Single-core high-resolution multi-mode spatial genomics

Abstract

Embodiments disclosed herein provide spatially labeled cell nucleuses compatible with any genomic or multiple groups of single cell / nucleus assays to allow for the generation of spatially resolved single cell sequencing libraries with single cell resolution.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 434,345, filed December 21, 2022. The entire contents of the above application are fully incorporated herein by reference.

[0003] Statement Regarding Federally Funded Research

[0004] This invention was made with government support under Grant Nos. HG010647, CA246632, and NS132135 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0005] Reference to an electronic sequence listing

[0006] The contents of the electronic sequence listing (BROD-5740WP_ST26.xml; size: 26,852, and created on December 21, 2023) are incorporated herein by reference in their entirety. Technical Field

[0007] The subject matter disclosed herein generally relates to spatially resolved single-nucleus sequencing libraries obtained using compositions and methods for labeling cell nuclei with spatial barcodes. Background Art

[0008] Recent technological innovations have enabled high-throughput quantification of gene expression and epigenetic regulation within individual cells, changing our understanding of how complex tissues are constructed. However, these measurements lack the ability to routinely and easily spatially locate dissected cells. Methods for spatially monitoring genomic data in single cells in tissue samples include traditional histological methods, i.e., fixing, staining, and evaluating tissue sections, such as whether individual transcripts are present in the visible area of a tissue section fixed on a microscope slide, and some newer in situ technologies for transcriptome monitoring, which have so far been laborious to apply, have low multiplexing levels, are technically difficult, and / or only provide low-resolution spatial capture on arrays (i.e., only provide a resolution of approximately 100-200 μm).

[0009] Spatially resolved capture of macromolecules from single nuclei is required. Current spatially resolved (multi)omics tools capture an uncertain fraction of each cell because they use thin tissue sections as input, during which incomplete cells / nuclei are captured (e.g., in situ sequencing, in situ capture). Furthermore, some technologies profile macromolecules in voxels containing multiple cells, requiring deconvolution (e.g., 10X Visium, Slide-seq, HOST, LCM), which limits some analyses. More recent technologies (e.g., sci-Space, XYZeq) allow capture of transcriptomic data from single nuclei while preserving spatial information, but are limited by low capture rates and low spatial resolution. Furthermore, they are limited to profiles based on a single composite index.

[0010] Therefore, there is a need for improved methods that provide spatial genomic profiling at a resolution approaching single-cell resolution. More generally, there is also a need for improved methods that provide spatial macromolecular abundance data (e.g., RNA expression, DNA and / or protein abundance) at a resolution approaching single-cell resolution.

[0011] Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention. Summary of the Invention

[0012] In one aspect, the present invention provides a method for generating spatially labeled cell nuclei for single-cell genomics, comprising: placing a tissue sample on a sequence-verified spatial array, wherein the spatial array comprises a nucleic acid sequence, the nucleic acid sequence comprising a spatial barcode coupled to the array by a cleavable linker, wherein the spatial barcode is the same for a single position on the array, but different for any other position on the spatial array; cleaving the linker and delivering the spatial barcode nucleic acid to the cell nuclei in the tissue sample; and isolating the labeled cell nuclei from the tissue sample. In certain embodiments, the method further comprises preparing a single-cell genomics sequencing library using the separated labeled cell nuclei, wherein each nucleic acid sequence comprises a cell barcode sequence that identifies the source cell and an optional unique molecular identifier (UMI) to capture the spatial barcode sequence from each spatially labeled cell nucleus to generate a nucleic acid sequence comprising a spatial barcode and a cell barcode, so that the genomic data of each single cell can be identified by the cell barcode, and the spatial position of the same single cell in the tissue can be identified by the same cell barcode.

[0013] In certain embodiments, the spatial barcode is delivered to the cell nucleus by diffusion. In certain embodiments, prior to step (a), the spatial array is sequence verified by in situ sequencing of the nucleic acid sequence comprising the spatial barcode, thereby generating an index of the spatial barcode on the array. In certain embodiments, in situ sequencing is performed by ligation sequencing or synthesis sequencing.

[0014] In certain embodiments, the spatial array comprises a solid support that is fixed to each position on the array and is coupled to the nucleic acid sequence comprising a spatial barcode via the cleavable linker, wherein the spatial barcode is identical for a single solid support but different for any other solid support in the spatial array. In certain embodiments, the solid support is covalently fixed to the spatial array. In certain embodiments, the solid support is fixed to the spatial array via a vinyl polymer. In certain embodiments, the solid support is a bead. In certain embodiments, the bead is a polystyrene bead. In certain embodiments, the bead has a diameter of 50 μm or less. In certain embodiments, the bead is 1, 3, 10, 15, or 20 μm.

[0015] In certain embodiments, the linker is a photocleavable, chemically cleavable, or enzymatically cleavable linker. In certain embodiments, the spatial barcode nucleic acid comprises a poly-A sequence for capture by a cellular barcode nucleic acid comprising a poly-T sequence. In certain embodiments, the tissue sample is treated to permeabilize the cell nucleus. In certain embodiments, the tissue sample is permeabilized in a buffer that increases the diffusion of the spatial barcode nucleic acid. In certain embodiments, the lengths of the spatial barcode nucleic acids are different. In certain embodiments, the spatial barcode nucleic acid further comprises one or more modifications that enhance diffusion into the cell nucleus. In certain embodiments, the spatial barcode nucleic acid is modified by adding one or more lipid or cholesterol groups. In certain embodiments, the spatial barcode nucleic acid further comprises one or more fluorescent labels that can be used to identify labeled cell nuclei.

[0016] In certain embodiments, the tissue sample is a fresh frozen tissue section. In certain embodiments, the tissue sample is a fresh unfixed tissue section. In certain embodiments, the tissue sample is a fixed tissue section.

[0017] In certain embodiments, the location of each cell in the tissue sample is computationally determined based on sequencing of the library. In certain embodiments, the cellular barcode nucleic acid comprises a UMI sequence, and the location of each cell in the tissue is determined based on the number of UMIs sequenced for each spatial barcode having the same cellular barcode sequence.

[0018] In certain embodiments, the single-cell genomics sequencing library is a single-nuclear RNA sequencing library (snRNA-seq). In certain embodiments, the single-cell genomics sequencing library is a single-cell DNA accessibility library. In certain embodiments, the single-cell genomics sequencing library is a single-cell ATAC sequencing library (ATAC-seq). In certain embodiments, the single-cell genomics sequencing library is a single-cell chromatin immunoprecipitation (ChIP) sequencing library. In certain embodiments, the single-cell genomics sequencing library is a single-cell genome sequencing library. In certain embodiments, the single-cell genomics sequencing library is a single-cell DNA methylation sequencing library. In certain embodiments, the single-cell genomics sequencing library is a single-cell Hi-C sequencing library. In certain embodiments, the single-cell genomics sequencing library is a single-cell enzyme-tethered chromatin profiling sequencing library. In certain embodiments, the single-cell genomics sequencing library is a single-cell genome and transcriptome sequencing library (G&T-seq). In certain embodiments, the single-cell genomics sequencing library is a single-cell proteome library.

[0019] On the other hand, the present invention provides a kit comprising a plurality of solid supports coupled to a nucleic acid sequence comprising a spatial barcode and a capture sequence via a cleavable linker, wherein the spatial barcode is the same for a single solid support but different for any other solid support in the spatial array, and wherein the capture sequence is the same on all the solid supports. In certain embodiments, the solid support further comprises a chemical binding portion for covalently fixing the solid support to the array. In certain embodiments, the solid support is a bead. In certain embodiments, the beads are polystyrene beads. In certain embodiments, the diameter of the beads is 50 μm or less. In certain embodiments, the beads are 1, 3, 10, 15 or 20 μm, preferably 10 μm. In certain embodiments, the linker is a photocleavable, chemically cleavable or enzyme-cleavable linker. In certain embodiments, the capture sequence comprises a poly-A sequence. In certain embodiments, the length of the spatial barcode nucleic acid coupled to the solid support is different. In certain embodiments, the spatial barcode nucleic acid further comprises one or more modifications that enhance diffusion into the cell nucleus. In certain embodiments, the spatial barcode nucleic acid is modified by the addition of one or more lipid or cholesterol groups. In certain embodiments, the spatial barcode nucleic acid further comprises one or more fluorescent markers that can be used to identify and label the cell nucleus.

[0020] These and other aspects, objects, features and advantages of the exemplary embodiments will become apparent to those of ordinary skill in the art after considering the following detailed description of the exemplary embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] An understanding of the features and advantages of the present invention will be gained by referring to the following detailed description and accompanying drawings which set forth illustrative embodiments in which the principles of the invention may be utilized, and in which:

[0022] Figure 1 - Diagram showing the Slide-tags protocol.

[0023] Figures 2A-2C -Slide-tag molecular reaction. Figure 2A .(SEQ ID NO:1-7) The oligonucleotides cleaved from the beads are used to label the cell nuclei, and the labeled cell nuclei are encapsulated in droplets with cell barcode beads, and the mRNA and spatial barcode are captured by the cell barcode beads. Figure 2B .(SEQ ID NO:6-12) Spatial barcodes and RNA separated by size. Figure 2C .(SEQ ID NO:13-21) Generation of sequencing library.

[0024] Figure 3 - Computational workflow for generating spatially resolved single-nucleus RNA-seq profiles.

[0025] Figure 4 - Comparison of Slide-tags with other spatial technologies.

[0026] Figures 5A-5C -Slide-tags results in mouse hippocampus. Figure 5A . UMAP plot showing cells identified by snRNA-seq. Figure 5B . Expression of marker genes projected onto the UMAP plot. Figure 5C .Mapping of cell types on tissue samples.

[0027] Figure 6 - Volcano plot showing the increase in signal and percentage of cells that can be spatially localized since the first slide-tag experiment.

[0028] Figures 7A-7B -Slide-tags results in mouse hippocampus. Figure 7A Slide-tag spatial plots compared to immunohistochemistry of sections from the same specimen using the same marker genes. Figure 7B .The spatial location of specific cell types.

[0029] Figures 8A-8G -Slide-tags results in mouse embryos. Figure 8AImages of embryonic sections and UMAP plots showing clustering of single nuclei based on snRNA-seq data from the sections. UMAP plots showing cell types and graphs showing percentages of cell types. Figure 8B .Spatial positioning of cell nuclei on sections. Figure 8C Spatial localization of brain regions identified with slide-tags compared to histological images. Figure 8D Images of slide-tags UMI in the sections and histological images of the sections' locations. Figure 8E .Slide-tags images of the spatial location of cell nuclei expressing the specified marker gene in the slice and histological images of the slice location. Figure 8F The spatial location of cell types in the slice. The spatial location of different neuronal clusters is also shown. Figure 8G .Spatial locations of individual neuronal clusters.

[0030] Figures 9A-9B -Slide-tags pseudo-time results. Figure 9A . Diagram showing dynamic events during neuronal development. Figure 9B UMAP plot and specific clustering by pseudotime analysis. Spatially resolved pseudotime analysis. Expression of specific markers during pseudotime.

[0031] Figure 10 -Slide-tags pseudo-time results. A UMAP plot showing the pseudo-time values and their spatial locations.

[0032] Figure 11 - Slide-tag results in mouse embryos. Spatial location of cell types in transverse slices. Also shown are the spatial locations of different neuronal clusters.

[0033] Figure 12 - A diagram describing 3D slide-tags.

[0034] Figures 13A-13C - Comparison of tissue section thickness. Figure 13A . Diagram showing cell nuclei in a tissue section. Figure 13B UMAP images of cell nuclei from 20 μm sections and graphs showing the percentage of each cell type. Figure 13C UMAP images of cell nuclei from 40 μm sections and graphs showing the percentage of each cell type.

[0035] Figures 14A-14B - Board-based slide-tag. Figure 14A .(SEQ ID NO:22) shows a flow chart for single-cell genome and transcriptome analysis. Figure 14BFlowchart showing the use of labeled nuclei to determine spatially resolved genomic and transcriptomic data.

[0036] Figures 15A-15D - Figure 15A Spatial barcodes and mRNA are captured in droplets (10X GEMS and beads). 10X beads contain a Capture Sequence 1 oligonucleotide for capturing spatial barcodes and a poly dT oligonucleotide for capturing mRNA. A template-switching oligonucleotide (TSO) adds an adapter sequence to the RT product. Spatial barcode sequencing libraries and gene expression libraries are generated. Figure 15B .Reverse transcription and extension are performed in GEM droplets. Figure 15C cDNA amplification of RT products. Figure 15D .Prepare sample index library.

[0037] Figures 16A-16B - Figure 16A Schematic diagram depicting a slide-tag experiment in the mouse hippocampus. The output of a snRNA-seq experiment is a cell x gene matrix and an accompanying matrix of spatial coordinates. Figure 16B . Dimensionality reduction plots highlighting populations of cell types are shown, with each of these cells having associated spatial barcode clusters.

[0038] Figure 17 Slide-tags enable single-nucleus spatial transcriptomics in the mouse hippocampus. Slide-tag experiments show the spatial localization of nuclei in the mouse hippocampus; cells are colored according to cell type annotation. The spatial expression of known marker genes is compared with in situ hybridization data from the Allen Mouse Brain Atlas (scale bar, normalized mean counts).

[0039] Figure 18 - Spatial resolution measurements in the mouse hippocampus and Slide-tags snRNA-seq enable characterization of deep and superficial sublayers in the CA1 region of the mouse hippocampus. 10 μm Nissl-stained sections (left) were obtained near the sections profiled by Slide-tags (right). The CA1 nuclei were subsetted in each case, and a line was fitted to measure the midpoint of this structure. For Slide-tags, nuclei were selected based on their cell type assignment, with 2 spatial outliers removed. For Nissl, nuclei were computationally segmented. The orthogonal distance to the midpoint was then calculated, and the points were colored by the distance.

[0040] Figure 19Spatial resolution measurements and Slide-tag snRNA-seq in the mouse hippocampus enabled characterization of deep and superficial sublayers in the CA1 region of the mouse hippocampus. PCA plots show cells from the CA1 cluster after subsetting, reprojection, and re-clustering. Cells are colored according to their new subcluster assignment. Cells are plotted according to their spatial location. Violin plots show gene expression differences between each subcluster and the spatial expression of these genes.

[0041] Figure 20 Slide-tags offer high molecular weight and spatial resolution. Schematic showing each nucleus receiving numerous spatially barcoded oligonucleotides from numerous different beads. Diagram showing an estimate of spatial resolution of less than 10 microns.

[0042] Figure 21 Comparative metrics plotting of snRNA-seq performed on serial sections compared to Slide-tags snRNA-seq. Plot cell type proportions and average UMI per cell by cell type. Determine the normalized average UMI count for each gene across all cells. Compare normalized average counts.

[0043] Figure 22 -Slide-tags generates high-quality spatial single-cell data. Log per nucleus (Slide-tags) or 20 μm spatial points (Slide-seqV2, DBiT-seq or Xenium) in mouse brain 10 Violin plots of converted genes and UMIs. Elbow plots of standard deviations of principal components from Slide-tags snRNA-seq, Slide-seqV2, DBiT-seq, and Xenium in mouse brain.

[0044] Figure 23 -Slide-tags amplify spatial genomics libraries at single-cell resolution. Schematic diagram showing slide-tagged nuclei that can be used for different single-cell techniques.

[0045] Figures 24A-24B Spatially resolved snRNA-seq in genetically heterogeneous samples. Slide tagging of human metastatic melanoma samples. Figure 24A . Schematic diagram showing the experimental design. H&E images of melanoma samples. Figure 24B .Transcriptome clustering and spatial location of single cells are shown.

[0046] Figures 25A-25B - Spatial separation of different tumor subpopulations. Figure 25A . Mapping of transcriptionally distinct melanoma subpopulations in space. Figure 25BInfer copy number variation from transcriptome data using inferCNV.

[0047] Figures 26A-26B - Tumor clusters have different spatial neighborhoods. Figure 26A The graph shows the proportion of neighbors that tumor cells have. The y-axis shows tumor clustering, and the x-axis shows immune cell types. Figure 26B Spatial maps of the fractional enrichment of CD8 T cells within each tumor neighborhood.

[0048] Figures 27A-27B -Differential immune status and receptors between tumor clones. Figure 27A . Showing differential expression in T cells between tumor lobes. Figure 27B (SEQ ID NO: 23-24) show T cell receptors (TCRs) recovered by slide-tags from Tumor 1 and Tumor 2. Spatial mapping of TCR clones on Tumor 1 and Tumor 2 is shown.

[0049] Figure 28 - HLA loci were downregulated in tumor 1. Copy number variation was inferred from transcriptome data using inferCNV (Chr6 boxed).

[0050] Figures 29A-29C -Spatial multi-omics sequencing using Slide-tags. Figure 29A Clustering single cells by RNA expression and chromatin accessibility using slide-tags. Figure 29B For each cell type, chromatin accessibility and single gene expression in single cell types are shown by violin plots. Figure 29C .Spatial map of a single cell.

[0051] Figures 30A-30C - Epigenomic differences between Tumor 1 and Tumor 2 populations. Figure 30A Figure 1 shows a comparison of ATAC-seq and RNA expression in single cells (TNC bold). Figure 30B For each tumor, chromatin accessibility and Tnc gene expression in each tumor are shown by violin plots. Figure 30C Spatial maps of single cells scored by TNC chromatin genes in Tumor 1 and Tumor 2.

[0052] Figures 31A-31B -Tumor 1 cell states driven by spatially clustered TF motifs. Figure 31A .Expression of mesenchymal-like cell state genes in tumor 1 and tumor 2. Figure 31B Figure 3 shows transcription factor motifs identified by ATAC-seq that are positively and negatively correlated with the mesenchymal score.

[0053] Figures 32A-32G- Slide-tag analysis of postmortem human cortex. Figure 32A .Shows slide-tags performed on a 5.5×5.5 area of the human prefrontal cortex. Figure 32B .Spatial maps showing all cortical cell types. Figure 32C . Spatial maps showing genetic markers of cortical cell types. Figure 32D .Spatial maps and plots showing all cortical layer types. Figure 32E .Spatial map showing CUX2 expression. Figure 32F .Spatial map showing RORB expression. Figure 32G .Spatial map showing FOXP2 expression.

[0054] Figures 33A-33B -Slide-tags analysis of human tonsils. Figure 33A snRNA-seq data from tonsil. Clustering by cell type is shown. Figure 33B Spatial maps of single cells and H and E staining of tissues.

[0055] Figures 34A-34C -Using spatially varying genes to identify dark and light zones in germinal centers. Figure 34A .Schematic diagram of a germinal center with light and dark areas. Figure 34B Slide-tags in germinal centers are used to cluster cell types. Figure 34C B cell clustering and marker gene expression. Spatial map of germinal center B cells.

[0056] Figures 35A-35B - Name spatially significant receptor-ligand interactions. Figure 35A .Previous methods for inferring receptor-ligand interactions using single-cell data. Figure 35B .Using spatial information to organize receptor-ligand interactions in germinal centers.

[0057] Figures 36A-36B -Slide-tags generate high-quality spatial multi-omics data. Figure 36A . Schematic diagram showing the experimental design for multimodal analysis of the P1 mouse brain. Figure 36B Single-cell ATAC-seq and snRNA-seq from the same single nucleus.

[0058] Figures 37A-37B -Slide-tags allow spatial ATAC and RNA-seq to be performed in the same single cells. Figure 37A. Clustering of single nuclei by RNA expression, accessible chromatin (ATAC) and weighted nearest neighbor (WNN) analysis (see, e.g., Hao Y, Hao S, Andersen-Nissen E et al., Integrated analysis of multimodal single-cell data. Cell. 2021; 184(13): 3573-3587. e29). Figure 37B .(A) Graph of the clusters in space.

[0059] Figures 38A-38D -Transcribed and open chromatin can define spatially distinct elements. Figure 38A Spatial map of the developing outer cortical layers (isocortex, retrosplenial cortex, and subiculum) mapped using RNA expression. Figure 38B . Representative top few RNA hits. Figure 38C Clustering using ATAC and the top few chromatin accessible hits. Figure 38D .Transcription factor motifs and top hits that vary along this axis.

[0060] Figure 39 -Oligonucleotide variations of Slide-tags. (SEQ ID NO: 2-3, 25-28) Capture sequence 1 can be a capture sequence on a spatial barcode oligonucleotide. Capture sequence 1 is complementary to the 10X Genomics chromium 3'v3.1 platform. Oligonucleotide variation 1 shows the addition of a capture sequence using 10X capture sequence 1 and a splint sequence. Oligonucleotide variation 2 shows a spatial barcode oligonucleotide with capture sequence 1 downstream of the spatial barcode. Oligonucleotide variation 3 shows the capture sequence as a polyA sequence for capturing a poly T sequence. Oligonucleotide variation 4 shows a chemical modification (such as cholesterol-TEG, squalene, fatty acids, or α-tocopherol) that can be present on any spatial barcode oligonucleotide to facilitate nuclear uptake or retention. Oligonucleotide variation 5 shows a chemical modification (such as a fluorescent dye, a trifunctional linker that can have a fluorescent dye and a combination of one of the chemical modifications listed above) that can be present on any spatial barcode oligonucleotide to facilitate detection and quantification of the spatial barcode.

[0061] The drawings herein are for illustration purposes only and are not necessarily drawn to scale. DETAILED DESCRIPTION

[0062] General Definition

[0063] 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 belongs. Definitions of common terms and techniques in molecular biology can be found in Molecular Cloning: A Laboratory Manual, 2nd edition (1989) (Sambrook, Fritsch and Maniatis); Molecular Cloning: A Laboratory Manual, 4th edition (2012) (Green and Sambrook); Current Protocols in Molecular Biology (1987) (FM Ausubel et al., eds.); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (1995) (MJ MacPherson, BD Hames and GR Taylor, eds.); Antibodies, A Laboratory Manual (1988) (Harlow and Lane, eds.); Antibodies A Laboratory Manual, 2nd edition 2013 (E.A. Greenfield, ed.); Animal Cell Culture (1987) (RI Freshney, ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlett, 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 Ltd., 1995 (ISBN 9780471185710); Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th Edition, John Wiley & Sons (New York, NY 1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2nd Edition (2011).

[0064] As used herein, the singular forms "a," "an," and "the" include both singular and plural referents unless the context clearly dictates otherwise.

[0065] The terms "optional" or "optionally" mean that the subsequently described event, circumstance, or substituent may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0066] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the corresponding range, as well as the recited endpoints.

[0067] As used herein, the terms "about" or "approximately" when referring to a measurable value such as a parameter, amount, duration, etc., are intended to encompass variations from the specified value and variations from the specified value, such as variations of + / -10% or less, + / -5% or less, + / -1% or less, and + / -0.1% or less from the specified value, as long as such variations are suitable for performing the disclosed invention. It is understood that the value to which the modifier "about" or "approximately" refers is also specifically and preferably disclosed itself.

[0068] As used herein, a "biological sample" may contain whole cells and / or living cells and / or cell fragments. A biological sample may contain (or be derived from) a "body fluid". The present invention encompasses embodiments in which the body fluid is selected from amniotic fluid, aqueous humor, vitreous humor, bile, serum, breast milk, cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudate, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, rhubarb, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vomitus, and a mixture of one or more thereof. A biological sample includes cell cultures, body fluids, cell cultures from body fluids. Body fluids can be obtained from mammalian organisms, for example, by puncture or other collection or sampling procedures.

[0069] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to a vertebrate, preferably a mammal, and more preferably a human. Mammals include, but are not limited to, rodents, monkeys, humans, farm animals, sports animals, and pets. Also included are tissues, cells, and progeny of biological entities obtained in vivo or cultured in vitro.

[0070] Various embodiments are described below. It should be noted that the specific embodiments are not intended to be exhaustive descriptions or to serve as limitations on the broader aspects discussed herein. An aspect described in conjunction with a specific embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiment. References throughout this specification to "one embodiment," "an embodiment," or "an exemplary embodiment" mean that the specific features, structures, or characteristics described in conjunction with that embodiment are included in at least one embodiment of the present invention. Therefore, the phrases "in one embodiment," "in an embodiment," or "an exemplary embodiment" appearing in different places throughout this specification do not necessarily refer to the same embodiment, but may refer to the same embodiment. In addition, in one or more embodiments, specific features, structures, or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art based on this disclosure. In addition, although some embodiments described herein include some but not other features included in other embodiments, combinations of features from different embodiments are intended to be within the scope of the present invention. For example, in the appended claims, any of the claimed embodiments may be used in any combination.

[0071] See U.S. Patent Application Publication No. US20210123040A1. See also “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, and Fei Chen, bioRxiv 2023.04.01.535228. See also Russell, AJC, Weir, JA, Nadaf, NM 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 into this document, as if each individual publication, published patent document or patent application was specifically and individually indicated to be incorporated by reference.

[0072] Overview

[0073] Embodiments disclosed herein provide spatially labeled nuclei compatible with any genome or multiple sets of single cell / nucleus assays to allow the generation of spatially resolved single cell sequencing libraries with single cell resolution. Previous methods were limited to detecting mRNA expression and were also limited by the amount of mRNA diffused from cells and captured (see, for example, 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 invention overcomes these limitations by labeling the nucleus with spatial barcodes so that the capture analyte does not depend on diffusion from the cell. The nucleus can be completely dissolved in the reaction volume to release all analytes (such as RNA) so that all RNA can be captured. In addition, in order to use slide-seq for multi-omics spatial research, each group measurement must be performed separately. The present invention does not need to perform each spatial experiment twice or multiple times to obtain multiple groups of spatial results. The present invention is based on the use of modified slide-seq arrays (polystyrene support beads with barcoded oligonucleotides) to deliver spatial barcodes to cell nuclei within fresh frozen thin tissue sections. The use of slide-seq arrays and the lack of tissue fixation distinguish the present invention from XYZeq and sci-Space. In addition, the architecture of the spatial barcodes allows for one of the following: plate-based, microfluidic-based, or nanopore-based capture of macromolecules and spatial barcodes from single nuclei, in contrast to XYZeq and sci-Space, which require split-pool indexed profiling.

[0074] Current methods only require the delivery of one or more spatial barcode tags to permeabilized cell nuclei. The labeled nuclei can then be stored or used directly for any single-cell genomics assay. In other words, the present invention unifies spatial profiling and single-cell sequencing methods. In addition, the present invention provides an array such that the distance between different spatial barcodes is less than the size of the cell, thereby achieving single-cell resolution. In addition, the use of fluorescently labeled spatial barcode tags allows only labeled nuclei to be used for subsequent single-cell genomics assays. Therefore, because a single labeled nucleus is used as input for single-nuclear / cell genome assays, the analyte capture efficiency approaches the detection efficiency of non-spatially resolved single-cell genome sequencing technologies. For example, the RNA capture rate is close to about 100% of single-nuclear RNA sequencing data.

[0075] Labeling cell nuclei in tissue samples with spatial barcoding

[0076] In an exemplary embodiment, a tissue sample is placed on an array containing spatial barcodes, and the spatial barcodes are released from the array to label cell nuclei in the tissue. The tissue sample is permeabilized to allow the spatial barcodes to label the cell nuclei. The labeled single nuclei are then isolated from the tissue sample.

[0077] Space Array

[0078] In an exemplary embodiment, the nuclei in the tissue sample are labeled with spatial barcode nucleic acids. Spatial barcode nucleic acids are nucleic acids that are connected or attached to an array at a specific position and include a barcode sequence. Therefore, spatial barcodes can identify positions in the array. As used herein, the term "array" refers to a group of features or sites that can be distinguished from each other based on relative position. Different molecules located at different sites of the array can be distinguished from each other based on the position of the sites in the array. A single site of the array can include one or more specific types of molecules. For example, a site can include a single target nucleic acid molecule with a specific sequence, or a site can include several nucleic acid molecules with the same sequence (and / or its complementary sequence). The sites of the array can be different features located on the same substrate. Exemplary features include, but are not limited to, holes in the substrate, beads (or other particles) in or on the substrate, protrusions from the substrate, ridges on the substrate, or channels in the substrate. The sites of the array can be separate substrates that each carry different molecules. Different molecules attached to a separate substrate can be identified based on the position of the substrate on the surface to which the substrate is bound or based on the position of the substrate in a liquid or gel. Exemplary arrays in which separate substrates are on a surface include, but are not limited to, those with beads in wells, beads arranged on a flat surface (e.g., a glass slide), beads optionally captured on a flat surface (e.g., a layer of beads adhered to or otherwise stably associated with a glass slide (e.g., a layer of beads adsorbed to an elastomeric surface to which the slide is attached), etc. In exemplary embodiments, the arrays of the invention include greater than 10,000 separate locations, each with a different spatial barcode. In exemplary embodiments, the arrays of the invention include from 10,000 to over 1 million separate locations, each with a different spatial barcode.

[0079] As used herein, the term "feature" means the location of a particular type of molecule in an array. A feature may contain only a single molecule, or it may contain a population of several molecules of the same type. The features of an array are typically discrete. Discrete features may be continuous or they may have spaces between each other. The size of the features and / or the spacing between the features may vary so that the array may be high density, medium density, or low density. A high density array is characterized by sites that are spaced less than about 15 μm apart. The sites of a medium density array are spaced about 15 to 30 μm apart, while the sites of a low density array are spaced greater than 30 μm apart. Arrays useful herein may have, for example, sites spaced less than 100 μm, 50 μm, 10 μm, 5 μm, 1 μm, or 0.5 μm apart. The apparatus or method disclosed herein can be used to detect arrays with a resolution sufficient to distinguish sites of the above-mentioned densities or density ranges.

[0080] As used herein, the term "attachment" refers to the state in which two objects are joined, fastened, adhered, connected or bound to each other. For example, an analyte (e.g., a nucleic acid) can be attached to a material (e.g., a gel or solid support) by covalent or non-covalent bonds. A covalent bond is characterized by the sharing of electron pairs between atoms. A non-covalent bond is a chemical bond that does not involve shared electron pairs and can include, for example, hydrogen bonds, ionic bonds, van der Waals forces, hydrophilic interactions, and hydrophobic interactions.

[0081] Spatial barcoded nucleic acids

[0082] In an exemplary embodiment, the spatial barcode nucleic acid is a nucleic acid sequence that includes a barcode sequence. In an exemplary embodiment, the spatial barcode nucleic acid is a nucleic acid sequence that includes a cleavable linker, a barcode sequence, and a capture sequence from 5' to 3'. In an exemplary embodiment, the spatial barcode nucleic acid is closed at the 3' end to prevent extension. Therefore, after being captured by the cell barcode, the cell barcode sequence triggers extension into the spatial barcode nucleic acid to add the spatial barcode sequence to the cell barcode sequence, while the spatial barcode nucleic acid is not extended. In an exemplary embodiment, the spatial barcode nucleic acid includes a barcode sequence that identifies a position on the array. The term "barcode" as used herein refers to a short nucleotide sequence (such as DNA or RNA) or a series of nucleotides in a nucleic acid that can be used to identify nucleic acids, features of nucleic acids (such as the identity and optional position of nucleic acids), or operations (such as disturbances) that have been performed on nucleic acids, which are used as identifiers of related molecules (such as target molecules and / or target nucleic acids), or as identifiers of the sources (such as source cells) of related molecules. As used herein, the term "spatial barcode" or "spatial tag" is intended to refer to a series of nucleotides in a nucleic acid that can be used to identify the position on the array to which the nucleic acid is fixed. As used herein, the term "spatial barcode" or "spatial tag" is also intended to refer to a nucleic acid having a sequence indicating a position. Typically, a nucleic acid is a synthetic molecule having a sequence that is not found in one or more biological samples to 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, such as in a biological sample to be used with the nucleic acid. The position indicated by the spatial tag can be a position in or on a biological sample, in or on a solid support, or a combination thereof. The barcode sequence can be used as a spatial tag. In an exemplary embodiment, only after a population of beads (each having a different barcode sequence) has been arranged on a solid support (optionally randomly arranged on a solid support) and sequencing of such a bead-related barcode sequence has been determined in situ on the solid support, the position identification of the tag used as the spatial tag is determined.

[0083] The barcode sequence can be a naturally occurring sequence or a sequence that does not naturally occur in the organism from which the barcoded nucleic acid is obtained or the labeled sample is obtained. The barcode sequence can be unique to a single nucleic acid species in a population, or the barcode sequence can be shared by several different nucleic acid species in a population (e.g., all nucleic acid species or single beads attached to an array at a defined position can have the same barcode sequence, while different defined positions or beads present different shared barcode sequences for identifying each such different position or bead). As another example, each nucleic acid probe in a population can include a barcode sequence that is different from all other nucleic acid probes in the population. Alternatively, each nucleic acid in a population can include a barcode sequence that is different from some or most other nucleic acids in the population. In a specific embodiment, one or more barcode sequences used with a biological sample (e.g., a tissue sample) are not present in the genome, transcriptome, or other nucleic acid of the biological sample. For example, the barcode sequence can have a sequence identity of less than 80%, 70%, 60%, 50%, or 40% with a nucleic acid sequence in a specific biological sample. As used herein, when used with respect to nucleic acids, the term "different" means that the nucleic acids have nucleotide sequences that are different from each other. The nucleotide sequences of two or more nucleic acids may be different over their entire lengths. Alternatively, the nucleotide sequences of two or more nucleic acids may differ along a substantial portion of their lengths. For example, two or more nucleic acids may have a target nucleotide sequence portion that is different for the two or more molecules while also having a common sequence portion that is the same on the two or more molecules.

[0084] As used herein, the term "biological sample" is intended to refer to one or more cells, tissues, organisms, or parts thereof. A biological sample can be obtained from any of a variety of organisms. Exemplary organisms include, but are not limited to, mammals, such as rodents, mice, rats, rabbits, guinea pigs, ungulates, horses, sheep, pigs, goats, cows, cats, dogs, primates (i.e., humans or non-human primates); plants, such as Arabidopsis thaliana, corn, sorghum, oats, wheat, rice, canola, or soybeans; algae, such as Chlamydomonas reinhardtii; nematodes, such as Caenorhabditis elegans; insects, such as Drosophila melanogaster, mosquitoes, fruit flies, honey bees, or spiders; fish, such as zebrafish; reptiles; amphibians, such as frogs or Xenopus laevis; Dictyostelium discoideum; fungi, such as Pneumocystis carinii, Aspergillus fumigatus, and Aspergillus niger. rubripes), yeast, Saccharamoyces cerevisiae, or Schizosaccharomyces pombe; or Plasmodium falciparum. The target nucleic acid can also be derived from a prokaryotic organism, such as bacteria, Escherichia coli, Staphylococci, or Mycoplasma pneumoniae; an archaeon; a virus, such as hepatitis C virus or human immunodeficiency virus; or a viroid. The sample can be derived from a homogenous culture or population of the above organisms or, alternatively, from a collection of several different organisms (e.g., a community or ecosystem).

[0085] In an exemplary embodiment, the spatial barcode nucleic acid includes a universal sequence for capturing the spatial barcode nucleic acid onto another nucleic acid sequence (e.g., a barcode nucleic acid that identifies a cell of origin). In some embodiments, the universal sequence is also referred to as a capture sequence or a disposal sequence, such as a connection, PCR, or index disposal sequence. In a preferred embodiment, the capture sequence is located at the 3' end of the spatial barcode nucleic acid. In an exemplary embodiment, 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 shared by two or more nucleic acid molecules, even if these molecules also have sequence regions that are different from each other. The universal sequence present in different members of the molecular set can allow a universal capture nucleic acid population that is complementary to the universal sequence to capture a variety of different nucleic acids. Similarly, the universal sequence present in different members of the molecular set can allow a universal primer population that is complementary to the universal sequence to replicate or amplify a variety of different nucleic acids. Therefore, the universal capture nucleic acid or universal primer includes a sequence that can specifically hybridize with the universal sequence. The target nucleic acid molecule can be modified to attach a universal adapter, for example, at one or both ends of different target sequences. Non-limiting examples of 3' universal sequences for capturing spatially barcoded nucleic acids include poly-A sequences for capture via poly-T sequences, or sequences complementary to commercially available capture sequences, such as tag fragmentation adapter sequences (e.g., the read 1 (read 1N) sequence on beads in the Chromium NextGEM Single Cell ATAC Kit v1.1 (10x Genomics, Pleasanton, CA, USA)).

[0086] As used herein, the term "poly-T or poly-A" when used in reference to a nucleic acid sequence is intended to refer to a series of two or more thiamine (T) or adenine (A) bases. The poly-T or poly-A may include at least about 2, 5, 8, 10, 12, 15, 18, 20, 25, 30 or more T or A bases, respectively. Alternatively or additionally, the poly-T or poly-A may include up to about 30, 25, 20, 18, 15, 12, 10, 8, 5 or 2 T or A bases, respectively.

[0087] In an exemplary embodiment, spatially barcoded nucleic acids do not require UMI sequences because the UMI sequences are present on the single-cell / nucleus genomics assay capture sequence, which also includes the cell-of-origin barcode sequence. Therefore, when spatial barcodes are captured in a single-cell assay, a UMI specific to each spatial barcode capture event will be present on each sequencing read.

[0088] In an exemplary embodiment, the length of the spatial barcode nucleic acid is about 50 to 250 nucleotides. In one example, the spatial barcode nucleic acid includes a connector, a primer binding sequence that is the same for all spatial barcode nucleic acids, a spatial barcode of about 6 to 50 nucleotides, preferably 6 to 20 nucleotides, and a capture sequence of about 6 to 50 nucleotides. The spatial barcode nucleic acid may also include additional sequences, for example, to change the length of the sequence. In an exemplary embodiment, the spatial barcode nucleic acid is single-stranded, preferably ssDNA. In some embodiments, single-stranded oligonucleotides diffuse into the cell nucleus better than double-stranded nucleotides. In an exemplary embodiment, single-stranded DNA (ssDNA) specifically stains the nuclei of permeabilized cells rather than intact cells.

[0089] In an exemplary embodiment, the spatial barcode nucleic acids on the spatial array have the same spatial barcode for each position, but have different lengths for each position. Spatial barcode nucleic acids with different lengths allow the spatial location of single cells to be identified in three dimensions, because shorter nucleic acids will diffuse farther into the tissue section than longer nucleic acids. In an exemplary embodiment, computational methods can be used to determine the location of single cells in a tissue section with multiple layers of cells, for example, by quantifying the number of spatial barcodes of different lengths in a single nucleus.

[0090] The barcode sequence can be any of a variety of lengths. Longer sequences can generally accommodate barcodes of greater numbers and types of populations. Typically, all probes in a plurality of probes will have barcodes of the same length (although with different sequences), but barcodes of different lengths can also be used for different probes. The length of the barcode sequence can be at least 2, 4, 6, 8, 10, 12, 15, 20 or more nucleotides. Alternatively or additionally, the length of the barcode sequence can be at most 20, 15, 12, 10, 8, 6, 4 or less nucleotides. Examples of usable barcode sequences are described in, for example, U.S. Patent Publication No. 2014 / 0342921A1 and U.S. Patent No. 8,460,865, each of which is incorporated herein by reference.

[0091] connector

[0092] In an exemplary embodiment, the spatial barcode nucleic acid includes a linker sequence for attachment to an array or solid support (e.g., beads further described herein). In a preferred embodiment, the linker is cleavable so that the spatial barcode nucleic acid can be released when in contact with or adjacent to a tissue sample. In an exemplary embodiment, the cleavable linker is chemically cleavable, photocleavable, or enzymatically cleavable.

[0093] In a preferred embodiment, the linker is photocleavable. Photocleavable linkers that can be released by UV irradiation are available. PC (photocleavable) spacers can be placed between DNA bases or between an oligonucleotide and a 5'-modifying group. The spacer arm can be cleaved by exposure to UV light in the 300-350 nm spectral range. Cleavage releases the oligonucleotide having a 5'-phosphate group. Exemplary photocleavable linkers are commercially available (Integrated DNA Technologies, Inc., Coralville, Iowa) and are shown below:

[0094]

[0095] In other exemplary embodiments, the spatial barcode nucleic acid can contain one or more cleavable linkers, e.g., that can be cleaved upon application of a suitable stimulus. For example, the cleavable sequence can be a photocleavable linker that can be cleaved by application of light, a chemically cleavable linker that can be cleaved by application of a suitable chemical, or an enzymatically cleavable linker that can be cleaved by application of an enzyme.

[0096] Oligonucleotides with photosensitive chemical bonds (such as photocleavable joints) have various advantages. They can be effectively and rapidly cracked (for example, in nanoseconds and milliseconds). In some cases, a photomask can be used so that only specific areas of the array are exposed to cleavable stimulation (for example, exposed to UV light, exposed to light, exposed to heat induced by laser). When using a photocleavable joint, the cleavable reaction is triggered by light, and the joint can be highly selective and therefore biorthogonal. Non-limiting examples of photosensitive chemical bonds that can be used for cleavage domains include Leriche et al., Bioorg Med Chem. January 15, 2012; 20 (2): 571-82; U.S. Publication No. 2017 / 0275669; and those described in WO2020190509A9.

[0097] Spatial barcoding nucleic acid modification

[0098] In an exemplary embodiment, the spatial barcode nucleic acid includes nucleotide modifications to enhance diffusion into the nucleus. In an exemplary embodiment, the spatial barcode nucleic acid is coupled to a lipophilic or amphiphilic portion. Lipophilic molecules can be associated with and / or inserted 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 its derivatives, lignoceric acid and palmitic acid. Other lipophilic molecules that can be used in the methods provided herein include amphiphilic molecules, wherein head groups (such as charge, aliphatic content and / or aromatic content) and / or fatty acid chain lengths (such as C12, C14, C16 or C18) can vary. For example, fatty acid side chains (such as C12, C14, C16 or C18) can be coupled to glycerol or glycerol derivatives (such as 3-tert-butyldiphenylsilylglycerol), which can also include, for example, cationic head groups. Spatial barcode nucleic acids disclosed herein can be coupled (directly or indirectly) to these amphiphilic molecules. Amphiphilic molecules can associate with and / or insert into membranes (eg, nuclear membranes).

[0099] Spatial barcode nucleic acid can be attached to a lipophilic part (e.g., a cholesterol molecule). Spatial barcode nucleic acid can be attached to a lipophilic part through a joint (e.g., a tetraethylene glycol (TEG) joint). Other exemplary joints include, but are not limited to, amino joint C6, amino joint C12, spacer C3, spacer C6, spacer C12, spacer 9, spacer 18. Spatial barcode nucleic acid can be attached to a lipophilic part or joint on the 5' end of the spatial barcode nucleic acid. The joint can be a diol or a derivative thereof. For example, the joint can be tetraethylene glycol (TEG) or polyethylene glycol (PEG). Spatial barcode nucleic acid can be releasably attached to a joint or a lipophilic part (e.g., as described elsewhere herein for the releasable attachment of nucleic acid molecules) so that the spatial barcode nucleic acid or a portion thereof can be released from the lipophilic molecule. In an exemplary embodiment, the lipophilic part (e.g., cholesterol) is indirectly coupled to the oligonucleotide (e.g., by hybridization or ligand-ligand interaction, such as biotin-streptavidin).

[0100] In an exemplary embodiment, the spatial barcode nucleic acid includes a fluorescent tag so that the labeled nucleus can be identified and sorted from the unlabeled nucleus. The efficiency of the nucleus labeling can also be determined by the percentage of the quantitatively labeled nucleus. In an exemplary embodiment, the lipophilic molecule can include a fluorescent moiety. Many different fluorophores can be easily attached to the oligonucleotide, such as fluorescein and its tetrachloro and hexachloro derivatives TET and HEX.

[0101] Sequence-verified arrays

[0102] In an exemplary embodiment, the spatial array is sequence verified. As used herein, "sequence verification" refers to knowing the sequence of the spatial barcode at each position or substantially all positions in the array, preferably greater than 50%, 60%, 70%, 80% or 90% of the positions. In an exemplary embodiment, the array is sequence verified because the spatial barcode attached to the array at each position is specifically placed at each position. In an exemplary embodiment, the sequence of the spatial barcode at each position is determined by in situ sequencing. In an exemplary embodiment, the array is sequence verified by in situ sequencing before the tissue sample is placed on the array. In an exemplary embodiment, in situ sequencing is performed by ligation sequencing or synthesis sequencing directly on the array and captured by a microscope.

[0103] In certain aspects of the present disclosure, in situ sequencing is performed on a surface-attached array, which can be performed by any art-recognized parallel (optionally massively parallel) in situ sequencing format, examples of which include, inter alia, the previously described SOLiD TM Method, which is a ligation sequencing technology that can be performed in situ on a solid support (see, for example, Voelkerding et al., Clinical Chem., 55-641-658, 2009; U.S. Patent Nos. 5,912,148; and 6,130,073, which are incorporated herein by reference in their entirety). In certain embodiments of the present disclosure, such sequencing can be performed on an array present on a standard microscope slide, optionally using a standard microscope equipped with sufficient computing power to track and associate individual sequences with their spatial positions during progressive rounds of detection. The present disclosure also employs fluidics, incubation times, enzyme mixtures, and imaging settings in performing in situ sequencing (see, for example, US20210123040A1).

[0104] Sequencing technology, such as synthetic sequencing (SBS) technology, is a useful method for determining barcode sequences. SBS can be performed as follows. In order to start the first SBS cycle, one or more labeled nucleotides, DNA polymerases, SBS primers, etc. can be contacted with one or more features (such as nucleic acid probes attached to the features of beads or other solid supports) on beads or other solid supports. Those features that the SBS primer extension causes the labeled nucleotide to be incorporated can be detected. Optionally, the nucleotides can include a reversible termination portion, and once the nucleotides have been added to the SBS primers, the reversible termination portion terminates further primer extension. For example, a nucleotide analog with a reversible terminator portion can be added to the primer so that subsequent extension cannot occur until a blocking agent is delivered to remove the portion. Therefore, for embodiments using reversible termination, a blocking agent can be delivered to beads or other solid supports (before or after detection occurs). Washing can be performed between each delivery step. Then, the cycle can be repeated n times so that the primers extend n nucleotides, thereby detecting a sequence with a length of n. Exemplary SBS procedures, fluidic systems, and detection platforms that can be readily adapted for use with the compositions, apparatus, or methods of the present disclosure are described, for example, in Bentley et al., Nature 456:53-59 (2008); PCT Publication 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. Pat. Pub. No. 2008 / 0108082, each of which is incorporated herein by reference.

[0105] Sequencing by ligation is a DNA sequencing method that uses the enzyme DNA ligase to identify the nucleotides present at a given position in a DNA sequence. Unlike most currently popular DNA sequencing methods, this method does not use DNA polymerase to generate the second strand. Instead, the mismatch sensitivity of DNA ligase 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. Patent Nos. 5,599,675 or 5,750,341, each of which is incorporated herein by reference. Some embodiments may include hybridization sequencing procedures, such as described in, for example, 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 ligation sequencing and hybridization sequencing procedures, the target nucleic acid (or its amplicon) present at the array site undergoes repeated cycles of oligonucleotide delivery and detection. The compositions, devices or methods described herein or in the references cited herein can be readily adapted for ligation sequencing or hybridization sequencing procedures. Typically, the oligonucleotides are fluorescently labeled and can be detected using fluorescence detectors similar to those described for the SBS procedures herein or in the references cited herein.

[0106] The method of the present disclosure can include the step of carrying out nucleic acid detection reaction on beads or other solid supports to determine the barcode sequence of the nucleic acid probe located on beads or other solid supports. In multiple embodiments, the probe is randomly located on beads or other solid supports, and the nucleic acid detection reaction provides information on locating each different probe. Exemplary nucleic acid detection methods include but are not limited to nucleic acid sequencing of probes, hybridization of nucleic acids with probes, connection of nucleic acids hybridized with probes, extension of nucleic acids hybridized with probes, extension of the first nucleic acid hybridized with probes, and then connection of the extended nucleic acid to the second nucleic acid hybridized with probes, or other methods known in the art, such as those described in U.S. Patent Nos. 8,288,103 or 8,486,625, each of which is incorporated herein by reference.

[0107] Solid support

[0108] In an exemplary embodiment, the spatial barcode nucleic acid is attached to a solid support via a cleavable linker. In one embodiment, the spatial barcode nucleic acid is directly attached to the solid support. In one embodiment, the spatial barcode nucleic acid is attached to a plurality of solid supports (e.g., beads), each of the plurality of solid supports having a unique spatial barcode sequence, which is further attached to a solid support (e.g., a glass slide). As used herein, the term "solid support" refers to a rigid substrate that is insoluble in aqueous liquids. The substrate can be non-porous or porous. The substrate may optionally be capable of absorbing liquids (e.g., due to porosity), but is typically sufficiently rigid so that the substrate does not substantially expand when absorbing liquids and does not substantially shrink when the liquid is removed by drying. Non-porous solid supports are generally impermeable to liquids or gases. Exemplary solid supports include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylic, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, Teflon TM , cyclic olefins, polyimides, etc.), nylon, ceramics, resins, Zeonor, silica or silica-based materials (including silicon and modified silicon), carbon, metals, inorganic glasses, fiber optic bundles, and polymers. Particularly useful solid supports for some embodiments are glass slides and beads that can be sorted / packed on the surface of a glass slide (e.g., beads with a large number of oligonucleotides attached).

[0109] Any of a variety of solid supports can be used in the methods, compositions or devices of the present disclosure. Particularly useful solid supports are those used for nucleic acid arrays. Examples include glass, modified glass, functionalized glass, inorganic glass, microspheres (e.g., inert and / or magnetic particles), plastics, polysaccharides, nylon, nitrocellulose, ceramics, resins, silica, silica-based materials, carbon, metals, optical fibers or fiber bundles, polymers and porous (e.g., microtiter) plates. Exemplary plastics include acrylic acid, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane and Teflon TM Exemplary silica-based materials include silicon and various forms of modified silicon.

[0110] In certain embodiments, the solid support can be within or a portion of a container, such as a well, tube, channel, cuvette, culture dish, bottle, or the like. Optionally, the container is a flow cell, such as described in WO 2014 / 142841 A1; U.S. Patent Application Publication No. 2010 / 0111768 A1 and U.S. Patent No. 8,951,781, or Bentley et al., Nature 456:53-59 (2008), each of which is incorporated herein by reference. An exemplary flow cell is commercially available from Illumina, Inc. (San Diego, Calif.) for use with sequencing platforms such as Genome or Optionally, the container is a well in a multiwell plate or a microtiter plate.

[0111] In an exemplary embodiment, the solid support may include a gel coating. For example, examples of nucleic acids attached to a solid support via a gel are available from Illumina (San Diego, Calif.) or described in U.S. Patent Application Publication Nos. 2011 / 0059865A1, 2014 / 0079923A1, or 2015 / 0005447A1; or the flow cell in PCT Publication No. WO 2008 / 093098, each of which is incorporated herein by reference. Exemplary gels that can be used for the methods and devices described herein include, but are not limited to, those having a colloidal structure, such as agarose; a polymeric network structure, such as gelatin; or a cross-linked polymer structure, such as polyacrylamide, SFA (see, for example, U.S. Patent Application Publication No. 2011 / 0059865A1, which is incorporated herein by reference) or PAZAM (see, for example, U.S. Patent Application Publication Nos. 2014 / 0079923A1 or 2015 / 0005447A1, each of which is incorporated herein by reference).

[0112] In some embodiments, the solid support can be configured as an array of features to which beads can be attached. Features can exist in any of a variety of desired formats. For example, features can be holes, pits, channels, ridges, raised areas, spikes, columns, etc. Exemplary features include holes present in substrates for commercial sequencing platforms sold by 454 Life Sciences (a subsidiary of Roche, Basel, Switzerland) or Ion Torrent (a subsidiary of Life Technologies, Carlsbad, Calif.). Other substrates having holes include, for example, etched optical fibers and other substrates described in U.S. Patent Nos. 6,266,459; 6,355,431; 6,770,441; 6,859,570; 6,210,891; 6,258,568; 6,274,320; U.S. Patent Application Publication Nos. 2009 / 0026082A1; 2009 / 0127589A1; 2010 / 0137143A1; 2010 / 0282617A1 or PCT Publication No. WO 00 / 63437, each of which is incorporated herein by reference. In some embodiments, the holes of the substrate can include a gel material (with or without beads), as described in U.S. Patent Application Publication No. 2014 / 0243224A1, which is incorporated herein by reference.

[0113] Features can appear on a solid support as a grid of spots or patches. Features can be located in a repeating pattern or an irregular non-repeating pattern. Optionally, the repeating pattern can include a hexagonal pattern, a rectilinear pattern, a grid pattern, a pattern with reflection symmetry, a pattern with rotational symmetry, etc. Asymmetric patterns can also be used. The spacing of the array can be the same between different pairs of nearest neighbor features, or the spacing can vary between different pairs of nearest neighbor features.

[0114] In certain embodiments, the features on the solid support may each have a diameter greater than about 100 nm. 2 , 250nm 2 , 500nm 2 , 1μm 2 , 2.5μm 2 , 5μm 2 , 10μm 2 or 50μm 2 Alternatively or additionally, the features may each have an area of less than about 50 μm 2 , 25μm 2 , 10μm 2 , 5μm 2 , 1μm 2 , 500nm 2 or 100nm 2The foregoing ranges may describe the apparent area of a bead or other particle on a solid support when viewed or imaged from above.

[0115] In an exemplary embodiment, the present disclosure provides a method for generating and using a spatially labeled micro-bead array to label cell nuclei with spatial barcode nucleic acids on frozen sectioned tissue samples at high image resolution. The method may include the following steps: (a) attaching different nucleic acid probes (spatial barcode nucleic acids) to beads, and then capturing the beads on a solid support to produce beads with randomly positioned probes on the solid support, wherein the different nucleic acid probes each include a barcode sequence (which is shared by all such nucleic acid probes of a single bead), and wherein each randomly positioned bead includes a barcode sequence different from other randomly positioned beads on the solid support; (b) performing a nucleic acid detection reaction on the solid support to determine the barcode sequence of the randomly positioned beads on the solid support; (c) contacting a biological sample with the solid support with the randomly positioned beads; (d) releasing the probes presented by the randomly positioned beads to label the biological sample adjacent to the randomly positioned beads; and (e) isolating the labeled cell nuclei, thereby spatially labeling the cell nuclei of the biological sample.

[0116] As used herein, "beads," "microbeads," "microspheres," or "particles," or grammatical equivalents, may include small discrete particles. The composition of the beads may vary depending on the type of capture probe, the method of synthesis, and other factors. In exemplary embodiments of the present disclosure, the size of the beads of the present disclosure tends to be in the range of 1 μm to 100 μm in diameter (all subranges within this range are expressly contemplated), depending, for example, on the degree of image resolution required, the nature of the solid support used for spatial bead array construction, the sequencing process to be adopted (e.g., flow cell sequencing), and other factors. In exemplary embodiments, the smaller the beads used, the tighter the space in which each spatial barcode is located, and the better the resolution. Using smaller beads, particularly when the distance between different spatial barcodes is less than the size of a single cell, allows the same cell nucleus to be labeled with more than one spatial barcode. This situation does not affect the resolution because single cells can be located at overlapping sites on the array. In the case where the beads are larger than a single cell, the resolution will be reduced because more than one cell nucleus is labeled with the same spatial barcode. In this case, single cells can only be located at positions on the array that are larger than a single cell.

[0117] Exemplary embodiments of the present disclosure employ a collection of beads or other particles to which oligonucleotides are attached. Bead sizes can range from nanometers (e.g., 100 nm) to millimeters (e.g., 1 mm), with beads typically ranging from about 0.2 μm to about 200 μm, and beads of about 5 to about 20 μm within the scope of the current examples, but in some embodiments, smaller or larger beads can be used. For example, beads are less than 50 μm, such as 1 μm, 3 μm, 10 μm, 15 μm, 20 μm, and particularly about 10 μm.

[0118] Suitable bead compositions include those used for peptide, nucleic acid and organic moiety synthesis, including but not limited to plastics, ceramics, glass, polystyrene, methylstyrene, acrylic polymers, paramagnetic materials, thorium oxide, carbon graphite, titanium dioxide, latex or cross-linked dextran such as Sepharose, cellulose, nylon, cross-linked micelles and Teflon can all be used. The "Microsphere Detection Guide" from Bangs Laboratories, FishersInd is a practical guide, the entire contents of which are incorporated herein by reference. The beads need not be spherical; irregular particles can be used. In addition, the beads can be porous, thereby increasing the surface area of the beads available for capture probe attachment or label attachment.

[0119] In an exemplary embodiment, the beads include any beads used in single cell genomics methods as further described herein. Non-limiting examples of beads include hydrogel particles (polyacrylamide, agarose, etc.), colloidal particles (polystyrene, magnetic or polymeric particles, etc.), any beads that can utilize phosphoramidate chemistry, such as those used in oligonucleotide synthesis known to those skilled in the art (e.g., methacrylate, polystyrene, polyacrylamide, polyethylene glycol), paramagnetic beads, and magnetic beads.

[0120] In an exemplary embodiment, the beads can be hydrogel particles (see, for example, International Patent Application Publication 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 monomers can be dispersed in droplets and then polymerized, for example, to form a gel.

[0121] In an exemplary embodiment, 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 copolymers of these and / or other polymers. Additionally, in some cases, the particles may be magnetic, which may allow for magnetic manipulation of the particles. For example, the particles may comprise iron or other magnetic materials. The particles may also be functionalized so that they may have other molecules attached, such as proteins, nucleic acids, or small molecules. In some embodiments, the particles may be fluorescent.

[0122] Beads containing spatial barcode nucleic acids of the present invention can be obtained by any previously described method. For example, spatial barcode nucleic acids can be synthesized directly on beads so that barcodes can be generated by random synthesis (see, for example, Macosko et al., 2015, "Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets" Cell 161, 1202-1214; and International Patent Application No. PCT / US2015 / 049178, published on March 17, 2016 as WO2016 / 040476). In an exemplary embodiment, beads are obtained by: 1) performing reverse phosphoramidite synthesis on the surface of the beads to synthesize the 5' end of the spatial barcode nucleic acid from the joint on the beads; 2) performing reverse phosphoramidite synthesis on the surface of the beads in a pool splitting manner so that in each synthesis cycle, the beads are split into four reactions with one of the four canonical nucleotides (T, C, G or A) or a unique oligonucleotide; 3) repeating the process multiple times, at least twice, and optimally more than twelve times, so 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., connecting) the 3' end of the spatial barcode nucleic acid containing a universal sequence for being captured by the cell barcode. In order to synthesize, the beads must be a material that can be maintained during organic synthesis. Non-limiting examples include any beads that can utilize phosphoramidite chemistry, such as those used in oligonucleotide synthesis known to those skilled in the art. Directly synthesizing oligonucleotides on beads is a preferred embodiment because direct synthesis allows for more diverse barcodes in more compact sequences.

[0123] In another example, spatial barcode nucleic acids can be synthesized by connecting oligonucleotides to beads, followed by split pool hybridization and extension to generate a unique cell barcode for each bead (see, for example, Klein et al., 2015, "Droplet Barcoding for Single-Cell Transcriptomics Applied to Embryonic Stem Cells" Cell 161, 1187-1201; and International Patent Application No. PCT / US2016 / 027734, published as WO2016168584A1 on October 20, 2016). In an exemplary embodiment, nucleic acid barcodes can be constructed in a combinatorial manner by combining randomly selected indexes (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 indexes selected from the sequence pool of each index). Each such index is a short nucleotide sequence (e.g., DNA, RNA, or a combination thereof) having a different sequence. The 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. Thus, in some embodiments, the possible barcodes used are formed from one or more individual "pools" of barcode elements, which are then joined together to generate the final barcode, for example using a split-pooling approach. The pools can 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, the first pool can contain x1 elements and the second pool can contain x2 elements; forming a barcode containing elements from the first pool and elements from the second pool can result in, for example, x1x2 possible barcodes that can be used. It should be noted that x1 and x2 can be equal or unequal. This process can be repeated any number of times; for example, a barcode can include elements from the first pool, the second pool, and the third pool (e.g., resulting in x1x2x3 possible barcodes), or from the first pool, the second pool, the third pool, and the fourth pool, etc. Thus, due to the potential number of combinations, even a relatively small number of barcode elements can be used to generate a large number of distinguishable barcodes.

[0124] In another example, spatially barcoded nucleic acids can be synthesized by ligating the 5' end of oligonucleotides containing adapter sequences to beads to produce functionalized beads, and then performing 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 / ncomms14049; International Patent Publication No. WO2014210353A2; and Zilionis et al., 2017, "Single-cell barcoding and sequencing using droplet microfluidics" Nat Protoc. Jan; 12(1): 44-73). In this example, each emulsion PCR reaction includes a single primer that hybridizes to the oligonucleotides on the functionalized beads and contains the barcode sequence. Thus, after several rounds of amplification, the barcode sequence is transferred to each oligonucleotide on the functionalized beads. This results in each bead having a barcode that is unique to that bead.

[0125] In exemplary embodiments, the beads can be suspended in a solution, or they can be located on the surface of a substrate (e.g., arranged on the surface of a solid support such as a glass slide). Art-recognized examples of arrays having beads located on a surface include those in which the beads are located in wells, such as BeadChip arrays (Illumina Inc., San Diego Calif.), substrates used in sequencing platforms from 454 LifeSciences (a subsidiary of Roche, Basel Switzerland), or substrates used in sequencing platforms from Ion Torrent (a subsidiary of Life Technologies, Carlsbad Calif.). Other solid supports having beads located on the surface are described in U.S. Patent Nos. 6,266,459; 6,355,431; 6,770,441; 6,859,570; 6,210,891; 6,258,568; 6,274,320; U.S. Patent Application Publication Nos. 2009 / 0026082A1; 2009 / 0127589A1; 2010 / 0137143A1; 2010 / 0282617A1 or PCT Publication No. WO 00 / 63437, each of which is incorporated herein by reference. Several of the above references describe methods of attaching nucleic acid probes to beads before loading the beads into or onto the solid support. Thus, a bead collection can include different beads, each with a unique (or sufficiently unique and / or nearly unique, as described elsewhere herein) probe attached. However, it should be understood that the beads can be made to include universal primers, which can then be loaded onto an array to form a universal array for use in the methods described herein. Solid supports typically used for bead arrays can be used without beads. For example, nucleic acids, such as probes or primers, can be directly attached to the wells or gel material in the wells. Thus, the above references illustrate materials, compositions, or devices that can be modified for use in the methods and compositions described herein.

[0126] In another embodiment, the present invention can adopt bead array, wherein different nucleic acid probes are attached to the different beads in the array.In this embodiment, each bead can be attached to different nucleic acid probes and beads can be randomly distributed on the solid support so that different nucleic acid probes are attached to the solid support effectively. Optionally, the solid support can include a hole with a size of accommodating no more than a single bead. In this configuration, the bead is attached to the hole owing to the force generated by the adaptation of the bead in the hole. As described elsewhere herein, attachment chemicals or capture materials (such as vinyl polymer liquid electrical tape) can also be used to make beads adhere to the solid support or otherwise stably associate, optionally including beads being maintained in the hole that may or may not be present on the solid support.

[0127] The nucleic acid probe attached to beads can include a barcode sequence. Bead colony can be configured so that each bead is only attached to a type of barcode (such as spatial barcode), and each many different beads with different barcodes are present in the colony. In this embodiment, beads are randomly distributed to solid supports so that the beads (and their corresponding barcode sequences) presenting nucleic acid probes are randomly positioned on the solid support. In some cases, there may be multiple beads with identical barcode sequences so that there is redundancy in the colony. However, randomly distributing the bead colony (particularly the bead colony with a capacity greater than the number of unique barcodes in the bead colony) comprising redundancy on the solid support will tend to cause the redundancy of barcode on the solid support, which will tend to reduce image resolution in the context of the present disclosure (that is, wherein the precise position of the barcoded beads cannot be distinguished due to the redundancy used by the barcode in the array bead colony, it is expected that such redundant positions will simply be eliminated from the final image produced by the method of the present disclosure, or other adjustment modes (such as, the normalization and / or average of values) can also be adopted to solve such redundancy). Alternatively, in preferred embodiments, the number of different barcodes in the bead population can exceed the capacity of the solid support to produce an array that is not redundant with respect to the population of barcodes on the solid support. In some embodiments, the capacity of the solid support will be determined by the number of features (e.g., wells occupied by a single bead) that can be attached or otherwise accommodated by beads.

[0128] The beads or other solid supports for presenting nucleic acids of the present disclosure may include or may be prepared by the methods described herein to attach a plurality of different nucleic acid probes. For example, the beads or other solid supports for presenting nucleic acids may include at least 10, 100, 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 Alternatively or in addition, beads or other solid supports presenting nucleic acids may comprise up to 1×10 9 , 1×10 8 , 1×10 7 , 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3, 100 or less different probes. It should be understood that each different probe can exist in several copies, for example, when the probe has been amplified to form a cluster. Therefore, the above ranges can describe the number of different nucleic acid clusters on the beads of the present disclosure or other solid supports for presenting nucleic acids. It should also be understood that the above ranges can describe the number of different barcodes, target capture sequences or other sequence elements described herein, which are unique (or unique enough) for a specific nucleic acid probe. Alternatively or additionally, the ranges can describe the number of extended probes or modified probes produced on the beads of the present disclosure or other solid supports for presenting nucleic acids using the methods described herein.

[0129] Before making beads or other solid supports contact with nucleic acid probes, features may be present on beads or other solid supports of the present disclosure. For example, in embodiments where probes are attached to beads or other solid supports by hybridization with primers, primers may be attached to features, and the gap region outside the features is substantially free of any primers. Nucleic acid probes may be captured at preformed features on beads or other solid supports, and optionally amplified on beads or other solid supports, such as by using the methods described in U.S. Patent Nos. 8,895,249 and 8,778,849 and / or U.S. Patent Publication No. 2014 / 0243224A1, each of which is incorporated herein by reference. Alternatively, beads or other solid supports may have a primer layer or may otherwise lack features. In this case, features may be formed by attaching nucleic acid probes to beads or other solid supports. Optionally, the captured nucleic acid probes may be amplified on beads or other solid supports so that the resulting clusters become features. Although attachment is exemplified above as capture between complementary portions of a primer and a probe, it should be understood that capture moieties other than primers can be present at preformed features or as a flat layer. Other exemplary capture moieties include, but are not limited to, chemical moieties capable of reacting with a nucleic acid probe to produce a covalent bond or receptors capable of non-covalently binding to a ligand on a nucleic acid probe.

[0130] The step of nucleic acid probe being attached to bead or other solid support can be carried out by providing the fluid of the mixture containing different nucleic acid probes and making described fluid mixture contact with bead or other solid support.Described contact can make fluid mixture contact with the surface that many different nucleic acid probes from fluid mixture will attach.Therefore, probe enters surface (no matter surface has the preformed feature that is configured to attach probe or is configured for the uniform surface of attachment) at random.Therefore, probe can be located at bead or other solid support at random.

[0131] The total number and type of different probes ultimately attached to a surface can be selected for a specific application or use. For example, in embodiments where a fluid mixture of different nucleic acid probes is contacted with beads or other solid supports for the purpose of attaching the probes to the support, the number of different probe types can exceed the occupancy rate of the beads or other solid supports for the probes. Thus, the number and type of different probes attached to the beads or other solid supports can be equal to the occupancy rate of the beads or other solid supports for the probes.

[0132] Alternatively, the number and variety of different probe species on the beads or other solid support can be less than the occupancy (i.e., there will be a redundancy of probe species such that the beads or other solid support can contain multiple features with the same probe species). Such redundancy can be achieved, for example, by contacting the beads or other solid support with a fluid mixture containing many and various probe species that are significantly less than the probe occupancy of the beads or other solid support.

[0133] Attachment of the nucleic acid probe can be mediated by hybridization of the nucleic acid probe to a complementary primer attached to a bead or other solid support, chemical bond formation between a reactive portion on the nucleic acid probe and the bead or other solid support (examples are described in U.S. Pat. Nos. 8,895,249 and 8,778,849 and U.S. Patent Publication No. 2014 / 0243224A1, each of which is incorporated herein by reference), affinity interaction of a portion on the nucleic acid probe with a portion bound to the bead or other solid support (e.g., between known receptor-ligand pairs such as streptavidin-biotin, antibody-epitope, lectin-carbohydrate, etc.), physical interaction of the nucleic acid probe with the bead or other solid support (e.g., hydrogen bonding, ionic forces, van der Waals forces, etc.), or other interactions known in the art for attaching nucleic acids to surfaces.

[0134] In certain embodiments, the attachment of nucleic acid probes is non-specific for any sequence difference between nucleic acid probes and other nucleic acid probes that are attached or will be attached to beads or other solid supports. For example, different probes can have universal sequences complementary to the primers attached to the surface, or different probes can have a common part that mediates and attaches to the surface. Alternatively, every kind of different probes (or a subgroup of different probes) can have unique (or unique enough) sequences complementary to the unique (or unique enough) primers on beads or other solid supports, or they can have unique (or unique enough) parts that interact with one or more different reactive moieties on beads or other solid supports. In this case, unique (or unique enough) primers or unique (or unique enough) parts can optionally be connected in predetermined locations to selectively capture specific probes or specific types of probes at corresponding predetermined locations.

[0135] One or more features on beads or other solid supports can each include a single molecule of a specific probe. In certain embodiments, features can be configured to accommodate no more than a single nucleic acid probe molecule. However, no matter whether a feature can accommodate more than one nucleic acid probe molecule, the feature can still include no more than a single nucleic acid probe molecule. Alternatively, a single feature can include a plurality of nucleic acid probe molecules, for example, a set of nucleic acid probe molecules with identical sequences to each other. In a specific embodiment, a set can be generated by amplifying from a single nucleic acid probe template to produce an amplicon, for example, as a cluster attached to a surface.

[0136] Methods described herein can use any of a variety of amplification techniques. The 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 priming amplification (RPA). In certain embodiments, amplification can be carried out in solution, such as when the feature of the array can contain amplicon in a volume with the desired capacity. In certain embodiments, the amplification technique used in the method of the present disclosure will be carried out on a solid phase. For example, one or more primer materials (for example, universal primers for one or more universal primer binding sites present in nucleic acid probes) can be attached to beads or other solid supports. In PCR embodiments, one or two of the primers for amplification can be attached to beads or other solid supports (for example, by gel). The form of two primers attached to beads or other solid supports is commonly referred to as bridge amplification because double-stranded amplicon forms a bridge-like structure between the primers attached to two surfaces, and the template sequence that the primer side joint has copied. Exemplary reagents and conditions that can be used for bridge amplification are described in, for example, U.S. Patent Nos. 5,641,658; 7,115,400; and 8,895,249; and / or U.S. Patent Publication Nos. 2002 / 0055100A1, 2004 / 0096853A1, 2004 / 0002090A1, 2007 / 0128624A1, and 2008 / 0009420A1, each of which is incorporated herein by reference. Solid-phase PCR amplification can also be performed with one of the amplification primers attached to a bead or other solid support and a second primer in solution. An exemplary format using a combination of surface-attached primers and soluble primers is that 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 Publication Nos. 2005 / 0130173A1 or 2005 / 0064460A1, each of which is incorporated herein by reference. Emulsion PCR is illustrative of the format, and it should be understood that for the purposes of the methods described herein, the use of an emulsion is optional, and indeed for several examples, no emulsion is used.

[0137] The RCA technique can be modified for use in the methods of the present disclosure. Exemplary components that can be used in RCA reactions and the principles of RCA amplicons are described in, for example, Lizardi et al., Nat. Genet. 19: 225-232 (1998) and U.S. Patent Publication No. 2007 / 0099208A1, each of which is incorporated herein by reference. The primers used for RCA can be in solution or attached to beads or other solid supports. The primers can be one or more of the universal primers described herein.

[0138] The MDA technique can be modified for use in the methods of the present disclosure. Some basic principles and useful conditions of MDA are described in, for example, 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. Patent Nos. 5,455,166; 5,130,238; and 6,214,587, each of which is incorporated herein by reference. The primers used for MDA can be attached to beads or other solid supports in solution or at the amplification site. Similarly, the primers can be one or more of the universal primers described herein.

[0139] In a specific embodiment, the combination of the amplification techniques of the above examples can be used. For example, RCA and MDA can be used in combination, wherein RCA is used to produce concatemer amplicons (for example, using solution phase primers) in solution. The primers (for example, universal primers) attached to beads or other solid supports can then be used to use the amplicon as a template for MDA. In this example, the amplicon produced after the RCA and MDA steps of the combination will be connected to beads or other solid supports.

[0140] In certain aspects of the present disclosure, a capture material is used to associate the bead array with a solid support (e.g., a glass slide). In some embodiments, the capture material is liquid electrical tape. An exemplary liquid electrical tape of the present disclosure is Permatex TM Liquid electrical tape is a weatherproof protectant for wiring and electrical connections. Liquid capture materials such as liquid tape can be applied as a liquid and then dry into a vinyl polymer that resists dirt, dust, chemicals, and moisture. The capture materials of the present disclosure can be applied by any of a number of methods, including brushing onto a solid support, spraying onto a solid support, or by immersing the solid support in the capture material. For some forms of liquid capture materials, the use of a brush-top applicator can allow for gapless coverage and can enable access to tight spaces, which in some embodiments provides advantages over forms of capture materials (i.e., tape) that are applied in a non-liquid state.

[0141] While liquid electrical tape has been exemplified as a capture material for use in the methods and compositions of the present disclosure, other capture materials are contemplated for such use, including any art-recognized glue or other agent that (a) is spreadable and / or depositable on a solid surface (e.g., on a glass slide, optionally on a slide that allows light to be transmitted through the slide, such as a microscope slide) and (b) is capable of binding or otherwise capturing a population of beads ranging in size from 1 to 100 μm. Exemplary other capture materials that are specifically contemplated include latexes, such as cis-1,4-polyisoprene and other rubbers, and elastomers (which are generally defined as polymers having viscoelastic properties (i.e., both viscosity and elasticity), very weak intermolecular forces, and generally a low Young's modulus and a high failure strain compared to other materials), including artificial elastomers (e.g., neoprene) and / or silicone elastomers. Acrylate polymers (e.g., Scotch tape) are also specifically contemplated, for example, for use as capture materials of the present disclosure. Exemplary other capture materials specifically contemplated include super glue adhesives (e.g., Loctite super glue), gels (e.g., agarose, acrylamide), rubberized sealant sprays (e.g., Gorilla Glue, PLASTIDIP Multipurpose Rubber Coating), silicone conformal coatings (e.g., Fine-L-Kote SR), or other conformal coatings (e.g., polyurethane resins, epoxies, acrylic resins, parylene). Additionally, bead oligonucleotides can be transferred to agarose or acrylamide gel substrates for subsequent release into tissue.

[0142] In an exemplary embodiment, the array is printed on a solid support. In one embodiment, the printed spatial barcode nucleic acid is amplified (e.g., bridge amplification) on a solid support as described herein. In an exemplary embodiment, the solid support is a glass slide or an array on a glass slide. As used herein, the term "glass slide" includes an "array," "substrate," or "surface" comprising a plurality of spatial barcode nucleic acids as described herein. For the spatial array-based analysis methods described herein, the substrate is used as a support for the direct or indirect attachment of spatial barcode nucleic acids to array features. In addition, in some embodiments, a substrate (e.g., the same substrate or a different substrate) can be used to provide support for biological samples, particularly, for example, thin tissue sections. Therefore, a "substrate" is a support that is insoluble in aqueous liquids and allows biological samples, analytes, features, and / or spatial barcode nucleic acids to be positioned on a substrate.

[0143] In addition, " substrate " as used herein, and when there is no modifier " chemical " in front, refers to a member with at least one surface, the surface is generally used for providing physical support for biological samples, analytes and / or any other chemical and / or physical parts, reagents and structures described herein. The substrate can be formed by various solid materials, gel-based materials, colloidal materials, semi-solid materials (such as at least partially cross-linked materials), completely or partially solidified materials and materials that undergo phase change or transformation to provide physical support. Examples of substrates that can be used for the methods and systems described herein include, but are not limited to, slides (for example, slides formed by various glasses, slides formed by various polymers), hydrogels, layers and / or films, membranes (such as porous membranes), flow cells, cuvettes, wafers, plates or combinations thereof. In some embodiments, the substrate can optionally include functional elements, such as recesses, protruding structures, microfluidic elements (for example, channels, reservoirs, electrodes, valves, seals) and various labels. Slides and arrays for spatial profiling have been described (see, e.g., Visium Spatial Capture Technology, 10X Genomics, Pleasanton, CA; WO2020047007A2; WO2020123317A2; WO2020047005A1; WO2020176788A1; and WO2020190509A9). Capture probes comprising spatial barcodes can be replaced with spatial barcode nucleic acids comprising spatial barcodes as described herein.

[0144] Slides containing spatially barcoded nucleic acids can be obtained by synthesizing spatially barcoded nucleic acids and attaching them to a slide or array. In an exemplary embodiment, spatially barcoded nucleic acids are added to specific locations of an array.

[0145] Arrays can be prepared by depositing features (e.g., droplets, beads) on the substrate surface to produce spatial barcoding arrays. Methods for depositing (e.g., droplet manipulation) features are known in the art (see, U.S. Patent Application Publication No. 2008 / 0132429; Rubina, AY et al., Biotechniques. May 2003; 34(5): 1008-14, 1016-20, 1022; and Vasiliskov et al., Biotechniques. September 1999; 27(3): 592-4, 596-8, 600 everywhere). A can print or deposit features (e.g., inkjet printing) at specific locations on a substrate. In some embodiments, each feature can have a spatial barcode nucleic acid. In some embodiments, an electric field can be used to print or deposit features at specific locations. Features can contain photocrosslinkable polymer precursors and oligonucleotides. In some embodiments, photocrosslinkable polymer precursors can be deposited into patterned features on a substrate (e.g., a hole). "Photocrosslinkable polymer precursors" refers to compounds that crosslink and / or polymerize when exposed to light. In some embodiments, one or more photoinitiators may also be included to induce and / or promote polymerization and / or crosslinking (see, e.g., Choi et al., Biotechniques. 2019 Jan; 66(1): 40-53).

[0146] Array can be prepared by a variety of methods. In certain embodiments, by synthesizing (such as in situ synthesis) oligonucleotides on the array or by jet printing or photolithography to prepare the array. In an exemplary embodiment, spatial barcode nucleic acid provides a deterministic patterning of synthesis. For example, the light-directed synthesis of high-density DNA oligonucleotides can be achieved by photolithography or solid-phase DNA synthesis. In order to implement photolithographic synthesis, the synthetic linker modified with a photochemical protecting group can be connected to a substrate, and a photolithographic mask (applied to a specific area of the substrate) and light can be used to modify the photochemical protecting group, thereby producing an array with local photodeprotection. Many of these methods are known in the art, and are described in, for example, Miller et al., " Basic concepts of microarrays and potential applications in clinical microbiology." Clinical Microbiology Reviews 22.4 (2009): 611-633; US201314111482A; US9593365B2; US2019203275; and WO2018091676.

[0147] organize

[0148] In certain embodiments, tissue sections are used. Tissue can be derived from a multicellular organism. Exemplary multicellular organisms include, but are not limited to, mammals, plants, algae, nematodes, insects, fish, reptiles, amphibians, fungi or Plasmodium falciparum. Exemplary substances are described herein or are known in the art. Tissue can be freshly excised from an organism, or it can be preserved in advance by, for example, freezing, embedding in materials such as paraffin (e.g., formalin-fixed paraffin-embedded samples (FFPE)), formalin fixation, infiltration, dehydration, etc. Optionally, tissue sections can be frozen and sectioned using techniques and compositions as described herein and known in the art. Alternatively, tissue can be permeabilized to allow the nucleus to be accessible to spatial barcode nucleic acids. As used herein, the term "tissue" is intended to refer to an aggregate of cells and optional intercellular substances. Typically, the cells in a tissue are not free-floating in a solution, but are attached to each other to form a multicellular structure. Exemplary tissue types include muscle, nerve, epidermis, and connective tissue. The tissue can also be from a subject with a disease, such as, but not limited to, an autoimmune disease or cancer (eg, tissue from irritable bowel disease (IBD) or MS, or tumor tissue).

[0149] The method of the present disclosure may include the step of contacting a biological sample (i.e., a frozen sectioned tissue sample) with beads or other solid supports having attached thereto a spatial barcode nucleic acid. In some embodiments, the spatial barcode nucleic acid is randomly located on the beads or other solid supports. Before the biological sample is contacted with the beads or other solid supports, the identity and position of the spatial barcode nucleic acid may have been decoded. Alternatively, the identity and position of the spatial barcode nucleic acid can be determined after the beads or other solid supports are contacted with the biological sample.

[0150] As used herein, the term "cryosection" refers to a piece of tissue, such as a biopsy, that has been obtained from a subject, rapidly frozen, embedded in an optimal cutting temperature embedding material, frozen, and cut into thin sections. In certain embodiments, the thin sections can be applied directly to a bead array captured on a solid support (e.g., a glass slide), or the thin sections can be fixed (e.g., fixed in methanol or paraformaldehyde) and applied to a planar surface presenting beads, such as a glass slide to which a layer of microbeads has been attached / arrayed.

[0151] In exemplary embodiments, tissues can be obtained from complex multicellular systems (e.g., organoids, tissue explants, or organs 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, RJ, Murray, GI and McLean, MH Current concepts in tumor-derived organoids. Br J Cancer123,1209-1218(2020).doi.org / 10.1038 / s41416-020-0993-5; Sontheimer-Phelps, A., Hassell, BA, and Ingber, DEModelling 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); 145,pii:dev156125(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; NeilJE, Brown MB, Williams AC. Human skin explant model for the investigation oftopical 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 patient-derived organoids (PDO) or patient-derived xenografts (PDX). .

[0152] Tissue can be prepared in any convenient or desired manner for use in the methods, compositions, or devices herein. Fresh, frozen, fixed, or unfixed tissue can be used. Tissue can be fixed or embedded using methods described herein or known in the art.

[0153] Tissue samples for this article can be fixed by deep freezing at a temperature (e.g., below -20°C) suitable for maintaining or keeping the structural integrity of the tissue. In another example, formalin fixation and paraffin embedding (FFPE) methods known in the art can be used to prepare tissue. Other fixatives and / or embedding materials can be used as needed. Fixed or embedded tissue samples can be sliced, i.e., thin slices, using known methods. For example, a freezing microtome or a cryostat can be used to slice the tissue sample, and the device is set at a temperature suitable for maintaining the structural integrity of the tissue sample and the chemical properties of the nucleic acid in the sample. Exemplary additional fixatives clearly considered include alcohol fixation (e.g., methanol fixation, ethanol fixation), glutaraldehyde fixation, and paraformaldehyde fixation.

[0154] In certain embodiments, before labeling the nucleus, the tissue sample will be processed to remove embedding material (e.g., paraffin or formalin) from the sample. This can be achieved by contacting the sample with a suitable solvent (e.g., dimethylbenzene and ethanol washes). Processing can be carried out before the tissue sample contacts a spatial array as described herein, or processing can be carried out when the tissue sample is positioned on a bead array captured by a solid support.

[0155] Exemplary methods for manipulating tissue for use with nucleic acid-attached solid supports are described in US Patent Application Publication No. 2014 / 0066318 Al, which is incorporated herein by reference.

[0156] The thickness of the tissue sample or other biological sample contacted with the bead array in the methods, compositions or devices described herein can be any suitable thickness desired. In representative embodiments, the thickness will be at least 0.1 μm, 0.25 μm, 0.5 μm, 0.75 μm, 1 μm, 5 μm, 10 μm, 50 μm, 100 μm or thicker. Alternatively or in addition, the thickness of the tissue sample contacted with the bead array will not exceed 100 μm, 50 μm, 10 μm, 5 μm, 1 μm, 0.5 μm, 0.25 μm, 0.1 μm or thinner.

[0157] A particularly relevant source of tissue samples is humans. Another source of tissue samples is animal models (eg mouse tissue samples). The sample can be derived from an organ, including, for example, an organ of the central nervous system, such as the brain, brainstem, cerebellum, spinal cord, cranial nerves or spinal nerves; an organ of the musculoskeletal system, such as muscle, bone, tendon or ligament; an organ of the digestive system, such as salivary glands, pharynx, esophagus, stomach, small intestine, large intestine, liver, gall bladder or pancreas; an organ of the respiratory system, such as the larynx, trachea, bronchi, lungs or diaphragm; an organ of the urinary system, such as the kidney, ureter, bladder or urethra; a reproductive organ, such as the ovary, fallopian tube, uterus, vagina, placenta, testicle, epididymis, vas deferens, seminal vesicle, prostate, penis or scrotum; an organ of the endocrine system, such as the pituitary gland, pineal gland, thyroid gland, parathyroid gland or adrenal gland; an organ of the circulatory system, such as the heart, artery, vein or capillary blood vessels; an organ of the lymphatic system, such as lymphatic vessels, lymph nodes, bone marrow, thymus gland or spleen; a sensory organ, such as the eye, ear, nose or tongue; or an integumentary organ, such as the skin, subcutaneous tissue or mammary gland. In some embodiments, the tissue sample is obtained from a body fluid or excretion, such as blood, lymph, tears, sweat, saliva, semen, vaginal secretions, earwax, fecal matter, or urine.

[0158] When used, samples from the mankind can be considered (or suspected) healthy or sick. In some cases, two samples can be used: the first is considered to be sick, and the second is considered to be healthy (e.g., used as a healthy control). Any of a variety of patient conditions can be assessed, including but not limited to autoimmune diseases, cancer, cystic fibrosis, aneuploidy, pathogenic infection, psychological condition, hepatitis, diabetes, sexually transmitted diseases, heart disease, stroke, cardiovascular disease, multiple sclerosis (MS) or muscular dystrophy. Some expected patient conditions include genetic conditions or the patient conditions associated with pathogens with identifiable genetic characteristics.

[0159] In an exemplary embodiment, tissue is prepared for nuclear labeling using steps from single-nucleus 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, No. 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 on September 28, 2017 as WO2017164936; International Patent Application No. PCT / US2018 / 060860, published on May 16, 2019 as WO / 2019 / 094984; International Patent Application No. PCT / US2019 / 055894, published on April 16, 2020 as WO / 2020 / 077236; 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 ... E, Smillie CS, Van Wittenberghe N, et al., The Human and MouseEnteric Nervous System at Single-Cell Resolution. Cell. 2020;182(6):1606-1622.e23; and Slyper, M., Porter, CBM, Ashenberg, O. et al., (2020). A single-cellandsingle-nucleus RNA-seq toolbox for fresh and frozen human tumors. NatureMedicine 26(5):792-802).In an exemplary embodiment, methods for preparing and isolating cell nuclei that retain the nuclear envelope and ribosomes (Ribosomes and Intact Single Nucleus (RAISIN) RNA-seq) or retain the rough ER on the outer nuclear membrane and its attached ribosomes (Intact Nucleus and Endoplasmic Reticulum from Single Cell (INNER Cell) RNA-seq) are applicable to labeled cell nuclei (see WO / 2020 / 077236).

[0160] In an exemplary embodiment, mononuclei are prepared from FFPE tissue sections (see, for example, WO / 2020 / 077236). For example, FFPE tissue samples are processed by dissolving paraffin in a solvent, preferably selected from xylene or mineral oil, wherein the tissue is dissolved at a temperature between 4°C and 90°C, preferably at room temperature (20 to 25°C) or 90°C; rehydrating the tissue using an ethanol gradient from 100% to 0% ethanol (EtOH); transferring the rehydrated tissue to a volume of a first buffer comprising a buffer, a detergent, and an ionic strength of 100 mM to 200 mM, optionally comprising a protease inhibitor or protease and / or BSA. In an exemplary embodiment, mononuclei are prepared from tissue samples using a buffer comprising 10 mM Tris, 0.49% CHAPS, 146 mM NaCl, 1 mM CaCl2, 21 mM MgCl2, and 0.01% BSA (CST) (see, for example, Risin-seq). In one exemplary embodiment, single nuclei are prepared from tissue samples using a buffer containing 10 mM Tris, 0.03% Tween-20, 146 mM NaCl, 1 mM CaCl2, 21 mM MgCl2, and 0.01% BSA (TST) (see, e.g., INNER Cell-seq). In one exemplary embodiment, a buffer is used during the extraction of nuclei from tissue.

[0161] In some embodiments, the biological sample can be permeabilized to facilitate the transfer of the spatial barcode nucleic acid into the sample. If the sample is not fully permeabilized, the amount of the spatial barcode nucleic acid entering the sample may be too low to be fully analyzed. Conversely, if the tissue sample is too permeabilized, the relative spatial relationship of the analytes within the tissue sample may be lost. Therefore, it is desirable that the tissue sample be permeabilized enough to obtain good signal intensity while still maintaining a balance between the spatial resolution of the analyte distribution in the sample.

[0162] Typically, a biological sample can be permeabilized by exposing the sample to one or more permeabilizing agents. Suitable reagents for this purpose include, but are not limited to, organic solvents (e.g., acetone, ethanol, and methanol), cross-linking agents (e.g., paraformaldehyde), detergents (e.g., saponin, Triton X-100, TM 、Tween-20TM or sodium dodecyl sulfate (SDS)) and an enzyme (e.g., trypsin, a protease (e.g., proteinase K)). In some embodiments, the detergent is an anionic detergent (e.g., SDS or a solution of N-lauroylsarcosine sodium salt). 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, such as SDS and / or a solution of N-lauroylsarcosine sodium salt) before or after enzymatic treatment (e.g., treatment with any enzyme described herein, such as trypsin, a protease (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.

[0163] Cleavage of the linker and delivery of the spatial barcode to the nucleus

[0164] In an exemplary embodiment, the spatial barcode nucleic acid in contact with the permeabilized tissue sample is delivered to the cell nucleus. In an exemplary embodiment, the tissue sample is incubated in a dissociation buffer as described herein (see Example 1 Dissociation Buffer (DB)). In an exemplary embodiment, the incubation is at 4-25°C, preferably about 4°C. The linker is then cleaved (for example, using light, chemicals or enzymes) to release the spatial barcode nucleic acid from the spatial array. In an exemplary embodiment, the spatial barcode nucleic acid is delivered to the cell nucleus by diffusion. In an exemplary embodiment, the spatial barcode nucleic acid is delivered to the cell nucleus using electroporation. Through electroporation, the spatial barcode nucleic acid can enter the cell through one or more holes in the cell membrane formed by the applied electricity. Based on the applied field strength and pulse duration, the pores of the membrane can be reversible. In an exemplary embodiment, the spatial barcode nucleic acid is delivered to the cell nucleus using sonoporation. Sound waves can be used to temporarily permeabilize the cell membrane, allowing the cell to take up the spatial barcode nucleic acid. In an exemplary embodiment, water perforation (hydroporation) is used to deliver the spatial barcode nucleic acid to the cell nucleus. The spatial barcode nucleic acid can be delivered to the cell by fluid dynamic pressure.

[0165] Isolation of labeled nuclei from tissue

[0166] In an exemplary embodiment, the nuclei are extracted from the labeled tissue by dissociating the tissue with an extraction buffer (see, e.g., Example 1, extraction buffer (ExB)). In an exemplary embodiment, the extraction buffer comprises a detergent. In an exemplary embodiment, the detergent is Triton X-100. In an exemplary embodiment, the detergent is CHAPS (3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate). In an exemplary embodiment, the detergent is Tween-20. In one embodiment, the tissue is dissociated by pipetting. In one embodiment, the tissue is dissociated by gently scraping. In an exemplary embodiment, where the spatial barcode nucleic acid comprises a fluorescent dye, the labeled nuclei can be sorted by FACS. Therefore, only the labeled nuclei will be used in the following steps. Sorting the nuclei can allow the same efficiency as using unlabeled nuclei.

[0167] Single-cell genomics assays

[0168] Single-cell genomic sequencing was first achieved for transcriptomes in 2009, followed by rapid development of technologies for profiling the genome, DNA methylome, 3D genome architecture, chromatin accessibility, histone modifications, etc. in individual cells (see, for example, Wen L, Tang F. Recent advances in single-cell sequencing technologies. Precis Clin Med. 2022; 5(1): pbac002. Published on January 31, 2022). In exemplary embodiments, the labeled nuclei produced by the methods of the present invention can be used with any of these single-cell genomic assays to enable spatial resolution of the assay.

[0169] In an exemplary embodiment, single-cell genomics assays typically include a barcode (i.e., a cell barcode) for identifying the source cell of a genomic analyte. For example, the analyte can be RNA, such as poly-A tail mRNA, genomic DNA sequence, connected DNA fragments, immunoprecipitated genomic DNA, or antibody-specific oligonucleotides. The cell barcode is also typically associated with a unique molecular identifier (UMI) so that the number of each analyte of each single cell can be counted (i.e., quantified). As used herein, the term "unique molecular identifier" (UMI) refers to a sequencing adapter or nucleic acid barcode subtype used in a method using a molecular tag to detect and quantify a unique amplification product (see, for example, Islam S et al., 2014. Nature Methods No: 11, 163-166). In an exemplary embodiment, the UMI is a random sequence of 4 to 20 bases. The UMI is used to distinguish the effect of a single clone from multiple clones (e.g., an amplification product from a single clone will include the same UMI as the original clone). As used herein, the term "clone" can refer to a single mRNA or target nucleic acid to be sequenced. Therefore, UMIs can also be used to determine the number of specific analytes or events that produced amplification products. In an exemplary embodiment, each cellular barcode of a single nucleus is identical, but each UMI associated with the cellular barcode is randomized so that each capture event has a unique UMI. For example, a spatially barcoded nucleic acid can be captured by a cellular barcoded nucleic acid that includes a UMI. The number of distinct UMIs sequenced for a spatial barcode in a single nucleus identified by a cellular barcode will indicate the number of spatial barcodes that single nuclei were labeled with (e.g., counting UMIs of sequence reads with the same cellular barcode but different spatial barcodes).

[0170] In an exemplary embodiment, if a single cell is adjacent to more than one location on the array or overlaps with more than one location on the array, then a nucleic acid sequence comprising a cellular barcode and a UMI can capture more than one spatial barcode. In an exemplary embodiment, a computational method can be used to resolve the location of a single cell in a spatial array, for example by counting UMIs. The computational method can use the distance on the array of different spatial barcodes. If the spatial barcodes are close on the array, then the location is most likely somewhere between locations or overlapping with a location. If the spatial barcodes are far away, then the spatial barcode nucleic acid is most likely to diffuse away from its location, and that location is the spatial barcode with the most UMIs, because the spatial barcode with the higher UMI is the main spatial barcode that diffuses into the single cell.

[0171] Single cell assays typically add cellular barcodes by one of three methods; adding single cells / nuclei to separate wells, each with a unique barcode or combination of barcodes; performing pool splitting indexing of intact nuclei / cells; and separating single cells / nuclei into separate reaction vessels, such as microwells, reaction chambers, or droplets, that include unique cellular barcodes. In an exemplary embodiment, for pool splitting indexing, the spatial barcode nucleic acid is modified to include a common universal sequence (e.g., a treatment sequence) present on each target analyte, such that both the spatial barcode and the analyte can capture the same index sequence at each pool splitting step). In an exemplary embodiment, for the droplet method, 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 the cellular barcode nucleic acid present in each droplet (e.g., typically on a bead). Single cell assays typically use a capture sequence specific to the analyte, such as an analyte with a poly-A tail or a specific adapter sequence added by a transposase (e.g., a tag fragmentation adapter). These sequences can be readily included in spatially barcoded nucleic acids.

[0172] The methods of the present invention can be adapted so that spatial barcodes can be captured by cellular barcodes for any single cell assay. Specific examples are provided below.

[0173] Spatially resolved single-nucleus / cell RNA sequencing

[0174] In an exemplary embodiment, the single-cell genomics sequencing library is a single-cell RNA sequencing library (see, e.g., Trombetta, JJ, Gennert, D., Lu, D., Satija, R., Shalek, A.K., and Regev, A. Preparation of Single-Cell RNA-Seq Libraries for Next Generation Sequencing. Curr Protoc Mol Biol. 107, 422 21-24 22 17, doi: 10.1002 / 0471142727.mb0422s107 (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., and Quake, SR Genomic Analysis at the Single-Cell Level. Annual review of genetics 45, 431-445, (2011); Kalisky, T. and Quake, SRSingle-cellgenomics. Nature Methods 8, 311-314 (2011); Islam, S. et al., Characterization of the single-cell transcriptional landscape by highly multiplex RNA-seq. GenomeResearch, (2011); Tang, F. et al., RNA-Seq analysis to capture the transcriptomelandscape 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., and Yanai, I. CEL-Seq: Single-Cell RNA-Seq by Multiplexed Linear Amplification. Cell Reports, Cell Reports, Vol. 2, No. 3, pp. 666-673, 2012). PMCID: 4338574).

[0175] In an exemplary embodiment, the present invention relates to 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 an exemplary embodiment, the present invention relates to 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 No. PCT / US2015 / 049178, published on March 17, 2016 as WO 2016 / 040476; Klein et al., 2015, “Droplet Barcoding for Single-Cell Transcriptomics Applied to Embryonic Stem Cells,” Cell 161, 1187-1201; International Patent Application No. PCT / US2016 / 027734, published on October 20, 2016 as WO 2016168584 A1; 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 / ncomms14049; International Patent Application No. WO2014210353A2; Zilionis et al., 2017, “Single-cell barcoding and sequencing using droplet microfluidics” Nat Protoc.Jan;12(1):44-73; Cao et al., 2017, “Comprehensive single cell transcriptional profiling of a multicellular organism by combinatorial indexing” bioRxiv preprint first posted online February 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 February 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 organization. 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-CellRNA-Seq Reveals Cellular States and Molecular Features of Human Skin Pathology" bioRxiv689273; doi:doi.org / 10.1101 / 689273, all contents and disclosures of each of which are incorporated herein by reference in their entirety.

[0176] In an exemplary embodiment, the present invention relates to single-nucleus RNA sequencing. In this regard, reference is made to Swiech et al., 2014, “In vivo interrogation of gene function in the 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, No. 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 on September 28, 2017, as WO2017164936; International patent application No. PCT / US2018 / 060860, published on May 16, 2019, as WO / 2019 / 094984; International patent application No. PCT / US2019 / 055894, published on April 16, 2020, as WO / 2020 / 077236; Drokhlyansky et al., “The entericnervous 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 is incorporated herein by reference in its entirety.

[0177] Single cell / nucleus RNA-seq typically uses a poly-T capture sequence and reverse transcription (RT) to capture mRNA with a poly-A tail. In an exemplary embodiment, labeled nuclei are generated according to the present invention using spatial barcode nucleic acids containing poly-A sequences. The labeled nuclei are then used in any of the RNA-seq methods described above. For example, the labeled nuclei can be used in a pool splitting method, or can be separated into separate wells or droplets. Both the spatial barcode nucleic acid and the mRNA are captured by the cellular barcode sequence. For plate- or microwell-based methods, reverse transcription and the use of template switching primers can add unique barcodes to the mRNA and spatial barcode nucleic acids.

[0178] Spatially resolved single-cell chromatin accessibility

[0179] In an exemplary embodiment, the single-cell genomics sequencing library is a single-cell sequencing library using the assay for transposase-accessible chromatin via sequencing (ATAC-seq). ATAC-seq can be used to identify accessible chromatin in cells (see, e.g., Buenrostro et al., Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosomeposition. Nature methods 2013; 10(12): 1213-1218). Using plate-based, droplet-based, or combinatorial indexing-based methods, thousands to hundreds of thousands of individual cells / nuclei can be profiled 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, DA, Daza, R., Adey, A., Pliner, H., Christiansen, L., Gunderson, KL, Steemers, FJ, Trapnell, C., and 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.aab1601. Epub 2015 May 7; Cusanovich DA, Hill AJ, Aghamirzaie D et al., A Single-Cell atlas of in vivo mammalian chromatinaccessibility. Cell. 2018; 174:1309-24; Lake BB, Chen S, Sos BC et al., Integrative single-cell analysis of transcriptional and epigenetic states in the humanadult brain. Nat Biotechnol.2018;36:70-80; Preissl S, Fang R, Huang H, et al., Single-nucleus analysis of accessible chromatin in mouse developing forebrainreveals cell-type-specific transcriptional regulation. Nat Neurosci. 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 cellexhaustion. 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 chromatinaccessibility. Nat Protoc. 2021; 16: 4084-107; US20160208323A1; US20160060691A1; and WO2017156336A1). Single-nucleus ATAC-seq can also be performed by partitioning nuclei into droplets and subsequently constructing snATAC-Seq libraries using the Chromium Next GEM Single Cell ATAC Kit v1.1 (10× 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).

[0180] In one exemplary embodiment, labeled nuclei are generated according to the present invention using spatially barcoded nucleic acids containing adapter sequences identical to those inserted into active chromatin by the Tn5 transposase. Barcoded beads can then be used to capture labeled chromatin fragments and spatially barcoded nucleic acids via cellular barcoding using combinatorial indexing or droplet-based approaches.

[0181] Spatially resolved single-cell Hi-C

[0182] In an exemplary embodiment, the single-cell genomics sequencing library is a single-cell Hi-C sequencing library. In situ Hi-C involves cross-linking cells with formaldehyde; permeabilizing them with intact nuclei; digesting DNA with a suitable 4-cutting restriction enzyme (e.g., MboI); filling in 5'-overhangs while incorporating biotinylated nucleotides; ligating the resulting blunt-ended fragments; shearing the DNA; capturing the biotinylated junction with 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 using various barcoding methods, such as combinatorial cell 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, Várnai C, et al., Cell-cycle dynamics of chromosomal organization at single-cell resolution. Nature. 2017; 547: 61-7).

[0183] In an exemplary embodiment, according to the present invention, labeled nuclei are generated using spatial barcode nucleic acids containing double-stranded adapter sequences, the labeled nuclei are cross-linked, and the DNA is digested with appropriate restriction enzymes. The nuclei are then distributed into 96 wells, where the first barcode is introduced by connecting a barcoded biotinylated double-stranded bridge adapter. The intact nuclei are then combined and adjacently ligated, then diluted and redistributed into a second 96-well plate. After dissolution, the second barcode is introduced by connecting a barcoded Y-adapter. In this example, both the fragmented DNA and the spatial barcode receive the same nucleus-specific barcode.

[0184] In an exemplary embodiment, according to the present invention, labeled cell nuclei are generated using spatial barcode nucleic acids containing adapter sequences, the labeled cell nuclei are cross-linked, the DNA is digested with appropriate restriction enzymes, the resulting fragments are ligated, and the DNA is fragmented using a transposase (Tn5) loaded with adapters in intact cell nuclei, and then the cell nuclei can be used for droplet-based single-cell sequencing methods using beads that capture the adapter sequences and add cellular barcodes.

[0185] Spatially resolved single-cell DNA methylation sequencing

[0186] In an exemplary embodiment, the single-cell genomics sequencing library is a single-cell DNA methylation sequencing library. The method for distinguishing DNA methylation includes (i) bisulfite conversion, (ii) Tet-assisted bisulfite conversion, (iii) Tet-assisted conversion with a substituted borane reducing agent, and (iv) protecting hmC, followed by Tet-assisted conversion with a substituted borane reducing agent (see, for example, U.S. Patent Application No. US20210115502A1). Methylation can also be detected using methylation-specific restriction enzymes or methylated DNA immunoprecipitation (MeDIP). In an exemplary embodiment, DNA methylation can be detected, wherein methylated cytosine (mC) and hydroxymethylated cytosine (hmC) are determined by the sequencer itself and independently of one or more reagents (e.g., using PacBio or nanopore sequencers).Single-cell DNA methylome sequencing technologies have been established using various strategies, including reduced bisulfite sequencing (RRBS)-based and post-bisulfite adapter 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 inmammalian 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. Nat Biotechnol. 2021; 39:1086-94).The sci-MET method has applied a combinatorial indexing strategy to increase throughput, in which the first and second rounds of barcoding are 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).

[0187] Spatially resolved single-cell chromatin immunoprecipitation (ChIP)

[0188] In an exemplary embodiment, the single-cell genomics sequencing library is a single-cell chromatin immunoprecipitation (ChIP) sequencing library. Chromatin immunoprecipitation (ChIP) is a widely used method for detecting histone modifications in chromatin nucleosomes. In single-cell ChIP-seq analysis, cell-specific barcodes are added before cells are aggregated for immunoprecipitation (Ai S, Xiong H, Li CC, et al., Profiling chromatin states using single-cellitChIP-seq. Nat Cell Biol. 2019; 21: 1164-72; Grosselin K, Durand A, Marsolier J, et al., High-throughput single-cell ChIP-seq identifies heterogeneity of chromatinstates in breast cancer. Nat Genet. 2019; 51: 1060-6; and Rotem A, Ram O, Shoresh N, et al., Single-cell ChIP-seq reveals cell subpopulations defined by chromatinstate. Nat Biotechnol. 2015; 33: 1165-72). Among these methods, Drop-ChIP and scChIP-seq add cellular barcodes through MNase digestion and connection with a droplet microfluidics workflow, while itChIP adds cellular barcodes through Tn5 transposase-tagged fragmentation with a chromatin opening step.

[0189] In an exemplary embodiment, the spatial barcode is designed to allow for tag fragmentation or digestion and ligation, resulting in a barcode sequence having a cellular barcode and a spatial barcode. For example, a single cell nucleus can be isolated into a single droplet or microwell containing a cellular barcode. The cellular barcode / spatial barcode hybrid can be amplified and sequenced from the input chromatin for immunoprecipitation. This provides the spatial location of each cell. The immunoprecipitated chromatin can then be assigned to the same cell based on the cellular barcode.

[0190] Spatially resolved profiling of enzyme-tethered chromatin in single cells

[0191] In an exemplary embodiment, the single-cell genomics sequencing library is a single-cell enzyme-tethered chromatin profiling sequencing library. Enzyme tethering represents a non-immunoprecipitation chromatin profiling method that is becoming increasingly popular and has been adapted for 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, Maehara K, 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 Biol. 2019; 10:1931). 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 technologies, Tn5 transposase, MNase, or adenine methyltransferase is tethered to protein A that is bound to an antibody, directly bound to an antibody, or directly bound to a target chromatin protein, allowing the labeling of genomic regions with 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, with Ca2+ for MNase and Mg2+ for Tn5 transposase, allows for activation of enzyme activity within a short time window after washing away nonspecifically bound enzyme.

[0192] In an exemplary embodiment, a single labeled nucleus according to the present invention is labeled with a spatial barcode nucleic acid comprising a sequence that is identical to the tag fragmentation adapter or ligation adapter used in the enzyme-tethered chromatin profiling method. The enzyme-tethered chromatin profiling method adds adapters to chromatin bound by antibodies specific to chromatin modifications or proteins. These nuclei can then be used in a single-cell sequencing method comprising barcoded beads that contain sequences that hybridize to the adapter sequence and add bead-specific barcodes to the labeled chromatin and spatial barcodes (e.g., a 10X ATAC-seq kit). In other words, the nuclei are labeled with spatial barcodes, the nuclei are isolated, antibodies are added to the nuclei, unbound antibodies are washed away, secondary antibodies with Tn5 or MNase are added, Ca is added, and the nuclei are labeled with a barcode. 2+ or Mg 2+ Buffer to label DNA bound by antibodies and perform compatible single-cell sequencing methods to add cellular barcodes to spatially barcoded and labeled chromatin.

[0193] Spatially resolved single-cell proteomics

[0194] In an exemplary embodiment, the single-cell genomics sequencing library comprises a single-cell proteomic 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):e1900226. doi:10.1002 / pmic.201900226; Single Cell Composition (US20180340939A), Single Cell Proteomic Assay Using Aptamers (US20180320224A1), and Methods for Identifying Multiple Epitopes in Cells (US20170321251A1)).

[0195] In an exemplary embodiment, the labeled cell nucleus is labeled with an antibody or aptamer linked to a barcode oligonucleotide, the nucleus is washed to remove unbound antibody or aptamer, and then the barcode oligonucleotide bound to the antibody or aptamer captures the cellular barcode sequence. The oligonucleotide-linked antibody or aptamer includes a universal sequence complementary to the capture sequence or treatment sequence for pool splitting indexing or for capture by cellular barcode beads. The spatial barcode nucleic acid is modified to include the same universal sequence to capture the spatial barcode to the cellular barcode sequence.

[0196] Spatially resolved single-cell multi-omics

[0197] In an exemplary embodiment, the single-cell genomics sequencing library comprises a single-cell multiomics sequencing library (see, e.g., Lee J, Hyeon DY, Hwang D. Single-cell multiomics: technologies and data analysis methods. Exp Mol Med. 2020; 52(9): 1428-1442. doi: 10.1038 / s12276-020-0420-2; 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, Lake BB, 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. NatCommun.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 MolBiol.2019;26:1063-70;Ma, S. et al., Chromatin potential identified by sharedsingle cell profiling of RNA and chromatin.bioRxiv2020.06.17.156943(2020)doi:10.1101 / 2020.06.17.156943; and Stoeckius, M. et al., Simultaneous epitope and transcriptome measurement in single cells. Nat. Methods 14, 865-868 (2017). In an exemplary embodiment, for a multi-omics assay, the spatial barcode only needs to be captured by the cell barcode of one omics portion of the multi-omics single-cell assay. For example, if the multi-omics assay includes transcriptome sequencing, the spatial barcode can be configured to be captured by the poly-T sequence.

[0198] Imaging / image assembly

[0199] Utilizing the spatial barcode of a single cell nucleus identified by a source cell barcode, and utilizing an analyte identified by a sequence that also includes a source cell barcode, a high-resolution image of the location of the analyte expression site can be easily constructed in a computer. In an exemplary embodiment, the spatial position of a large number of sites (e.g., beads) within the array can first be assigned to the image position, and all relevant analyte expression data are also assigned to the position (optionally, once the spatial barcode has been used to assign analyte sequence information to the array position, the spatial barcode is effectively decoupled from the array / matrix of the analyte sequence information associated with a given site / bead). High-resolution images of the range of individual or grouped analytes representing the various spatial positions of the array can then be generated using potential analyte sequence information. Any (or any number of) techniques recognized in the art that one of ordinary skill in the art deems appropriate can be used to adjust and / or normalize the image (i.e., pixel coloring and / or intensity).

[0200] In an exemplary embodiment, a high resolution image of the present disclosure is one in which discrete features (e.g., pixels) of the image are spaced 50 μm or less apart. In some embodiments, the spacing of discrete features within the image is 40 μm or less, optionally 30 μm or less, optionally 20 μm or less, optionally 15 μm or less, optionally 10 μm or less, optionally 9 μm or less, optionally 8 μm or less, optionally 7 μm or less, optionally 6 μm or less, optionally 5 μm or less, optionally 4 μm or less, optionally 3 μm or less, optionally 2 μm or less, or optionally 1 μm or less.

[0201] 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. The biological sample can be stained before imaging to provide contrast between different regions or cells. In some embodiments, more than one staining agent can be used to image different aspects of the sample (e.g., different regions of an organization, different cells, specific subcellular components, etc.). In other embodiments, the biological sample can be imaged without staining.

[0202] In certain embodiments, a fluorescence microscope (e.g., a confocal fluorescence microscope) can be used to detect biological samples that fluoresce, for example, by virtue of fluorescent labels. Fluorescent samples can also be imaged using a nucleic acid sequencing device with fluorescence detection optics, such as the Genome or Platform device; or SOLiD commercialized by Life Technologies (Carlsbad, Calif.) TM Sequencing platform. Other imaging optical devices that can be used include those found in the detection devices described in Bentley et al., Nature 456:53-59 (2008); PCT Publication Nos. WO 91 / 06678, WO 04 / 018497, or WO 07 / 123744; U.S. Patent 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, each of which is incorporated herein by reference.

[0203] Images of biological samples can be obtained at a desired resolution, for example, to distinguish tissue, cellular, or subcellular components. Thus, the resolution can be sufficient to distinguish and separate components of a biological sample that are at least 0.5 μm, 1 μm, 5 μm, 10 μm, 50 μm, 100 μm, 500 μm, 1 mm, or larger. Alternatively or additionally, the resolution can be set to distinguish and separate components of a biological sample that are at least 1 mm, 500 μm, 100 μm, 50 μm, 10 μm, 5 μm, 1 μm, 0.5 μm, or smaller.

[0204] Methods described herein can include the step of associating the position in the image of the biological sample with the barcode sequence of a nucleic acid probe, which is attached to a separate bead that is in contact with, has been in contact with, or will be in contact with the biological sample. Therefore, the characteristics of the biological sample that can be identified in the image can be associated with the nucleic acid that is found to be present in its vicinity. Any of a variety of morphological characteristics can be used for this correlation, including, for example, cell shape, cell size, tissue shape, staining pattern, the presence of specific proteins (e.g., detected by immunohistochemical staining) or other features routinely assessed in pathology or research applications. Therefore, the biological state of the tissue or its components determined by visual observation can be associated with the molecular biological characteristics determined by spatially resolved nucleic acid analysis.

[0205] The solid support on which the biological sample is imaged may include a fiducial marker to facilitate determining the orientation of the sample or its image relative to the probe attached to the solid support. Exemplary fiducials include, but are not limited to, beads (with or without a fluorescent moiety or a portion that a labeled probe can bind, such as a nucleic acid), fluorescent molecules attached at known or determinable features, or structures combining a morphological shape with a fluorescent moiety. Exemplary fiducials are disclosed in U.S. Patent Application Publication No. 2002 / 0150909A1 or U.S. Patent Application Serial No. 14 / 530,299, each of which is incorporated herein by reference. When obtaining an image of a biological sample, one or more fiducials are preferably visible. Preferably, the solid support includes at least 2, 3, 4, 5, 10, 25, 50, 100 or more fiducial markers. The fiducials may be provided in a pattern, such as along the outer edge of the solid support or the periphery of the biological sample location. In one embodiment, one or more fiducials are detected using the same imaging conditions used to visualize the biological sample. However, if desired, separate images (e.g., one image of the biological sample and another image of the fiducial) may be obtained, and the images may be aligned with each other.

[0206] In an exemplary embodiment, spatial filtering can be used to clean up the data. In one example, specific beads that are missing are filtered out. The total spatial barcode nUMI can be used to remove beads.

[0207] In an exemplary embodiment of Slide-tags, spatial barcode oligonucleotides are released into tissue sections to "tag" cell nuclei. The nuclei are then isolated from the tissue and sequenced for the spatial barcodes, as well as other patterns of molecular information. Importantly, each nucleus receives many spatial barcode oligonucleotides from different locations on the bead array. Therefore, the nuclei must be computationally located in space based on the distribution of the spatial barcodes received by each nucleus. In an example embodiment, density-based spatial clustering with noise (DBSCAN) is used to locate cell nuclei to their spatial locations (see, for example, Ester, M., Kriegel, H.-P., Sander, J., and Xu, XA density-based algorithm for discovering clusters in large spatial databases with noise. In Proc. Second International Conference on Knowledge Discovery and Data Mining (Simoudis, E. et al., eds.) 226-231 (AAAIPress, 1996); and Hahsler, M., Piekenbrock, M., and Doran, D. dbscan: fast density-based clustering with RJStat. Softw. 91, 1-30 (2019)). DBSCAN is applied to distinguish "signal" spatial barcodes (those that may provide value in locating cell nuclei) from background "noise" spatial barcodes (that may confuse cell nucleus localization). DBSCAN outputs a cluster assignment for each spatial barcode. Cluster = 0 represents a noise barcode, and cluster > 0 represents a signal barcode that is grouped with other signal barcodes clustered in space. The spatial positions of all spatial barcodes indicated as noise are not assigned to a nucleus, or are assigned to nuclei with multiple signal clusters. From the remaining nuclei with a different spatial barcode signal cluster, the weighted centroid of the spatial barcode coordinates in the signal cluster is obtained, where the weight is the number of unique molecular identifiers (UMIs) of the sequenced barcode. Importantly, DBSCAN requires two parameters: minPts and eps (effectively, radius). To determine the optimal parameter set for each Slide-tags run, we iterated 15 different minPts parameters and selected the parameter set with the highest proportion of nuclei assigned spatial positions (one DBSCAN signal cluster).

[0208] While DBSCAN localizes nuclei with relatively high sensitivity and specificity, it is not the only method for determining locations from a set of spatial barcode coordinates. Example alternatives include assigning the location of the nucleus to its highest UMI spatial barcode or taking the weighted 2-dimensional median of the spatial barcode coordinates. Other methods for distinguishing signal barcodes from noise barcodes include (1) K-means clustering, (2) affinity propagation, (3) mean shift, (4) spectral clustering, (5) agglomerative clustering, and (6) DBSCAN extensions such as HDBSCAN and OPTICS.

[0209] In an exemplary embodiment, the position of the cell nucleus in the Z-plane can be measured by using the following, alone or in combination: the count of spatial barcode UMIs per cell nucleus, the distribution of spatial barcode coordinates per cell nucleus (e.g., median pairwise distance or similar distance), and a measurement of the fluorescence of the fluorescently labeled spatial barcodes retained in the cell nucleus.

[0210] Reagent test kit

[0211] The present disclosure also provides a kit containing reagents of the present disclosure for the methods of the present disclosure. The kit of the present disclosure may include one or more containers comprising reagents of the present disclosure (e.g., capture materials, such as liquid electrical tape) and / or compositions (e.g., bead arrays captured by a slide). In an exemplary embodiment, the kit includes beads comprising spatial barcode nucleic acids as described herein. In an exemplary embodiment, the beads are configured for specific single-cell genomics assays as described herein. In some embodiments, the kit also includes instructions for use according to the methods of the present disclosure. In some embodiments, the instructions include how to create a tissue cryosection, form a spatially defined (or simply spatially definable, steps to be performed to limit the spatial resolution of the bead array), contact the tissue cryosection with the spatially defined bead array, and a description of how to use the labeled nuclei for subsequent single-cell assays.

[0212] The instructions provided in the kit of the present disclosure are typically written instructions on a label or package insert (e.g., a paper included in the kit), but machine-readable instructions (e.g., instructions carried on a storage disk or instructions provided on the Internet) are also acceptable. The label or package insert indicates that the composition is used to stage frozen sections and / or diagnose analyte patterns in frozen sections. Instructions for practicing any of the methods described herein can be provided.

[0213] The kits of the present 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), etc. The container may also contain a pharmaceutically active agent.

[0214] The kit may optionally provide additional components such as buffers, and explanatory information. Typically, the kit comprises a container and a label or package insert on or associated with the container.

[0215] Other embodiments are described in the following examples, which are for illustrative purposes only and are not intended to limit the scope of the invention.

[0216] Examples

[0217] Example 1 - Slide-tag process

[0218] preparation:

[0219] Prepare 500 mL of Dissociation Buffer (DB) stock solutions using ultrapure nuclease-free water and these reagents:

[0220] Na2SO4 -5.83g

[0221] K2so4 -2.615g

[0222] glucose -0.905g

[0223] HEPES -1.2g

[0224] Mgcl2 -2.5mL

[0225] Extraction buffer: ExB

[0226] DB-15mL

[0227] 1% Kollidon - 0.150g

[0228] 1% TX-100-150 μl

[0229] 10% BSA - 15 μl

[0230] 1 bottle of ribonuclease inhibitor (lucigen)

[0231] Washing buffer: WB (20 ml per sample)

[0232] DB-20mL

[0233] 10% BSA - 20 μl

[0234] 50 μl RNase inhibitor

[0235] step:

[0236] 1. Prepare 3 mm 20 μm tissue sections.

[0237] Note: Use a brush to gently press the sliced tissue to flatten it.

[0238] 2. Transfer the tissue to a 3 mm disc and thaw the tissue with your fingers.

[0239] Note: Place the disc on a glass slide. Keep the slide on ice until you transfer the tissue. Place the slide in the cryostat just before transferring the tissue from the disc. Transfer the sectioned tissue onto the disc, center the tissue on the disc with a brush, and very gently tap the tissue and thaw it with your fingers. This step must be done as quickly as possible, otherwise the tissue will begin to fold.

[0240] 3. 5 μl of DB was placed on the disc, photolyzed for 3 minutes, and then incubated on ice for 5 minutes.

[0241] 4. Transfer the disc to the well plate.

[0242] NOTE: Use fine-tipped tweezers to grip the transfer disc by the edge.

[0243] 5. Dissociate the tissue from the disc using extraction buffer using a 200 / 1000 μl pipette.

[0244] Note: Use a 200 / 1000 μl pipette to apply extraction buffer to the disc. The total volume of extraction buffer used is 2 ml. Pipetting pressure should be moderate. Observe the tissue under a microscope every 15-20 pipetting cycles to confirm dissociation. Repeat this process until the tissue is dissociated.

[0245] 6. After tissue dissociation, remove the disc from the well.

[0246] 7. Transfer the dissociated sample into 20 ml of wash buffer; divide it into two 50 ml tubes.

[0247] 8. Centrifuge at 600 g for 10 minutes at 4°C.

[0248] 9. Remove the supernatant and leave 500 μl of the pellet in each tube; combine the pellets to make up to 1 ml.

[0249] 10. Filter the solution using a 40 micron filter.

[0250] 11. Add 1:1000 DAPI to the filtered solution and incubate it for 8-10 minutes.

[0251] 12. Centrifuge at 200 g for 10 minutes at 4°C.

[0252] 13. Remove the supernatant, leaving 50 μl of the centrifuge pellet.

[0253] 14. Count the nuclei using a hemocytometer and load 43.3 μl onto the 10X.

[0254] Example 2 - Slide-tags description and results

[0255] Figure 1 , 2 and 3 describe the slide tag method. In the first step of slide-tags, thin sections of fresh frozen tissue are placed on a barcoded bead array, where each bead contains tens of thousands of oligonucleotides with the same spatial barcode, and the sequence and position of each spatial bead barcode are known. These oligonucleotides are photocleaved to release them into the tissue, where they "tag" cell nuclei. These labeled nuclei are then isolated. Finally, these nuclei can be used as input for established single-cell sequencing technologies. This provides users with true single-cell resolution, with data quality indistinguishable from snRNA-seq experiments alone, while also providing the spatial coordinates of each nucleus. This process is easy to implement, with the labeling process adding only about 10 minutes to the workflow time.

[0256] Slide-tags has many advantages over other real-space methods ( Figure 4 Slide-tags are optimized to increase the percentage of cells that can be spatially located ( Figure 6 Slide-tags accurately dissected the mouse hippocampus (Figures 5 and 7). Slide-tags recovered embryonic cell types with a high confidence percentage of assigned positions and recapitulated known anatomical structures, revealing distinct regional patterns through de novo clustering (Figures 8 and 9). Figure 11 Slide-tags can summarize dynamic events during development (Figure 9, Figure 10 ). Slide-tags can provide 3D spatial data ( Figure 12 Spatial distribution is higher in thinner tissue sections (Figure 13). Slide-tags can be used for spatially resolved single-cell multi-omics experiments (Figure 14).

[0257] Slide-tags can be used together with 10X single-cell sequencing technology (Figure 15). In order to benchmark slide-tags, the applicant applied it to the mouse hippocampus for analysis, and the mouse hippocampus has a highly fixed architecture, thereby allowing the performance of this technology to be clearly evaluated (Figure 16). The standard output of the snRNA-seq experiment is a cell x gene matrix, and the applicant can thus construct a dimensionality reduction map to highlight the cell type population, which is shown here by its coloring. Using Slide-tags, each of these cells has an associated spatial barcode cluster. The applicant can then take the weighted center of these points to obtain the precise position of the cell. This allows each slide-tags experiment to have an accompanying spatial coordinate matrix. Slide-tags can realize single-nuclear spatial transcriptomics in the mouse hippocampus ( Figure 17 Slide-tags recapitulate the expected organizational architecture of the mouse hippocampus after spatially mapping all profiled cells.

[0258] To quantify spatial localization accuracy, the applicants first compared the width of the hippocampal subregion cornu ammonis area 1 (CA1) in Slide-tags with serial sections stained with Nissl and found that the width of the Slide-tag features was consistent with the Nissl images ( Figure 18 ). CA1 is divided into deep sublayers and superficial sublayers. The deep sublayers and superficial sublayers have different gene expression patterns (Dong HW, Swanson LW, Chen L, Fanselow MS, Toga AW. Genomic-anatomic evidence for distinct functional domains in hippocampal field CA1. Proc Natl Acad Sci U S A. 2009; 106(28): 11794-11799; Cid E, Marquez-Galera A, Valero M et al., Sublayer-and cell-type-specific neurodegenerative transcriptional trajectories in hippocampal sclerosis. Cell Rep. 2021; 35(10): 109229). In addition, the applicant can accurately locate the subcell types in the deep and superficial layers of CA1 ( Figure 19 ).

[0259] To test whether Slide-tags affect transcriptome data quality, we compared a standard snRNA-seq experiment with snRNA-seq performed after Slide-tags. We found that the data quality was almost indistinguishable in terms of cell type ratio, average UMI per cell, and average gene expression ( Figure 21 Furthermore, since each nucleus receives many spatial barcode oligonucleotides from many different beads, the spatial resolution is not limited to the 10 micron size of the beads ( Figure 20 For example, cell X receives 4 spatial barcodes from two beads, 3 spatial barcodes from another bead, and 1 barcode from each of the two other beads. Using this information, the position of cell X can be determined to within less than 10 microns. In contrast, applicants estimate that the spatial resolution of Slide-tags will be less than 10 microns, between 3 and 4 microns ( Figure 20 Slide-tags generates high-quality spatial single-cell data that outperforms previous spatial methods ( Figure 22 Slide-tags are compatible with existing single-nucleus sequencing technologies and analysis tools ( Figure 23 ). The applicant has demonstrated this through the application shown.

[0260] The applicant applied slide tags to a human metastatic melanoma sample ( FIG. 24 ). The H&E image shows two histopathological lobes divided by a line. As previously described, the applicant can identify transcriptome clusters and see three tumor cell clusters here, but using Slide-tags, these clusters are localized. The applicant observed that the dissected cancer cells had two transcriptionally distinct subpopulations. The applicant mapped these subpopulations in space and found that they were spatially separated ( Figure 25A We investigated whether there were cytogenetic differences between the subpopulations and used inferCNV, a method for inferring copy number variation from transcriptome data. We found many shared copy number variations between the subpopulations, but also some inferred alterations that were unique to each subpopulation ( Figure 25B Using single-cell data, we can interrogate how this heterogeneity might be related to the cell-cell interactions that form these tumor clusters. We calculated the variation in the proportion of neighbors that these tumor cells have and identified that CD8 T cells are enriched in the neighborhood of tumor_2, depleted in tumor_1b, and neither enriched nor depleted in tumor_1a ( Figure 26A Applicants demonstrated this spatially on a single-cell basis when mapping the enrichment of the CD8_T cell fraction within each tumor neighborhood ( Figure 26B ).

[0261] The applicants asked what the T cell states and receptors were in different neighborhoods. The applicants observed that T cells in tumor 2 had a more cytotoxic phenotype (i.e., cell state) ( Figure 27A The applicant sequenced TCR receptors from slide-tags and observed that TCR clones had different spatial localizations ( Figure 27B Slide-tags are compatible with sequencing TCR receptors from 3' single-cell libraries (Liu S, Iorgulescu JB, LiS et al., Spatialmaps of T cell receptors and transcriptomes reveal distinct immune niches and interactions in the adaptive immune response. Immunity. 2022; 55(10): 1940-1952.e5).

[0262] Applicants observed differences in T cell infiltration and status between Tumor 1 and Tumor 2. Applicants hypothesized that the HLA locus would be downregulated in the presence of copy number loss on Chr6 in Tumor 1 but not in Tumor 2. Applicants observed inferred Chr6 copy number loss ( Figure 28 ). In addition, gene set enrichment analysis of differentially expressed genes between tumor populations revealed that tumor 1 is downregulated in the endogenous antigen presentation machinery. Slide-tags multiomics recovered expression and epigenomic profiles ( Figure 29A -C).

[0263] Another hypothesis is that the melanoma cells in tumor 1 are undergoing a cell state transition, or dedifferentiation, with loss of endogenous antigen expression. Applicants observed differentially expressed and differentially accessible genes between the two tumor populations in both RNA and ATAC data and discovered TNC, a gene associated with a cell state transition to a more aggressive phenotype in melanoma ( Figure 30A -C). Applicants hypothesized that tumor 1 was losing its melanocyte identity and associated endogenous antigens, resulting in a more suppressive T cell microenvironment. Applicants scored tumor cells based on the expression of mesenchymal-like cell state genes ( Figure 31A). Applicants found that, as hypothesized, tumor 1 was enriched for a more mesenchymal-like phenotype compared to tumor 2 and began to lose melanocyte markers such as PMEL and MLANA, while gaining TNC and AXL expression. Next, applicants went back to the ATAC data in these same cell nuclei to identify transcription factor motifs associated with this mesenchymal-like state. Applicants found that transcription factors such as FOS, JUN, and IRF9 were positively correlated with the mesenchymal-like score, while TFs such as MITF were negatively correlated with it (or more correlated with the melanocyte-like score) ( Figure 31B Finally, mapping these TF motif scores in space, we found that some TF motif-associated mesenchymal-like cell states clustered together spatially, suggesting tumor microenvironmental stress or epigenetic inheritance through cell division.

[0264] Human tissue often presents unique challenges to technological development. For example, the nuclei of the human brain are more sparsely distributed, so it is necessary to change the scale of the array for barcoding. The applicant performed slide-tags on a 5.5 × 5.5 area of the human prefrontal cortex, where there is a well-characterized laminar structure (Figure 32). The applicant sequenced a total of 14,000 nuclei, of which 6500 nuclei were able to be placed in space. The applicant has restored all known cortical layer types (see, for example, Bakken TE, Jorstad NL, Hu Q et al., Comparative cellular analysis of motor cortex in human, marmoset and mouse [see Nature. April 2022 for publication of corrections; 604 (7904): E8]. Nature. 2021; 598 (7879): 111-119).

[0265] Human tonsil is a highly dynamic, dense organ in which there are many simultaneous cell differentiation events. Human tonsil includes germinal centers where B cell maturation occurs. Memory B cells stimulate germinal centers, while initial cells are excluded from germinal centers. Tonsils have a rich library of immune receptor information. Tonsils are traditionally difficult to analyze with spatial tools because their cell types are closely juxtaposed and relatively small. The practicality of using slide-tags is shown (Figure 33). The applicant uses spatially varying genes to identify dark and bright areas in germinal centers (Figure 34).

[0266] Slide-tags can be used to name spatially significant receptor-ligand interactions (Figure 35). Previous methods that use single-cell data to infer receptor-ligand interactions cannot determine whether single cells are spatially adjacent ( Figure 35AUsing spatial information to organize receptor-ligand interactions in germinal centers allows identification of neighboring single cells expressing ligand-receptor pairs ( Figure 35B ). For example, single cells expressing CD40LG and CD40 in the bright area.

[0267] We next explored whether we could perform spatial multi-omics sequencing by testing ATAC and RNA-seq on the same single cell using slide-tags (Figure 36). We dissected regions of the mouse p1 brain ( Figure 36A The applicants collected high-quality ATAC data, including: TSS enrichment, fragment lengths corresponding to nucleosome peaks, and high counts of unique fragments per cell ( Figure 36B ).

[0268] Applicants can perform embedding analysis of cells by RNA expression and ATAC peak profiles, but now applicants can also use combined variation (WNN) to better inform cell clustering ( Figure 37A ). Applicants can plot these clusters in space, revealing many spatially defined clusters ( Figure 37B ).

[0269] Applicants used slide-tags to identify spatially restricted transcriptomic and epigenetic signatures in the developing L 2 / 3 ( FIG. 38 ). Applicants zoomed in on one of these clusters, which belongs to the outer layers of the developing cortex, where the layers have been defined but the regions along the layers are still being further defined. Applicants used RNA data to identify these broad regions by performing autocorrelation analysis: isocortex, retrosplenial cortex, and subiculum, with some representative top hits shown ( Figure 38A and B). Applicants then asked the same question in the ATAC data and found a peak that identified a subregion of the isocortex ( Figure 38C Applicants sought to further understand this by identifying transcription factor motifs that vary along this axis and discovered four transcription factors, three of which have known roles in cortical development ( Figure 38D ).

[0270] ***

[0271] Without departing from the scope and spirit of the present invention, various modifications and variations of the methods, pharmaceutical compositions and kits described herein will be readily apparent to those skilled in the art. Although the present invention has been described in conjunction with specific embodiments, it will be understood that the present invention is capable of further modifications, and the claimed invention should not be unduly limited to such specific embodiments. In fact, various modifications of the described modes for implementing the present invention that are apparent to those skilled in the art are intended to be within the scope of the present invention. This application is intended to encompass any variation, use or adaptation of the present invention, which generally follows the principles of the present invention and includes deviations from the present disclosure that occur within the scope of known and customary practice in the art to which the present invention relates, and can be applied to the basic features previously set forth herein.

Claims

1. A method for generating spatially labeled nuclei for single-cell genomics, comprising: a) placing a tissue sample on a sequence-verified spatial array, wherein the spatial array comprises a nucleic acid sequence comprising a spatial barcode coupled to the array via a cleavable linker, wherein the spatial barcode is the same for a single position on the array but different for any other position on the spatial array; b) cleaving the linker and delivering the spatial barcode nucleic acid to the nuclei of cells in the tissue sample; and c) isolating the labeled cell nuclei from the tissue sample.

2. The method according to claim 1 further comprises preparing a single-cell genomics sequencing library using the isolated labeled cell nuclei, wherein each nucleic acid sequence comprises a cellular barcode sequence that identifies the cell of origin and an optional unique molecular identifier (UMI), and capturing the spatial barcode sequence from each spatially labeled cell nucleus to generate a nucleic acid sequence comprising a spatial barcode and a cellular barcode, so that the genomics data of each single cell can be identified by the cellular barcode, and the spatial location of the same single cell in the tissue can be identified by the same cellular barcode. The method according to claim 1 , wherein the spatial barcode is delivered to the cell nucleus by diffusion.

4. The method according to any one of claims 1 to 3, wherein before step (a), the spatial array is sequence verified by in situ sequencing of the nucleic acid sequence containing the spatial barcode, thereby generating an index of the spatial barcode on the array. The method according to claim 4 , wherein the in situ sequencing is performed by ligation sequencing or synthesis sequencing.

6. A method according to any one of claims 1 to 5, wherein the spatial array comprises a solid support, which is fixed to each position on the array and coupled to the nucleic acid sequence comprising a spatial barcode via the cleavable linker, wherein the spatial barcode is the same for a single solid support but different for any other solid support in the spatial array. The method of claim 6 , wherein the solid support is covalently fixed to the spatial array.

8. The method of claim 6, wherein the solid support is fixed to the spatial array via a vinyl polymer.

9. The method according to any one of claims 6 to 8, wherein the solid support is a bead.

10. The method of claim 9, wherein the beads are polystyrene beads.

11. The method according to claim 9 or 10, wherein the beads have a diameter of 50 μm or less.

12. The method of claim 11, wherein the beads are 1, 3, 10, 15 or 20 μm.

13. The method of any one of claims 1 to 12, wherein the linker is a photocleavable, chemically cleavable, or enzymatically cleavable linker.

14. The method according to any one of claims 2 to 13, wherein the spatial barcode nucleic acid comprises a poly-A sequence for capture by a cellular barcode nucleic acid comprising a poly-T sequence.

15. The method of any one of claims 1 to 14, wherein the tissue sample is treated to permeabilize the cell nuclei.

16. The method of claim 15, wherein the tissue sample is permeabilized in a buffer that increases diffusion of the spatially barcoded nucleic acid.

17. The method according to any one of claims 1 to 16, wherein the lengths of the spatial barcode nucleic acids are different.

18. The method of any one of claims 1 to 17, wherein the spatially barcoded nucleic acid further comprises one or more modifications that enhance diffusion into the cell nucleus.

19. The method of claim 18, wherein the spatial barcode nucleic acid is modified by adding one or more lipid or cholesterol groups.

20. The method according to any one of claims 1 to 19, wherein the spatial barcode nucleic acid further comprises one or more fluorescent labels that can be used to identify and label cell nuclei.

21. The method of any one of claims 1 to 20, wherein the tissue sample is a fresh frozen tissue section.

22. The method of any one of claims 1 to 20, wherein the tissue sample is a fresh unfixed tissue section.

23. The method of any one of claims 1 to 20, wherein the tissue sample is a fixed tissue section.

24. The method of any one of claims 2 to 23, wherein the position of each cell in the tissue sample is determined computationally based on sequencing of the library.

25. The method of claim 24, wherein the cellular barcode nucleic acid comprises a UMI sequence, and the location of each cell in the tissue is determined based on the number of UMIs sequenced for each spatial barcode having the same cellular barcode sequence.

26. The method of any one of claims 2 to 25, wherein the single-cell genomics sequencing library is a single-nucleus RNA sequencing library (snRNA-seq).

27. The method of any one of claims 2 to 25, wherein the single-cell genomics sequencing library is a single-cell DNA accessibility library.

28. The method of any one of claims 2 to 25, wherein the single-cell genomics sequencing library is a single-cell ATAC sequencing library (ATAC-seq).

29. The method of any one of claims 2 to 25, wherein the single-cell genomics sequencing library is a single-cell chromatin immunoprecipitation (ChIP) sequencing library.

30. The method of any one of claims 2 to 25, wherein the single-cell genomics sequencing library is a single-cell genome sequencing library.

31. The method according to any one of claims 2 to 25, wherein the single-cell genomics sequencing library is a single-cell DNA methylation sequencing library.

32. The method of any one of claims 2 to 25, wherein the single-cell genomics sequencing library is a single-cell Hi-C sequencing library.

33. The method of any one of claims 2 to 25, wherein the single-cell genomics sequencing library is a single-cell cut-and-tag sequencing library.

34. The method of any one of claims 2 to 25, wherein the single-cell genomics sequencing library is a single-cell genome and transcriptome sequencing library (G&T-seq).

35. The method of any one of claims 2 to 25, wherein the single-cell genomics sequencing library is a single-cell proteomic library.

36. A kit comprising a plurality of solid supports coupled via cleavable linkers to a nucleic acid sequence comprising a spatial barcode and a capture sequence, wherein the spatial barcode is the same for an individual solid support but different for any other solid support in the spatial array, and wherein the capture sequence is the same on all of the solid supports.

37. The kit of claim 36, wherein the solid support further comprises a chemical binding moiety for covalently fixing the solid support to an array.

38. The kit of claim 36 or 37, wherein the solid support is a bead.

39. The kit of claim 38, wherein the beads are polystyrene beads.

40. The kit of claim 38 or 39, wherein the beads have a diameter of 50 μm or less.

41. The kit of claim 40, wherein the beads are 1, 3, 10, 15 or 20 μm, preferably 10 μm.

42. The kit of any one of claims 36 to 41, wherein the linker is a photocleavable, chemically cleavable, or enzymatically cleavable linker.

43. The kit of any one of claims 36 to 42, wherein the capture sequence comprises a poly-A sequence.

44. The kit according to any one of claims 36 to 43, wherein the lengths of the spatial barcode nucleic acids coupled to the solid support are different.

45. The kit according to any one of claims 36 to 44, wherein the spatially barcoded nucleic acid further comprises one or more modifications that enhance diffusion into the cell nucleus.

46. The kit of claim 45, wherein the spatial barcode nucleic acid is modified by the addition of one or more lipid or cholesterol groups.

47. The method according to any one of claims 36 to 46, wherein the spatial barcode nucleic acid further comprises one or more fluorescent labels that can be used to identify and label cell nuclei.

Images