the spatial barcode nucleic acids can be directly synthesized on the beads, such that barcodes can be generated by random synthesis (see, e.g., Macosko et al., 2015, “Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets” Cell 161, 1202-1214; and International patent application number PCT / US2015 / 049178, published as WO 2016 / 040476 on March 17, 2016). In example embodiments, beads are obtained by 1) performing reverse phosphoramidite synthesis on the surface of the bead to synthesize the 5' end of the spatial barcode nucleic acids from a linker on the bead; 2) performing reverse phosphoramidite synthesis on the surface of the bead in a pool-and-split fashion, such that in each cycle of synthesis the beads are split into four reactions with one of the four canonical nucleotides (T, C, G, or A) or unique oligonucleotides; 3) repeating this process a large number of times, at least two, and optimally more than twelve, such that, in the latter, there are more than 16 million unique spatial barcodes on the surface of each bead in the pool; and 4) synthesizing or attaching (e.g., ligating) the 3' end of the spatial barcode nucleic acids comprising a universal sequence for capture by a cell barcode. For synthesis, the bead has to be a material that can be maintained during organic synthesis. Non-limiting examples include any bead which can leverage phosphoramidate chemistry such as those used in oligonucleotide synthesis known to those skilled in the art. Direct synthesis of oligonucleotides on beads is a preferred embodiment because direct synthesis allows more diverse barcodes in a more compact sequence.

[0132] In another example, the spatial barcode nucleic acids can be synthesized by linking oligonucleotides to beads followed by split-pool hybridization and extension to generate unique cell barcodes for each bead (see, e.g., Klein et al., 2015, “Droplet Barcoding for Single-Cell Transcriptomics Applied to Embryonic Stem Cells” Cell 161, 1187-1201; and International patent application number PCT / US2016 / 027734, published as WO 2016 / 168584A1 on October 20, 2016). In example embodiments, a nucleic acid barcode can be constructed in combinatorial fashion by combining randomly selected indices (for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 indexes selected from a pool of sequences for each index). Each such index is a short sequence of nucleotides (for example, DNA, RNA, or a combination thereof) having a distinct sequence.An index can have a length of about, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bp or nt. Accordingly, in some embodiments, the possible barcodes that are used are formed from one or more separate "pools" of barcode elements that are then joined together to produce the final barcode, e.g., using a split- and-pool approach. A pool may contain, for example, at least about 300, at least about 500, at least about 1,000, at least about 3,000, at least about 5,000, or at least about 10,000 distinguishable barcodes. For example, a first pool may contain xi elements and a second pool may contain X2 elements; forming a barcode containing an element from the first pool and an element from the second pool may yield, e.g., xix2 possible barcodes that could be used. It should be noted that xi and X2 may or may not be equal. This process can be repeated any number of times; for example, the barcode may include elements from a first pool, a second pool, and a third pool (e.g., producing X1X2X3 possible barcodes), or from a first pool, a second pool, a third pool, and a fourth pool, etc. Accordingly, due to the potential number of combinations, even a relatively small number of barcode elements can be used to produce a much larger number of distinguishable barcodes.

[0133] In another example, the spatial barcode nucleic acids can be synthesized by linking the 5' end of oligonucleotides containing adaptor sequences to beads to generate functionalized beads followed by emulsion PCR using primers containing unique barcode sequences (see, e.g., Zheng, et al., 2016, “Haplotyping germline and cancer genomes with high-throughput linked-read sequencing” Nature Biotechnology 34, 303-311; Zheng, et al., 2017, “Massively parallel digital transcriptional profiling of single cells” Nat. Commun. 8, 14049 doi: 10.1038 / ncommsl4049; International patent publication number WO 2014 / 210353 A2; and Zilionis, et al., 2017, “Singlecell barcoding and sequencing using droplet microfluidics” Nat Protoc. Jan;12(l):44-73). In this embodiment, each emulsion PCR includes a single primer that can hybridize to oligonucleotides on the functionalized beads and comprise a barcode sequence. Thus, after several rounds of amplification the barcode sequence is transferred to every oligonucleotide on the functionalized beads. This results in beads each having a barcode unique to that bead.

[0134] In example embodiments, beads can be suspended in a solution, or they can be located on the surface of a substrate (e.g., arrayed upon the surface of a solid support, such as a glass slide). Art-recognized 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.), substratesused 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 00 / 63437, each of which is incorporated herein by reference. Several of the above references describe methods for attaching nucleic acid probes to beads prior to loading the beads in or on a solid support. As such, the collection of beads can include different beads each having a unique (or sufficiently unique and / or near-unique, as described elsewhere herein) probe attached. It will, however, be understood that the beads can be made to include universal primers, and the beads can then be loaded onto an array, thereby forming universal arrays for use in a method set forth herein. The solid supports typically used for bead arrays can be used without beads. For example, nucleic acids, such as probes or primers can be attached directly to the wells or to 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.

[0135] Accordingly, the instant methods can employ an array of beads, wherein different nucleic acid probes are attached to different beads in the array. In this embodiment, each bead can be attached to a different nucleic acid probe 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. 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. As described elsewhere herein, it is also possible to use attachment chemistries or capture materials (e.g., a vinyl polymer liquid electrical tape) to adhere or otherwise stably associate the beads with a solid support, optionally including holding the beads in wells that may or may not be present on a solid support.

[0136] Nucleic acid probes that are attached to beads can include barcode sequences. A population of the beads can be configured such that each bead is attached to only one type of barcode (e.g., a spatial barcode) and many different beads each with a different barcode are present in the population. In this embodiment, randomly distributing the beads to a solid supportwill result in randomly locating the nucleic acid probe-presenting beads (and their respective barcode sequences) on the solid support. In some cases, there can be multiple beads with the same barcode sequence such that there is redundancy in the population. However, randomly distributing a redundancy-comprising population of beads on a solid support — especially one that has a capacity that is greater than the number of unique barcodes in the bead population — will tend to result in redundancy of barcodes on the solid support, which will tend to reduce image resolution in the context of the instant disclosure (i.e., where the precise location of a barcoded bead cannot be resolved due to redundancy of barcode use within an arrayed population of beads, it is contemplated that such redundant locations will simply be eliminated from an ultimate image produced by methods of the instant disclosure, or other modes of adjustment (e.g., normalization and / or averaging of values) may also be employed to address such redundancies). Alternatively, in preferred embodiments, the number of different barcodes in a population of beads can exceed the capacity of the solid support in order to produce an array that is not redundant with respect to the population of barcodes on the solid support. The capacity of the solid support will be determined in some embodiments by the number of features (e.g., single-bead occupancy wells) that attach or otherwise accommodate a bead.

[0137] A bead or other nucleic acid-presenting solid support of the instant disclosure can include, or can be made by the methods set forth herein to attach, a plurality of different nucleic acid probes. For example, a bead or other nucleic acid-presenting solid support can include at least 10, 100, IxlO3, IxlO4, IxlO5, IxlO6, IxlO7, IxlO8, IxlO9or more different probes. Alternatively or additionally, a bead or other nucleic acid-presenting solid support can include at most lx109, IxlO8, IxlO7, IxlO6, IxlO5, IxlO4, IxlO3, 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 bead or other nucleic acid-presenting solid support of the instant disclosure. 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 (or sufficiently unique) to particular nucleic acid probes. Alternatively or additionally, the ranges can describe the number of extended probes or modified probes created on a bead or other nucleic acid-presenting solid support of the instant disclosure using a method set forth herein.

[0138] Features may be present on a bead or other solid support of the instant disclosure prior to contacting the bead or other solid support with nucleic acid probes. For example, in embodiments where probes are attached to a bead or other solid 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 bead or other solid support, and optionally amplified on the bead or other solid support, e.g., using methods set forth in U.S. Pat. Nos. 8,895,249 and 8,778,849 and / or U.S. Patent Application Publication No. 2014 / 0243224 Al, each of which is incorporated herein by reference. Alternatively, a bead or other solid support may have a lawn of primers or may otherwise lack features. In this case, a feature can be formed by virtue of attachment of a nucleic acid probe on the bead or other solid support. Optionally, the captured nucleic acid probe can be amplified on the bead or other solid support such that the resulting cluster becomes a feature. Although attachment is exemplified above as 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.

[0139] A step of attaching nucleic acid probes to a bead or other 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 bead or other solid support. The contact can result in the fluidic mixture 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 bead or other solid support.

[0140] 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 embodiments where a fluidic mixture of different nucleic acid probes is contacted with a bead or other solid support for purposes of attaching the probes to the support, the number of different probe species can exceed the occupancy of the bead or other solid support for probes. Thus, the number and variety ofdifferent probes that attach to the bead or other solid support can be equivalent to the probe occupancy of the bead or other solid support.

[0141] Alternatively, the number and variety of different probe species on the bead or other solid support can be less than the occupancy (i.e., there will be redundancy of probe species such that the bead or other solid support may contain multiple features having the same probe species). Such redundancy can be achieved, for example, by contacting the bead or other 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 bead or other solid support.

[0142] Attachment of the nucleic acid probes can be mediated by hybridization of the nucleic acid probes to complementary primers that are attached to the bead or other solid support, chemical bond formation between a reactive moiety on the nucleic acid probe and the bead or other solid support (examples are set forth in U.S. Pat. Nos. 8,895,249 and 8,778,849, and in U.S. Patent Application Publication No. 2014 / 0243224 Al, each of which is incorporated herein by reference), affinity interactions of a moiety on the nucleic acid probe with a bead- or other 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 bead or other 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.

[0143] In some embodiments, attachment of a nucleic acid probe is non-specific with regard to any sequence differences between the nucleic acid probe and other nucleic acid probes that are or will be attached to the bead or other solid support. For example, different probes can have a universal sequence that complements surface-attached primers, or the different probes can have a common moiety that mediates attachment to the surface. Alternatively, each of the different probes (or a subpopulation of different probes) can have a unique (or sufficiently unique) sequence that complements a unique (or sufficiently unique) primer on the bead or other solid support, or they can have a unique (or sufficiently unique) moiety that interacts with one or more different reactive moiety on the bead or other solid support. In such cases, the unique (or sufficiently unique) primers or unique (or sufficiently unique) moieties can, optionally, be attached at predefined locations in order to selectively capture particular probes, or particular types of probes, at the respective predefined locations.

[0144] One or more features on a bead or other solid support can each include a single molecule of a particular probe. The features can be configured, in some embodiments, to accommodate no more than a single nucleic acid probe molecule. However, whether or not the feature can accommodate more than one nucleic acid probe molecule, the feature may nonetheless include no more than a single nucleic acid probe molecule. Alternatively, an individual feature can include a plurality of nucleic acid probe molecules, for example, an ensemble of nucleic acid probe molecules having the same sequence as each other. In particular embodiments, the ensemble can be produced by amplification from a single nucleic acid probe template to produce amplicons, for example, as a cluster attached to the surface.

[0145] A method set forth herein can use any of a variety of amplification techniques. Exemplary techniques that can be used include, but are not limited to, polymerase chain reaction (PCR), rolling circle amplification (RCA), multiple displacement amplification (MDA), or random prime amplification (RPA). In some embodiments the amplification can be carried out in solution, for example, when features of an array are capable of containing amplicons in a volume having a desired capacity. In certain embodiments, an amplification technique used in a method of the present disclosure will be carried out on solid phase. For example, one or more primer species (e.g., universal primers for one or more universal primer binding site present in a nucleic acid probe) can be attached to a bead or other solid support. In PCR embodiments, one or both of the primers used for amplification can be attached to a bead or other solid support (e g., via a gel). Formats that utilize two species of primers attached to a bead or other solid support are often referred to as bridge amplification because double stranded amplicons form a bridge-like structure between the two surface attached primers that flank the template sequence that has been copied. Exemplary reagents and conditions that can be used for bridge amplification are described, for example, in U.S. Pat. Nos. 5,641,658; 7,115,400; and 8,895,249; and / or U.S. Patent Application Publication Nos. 2002 / 0055100 Al, 2004 / 0096853 Al, 2004 / 0002090 Al, 2007 / 0128624 Al and 2008 / 0009420 Al, each of which is incorporated herein by reference. Solid-phase PCR amplification can also be carried out with one of the amplification primers attached to a bead or other solid support and the second primer in solution. An exemplary format that uses a combination of a surface attached primer and soluble primer is the format used in emulsion PCR as described, for example, in Dressman et al., Proc. Natl. Acad. Sci. USA 100:8817-8822 (2003),WO 05 / 010145, or U.S. Patent Application Publication Nos. 2005 / 0130173 Al or 2005 / 0064460 Al, each of which is incorporated herein by reference. Emulsion PCR is illustrative of the format, and it will be understood that for purposes of the methods set forth herein the use of an emulsion is optional and indeed for several embodiments an emulsion is not used.

[0146] RCA techniques can be modified for use in a method of the present disclosure. Exemplary components that can be used in an RCA reaction and principles by which RCA produces amplicons are described, for example, in Lizardi et al., Nat. Genet. 19:225-232 (1998) and U.S. Patent Application Publication No. 2007 / 0099208 Al, each of which is incorporated herein by reference. Primers used for RCA can be in solution or attached to a bead or other solid support. The primers can be one or more of the universal primers described herein.

[0147] MDA techniques can be modified for use in a method of the present disclosure. Some basic principles and useful conditions for MDA are described, for example, in Dean et al., Proc Natl. Acad. Sci. USA 99:5261-66 (2002); Lage et al., Genome Research 13:294-307 (2003); Walker et al., Molecular Methods for Virus Detection, Academic Press, Inc., 1995; Walker et al., Nucl. Acids Res. 20:1691-96 (1992); U.S. Pat. Nos. 5,455,166; 5,130,238; and 6,214,587, each of which is incorporated herein by reference. Primers used for MDA can be in solution or attached to a bead or other solid support at an amplification site. Again, the primers can be one or more of the universal primers described herein.

[0148] In particular embodiments a combination of the above-exemplified amplification techniques can be used. For example, RCA and MDA can be used in a combination wherein RCA is used to generate a concatameric amplicon in solution (e.g., using solution-phase primers). The amplicon can then be used as a template for MDA using primers that are attached to a bead or other solid support (e.g., universal primers). In this example, amplicons produced after the combined RCA and MDA steps will be attached to the bead or other solid support.

[0149] In certain aspects of the instant disclosure, a capture material is employed to associate a bead array with a solid support (e.g., a glass slide). In some embodiments, the capture material is a liquid electrical tape. An exemplary liquid electrical tape of the instant disclosure is Permatex™ liquid electrical tape, which is a weatherproof protectant for wiring and electrical connections. Liquid capture material such as liquid tape can be applied as a liquid, which then dries to a vinyl polymer that resists dirt, dust, chemicals, and moisture. Capture materials of theinstant disclosure can be applied by any of a number of methods, including brushed onto the solid support, sprayed onto the solid support, or the like, or via submersion of the solid support in the capture material. For certain forms of liquid capture material, use of a brush top applicator can allow coverage without gaps and can enable access to tight spaces, which offers advantages in certain embodiments over forms of capture material (i.e., tape) that are applied in a non-liquid state.

[0150] While liquid electrical tape has been exemplified as a capture material for use in the methods and compositions of the instant disclosure, other capture materials are also contemplated for such use, including any art-recognized glue or other reagent that is (a) spreadable and / or depositable upon a solid surface (e.g., upon a slide, optionally a slide that allows for light transmission through the slide, e.g., a microscope slide) and (b) capable of binding or otherwise capturing a population of beads of 1-100 pm size. Other exemplary capture materials that are expressly contemplated include latex such as cis-l,4-polyisoprene and other rubbers, as well as elastomers (which are generally defined as polymers that possess viscoelasticity (i.e., both viscosity and elasticity), very weak inter-molecular forces, and generally low Young's modulus and high failure strain compared with other materials), including artificial elastomers (e.g., neoprene) and / or silicone elastomers. Acrylate polymers (e.g., scotch tape) are also expressly contemplated, e.g., for use as a capture material of the instant disclosure. Exemplary other capture materials that are expressly contemplated include super glue adhesives (e.g. Loctite™ super glue), gels (e.g. agarose, acrylamide), rubberized sealant sprays (e.g. Gorilla Glue™, PLASTI DIP™ multipurpose rubber coating), silicone conformal coating (e.g. Fine-L-Kote SR™), or other conformal coatings (e.g. Urethane resin, Epoxy resin, Acrylic resin, Parylene). Additionally, bead oligos may be transferred to agarose or acrylamide gel substrates for subsequent release into tissues.

[0151] In example embodiments, the array is printed on a solid support. In one embodiment, the printed spatial barcode nucleic acids are amplified on the solid support as described herein (e.g., bridge amplification). In example embodiments, the solid support is a slide or an array on a slide. As used herein the term "slide" includes an "array", "substrate" or "surface" including a plurality of spatial barcode nucleic acids as described herein. For the spatial array-based analytical methods described herein, a substrate functions as a support for direct or indirect attachment ofspatial barcode nucleic acids to features of the array. In addition, in some embodiments, a substrate (e.g., the same substrate or a different substrate) can be used to provide support to a biological sample, particularly, for example, a thin tissue section. Accordingly, a “substrate” is a support that is insoluble in aqueous liquid and which allows for positioning of biological samples, analytes, features, and / or spatial barcode nucleic acids on the substrate.

[0152] Further, a “substrate” as used herein, and when not preceded by the modifier “chemical”, refers to a member with at least one surface that generally functions to provide physical support for biological samples, analytes, and / or any of the other chemical and / or physical moieties, agents, and structures described herein. Substrates can be formed from a variety of solid materials, gel-based materials, colloidal materials, semi-solid materials (e.g., materials that are at least partially cross-linked), materials that are fully or partially cured, and materials that undergo a phase change or transition to provide physical support. Examples of substrates that can be used in the methods and systems described herein include, but are not limited to, slides (e.g., slides formed from various glasses, slides formed from various polymers), hydrogels, layers and / or films, membranes (e.g., porous membranes), flow cells, cuvettes, wafers, plates, or combinations thereof. In some embodiments, substrates can optionally include functional elements such as recesses, protruding structures, microfluidic elements (e.g., channels, reservoirs, electrodes, valves, seals), and various markings. Slides and arrays for spatial profiling have been described (see, e.g., Visium Spatial Capture Technology, 10X Genomics, Pleasanton, CA; WO 2020 / 047007A2; WO 2020 / 123317A2; WO 2020 / 047005A1; WO 2020 / 176788A1; and WO 2020 / 190509A9). The capture probes comprising spatial barcodes can be replaced with the spatial barcode nucleic acids comprising spatial barcodes as described herein.

[0153] Slides comprising spatial barcode nucleic acids can be obtained by synthesizing spatial barcode nucleic acids and attaching them to a slide or array. In an example embodiment, spatial barcode nucleic acids are added to specific locations of an array.

[0154] Arrays can be prepared by depositing features (e.g., droplets, beads) on a substrate surface to produce a spatially-barcoded array. Methods of depositing (e.g., droplet manipulation) features are known in the art (see, U.S. Patent Application Publication No. 2008 / 0132429; Rubina, A.Y., et al., Biotechniques.2003 May; 34(5): 1008-14, 1016-20, 1022; and Vasiliskov et al. Biotechniques.1999 September; 27(3):592-4, 596-8, 600 passim). A feature can be printed ordeposited at a specific location on the substrate (e.g., inkjet printing). In some embodiments, each feature can have a spatial barcode nucleic acid. In some embodiments, a feature can be printed or deposited at the specific location using an electric field. A feature can contain a photo- crosslinkable polymer precursor and an oligonucleotide. In some embodiments, the photo- crosslinkable polymer precursor can be deposited into a patterned feature on the substrate (e.g., well). A “photo-crosslinkable polymer precursor” refers to a compound that cross-links and / or polymerizes upon exposure to light. In some embodiments, one or more photoinitiators may also be included to induce and / or promote polymerization and / or cross- linking (see, e.g., Choi et al. Biotechniques. 2019 Jan;66(l):40-53).

[0155] Arrays can be prepared by a variety of methods. In some embodiments, arrays are prepared through the synthesis (e.g., in situ synthesis) of oligonucleotides on the array, or by jet printing or lithography. In example embodiments, spatial barcode nucleic acids are deterministically patterned via synthesis. For example, light-directed synthesis of high-density DNA oligonucleotides can be achieved by photolithography or solid-phase DNA synthesis. To implement photolithographic synthesis, synthetic linkers modified with photochemical protecting groups can be attached to a substrate and the photochemical protecting groups can be modified using a photolithographic mask (applied to specific areas of the substrate) and light, thereby producing an array having localized photo-deprotection. Many of these methods are known in the art, and are described e.g., in Miller et al., “Basic concepts of microarrays and potential applications in clinical microbiology.” Clinical Microbiology Reviews 22.4 (2009): 611-633; US 2013 / 14111482A; US 9593365B2; US 2019 / 203275; and WO 2018 / 091676.Tissues

[0156] In some embodiments, a tissue section is employed. The tissue can be derived from a multicellular organism. Exemplary multicellular organisms include, but are not limited to a mammal, plant, algae, nematode, insect, fish, reptile, amphibian, fungi o Plasmodium falciparum. Exemplary species are set forth previously herein or known in the art. 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 (FFPE)), formalin fixation, infiltration, dehydration or the like. Optionally, a tissue section can be cryosectioned, using techniques and compositions as described herein and as known in the art.As a further option, a tissue can be permeabilized to allow nuclei to be accessible by spatial barcode nucleic acids. As used herein, the term “tissue” is intended to mean an aggregation of cells, and, optionally, intercellular matter. Typically, 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. Tissues can also be from a diseased subject, such as but not limited to an autoimmune disease or cancer (e.g., a tissue from irritable bowel disease (IBD) or MS, or a tumor tissue).

[0157] A method of the present disclosure can include a step of contacting a biological specimen (i.e., a cryosectioned tissue sample) with a bead or other solid support that has spatial barcode nucleic acids attached thereto. In some embodiments, the spatial barcode nucleic acids are randomly located on the bead or other solid support. The identity and location of the spatial barcode nucleic acids may have been decoded prior to contacting the biological specimen with the bead or other solid support. Alternatively, the identity and location of the spatial barcode nucleic acids can be determined after contacting the bead or other solid support with the biological specimen.

[0158] As used herein, the term “cryosection” refers to a piece of tissue, e.g. a biopsy, that has been obtained from a subject, snap frozen, embedded in optimal cutting temperature embedding material, frozen, and cut into thin sections. In certain embodiments, the thin sections can be directly applied to an array of beads captured upon a solid support (e.g., a slide), or the thin sections can be fixed (e.g. in methanol or paraformaldehyde) and applied to a bead-presenting planar surface, e.g., a slide upon which a layer of microbeads has been attached / arrayed.

[0159] In example embodiments, the tissue can be obtained from a complex multicellular system (e.g., organoid, tissue explant, or organ on a chip) (see, e.g., Yin X, Mead BE, Safaee H, Langer R, Karp JM, Levy O. Engineering Stem Cell Organoids. Cell Stem Cell. 2016; 18(1):25-38; Clevers, Modeling Development and Disease with Organoids, Cell. 2016 Jun 16; 165(7): 1586-1597; Porter, R.J., Murray, G.I. & McLean, M.H. Current concepts in tumour-derived organoids. Br J Cancer 123, 1209-1218 (2020). doi.org / 10.1038 / s41416-020-0993-5; Sontheimer-Phelps, A., Hassell, B. A. & Ingber, D. E. Modelling cancer in microfluidic human organs-on-chips. Nat. Rev. Cancer 19, 65-81 (2019); and Wu, Q., Liu, J., Wang, X. et al. Organ-on-a-chip: recent breakthroughs and future prospects. BioMed Eng OnLine 19, 9 (2020); Ingber, D. E.Developmentally inspired human ‘organs on chips’. Development 145, pii:devl56125 (2018); Ghosh S, Prasad M, Kundu K, et al. Tumor Tissue Explant Culture of Patient-Derived Xenograft as Potential Prioritization Tool for Targeted Therapy. Front Oncol. 2019;9:17; Neil JE, Brown MB, Williams AC. Human skin explant model for the investigation of topical therapeutics. Sci Rep. 2020; 10(1 ):21192; and Grivel JC, Margolis L. Use of human tissue explants to study human infectious agents. Nat Protoc. 2009;4(2):256-269). Tissues or complex multicellular systems include a patient derived organoid (PDO) or patient derived xenograft (PDX).

[0160] A tissue can be prepared in any convenient or desired way for its use in a method, composition or apparatus herein. Fresh, frozen, fixed or unfixed tissues can be used. A tissue can be fixed or embedded using methods described herein or known in the art.

[0161] A tissue sample for use herein, can be fixed by deep freezing at temperature suitable to maintain or preserve the integrity of the tissue structure, e.g., less than -20° C. In another example, a tissue can be prepared using formalin-fixation and paraffin embedding (FFPE) methods which are known in the art. Other fixatives and / or embedding materials can be used as desired. A fixed or embedded tissue sample can be sectioned, i.e. thinly sliced, using known methods. For example, a tissue sample can be sectioned using a chilled microtome or cryostat, set at a temperature suitable to maintain both the structural integrity of the tissue sample and the chemical properties of the nucleic acids in the sample. Exemplary additional fixatives that are expressly contemplated include alcohol fixation (e.g., methanol fixation, ethanol fixation), glutaraldehyde fixation and paraformaldehyde fixation.

[0162] In some embodiments, a tissue sample will be treated to remove embedding material (e.g., to remove paraffin or formalin) from the sample prior to tagging nuclei. This can be achieved by contacting the sample with an appropriate solvent (e.g., xylene and ethanol washes). Treatment can occur prior to contacting the tissue sample with a spatial array as set forth herein or the treatment can occur while the tissue sample is on the solid support-captured bead array.

[0163] Exemplary methods for manipulating tissues for use with solid supports to which nucleic acids are attached are set forth in US Pat. App. Publ. No. 2014 / 0066318 Al, which is incorporated herein by reference.

[0164] The thickness of a tissue sample or other biological specimen that is contacted with a bead array in a method, composition or apparatus set forth herein can be any suitable thicknessdesired. In representative embodiments, the thickness will be at least 0.1 pm, 0.25 pm, 0.5 pm, 0.75 pm, 1 pm, 5 pm, 10 pm, 50 pm, 100 pm or thicker. Alternatively or additionally, the thickness of a tissue sample that is contacted with bead array will be no more than 100 pm, 50 pm, 10 pm, 5 pm, 1 pm, 0.5 pm, 0.25 pm, 0.1 pm or thinner.

[0165] A particularly relevant source for a tissue sample is a human being. Another source of a tissue sample is an animal model (e.g., a mouse tissue sample). The sample can be derived from an organ, including for example, an organ of the central nervous system such as brain, brainstem, cerebellum, spinal cord, cranial nerve, or spinal nerve; an organ of the musculoskeletal system such as muscle, bone, tendon or ligament; an organ of the digestive system such as salivary gland, pharynx, esophagus, stomach, small intestine, large intestine, liver, gallbladder or pancreas; an organ of the respiratory system such as larynx, trachea, bronchi, lungs or diaphragm; an organ of the urinary system such as kidney, ureter, bladder or urethra; a reproductive organ such as ovary, fallopian tube, uterus, vagina, placenta, testicle, epididymis, vas deferens, seminal vesicle, prostate, penis or scrotum; an organ of the endocrine system such as pituitary gland, pineal gland, thyroid gland, parathyroid gland, or adrenal gland; an organ of the circulatory system such as heart, artery, vein or capillary; an organ of the lymphatic system such as lymphatic vessel, lymph node, bone marrow, thymus or spleen; a sensory organ such as eye, ear, nose, or tongue; or an organ of the integument such as skin, subcutaneous tissue or mammary gland. In some embodiments, a tissue sample is obtained from a bodily fluid or excreta, such as blood, lymph, tears, sweat, saliva, semen, vaginal secretion, ear wax, fecal matter or urine.

[0166] A sample from a human can be considered (or suspected) healthy or diseased when used. In some cases, two samples can be used: a first being considered diseased and a second being considered as healthy (e.g., for use as a healthy control). Any of a variety of conditions can be evaluated, including but not limited to, an autoimmune disease, cancer, cystic fibrosis, aneuploidy, pathogenic infection, psychological condition, hepatitis, diabetes, sexually transmitted disease, heart disease, stroke, cardiovascular disease, multiple sclerosis (MS) or muscular dystrophy. Certain contemplated conditions include genetic conditions or conditions associated with pathogens having identifiable genetic signatures.

[0167] In example embodiments, tissue is prepared for nuclei tagging using steps from single nuclei sequencing methods (e.g., snRNA-seq) (see, e.g., Habib et al., 2016, “Div-Seq: Single-nucleus RNA-Seq reveals dynamics of rare adult newborn neurons” Science, Vol. 353, Issue 6302, pp. 925-928; Habib et al., 2017, “Massively parallel single-nucleus RNA-seq with DroNc-seq” Nat Methods. 2017 Oct;14(10):955-958; International Patent Application No. PCT / US2016 / 059239, published as WO 2017 / 164936 on September 28, 2017; International Patent Application No. PCT7US2018 / 060860, published as WO / 2019 / 094984 on May 16, 2019; International Patent Application No. PCT / US2019 / 055894, published as W 0 / 2020 / 077236 on April 16, 2020; Drokhlyansky, et al., “The enteric nervous system of the human and mouse colon at a single-cell resolution,” bioRxiv 746743; doi: doi.org / 10.1101 / 746743; Drokhlyansky E, Smillie CS, Van Wittenberghe N, et al. The Human and Mouse Enteric Nervous System at SingleCell Resolution. Cell. 2020;182(6):1606-1622.e23; and Slyper, M., Porter, C.B.M., Ashenberg, O. et al. (2020). A single-cell and single-nucleus RNA-seq toolbox for fresh and frozen human tumors. Nature Medicine 26(5):792-802). In example embodiments, methods for preparing and isolating nuclei that preserve the nuclear envelope and ribosomes (RAISIN (Ribosomes And Intact Single Nucleus) RNA-seq) or that preserve both the rough ER and its attached ribosomes on the outer nuclear membrane (INNER Cell (INtact Nucleus and Endoplasmic Reticulum from a single Cell) RNA-seq) are applicable to tagged nuclei (see, WO / 2020 / 077236).

[0168] In an example embodiment, single nuclei are prepared from FFPE tissue sections (see, e.g., W 0 / 2020 / 077236). For example, an FFPE tissue sample is processed by dissolving paraffin in a solvent, preferably the solvent is selected from xylene or mineral oil, wherein the tissue is dissolved at a temperature between 4°C to 90°C, preferably room temperature (20° to 25°C) or 90°C; rehydrating the tissue using a gradient of ethanol from 100% to 0% ethanol (EtOH); transferring the rehydrated tissue to a volume of a first buffer comprising a buffering agent, a detergent and an ionic strength between lOOmM and 200mM, optionally the first buffer comprises protease inhibitors or proteases and / or BSA. In an example embodiment, single nuclei are prepared from tissue samples (see, e.g., Raisin-seq) using buffer comprising 10 mM Tris, 0.49% CHAPS, 146 mM NaCl, 1 mM CaCh, 21 mM MgCh, and 0.01% BSA (CST). In an example embodiment, single nuclei are prepared from tissue samples (see, e.g., INNER Cell-seq) using buffer comprising 10 mM Tris, 0.03% Tween-20, 146 mM NaCl, ImM CaCh, 21 mM MgCh, and 0.01% BSA (TST). In one example embodiment, the buffers are used during extraction of nuclei from the tissue.

[0169] In some embodiments, a biological sample can be permeabilized to facilitate transfer of spatial barcode nucleic acids into the sample. If a sample is not permeabilized sufficiently, the amount of spatial barcode nucleic acids into the sample may be too low to enable adequate analysis. Conversely, if the tissue sample is too permeable, the relative spatial relationship of the analytes within the tissue sample can be lost. Hence, a balance between permeabilizing the tissue sample enough to obtain good signal intensity while still maintaining the spatial resolution of the analyte distribution in the sample is desirable.

[0170] In general, a biological sample can be permeabilized by exposing the sample to one or more permeabilizing agents. Suitable agents for this purpose include, but are not limited to, organic solvents (e.g., acetone, ethanol, and methanol), cross-linking agents (e.g., paraformaldehyde), detergents (e.g., saponin, Triton X-100™, Tween-20™, or sodium dodecyl sulfate (SDS)), and enzymes (e.g., trypsin, proteases (e.g., proteinase K). In some embodiments, the detergent is an anionic detergent (e.g., SDS or N-lauroylsarcosine sodium salt solution). In some embodiments, the biological sample can be permeabilized using any of the methods described herein (e.g., using any of the detergents described herein, e.g., SDS and / or N- lauroylsarcosine sodium salt solution) before or after enzymatic treatment (e.g., treatment with any of the enzymes described herein, e.g., trypsin, proteases (e.g., pepsin and / or proteinase K)). Additional methods for sample permeabilization are described, for example, in Jamur et al., Method Mol. Biol.588:63-66, 2010, the entire contents of which are incorporated herein by reference.Cleaving linkers and delivering spatial barcodes to nuclei

[0171] In example embodiments, the spatial barcode nucleic acids in contact with the permeabilized tissue sample are delivered to the nuclei. In example embodiments, the tissue sample is incubated in a dissociation buffer as described herein (see, Example 1 Dissociation Buffer (DB)). In example embodiments, the incubation is at 4-25 °C, preferably about 4 °C. The linkers are then cleaved (e.g., using light, chemical or an enzyme) to release the spatial barcode nucleic acids from the spatial array. In example embodiments, the spatial barcode nucleic acids are delivered to the nuclei by diffusion. In example embodiments, the spatial barcode nucleic acids are delivered to the nuclei using electroporation. With electroporation, spatial barcodenucleic acids can enter a cell through one or more pores in the cellular membrane formed by applied electricity. The pore of the membrane can be reversible based on the applied field strength and pulse duration. In example embodiments, the spatial barcode nucleic acids are delivered to the nuclei using sonoporation. Cell membranes can be temporarily permeabilized using sound waves, allowing cellular uptake of spatial barcode nucleic acids. In example embodiments, the spatial barcode nucleic acids are delivered to the nuclei using hydroporation. Spatial barcode nucleic acids can be delivered to cells via hydrodynamic pressure.Isolating tagged nuclei from tissue

[0172] In example embodiments, nuclei are extracted from the tagged tissue by dissociating the tissue with extraction buffer. In example embodiments, the extraction buffer includes a detergent. In example embodiments, the detergent is Triton X-100. In example embodiments, the detergent is CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-l -propanesulfonate). In example embodiments, the detergent is Tween-20. In one embodiment, the tissue is dissociated by pipetting. In one embodiment, the tissue is dissociated by gentle scraping. In example embodiments, the tagged nuclei can be sorted by FACS in the case where the spatial barcode nucleic acids include a fluorescent dye. Thus, only tagged nuclei will be used in the following steps. Sorting nuclei can allow the same efficiency as using untagged nuclei.Single cell genomics assays

[0173] Single-cell omics sequencing was first achieved for the transcriptome in 2009, which was followed by fast development of technologies for profiling the genome, DNA methylome, 3D genome architecture, chromatin accessibility, histone modifications, etc., in an individual cell (see, e.g., Wen L, Tang F. Recent advances in single-cell sequencing technologies. Precis Clin Med. 2022;5(l):pbac002. Published 2022 Ian 31). In example embodiments, the tagged nuclei generated by the present methods can be used with any of these single cell genomics assays to make the assays spatially resolved.

[0174] In example embodiments, single cell genomics assays generally include barcodes used to identify the cell of origin of the genomic analyte (i.e., cell barcodes). For example, the analyte can be RNA, such as a poly-A tailed mRNA, a genomic DNA sequence, joined DNA fragments, immunoprecipitated genomic DNA, or antibody specific oligonucleotides. The cell barcodes alsogenerally are associated with a unique molecular identifier (UMI), such that the number of each analyte for each single cell can be counted (i.e., quantified). The term “unique molecular identifiers” (UMI) as used herein refers to a sequencing linker or a subtype of nucleic acid barcode used in a method that uses molecular tags to detect and quantify unique amplified products (see e.g., Islam S. et al., 2014. Nature Methods No:l 1, 163-166). In example embodiments, a UMI is a random sequence of between 4 and 20 bases. A UMI is used to distinguish effects through a single clone from multiple clones (e.g., an amplified product from a single clone will include the same UMI as the original clone). The term “clone” as used herein may refer to a single mRNA or target nucleic acid to be sequenced. Thus, the UMI may also be used to determine the number of a specific analyte or events that gave rise to an amplified product. In an example embodiment, each cell barcode for a single nucleus is the same, but each UMI associated with the cell barcode is randomized, such that each capture event has a unique UMI. For example, a spatial barcode nucleic acid can be captured by a cell barcode nucleic acid that includes a UMI. The number of different UMIs sequenced for a spatial barcode in a single nucleus identified by a cell barcode would indicate the number of spatial barcodes that the single nuclei was tagged with (e.g., counting UMIs for sequence reads having the same cell barcode, but different spatial barcodes).

[0175] In example embodiments, nucleic acid sequences comprising a cell barcode and UMI can capture more than one spatial barcode if the single cell was in proximity to or overlapping more than one location on the array. In example embodiments, computational methods can be used to resolve the location of single cells in the spatial array, such as by counting UMIs. The computational methods can use the distance on the array of the different spatial barcodes. If the spatial barcodes are close on the array, the location is most likely somewhere in between or overlapping the locations. If the spatial barcodes are far away, then most likely, the spatial barcode nucleic acids diffused away from their location and the location is the spatial barcode with the most UMIs because the spatial barcode with higher UMIs was the predominant spatial barcode that diffused into that single cell.

[0176] Single cell assays generally add the cell barcode by one of three methods: adding single cells / nuclei to individual wells each having a unique barcode or combination of barcodes; pool and split indexing of intact nuclei / cells; and segregating single cells / nuclei into separate reaction vessels that include unique cell barcodes, such as microwells, reaction chambers, or droplets. Inan example embodiment, for pool and split indexing, the spatial barcode nucleic acid is modified to include a common universal sequence (e.g., handle sequence) present on each target analyte, such that both the spatial barcode and analyte can capture the same index sequence at each pool and split step. In an example embodiment, for droplet methods, the spatial barcode nucleic acid is modified to include a common universal sequence present on each target analyte, such that both can be captured by a cell barcode nucleic acid present in each droplet (e.g., usually on a bead). Single cell assays generally use a capture sequence specific to an analyte, such as an analyte having a poly-A tail or a specific adapter sequence added by a transposase (e.g., tagmentation adapter). These sequences can easily be included in a spatial barcode nucleic acid.

[0177] The method of the present disclosure can be adapted, such that the spatial barcode can be captured by the cell barcode for any single cell assay. Specific examples are provided below.Spatially resolved single nucleus / cell RNA-sequencing

[0178] In example embodiments, the single cell genomics sequencing library includes a single cell RNA sequencing library (see, e.g., Trombetta, J. J., Gennert, D., Lu, D., Satija, R., Shalek, A. K. & Regev, A. Preparation of Single-Cell RNA-Seq Libraries for Next Generation Sequencing. Curr Protoc Mol Biol. 107, 42221-242217, doi: 10.1002 / 0471142727.mb0422sl07 (2014); Qi Z, Barrett T, Parikh AS, Tirosh I, Puram SV. Single-cell sequencing and its applications in head and neck cancer. Oral Oncol. 2019;99: 104441; Kalisky, T., Blainey, P. & Quake, S. R. Genomic Analysis at the Single-Cell Level. Annual review of genetics 45, 431-445, (2011); Kalisky, T. & Quake, S. R. Single-cell genomics. Nature Methods 8, 311-314 (2011); Islam, S. et al. Characterization of the single-cell transcriptional landscape by highly multiplex RNA-seq. Genome Research, (2011); Tang, F. et al. RNA-Seq analysis to capture the transcriptome landscape of a single cell. Nature Protocols 5, 516-535, (2010); Tang, F. et al. mRNA-Seq whole-transcriptome analysis of a single cell. Nature Methods 6, 377-382, (2009); Ramskold, D. et al. Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells. Nature Biotechnology 30, 777-782, (2012); and Hashimshony, T., Wagner, F., Sher, N. & Yanai, I. CEL-Seq: Single-Cell RNA-Seq by Multiplexed Linear Amplification. Cell Reports, Cell Reports, Volume 2, Issue 3, p666-673, 2012). PMCID:4338574), to allow for coordinated assessment of both genomic DNA methylation status and RNA expression levels at single-cell resolution.

[0179] In example embodiments, the disclosure includes plate based single cell RNA sequencing (see, e.g., Picelli, S. et al., 2014, “Full-length RNA-seq from single cells using Smart-seq2” Nature protocols 9, 171-181, doi:10.1038 / nprot.2014.006). In example embodiments, the disclosure includes high-throughput single-cell RNA-seq. In this regard, reference is made to Macosko et al., 2015, “Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets” Cell 161, 1202-1214; International patent application number PCT / US2015 / 049178, published as WO 2016 / 040476 on March 17, 2016; Klein et al., 2015, “Droplet Barcoding for Single-Cell Transcriptomics Applied to Embryonic Stem Cells” Cell 161, 1187-1201; International patent application number PCT / US2016 / 027734, published as WO 2016 / 168584A1 on October 20, 2016; Zheng, et al., 2016, “Haplotyping germline and cancer genomes with high-throughput linked-read sequencing” Nature Biotechnology 34, 303-311; Zheng, et al., 2017, “Massively parallel digital transcriptional profiling of single cells” Nat. Commun. 8, 14049 doi: 10.1038 / ncommsl4049; International patent publication number WO2014210353A2; Zilionis, et al., 2017, “Single-cell barcoding and sequencing using droplet microfluidics” Nat Protoc. Jan;12(l):44-73; Cao et al., 2017, “Comprehensive single cell transcriptional profiling of a multicellular organism by combinatorial indexing” bioRxiv preprint first posted online Feb. 2, 2017, doi: dx.doi.org / 10.1101 / 104844; Rosenberg et al., 2017, “Scaling single cell transcriptomics through split pool barcoding” bioRxiv preprint first posted online Feb.2, 2017, doi: dx. doi. org / 10.1101 / 105163; Rosenberg et al., “Single-cell profiling of the developing mouse brain and spinal cord with split-pool barcoding” Science 15 Mar 2018; Vitak, et al., “Sequencing thousands of single-cell genomes with combinatorial indexing” Nature Methods, 14(3):302-308, 2017; Cao, et al., Comprehensive single-cell transcriptional profiling of a multicellular organism. Science, 357(6352):661-667, 2017; Gierahn et al., “Seq-Well: portable, low-cost RNA sequencing of single cells at high throughput” Nature Methods 14, 395-398 (2017); and Hughes, et al., “Highly Efficient, Massively-Parallel Single-Cell RNA-Seq Reveals Cellular States and Molecular Features of Human Skin Pathology” bioRxiv 689273; doi: doi. org / 10.1101 / 689273, all the contents and disclosure of each of which are herein incorporated by reference in their entirety.

[0180] In example embodiments, the disclosure can include single nucleus RNA sequencing. In this regard, reference is made to Swiech et al., 2014, “In vivo interrogation of gene function inthe mammalian brain using CRISPR-Cas9” Nature Biotechnology Vol. 33, pp. 102-106; Habib et al., 2016, “Div-Seq: Single-nucleus RNA-Seq reveals dynamics of rare adult newborn neurons” Science, Vol. 353, Issue 6302, pp. 925-928; Habib et al., 2017, “Massively parallel single-nucleus RNA-seq with DroNc-seq” Nat Methods. 2017 Oct;14(10):955-958; International Patent Application No. PCT / US2016 / 059239, published as WO2017164936 on September 28, 2017; International Patent Application No.PCT / US2018 / 060860, published as WO / 2019 / 094984 on May 16, 2019; International Patent Application No. PCT / US2019 / 055894, published as WO / 2020 / 077236 on April 16, 2020; Drokhlyansky, et al., “The enteric nervous system of the human and mouse colon at a single-cell resolution,” bioRxiv 746743; doi: doi.org / 10.1101 / 746743; and Drokhlyansky E, Smillie CS, Van Wittenberghe N, et al., The Human and Mouse Enteric Nervous System at Single-Cell Resolution. Cell. 2020;182(6):1606- 1622.e23, which are herein incorporated by reference in their entirety.

[0181] Single cell / nuclei RNA-seq generally uses a poly-T capture sequence and reverse transcription (RT) to capture mRNAs having a poly-A tail. In an example embodiment, tagged nuclei are generated according to the present disclosure with spatial barcode nucleic acids comprising a poly-A sequence. The tagged nuclei are then used in any RNA-seq method above. For example, tagged nuclei can be used in a pool and split method or can be segregated to individual wells or droplets. The spatial barcode nucleic acids and mRNA are both captured by a cell barcode sequence. For plate or microwell based methods, reverse transcription and use of a template switching primer can add a unique barcode to mRNA and the spatial barcode nucleic acid.Spatially resolved single cell chromatin accessibility

[0182] In example embodiments, the single cell genomics sequencing library is a single cell Assay for Transposase Accessible Chromatin using sequencing (ATAC-seq) sequencing library. ATAC-seq can be used to identify accessible chromatin in a cell (see, e.g., Buenrostro, et al., Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nature Methods 2013; 10 (12): 1213-1218). Using plate-, droplet-, or combinatorial indexing-based methods, thousands to hundreds of thousands of individual cells / nuclei can be analyzed in a single sample (see, e.g., Buenrostro et al., Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-490 (2015); Cusanovich, D. A., Daza, R., Adey, A., Pliner, H., Christiansen, L., Gunderson, K. L., Steemers, F. J., Trapnell, C. & Shendure, J. Multiplex single-cell profiling of chromatin accessibility by combinatorial cellular indexing. Science. 2015 May 22;348(6237):910-4. doi: 10.1126 / science.aabl601. Epub 2015 May 7; Cusanovich DA, Hill AJ, Aghamirzaie D, et al. A Single-Cell atlas of in vivo mammalian chromatin accessibility. Cell. 2018; 174:1309-24; Lake BB, Chen S, Sos BC, et al. Integrative single-cell analysis of transcriptional and epigenetic states in the human adult brain. Nat Biotechnol. 2018; 36:70-80; Preissl S, Fang R, Huang H, et al. Single-nucleus analysis of accessible chromatin in developing mouse forebrain reveals cell-type- specific transcriptional regulation. NatNeurosci. 2018; 21:432-9; Satpathy AT, Granja JM, Yost KE, et al. Massively parallel single-cell chromatin landscapes of human immune cell development and intratumoral T cell exhaustion. Nat Biotechnol. 2019; 37:925-36; Xu W, Wen Y, Liang Y, et al. A plate-based single-cell ATAC-seq workflow for fast and robust profiling of chromatin accessibility. Nat Protoc. 2021; 16:4084-107; US 2016 / 0208323A1; US 2016 / 0060691A1; and WO 2017 / 156336A1). Single nuclei ATAC-seq can also be performed by partitioning nuclei in droplets and subsequent snATAC-Seq library construction using the Chromium Next GEM Single Cell ATAC Reagent Kit vl.l (10 x Genomics, Pleasanton, CA, USA) (see, e.g., Briel N, Ruf VC, Pratsch K, et al. Single-nucleus chromatin accessibility profiling highlights distinct astrocyte signatures in progressive supranuclear palsy and corticobasal degeneration. Acta Neuropathol. 2022;144(4):615-635).

[0183] In an example embodiment, tagged nuclei are generated according to the present disclosure with spatial barcode nucleic acids comprising an adapter sequence that is the same as the adapters inserted into active chromatin by Tn5 transposase. The tagged chromatin fragments and spatial barcode nucleic acids can then be captured by cell barcodes using combinatorial indexing or droplet-based method using barcoded beads.Spatially resolved single cell Hi-C

[0184] In example embodiments, the single cell genomics sequencing library is or includes a single cell Hi-C sequencing library. In situ Hi-C involves cross-linking cells with formaldehyde; permeabilizing them with nuclei intact; digesting DNA with a suitable 4-cutter restriction enzyme (such as Mbol); fdling the 5'-overhangs while incorporating a biotinylated nucleotide; ligating the resulting blunt-end fragments; shearing the DNA; capturing the biotinylated ligation junctionswith streptavidin beads; and analyzing the resulting fragments with paired-end sequencing (see, e.g., Rao SS, Huntley MH, Durand NC, et al. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell. 2014;159(7):1665-1680). Hi-C can also be performed in single cells with various barcoding methods, such as combinatorial cellular indexing (see, e.g., Ramani, et al., Sci-Hi-C: A single-cell Hi-C method for mapping 3D genome organization in large number of single cells Methods. 2020 Jan 1; 170: 61-68; Ramani V, Deng X, Qiu R, et al. Massively multiplex single-cell Hi-C. Nat Methods. 2017;14(3):263-266; and Nagano T, Lubling Y, Varnai C, et al. Cell-cycle dynamics of chromosomal organization at single-cell resolution. Nature. 2017; 547:61-7).

[0185] In an example embodiment, tagged nuclei are generated according to the present disclosure with spatial barcode nucleic acids comprising double stranded adapter sequences, tagged nuclei are crosslinked, and DNA is digested with a suitable restriction enzyme. Nuclei are then distributed to 96 wells, wherein the first barcode is introduced through ligation of barcoded biotinylated double-stranded bridge adaptors. Intact nuclei are then pooled and subjected to proximity ligation, followed by dilution and redistribution to a second 96-well plate. Following lysis, a second barcode is introduced through ligation of barcoded Y-adaptors. In this example, both the fragmented DNA and the spatial barcodes receive the same nuclei specific barcode.

[0186] In an example embodiment, tagged nuclei are generated according to the present disclosure with spatial barcode nucleic acids comprising adapter sequences, tagged nuclei are crosslinked, DNA is digested with a suitable restriction enzyme, the resulting fragments are ligated, the DNA is fragmented with a transposase (Tn5) loaded with adapters in the intact nuclei, and the nuclei can then be used in a droplet based single cell sequencing method using beads that capture the adapter sequences and add a cell barcode.Spatially resolved single cell DNA-methylation sequencing

[0187] In example embodiments, the single cell genomics sequencing library is or includes a single cell DNA methylation sequencing library. Methods for distinguishing DNA methylation include (i) bisulfite conversion, (ii) Tet-assisted bisulfite conversion, (iii) Tet-assisted conversion with a substituted borane reducing agent, and (iv) protection of hmC followed by Tet-assisted conversion with a substituted borane reducing agent (see, e.g., US Patent Application No. US 2021 / 0115502A1). Methylation can also be detected using methylation specific restrictionenzymes or methylated DNA immunoprecipitation (MeDIP) Tn example embodiments, DNA methylation can be detected where methylated cytosines (mC) and hydroxymethylated cytosines (hmC) are determined by the sequencer itself and independent of one or more agents (e.g., using PacBio or Nanopore sequencers). Single-cell DNA methylome sequencing techniques have been established using various strategies including the reduced representation bisulfite sequencing (RRBS)- and the post-bi sulfite adaptor tagging (PBAT)-based methods (see, e.g., Ahn J, Heo S, Lee J, Bang D. Introduction to Single-Cell DNA Methylation Profiling Methods. Biomolecules.2021; 11(7): 1013; Guo H, Zhu P, Wu X, et al. Single-cell methylome landscapes of mouse embryonic stem cells and early embryos analyzed using reduced representation bisulfite sequencing. Genome Res. 2013; 23:2126-35; Luo C, Keown CL, Kurihara L, et al. Single-cell methylomes identify neuronal subtypes and regulatory elements in mammalian cortex. Science.2017; 357:600-4; Smallwood SA, Lee HJ, Angermueller C, et al. Single-cell genome-wide bisulfite sequencing for assessing epigenetic heterogeneity. Nat Methods. 2014; 11:817-20; and Shareef SJ, Bevill SM, Raman AT, et al. Extended-representation bisulfite sequencing of gene regulatory elements in multiplexed samples and single cells. NatBiotechnol. 2021; 39:1086-94). A sci-MET method has applied a combinatorial indexing strategy for increasing the throughput, with the first and second rounds of barcodes being incorporated by Tn5 transposon and random priming, respectively (Mulqueen RM, Pokholok D, Norberg SJ, et al. Highly scalable generation of DNA methylation profiles in single cells. Nat Biotechnol. 2018; 36:428-31).Spatially resolved single cell chromatin immunoprecipitation (ChIP)

[0188] In example embodiments, the single cell genomics sequencing library is or can include a single cell chromatin immunoprecipitation (ChIP) sequencing library. Chromatin immunoprecipitation (ChIP) is a widely used method for detecting modifications of histones in nucleosomes of chromatin. In single-cell ChlP-seq analysis, cell-specific barcodes are added before aggregating the cells for immunoprecipitation (Ai S, Xiong H, Li CC, et al. Profiling chromatin states using single-cell itChlP-seq. Nat Cell Biol. 2019;21:1164-72; Grosselin K, Durand A, Marsolier J, et al. High-throughput single-cell ChlP-seq identifies heterogeneity of chromatin states in breast cancer. Nat Genet. 2019; 51:1060-6; and Rotem A, Ram O, Shoresh N, et al. Single-cell ChlP-seq reveals cell subpopulations defined by chromatin state. Nat Biotechnol. 2015; 33:1165-72). Among these methods, Drop-ChIP and scChlP-seq add cellbarcodes by MNase digestion and ligation with a droplet microfluidics workflow, while itChIP adds cell barcodes by Tn5 transposase tagmentation with a chromatin opening step.

[0189] In example embodiments, the spatial barcode is designed to allow tagmentation or digestion and ligation such that a barcode sequence is generated having a cell barcode and spatial barcode. For example, single nuclei can be segregated into individual droplets or microwells containing the cell barcodes. The cell barcode / spatial barcode hybrids can be amplified from the input chromatin used for immunoprecipitation and sequenced. This provides the spatial location of each cell. The immunoprecipitated chromatin can then be assigned to the same cells based on the cell barcode.Spatially resolved single cell enzyme-tethering chromatin profiling

[0190] In example embodiments, the single cell genomics sequencing library is or includes a single cell enzyme-tethering chromatin profiling sequencing library. Enzyme-tethering represents a non-immunoprecipitation chromatin profiling approach that is becoming increasingly popular and has been adapted to single-cell analysis (see, e.g., Carter B, Ku WL, Kang JY, et al. Mapping histone modifications in low cell number and single cells using antibody-guided chromatin tagmentation (ACT-seq). Nat Commun. 2019; 10:3747; Harada A, MaeharaK, Handa T, et al. A chromatin integration labelling method enables epigenomic profiling with lower input. Nat Cell Biol. 2019; 21:287-96; Kaya-Okur HS, Wu SJ, Codomo CA, et al. CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nat Commun. 2019; 10: 1930; Schmid M, Durussel T, Laemmli UK. ChIC and ChEC; genomic mapping of chromatin proteins. Mol Cell.2004; 16:147-57; Skene PJ, Henikoff S. An efficient targeted nuclease strategy for high- resolution mapping of DNA binding sites. Elife. 2017; 6:e21856; and Wang Q, Xiong H, Ai S, et al. CoBATCH for High-Throughput Single-Cell Epigenomic Profiling. Mol Cell. 2019; 76:206- 16. e7). In these techniques, Tn5 transposase, MNase, or adenine methyltransferase is tethered to protein A that binds to the antibody, directly to the antibody, or directly to the target chromatin protein, which allows marking of the genomic regions having specific histone marks. ChIC, CUT&RUN, and scChIC use MNase, while scCUT&Tag, COBATCH, ACT-seq, and ChIL-seq use Tn5 transposase. A key cation-activation step, Ca2+ for MNase and Mg2+ for Tn5 transposase, allows activation of the enzyme activity in a short time window after washing off the nonspecifically-bound enzyme.

[0191] In an example embodiment, single tagged nuclei according to the present disclosure are tagged with spatial barcode nucleic acids including the same sequence as tagmentation adapters or ligation adapters used in the enzyme-tethering chromatin profding method. The enzymetethering chromatin profding method adds the adapters to chromatin bound by an antibody specific for a chromatin modification or protein. These nuclei can then be used in a single cell sequencing method that include barcoded beads that comprise sequences that hybridize to the adapter sequences and add a bead specific barcode to the tagged chromatin and spatial barcodes (e.g., 10X ATAC-seq kit). In other words, nuclei are tagged with spatial barcodes, nuclei are isolated, antibodies are added to nuclei, unbound antibodies are washed away, secondary antibodies with Tn5 or MNase are added, Ca2+or Mg2+buffer is added to tag DNA bound by the antibody, and a compatible single cell sequencing method is performed to add cell barcodes to the spatial barcodes and marked chromatin.Spatially resolved single cell proteomics

[0192] In certain embodiments, the single cell genomics sequencing library may comprise a single cell proteomics sequencing library (see, e.g., Yang L, George J, Wang J. Deep Profiling of Cellular Heterogeneity by Emerging Single-Cell Proteomic Technologies. Proteomics. 2020; 20(13):el900226. doi:10.1002 / pmic.201900226; single cell constituents (US 2018 / 0340939A), single-cell proteomic assay using aptamers (US 2018 / 0320224A1), and methods of identifying multiple epitopes in cells (US 2017 / 0321251 Al)).

[0193] In certain embodiments, the tagged nuclei are labeled with barcode oligonucleotide linked antibodies or aptamers, the nuclei are washed to remove unbound antibodies or aptamers, and the barcode oligonucleotides of the bound antibodies or aptamers then capture the cell barcode sequence. The oligonucleotide linked antibodies or aptamers include a universal sequence complementary to a capture sequence or handle for pool and split indexing or for capture by cell barcode beads. The spatial barcode nucleic acid is modified to include the same universal sequence, such as to capture the spatial barcode to the cell barcode sequence.Spatially resolved single cell multiomics

[0194] In example embodiments, the single cell genomics sequencing library comprises a single cell multiomics sequencing library (see, e.g., Lee J, Hyeon DY, Hwang D. Single-cellmultiomics: technologies and data analysis methods. Exp Mol Med. 2020;52(9): 1428-1442. doi:10.1038 / sl2276-020-0420-2; Wen L, Tang F. Single cell epigenome sequencing technologies. Mol Aspects Med. 2018; 59:62-69; Cao J, Cusanovich DA, Ramani V, et al. Joint profiling of chromatin accessibility and gene expression in thousands of single cells. Science.2018; 361:1380-5; Chen S, LakeBB, Zhang K. High-throughput sequencing of the transcriptome and chromatin accessibility in the same cell. Nat BiotechnoL 2019; 37:1452-7; Liu L, Liu C, Quintero A, et al. Deconvolution of single-cell multi-omics layers reveals regulatory heterogeneity. Nat Commun. 2019; 10:470; Zhu C, Yu M, Huang H, et al. An ultra high- throughput method for single-cell joint analysis of open chromatin and transcriptome. Nat Struct Mol Biol. 2019; 26:1063-70; Ma, S. et al. Chromatin potential identified by shared single cell profiling of RNA and chromatin. bioRxiv 2020.06.17.156943 (2020) doi: 10.1101 / 2020.06.17.156943; and Stoeckius, M. etal. Simultaneous epitope and transcriptome measurement in single cells. Nat. Methods 14, 865-868 (2017). In example embodiments, for multiomic assays, the spatial barcode needs to be captured by a cell barcode for only one of the omic parts of the multiomic single cell assay. For example, the spatial barcode can be configured to be captured by a poly-T sequence if the multiomic assay includes transcriptome sequencing.Itnaging / image assembly

[0195] With spatial barcodes of individual nuclei identified by cell of origin barcodes, and with analytes (e.g., including genomic DNA, surveyed for methylation status) identified by sequences also including the cell of origin barcodes also identified, high-resolution images that localize sites of analyte expression and / or methylation content / patterning can be readily constructed in silico. In example embodiments, the spatial locations of a large number of sites (e.g., beads) within an array can first be assigned to an image location, with all associated analyte expression and / or methylation status, etc. data also assigned to that position (optionally, effectively de-coupling the spatial barcode from the array / matrix of analyte sequence information associated with a given site / bead, once the spatial barcode has been used to assign the analyte sequence information to an array position). High resolution images representing the extent of individual or grouped analytes across the various spatial positions of the arrays can then be generated using the underlying analyte sequence information. Images (i.e., pixel coloring and / orintensities) can be adjusted and / or normalized using any (or any number of) art-recognized technique(s) deemed appropriate by one of ordinary skill in the art.

[0196] In example embodiments, a high-resolution image of the instant disclosure is an image in which discrete features (e.g., pixels) of the image are spaced at 50 pm or less. In some embodiments, the spacing of discrete features within the image is at 40 pm or less, optionally 30 pm or less, optionally 20 pm or less, optionally 15 pm or less, optionally 10 pm or less, optionally 9 pm or less, optionally 8 pm or less, optionally 7 pm or less, optionally 6 pm or less, optionally 5 pm or less, optionally 4 pm or less, optionally 3 pm or less, optionally 2 pm or less, or optionally 1 pm or less.

[0197] Images can be obtained using detection devices known in the art. Examples include microscopes configured for light, bright field, dark field, phase contrast, fluorescence, reflection, interference, or confocal imaging. A biological specimen can be stained prior to imaging to provide contrast between different regions or cells. In some embodiments, more than one stain can be used to image different aspects of the specimen (e.g. different regions of a tissue, different cells, specific subcellular components or the like). In other embodiments, a biological specimen can be imaged without staining.

[0198] In particular embodiments, a fluorescence microscope (e.g., a confocal fluorescent microscope) can be used to detect a biological specimen that is fluorescent, for example, by virtue of a fluorescent label. Fluorescent specimens can also be imaged using a nucleic acid sequencing device having optics for fluorescent detection such as a Genome Analyzer®, MiSeq®, NextSeq® or HiSeq® platform device commercialized by Illumina, Inc. (San Diego, Calif.); or a SOLiD™ sequencing platform commercialized by Life Technologies (Carlsbad, Calif.). Other imaging optics that can be used include those that are found in the detection devices described in Bentley et al., Nature 456:53-59 (2008), PCT Publ. Nos. WO 91 / 06678, WO 04 / 018497 or WO 07 / 123744; U.S. 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.

[0199] An image of a biological specimen can be obtained at a desired resolution, for example, to distinguish tissues, cells or subcellular components. Accordingly, the resolution can be sufficient to distinguish components of a biological specimen that are separated by at least 0.5 pm, 1 pm, 5 pm, 10 pm, 50 pm, 100 pm, 500 pm, 1 mm or more. Alternatively or additionally,the resolution can be set to distinguish components of a biological specimen that are separated by at least 1 mm, 500 pm, 100 pm, 50 pm, 10 pm, 5 pm, 1 pm, 0.5 pm or less.

[0200] A method set forth herein can include a step of correlating locations in an image of a biological specimen with barcode sequences of nucleic acid probes that are attached to individual beads to which the biological specimen is, was or will be contacted. Accordingly, characteristics of the biological specimen that are identifiable in the image can be correlated with the nucleic acids that are found to be present in their proximity. Any of a variety of morphological characteristics can be used in such a correlation, including for example, cell shape, cell size, tissue shape, staining patterns, presence of particular proteins (e.g., as detected by immunohistochemical stains) or other characteristics that are routinely evaluated in pathology or research applications. Accordingly, the biological state of a tissue or its components as determined by visual observation can be correlated with molecular biological characteristics as determined by spatially resolved nucleic acid analysis.

[0201] A solid support upon which a biological specimen is imaged can include fiducial markers to facilitate determination of the orientation of the specimen or the image thereof in relation to probes that are attached to the solid support. Exemplary fiducials include, but are not limited, to beads (with or without fluorescent moieties or moi eties such as nucleic acids to which labeled probes can be bound), fluorescent molecules attached at known or determinable features, or structures that combine morphological shapes with fluorescent moieties. Exemplary fiducials are set forth in US Pat. App. Publ. No. 2002 / 0150909 Al or U.S. patent application Ser. No.14 / 530,299, each of which is incorporated herein by reference. One or more fiducials are preferably visible while obtaining an image of a biological specimen. Preferably, the solid support includes at least 2, 3, 4, 5, 10, 25, 50, 100 or more fiducial markers. The fiducials can be provided in a pattern, for example, along an outer edge of a solid support or perimeter of a location where a biological specimen resides. In one embodiment, one or more fiducials are detected using the same imaging conditions used to visualize a biological specimen. However, if desired, separate images can be obtained (e.g., one image of the biological specimen and another image of the fiducials) and the images can be aligned to each other.

[0202] In example embodiments, spatial filtering can be used to clean up data. In one example, specific beads that are lost are filtered out. Total spatial barcode nUMIs can be used to remove beads.

[0203] In example embodiments of Slide-tag implementations, spatial barcode oligonucleotides are released into tissue sections to ‘tag’ nuclei. Nuclei are then isolated from the tissue and the spatial barcodes are sequenced, alongside other modalities of molecular information (e.g., genomic DNA methylation status / content). Importantly, each nucleus receives many spatial barcode oligonucleotides from different locations on the bead arrays. Therefore, nuclei must be computationally positioned in space from the distribution of spatial barcodes each nucleus receives. In example embodiments, density-based spatial clustering of applications with noise (DBSCAN) is used to localize nuclei to their spatial positions (see, e.g., Ester, M., Kriegel, H -P., Sander, J. & Xu, X. A density-based algorithm for discovering clusters in large spatial databases with noise. In Proc. Second International Conference on Knowledge Discovery and Data Mining (eds Simoudis, E. et al.) 226-231 (AAAI Press, 1996); and Hahsler, M., Piekenbrock, M. & Doran, D. dbscan: fast density -based clustering with R. J. Stat. Softw. 91, 1— 30 (2019)). DBSCAN is applied to distinguish “signal” spatial barcodes (those likely to provide value in positioning nuclei) from background “noise” spatial barcodes (likely to confound nuclei positioning). DBSCAN outputs a cluster assignment for each spatial barcode. Cluster = 0 denotes noise barcodes, and cluster > 0 denotes signal barcodes grouped with other signal barcodes that cluster in space. Spatial positions with all spatial barcodes denoted noise are not assigned to nuclei, or assigned to nuclei with multiple signal clusters. From the remaining nuclei with one distinct spatial barcode signal cluster, a weighted centroid of spatial barcode coordinates in the signal cluster is taken, where weights are the number of unique molecular identifiers (UMIs) for sequenced barcodes. Importantly, DBSCAN requires two parameters: minPts and eps (effectively, radius). To determine the optimal parameter set for each Slide-tags run, 15 different minPts parameters are iterated through, and the parameter set with the highest proportion of nuclei that are assigned a spatial position is chosen (one DBSCAN signal cluster).

[0204] While DBSCAN positions nuclei with relatively high sensitivity and specificity, it is not the only approach for determining positions from a set of spatial barcode coordinates. Example alternatives include assigning a nucleus the position of its highest UMI spatial barcodeor taking a weighted 2-dimensional median of spatial barcode coordinates. Other methods to distinguish signal barcodes from noise barcodes include (1) K-means clustering, (2) Affinity propagation, (3) Mean Shift, (4) Spectral Clustering, (5) Agglomerative Clustering, (6) DBSCAN extensions such as HDBSCAN and OPTICS.

[0205] In example embodiments, the position of nuclei in the Z-plane may be measured by using individually, or in combination: the counts of spatial barcode UMIs per nucleus, the spread of spatial barcode coordinates per nucleus (e.g., median pairwise distance or similar), and / or fluorescence measurements fluorescently labelled spatial barcodes retained in the nuclei.Kits

[0206] The instant disclosure also provides kits containing agents of this disclosure for use in the methods of the present disclosure. Kits of the instant disclosure may include one or more containers comprising an agent (e.g., a capture material, such as liquid electrical tape) and / or composition (e.g., a slide-captured bead array) of this disclosure. In example embodiments, the kit includes beads comprising spatial barcode nucleic acids as described herein. In example embodiments, the beads are configured for a specific single cell genomics assay as described herein. In some embodiments, the kits further include instructions for use in accordance with the methods of this disclosure. In some embodiments, the instructions comprise a description of how to create a tissue cryosection, form a spatially-defined (or simply spatially definable, pending performance of a step that defines the spatial resolution of the bead array) bead array, contact a tissue cryosection with a spatially-defined bead array, and how to use the tagged nuclei for subsequent single cell assays.

[0207] Instructions supplied in the kits of the instant disclosure are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine- readable instructions (e.g., instructions carried on a storage drive or provided on the internet) are also acceptable. The label or package insert indicates that the composition is used for staging a cryosection and / or diagnosing an analyte pattern in a cryosection. Instructions may be provided for practicing any of the methods described herein.

[0208] The kits of this disclosure are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. The container may further comprise a pharmaceutically active agent.

[0209] Kits may optionally provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container.

[0210] Further embodiments are illustrated in the following Examples which are given for illustrative purposes only and are not intended to limit the scope of the disclosure.EXAMPLESExample 1 - MethodsdropMe - xSDS EM-seq

[0211] Certain aspects of the disclosure provide methods for DNA methylation profding for use with droplet-based cell barcoding ("dropMe"; FIG. 1 A-C). Nuclei were isolated from a 30pm- thick fresh frozen tissue sections in a 1.5mL nuclei isolation buffer (NIB; 20mM HEPES, lOmM NaCl, 3mM MgC12, 1% NP-40 alternative, 1% Tween-20). Samples were spun at 400g for 10 minutes at 4°C, and supernatant was removed to lOOpL. To deplete nucleosomes, samples were fixed with 0.75% PFA (150pL total) for 10 minutes at room temperature, quenched with 250mM Tris-HCl pH 7.5 (200pL total), diluted to 1.5mL with NIB, spun at 400g for 10 minutes at 4°C, concentrated to lOOpL, treated with 0.3% SDS (150pL total) for 20 minutes at 37°C, diluted 1 / 10 to 1.5mL with NIB with 1% BSA, spun at 600g for 10 minutes at 10°C, concentrated to 150pL, diluted 1 / 10 to 1.5mL with lOx nuclei buffer with BSA (IX Nuclei Buffer, lOmM DTT, lU / pL RNase inhibitor, 1% BSA), spun at 600g for 10 minutes at 10°C, concentrated to 5pL, and used as input to the lOx Multi ome kit.

[0212] lOx Multi ome tagmentation, GEM formation, post-GEM incubation, and GEM recovery were performed according to the manufacturer’s recommendations. Following GEM recovery, samples were gap-filled (50pM 5mdCTP, 50pM dATP, 50pM dGTP, 50 pM dTTP, IX ThermoPol buffer, 0.05U / pL Taq) at 72° for 10 minutes and SPRI-purified. Conversion was performed with EM-seq according to manufacturer’s instructions with the following modification: a 1.8X SPRI was performed following the APOBEC reaction. Samples were amplified and indexed using Q5U, and sequenced on a NovaSeq X using custom index 1 and read 2 primers complementary to the converted Nextera Read 2 handle. Single-cell clustering and dimensionality reduction results were obtained (FIG. 2). The current processes can also be appliedto DNA methylation profiling (DNAme) that involves linear amplification and / or random priming, as well as droplet-based cell barcoding.dropMe - LAND SEM-seq

[0213] Alternative nucleosome depletion and conversion strategies were substituted to increase per-cell library complexity. To deplete nucleosomes, samples were concentrated to 10pL, treated with 12.5mM LIS (12pL total) for 5 minutes at 4°C, diluted 1 / 10 to 120pL with NIB with 1% BSA, and 5pL of the resulting sample was used as input into the lOx Multi ome kit. Conversion was performed with SEM-seq.Slide-tags dropMe

[0214] Nuclei in 30pm-thick frozen tissue sections were tagged using lOx Multiome- compatible spatial barcode bead arrays as described by Russell et al. (2023). Nuclei were then isolated and processed as in the dropMe protocol. Following GEM recovery, samples were split into equal halves. One half was processed according to the dropMe protocol, while the other half was used to amplify spatial barcodes and RNA. Nuclei positions were reconstructed as described by Russell et al. (2023). The "Slide-tags" process (FIG. 3) was previously disclosed. DNA methylation clustering and dimensionality reduction results (FIG. 4A) were obtained and spatial reconstruction of the positions of the nuclei (FIG. 4B) was performed, using the current "dropMe" adaptation of the "Slide-tags" method.Example 2 -"dropMe" assessment of single-cell genomic DNA methylation state

[0215] Single-cell ATAC libraries were generated using a lOx Single Cell Multiome kit, in which accessible DNA fragments from a tagmented nucleus were ligated to ATAC cell-barcoded oligos (aCBOs) released from a GEM bead within a co-encapsulating droplet. The resulting pool of cell-barcoded unamplified genomic DNA (gDNA) fragments was used as input for DNA methylation profiling methods that label unmethylated cytosine residues via conversion to uracil. To prevent C-to-U conversion within cell barcodes, which would cause significant overlap between previously distinct barcode sequences, a gap-filling step involving introduction of methylated dCTPs was performed, to generate a conversion-protected cell complementary barcode sequence copy in the newly polymerized sequence. The strand containing thecomplementary barcode sequence copy can be isolated and selectively amplified following conversion (FIG. 1A-C). To generate genome-wide DNA methylation profiles, a nucleosome depletion step was further introduced prior to tagmentation.Example 3 - dropMe xSDS EM-seq

[0216] As proof of principle, approximately 3,000 nuclei isolated from a mouse brain (hippocampus) sample were profiled for methylation status. Nucleosome depletion was performed by crosslinking with SDS (xSDS) and conversion using the NEB enzymatic methyl- sequencing (EM-seq) kit. Clustering on mean %mCpG across 100-kb genomic bins clearly separated neuronal (mCpH-high) and non-neuronal (mCpH-low) cell clusters. Averaged methylation profiles surrounding genomic features such as gene bodies, CGIs, and CTCF ChlP- seq peaks recovered stereotyped patterns of mCG enrichment / depletion, further confirming data quality (FIG. 2; FIGs. 4A-4C).Example 4 - dropMe LAND SEM-seq

[0217] Despite significant improvements in ease-of-use, an initial implementation of dropME confronted two significant challenges. First, per-cell library complexity in attempting to implement dropME LAND SEM-seq was comparatively low: x CpGs per cell were profiled on average at near-sequencing-saturation, compared to the y-z attainable with the latest iterations of snmC-seq* or sciMET*. Second, several plate-based scDNAme-seq methods are capable of concurrent transcriptome, chromatin accessibility, and / or chromatin conformation measurements, which can provide important contextualization to DNA methylation profiles. A single-cell RNA sequencing (scRNA-seq) approach that employs droplet-based cell barcoding ("dropMe") was designed and was used herein.

[0218] To increase per-cell library size, (1) xSDS was replaced with lithium-assisted nucleosome depletion (LAND), which has been previously demonstrated to yield higher- complexity, and (2) EM-seq was replaced with SEM-seq, which requires fewer purification steps through use of a deaminase specific to unmethylated cytosines that is active on double- stranded DNA. To simultaneously profile DNA methylation and RNA, cDNA molecules were physically separated from gDNA prior to conversion via streptavidin pull-down of a biotin moiety introduced during template switching (FIG. 2).

[0219] This improved protocol was applied to a glioblastoma (GBM) sample and produced significantly improved results. Spatial reconstruction of RNA clusters, DNA methylation clusters (FIGs. 5A-5C), and percent methylated CpG across gene expression profiles were assessed using Uniform Manifold Approximation and Projection (UMAP). A heatmap of global DNA methylation levels and CpG sites profiled per cell was also generated. The spatial distribution of RNA clusters, spatial distribution of DNA methylation clusters, and the spatial distribution of percent methylated CpGs was successfully assessed. The spatial distribution and localization of various RNA clusters within different spatial regions was further assessed, and expression of individual biomarkers MOG, CSF1R, EGFR, and CDH5 was also spatially reconstructed using UMAP. Spatial reconstruction of RNA clusters, DNA methylation clusters, and percent methylated CpG across the gene expression profile of a tumor sample was additionally performed using UMAP, with seven distinct RNA subclusters identified within a spatial reconstruction of a gene expression profile of the tumor sample. Spatial reconstruction of individual biomarkers OPCI, CC1, AC1, MES1, NPC1, and EXN1 was further performed in the tumor sample using UMAP, with additional focus upon MES1 results. DNA methylation patterns were also observed to cluster, with distinct spatial reconstructions obtained for each DNA methylation cluster.Example 5 - Slide-tags dropMe of the developing mouse brain

[0220] The developing mammalian brain undergoes dramatic methylome remodeling. Both mCG and mCH play key roles in the specification of diverse neuronal and glial lineages, and disruption of methylation machinery is linked to a variety of neurodevelopmental disorders. To explore how DNA methylation patterns are spatially and transcriptionally organized during neurodevelopment, Slide-tags dropMe was applied to the E16.5 mouse forebrain.

[0221] To improve DNAme and RNA library complexity, Applicant replaced xSDS with DTSSP fixation and lithium diiodosalicylate-assisted nucleosome depletion (LAND), thereby maintaining nuclei integrity through nucleosome depletion without crosslinking nucleic acids. In this experiment, 15% of barcoded nucleic acids were diverted for transcriptional and spatial profiling. These optimizations enabled high quality multi-modal profiling, yielding 6,408 spatially mapped methylome-transcriptomes. Applicant profiled a mode of 3.0E+5 CG sites (3.8E+5 DNA fragments) and a median of 2.0E+3 RNA transcripts per cell, with theoreticalmaximum library sizes (accounting for sequencing saturation and nucleic acid splitting) of 9.4E+5 fragments (mode) and 1.3E+4 transcripts (median), consistent with the modifications having increased library complexity. Dimensionality reduction and clustering on either RNA or mCG yielded spatially structured clusters corresponding to expected cell types and structures of the E16.5 forebrain (FIGS 6A-6B).

[0222] Corticogenesis involves both laminar organization of neurons along the ventral-dorsal (DV) axis and regional patterning along the rostrocaudal (RC) axis of the developing pallium. To investigate how spatial patterning of CG methylation and gene expression jointly contribute to these developmental processes, Applicant subclustered on cortical excitatory neurons and calculated diffusion-based pseudotime scores across mCG and RNA modalities. RNA pseudotime was strongly correlated with the DV axis (p = 0.88), highlighting laminar specification as a dominant source of variation across transcriptomes. In contrast, mCG pseudotime correlated along both the ventral-dorsal axis (p = 0.57) and the rostrocaudal axis (p = -0.42), suggesting a greater proportion of mCG variation reflects regional rostrocaudal patterning.

[0223] To identify the features driving these correlations, Applicant calculated Spearman correlations of individual genes and variably methylated regions (VMRs) along each axis. Consistent with the pseudotime results, the majority of significantly correlated spatially variably expressed genes (SVGs) were associated with the ventral-dorsal axis (86% DV vs. 14% RC), whereas significantly correlated spatially variably methylated regions (SVMRs) were more evenly distributed (69% DV vs. 31% RC). SVG gradient patterns could be clustered into several distinct groups along the dorsoventral axis, reflecting changes in expression at distinct cell type boundaries (e.g. Bell la, upregulated in post-mitotic cortical neurons), and along the rostrocaudal axis, marking different cortical regions (e.g. Lefl and Lhx2, highly expressed in the developing hippocampus). SVMRs, by contrast, clustered primarily according to correlation sign.

[0224] Next, Applicant identified genes potentially regulated by large CG methylation domains in the cortical region by correlating each gene’s expression with Mean Squared Residue (MSR) scores of all VMRs within 50 kb of its TSS, merging significant like-signed cis-VMRs into aggregated methylation domains. This analysis identified 39 genes with >4.9 kb of negatively correlated cis-VMRs and 39 genes with >3.9 kb of positively correlated cis-VMRs (FIG. 6C; FDR < 0.05). Applicant designated these regions as methylation-defined domains of regulatorychromatin (mDORCs). Both negative mDORCs and positive mDORCs were strongly enriched for spatially correlated genes (35 / 39 negative, 23 / 39 positive) and for GO terms relating to neurodevelopment (FIG. 6D).

[0225] Negatively correlated mDORCs included key neurodevelopmental transcription factors, including those with both DV and RC gradients (e.g., Meis2, Bell la, Nfix), consistent with a prominent role for mCG in repression of these genes. To identify transcription factors contributing to these correlations, Applicant used Cistrome58 to identify factors enriched for binding in negative and positive mDORCs. This analysis identified enrichment of Chd4, a subunit of the repressive NuRD chromatin remodeling complex, within negative mDORCs, whereas Ctcf was enriched within positive mDORCs, suggesting altered chromatin conformation as a mechanism by which CG methylation in these regions might derepress nearby genes.

[0226] CH methylation refers to the process of adding a methyl group (CH3) to cytosine at the carbon 5 position within DNA's CpG dinucleotide sequence. This process is catalyzed by DNA methyltransferase and alters gene expression without changing the DNA sequence itself. CH methylation predominantly accumulates in neurons during early postnatal development, where it is believed to play an important role in stabilizing mature subtype-specific gene expression programs. However, its role in prenatal brain development is much less established. mCH is nearly undetectable in bulk embryonic forebrain, with only slight accumulation in midbrain and hindbrain at later stages. Nonetheless, domains of slightly elevated CH methylation spanning hundreds of kilobases have been identified in early embryonic tissues. These frequently overlap key lineage transcription factors, and have been proposed to help silence pathways active during early fetal development. To explore the possibility of mCH regulatory function in the data, Applicant subclustered on neuronal cell types and projected single-cell genome-wide mCH levels in space. Global %mCH was minimal in most telencephalic neurons, as expected, but was appreciably elevated in diencephalic regions (P < le-4), within which it displayed highly significant spatial autocorrelation (P < le-4) partly but not entirely explained by cluster identity. Differentially CH-methylated genes were enriched for functions in neurodevelopmental programs and transcriptional regulation, suggestive of a functional regulatory role. The relationship between CH methylation and gene expression across diencephalic, pallial, and subpallial regions varied by locus (FIG. 6E); certain genes-such as Zeb2, a key developmental transcription factorwhose disruption in humans leads to the severe neurodevel opmental disorder Mowat-Wilson syndrome 60-displayed focal mCH elevation that anti correlated with gene expression, while others-like Temn2, a transmembrane adhesion / signaling protein that regulates neuronal connectivity and was highly spatially variable in the adult mouse dataset-fell within a broadly elevated mCH domain in the diencephalon and, yet, was robustly expressed in diencephalic regions. These results suggest that diencephalic neurons begin acquiring functionally relevant CH methylation earlier in development than previously recognized, possibly with multiple distinct modes of regulation, and motivate further investigation into the timing, localization, and function of CH methylation during early brain patterning.

[0227] In this work, Applicant has disclosed dropMe and Slide-tags dropMe, complementary technologies that enable scalable, spatially resolved single-cell methylomics and multi-omics. Together, these methods expand the single-cell epigenomics toolkit by enabling both broad accessibility and, for the first time, spatially resolved single-cell DNA methylation measurements. The present disclosure shows that dropMe rapidly generates high-quality single-cell methylomes, dropMe Slide-tags enables precise positioning of these profiles within a tissue, and integration with RNA profiling further reveals DNA methylation heterogeneity across spatial and transcriptional contexts.

[0228] Using these approaches, Applicant generated thousands of spatially localized methylomes and methylome-transcriptomes across adult and embryonic mouse brains and human glioblastoma. These data uncovered spatially organized methylation patterns inaccessible to dissociated samples. In the adult dentate gyrus, Applicant identified an mCH gradient consistent with neuronal birthdate layering, providing high-resolution validation of a longstanding developmental hypothesis. In glioblastoma, Applicant observed global hypomethylation, increased methylation disorder, and clinically relevant MGMT promoter hypomethylation, revealing heterogeneity among transcriptionally defined, spatially structured subclusters. In the embryonic cortex, Applicant found abundant developmental mCG gradients along ventral-dorsal and rostral-caudal axes, while RNA gradients were dominated by laminar patterning, suggestive of distinct modes of spatiotemporal regulation. Applicant also detected elevated, spatially nonrandom mCH in diencephalic neurons that frequently overlapped neurodevelopmental regulators, suggesting an earlier role for mCH regulation in this structure’s development than previouslyrecognized. Collectively, these results illustrate the power of the instant approach to resolve informative DNA methylation patterns across diverse length scales and tissue contexts.

[0229] The methods described herein are compatible with emerging conversion reagents (such as the MsddA enzyme, which is a modification-sensitive double-stranded DNA deaminase, and other deaminases as described in Vaisvila, Romualdas, et al. "Discovery of cytosine deaminases enables base-resolution methylome mapping using a single enzyme." Molecular Cell 84.5 (2024): 854-866, incorporated herein in its entirety) employed in SEM-seq-potentially boosting per-cell library size by reducing handling steps. Bisulfite conversion may also be feasible if paired with strategies to mitigate DNA degradation or recover cleaved fragments. In some embodiments, during present methods, RNA can be physically separated from gDNA prior to conversion. Lastly, present methods can be used to capture additional modalities, such as hydroxymethylcytosine (hmC) profiles, chromatin accessibility, protein-DNA interactions, chromosome conformation, and copy number variation.***

[0230] Various modifications and variations of the described methods, pharmaceutical compositions, and kits of the disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it will be understood that it is capable of further modifications and that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure come within known customary practice within the art to which the invention pertains and may be applied to the essential features herein before set forth.

[0231] Features described above as well as those claimed below may be combined in various ways without departing from the scope thereof. The following examples illustrate some possible, non-limiting combinations:(Al) A method for assessing DNA methylation in single nuclei, the method comprising: fragmenting DNA to generate a population of DNA fragments, wherein for a plurality of the DNA fragments of the population, an adapter sequence is ligated to the DNA fragment, thereby generating a population of adapted DNA fragments, each comprising a top strand comprising a 5'-phosphate group at the 5'-end and a bottom strand; co-encapsulating solid supports bound to ligation-based cell barcode oligonucleotides (L-CBOs) with the adapted DNA fragments in individual discrete volumes; ligating the L-CBOs to the 5’-ends- of the top strands of the adapted DNA fragments in the individual discrete volumes, thereby generating a population of barcoded DNA fragments, each with adapter and L-CBO sequences at the 5 ’-end of the top strand; pooling the population of barcoded DNA fragments; and generating single-nucleus DNA methylation profdes from the population of barcoded DNA fragments, thereby assessing DNA methylation in single nuclei.(A2) For the method denoted in (Al), the DNA is genomic DNA (gDNA).(A3) For the method denoted in (Al) through (A2), the DNA is isolated from single cells or fragments thereof, nuclei, mitochondria, or chromosomes.(A4) For the method denoted in (Al) through (A3), further comprising isolating nuclei containing the DNA and treating the nuclei with a nucleosome depletion agent (e.g. SDS, LIS) prior to the fragmenting step.(A5) For the method denoted in (Al) through (A4), the fragmenting step comprises tagmentation, chemical fragmentation, or restriction enzyme digestion.(A6) For the method denoted in (Al) through (A5), the step of generating single-nucleus DNA methylation profdes from the population of barcoded DNA fragments comprises: extending the bottom strand using a DNA polymerase enzyme and dNTPs with 5-methyl-dCTP substituted for dCTP to generate an extended bottom strand, wherein 5-methylcytosines are incorporated into the extended bottom strand at all positions complementary to guanine residues of the L-CBO sequence of the top strand; converting cytosine residues into uracil residues in the extended bottom strand, wherein unmethylated cytosine residues are converted to uracil residues, and 5'-methylcytosine residues are not converted into uracil residues, thereby generating a converted extended bottom strand; amplifying the converted extended bottom strand; sequencing the amplified converted extended bottom strand; and detecting methylation sites associated with each L-CBO sequence.(A7) For the method denoted in (A6), the converting step comprises bisulfite-seq (BS-seq), Enzymatic methyl-sequencing (EM-seq), or single-enzyme 5-methylcytosine sequencing (SEM-seq).(A8) For the method denoted in (A6), the converting step uses a cytidine deaminase (e.g. APOBEC1 orMsddA).(A9) For the method denoted in (A6), the converting step uses a chemical reagent (e.g. sodium bisulfite, ammonium bisulfite) to deaminate unmethylated cytosines.(A 10) For the method denoted in (Al) through (A9), the ligating step is performed using the lOx Single Cell Multiome kit.(Al 1) For the method denoted in (Al) through (A10), fragmenting comprises tagmentation using a transposase enzyme (e.g., hyperactive Tn5).(A12) For the method denoted in (Al) through (Al 1), the solid support is a bead.(A13) For the method denoted in (Al) through (A12), the individual discrete volume is a droplet. (A14) For the method denoted in (A4) through (A13), the nucleosome depletion agent is a detergent (e.g. sodium dodecyl sulfate (SDS)) or a chaotropic agent (e.g. lithium diiodosalicylate (LIS)).(Al 5) For the method denoted in (A4) through (A14), further comprising treating the nuclei with a fixative agent (e.g. formaldehyde, dithio-bis(succinimidyl propionate)) prior to treating the nuclei with a nucleosome depletion agent.(Al 6) For the method denoted in (A6) through (Al 5), the fragmenting step is performed using a mixture of: (1) a transposase loaded with a first adapter sequence comprising a 5 ’-phosphorylated single-stranded 5 ’-overhang, and (2) a transposase loaded with a second adapter sequence, to generate DNA fragments with the first adapter sequence attached to the 5 ’-end of the top strand and the second adapter sequence attached to the 5 ’-end of the bottom strand; the ligating step comprises ligating the L-CBOs to the 5’-overhang of the first adapter; and the amplifying step comprises amplifying the converted extended bottom strand using a primer specific to the L-CBO 5 ’-end and a primer specific to a derivative sequence comprising the second adapter sequence, wherein the cytosines are replaced with uracils.(Al 7) For the method denoted in (A6) through (Al 5), the fragmenting step is performed using a mixture of: (1) a transposase loaded with a first adapter sequence comprising a 5 ’-phosphorylatedsingle-stranded 5 ’-overhang, and (2) a transposase loaded with a second adapter sequence to generate a population of adapted DNA fragments with the first adapter sequence attached to the 5 ’-end of the top strand and the second adapter sequence attached to the 5 ’-end of the bottom strand; the ligating step comprises ligating the L-CBOs to the 5 ’-overhang of the first adapter; and the amplifying step comprises using a primer specific to the L-CBO 5 ’-end and a primer specific to a derivative sequence comprising the second adapter sequence, wherein the cytosines are replaced with uracils.(Al 8) For the method denoted in (Al 7), the ligating step is performed via a splint oligonucleotide with a region complementary to the 5 ’-end of the first adapter sequence and a region complementary to the 3 ’-end of the L-CBO.(A19) For the method denoted in (Al) through (A5), the fragmenting step is performed using a transposase loaded with a first adapter sequence comprising a 5’ -phosphorylated single-stranded 5 ’-overhang to generate a population of adapted DNA fragments with the first adapter sequence attached to the 5 ’-end of the top and bottom strands, wherein at an end of the adapted DNA fragments, the strand with the 5 ’-end is the top strand and the strand with the 3 ’-end is the bottom strand; the ligating step comprises ligating the L-CBO to the 5’-overhang of the first adapter at both ends of each adapted DNA fragment; the step of generating single-nucleus DNA methylation profiles from the population of barcoded DNA fragments comprises: extending the bottom strand using a DNA polymerase enzyme and dNTPs with 5-methyl-dCTP substituted for dCTP to generate an extended bottom strand wherein 5-methylcytosines are incorporated into the extended bottom strand at all positions complementary to guanine residues of the L-CBO sequence of the top strand; converting cytosine residues into uracil residues in the extended bottom strand, wherein unmethylated cytosine residues are converted to uracil residues, and 5'-methylcytosine residues are not converted into uracil residues, thereby generating a converted extended bottom strand; linearly amplifying the converted extended bottom strand using a primer specific to the L-CBO sequence at the 5’ end to generate reverse-complemented copies of the converted extended bottom strand; adding a second adapter sequence to the reverse-complemented copies of the converted extended bottom strand via (a) random priming or (b) adaptase chemistry; amplifying the reverse-complemented copies of the converted extended bottom strand comprising the second adapter sequence using a primer specific to the L-CBO 5 ’-end and a primer specific to the second adapter;sequencing the amplified reverse-complemented copies; and detecting methylation sites associated with each L-CBO sequence.(A20) For the method denoted in (A19), the ligating step is performed via a splint oligonucleotide with a region complementary to the 5 ’-end of the first adapter and a region complementary to the 3 ’-end of the L-CBO.(Bl) A method for assessing DNA methylation and gene expression in single nuclei, the method comprising: isolating nuclei containing gDNA and mRNA; treating nuclei with a nucleosome depletion agent (e.g. SDS, LIS); fragmenting nuclei to generate a population of gDNA fragments, wherein for a plurality of the gDNA fragments of the population, an adapter sequence is ligated to the gDNA fragment, thereby generating a population of adapted gDNA fragments, each comprising a top strand comprising a 5'-phosphate group at the 5'-end and a bottom strand; coencapsulating solid supports bound to ligation-based cell barcode oligonucleotides (L-CBOs) and reverse transcription-based cell barcode oligonucleotides (RT-CBOs) with the adapted gDNA fragments in individual discrete volumes; cell-barcoding gDNA fragments and mRNA by: ligating L-CBOs to the 5 ’-ends- of the top strands of the adapted gDNA fragments in the individual discrete volumes, thereby generating a population of barcoded gDNA fragments, each with adapter and L-CBO sequences at the 5’-end of the top strand; reverse-transcribing the mRNA using RT-CBOs as primers, thereby generating a population of cell-barcoded first-strand cDNA molecules; pooling the population of barcoded gDNA fragments and the population of cell-barcoded first-strand cDNA molecules; generating single-nucleus DNA methylation profiles by: extending the bottom strand of the gDNA fragments using a DNA polymerase enzyme and dNTPs with 5-methyl-dCTP substituted for dCTP to generate extended bottom strands wherein 5-methylcytosines are incorporated into the extended bottom strands at all positions complementary to guanine residues of the L-CBO sequence of the top strand; converting cytosine residues into uracil residues in the extended bottom strands, wherein unmethylated cytosine residues are converted to uracil residues, and 5-methylcytosine residues are not converted into uracil residues, thereby generating converted extended bottom strands; amplifying the converted extended bottom strands; sequencing the amplified converted extended bottom strands; detecting methylation sites associated with each discrete L-CBO sequence; generating single-nucleus gene expression profiles by: amplifying thecell-barcoded first strand cDNA molecules; sequencing the amplified cell-barcoded first strand cDNA molecules; thereby assessing DNA methylation and gene expression of a single cell. (B2) For the method denoted in (Bl), further comprising, prior the step of amplifying the cell-barcoded first strand cDNA molecules, performing template-switching reactions with Template-Switching Oligonucleotides (TSOs) within the individual discrete volumes, thereby generating a population of cell-barcoded first-strand cDNA molecules comprising 3 ’-terminal TSO sequences. (B3) For the method denoted in (Bl) or (B2), wherein the TSO comprises a 5’-biotin, and the cell-barcoded first-strand cDNA molecules comprise 3 ’-terminal TSO sequences and biotin, and the method further comprising, after the pooling step, separating the population of barcoded gDNA fragments and cell-barcoded first-strand cDNA molecules comprising 3’-terminal TSO sequences and biotin via binding of the biotin to streptavidin beads.(Cl) A method for assessing DNA methylation, gene expression, and spatial location in single nuclei, the method comprising: contacting a spatial array with a tissue section, wherein the spatial array includes spatial barcode oligonucleotides (SBOs) comprising spatial barcode sequences coupled to individual solid supports of the array via cleavable linkers, wherein the spatial barcode sequences are the same for an individual solid support of the array but differ between individual solid supports of the array, and wherein the spatial location and spatial barcode sequence of each individual solid support is known; cleaving the linkers, thereby delivering the SBOs to cells of the tissue section and generating tagged nuclei containing gDNA molecules and RNA molecules; isolating the tagged nuclei containing gDNA molecules and RNA molecules from the tissue section; treating nuclei with a nucleosome depletion agent (e.g. SDS, LIS); fragmenting nuclei to generate a population of gDNA fragments, wherein for a plurality of the gDNA fragments of the population, an adapter sequence is ligated to the gDNA fragment, thereby generating a population of adapted gDNA fragments, each comprising a top strand comprising a 5'-phosphate group at the 5'-end and a bottom strand; co-encapsulating solid supports bound to ligation-based cell barcode oligonucleotides (L-CBOs) and reverse transcription-based cell barcode oligonucleotides (RT-CBOs) with the adapted gDNA fragments in individual discrete volumes; cell-barcoding gDNA fragments, RNA molecules, and SBOs by: ligating L-CBOs to the 5’-ends- of the top strands of the adapted gDNA fragments in the individual discrete volumes, thereby generating a population of barcoded gDNA fragments, each with adapter and L-CBO sequences at the 5’-end of the topstrand; reverse-transcribing the RNA molecules using released RT-CBOs as primers, thereby generating a population of cell-barcoded first-strand cDNA molecules; extending SBOs using RT-CBOs as primers, thereby generating a population of cell-barcoded SBOs; pooling the population of cell-barcoded gDNA fragments, the population of cell-barcoded first-strand cDNA molecules, and the population of cell-barcoded SBOs; generating single-nucleus DNA methylation profiles by: extending the bottom strand of the gDNA fragments using a DNA polymerase enzyme and dNTPs with 5-methyl-dCTP substituted for dCTP to generate extended bottom strands wherein 5-methylcytosines are incorporated into the extended bottom strands at all positions complementary to guanine residues of the L-CBO sequence of the top strand; converting cytosine residues into uracil residues in the extended bottom strands, wherein unmethylated cytosine residues are converted to uracil residues, and 5-methylcytosine residues are not converted into uracil residues, thereby generating converted extended bottom strands; amplifying the converted extended bottom strands; sequencing the amplified converted extended bottom strands; detecting methylation sites associated with each discrete single-cell barcode sequence; generating single-nucleus gene expression profiles by: amplifying the cell-barcoded first strand cDNA molecules; sequencing the amplified cell-barcoded first strand cDNA molecules; generating single-nucleus spatial positions by: amplifying the cell-barcoded SBOs; sequencing the cell-barcoded SBO; counting the number of SBOs from each solid support in the spatial array associated with each nucleus to generate a distribution of SBOs; and determining the spatial locations of each nucleus within its original tissue section as the spatial centroid of the distribution, thereby assessing DNA methylation, gene expression, and spatial location of a single cell.(C2) For the method denoted in (Cl), wherein the spatial array is sequence-verified by in situ sequencing of nucleic acid sequences comprising spatial barcode sequences, and the method further comprises generating, prior to the step of contacting the sequence-verified spatial array with a tissue section, an index of the spatial barcode sequences.

Claims

CLAIMSWhat is claimed is:

1. A method for assessing DNA methylation in single nuclei, the method comprising:fragmenting DNA to generate a population of DNA fragments, wherein for a plurality of the DNA fragments of the population, an adapter sequence is ligated to the DNA fragment, thereby generating a population of adapted DNA fragments, each comprising a top strand comprising a 5'-phosphate group at the 5'-end and a bottom strand;co-encapsulating solid supports bound to ligation-based cell barcode oligonucleotides (L-CBOs) with the adapted DNA fragments in individual discrete volumes;ligating the L-CBOs to the 5 ’-ends of the top strands of the adapted DNA fragments in the individual discrete volumes, thereby generating a population of barcoded DNA fragments, each including adapter sequences and L-CBO sequences in the 5 ’-end region of the top strand;pooling the population of barcoded DNA fragments; andgenerating single-nucleus DNA methylation profiles from the population of barcoded DNA fragments,thereby assessing DNA methylation in single nuclei.

2. The method of claim 1, wherein the step of generating single-nucleus DNA methylation profiles from the population of barcoded DNA fragments comprises:extending the bottom strands using a DNA polymerase enzyme and dNTPs with 5-methyl-dCTP substituted for dCTP to generate extended bottom strands, wherein 5-methylcytosines are incorporated into the extended bottom strands at all positions complementary to guanine residues of the L-CBO sequences of the top strands;converting cytosine residues into uracil residues in the extended bottom strands, wherein unmethylated cytosine residues are converted to uracil residues, and 5'-methylcytosine residues are not converted into uracil residues, thereby generating converted extended bottom strands; amplifying the converted extended bottom strands;sequencing the amplified converted extended bottom strands; anddetecting methylation sites in the converted extended bottom strand sequences and associating said methylation sites with individual L-CBO sequences of the converted extended bottom strands.

3. The method of claim 2, wherein the converting step comprises bisulfite-seq (BS-seq), Enzymatic methyl-sequencing (EM-seq), or single-enzyme 5 -methyl cytosine sequencing (SEM-seq).

4. The method of claim 2, wherein the converting step uses a cytidine deaminase.

5. The method of claim 4, wherein the cytidine deaminase is APOB EC 1 or MsddA.

6. The method of claim 2, wherein the converting step uses a chemical reagent to deaminate unmethylated cytosines.

7. The method of claim 6, wherein the chemical reagent is sodium bisulfite or ammonium bisulfite.

8. The method of claim 1, wherein the DNA is genomic DNA (gDNA).

9. The method of claim 1, wherein the DNA is isolated from single cells or fragments thereof, nuclei, mitochondria, or chromosomes.

10. The method of claim 1, further comprising isolating nuclei containing the DNA and treating the nuclei with a nucleosome depletion agent prior to the fragmenting step.

11. The method of claim 10, wherein the nucleosome depletion agent is sodium dodecyl sulfate (SDS) or lithium diiodosalicylate (LIS).

12. The method of claim 1, wherein the fragmenting step comprises tagmentation, chemical fragmentation, or restriction enzyme digestion.

13. The method of claim 1, wherein the ligating step is performed using a lOx Single Cell Multiome kit.

14. The method of claim 1, wherein fragmenting comprises tagmentation using a transposase enzyme.

15. The method of claim 14, wherein the transposase enzyme is hyperactive Tn5.

16. The method of claim 1, wherein the solid support is a bead.

17. The method of claim 1, wherein the individual discrete volume is a droplet.

18. The method of claim 17, wherein the nucleosome depletion agent is a detergent or a chaotropic agent.

19. The method of claim 18, wherein the detergent is sodium dodecylsulfate (SDS).

20. The method of claim 18, wherein the chaotropic agent is lithium diiodosalicylate (LIS).

21. The method of claim 10, further comprising treating the nuclei with a fixative agent prior to treating the nuclei with the nucleosome depletion agent.

22. The method of claim 21, wherein the fixative agent is formaldehyde or dithio-bis(succinimidyl propionate).

23. The method of claim 2, wherein:the fragmenting step is performed using a mixture of (1) a transposase loaded with a first adapter sequence comprising a 5 ’-phosphorylated single-stranded 5’-overhang, and (2) a transposase loaded with a second adapter sequence to generate a population of adapted DNA fragments, each having the first adapter sequence attached to the 5 ’-end of the top strand and the second adapter sequence attached to the 5 ’-end of the bottom strand;the ligating step comprises ligating the L-CBOs to the 5’-overhang of the first adapter sequences; andthe amplifying step comprises using a primer specific to the L-CBO 5 ’-end and a primer specific to a derivative sequence comprising the second adapter sequence, wherein the cytosines are replaced with uracils.

24. The method of claim 23, wherein the ligating step is performed via a splint oligonucleotide having (i) a region complementary to the 5 ’-end of the first adapter sequence and (ii) a region complementary to the 3 ’-end of the L-CBO sequence.

25. The method of claim 1, wherein:the fragmenting step is performed using a transposase loaded with a first adapter sequence comprising a 5 ’-phosphorylated single-stranded 5’-overhang, thereby generating a population of adapted DNA fragments having the first adapter sequence attached to the original 5 ’-end of the top strand, wherein for any adapted DNA fragment, the strand with the 5 ’-overhang is the top strand and the strand complementary to the top strand is the bottom strand;the ligating step comprises ligating the L-CBO to the 5’-overhang of the first adapter sequence;the step of generating single-nucleus DNA methylation profiles from the population of barcoded DNA fragments comprises:extending the bottom strand using a DNA polymerase enzyme and dNTPs with 5-methyl-dCTP substituted for dCTP, thereby generating an extended bottom strand wherein 5-methylcytosines are incorporated into the extended bottom strand at all positions complementary to guanine residues of the L-CBO sequence of the top strand;converting cytosine residues into uracil residues in the extended bottom strand, wherein unmethylated cytosine residues are converted to uracil residues, and 5'-methylcytosine residues are not converted into uracil residues, thereby generating a converted extended bottom strand; linearly amplifying the converted extended bottom strand using a primer specific to the L-CBO sequence at the 5’ end, thereby generating a double- stranded product comprising the converted extended bottom strand and its complement;adding a second adapter sequence to the double-stranded product comprising the converted extended bottom strand and its complement via (a) random priming or (b) adaptase chemistry, thereby generating a second adapter-comprising product;amplifying the second adapter-comprising product using a primer specific to the L-CBO sequence and a primer specific to the second adapter;sequencing the amplified second adapter-comprising product; anddetecting methylation sites associated with each L-CBO sequence.

26. The method of claim 25, wherein the ligating step is performed via a splint oligonucleotide having a first region complementary to the 5 ’-end of the first adapter and a second region complementary to the 3 ’-end of the L-CBO.

27. A method for assessing DNA methylation and gene expression in single nuclei, the method comprising:treating nuclei comprising gDNA and mRNA with a nucleosome depletion agent; fragmenting the gDNA of the nuclei, thereby generating a population of gDNA fragments, wherein for a plurality of the gDNA fragments of the population, an adapter sequence is ligated to the gDNA fragment, thereby generating a population of adapted gDNA fragments, each comprising a top strand comprising a 5'-phosphate group at the 5'-end and a bottom strand;co-encapsulating in individual discrete volumes solid supports bound to ligation-based cell barcode oligonucleotides (L-CBOs) and reverse transcription-based cell barcode oligonucleotides (RT-CBOs) with individual nuclei comprising the adapted gDNA fragments and mRNA;cell-barcoding gDNA fragments and mRNA by a process comprising:ligating L-CBOs to the 5 ’-ends- of the top strands of the adapted gDNA fragments in the individual discrete volumes, thereby generating a population of barcoded gDNA fragments, each with adapter and L-CBO sequences at the 5 ’-end of the top strand; andreverse-transcribing the mRNA using RT-CBOs as primers, thereby generating a population of cell-barcoded first-strand cDNA molecules; pooling the population of barcoded gDNA fragments and the population of cell-barcoded first-strand cDNA molecules;generating single-nucleus DNA methylation profiles by a process comprising:extending the bottom strand of the gDNA fragments using a DNA polymerase enzyme and dNTPs with 5-methyl-dCTP substituted for dCTP to generate extended bottom strands wherein 5-methylcytosines are incorporated into the extended bottom strands at all positions complementary to guanine residues of the L-CBO sequence of the top strand;converting cytosine residues into uracil residues in the extended bottom strands, wherein unmethylated cytosine residues are converted to uracil residues, and 5-methylcytosine residues are not converted into uracil residues, thereby generating converted extended bottom strands;amplifying the converted extended bottom strands;sequencing the amplified converted extended bottom strands; and detecting methylation sites in the converted extended bottom strand sequences and associating said methylation sites with individual L-CBO sequences of the converted extended bottom strands; andgenerating single-nucleus gene expression profiles by a process comprising:amplifying the cell-barcoded first strand cDNA molecules; sequencing the amplified cell-barcoded first strand cDNA molecules and associating sequenced cDNAs with individual RT-CBO sequences; thereby assessing DNA methylation and gene expression in single nuclei.

28. The method of claim 27, further comprising, prior the step of amplifying the cell-barcoded first strand cDNA molecules, performing template-switching reactions using Template-Switching Oligonucleotides (TSOs) within the individual discrete volumes, thereby generating a population of cell-barcoded first-strand cDNA molecules comprising 3 ’-terminal TSO sequences.

29. The method of claim 28, wherein:the TSO comprises a 5’-biotin;the cell-barcoded first-strand cDNA molecules comprise 3 ’-terminal TSO sequences and biotin; and / orthe method further comprises, after the pooling step, separating the cell-barcoded first-strand cDNA molecules comprising 3 ’-terminal TSO sequences and biotin from the population of barcoded gDNA fragments via binding of the biotin to streptavidin beads.

30. A method for assessing DNA methylation, gene expression, and spatial location in single nuclei, the method comprising:contacting a spatial array with a tissue section, wherein the spatial array includes spatial barcode oligonucleotides (SBOs) comprising spatial barcode sequences coupled via cleavable linkers to individual solid supports of the array, wherein the spatial barcode sequences are the same for an individual solid support of the array but differ between individual solid supports of the array, and wherein the spatial location and spatial barcode sequence of each individual solid support is known;cleaving the linkers, thereby delivering the SBOs to cells of the tissue section and generating tagged nuclei comprising gDNA molecules and RNA molecules;isolating the tagged nuclei comprising gDNA molecules, RNA molecules, and SBOs from the tissue section;treating the tagged nuclei comprising gDNA molecules, RNA molecules, and SBOs with a nucleosome depletion agent;fragmenting the gDNA molecules of the nuclei, thereby generating a population of gDNA fragments, wherein for a plurality of the gDNA fragments of the population, an adapter sequenceis ligated to the gDNA fragment, thereby generating a population of adapted gDNA fragments, each comprising a top strand comprising a 5'-phosphate group at the 5'-end and a bottom strand;co-encapsulating in individual discrete volumes solid supports bound to ligation-based cell barcode oligonucleotides (L-CBOs) and reverse transcription-based cell barcode oligonucleotides (RT-CBOs) with individual nuclei comprising the adapted gDNA fragments, RNA molecules, and SBOs;cell-barcoding gDNA fragments, RNA molecules, and SBOs by a process comprising:ligating L-CBOs to the 5 ’-ends- of the top strands of the adapted gDNA fragments in the individual discrete volumes, thereby generating a population of barcoded gDNA fragments, each with adapter and L-CBO sequences at the 5 ’-end of the top strand; andreverse-transcribing the RNA molecules using RT-CBOs as primers, thereby generating a population of cell-barcoded first-strand cDNA molecules; and extending SBOs using RT-CBOs as primers, thereby generating a population of cell-barcoded SBOs;pooling the population of barcoded gDNA fragments, the population of cell-barcoded first-strand cDNA molecules, and the population of cell-barcoded SBOs;generating single-nucleus DNA methylation profiles by a process comprising:extending the bottom strand of the gDNA fragments using a DNA polymerase enzyme and dNTPs with 5-methyl-dCTP substituted for dCTP to generate extended bottom strands wherein 5-methylcytosines are incorporated into the extended bottom strands at all positions complementary to guanine residues of the L-CBO sequence of the top strand;converting cytosine residues into uracil residues in the extended bottom strands, wherein unmethylated cytosine residues are converted to uracil residues, and 5-methylcytosine residues are not converted into uracil residues, thereby generating converted extended bottom strands;amplifying the converted extended bottom strands;sequencing the amplified converted extended bottom strands; anddetecting methylation sites in the converted extended bottom strand sequences and associating said methylation sites with individual L-CBO sequences of the converted extended bottom strands;generating single-nucleus gene expression profiles by a process comprising:amplifying the cell-barcoded first strand cDNA molecules; sequencing the amplified cell-barcoded first strand cDNA molecules and associating sequenced cDNAs with individual RT-CBO sequences; and generating single-nucleus spatial positions by a process comprising:amplifying and sequencing the cell-barcoded SBOs;counting the number of SBOs from each solid support in the spatial array associated with each nucleus, thereby generating a distribution of SBOs; and determining the spatial locations of each nucleus within the original tissue section as the spatial centroid of the distribution for the single-nucleus DNA methylation profile and the single-nucleus gene expression profile for the nucleus, thereby assessing DNA methylation, gene expression, and spatial location of single nuclei.

31. The method of claim 30, wherein, prior to the step of contacting the spatial array with the tissue section, the spatial array is sequence-verified by a process comprising in situ sequencing of nucleic acid sequences comprising spatial barcode sequences, thereby generating an index of the spatial barcode sequences.

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