Spatial differentiation of biological samples, multiplex nucleic acid analysis

Abstract

The present application relates to spatially distinguishing and multiplexed nucleic acid analysis of biological samples, including a method for spatially labeling nucleic acids of biological samples, which comprises the following steps: (a) providing a solid support comprising different nucleic acid probes randomly positioned on the solid support, wherein the different nucleic acid probes each comprise a barcode sequence different from the barcode sequence of other randomly positioned probes on the solid support; (b) performing a nucleic acid detection reaction on the solid support to locate the barcode sequence on the solid support; (c) contacting a biological sample with the solid support having the randomly positioned probes; (d) hybridizing the randomly positioned probes with a target nucleic acid from a portion of the biological sample; and (e) modifying the randomly positioned probes hybridized with the target nucleic acid, thereby producing a modified probe comprising the barcode sequence and a target-specific modification, thereby spatially labeling the nucleic acids of the biological sample.

Description

[0001] This application is a divisional application of an invention patent application with an application date of April 4, 2016, application number 201680020206.8, and invention name “Spatial differentiation of biological samples, multiple nucleic acid analysis”. Technical Field

[0002] The present invention relates to spatial differentiation and multiplex nucleic acid analysis of biological samples, including a method for spatially tagging nucleic acids of biological samples. Background Art

[0003] One in four men will die from cancer. Further statistics from the American Cancer Society predict that one in five women will suffer the same fate. Treatments are available for many cancers. However, success largely relies on early detection.

[0004] Cancer is now known as a genomic disease. Many oncologists and cancer researchers hope that advances in genomic analysis tools will provide early detection and treatment pathways. However, these tools are used more in research laboratories and are not yet mature enough for most oncologists to use easily. Improvements are needed.

[0005] It is said that at the time of diagnosis, all cancer patients are chimeras. They are chimeras because they have at least two different genomes: the genome they are born with, and the genome they unwillingly acquire due to cancer. In addition, as the tumor grows, different populations of cancer cells become apparent. This leads to even more complex chimeras within the tumor. This cancer cell heterogeneity usually leads to cell subpopulations that respond differently to cancer therapy. The final result is usually an initial positive response of a cell subpopulation, resulting in the observation that the patient's tumor shrinks, but then the tumor tissue will grow again and in some cases will metastasize. Despite early detection of cancer, the inability to identify cell subpopulations that are resistant to treatment can lead to the loss of time required to treat invasive cancers. This can have adverse consequences for patients both emotionally and physically.

[0006] There is a need for genomic tools that can distinguish cancer cell subpopulations in tumors. The present disclosure satisfies this need and also provides other advantages. Summary of the invention

[0007] The present disclosure provides a method for labeling nucleic acids of biological samples in a spatial manner. The method may include the following steps: (a) providing a solid support comprising a plurality of different nucleic acid probes randomly positioned on a solid support, wherein each of the different nucleic acid probes comprises a barcode sequence different from the barcode sequence of other randomly positioned probes on the solid support; (b) performing a nucleic acid detection reaction on the solid support to locate the barcode sequence on the solid support; (c) contacting the biological sample with the solid support having the randomly positioned probes; (d) hybridizing the randomly positioned probes with target nucleic acids from a portion of the biological sample near the randomly positioned probes; and (e) modifying the randomly positioned probes hybridized with the target nucleic acids, thereby generating a modified probe comprising a barcode sequence and a target-specific modification, thereby labeling the nucleic acids of the biological sample in a spatial manner.

[0008] The present disclosure further provides a method for spatially labeling nucleic acids in a biological sample, the method comprising the following steps: (a) attaching different nucleic acid probes to a solid support to produce randomly positioned probes on the solid support, wherein the different nucleic acid probes each include a barcode sequence, and wherein the randomly positioned probes each include a barcode sequence different from other randomly positioned probes on the solid support; (b) performing a nucleic acid detection reaction on the solid support to determine the barcode sequence of the randomly positioned probes on the solid support; (c) contacting the biological sample with the solid support having the randomly positioned probes; (d) hybridizing the randomly positioned probes with a target nucleic acid from a portion of the biological sample proximal to the randomly positioned probes; and (e) extending the randomly positioned probes to produce extended probes including the barcode sequence and a sequence from the target nucleic acid, thereby spatially labeling the nucleic acids in the biological sample.

[0009] Also provided is a method for spatially labeling nucleic acids of a biological sample, the method comprising the following steps: (a) providing a plurality of nucleic acid primers attached to a solid support, wherein the nucleic acid primers in the plurality include a universal primer sequence common to the nucleic acid primers in the plurality; (b) binding a population of nucleic acid probes to the plurality of nucleic acid primers, wherein the nucleic acid probes include a universal primer binding sequence that hybridizes to the universal primer sequence, a target capture sequence, and a barcode sequence that is different from the barcode sequence of other nucleic acid probes in the population, thereby attaching different nucleic acid probes to randomly located locations on the solid support; and (c) Amplifying different nucleic acid probes by extending nucleic acid primers, thereby generating nucleic acid clusters having copies of a barcode sequence and a target capture sequence at randomly positioned positions on a solid support; (d) performing a sequencing reaction to determine the barcode sequence at the randomly positioned positions on the solid support; (e) contacting a biological sample with the nucleic acid cluster on the solid support; (f) hybridizing the target capture sequence of the cluster with a target nucleic acid from a portion of the biological sample proximal to the cluster; and (g) extending the target capture sequence to generate an extended probe comprising a sequence from the target nucleic acid and a copy of the barcode sequence, thereby labeling the nucleic acid of the biological sample.

[0010] The present disclosure further provides a method for spatially labeling nucleic acids in a biological sample, the method comprising the following steps: (a) providing a bead array on a solid support, wherein different nucleic acid probes are attached to different beads in the array, wherein the different nucleic acid probes each include a barcode sequence, wherein each bead includes a barcode sequence different from that of other beads on the solid support, and wherein the different nucleic acid probes each include a target capture sequence; (b) performing a decoder probe hybridization reaction on the solid support to determine the barcode sequence of the randomly positioned probes on the solid support; (c) contacting the biological sample with the bead array; (d) hybridizing the different nucleic acid probes with a target nucleic acid from a portion of the biological sample proximal to the bead; and (e) extending the different nucleic acid probes to produce extended probes including a sequence from the target nucleic acid and a barcode sequence, thereby labeling the nucleic acids in the biological sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 A schematic representation showing the steps and reagents that can be used to generate barcoded oligo dT probes on an Illumina flow cell, produce extended barcoded probes with mRNA sequences, and release the extended probes from the flow cell.

[0012] Figure 2 Shown are data indicating the availability of the oligo dT capture sequence on the probe after bridge amplification of the probe and restriction enzyme digestion with BspH1 to remove one of the primer binding sites used for bridge amplification.

[0013] Figure 3 Showing the example 1 and Figure 2Sequencing metrics for the flow cell shown in .

[0014] Figure 4 Showing the example 1 and Figure 2 The number of unique barcodes determined in the 21 tiles of the flow cell shown in FIG.

[0015] Figure 5 Shown are images of cells captured on a patterned flow cell (Panel A) and cell count data (Panel B).

[0016] Figure 6 Cells remaining adhered to the flow cell under different conditions are shown.

[0017] Figure 7 A schematic representation of the steps and reagents used to generate probes attached to a gel (Panel A), a schematic representation of the steps and reagents used to capture target nucleic acids using the gel-attached probes and fluorescently label the probes (Panel B), and images generated from the fluorescently labeled target nucleic acids after capture with the probes and removal of the tissue from the gel.

[0018] Figure 8 Displays the BeadArray TM Schematic representation of the steps and reagents for attaching probes to capture target nucleic acids and fluorescently labeling the probes (Figure A), as well as for capturing and quantifying nucleic acids from BeadArray using probes. TM Images produced by fluorescently labeled target nucleic acids after tissue removal. DETAILED DESCRIPTION

[0019] The present disclosure provides compositions, equipment and methods for preserving spatial information when performing multiple nucleic acid analysis of biological samples. A variety of tools can be used for multiple nucleic acid analysis, including, for example, nucleic acid microarrays and so-called "next generation" sequencing platforms. The tool allows parallel detection of extremely large and complex nucleic acid collections, including, for example, DNA collections representing all or almost all genetic materials of an organism (i.e., 'genomes'), RNA (or cDNA) collections representing all or almost all complements of the expressed genes of an organism (i.e., 'transcriptomes'), and in some cases, the collection may include several genomes and / or transcriptomes (e.g., metabolites from communities or ecosystems or biomes) from several different organisms. Although these tools provide a large amount of information about what kind of nucleic acid sequence is present in the biological sample being evaluated, it is inherently unable to distinguish where any particular nucleic acid resides in the biological sample. In fact, most samples applied to multiple nucleic acid analysis tools are homogenates of mixtures of multiple different cells derived from biological samples. Therefore, spatial information is lost and the results obtained from these tools constitute the average transcriptome or average genome of the sample, and the important differences between individual cells are lost.

[0020] In certain embodiments, the present disclosure provides new and useful modifications to existing multiplex nucleic acid analysis tools to allow preservation of spatial information of biological samples from which nucleic acids are obtained. For example, solid supports commonly used in multiplex synthesis sequencing (SBS) techniques can be modified to capture nucleic acids from biological samples and label them spatially. In alternative examples, bead arrays (such as those used for genotyping or gene expression analysis) can be used to capture nucleic acids from biological samples and label them spatially. As described in the examples below, SBS or BeadArray commercialized by Illumina (San Diego, California) can be used to capture nucleic acids from biological samples and label them spatially. TM The solid support of the platform can be modified for spatial labeling. However, it should be understood that any of a variety of solid supports can be made and used according to the teachings herein. Spatially labeled nucleic acids can be removed from the solid support, pooled together and attached to a second solid support for detection in any of a variety of multiplex nucleic acid analysis systems, including, for example, sequencing platforms or microarray platforms described herein.

[0021] The spatial information provided by the methods, compositions or devices herein may include, for example, the location of one or more cells in a tissue (or other sample), the cells having specific alleles (e.g., genotyping) at one or more loci, having specific structural variations (e.g., fusions, insertions, deletions, rearrangements, etc.) in the genome, having specific epigenetic imprints (e.g., methylation), expressing specific genes, expressing specific alleles of genes, expressing specific splicing variants of genes, or the like. In addition to identifying nucleic acids based on their spatial location in biological samples, the methods, compositions or devices disclosed herein may be used to quantify one or more nucleic acids based on spatial location. For example, the spatial information of one or more cells in a tissue (or other sample) may include the amount of a specific allele or chromosomal region in the genome (e.g., ploidy); the amount of epigenetic modifications (e.g., methylation) in a genetic locus; the expression level of a specific gene, allele or splicing variant; or the like. The amount may be an absolute amount or a relative amount based on similar measurements obtained in the industry for mixed or non-spatially labeled samples.

[0022] The methods described herein can be used to locate nucleic acids in biological samples. In some embodiments, a method can be used to identify or characterize all transcriptomes or genomes of a biological sample. Alternatively, a method can be used to identify or characterize only a portion of the transcriptome or genome of a sample. The subset of transcripts or genes evaluated in the methods herein can be related to a specific disease or condition.

[0023] The methods described herein can be used to locate or spatially detect nucleic acids (DNA or RNA) in biological samples. Thus, one or more RNA or DNA molecules can be located within a cell or tissue or other biological sample relative to their natural location or location. For example, one or more nucleic acids can be located in a cell or adjacent cell group or a class of cells, or in a specific region of a region within a tissue sample. The natural location or position of individual RNA or DNA molecules can be determined using the methods, devices or compositions disclosed herein.

[0024] Unless otherwise indicated, the terms used herein should be understood to take their ordinary meanings in the relevant art. Several terms used herein and their meanings are set out below.

[0025] As used herein, the term "amplicon" means the product of replicating nucleic acid when used with respect to nucleic acid, wherein the product has a nucleotide sequence identical or complementary to at least a portion of the nucleotide sequence of the nucleic acid. Amplicon can be produced by any of a variety of amplification methods using nucleic acid or its amplicon as a template, including, for example, polymerase extension, polymerase chain reaction (PCR), rolling circle amplification (RCA), multiple displacement amplification (MDA), connection extension or connection chain reaction. Amplicon can be a nucleic acid molecule with a specific nucleotide sequence (such as PCR product) or a multi-copy nucleotide sequence (such as a concatemer (concatameric) product of RCA) of a single copy. The first amplicon of a target nucleic acid is generally a complementary copy. Subsequent amplicon is a copy produced from the target nucleic acid or from the first amplicon after generating the first amplicon. Subsequent amplicon can have a sequence that is substantially complementary to the target nucleic acid or substantially consistent with the target nucleic acid.

[0026] As used herein, the term "array" refers to a population of features or sites that can be distinguished from each other based on relative positioning. Different molecules at different sites of an array can be distinguished from each other based on the positioning of the sites in the array. Individual sites of an array may include one or more molecules of a specific type. For example, a site may include a single target nucleic acid molecule with a specific sequence, or a site may include several nucleic acid molecules with the same sequence (and / or its complementary sequence). The sites of an array may be different features positioned on the same substrate. Exemplary features include, but are not limited to, holes in a substrate, beads (or other particles) in or on a substrate, protrusions of a substrate, protuberances on a substrate, or trenches in a substrate. The sites of an array may be separate substrates each with different molecules. Different molecules attached to a separate substrate may be identified based on the positioning of the substrate on a surface bound to the substrate or based on the positioning of the substrate in a liquid or gel. Exemplary arrays in which separate substrates are positioned on surfaces include, but are not limited to, arrays with beads in holes.

[0027] As used herein, the term "attachment" refers to the state in which two objects are joined, fastened, adhered, connected or combined with each other. For example, an analyte (e.g., 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. Non-covalent bonds are chemical bonds that do not involve sharing of electron pairs and may include, for example, hydrogen bonds, ionic bonds, van der Waals forces, hydrophilic interactions, and hydrophobic interactions.

[0028] As used herein, the term "barcode sequence" is intended to mean a series of nucleotides in a nucleic acid that can be used to identify a nucleic acid, a feature of a nucleic acid, or an operation that has been performed on a nucleic acid. A barcode sequence can be a natural sequence or a sequence that does not naturally occur in the organism from which the barcoded nucleic acid is obtained. A barcode sequence can be unique to a single nucleic acid species in a population, or a barcode sequence can be shared by several different nucleic acid species in a population. For example, each nucleic acid probe in a population can include a different barcode sequence from all other nucleic acid probes in the population. Alternatively, each nucleic acid probe in a population can include a different barcode sequence from some or most other nucleic acid probes in the population. For example, even if probes with a common barcode differ from each other in other sequence regions along their length, each probe in a population can have a barcode that is present in several different probes in the population. In particular embodiments, one or more barcode sequences for a biological sample are not present in the genome, transcriptome, or other nucleic acid of the biological sample. For example, a barcode sequence can have less than 80%, 70%, 60%, 50%, or 40% sequence identity with a nucleic acid sequence in a particular biological sample.

[0029] As used herein, the term "biological sample" is intended to mean 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, bees, or spiders; fish, such as zebrafish; reptiles; amphibians, such as frogs or Xenopus laevis; Dictyostelium discoideum; fungi, such as Pneumocystis carinii, Takifugu rubripes); yeast, Saccharamoyces cerevisiae or Schizosaccharomyces pombe; or Plasmodium falciparum. The target nucleic acid may also be derived from a prokaryotic organism, such as bacteria, Escherichia coli, Staphylococci pneumoniae, or Mycoplasma pneumoniae; an archaea; a virus, such as hepatitis C virus or human immunodeficiency virus; or a viroid. The sample may be derived from a homogenous culture or population of the above organisms or alternatively from a collection of several different organisms, such as in a community or ecosystem.

[0030] As used herein, the term "cleavage site" is intended to mean a location in a nucleic acid molecule where bond breakage is prone to occur. The location can be specific to a particular chemical, enzyme, or physical process that causes bond breakage. For example, the location can be an abasic nucleotide or a nucleotide with a base that is easily removed to produce an abasic site. Examples of nucleotides that are easily removed include uracil and 8-oxo-guanine as set forth in more detail below. The location can also be at or near the recognition sequence of a restriction endonuclease (e.g., nickase).

[0031] As used herein, the term "cluster" refers to a nucleic acid population attached to a solid support to form a feature or site when used with respect to nucleic acid. The nucleic acid is generally a member of a single species, thereby forming a monoclonal cluster. A "monoclonal population" of nucleic acid is a population homologous to a specific nucleotide sequence. A cluster does not need to be monoclonal. Rather, for some applications, a cluster can be mainly colonized with an amplicon from a first nucleic acid, and may also have a low level of contamination amplicon from a second nucleic acid. For example, when a cluster array is to be used in a detection application, an acceptable level of contamination may be a level that does not affect the signal-to-noise ratio or resolution of the detection technology in an unacceptable manner. Therefore, obvious clonality will generally be associated with a specific use or application of an array manufactured by the methods described herein. Example contamination levels acceptable in individual clusters include, but are not limited to, up to 0.1%, 0.5%, 1%, 5%, 10%, 5 25% or 35% contamination amplicon. The nucleic acid in the cluster is generally, for example, covalently attached to a solid support by its 5' end, but other attachment methods are possible in some cases. The nucleic acid in the cluster may be single-stranded or double-stranded. In some but not all embodiments, clusters are made by a solid phase amplification method known as bridge amplification. Exemplary configurations of clusters and methods of producing them are set forth, for example, in U.S. Pat. No. 5,641,658, U.S. Pat. Publication No. 2002 / 0055100, U.S. Pat. No. 7,115,400, U.S. Pat. Publication No. 2004 / 0096853, U.S. Pat. Publication No. 2004 / 0002090, U.S. Pat. Publication No. 2007 / 0128624, and U.S. Pat. Publication No. 2008 / 0009420, each of which is incorporated herein by reference.

[0032] As used herein, the term "different" when used with respect to nucleic acids means that the nucleic acids have nucleotide sequences that are not identical to each other. Two or more nucleic acids may have nucleotide sequences that are different along their entire length. Alternatively, two or more nucleic acids may have nucleotide sequences that are different along a majority of their length. For example, two or more nucleic acids may have a target nucleotide sequence portion that is different for two or more molecules, while also having a common sequence portion that is the same on two or more molecules. Two beads may differ from each other by being attached to different nucleic acids.

[0033] As used herein, the term "each" when used with respect to a collection of items is intended to identify an individual item in the collection, but does not necessarily refer to each item in the collection. Exceptions exist if explicitly disclosed or context clearly indicates otherwise.

[0034] As used herein, the term "extension" is intended to mean the addition of at least one nucleotide or oligonucleotide to a nucleic acid when used with respect to a nucleic acid. In a particular embodiment, one or more nucleotides may be added to the 3' end of a nucleic acid, for example, by polymerase catalysis (e.g., DNA polymerase, RNA polymerase, or reverse transcriptase). One or more nucleotides may be added to the 3' or 5' end of a nucleic acid using chemical or enzymatic methods. One or more oligonucleotides may be added to the 3' or 5' end of a nucleic acid, for example, by chemical or enzymatic (e.g., ligase catalysis) methods. Nucleic acids may be extended in a template-directed manner, whereby the extension product is complementary to a template nucleic acid hybridized to the extended nucleic acid.

[0035] 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 adjacent or they may have intervals between each other. The size of the feature and / or the intervals between the features may be variable, so that the array may have a high density, a medium density, or a relatively low density. A high-density array is characterized by sites being less than about 15 μm apart. The sites of a medium-density array are about 15 to 30 μm apart, while the sites of a low-density array are greater than 30 μm apart. The sites of the arrays available herein may, for example, be less than 100 μm, 50 μm, 10 μm, 5 μm, 1 μm, or 0.5 μm apart. The apparatus or method disclosed herein may be used to detect arrays with a resolution sufficient to distinguish sites having the above density or density range.

[0036] As used herein, the term "fluid mixture" is intended to mean two or more different items that are present in a solution simultaneously. Typically, the two or more items can diffuse freely in a solution. The two or more items can be different types of items (e.g., nucleic acids and proteins, which are different types of molecules), or they can be different types of items of the same type (e.g., two nucleic acid molecules with different sequences). Exemplary items that can be in a fluid mixture include, but are not limited to, molecules, cells, or beads.

[0037] As used herein, the term "flow cell" is intended to mean a container having a chamber in which a reaction can be performed, an inlet for delivering reagents to the chamber, and an outlet for removing reagents from the chamber. In some embodiments, the chamber is configured to detect a reaction occurring in the chamber. For example, the chamber may include one or more transparent surfaces that allow optical detection of a biological sample, an optically labeled molecule, or the like in the chamber. Exemplary flow cells include, but are not limited to, those used in nucleic acid sequencing devices, such as the Genome 100 commercialized by ILUMINA (San Diego, CA). or or for the SOLiD flow cell commercialized by Life Technologies (Carlsbad, CA)TM or Ion Torrent TM Flow cells for sequencing platforms. Exemplary flow cells and methods of making and using the same are also described in, for example, WO 2014 / 142841 A1; US ​​Patent Application Publication No. 2010 / 0111768 A1 and US Patent No. 8,951,781, each of which is incorporated herein by reference.

[0038] As used herein, the term "gel" is intended to mean a semi-rigid material that is permeable to liquids and gases. Typically, gel materials can expand when absorbing liquids and can shrink when the liquid is removed by drying. Exemplary gels include, but are not limited to, those having a colloidal structure, such as agarose; a polymer mesh structure, such as gelatin; or a cross-linked polymer structure, such as polyacrylamide, SFA (see, e.g., U.S. Patent Application Publication No. 2011 / 0059865 A1, which is incorporated herein by reference) or PAZAM (see, e.g., U.S. Patent Application Publication No. 2014 / 0079923 A1, which is incorporated herein by reference). Particularly useful gel materials will conform to the shape of the hole or other recessed feature in which they reside.

[0039] As used herein, the terms "nucleic acid" and "nucleotide" are intended to be consistent with their use in the art and to include natural species or functional analogs thereof. Particularly useful functional analogs of nucleic acids can hybridize with nucleic acids in a sequence-specific manner or can be used as templates for replicating specific nucleotide sequences. Natural nucleic acids generally have a backbone containing phosphodiester bonds. The analog structure can have alternative backbone links, including any of a variety of links known in the art. Natural nucleic acids generally have deoxyribose (such as found in deoxyribonucleic acid (DNA)) or ribose (such as found in ribonucleic acid (RNA)). Nucleic acids can contain nucleotides having any of a variety of analogs of these sugar moieties known in the art. Nucleic acids can include natural or non-natural nucleotides. In this regard, natural deoxyribonucleic acids can have one or more bases selected from the group consisting of adenine, thymine, cytosine or guanine, and ribonucleic acids can have one or more bases selected from the group consisting of uracil, adenine, cytosine or guanine. Useful non-natural bases that can be included in nucleic acids or nucleotides are known in the art. The term "probe" or "target" when used in relation to a nucleic acid or nucleic acid sequence is intended to be a semantic identifier of the nucleic acid or sequence in the context of the methods or compositions described herein, and is not necessarily limited to the structure or function of the nucleic acid or sequence except where otherwise expressly indicated. The terms "probe" and "target" may be similarly applied to other analytes, such as proteins, small molecules, cells, or the like.

[0040] As used herein, the term "pitch" is intended to refer to the center-to-center spacing of adjacent features when used with respect to array features. The pattern of features can be characterized according to the average pitch. The pattern can be ordered so that the coefficient of variation near the average pitch is small, or the pattern can be random, in which case the coefficient of variation can be relatively large. In either case, the average pitch can be, for example, at least about 10nm, 0.1 μm, 0.5 μm, 1 μm, 5 μm, 10 μm, 100 μm or larger. Alternatively or in addition, the average pitch can be, for example, at most about 100 μm, 10 μm, 5 μm, 1 μm, 0.5 μm, 0.1 μm or smaller. Of course, the average pitch of a particular feature pattern can be between one of the lower values ​​selected from the above range and one of the higher values.

[0041] As used herein, the term "poly T or poly A" is intended to mean a series of two or more thymine (T) or adenine (A) bases, respectively, when used with respect to a nucleic acid sequence. Poly T or poly A may include at least about 2, 5, 8, 10, 12, 15, 18, 20 or more T or A bases, respectively. Alternatively or additionally, poly T or poly A may include up to about 30, 20, 18, 15, 12, 10, 8, 5 or 2 T or A bases, respectively.

[0042] As used herein, the term "random" can be used to refer to the composition of spatial arrangement or positioning on the surface. For example, the array described herein has at least two types of order, the first type is about the spacing and relative positioning of features (also called "sites"), and the second type is about the properties or predetermined knowledge of the molecules of a specific type present at a specific feature. Therefore, the array features can be randomly spaced so that the nearest adjacent features have a variable spacing between each other. Alternatively, the spacing between features can be ordered, such as forming a regular pattern, such as a rectilinear grid or a hexagonal grid. In another aspect, the array features can be random about the properties or predetermined knowledge of the type of analyte (such as a specific sequence of nucleic acids) occupying each feature, regardless of whether the spacing produces a random pattern or an ordered pattern. The array described herein can be ordered in one aspect and random in another aspect. For example, in some embodiments described herein, the surface is contacted with a nucleic acid population under conditions in which nucleic acids are attached to sites, and the sites are ordered about their relative positioning, but the knowledge of the sequence of the nucleic acid species present at any particular site is 'randomly positioned'. In referring to nucleic acids being "randomly distributed" at locations on a surface, it is intended that there is no knowledge or prior determination as to which nucleic acids will be captured at said locations (regardless of whether said locations are arranged in an ordered pattern).

[0043] As used herein, the term "solid support" refers to a rigid substrate that is insoluble in aqueous liquids. The substrate may be nonporous or porous. The substrate may optionally be capable of absorbing liquids (e.g., due to porosity), but will generally be sufficiently rigid so that the substrate does not significantly expand when absorbing liquids and does not significantly shrink when the liquids are removed by drying. Nonporous 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 acrylics, polystyrene and copolymers of styrene with 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, optical fiber bundles, and polymers. Particularly useful solid supports for some embodiments are located within a flow cell device. Exemplary flow cells are described in more detail herein.

[0044] As used herein, the term "spatially tagged" is intended to mean a nucleic acid having a sequence that indicates a location. Typically, a nucleic acid is a synthetic molecule having a sequence that is not found in one or more biological samples to be used for the nucleic acid. However, in some embodiments, the nucleic acid molecule may be of natural origin or the nucleic acid sequence may be naturally present in a biological sample, such as for nucleic acid. The location indicated by the spatial tag may be a location in or on a biological sample, in or on a solid support, or a combination thereof. A barcode sequence may be used as a spatial tag.

[0045] As used herein, the term "tissue" is intended to mean an aggregate of cells and optionally intercellular material. Typically, 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.

[0046] As used herein, the term "universal sequence" refers to a series of nucleotides common to two or more nucleic acid molecules, even if the molecules also have sequence regions that are different from each other. Universal sequences present in different members of a molecular set allow the use of a universal capture nucleic acid population that is complementary to the universal sequence to capture a variety of different nucleic acids. Similarly, universal sequences present in different members of a molecular set allow the use of a universal primer population that is complementary to the universal sequence to replicate or amplify a variety of different nucleic acids. Therefore, universal capture nucleic acids or universal primers include sequences that can specifically hybridize with universal sequences. Target nucleic acid molecules can be modified to attach universal adapters at one or both ends of, for example, different target sequences.

[0047] The embodiments described below and recited in the claims are to be understood in light of the above definitions.

[0048] The present disclosure provides a method for labeling nucleic acids of biological samples in a spatial manner. The method may include the following steps: (a) attaching different nucleic acid probes to a solid support to generate random positioning probes on the solid support, wherein the different nucleic acid probes each include a barcode sequence, and wherein the random positioning probes each include a barcode sequence different from other random positioning probes on the solid support; (b) performing a nucleic acid detection reaction on the solid support to determine the barcode sequence of the random positioning probe on the solid support; (c) contacting the biological sample with the solid support having the random positioning probe; (d) hybridizing the random positioning probe with a target nucleic acid from a portion of the biological sample near the random positioning probe; and (e) extending the random positioning probe to generate an extended probe including a barcode sequence and a sequence from the target nucleic acid, thereby labeling the nucleic acids of the biological sample in a spatial manner.

[0049] 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 optical fiber bundles, polymers, and porous (e.g., microtiter) plates. Exemplary plastics include acrylics, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, and Teflon. TM Exemplary silica-based materials include silicon and various forms of modified silicon.

[0050] In certain embodiments, the solid support can be within or part of a container, such as a well, tube, channel, cuvette, Petri plate, bottle, or the like. A particularly useful 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 a flow cell available from ILUMINA (San Diego, CA) for use in sequencing platforms such as Genome or Another particularly useful container is the wells in a multiwell plate or microtiter plate.

[0051] Optionally, the solid support may include a gel coating. Attachment of nucleic acids to a solid support by gel is exemplified by a flow cell available from ILUMINA (San Diego, CA) or described in U.S. Patent Application Publication Nos. 2011 / 0059865 A1, 2014 / 0079923 A1, or 2015 / 0005447 A1; or PCT Publication No. WO 2008 / 093098, each of which is incorporated herein by reference. Exemplary gels that can be used in the methods and devices described herein include, but are not limited to, gels having a colloidal structure, such as agarose; a polymer network structure, such as gelatin; or a cross-linked polymer structure, such as polyacrylamide, SFA (see, e.g., U.S. Patent Application Publication No. 2011 / 0059865 A1, which is incorporated herein by reference) or PAZAM (see, e.g., U.S. Patent Application Publication No. 2014 / 0079923 A1 or 2015 / 0005447 A1, each of which is incorporated herein by reference).

[0052] In some embodiments, the solid support can be configured as an array of features to which nucleic acids can be attached. Features can exist in any of a variety of desired formats. For example, features can be holes, recesses, channels, protuberances, raised areas, piles, columns, or the like. In some embodiments, features can contain beads. However, in certain embodiments, features do not need to contain beads or particles. Exemplary features include holes present in substrates for commercially available sequencing platforms sold by 454 Life Sciences (454 Life Sciences, a subsidiary of Roche, Basel, Switzerland) or Aintorent (a subsidiary of Life Technologies, Carlsbad, California). Other substrates having holes include, for example, etched fiber optic substrates and other substrates described in the following documents: 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 / 0026082 Al, 2009 / 0127589 Al, 2010 / 0137143 Al, 2010 / 0282617 Al, or PCT Publication No. WO 00 / 63437, each of which is incorporated herein by reference. In some embodiments, the wells of the substrate may include a gel material (with or without beads) as described in U.S. Patent Application Publication No. 2014 / 0243224 Al, which is incorporated herein by reference.

[0053] The features on the solid support can be metallic features on a non-metallic surface (e.g., glass, plastic, or other materials exemplified above). The metal layer can be deposited on the surface using methods known in the art, such as wet plasma etching, dry plasma etching, atomic layer deposition, ion beam etching, chemical vapor deposition, vacuum sputtering, or the like. If appropriate, any of a variety of commercially available instruments can be used, including, for example, Ionfab or Optofab System (Oxford Instruments, UK). Metal layers can also be deposited by electron beam evaporation or sputtering, as described in Thornton, Ann. Rev. Mater. Sci. 7:239-60 (1977), which is incorporated herein by reference. Metal layer deposition techniques (such as those exemplified above) can be combined with photolithography techniques to produce metal areas or patches on a surface. Exemplary methods of combining metal layer deposition techniques and photolithography techniques are provided in U.S. Patent No. 8,895,249 or U.S. Patent Application Publication No. 2014 / 0243224A1, each of which is incorporated herein by reference.

[0054] Features can be shown as a grid of plaques or patches on a solid support. Features can be positioned with a repeating pattern or an irregular non-repeating pattern. Particularly useful repeating patterns are hexagonal patterns, rectilinear patterns, grid patterns, patterns with reflection symmetry, patterns with rotational symmetry, or the like. Asymmetric patterns can also be used. Pitch can be the same between different nearest neighbor feature pairs, or pitch can be variable between different nearest neighbor feature pairs.

[0055] High density arrays are characterized by an average pitch of less than about 15 μm. Medium density arrays have an average pitch of about 15 to 30 μm, while low density arrays have an average pitch of greater than 30 μm. Arrays useful in the present invention may have an average pitch of less than 100 μm, 50 μm, 10 μm, 5 μm, 1 μm, or 0.5 μm. The average pitch values ​​and ranges stated above and elsewhere herein are intended to apply to either ordered or random arrays.

[0056] 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 , 100μm 2 or 500μm 2Alternatively or additionally, the features may each have an area of ​​less than about 1 mm 2 , 500μm 2 , 100μm 2 , 25μm2, 10μm 2 , 5μm 2 , 1μm 2 , 500nm 2 or 100nm 2 The above ranges may describe the apparent area of ​​a bead or other particle on a solid support when viewed or imaged from above.

[0057] In certain embodiments, the solid support may include a collection of beads or other particles. The particles may be suspended in a solution or may be positioned on a substrate surface. Examples of arrays with beads positioned on the surface include those arrays in which beads are positioned in holes, such as BeadChip arrays (Illumina, San Diego, CA), substrates for sequencing platforms from 454 Life Sciences (a subsidiary of Roche, Basel, Switzerland), or substrates for sequencing platforms from Iron Torrent (a subsidiary of Life Technologies, Carlsbad, California). Other solid supports with beads positioned on the surface are described in the following documents: U.S. Patent Nos. 6,266,459, 6,355,431, 6,770,441, 6,859,570, 6,210,891, 6,258,568 or 6,274,320; U.S. Patent Application Publication Nos. 2009 / 0026082 A1, 2009 / 0127589 A1, 2010 / 0137143 A1 or 2010 / 0282617 A1 or PCT Publication No. WO 00 / 63437, each of which is incorporated herein by reference. Several documents in the above cited documents describe methods of attaching nucleic acid probes to beads before loading beads in or on a solid support. Therefore, a collection of beads may include different beads each with a unique probe attached thereto. However, it should be understood that the beads can be made to include universal primers, and the beads can then be loaded onto an array, thereby forming a universal array for use in the methods described herein. As previously described, solid supports typically used for bead arrays can be used without beads. For example, nucleic acids (e.g., probes or primers) can be attached directly to the wells or to a gel material in the wells. Therefore, the above references illustrate materials, compositions, or devices that can be modified for use in the methods and compositions described herein.

[0058] Therefore, the solid support used in the methods described herein may include a bead array, wherein different nucleic acid probes are attached to different beads in the array. In this embodiment, each bead can be attached to a different nucleic acid probe, and the beads can be randomly distributed on the solid support to effectively attach different nucleic acid probes to the solid support. Optionally, the solid support may include a hole with a size that accommodates no more than a single bead. In this configuration, the beads can be attached to the hole by the force generated by the cooperation of the beads in the hole. It is also possible to use an attachment chemical or an adhesive to hold the beads in the hole.

[0059] The nucleic acid probe attached to the bead may include a barcode sequence. The bead population may be configured so that each bead is attached to only one type of barcode and there are a variety of different beads each having different barcodes in the population. In this embodiment, the beads are randomly distributed to the solid support and will result in the nucleic acid probe (and its corresponding barcode sequence) being randomly positioned on the solid support. In some cases, multiple beads may have the same barcode sequence so that there is redundancy in the population. The redundant population of beads is randomly distributed on a solid support having a capacity greater than the number of unique barcodes in the bead population and will result in redundancy of barcodes on the solid support. Alternatively, the number of different barcodes in the bead population may exceed the capacity of the solid support to produce an array that is not redundant with respect to the barcode population on the solid support. In certain embodiments, the capacity of the solid support will be determined according to the number of features (such as single beads occupying holes) of the beads attached or otherwise accommodated.

[0060] The solid support may include a plurality of different nucleic acid probes, or may be manufactured by the methods described herein to attach a plurality of different nucleic acid probes. For example, the solid support 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 or more different probes. Alternatively or additionally, the solid support may include 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, for example, when the probe has been amplified to form a cluster, the different probes can each exist in several copies. Therefore, the above range can describe the number of different nucleic acid clusters on the solid support. It will also be understood that the above range can describe the number of different barcodes, target capture sequences or other sequence elements stated herein as unique to a specific nucleic acid probe. Alternatively or in addition, the range can describe the number of extended probes or modified probes produced on a solid support using the methods described herein.

[0061] Features may be present on the solid support before the solid support is contacted with the nucleic acid probe. For example, in the embodiment in which the probe is attached to the carrier by hybridization with a primer, the primer may be attached to the feature, and the void region outside the feature is substantially free of any primer. The nucleic acid probe may be captured at the preformed features on the solid support using the method stated in the following document, and optionally amplified on the solid support: U.S. Patent No. 8,895,249, U.S. Patent No. 8,778,849 or U.S. Patent Application Publication No. 2014 / 0243224 A1, each of which is incorporated herein by reference. Alternatively, the solid support may have a primer lawn (lawn) or may lack features in addition. In this case, features may be formed by attaching nucleic acid probes on the solid support. Optionally, the captured nucleic acid probe may be amplified on the solid support so that the resulting cluster becomes a feature. Although attached to the capture illustrated above as the capture between the complementary part of the primer and the probe, it should be understood that the capture part that is not the primer may be present at the preformed features or exist as lawn. Other exemplary capture moieties include, but are not limited to, chemical moieties that react with the nucleic acid probe to form a covalent bond or receptors that non-covalently bind to a ligand on the nucleic acid probe.

[0062] The step of attaching nucleic acid probe to solid carrier can be implemented by providing the fluid of the mixture containing different nucleic acid probes and making this fluid mixture contact with solid carrier.Described contact can cause fluid mixture to contact with surface, and a variety of different nucleic acid probes from fluid mixture will be attached to described surface.Therefore, probe arrives at described surface (described surface has the preformed feature configured to attach probe or the uniform surface configured for attachment) at random.Therefore, probe can be randomly positioned on solid carrier.

[0063] The total number and the category of the different probes finally attached to the surface can be selected for a specific application or purpose.For example, in the embodiment in which the fluid mixture of different nucleic acid probes is contacted with a solid carrier for the purpose of probe being attached to the carrier, the number of different probe types can exceed the probe occupation number of the solid carrier.Therefore, the number and category of the different probes attached to the solid carrier can be equivalent to the probe occupation number of the solid carrier.Or, the number and category of the different probe types on the solid carrier can be less than the number of occupations (i.e., there will be redundancy in the probe types, so that the solid carrier can contain multiple features with the same probe types).Described redundancy can be realized, for example, by making the solid carrier contact the fluid mixture containing the number and category of the probe types that are significantly lower than the probe occupation number of the solid carrier.

[0064] Attachment of the nucleic acid probe can be mediated by hybridization of the nucleic acid probe to a complementary primer attached to a solid support, chemical bond formation between a reactive portion on the nucleic acid probe and the solid support (examples are set forth in U.S. Pat. No. 8,895,249, U.S. Pat. No. 8,778,849, or U.S. Patent Application Publication No. 2014 / 0243224 A1, each of which is incorporated herein by reference), affinity interactions of a portion on the nucleic acid probe with a binding portion of the solid support (e.g., between known receptor-ligand pairs, such as streptavidin-biotin, antibody-epitope, lectin-carbohydrate, etc.), physical interactions of the nucleic acid probe with the solid support (e.g., hydrogen bonding, ionic forces, van der Waals forces, etc.), or other interactions known in the art to attach nucleic acids to surfaces.

[0065] In certain embodiments, the attachment of nucleic acid probe is non-specific about any sequence difference between nucleic acid probe and other nucleic acid probes attached to or to be attached to solid carrier.For example, different probes can have universal sequences complementary to primers attached to surface, or different probes can have common parts of attachment mediated with surface.Or, different probes (or subgroups of different probes) can each have unique sequences complementary to unique primers on solid carrier, or it can have unique parts interacting with one or more different reactive parts on solid carrier.In the described situation, unique primer or unique part can be optionally attached to predefined positioning, to selectively capture specific probe or specific type of probe in corresponding predefined positioning.

[0066] One or more features on a solid support may each include a single molecule of a particular probe. In some embodiments, a feature may be configured to accommodate no more than a single nucleic acid probe molecule. However, regardless of whether a feature can accommodate more than one nucleic acid probe molecule, a feature may only include a single nucleic acid probe molecule. Alternatively, an individual feature may include a plurality of nucleic acid probe molecules, such as an ensemble of nucleic acid probe molecules having the same sequence as one another. In particular embodiments, the ensemble may be generated by amplifying from a single nucleic acid probe template to produce, for example, an amplicon in a cluster attached to a surface.

[0067] Methods described herein can use any of a variety of amplification techniques. Exemplary techniques that can be used include, but are not limited to, polymerase chain reaction (PCR), rolling circle amplification (RCA), multiple displacement amplification (MDA) or random primer amplification (RPA). In certain embodiments, when the feature of the array can contain an amplicon in a volume with the desired capacity, amplification can be implemented in a solution. Preferably, the amplification technique used in the method of the present disclosure will be implemented on a solid phase. For example, one or more primer types (such as universal primers for one or more universal primer binding sites present in a nucleic acid probe) can be attached to a solid support. In a PCR embodiment, one or two primers for amplification can be attached to a solid support (such as by gel). The format of two primer types attached to a solid support is generally referred to as bridge amplification, because the double-stranded amplicon forms a bridge-like structure between the primers attached to the two surfaces flanking the replicated template sequence. Exemplary reagents and conditions that can be used for bridge amplification are described, for example, in U.S. Pat. Nos. 5,641,658, 7,115,400, or 8,895,249; or U.S. Patent Publication Nos. 2002 / 0055100 A1, 2004 / 0096853 A1, 2004 / 0002090 A1, 2007 / 0128624 A1, or 2008 / 0009420 A1, each of which is incorporated herein by reference. Solid phase PCR amplification can also be performed using one amplification primer attached to a solid support and a second primer in solution. An exemplary format using a combination of surface-attached primers and soluble primers is a format used in emulsion PCR, such as described in Dressman et al., Proc. Natl. Acad. Sci. USA 100:8817-8822 (2003); WO 05 / 010145; or U.S. Patent Application Publication No. 2005 / 0130173 A1 or No. 2005 / 0064460 A1, each of which is incorporated herein by reference. Emulsion PCR illustrates the format and it is understood that for the purposes of the methods described herein, the use of an emulsion is optional and in fact for several embodiments, an emulsion is not used.

[0068] RCA technology can be modified for the disclosed method. Exemplary components that can be used in RCA reactions and the principles by which RCA produces amplicons are described, for example, in Lizardi et al., Nat. Genet. 19: 225-232 (1998) and U.S. Patent Application Publication No. 2007 / 0099208 A1, each of which is incorporated herein by reference. Primers for RCA can be in solution or attached to a solid support. Primers can be one or more of the universal primers described herein.

[0069] MDA technology can be modified for use in the disclosed method. Some basic principles and available conditions of MDA are described in, for example, the following documents: 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); US 5,455,166; US 5,130,238; and US 6,214,587, each of which is incorporated herein by reference. Primers for MDA can be attached to a solid support in solution or at the amplification site. Likewise, the primer may be one or more of the universal primers described herein.

[0070] In a particular embodiment, a combination of the amplification techniques illustrated above can be used. For example, RCA and MDA can be used in combination, wherein RCA is used to generate concatemer amplicons in solution (e.g., using solution phase primers). The amplicon can then be used as a template for MDA using a primer (e.g., universal primer) attached to a solid support. In this example, the amplicon produced after the combined RCA and MDA steps will be attached to a solid support.

[0071] The nucleic acid probes used in the methods described herein or present in the devices or compositions of the present disclosure may include barcode sequences, and for embodiments that include a plurality of different nucleic acid probes, the probes may each include a barcode sequence that is different from the other probes in the plurality of probes. The barcode sequences may have any of a variety of lengths. For a population, longer sequences generally can accommodate a larger number and variety of barcodes. Typically, all of the probes in a plurality of probes will have barcodes of the same length (although with different sequences), but it is also possible to use barcodes of different lengths 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 fewer nucleotides. Examples of barcode sequences that can be used are set forth, for example, in U.S. Patent Application Publication No. 2014 / 0342921 A1 and U.S. Patent No. 8,460,865, each of which is incorporated herein by reference.

[0072] The methods of the present disclosure may include the step of performing a nucleic acid detection reaction on a solid support to determine the barcode sequence of a nucleic acid probe that is located on the solid support. In a plurality of embodiments, the probes are randomly positioned on the solid support and the nucleic acid detection reaction provides information that locates each different probe. Exemplary nucleic acid detection methods include, but are not limited to, nucleic acid sequencing of the probe, hybridization of a nucleic acid to the probe, ligation of a nucleic acid that hybridizes to the probe, extension of a nucleic acid that hybridizes to the probe, ligation of an extended nucleic acid to a second nucleic acid that hybridizes to the probe after extending a first nucleic acid that hybridizes to the probe, or other methods known in the art, such as the methods set forth in U.S. Patent No. 8,288,103 or 8,486,625, each of which is incorporated herein by reference.

[0073] Sequencing techniques (e.g., synthetic sequencing (SBS) techniques) are particularly useful methods for determining barcode sequences. SBS can be implemented as follows. To initiate the first SBS cycle, one or more labeled nucleotides, DNA polymerases, SBS primers, etc. can be contacted with one or more features on a solid support (e.g., features where a nucleic acid probe is attached to a solid support). Features in which the labeled nucleotides are incorporated can be detected by SBS primer extension. Optionally, the nucleotides can include a reversible termination portion that immediately terminates further primer extension once the nucleotides are added to the SBS primer. For example, a nucleotide analog with a reversible terminator portion can be added to the primer so that subsequent extension does not occur before the unblocking agent is delivered to remove the portion. Therefore, for embodiments using reversible termination, unblocking agents can be delivered to the solid support (before or after detection). Washing can be implemented between multiple delivery steps. The cycle can then be repeated n times to extend the primer by n nucleotides, thereby detecting a sequence of length n. Exemplary SBS procedures, fluid systems, and detection platforms that can be readily adapted for 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. Patent Application Publication No. 2008 / 0108082, each of which is incorporated herein by reference.

[0074] Other sequencing procedures using cyclic reactions can be used, such as pyrophosphate sequencing. Pyrophosphate sequencing detects the release of inorganic pyrophosphate (PPi) when a specific nucleotide is incorporated into a nascent nucleic acid chain (Ronaghi et al., Analytical Biochemistry 242 (1), 84-9 (1996); Ronaghi, Genome Res. 11 (1), 3-11 (2001); Ronaghi et al., Science 281 (5375), 363 (1998); or U.S. Pat. Nos. 6,210,891, 6,258,568, or 6,274,320, each of which is incorporated herein by reference). In pyrophosphate sequencing, the released PPi can be detected by immediate conversion to adenosine triphosphate (ATP) by ATP sulfurylase, and the generated ATP level can be detected by photons generated by luciferase. Therefore, the sequencing reaction can be monitored by a luminescent detection system. An excitation radiation source for a fluorescence-based detection system is not necessary for the pyrophosphate sequencing procedure. Useful fluid systems, detectors, and procedures that can be used to apply pyrophosphate sequencing to the apparatus, compositions, or methods of the present disclosure are described, for example, in PCT Patent Application Publication No. WO2012 / 058096, U.S. Patent Application Publication No. 2005 / 0191698 A1, or U.S. Patent Nos. 7,595,883 or 7,244,559, each of which is incorporated herein by reference.

[0075] Ligation sequencing reactions may also be used, including, for example, those described in Shendure et al., Science 309: 1728-1732 (2005); or U.S. Pat. No. 5,599,675 or 5,750,341, each of which is incorporated herein by reference. Some embodiments may include hybridization sequencing procedures, such as described in Bains et al., Journal of Theoretical Biology 135(3), 303-7 (1988); Drmanac et al., Nature Biotechnology 16, 54-58 (1998); Fodor et al., Science 251(4995), 767-773 (1995); or PCT Patent Application Publication No. WO 1989 / 10977, each of which is incorporated herein by reference. In both 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. Compositions, devices or methods described herein or in the references cited herein can be easily adjusted for ligation sequencing or hybridization sequencing procedures. Typically, oligonucleotides are fluorescently labeled and can be detected using a fluorescence detector similar to those described for the SBS procedures herein or in the references cited herein.

[0076] Some sequencing embodiments may utilize methods involving real-time monitoring of DNA polymerase activity. For example, nucleotide incorporation can be detected by fluorescence resonance energy transfer (FRET) interactions between a polymerase carrying a fluorophore and a γ-phosphate labeled nucleotide or with a zero-mode waveguide (ZMW). Techniques and reagents for FRET-based sequencing are described, for example, in the following literature: Levene et al., Science 299, 682–686 (2003); Lundquist et al., Optics Express (Opt. Lett.) 33, 1026–1028 (2008); Korlach et al., Proceedings of the National Academy of Sciences of the United States of America 105, 1176–1181 (2008), each of which is incorporated herein by reference.

[0077] Some sequencing embodiments include detecting protons released when nucleotides are incorporated into extension products. For example, sequencing based on detection of released protons can use an electrical detector and related technology available from Iron Torrent (Guilford, Connecticut, a subsidiary of Life Technologies and Thermo Fisher Scientific (Thermo Fisher)) or sequencing methods and systems described in the following documents: U.S. Patent Application Publication Nos. 2009 / 0026082 A1; 2009 / 0127589 A1; 2010 / 0137143 A1; or US 2010 / 0282617 A1, each of which is incorporated herein by reference.

[0078] Nucleic acid hybridization techniques are also methods that can be used to determine barcode sequences. In some cases, combined hybridization methods can be used, such as those used to decode multiple bead arrays (see, for example, U.S. Pat. No. 8,460,865, which is incorporated herein by reference). The method utilizes a labeled nucleic acid decoder probe that is complementary to at least a portion of the barcode sequence. The hybridization reaction can be implemented using a decoder probe with a known label so that the final positioning of the label on the solid support identifies the nucleic acid probe according to the principle of nucleic acid complementarity. In some cases, a plurality of pools of different probes with distinguishable labels are used, thereby allowing multiple decoding operations. The number of different barcodes determined in the decoding operation may exceed the number of labels used for the decoding operation. For example, decoding can be implemented in several stages, each of which constitutes a hybridization with different pools of decoder probes. The same decoder probe may be present in different pools, but the labels present on each decoder probe may be different between pools (i.e., each decoder probe is in a different "state" when in different pools). Different combinations of these states and stages can be used to expand the number of decodable barcodes to far more than the number of different labels that can be used for decoding. Such combinatorial methods are described in more detail in U.S. Patent No. 8,460,865 or Gunderson et al., Genome Research 14:870-877 (2004), each of which is incorporated herein by reference.

[0079] The disclosed method may include the step of contacting the biological sample with a solid support to which a nucleic acid probe is attached. In certain embodiments, the nucleic acid probe is randomly positioned on the solid support. The properties and positioning of the nucleic acid probe may be decoded before contacting the biological sample with the solid support. Alternatively, the properties and positioning of the nucleic acid probe may be determined after contacting the solid support with the biological sample.

[0080] In some embodiments, the biological sample is one or more cells. The cell can be an individual cell and does not contain any tissue or multicellular structure when in contact with a solid carrier. For example, the cell can be present in a fluid (for example, when there are multiple different cells, the fluid can be a fluid mixture of different cells) and the fluid can be contacted with a solid carrier to which different probes are attached. Any of a variety of cells can be used, including, for example, cells from prokaryotes, archaebacteria, or eukaryotes. One or more cells used in the method, composition, or device of the present disclosure can be a unicellular organism or from a multicellular organism. Exemplary organisms from which one or more cells can be obtained include, but are not limited to, mammals, plants, algae, nematodes, insects, fish, reptiles, amphibians, fungi, or Plasmodium falciparum. Exemplary species are stated in the foregoing or are known in the industry.

[0081] The disclosed embodiments may also use one or more subcellular components as biological samples. For example, the fluid mixture may include one or more nuclei, golgi apparatus, mitochondria, chloroplasts, membrane fractions, vesicles, endoplasmic reticulum, or other components known in the art. Other useful types of biological samples are one or more viruses or viroids.

[0082] It should be understood that a biological sample can be a homogenous culture or population of the above cells, subcellular components, viruses or viroids. Alternatively, a biological sample can be a non-homogeneous collection of cells, subcellular components, viruses or viroids, for example, derived from several different organisms in a community or ecosystem. An example community is a collection of bacteria present in the digestive system, lungs or other organs of a multicellular organism (e.g., a mammal).

[0083] One or more cells, subcellular components, viruses or viroids contacted with solid carriers in the methods described herein can be attached to solid carriers. Attachment can be realized using methods known in the industry, such as those methods illustrated herein about attaching nucleic acid to solid carriers. In certain embodiments, attachment is selective for cells, subcellular components, viruses or viroids of a particular type. For example, solid carriers may include antibodies or other receptors, which are selective for epitopes or ligands present in one or a subgroup of different cells, subcellular components, viruses or viroids present in fluid mixtures. In other embodiments, attachment of cells, subcellular components, viruses or viroids can be mediated by non-selective moieties, such as chemical moieties with broad reactivity.

[0084] In certain embodiments, one or more cells, subcellular components, viruses or viroids that have been contacted with a solid support can be lysed to release the target nucleic acid. Lysis can be performed using methods known in the art, such as chemical treatment, enzyme treatment, electroporation, heating, hypotonic treatment, sonication, or one or more of the like. Exemplary lysis techniques are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ansubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1999).

[0085] In certain embodiments, biological sample is tissue section.Tissue can be derived from multicellular organisms, such as those exemplified above about cells.Tissue section can be contacted with solid carrier, such as by tissue being laid on the solid carrier surface.Tissue can be just excised from organism, or it can be stored in advance, such as by the following manner: freezing, embedding in materials such as paraffin (such as paraffin embedded samples fixed by formalin), formalin fixation, infiltration, dehydration or the like.Optionally, tissue section can be attached to solid carrier, such as by using the technology and composition exemplified herein about nucleic acid, cell, virus, bead or the like attached to solid carrier.As another option, tissue can be treated with infiltration and the cells in tissue are cracked when tissue contacts with solid carrier.Any one of multiple treatments can be used, such as those described above about lysed cells.The target nucleic acid released from the tissue treated with infiltration can be captured by nucleic acid probes on the surface.

[0086] The tissue can be prepared in any convenient or desired manner for its 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.

[0087] Tissue samples used herein can be fixed by deep freezing at a temperature suitable for maintaining or preserving the structural integrity of the tissue (e.g., below -20°C). In another example, the tissue can be prepared using formalin fixation and paraffin embedding (FFPE) methods known in the art. Other fixatives and / or embedding materials can be used as needed. Fixed or embedded tissue samples can be sliced, i.e., cut into thin slices, using known methods. For example, a freezing microtome or cryostat set at a temperature suitable for maintaining both the structural integrity of the tissue sample and the chemical properties of the nucleic acids in the sample can be used to slice the tissue sample.

[0088] In certain embodiments, the tissue sample will be treated to remove embedding material (e.g., paraffin or formalin) from the sample before the release, capture or modification of nucleic acid. This can be achieved by contacting the sample with a suitable solvent (e.g., dimethylbenzene and ethanol washings). The process can be performed before contacting the tissue sample with a solid carrier as described herein, or the process can be performed when the tissue sample is located on a solid carrier. An exemplary method for operating a tissue used together with a solid carrier attached with nucleic acid is set forth in U.S. Patent Application Publication No. 2014 / 0066318A1, which is incorporated herein by reference.

[0089] The thickness of the tissue sample or other biological sample contacted with the solid carrier in the methods, compositions or devices described herein can be any suitable thickness required. 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 biological sample contacted with the solid carrier will not be greater than 100 μm, 50 μm, 10 μm, 5 μm, 1 μm, 0.5 μm, 0.25 μm, 0.1 μm or thinner.

[0090] A particularly relevant source of biological samples is human. The sample may originate from an organ, including, for example, an organ of the musculoskeletal system, such as a muscle, bone, tendon or ligament; an organ of the digestive system, such as a salivary gland, pharynx, esophagus, stomach, small intestine, large intestine, liver, gall bladder or pancreas; an organ of the respiratory system, such as a larynx, trachea, bronchi, lungs or diaphragm; an organ of the urinary system, such as a kidney, ureter, bladder or urethra; a reproductive organ, such as an ovary, fallopian tube, uterus, vagina, placenta, testis, epididymis, vas deferens, seminal vesicle, prostate, penis or scrotum; an organ of the endocrine system, such as a pituitary gland, pineal gland, thyroid gland, parathyroid gland or adrenal gland; an organ of the circulatory system, such as a heart, artery, vein or capillary; an organ of the lymphatic system, such as a lymphatic vessel, lymph node, bone marrow, thymus gland or spleen; an organ of the central nervous system, such as the brain, brain stem, cerebellum, spinal cord, cranial nerves or spinal nerves; a sensory organ, such as an eye, ear, nose or tongue; or a skin organ, such as the skin, subcutaneous tissue or breast. In some embodiments, the biological sample is obtained from a body fluid or excretion, such as blood, lymph, tears, sweat, saliva, semen, vaginal secretions, earwax, fecal matter, or urine.

[0091] Samples from humans can be considered (or suspected) healthy or sick when used. In some cases, two samples can be used: the first sample is considered sick, and the second sample is considered healthy (e.g., used as a healthy control). Any of a variety of conditions can be assessed, including but not limited to autoimmune diseases, cancer, cystic fibrosis, aneuploidy, pathogenic infections, psychological conditions, hepatitis, diabetes, sexually transmitted diseases, heart disease, stroke, cardiovascular disease, multiple sclerosis, or muscular dystrophy. Particularly relevant conditions are genetic conditions or conditions associated with pathogens with identifiable genetic imprints.

[0092] As described above, flow cell provides a convenient device for the methods described herein. For example, flow cell is a convenient device for accommodating a solid carrier, and the solid carrier will be processed with a variety of fluid reagents (such as repeated fluid delivery) for some nucleic acid sequencing schemes or some nucleic acid hybridization schemes. In certain embodiments, for example, when a fluid mixture of a cell, a subcellular component, a virus or a viroid is delivered to a solid carrier, a biological sample can be delivered to the solid carrier in the flow cell. In certain embodiments, the flow cell can be preferably opened to expose the solid carrier inside or to remove the solid carrier from the flow cell to allow the biological sample to be conveniently delivered to the solid carrier. For example, opening the flow cell or removing the solid carrier can allow the user or a robotic device to lay a tissue slice on the solid carrier. Opening the flow cell or removing the solid carrier from the flow cell can be temporary. Therefore, the flow cell can be closed subsequently or the solid carrier can be returned to the flow cell to proceed with one or more subsequent steps of the methods described herein.

[0093] In some embodiments, the flow cell may have a structure that allows it to be opened or disassembled. For example, when performing a sequencing reaction to, for example, decode a barcode, the flow cell may be in a closed state. The flow cell may then be disassembled so that the tissue can be placed on the flow cell surface. The flow cell may be held together by an adhesive so that one or more surfaces may be removed to open the flow cell. For example, the flow cell may have a spacer having an adhesive surface (similar to a single-sided or double-sided adhesive tape) on the top or bottom and this spacer may be present between two solid carriers. One or two solid carriers may be configured to attach nucleic acids and support biological samples as described herein. The spacer may have an open area (e.g., produced by laser cutting of the spacer material), which produces a fluid channel bounded by two solid carriers and the spacer. Therefore, one or two solid carriers may be non-permanently adhered to the spacer to allow removal of one or both of them, to allow access to the surface when a tissue or other sample is placed thereon.

[0094] The nucleic acid probe used in the compositions, devices or methods described herein may include a target capture portion. In a particular embodiment, the target capture portion is a target capture sequence. The target capture sequence is generally complementary to the target sequence so that target capture is performed by forming a probe-target hybrid complex. The target capture sequence may have any of a variety of lengths, including, for example, the lengths exemplified above in the case of a barcode sequence.

[0095] In various embodiments, a plurality of different nucleic acid probes may include different target capture sequences that hybridize with different target nucleic acid sequences from a biological sample. Different target capture sequences may be used to selectively bind to one or more desired target nucleic acids from a biological sample. In some cases, different nucleic acid probes may include a target capture sequence that is common to all probes or probe subsets on a solid support. For example, the nucleic acid probe on a solid support may have a poly-A or poly-T sequence. The probe or its amplicon may hybridize with an mRNA molecule, a cDNA molecule, or its amplicon having a poly-A or poly-T tail. Although the mRNA or cDNA species will have different target sequences, capture will be mediated by a common poly-A or poly-T sequence region.

[0096] Any of a variety of target nucleic acids can be captured and analyzed in the methods described herein, including but not limited to messenger RNA (mRNA), replicating DNA (cDNA), genomic DNA (gDNA), ribosomal RNA (rRNA) or transfer RNA (tRNA). Specific target sequences can be selected from a database and appropriate capture sequences designed using techniques and databases known in the art.

[0097] Other target capture moieties that can be used include, for example, those set forth herein as being useful for attaching nucleic acid probes to solid supports.

[0098] The method described herein may include a step of hybridizing a nucleic acid probe on a solid support with a target nucleic acid from a portion near the probe in a biological sample. Typically, the target nucleic acid will diffuse from the biological sample region to the region of the solid support near the region in the sample. Here, the target nucleic acid will interact with the nucleic acid probe near the region in the sample where the target nucleic acid is released. A target-probe hybrid complex can be formed, in which the target nucleic acid encounters a complementary target capture sequence on the nucleic acid probe. The positioning of the target-probe hybrid complex will generally be associated with the region in which the target nucleic acid is produced in the biological sample. In a number of embodiments, the solid support will include a plurality of nucleic acid probes, the biological sample will release a plurality of target nucleic acids, and a plurality of target-probe hybrids will be formed on the solid support. The sequence of the target nucleic acid and its positioning on the support will provide spatial information about the nucleic acid content of the biological sample. Although the above examples are described in the context of a target nucleic acid released from a biological sample, it should be understood that the target nucleic acid does not need to be released. On the contrary, for example, when the target nucleic acid is attached to the exposed surface of the biological sample in a manner such that the target nucleic acid can also be bound to an appropriate nucleic acid probe on a solid support, the target nucleic acid can remain in contact with the biological sample.

[0099] The disclosed method may include the step of extending the solid support attachment probe hybridized with the target nucleic acid. In the embodiment where the probe includes a barcode sequence, the resulting extension probe will include the barcode sequence and the sequence from the target nucleic acid (even if in a complementary form). The extension probe is a form of spatially labeled target nucleic acid from a biological sample. The sequence of the extension probe identifies which nucleic acid is present in the biological sample and where the target nucleic acid is located in the biological sample. It should be understood that other sequence elements present in the nucleic acid probe may also be included in the extension probe. The element includes, for example, a primer binding site, a cleavage site, other tag sequences (such as sample identification tags), a capture sequence, a nucleic acid binding protein or a recognition site for a nuclease or the like.

[0100] The extension of the probe can be implemented using the methods exemplified herein or other methods known in the industry for nucleic acid amplification or nucleic acid sequencing. In a specific embodiment, one or more nucleotides can be added to the 3' end of the nucleic acid, for example, by polymerase catalysis (e.g., DNA polymerase, RNA polymerase, or reverse transcriptase). One or more nucleotides can be added to the 3' or 5' end of the nucleic acid using a chemical or enzymatic method. One or more oligonucleotides can be added to the 3' or 5' end of the nucleic acid, for example, by a chemical or enzymatic (e.g., ligase catalysis) method. The nucleic acid can be extended in a template-guided manner, whereby the extension product is complementary to the template nucleic acid hybridized to the extended nucleic acid. In some embodiments, the DNA primer is extended using an RNA template by a reverse transcriptase, thereby producing cDNA. Therefore, the extension probe manufactured in the method described herein can be a reverse transcribed DNA molecule. Exemplary methods for extending nucleic acids are set forth in U.S. Patent Application Publication No. US 2005 / 0037393 A1 or U.S. Patent No. 8,288,103 or No. 8,486,625, each of which is incorporated herein by reference.

[0101] The whole or part of the target nucleic acid hybridized with the nucleic acid probe can be replicated by extension.For example, the extension probe can include at least 1,2,5,10,25,50,100,200,500,1000 or more nucleotides replicated from the target nucleic acid.The length of the extension product can be controlled by, for example, using the nucleotides of reversible termination and running a limited number of extension cycles in the extension reaction.The circulation can be run as illustrated for the SBS technology, and it is not necessary to use labeled nucleotides.Therefore, the extension probe produced in the method described herein can include no more than 1000,500,200,100,50,25,10,5,2 or 1 nucleotides replicated from the target nucleic acid.Certainly, the extension probe can have any length within the scope described above or outside the scope.

[0102] Although the disclosed method is exemplified as extending the probe hybridized with the target nucleic acid to replicate at least a portion of the target nucleic acid, it should be understood that the probe can be modified in an alternative manner. The probe hybridized with the target nucleic acid can undergo a reaction that produces a target-specific modification of the probe. The target-specific modification will only be obtained when the probe, for example, interacts with the target nucleic acid by hybridization based on complementarity. In a number of embodiments, the target-specific modification will have specificity to the sequence of the specific target nucleic acid that interacts with the probe. Examples of available target-specific modifications include, but are not limited to, inserting or adding sequences by connection or displacement (see, for example, U.S. Patent Application Publication No. 2010 / 0120098 A1, incorporated herein by reference), chemical modification (such as psoralen cross-linking or adding a detectable label portion), nuclease modification, connection of hairpin connectors or U.S. Patent Application Publication No. US2005 / 0037393 A1 or U.S. Patent No. 8,288,103 or No. 8,486,625 Other modifications stated in nucleic acid analysis, each of which is incorporated herein by reference.

[0103] It should be understood that the probes used in the methods, compositions or devices described herein need not be nucleic acids. Other molecules, such as proteins, carbohydrates, small molecules, particles or the like can be used. The probe can be a combination of a nucleic acid component (e.g., having a barcode, primer binding site, cleavage site and / or other sequence elements described herein) and another part (e.g., a part that captures or modifies the target nucleic acid).

[0104] Methods described herein may further include the step of obtaining an image of a biological sample in contact with a solid carrier. The solid carrier may be in any of the various states described herein. For example, the solid carrier may include an attached nucleic acid probe or a cluster derived from an attached nucleic acid probe. Alternatively, the solid carrier may not include the nucleic acid probe, but may be in a state before the attached nucleic acid probe or in a state after the nucleic acid probe is removed from the solid carrier. Therefore, an image may be obtained at any of the multiple points in the methods described herein.

[0105] 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. Biological samples 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 tissue, different cells, specific subcellular components, or the like). In other embodiments, biological samples can be imaged without staining.

[0106] In certain embodiments, a fluorescent microscope (e.g., a confocal fluorescence microscope) can be used to detect biological samples that fluoresce, for example, with the aid of fluorescent markers. Fluorescent samples can also be imaged using a nucleic acid sequencing device with fluorescence detection optics, such as the Genome Imaging System commercialized by ILUMINA Corporation (San Diego, CA). or Platform device; or SOLiD commercialized by Life Technologies (Carlsbad, CA) TM Sequencing platform. Other imaging optics that can be used include those found in 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. Pat. Nos. 7,057,026, 7,329,492, 7,211,414, 7,315,019, or 7,405,281; and U.S. Patent Application Publication No. 2008 / 0108082, each of which is incorporated herein by reference.

[0107] An image of a biological sample can be obtained at a desired resolution, for example to distinguish tissue, cell or subcellular components. Thus, the resolution can be sufficient to distinguish 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 more apart. Alternatively or additionally, the resolution can be set to distinguish 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 more apart.

[0108] The methods described herein may include a step of associating the location in the image of the biological sample with a barcode sequence of a nucleic acid probe attached to a surface with which the biological sample is, has been, or will be in contact. Thus, the features of the biological sample identifiable in the image may be associated with nucleic acids found to be present in its vicinity. Any of a variety of morphological features may be used in this association, including, for example, cell shape, cell size, tissue shape, staining pattern, the presence of a specific protein (e.g., as detected by immunohistochemical staining) or other features routinely assessed in pathology or research applications. Thus, the biological state of a tissue or its components as determined by visual observation may be associated with molecular biological features as determined by spatially resolved nucleic acid analysis.

[0109] The solid support on which the biological sample is imaged may include a fiducial marker to help determine the orientation of the sample or its image relative to the probe attached to the solid support. Example benchmarks include, but are not limited to, beads (with or without a fluorescent moiety or, for example, a portion of a nucleic acid to which a labeled probe can be bound), fluorescent molecules attached to known or measurable features, or structures of combined morphological shapes and fluorescent moieties. Example benchmarks are set forth in U.S. Patent Application Publication No. 2002 / 0150909A1 or U.S. Patent Application No. 14 / 530,299, each of which is incorporated herein by reference. One or more benchmarks are preferably visible when obtaining an image of a biological sample. Preferably, the solid support includes at least 2, 3, 4, 5, 10, 25, 50, 100 or more fiducial markers. The benchmark may be provided in a pattern, such as provided along the outer edge of the solid support or the periphery of the location where the biological sample resides. In a preferred embodiment, one or more benchmarks are detected using the same imaging conditions as for visualizing the biological sample. However, if desired, a separate image (such as an image of a biological sample and another benchmark image) may be obtained and the images may be aligned to each other.

[0110] Optionally, the biological sample can be removed from the solid support after the image is obtained and after the nucleic acid probe on the solid support captures the target nucleic acid. Therefore, the disclosed method may include washing the solid support to remove the cell, tissue or other material from the biological sample. The removal of the sample can be carried out using any suitable technology and will depend on the tissue sample. In some cases, the solid support can be washed with water. The water may contain various additives, such as surfactants (such as detergents), enzymes (such as proteases and collagenases), cleavage reagents or the like, to help remove the sample. In some embodiments, the solid support is treated with a solution comprising a protease. Alternatively or in addition, the solution may include cellulase, hemicellulase or chitinase (such as if it is necessary to remove the tissue sample from a plant or fungal source). In some cases, the temperature of the washing solution will be at least 30°C, 35°C, 50°C, 60°C or 90°C. Conditions may be selected to remove the biological sample while the heterocomplex formed between the nucleic acid probe attached to the target nucleic acid and the solid support is not denatured.

[0111] The disclosed method may further include the step of removing one or more extension probes from the solid support. In a particular embodiment, the probe will already include a cleavage site so that the product of the extended probe will also include the cleavage site. Alternatively, the cleavage site may be introduced into the probe during the modification step. For example, the cleavage site may be introduced into the extended probe during the extension step.

[0112] Exemplary cleavage sites include, but are not limited to, portions that are sensitive to chemical, enzymatic or physical processes that cause bond breakage. For example, positioning can be a nucleotide sequence recognized by an endonuclease. Suitable endonucleases and their recognition sequences are well known in the industry and are even commercially available in many cases (e.g., from New England Biolabs, Beverly, Massachusetts; ThermoFisher, Waltham, Massachusetts; or Sigma Aldrich, St. Louis, Missouri). Particularly useful endonucleases will break the bond of the site 3' away from its binding site in the nucleic acid chain, examples of which include type II or type IIs restriction endonucleases. In certain embodiments, an endonuclease will cut only one chain (e.g., nickase) in a diploid nucleic acid. Examples of endonucleases that only cleave one chain include Nt.BstNBI and Nt.AlwI.

[0113] In some embodiments, the cleavage site is an abasic site or a nucleotide with a base that is easily removed to produce an abasic site. Examples of nucleotides that are easily removed to produce an abasic site include uracil and 8-oxo-guanine. Abasic sites can be produced by using chemical or enzymatic reagents to hydrolyze nucleotide residues. Once formed, abasic sites can be cleaved (e.g., by treating with endonucleases or other single-stranded cleavage enzymes, being exposed to heat or alkali), providing a site-specific cleavage mode of nucleic acid. Abasic sites can be produced in uracil nucleotides on one strand of nucleic acid. Uracil DNA glycosidase (UDG) can be used to remove uracil bases and generate abasic sites on the strand. Subsequently, nucleic acid chains with abasic sites can be cleaved at abasic sites by treating with endonucleases (e.g., EndoIV endonucleases, AP cleavage enzymes, FPG glycosidase / AP cleavage enzymes, EndoVIII glycosidase / AP cleavage enzymes), heat or alkali. In a specific embodiment, USER DNA cleavage enzymes available from New England Biolabs are used. TM The reagent creates a single nucleotide vacancy at a uracil base in a nucleic acid.

[0114] Abasic sites can also be generated at non-natural / modified deoxyribonucleotides that are not uracil and cleaved in a similar manner by treatment with endonucleases, heat or alkali. For example, 8-oxo-guanine can be converted to an abasic site by exposure to FPG glycosidase. Deoxyinosine can be converted to an abasic site by exposure to AlkA glycosidase. The abasic site thus generated can then be cleaved, typically by treatment with a suitable endonuclease (e.g., EndoIV or AP lyase).

[0115] Other examples of cleavage sites and methods that can be used to cleave nucleic acids are set forth, for example, in U.S. Pat. No. 7,960,120, which is incorporated herein by reference.

[0116] The modified nucleic acid probes (e.g., extended nucleic acid probes) released from the solid support can be collected to form a fluid mixture. The mixture can include, for example, 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 or more different modified probes. Alternatively or additionally, the fluid mixture may include up to 1×10 9 , 1×10 8 , 1×10 7 , 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 , 100, 10 or fewer different modified probes. The fluid mixture can be manipulated to allow detection of modified nucleic acid probes. For example, the modified nucleic acid probes can be spatially separated on a second solid support (i.e., different from the solid support from which the nucleic acid probes were released after contact with the biological sample and modified), or the probes can be separated in time in the fluid stream.

[0117] In the capture or detection methods (such as those stated above) of microarray-based technology or nucleic acid sequencing technology, modified nucleic acid probes (such as extended nucleic acid probes) can be separated on a solid support. For example, modified probes can be attached to a microarray by hybridization with complementary nucleic acids. Modified probes can be attached to beads or to the surface of a flow tank and optionally amplified, as implemented in multiple nucleic acid sequencing platforms. Modified probes can be separated in a fluid stream using a microfluidic device, a droplet operating device or a flow cytometer. Typically, detection is implemented on these separation devices, but detection is not necessary in all embodiments.

[0118] Particularly useful droplet manipulation devices are droplet actuators, as described, for example, in U.S. Pat. No. 8,637,242, U.S. Pat. No. 6,911,132, entitled “Apparatus for Manipulating Droplets by Electrowetting-Based Techniques,” issued June 28, 2005; Pamula et al., U.S. Patent Publication No. 20060194331, entitled “Apparatuses and Methods for Manipulating Droplets on a Printed Circuit Board” issued June 28, 2006. Board), published on August 31, 2006; Pollack et al., International Patent Publication No. WO / 2007 / 120241, entitled “Droplet-Based Biochemistry,” published on October 25, 2007; Shenderov, U.S. Patent No. 6,773,566, entitled “Electrostatic Actuators for Microfluidics and Methods for Using Same,” issued on August 10, 2004; Shenderov, U.S. Patent No. 256,565,727, entitled “Actuators for Microfluidics Without Moving Parts,” issued on May 20, 2003; Kim et al., U.S. Patent Publication No. 20030205632, entitled “Electrowetting driven micropumping =U.S. Patent No. 20060164490, entitled "Method and Apparatus for Promoting the Complete Transfer of Liquid Drops from a Nozzle", published on November 6, 2003; Jin et al., U.S. Patent No. 20060164490, entitled "Method and Apparatus for Promoting the Complete Transfer of Liquid Drops from a Nozzle", published on July 27, 2006; Jin et al., U.S. Patent No. 20070023292, entitled "Small Object Moving on Printed Circuit Board", published on February 1, 2007;Shah et al., U.S. Patent Publication No. 20090283407, entitled “Method for Using Magnetic Particles in Droplet Microfluidics,” published on November 19, 2009; Kim et al., U.S. Patent Publication No. 20100096266, entitled “Method and Apparatus for Real-time Feedback Control of Electrical Manipulation of Droplets on Chip,” published on April 22, 2010; Velev, U.S. Patent No. 7,547,380, entitled “Droplet Transportation Devices and Methods Having a Fluid Surface Surface), issued June 16, 2009; Sterling et al., U.S. Patent No. 7,163,612, entitled “Method, Apparatus and Article for Microfluidic Control via Electrowetting, for Chemical, Biochemical and Biological Assays and the Like,” issued January 16, 2007; Becker et al., U.S. Patent No. 7,641,779, entitled “Method and Apparatus for Programmable Fluidic Processing Becker et al., U.S. Patent No. 6,977,033, entitled “Method and Apparatus for Programmable Fluid Processing,” issued on January 5, 2010; Becker et al., U.S. Patent No. 6,977,033, entitled “Method and Apparatus for Programmable Fluid Processing,” issued on December 20, 2005; Decre et al., U.S. Patent No. 7,328,979, entitled “System for Manipulation of a Body of Fluid,” issued on February 12, 2008;Yamakawa et al., U.S. Patent Publication No. 15, 20060039823, entitled “Chemical Analysis Apparatus,” published on February 23, 2006; Wu, U.S. Patent Publication No. 20110048951, entitled “Digital Microfluidics Based Apparatus for Heat-exchanging Chemical Processes,” published on March 3, 2011; Fouillet et al., U.S. Patent Publication No. 20090192044, entitled “Electrode Addressing Method,” published on July 30, 2009; Fouillet et al., U.S. Patent No. 7,052,244, entitled “Device for Displacement of Small Liquid Volumes Along a Micro-catenary Line by Electrostatic Force Forces", issued May 30, 2006; Marchand et al., U.S. Patent Publication No. 20080124252, entitled "Droplet Microreactor", issued May 29, 2008; Adachi et al., U.S. Patent Publication No. 20090321262, entitled "Liquid Transfer Device", issued December 31, 2009; Roux et al., U.S. Patent Publication No. 20050179746, entitled "Device for Controlling the Displacement of a Drop Between Two or Several Solid Substrates", issued August 18, 2005; and Dhindsa et al., "Virtual Electrowetting Channels: Electronic Liquid Transport with Continuous Channel Functionality". Channel Functionality", Lab Chip, 10:832–836 (2010), each of which is incorporated herein by reference.;

[0119] Modified probes (e.g., extended nucleic acid probes) can be detected, for example, using methods described above or known in the industry after separation from a fluid mixture. In a particular embodiment, the modified probes separated on a second solid support (i.e., a solid support different from the first solid support in which contact is made between the probe and the biological sample) can be detected using microarray-based techniques or nucleic acid sequencing techniques, such as those described above. Probes separated in a fluid stream can be detected using optical, electrical, or other detectors equipped in a known microfluidic device, a droplet handling device, or a flow cytometer. Detection methods can be used to determine the target nucleic acid sequence, barcode sequence, or other sequence region of the extended probe.

[0120] Several embodiments have been exemplified with respect to removing modified probes from a solid support in which the probes are produced. However, it should be understood that the probes on the solid support can be contacted with a biological sample, modified on the solid support in the presence of a target nucleic acid from the sample, and then the modified probes can be detected on the solid support. In this embodiment, the biological sample can be removed from the solid support prior to the detection step.

[0121] In a specific embodiment, the present disclosure provides a method for spatially labeling nucleic acids in a biological sample, which comprises the following steps: (a) providing a plurality of nucleic acid primers attached to a solid support, wherein the nucleic acid primers in the plurality include a universal primer sequence common to the nucleic acid primers in the plurality; (b) binding a population of nucleic acid probes to the plurality of nucleic acid primers, wherein the nucleic acid probes include a universal primer binding sequence that hybridizes to the universal primer sequence, a target capture sequence, and a barcode sequence that is different from the barcode sequences of other nucleic acid probes in the population, thereby attaching different nucleic acid probes to randomly positioned locations on the solid support. ; (c) amplifying different nucleic acid probes by extending nucleic acid primers, thereby generating nucleic acid clusters having copies of a barcode sequence and a target capture sequence at randomly positioned positions on a solid support; (d) performing a sequencing reaction to determine the barcode sequence at the randomly positioned positions on the solid support; (e) contacting a biological sample with the nucleic acid cluster on the solid support; (f) hybridizing the target capture sequence of the cluster with a target nucleic acid from a portion of the biological sample proximal to the cluster; and (g) extending the target capture sequence to generate an extended probe comprising a sequence from the target nucleic acid and a copy of the barcode sequence, thereby labeling the nucleic acid of the biological sample.

[0122] As exemplified above, a plurality of nucleic acid primers can be attached to a solid support, wherein the nucleic acid primers in the plurality include a universal primer sequence common to the nucleic acid primers in the plurality. In this embodiment, a second plurality of nucleic acid primers can be attached to a solid support, and the nucleic acid primers in the second plurality can have a second universal primer sequence common to the nucleic acid primers in the second plurality. In this embodiment, a plurality of different nucleic acid probes contacted with a carrier can include a universal primer binding sequence hybridized with the universal primer on the solid support, as described above, and different nucleic acid probes can also include a second universal primer binding sequence hybridized with the second universal primer sequence. This configuration of universal primers and universal primer binding sites can be particularly useful for amplifying different nucleic acid probes by bridge amplification, wherein the nucleic acid primers in the first and second plurality are extended.

[0123] Typically, when the nucleic acid probe contains the first and second universal primer binding sites, it will be positioned at the probe end. In certain embodiments, it may be necessary to remove at least one primer binding site from the nucleic acid probe or from the amplicon produced by the probe. Therefore, the nucleic acid probe may optionally include a cleavage site between a target capture sequence and a universal primer binding sequence. In this case, a cleavage reaction can be performed to separate the universal primer binding site from the target capture sequence. Typically, the part containing the target capture sequence in the probe (or its amplicon) will be attached to a solid support, resulting in the removal of the primer binding site from the solid support and retaining the target capture sequence. Therefore, the cleaved probe can be used for hybridization target nucleic acid and the cleaved probe can be extended using the method described above.

[0124] In certain embodiments, nucleic acid probe will include two different cleavage sites. The first cleavage site will be positioned between the first primer binding site and one or more other sequence elements of the probe. The second cleavage site can be positioned between the second primer binding site and one or more other sequence elements of the probe. The cleavage site can be reactive to different cleavage reactions, so that each site of selective cleavage can be cracked without the need for cracking other sites. Therefore, the first cleavage site can be cracked before the modified probe (for example, before the extension probe is generated), thus separating the first primer binding site from one or more other sequence elements that remain attached to the solid support. The second cleavage site can be cracked after the modified probe (for example, after the extension probe is generated), thus releasing the modified probe for subsequent detection.

[0125] Alternatively, the nucleic acid probe may include a first cleavage site, and the primer for capturing or amplifying the nucleic acid probe may include a second cleavage site. In this configuration, the first cleavage site may be positioned between the first primer binding site and one or more other sequence elements of the probe, so that the first primer binding site is separated from the one or more other sequence elements of the probe that are attached to the solid support by cleavage. Similarly, this first cleavage step will usually be implemented before the modified probe (for example, before the extension probe is generated). The second cleavage step may be implemented to crack the second cleavage site after the modified probe (for example, after the extension probe is generated), thereby releasing the modified probe for subsequent detection.

[0126] The above two embodiments illustrate a cleavage site positioned between the attachment point of a nucleic acid probe (or modified nucleic acid probe) and one or more sequences (e.g., a spatial barcode or a target sequence) of the probe (or modified probe) containing information. Thus, this cleavage site can be used to release a modified probe (e.g., an extended probe) to detect sequence information and determine what sequence is present in a biological sample and where the sequence is present in the sample.

[0127] In some embodiments, one or more probes contacted with a solid support in the methods described herein may include a sequencing primer binding site. Thus, a modified probe (e.g., an extended probe) may be detected in a sequencing technique comprising a step of hybridizing a sequencing primer to a sequencing primer binding site. The sequencing primer binding site may be positioned in the probe such that cleavage of a modified form of the probe (e.g., an extended probe) will produce a released probe comprising a sequencing primer binding site. The sequencing primer binding site may be a universal sequencing primer binding site such that a plurality of different probes (e.g., with different barcodes and / or target sequences) will have the same sequencing primer binding site.

[0128] The present disclosure further provides a method for spatially labeling nucleic acids in a biological sample, the method comprising the following steps: (a) providing a bead array on a solid support, wherein different nucleic acid probes are attached to different beads in the array, wherein the different nucleic acid probes each include a barcode sequence, wherein each bead includes a barcode sequence different from that of other beads on the solid support, and wherein the different nucleic acid probes each include a target capture sequence; (b) performing a decoder probe hybridization reaction on the solid support to determine the barcode sequence of the randomly positioned probes on the solid support; (c) contacting the biological sample with the bead array; (d) hybridizing the different nucleic acid probes with a target nucleic acid from a portion of the biological sample proximal to the bead; and (e) extending the different nucleic acid probes to produce extended probes including a sequence from the target nucleic acid and a barcode sequence, thereby labeling the nucleic acids in the biological sample.

[0129] It will be appreciated that manipulations of solid supports or nucleic acids attached to solid supports can be performed using beads as solid supports. The beads can be attached to a surface (e.g., a well array, such as in the BeadArray from Illumina) before or after performing the manipulations. TM For example, nucleic acid probes can be captured on beads before or after the beads are distributed on the array, nucleic acid probes can be amplified to produce amplicons on the beads before or after the beads are distributed on the array, and so on.

[0130] Example I

[0131] Spatial tagging of mRNA from tissue samples using the ILUMINA flow cell

[0132] Methods for generating clusters containing barcoded oligo-dT and then revealing the barcoded oligo-dT by restriction enzyme digestion followed by sequencing are described in Figure 1 A single-stranded barcoded oligo-dA, P5', P7, SBS3 sequencing primer binding site and BspHI restriction enzyme site (shown in Figure 2) were prepared by oligo synthesis (Integrated DNA Technologies). Figure 1 The barcodes were 27-mers and randomly generated during synthesis. A binding site for the SBS3 sequencing primer was included for decoding the barcodes by sequencing. An oligo-dA segment was included to generate oligo-dT sites after clustering and linearization. Bridge amplification and clustering were performed according to standard cluster chemistry (Illumina TruSeq PE Cluster Kit v3 cBot P / N: 15037931) on an Illumina GA flow cell using the manufacturer's recommended protocol.

[0133] After bridge amplification and clustering, the cluster was linearized by cleaving the 8-oxo-G in the P7 primer using the formamidopyrimidine DNA glycosidase (Fpg) enzyme provided in the TruSeq PE cluster kit. This was followed by restriction enzyme digestion with 200 units / mL BspH1 (NEB catalog number R0517L) at 37°C for 15 min to remove P7' from the P5 adapter anchor strand of the cluster to reveal an oligo-dT segment for subsequent extension in the presence of mRNA. Enzyme concentrations ranging from 100-400 U / mL for 15 or 30 min have been tested. Decoding of the barcode was initiated with the SBS3 sequencing primer.

[0134] like Figure 1As shown in the figure below, after barcode decoding, oligo-dT sequences in the cluster are used to capture poly A+ RNA. Barcoded cDNA is generated by extending the oligo-dT strand of the cluster using the TruSeq RNA Sample Preparation Kit (Illumina P / N: 15012997) and the MMLV Reverse Transcriptase 1st Strand cDNA Synthesis Kit (Epicentre P / N: MM070150) according to the manufacturer's recommended conditions. The captured RNA is used as a template. The barcoded cDNA is released from the P5 sequence of the flow cell using Illumina's Uracil Specific Excision Reagent (USER) (Illumina's TruSeq PE Cluster Kit), thereby releasing the barcoded cDNA library for sequencing on a second Illumina flow cell.

[0135] The availability of the oligo dT capture sequence after restriction enzyme digestion with BspH1 was confirmed by hybridizing the linearized cluster with Cy5-labeled polyA (24-mer), as shown in Figure 2 . Briefly, after restriction enzyme digestion, the clusters were treated with 0.1 N NaOH and washed with HT2 low salt buffer to remove the second strand on the flow cell. Subsequently, 500 nM Cy5 oligo-dA (24-mer) was flowed through the linearized and denatured clusters at a rate of 30 μl / min and incubated at 40°C for 5 min before imaging. The hybridization of Cy5-labeled poly-A with oligo-dT was detected in lanes 2-7 of the GA flow cell in which the BODT-1 library containing oligo-dT was present (see Figure 2 As can be seen from the flow cell image (B) and stick graph (C), the control PhiX library (lanes 1 and 8 of the flow cell) showed very low fluorescence in the Cy5 signal. These results show that oligo-dT sites can be generated in clusters that can specifically bind to Cy5 polyA (24-mer) after linearization.

[0136] The sequencing metrics for the above flow cell using a 3.2 pM BODT-1 library are given in Figure 3 Millions of reads were detected in 21 tiles from GA sequencing. After sequencing, the number of unique barcodes was determined, as Figure 4 This was performed by assuming that each passing filter (PF) read was a barcode and determining the number of unique reads (barcodes) in each lane. After sequencing the tiles compared to the PhiX control library, between 5 and 11 million unique barcoded clusters were detected. These results show that sequence decoding of a barcoded oligo-dT sequence library is feasible and generates millions of unique barcodes.

[0137] Example II

[0138] Cell Adhesion on ILUMINA Flow Cells

[0139] Single cells were captured on a patterned flow cell (HiSeq X10 flow cell, ILLUMINA). All reagent flow steps were performed using a peristaltic pump or cBOT cluster generation instrument (ILLUMINA). Briefly, nuclease-free water was flowed over all lanes of the patterned flow cell, followed by a flow rate of 100 μl / min for 8 min for 30-70K poly-D-lysine solutions (100 μg / ml and 20 μg / ml). Heat-inactivated fetal bovine serum (Life Technologies No. 10082-139) was also tested as an adhesive. The adhesive was incubated on the flow cell lanes for 1 hr, followed by washing with 1X PBS + 0.5% Pluronic F-68 (Life Technologies No. 24040-032). The cells were then attached to the coated flow cell by flowing 5 to 50 cells / μl or about 100–1000 cells / lane at 100 μl / min, followed by a 60 min incubation step to bind the cells. The flow cell was washed with 1X PBS / 0.5% Pluronic at 75 μl / min. If the cells were fixed on the flow cell, 1% paraformaldehyde (PFA) was flowed on the flow cell and incubated for 15 min after flowing the cells as described above, followed by a 1X PBS / 0.5% Pluronic wash step. The flow cell was removed and the number of cells per lane was counted using a microscope.

[0140] Figure 5 Panel A shows an image of cells captured on a patterned flow cell. Figure 5 The cell counting data shown in Figure B of confirm that poly-D-lysine-coated flow cells facilitate cell adhesion compared to BSA-coated controls or controls not treated with adhesives. Figure 6 As shown in , adherent cells can be successfully fixed with 1% PFA.

[0141] Example III

[0142] Spatial capture of target mRNA by probes attached to the gel surface

[0143] This example describes generating a lawn of poly-T probes on a gel-coated slide, placing tissue sections on top of the lawn of poly-T probes, releasing RNA from the tissue sections, capturing the released mRNA with the poly-T probe, reverse transcribing to label the poly-T probe with Cy3, removing the tissue and imaging the slide.

[0144] Figure 7Figure A of shows a schematic representation of the steps and reagents used to generate probes attached to the gel. Briefly, microscope slides were coated with silane-free acrylamide (SFA), P5 and P7 primers were attached (see U.S. Patent Application Publication No. 2011 / 0059865 A1, which is incorporated herein by reference), probes having poly-A sequences and P5 or P7 complementary sequences were hybridized to the P5 and P7 primers, respectively, and the P5 and P7 primers were extended to generate poly-T sequence extensions. The quality control step was performed by hybridizing Cy5-labeled poly-A oligonucleotides to the extended primers and imaging the surface using an Axon imager.

[0145] like Figure 7 As shown in Figure B of , tissue sections are placed on a gel with poly-T extension primers. Tissue is processed to release mRNA and the poly-A tail of the released mRNA is hybridized with the poly-T sequence of the extension primer. The captured mRNA is used as a template to extend the poly-T sequence and selectively labeled with Cy3 through the extension primer. Tissue is removed from the gel and the surface is imaged to detect Cy3 fluorescence.

[0146] like Figure 7 As shown in the image of , areas of the gel near areas of tissue that released mRNA species appear fluorescent, while areas that did not release mRNA appear darker in the image. Thus, the captured mRNA produces a fingerprint-like image of the tissue.

[0147] Example IV

[0148] By attaching to the BeadArray TM The probes on the surface capture the target mRNA spatially

[0149] This example describes placing tissue sections on a BeadArray with poly-T probes. TM On top of the , RNA is released from tissue sections, the released mRNA is captured with a poly-T probe, reverse transcribed to label the poly-T probe with Cy5, the tissue is removed and loaded onto the BeadArray TM Imaging.

[0150] like Figure 8 As shown in Figure A, a mouse olfactory organ tissue section is placed on a BeadArray with a poly-T probe. TM The tissue is processed to release mRNA and the poly-A tail of the released mRNA is hybridized to the poly-T sequence of the probe. The poly-T sequence is extended using the captured mRNA as a template and the extension primer is selectively labeled with Cy5. TM Remove tissue and assemble BeadArray TM Imaging was performed to detect Cy5 fluorescence.

[0151] like Figure 7 As shown in Figure B, BeadArray TM Areas of the tissue close to areas where mRNA species are released appear fluorescent, while areas where mRNA is not released appear darker in the image. Thus, the captured mRNA produces a fingerprint-like image of the tissue.

[0152] Throughout this application, various publications, patents or patent applications have been mentioned. The disclosures of these publications in their entirety are incorporated herein by reference.

[0153] The term "comprising" is intended herein to be open-ended, including not only the listed elements but also further encompassing any additional elements.

[0154] Several embodiments have been described herein. However, it should be understood that various modifications may be made. Therefore, other embodiments are within the scope of the following claims.

Claims

1. A method for spatially labeling a target nucleic acid of a biological sample, comprising: (a) providing a solid support comprising a plurality of nucleic acids randomly positioned at a plurality of features of the solid support, wherein the nucleic acids comprise a barcode sequence, and wherein the barcode sequence is different from the barcode sequences of other nucleic acids in the plurality of nucleic acids; (b) performing a sequencing reaction to determine the barcode sequence of the nucleic acids of the plurality of features randomly located on the solid support, or a complementary sequence of the barcode sequence, thereby determining the position of the barcode sequence on the solid support; (c) contacting a biological sample with the solid support comprising the nucleic acid; (d) hybridizing a target nucleic acid from the biological sample to a capture sequence on a nucleic acid proximal to the target nucleic acid; and (e) extending the nucleic acid to generate an extended probe, wherein the extended probe comprises the barcode sequence, or a complementary sequence to the barcode sequence, and all or a portion of the target nucleic acid, or a complementary sequence to all or a portion of the target nucleic acid, thereby spatially tagging the target nucleic acid from the biological sample.

2. The method according to claim 1, wherein the nucleic acid further comprises a primer binding sequence, in, The primer binding sequence is identical for the plurality of nucleic acids.

3. The method of claim 1 or 2, wherein the capture sequence on the nucleic acid comprises different capture sequences that hybridize to different target nucleic acids from the biological sample.

4. The method of claim 1 or 2, wherein the capture sequence on the nucleic acid comprises a consensus capture sequence, and the consensus capture sequence comprises a poly-T or poly-A sequence.

5. The method of claim 1 or 2, wherein the plurality of features are selected from the group consisting of holes or channels.

6. The method of claim 5, wherein the plurality of features have an average pitch of less than 10 microns, less than 5 microns, or less than 1 micron.

7. The method of claim 1 or 2, wherein the solid support comprises one or more fiducial markers.

8. The method according to claim 1 or 2, wherein the target nucleic acid is selected from the group consisting of mRNA, rRNA, gDNA and cDNA.

9. The method according to claim 1 or 2 further comprises removing the extension probe from the solid support, amplifying the extension probe to produce a plurality of amplified extension probes, providing the amplified extension probes to a second solid support, and determining the spatially tagged target nucleic acid, or the complementary sequence of the spatially tagged target nucleic acid, and the barcode sequence, or the complementary sequence of the barcode sequence, on the second solid support, thereby determining the positioning of the spatially tagged target nucleic acid on the solid support.

10. The method of claim 9, wherein the amplification is polymerase chain reaction, rolling circle amplification, multiple displacement amplification, or random primer amplification. The method according to claim 1 or 2 , wherein the biological sample is a mixture of cells.

12. The method of claim 1 or 2, wherein the biological sample is a tissue.

13. The method according to claim 1 or 2, further comprising the step of acquiring an image of the biological sample in contact with the solid support.

14. The method of claim 1 or 2, wherein the solid support is a flow cell.

15. The method of claim 1 or 2, wherein the method further comprises staining the biological sample.

16. A method for determining the spatial localization of a target nucleic acid in a biological sample, comprising: (a) providing a solid support, the solid support comprising a plurality of nucleic acids randomly positioned at a plurality of features on the solid support, wherein the nucleic acids comprise a spatial tag sequence and a primer binding sequence, and wherein the spatial tag sequence is different from the spatial tag sequences of other nucleic acids in the plurality of nucleic acids; (b) performing a sequencing reaction to determine the spatial tag sequences of the nucleic acids of the plurality of features randomly positioned on the solid support, thereby determining the positions of the spatial tag sequences on the solid support; (c) adding a capture sequence to the nucleic acid; (d) contacting a biological sample with the solid support comprising the nucleic acid; (e) hybridizing a target nucleic acid from the biological sample to the capture sequence on a nucleic acid proximal to the target nucleic acid; (f) generating a nucleic acid comprising a sequence complementary to a target nucleic acid hybridized to the capture sequence, or a portion thereof, and the spatial tag sequence, or a complementary sequence to the spatial tag sequence, and the primer binding sequence, or a complementary sequence to the primer binding sequence; (g) removing the nucleic acid of step (f) and attaching the nucleic acid to a second solid support; and (h) determining the sequence of the nucleic acid attached to the second solid support, thereby determining the spatial location of the target nucleic acid in the biological sample, The methods described herein are not intended for diagnostic purposes.

17. The method of claim 16, wherein the primer binding sequence is identical across the plurality of nucleic acids.

18. The method of claim 16 or 17, wherein the capture sequence on the nucleic acid comprises different capture sequences that hybridize to different target nucleic acids from the biological sample.

19. The method of claim 16 or 17, wherein the capture sequence on the nucleic acid comprises a consensus capture sequence, and the consensus capture sequence comprises a poly-T or poly-A sequence.

20. The method of claim 16 or 17, wherein the plurality of features are selected from the group consisting of: holes, channels, or beads.

21. The method of claim 20, wherein the solid support comprises a gel coating.

22. The method of claim 20, wherein the plurality of features have an average pitch of less than 10 microns, less than 5 microns, or less than 1 micron.

23. The method of claim 16 or 17, wherein the solid support comprises one or more fiducial markers.

24. The method of claim 16 or 17, wherein the target nucleic acid is selected from the group consisting of mRNA, rRNA, gDNA and cDNA.

25. A method according to claim 16 or 17, wherein generating a nucleic acid comprises extending the nucleic acid to generate an extended probe comprising the spatial tag sequence, or a complementary sequence to the spatial tag sequence, and all or part of the target nucleic acid, or all or part of the complementary sequence to the target nucleic acid, and wherein step (g) further comprises amplifying the extended probe to generate a plurality of amplified and extended nucleic acids, and attaching the amplified and extended nucleic acids to a second solid support.

26. The method of claim 25, wherein the amplification is polymerase chain reaction, rolling circle amplification, multiple displacement amplification or random primer amplification.

27. The method of claim 16 or 17, wherein the biological sample is a mixture of cells.

28. The method of claim 27, wherein step (c) further comprises contacting the cell with the solid support and / or lysing the cell to release the target nucleic acid from the cell.

29. The method of claim 16 or 17, wherein the biological sample is tissue.

30. The method of claim 29, wherein step (c) further comprises contacting the tissue with the solid support and / or permeabilizing the tissue to release the nucleic acid from the tissue.

31. The method of claim 16 or 17, further comprising the step of acquiring an image of the biological sample in contact with the solid support.

32. The method of claim 31, further comprising the step of associating the spatial tag sequence from the randomly located nucleic acid, or the complement of the spatial tag sequence, with a location in the image of the biological sample.

33. The method of claim 16 or 17, wherein the solid support is a flow cell.

34. The method of claim 33, wherein the second solid support is a flow cell.

35. The method of claim 16 or 17, wherein the method further comprises staining the biological sample.

36. A method for determining the spatial location of a target nucleic acid in a biological sample, comprising: (a) providing a solid support, the solid support comprising a plurality of nucleic acids randomly positioned at a plurality of features of the solid support, wherein the nucleic acids comprise a spatial tag sequence and a primer binding sequence, and wherein the spatial tag sequence is different from the spatial tag sequences of other nucleic acids in the plurality of nucleic acids; (b) performing a sequencing reaction to determine the spatial tag sequence of the nucleic acid of the plurality of features located on the solid support, or a complementary sequence of the spatial tag sequence, thereby determining the position of the spatial tag sequence on the solid support; (c) contacting the biological sample with the solid carrier; (d) hybridizing a target nucleic acid from the biological sample to a capture sequence on the nucleic acid randomly positioned proximal to the target nucleic acid; (e) extending the capture sequence, wherein the extended capture sequence comprises a complementary sequence to the target nucleic acid or a portion thereof, (f) amplifying the extended capture sequence, the spatial tag sequence or the complementary sequence of the spatial tag sequence, and the primer binding sequence with a primer complementary to the primer binding site, thereby generating an amplification product comprising the target nucleic acid or the complementary sequence of the target nucleic acid or a portion of the target nucleic acid or the complementary sequence of a portion of the target nucleic acid, and the spatial tag or the complementary sequence of the spatial tag; and (g) performing a nucleic acid detection reaction on the amplified product to determine all or part of the target nucleic acid sequence or all or part of the complementary sequence of the target nucleic acid sequence, and the spatial tag sequence or the complementary sequence of the spatial tag sequence, thereby determining the spatial location of the target nucleic acid in the biological sample, The methods described therein are not intended for diagnostic purposes.

37. The method of claim 36, wherein the sequencing reaction comprises sequencing by synthesis, sequencing by hybridization, or sequencing by ligation.

38. The method of claim 36, wherein the primer binding sequence is identical across the plurality of nucleic acids.

39. The method of claim 36, wherein the capture sequences on the randomly positioned nucleic acids comprise different target capture sequences that hybridize to different target nucleic acids from the biological sample.

40. The method of claim 36, wherein the capture sequence on the randomly positioned nucleic acids comprises a common target capture sequence, and the common target capture sequence comprises a poly-T or poly-A sequence.

41. The method of claim 36, wherein the solid support comprises a gel coating.

42. The method of claim 36, wherein the plurality of features are selected from the group consisting of: holes, channels, or beads.

43. The method of claim 42, wherein the plurality of features on the solid support have an average pitch of less than 10 microns.

44. The method of claim 42, wherein the plurality of features on the solid support have an average pitch of less than 5 microns.

45. The method of claim 42, wherein the plurality of features on the solid support have an average pitch of less than 1 micron.

46. ​​The method of claim 36, wherein the solid support comprises a fiducial marker.

47. A method according to claim 36, wherein the method further comprises the step of acquiring an image of the biological sample in contact with the solid carrier, and the step of associating the determined spatial tag sequence, or the complementary sequence of the spatial tag sequence, with the position in the image of the biological sample.

48. The method of claim 36, wherein the nucleic acid detection reaction in step (g) comprises sequencing.

49. The method of claim 48, wherein the sequencing reaction comprises sequencing by synthesis, sequencing by hybridization, or sequencing by ligation.

50. The method of claim 36, wherein the method, between steps (f) and (g), further comprises the step of removing the amplification product from the solid support.

51. The method of claim 50, wherein the method further comprises pooling the amplification products to form a mixture of the amplification products that have been removed from the solid support.

52. The method of claim 51, wherein the method further comprises providing the mixture of amplification products removed from the solid support to a second solid support.

53. The method of claim 52, wherein the method further comprises determining the sequence of the mixture of amplification products on the second solid support.

54. The method of claim 36, wherein the solid support is positioned in or on a flow cell.

55. The method of claim 36, wherein the biological sample is a cell mixture, and step (c) further comprises contacting the cells with the solid support and / or lysing the cells to release the target nucleic acid from the cells.

56. The method of claim 36, wherein the biological sample is a tissue, and step (c) further comprises permeabilizing the tissue to release the target nucleic acid from the tissue.

57. The method of claim 36, wherein the target nucleic acid is RNA.

58. The method of claim 57, wherein the RNA is mRNA.

59. The method of claim 36, wherein the target nucleic acid is DNA.

60. The method of claim 59, wherein the DNA is genomic DNA.

61. The method of claim 36, wherein the amplification in step (f) is selected from the group consisting of: polymerase chain reaction, rolling circle amplification, multiple displacement amplification, and random primer amplification.

62. The method of claim 61, wherein the amplifying comprises polymerase chain reaction.

63. The method of claim 61, wherein the amplification comprises rolling circle amplification.

64. The method of claim 36, wherein the method further comprises the step of staining the biological sample.

65. The method of claim 52, wherein the second solid support is a flow cell.

66. A method for determining the spatial location of a target nucleic acid in a biological sample, comprising: (a) providing a solid support, the solid support comprising a plurality of nucleic acids randomly positioned at a plurality of features on the solid support, wherein the nucleic acids comprise a spatial tag sequence and a primer binding sequence, and wherein the spatial tag sequence is different from the spatial tag sequences of other nucleic acids in the plurality of nucleic acids; (b) performing a sequencing reaction to determine the spatial tag sequence of the nucleic acid of the plurality of features located on the solid support, or a complementary sequence of the spatial tag sequence, thereby determining the position of the spatial tag sequence on the solid support; (c) contacting the biological sample with the solid carrier; (d) hybridizing a target nucleic acid from the biological sample to a capture sequence on a randomly positioned nucleic acid proximal to the target nucleic acid; (e) generating a nucleic acid comprising a sequence complementary to a target nucleic acid hybridized to the capture sequence, or a portion thereof, a spatial tag sequence or a complementary sequence to the spatial tag sequence, and a primer binding sequence or a complementary sequence to the primer binding sequence; and (f) determining the spatial tag sequence or the complementary sequence of the spatial tag sequence, and all or part of the target nucleic acid sequence or the complementary sequence of all or part of the target nucleic acid sequence, thereby determining the spatial location of the target nucleic acid in the biological sample, The methods described therein are not intended for diagnostic purposes.

67. The method of claim 66, wherein the plurality of features are selected from the group consisting of: holes, channels, or beads.

68. The method of claim 67, wherein the plurality of features on the solid support have an average pitch of less than 10 microns.

69. The method of claim 67, wherein the plurality of features on the solid support have an average pitch of less than 5 microns.

70. The method of claim 67, wherein the plurality of features on the solid support have an average pitch of less than 1 micron.

71. The method of claim 66, wherein the solid support comprises a fiducial marker.

72. The method of claim 66, wherein the solid support comprises a gel coating.

73. The method of claim 66, wherein the solid support is positioned in or on a flow cell.

74. The method of claim 66, wherein the capture sequence on the nucleic acid comprises different target capture sequences that hybridize to different target nucleic acids from the biological sample.

75. The method of claim 66, wherein the capture sequence on the nucleic acid comprises a consensus target capture sequence, and the consensus target capture sequence comprises a poly-T or poly-A sequence.

76. A method according to claim 66, wherein the method further comprises the step of acquiring an image of the biological sample in contact with the solid carrier, and the step of associating the measured spatial tag sequence at a randomly positioned position on the solid carrier, or the complementary sequence of the measured spatial tag sequence, with the position in the image of the biological sample.

77. The method of claim 66, wherein generating the nucleic acid in step (e) comprises extending the capture sequence using the captured target nucleic acid as a template.

78. The method according to claim 77 further comprises amplifying the nucleic acid using a primer complementary to the primer binding sequence, thereby producing an amplification product, wherein the amplification product comprises all or part of the target nucleic acid, or a complementary sequence of all or part of the target nucleic acid, and the spatial tag sequence, or a complementary sequence of the spatial tag sequence.

79. The method of claim 78, wherein amplification is selected from the group consisting of polymerase chain reaction, rolling circle amplification, multiple displacement amplification, and random primer amplification.

80. The method of claim 79, wherein the amplification is polymerase chain reaction.

81. The method of claim 79, wherein the amplification is a rolling circle amplification.

82. The method of claim 66, wherein determining the sequence in step (f) comprises sequencing.

83. The method of claim 82, wherein the sequencing comprises sequencing by synthesis, sequencing by hybridization, or sequencing by ligation.

84. The method of claim 66, wherein the biological sample contacted with the solid support is a cell mixture, and step (c) further comprises contacting the cells with the solid support and / or lysing the cells to release the target nucleic acid from the cells.

85. The method of claim 66, wherein the biological sample contacted with the solid support is a tissue, and step (c) further comprises contacting the tissue with the solid support and / or permeabilizing the tissue to release the target nucleic acid from the tissue.

86. The method of claim 66, wherein the target nucleic acid is RNA.

87. The method of claim 86, wherein the RNA is mRNA.

88. The method of claim 66, wherein the target nucleic acid is DNA.

89. The method of claim 88, wherein the DNA is genomic DNA.

90. The method of claim 66, wherein the method further comprises staining the biological sample.

91. The method of claim 1 or 2, wherein the plurality of features comprises beads.

92. The method of claim 1 or 2, wherein the solid support comprises a gel coating.

93. The method of claim 11, wherein step (c) further comprises contacting the cell with the solid support and / or lysing the cell to release the target nucleic acid from the cell.

94. The method of claim 12, wherein step (c) further comprises contacting the tissue with the solid support and / or permeabilizing the tissue to release the nucleic acid from the tissue.

95. The method of claim 13, further comprising the step of associating the barcode sequence, or the complement of the barcode sequence, from the randomly located nucleic acid with a location in the image of the biological sample.

96. The method of claim 9, wherein the second solid support is a flow cell.

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