Spatial mapping by sequential primer extension

Abstract

Provided herein is a probe system comprising a population of nucleic acid molecules having an extendable end; a first set of barcoded particles, each having a nucleotide sequence comprising (i) a binding sequence complementary to the extendable end of the nucleic acid molecule, (ii) a unique particle identifier sequence, and (iii) a first template sequence; and a second set of barcoded particles, each having a nucleotide sequence comprising (i) the first template sequence, and (ii) a unique particle identifier sequence. In use, extension of the nucleic acid molecule using the first set of barcoded particles as templates produces extension products that contain the complement of the particle's unique particle identifier sequence and the complement of the first template sequence. Also provided is a method of using the probe system to map binding events in or on a cell sample.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 168,132, filed March 30, 2021, which is incorporated by reference in its entirety. [Background technology]

[0002] Sample and molecular indexes are commonly used in many of today's genomics workflows. Sample indexing is a commonly used approach that allows samples to be sequenced and analyzed in a multiplexed manner. In sample indexing methods, all nucleic acids in a particular sample are labeled with the same sequence tag, the tagged library is pooled with other libraries tagged with different barcodes, and the pool is sequenced in parallel in a single sequencing run. Then, during analysis, the sample-specific index allows the software to separate the multiplexed sequence data into sample-specific data sets. The goal of molecular indexing, on the other hand, is to tag each molecule in a sample with a unique sequence before PCR amplification. In these methods, each nucleic acid in the starting sample is tagged with a unique molecular index, and the sequence analysis software filters out duplicate reads (i.e., reads from copies of the same molecule) and uses the index to eliminate PCR / sequencing errors.

[0003] The present disclosure uses the index to provide spatial information, which appears to be a novel application of molecular indexes. Summary of the Invention

[0004] In particular, the present disclosure provides a probe system comprising: (a) a population of nucleic acid molecules having extendable ends; (b) (i) a first set of barcoded particles, each having a nucleotide sequence comprising a binding sequence complementary to the extendable ends of the nucleic acid molecules of (a), (ii) a unique particle identifier sequence, and (iii) a first template sequence; and (c) a second set of barcoded particles, each having a nucleotide sequence comprising (i) the first template sequence, and (ii) a unique particle identifier sequence, in which extension of the nucleic acid molecules of (a) using the first set of barcoded particles of (b) as templates produces extension products that contain the complement of the unique particle identifier sequence of the particles of (a)(ii) and the complement of the first template sequence.

[0005] In the description that follows, the nucleic acid molecule having an extendable end is a primer, and the method is carried out using primer extension. In alternative embodiments (illustrated in Figures 12-14), the method can be carried out using gap-filling / ligation or ligation-based methods. Thus, the present system and method should not be limited to only primer extension-based methods.

[0006] In some embodiments, the method includes hybridizing a first population of primer molecules to a first set of barcoded particles; extending the hybridized primer molecules using nucleotide sequences of the first set of barcoded particles as templates to produce first primer extension products that contain complements of unique particle identifier sequences from barcoded particles in the first set of barcoded particles and complements of the first template sequences; removing the first set of barcoded particles; and hybridizing the first primer extension products to a second set of barcoded particles. and hybridizing a nucleotide sequence of the second set of barcoded particles to a nucleotide sequence of the first primer extension product, the nucleotide sequence of the second set of barcoded particles being hybridized to the first template sequence in the second set of barcoded particles; and extending the first primer extension product using the nucleotide sequence of the second set of barcoded particles as a template to produce a second primer extension product containing a unique particle identifier sequence from a barcoded particle in the first set of barcoded particles, the first template sequence, and a unique particle identifier sequence from a barcoded particle in the second set of barcoded particles.

[0007] The probe system may be used in a method for mapping binding events in a cell sample. In an embodiment, the method includes the steps of: (a) obtaining a sample containing primer molecules bound to sites in or on cells; (b) obtaining a first set of barcoded particles, each of which has a nucleotide sequence comprising (i) a primer binding sequence complementary to the 3' end of the primer molecule, (ii) a unique particle identifier sequence, and (iii) a first template sequence; and (c) obtaining a second set of barcoded particles, each of which has a nucleotide sequence comprising (i) the first template sequence, and (ii) the unique particle identifier sequence; (d) specifically hybridizing the first set of barcoded particles to the sample, wherein at least a portion of the nucleotide sequence of the first set of barcoded particles hybridizes to at least two primer molecules; and (e) extending the primers hybridized to the barcoded particles in step (b) using the nucleotide sequences to which the primers hybridized as templates to obtain the first unique particle identifier sequence. (d) removing the first set of barcoded particles from the sample; (e) specifically hybridizing a second set of barcoded particles to the first primer extension products of (c), wherein a nucleotide sequence of at least some of the barcoded particles of the second set hybridize to at least two molecules of the primer extension products; (f) extending the first primer extension products hybridized to the barcoded particles in step (e) using the nucleotide sequence to which the primer hybridized as a template to produce second primer extension products comprising two unique particle identifier sequences; (g) determining which unique particle identifier sequences or their complements are present in the second primer extension products; and (h) using the unique particle identifier sequences present in the second primer extension products to map the relative positions of the primers.

[0008] An alternative embodiment involves a probe system comprising: (a) a population of primer molecules; (b) a set of barcoded particles, each having a nucleotide sequence comprising (i) a primer binding sequence complementary to the 3' end of the primer molecule of (a), (ii) a unique particle identifier sequence, and (iii) a first template sequence; and (c) a ligation splint comprising a first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide comprises a first sequence and the first template sequence; and the second oligonucleotide comprises a second sequence complementary to the first sequence, and the first template sequence.

[0009] An alternative probe system includes the steps of: i. hybridizing the set of barcoded particles of (b) with the population of primer molecules of (a); ii. extending the hybridized primer molecules using a nucleotide sequence as a template to produce a first primer extension product that contains i. a complement of the unique particle identifier sequence from the barcoded particle and ii. a complement of the first template sequence; iii. removing the barcoded particle; and iv. hybridizing the first primer extension product with a ligation splint, which generates two The present invention may be used in a method comprising the steps of: hybridizing the complement of the first template sequence in the proximal first primer extension product to the first and second sequences of the ligation splint; and ligating at least one of the first or second oligonucleotides of the hybridized ligation splint to the first primer extension product and extending the 3' end of the ligated first or second oligonucleotide in the splint using the first primer extension product in the ligation product as a template, thereby adding two unique particle identifier sequences to the primer.

[0010] Those skilled in the art will understand that the drawings, described below, are for illustrative purposes only and are not intended to limit the scope of the present teachings in any way. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 illustrates diagrammatically the probe system of the present disclosure.

[0012] [Diagram 2] FIG. 2 shows diagrammatically how the probe system can be used.

[0013] [Diagram 3] FIG. 3 shows diagrammatically a probe system that has a primer binding site, thereby allowing the extension product to be amplified.

[0014] [Figure 4] FIG. 4 shows a schematic of one embodiment of how the primers are tethered.

[0015] [Diagram 5] FIG. 5 shows diagrammatically how the probe system can be used to produce an array.

[0016] [Figure 6] FIG. 6 also shows how the probe system can be used to produce an array.

[0017] [Figure 7] FIG. 7 shows how a sample can be analyzed using the arrays shown in FIGS.

[0018] [Figure 8] FIG. 8 shows diagrammatically how the binding site can be spatially reorganized.

[0019] [Figure 9A] 9A-9C show schematic diagrams of further details of one embodiment of this method. [Figure 9B] 9A-9C show schematic diagrams of further details of one embodiment of this method. [Figure 9C] 9A-9C show schematic diagrams of further details of one embodiment of this method.

[0020] [Figure 10] FIG. 10 illustrates diagrammatically an alternative probe system and method of using it.

[0021] [Figure 11] FIG. 11 shows diagrammatically how the alternative probe system shown in FIG. 10 can be used.

[0022] [Figure 12] FIG. 12 shows a schematic of a gap-filling / ligation implementation of the method.

[0023] [Figure 13] FIG. 13 shows a schematic of a ligation-based version of the method, in which a nucleic acid molecule is attached to a "pre-made" barcode.

[0024] [Figure 14] FIG. 14 shows a schematic of a gap-filling / ligation version of the method that can be used to add barcodes to cDNA.

[0025] [Figure 15] FIG. 15 shows diagrammatically how the method can be carried out using rolling circle amplification products that utilize hairpins in each of the repeats.

[0026] [Figure 16] FIG. 16 is a continuation of FIG. 15 and shows one example of an assay can be designed such that the primer extension product terminates at a defined nucleotide, thereby allowing it to be used as a primer in the next step of the method.

[0027] [Figure 17]FIG. 17 shows diagrammatically an alternative example of how an assay can be designed such that the primer extension product terminates at a defined nucleotide, thereby allowing it to be used as a primer in the next step of the method.

[0028] [Figure 18A-C] Figures 18A-18C show correlation plots of marker counts within each cluster. The poor correlation between CD3 and HLA-DR suggests that each cluster represents a single cell.

[0029] [Figure 19] Figure 19 is a visualization of the graph components (clusters) using a force-generated layout algorithm. Different antibody types can be distinguished.

[0030] definition Before describing the exemplary embodiments in more detail, the following definitions are set forth to illustrate and define the meaning and scope of terms used in the description.

[0031] Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994), and Hale&Markham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide those skilled in the art with the general meaning of many of the terms used herein. Furthermore, for clarity and ease of reference, certain terms are defined below.

[0033] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "primer" refers to one or more primers, i.e., a single primer and multiple primers. It should be further noted that the claims may be drafted to exclude any element. Thus, this statement is intended to serve as a predicate for use of exclusive terminology, such as "solely," "only," and the like, in connection with the recitation of claim elements or the use of a "negative" limitation.

[0034] The term "nucleotide" is intended to include moieties that contain not only the known purine and pyrimidine bases, but also modified other heterocyclic bases. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, alkylated ribose or other heterocycles. In addition, the term "nucleotide" includes moieties that contain hapten or fluorescent labels and may contain other sugars as well as traditional ribose and deoxyribose sugars. Modified nucleosides or nucleotides also include modifications of the sugar moiety, for example, where one or more of the hydroxyl groups are replaced with halogen atoms or aliphatic groups, functionalized as ethers, amines, and the like.

[0035] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to describe a polymer of any length, e.g., greater than about 2 bases, greater than about 10 bases, greater than about 100 bases, greater than about 500 bases, greater than 1000 bases, up to about 10,000 or more bases, composed of nucleotides, e.g., deoxyribonucleotides or ribonucleotides, which can be produced enzymatically or synthetically (e.g., PNAs, as described in U.S. Pat. No. 5,948,902 and references cited therein), and which can hybridize to naturally occurring nucleic acids in a sequence-specific manner similar to the sequences of two naturally occurring nucleic acids, e.g., participate in Watson-Crick base pairing interactions. Naturally occurring nucleotides include guanine, cytosine, adenine, thymine, uracil (G, C, A, T, and U, respectively). DNA and RNA have deoxyribose and ribose sugar backbones, respectively, while the backbone of PNAs is composed of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. In PNA, various purine and pyrimidine bases are linked to the backbone by methylene carbonyl bonds. Locked nucleic acids (LNA), often referred to as inaccessible RNA, are modified RNA nucleotides. The ribose moiety of LNA nucleotides is modified with an extra bridge connecting the 2' oxygen and the 4' carbon. The bridge "locks" the ribose in the 3'-endo (north) conformation that is prevalent in A-form duplexes. LNA nucleotides can be mixed with DNA or RNA residues in oligonucleotides whenever desired. The term "unstructured nucleic acid" or "UNA" refers to nucleic acids that contain non-natural nucleotides that bind to each other with reduced stability. For example, unstructured nucleic acids can contain G' and C' residues, which correspond to non-naturally occurring forms, i.e., analogs of G and C that base pair with each other with reduced stability but retain the ability to base pair with naturally occurring C and G residues, respectively. Unstructured nucleic acids are described in US Patent Publication No. 20050233340, which is incorporated by reference herein for its disclosure of UNAs.

[0036] The term "oligonucleotide" as used herein refers to a single-stranded multimer of nucleotides between about 2 and 200 nucleotides, up to 500 nucleotides in length. Oligonucleotides may be synthetic or enzymatically prepared, and in some embodiments are between 30 and 150 nucleotides in length. Oligonucleotides may contain ribonucleotide monomers (i.e., may be oligoribonucleotides) or deoxyribonucleotide monomers. Oligonucleotides may be, for example, 10-20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 80-100, 100-150, or 150-200 nucleotides in length.

[0037] The term "primer" as used herein refers to an oligonucleotide capable of acting as an initiation point for synthesis when placed under conditions in which synthesis of a primer extension product complementary to a nucleic acid strand is induced, i.e., in the presence of nucleotides and an inducing agent, such as a DNA polymerase, at a suitable temperature and pH. The primer may be single-stranded and must be of sufficient length to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend on many factors, including temperature, source of primer, and use of the method. For example, in diagnostic applications, depending on the complexity of the target sequence or fragment, oligonucleotide primers typically contain 15-25 or more nucleotides, but may contain fewer nucleotides. Primers herein are selected to be substantially complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Thus, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment can be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, the primer can be interspersed with non-complementary bases or longer sequences, so long as the primer sequence has sufficient complementarity to hybridize with a sequence on the strand and thereby form a template for the synthesis of extension products.

[0038] The term "primer extension product" refers to the product of extension of a primer or the product of extension of a molecule that is itself a primer extension product. The term "first primer extension product" refers to a molecule that is the product of extension of a primer. The term "second primer extension product" refers to the product obtained by extending the first primer extension product. When the second primer extension product is sequenced, the entire molecule (or most of it) may be sequenced, and the sequence includes at least the sequence added to the primer in the first primer extension reaction and the sequence added to the first primer extension product in the second primer extension reaction.

[0039] The term "hybridization" or "hybridizing" refers to the process by which a nucleic acid strand anneals to and forms a stable duplex, either a homoduplex or a heteroduplex, under normal hybridization conditions with a second complementary nucleic acid strand, and does not form a stable duplex with an unrelated nucleic acid molecule under the same normal hybridization conditions. The formation of a duplex is achieved by annealing two complementary nucleic acid strands in a hybridization reaction. Hybridization reactions can be made highly specific by adjusting the hybridization conditions (often called hybridization stringency) under which the hybridization reaction occurs, so that hybridization between two nucleic acid strands will not form a stable duplex, e.g., a duplex that retains a double-stranded region under normal stringency conditions, unless the two nucleic acid strands contain a certain number of nucleotides that are substantially or completely complementary in a particular sequence. "Normal hybridization or normal stringency conditions" are easily determined for any given hybridization reaction. See, e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press. As used herein, the term "hybridize" or "hybridization" refers to any process by which a strand of nucleic acid joins with a complementary strand through base pairing.

[0040] A nucleic acid is considered to be "selectively hybridizable" to a reference nucleic acid sequence if the two sequences specifically hybridize to each other under moderate to high stringency hybridization and washing conditions. Moderate and high stringency hybridization conditions are known (see, e.g., Ausubel, et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons 1995, and Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition, 2001 Cold Spring Harbor, NY). An example of high stringency conditions includes hybridization in 50% formamide, 5X SSC, 5X Denhardt's solution, 0.5% SDS, and 100 ug / ml denatured carrier DNA at about 42°C, followed by two washes in 2X SSC and 0.5% SDS at room temperature and two additional washes in 0.1X SSC and 0.5% SDS at 42°C.

[0041] As used herein, the term "sequencing" refers to a method in which the identity of at least 10 consecutive nucleotides of a polynucleotide (e.g., the identity of at least 20, at least 50, at least 100, or at least 200 or more consecutive nucleotides) is obtained.

[0042] The term "next generation sequencing" refers to so-called parallelized sequencing-by-synthesis or sequencing-by-ligation platforms currently used by, for example, Illumina, Life Technologies, BGI Genomics (Complete Genomics technology), and Roche. Next generation sequencing methods can also include nanopore sequencing methods or methods based on electronic detection, such as the Ion Torrent technology commercialized by Life Technologies.

[0043] The term "duplex" or "double stranded" as used herein describes two complementary polynucleotides that are base-paired, i.e., hybridized to one another.

[0044] The terms "determining," "measuring," "assessing," "investigating," "assaying," and "analyzing" are used interchangeably herein to refer to forms of measurement, including determining whether an element is present or not. These terms include both quantitative and / or qualitative determinations. Investigating may be relative or absolute.

[0045] As used herein, the term "ligating" refers to the enzyme-catalyzed joining of the terminal nucleotide at the 5' end of a first DNA molecule with the terminal nucleotide at the 3' end of a second DNA molecule.

[0046] The terms "plurality," "set," and "population" are used interchangeably to refer to something that contains at least two members. In certain cases, a plurality can have at least 10, at least 100, at least 1000, at least 10,000, or at least 100,000 members.

[0047] "Primer binding site" refers to a site on a target polynucleotide or fragment to which an oligonucleotide hybridizes. If an oligonucleotide "provides" a binding site for a primer, the primer may hybridize to that oligonucleotide or its complement.

[0048] The term "chain" as used herein refers to a nucleic acid made up of nucleotides covalently linked to one another by covalent bonds, e.g., phosphodiester bonds.

[0049] The term "extending" as used herein refers to the extension of a nucleic acid by ligation or addition of nucleotides using a polymerase. When a nucleic acid annealed to a polynucleotide is extended, the polynucleotide acts as a template for the extension reaction. In these embodiments, the nucleic acid may be extended by a template-dependent polymerase or by ligation to an oligonucleotide complementary to the polynucleotide, which acts as a splint.

[0050] The term "extending" includes extension at the 3' or 5' end. Primer extension, ligation and gap-filling ligation reactions are types of extension.

[0051] The term "extendable 5' or 3' end" refers to the 5' phosphate and 3' hydroxyl, respectively, which are both extendable by ligation. The 3' hydroxyl is also extendable by a polymerase.

[0052] The term "as a template" as used herein refers to: (a) a primer extension reaction in which one strand acts as a template for the addition of nucleotides by a polymerase; (b) a splint ligation in which one strand acts as a template (or "sprint") to ligate two nucleic acid molecules together. In a ligation reaction, both molecules hybridize to the template and are ligated. Ligation can occur at the 5' end of the nucleic acid molecule or the 3' end of the nucleic acid molecule. And (c) a gap-filling / ligation reaction. In a gap-filling / ligation reaction, two nucleic acids hybridize to the template with a gap between them. One nucleic acid molecule is extended towards the other nucleic acid molecule by primer extension, and then the 3' end of the product is ligated to the other nucleic acid. As used herein, the term "rolling circle amplification" or "RCA" for short refers to an isothermal amplification that uses a strand-displacing polymerase to generate linear concatemerized copies of a circular nucleic acid template. RCA is well known in the field of molecular biology and has been described in various publications, including but not limited to Lizardi et al (Nat. Genet. 1998 19:225-232), Schweitzer et al (Proc. Natl. Acad. Sci. 2000 97:10113-10119), Wiltshire et al (Clin. Chem. 2000 46:1990-1993), and Schweitzer et al (Curr. Opin. Biotech 2001 12:21-27), which are incorporated herein by reference.

[0053] As used herein, the term "rolling circle amplification product" refers to the concatemerized products of a rolling circle amplification reaction.

[0054] As used herein, the term "surface" refers to any solid material (e.g., glass, metal, ceramic, organic polymer surface, or gel) that may contain cells or any combination of biomolecules derived from cells, such as proteins, nucleic acids, lipids, oligo / polysaccharides, biomolecule complexes, cell organelles, cell debris, or waste products (exosomes, microvesicles). Tissue blots, Western blots, and glass slides are examples of solid materials with surfaces. A suspension of cells, e.g., mammalian cells, is another example of a surface.

[0055] As used herein, the term "splint" refers to an oligonucleotide that hybridizes to the ends of two other oligonucleotides, bringing their ends together to produce a ligatable junction, or a gap that can be filled by a gap-filling / ligation reaction.

[0056] As used herein, the term "barcoded particles" is intended to refer to both barcoded RCA products and barcoded nanoparticles, where the particles in a population of barcoded particles are each separately barcoded with a unique particle identifier sequence, i.e., a sequence that is unique to each particle such that the particles can be distinguished from one another by their unique identifier sequence.

[0057] Other term definitions may appear throughout the specification. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0058] Before various embodiments are described, it is to be understood that the teachings of the present disclosure are not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present teachings will be limited only by the appended claims.

[0059] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described in any way. While the present teachings have been described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those skilled in the art.

[0060] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present teachings, some exemplary methods and materials are described herein.

[0061] The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present claims are not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0062] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has individual components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present teachings. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0063] All patents and publications referred to herein, including all sequences disclosed within such patents and publications, are expressly incorporated by reference.

[0064] Before certain aspects of the invention are described in more detail, it is important to note that the figures show embodiments of the present probe system and method using RCA products. As described below, the principles shown in the figures can be easily applied to barcoded nanoparticles, and therefore the invention should not be limited to those shown in the figures. Additionally, only one RCA product repeat is shown in the figures. As is well known, barcoded RCA products (and barcoded nanoparticles) can contain at least 10, at least 50, at least 100, at least 500, or at least 1,000 repeats of molecules (linked in the RCA product or tethered to the surface of the nanoparticle).

[0065] Further, it will be appreciated that the present probe system and method need not be performed as a primer extension assay (as shown in FIG. 2). As shown in FIGS. 12-14, the method may be performed using a gap-filling / ligation or ligation assay, both of which can involve extending the 5' end of a nucleic acid molecule using a barcoded particle as a template. As such, while the following description focuses on an embodiment that uses primer extension, it should be appreciated that the primer extension-based embodiment described below is merely an example of how the method may be performed.

[0066] Some principles of the present probe system are illustrated in FIG. 1. With reference to FIG. 1, the probe system may include a population of primer molecules 2 (as shown, having a 3′ end of sequence C-BS1′). As described in more detail below, the primer molecules may be, for example, synthetic oligonucleotides or cDNA molecules with a primer sequence at their 3′ end. In some embodiments, the primer molecules may be linked to a binding agent (e.g., an oligonucleotide probe, an antibody, an aptamer, etc.), while in other embodiments, the primer molecules may be linked to a flat substrate as a lawn, with the 3′ end of the oligonucleotide distal to the substrate and capable of being extended by a polymerase. As shown, the probe system also includes a first set of barcoded particles 4 and a second set of barcoded particles 12. As shown, each of the particles in the first set of barcoded particles 4 has a nucleotide sequence that includes a unique particle identifier sequence 8 (UMI1), and a first template sequence 10 (C-BS2), in addition to a primer binding sequence 6 (C-BS1) that is complementary to the 3′ end of the primer molecules 2. Each of the particles in the second set of barcoded particles 12 has a nucleotide sequence that includes the first template sequence 10 (i.e., the same template sequence as in the first set of particles, C-BS2), and a unique particle identifier sequence 14 (UMI2). In the embodiment shown, the nucleotide sequence of the second set of barcoded particles does not contain a primer binding sequence 6 (CBS1). However, in other embodiments, the nucleotide sequence of the second set of barcoded particles may contain a primer binding sequence 6 (CBS1). In this probe system (shown in FIG. 2), extension of the primer molecule 2 using the first set of barcoded particles 4 as a template produces a first primer extension product 16 that contains a complement 18 of the unique particle identifier sequence of the particle of the first set of barcoded particles (i.e., the complement of UMI1, or UMI1′) and a complement 20 of the first template sequence (C-BS2′, which is the complement of C-BS2).As described in more detail below, the 3' end of the primer extension product 16 can hybridize to a first template sequence 10 of a second set of particles. As shown, extension of the 3' end of the primer extension product 16 using a particle from the second set of barcoded particles as a template results in a second primer extension product 22 containing a complement 18 of the unique particle identifier sequence from the first set of particles, a complement 20 of the first template sequence, and a complement 24 of the unique particle identifier sequence from the second set of particles. In some embodiments, there may be an internal hairpin immediately downstream of the first template sequence.

[0067] In some embodiments, the first and second sets of barcoded particles can be rolling circle amplification (RCA) products or barcoded nanoparticles. For example, in some embodiments, the first and second sets of barcoded particles can be RCA products, the first and second sets of barcoded particles can be barcoded nanoparticles, or the first set of barcoded particles can be RCA products or barcoded nanoparticles and the second set of barcoded particles can be other types.

[0068] In embodiments where the barcoded particles are RCA products, each of the RCA products contains a unique sequence that is in the repeat sequence. In other words, if there are 1,000 RCA products, each product has a unique sequence (referred to herein as a unique molecular identifier "UMI" or unique identifier "UID"). The UID for one particle is different from the UID for other particles. RCA products can be prepared, for example, by synthesizing initial oligonucleotides with degenerate sequences, circularizing the initial oligonucleotides using a splint, and amplifying the circularized oligonucleotides by RCA. In some embodiments, the initial oligonucleotides can contain a degenerate (e.g., random) sequence of 6-10 nucleotides, or even more random nucleotides, depending on the number of unique RCA products required. Amplification of the circularized oligonucleotides with degenerate sequences should produce a population of RCA products, each with a unique identifier (i.e., a sequence that is different from other RCA products in the population). Methods for generating RCA products with unique identifiers are described, for example, in Wu et al. (Nat.Comm.2019 10:3854) and US Patent Publication No. 20160281134, and are readily adapted for use herein. In some embodiments, the different oligonucleotides used to prepare the first and second sets of RCA products are prepared separately and then mixed together. In other embodiments, the different oligonucleotides can be prepared in parallel on a planar support in the form of an array and then cleaved from the array. Examples of such methods are described, for example, in Cleary et al. (Nature Methods 2004 1:241-248) and LeProust et al. (Nucleic Acids Research 2010 38:2522-2540).In some embodiments, one or both of the RCA products may contain uracil or thymine, thereby allowing them to be enzymatically degraded by a USER containing UDG (uracil DNA glycosylase) and AP lyase that cleaves the phosphodiester backbone at apurinic sites (see, e.g., Bitinaite et al Nucleic Acids Res. 2007 35:1992-2002).

[0069] In embodiments where the barcoded particles are barcoded nanoparticles, the barcoded nanoparticles are small beads or metal particles, etc., coated in oligonucleotides, where the oligonucleotides tethered to the surface on each particle have a unique sequence that is different from the sequences in the oligonucleotides tethered to other particles in the population. In other words, if there are 1,000 barcoded particles, the oligonucleotides tethered to each particle have a unique sequence (referred to herein as unique molecular identifiers "UMI" or unique identifiers "UID"). The UID for one particle is different from the UID for other particles. These particles can be of any suitable size, material, and shape. In many embodiments, the particles have a size between 10 nm and 200 nm. Gold particles (which can be easily sized to any diameter within the range of, for example, 1.8 nm to 1500 nm) can be used, but the particles can also be made from silver, silica, titanium dioxide, carbon, polymers (e.g., polystyrene, polyacrylate, etc.), agarose, etc. Magnetic particles of iron and various alloys can also be used (Creative Diagnostics, Shirley, NY, USA). The particles do not have to be magnetic, but magnetic nanospheres can be used in some cases (Creative Diagnostics, Shirley, NY, USA). There are several surface chemistries to functionalize metal surfaces so that they can be conjugated to nucleic acids. For example, particles can be modified to contain reactive groups, including, but not limited to, N-hydroxysuccinimidyl esters, sulfo-N-hydroxysuccinimidyl esters, halo-substituted phenol esters, pentafluorophenol esters, nitro-substituted phenol esters, anhydrides, isocyanates, isothiocyanates, imidoesters, maleimides, iodoacetyls, hydrazides, aldehydes, or epoxides. Other suitable groups are known in the art and can be, for example, those described in Hermanson, "Bioconjugate Techniques" Academic Press, 2nd Ed., 2008.The most commonly used capture agent reactive groups are NHS (amine reactive) and maleimide (sulfhydryl reactive), but many others can be used. Particles can also be coated in streptavidin, which can bind to biotinylated nucleic acids. In some embodiments, barcoded particles can be prepared by emulsion PCR, a method that has been successfully used for other applications and is described, for example, in Kanagal-Shamanna et al (Methods Mol Biol 2016 1392:33-42) and Shao et al (PlosOne 2011 0024910). In some embodiments, the nucleic acids of the barcoded particles can contain uracil, thereby allowing them to be cleaved using USER, as discussed above.

[0070] In any embodiment, primer molecule 2 may contain a 5' tail that does not hybridize to any of the nucleic acids of the barcoded particle. The 5' tail may contain a primer binding site (e.g., for a forward primer) and a target identifier sequence that may be used later in the protocol. As described above, the primer molecule may be a synthetic, artificial oligonucleotide of 10-200 nucleotides in length. In some of these embodiments, the primer molecule may be linked to a binding agent (e.g., an oligonucleotide probe, an antibody, an aptamer, etc.). In these embodiments, the primer molecule is not a cDNA. In some embodiments, the primer molecule may contain a primer sequence at the 3' end, i.e., a cDNA molecule with an added sequence that can serve as a primer. In these embodiments, cDNA can be prepared, for example, by hybridizing a reverse transcription primer (e.g., a primer having a 3' end comprised of oligo(dT), random or gene specific sequence, and optionally having a 5' end that does not hybridize to the RNA, and may contain a sequence that provides a binding site for a PCR primer) to RNA, e.g., RNA in a sample in situ. The reverse transcription primer can be extended in situ (in a reaction containing NTPs, reverse transcriptase and any other necessary reagents) to produce a first strand cDNA to produce a cDNA product. The primer sequence can be added to the cDNA by template switching (see, e.g., Zhu et al BioTechniques 2001 30:892-7), ligation of a 3' adapter, or tailing, e.g., by terminal transferase. As indicated above, in cDNA embodiments, the cDNA can be prepared in situ, so that the primer can be in or on the tissue section. In other embodiments, the primer molecules can be linked via their 5' ends to a planar substrate, for example to form a "lawn."

[0071] In embodiments in which the primer molecule may be linked to a binder, the binder may be, for example, an oligonucleotide probe, an antibody, or an aptamer. In these embodiments, the primer molecule may additionally contain a sequence that identifies the target to which the binder binds. For example, if the primer is linked to an antibody, the primer may additionally contain the name of the antibody or a target identifier sequence at the 5' end of the primer (downstream of any primer sequence) that identifies the target (e.g., epitope) to which the antibody binds. Thus, in some embodiments, the primer may be part of a capture agent-primer conjugate in which the primer is non-covalently (e.g., via a streptavidin / biotin interaction) or covalently (e.g., via a click reaction, etc.) linked to a capture agent, e.g., an antibody or aptamer, and a single-stranded primer such that the capture agent can still bind to its binding site. The oligonucleotide and capture agent may be linked via several different methods, including using maleimide or halogen-containing groups that are cysteine ​​reactive. The capture agent and oligonucleotide may be linked proximal or at the 5' end of the oligonucleotide, proximal or at the 3' end of the oligonucleotide, or anywhere in between. In some embodiments, the oligonucleotide may be linked to the capture agent by a linker that separates the oligonucleotide from the capture agent. The oligonucleotide may be linked to the capture agent using any convenient method (see, for example, Gong et al., Bioconjugate Chem. 2016 27:217-225, and Kazane et al. Proc Natl Acad Sci 2012 109:3731-3736). As described above, the sequence of the primer conjugated to the binding agent uniquely identifies the epitope or sequence to which the binding agent binds. For example, if the method is carried out using 10 different antibodies, each antibody is tethered to a different primer that contains a sequence that identifies the epitope to which the antibody binds.This feature allows the method to be multiplexed, and in some embodiments, the method can use at least 5, at least 10, at least 20, or at least 50 different antibodies that bind to different markers within or on the surface of a cell. Each antibody is conjugated to a different target identifier sequence, and the antibody identifier sequence allows for mapping of the binding event of a particular antibody. Such tagged antibodies are described, for example, in Wu et al. (Nat.Comm.2019 10:3854) and US Patent Publication No. 20160281134.

[0072] In embodiments in which the primer molecule may be linked to a flat substrate, the substrate may be prepared by synthetically preparing the primer molecule, e.g., using phosphoramidite chemistry, and tethering the primer molecule to a flat support, e.g., a glass slide, by its 5' end, as such chemistry is well known.

[0073] In any embodiment, primer molecule 2 may have a 5' tail with a forward primer sequence 30, and the nucleotide sequence of the second set of barcoded particles 12 has a reverse primer sequence 32 downstream of the unique particle identifier sequence 14, as shown in Figure 3. In this embodiment (and as shown in Figure 4), the second primer extension product has both the forward and reverse primer sequences, allowing the product to be amplified by PCR.

[0074] The lengths of the component portions of the nucleic acid molecules described herein can vary. In some embodiments, the portion of the primer that hybridizes to primer binding sequence 6 (C-BS1) can be at least 10 nucleotides in length, e.g., 12-50 nucleotides in length, the UMI sequence can be at least 5 nucleotides in length, e.g., 6-20 nucleotides in length, and the first template sequence 10 can be at least 10 nucleotides in length, e.g., 12-50 nucleotides in length.

[0075] In any embodiment, each of the first and second sets of barcoded particles can include at least 10 members (e.g., at least 100, at least 1,000, at least 10,000, at least 100,000, at least 1 M, at least 10 M, at least 100 M, at least 1 B, or at least 10 B members), each uniquely identifiable by their particle identifier sequence. In some embodiments, the nucleic acid sequences of the barcoded particles in the first and second sets of barcoded particles can be identical to one another except for their particle identifier sequences.

[0076] Methods of attaching unique particle identifier sequences to primers using the present probe system are also provided. The principle of the method is illustrated in Figure 2. In these embodiments, the method may include hybridizing a first set of barcoded particles 4 with a population of primer molecules 2 and extending the hybridized primer molecules using the nucleotide sequences of the first set of barcoded particles as templates to produce first primer extension products 16 that contain i. a complement 18 of the unique particle identifier sequence from a barcoded particle in the first set of barcoded particles and ii. a complement 20 of the first template sequence. The first set of barcoded particles are then removed, e.g., by denaturation and washing, or by incorporating uracil residues in place of thymidines in the first barcoded particles and enzymatically treating the products, e.g., using USER (NEB), which contains a uracil-n-glycosylase enzyme and an AP lyase that can cleave phosphodiester bonds specifically at apurinic base sites, thereby degrading the first barcoded particles and releasing the extended primer; in these embodiments, primer 2 (and thus primer extension product 16) can be tethered (e.g., via a capture agent) to the support or sample, such that the first set of barcoded particles can be subjected to stringent washing conditions without removing the primers or primer extension products from the sample or support. Additionally, the first primer extension product may be terminated at a precise location (i.e., at the end of the first template sequence) by engineering a site for a restriction enzyme at that location and then digesting the primer extension product (which is double-stranded) with the restriction enzyme after it is produced, or by hybridizing a blocking oligonucleotide to a sequence immediately downstream of the first template sequence, or by designing an internal hairpin immediately downstream of the first template sequence that causes the polymerase to stall. In these latter embodiments, the polymerase (which should be a non-strand displacing polymerase) should terminate synthesis upon entering the blocking oligonucleotide or hairpin.Alternative methods for terminating nucleic acid synthesis at defined sites are shown diagrammatically in Figures 15-17. Figure 15 shows RCA products containing hairpin loops. These hairpin loops can be cleaved by restriction enzymes to release 5' and 3' ends as blunt ends or with overhangs. Figure 16 shows how primer extension can be terminated at defined sites in the product. In this embodiment, the hairpins are digested to produce blunt ends or overhangs, but the RCA products stay together as a complex. The RCA products are then hybridized to a conjugated oligonucleotide, and a strand-displacing DNA polymerase (e.g., Klenow fragment of E. coli DNA polymerase I) copies the barcode from the RCA product, displacing the hairpin and naturally terminating at the template end. The primer extension products can then be used as primers in the next set of pixels. Restriction enzyme cleavage of the hairpins can be performed before or after hybridization of the RCA products to the oligonucleotides. FIG. 17 shows how defined termini can be produced by digesting the extension product after it has been prepared, while still being double-stranded.

[0077] The method may then include hybridizing the first primer extension product 16 to a second set of barcoded particles 12, where the complement 20 of the first template sequence in the first primer extension product hybridizes to the first template sequence in the second set of barcoded particles. The method then includes extending the first primer extension product using the nucleotide sequence of the second set of barcoded particles as a template to produce a second primer extension product containing the unique particle identifier sequence 18 from a barcoded particle in the first set of barcoded particles, the first template sequence 20, and the unique particle identifier sequence 20 from a barcoded particle in the second set of barcoded particles.

[0078] As shown in Figure 3, primer molecule 2 may have a forward primer sequence and the nucleotide sequence of the second set of barcoded particles 12 may have a reverse primer sequence downstream of the unique particle identifier sequence. In these embodiments, the first primer extension product 16 should contain the forward primer sequence at the 5' end and the second primer extension product 22 should contain the forward primer sequence at the 5' end and the reverse primer sequence at the 3' end. In this embodiment, the method may include amplifying the second primer extension product 22 by PCR using primers that target the forward and reverse primer sequences. Figure 4 shows an embodiment of the method in which the primers are tethered to either the support or the sample.

[0079] In some embodiments, the method may include sequencing the second primer extension product, or an amplified copy thereof. In these embodiments, each second primer extension product should contain a unique identifier for the two barcoded particles, thereby identifying which barcoded particle hybridized with the primer in the different hybridization steps. In some embodiments, as described in more detail below, the method may include mapping the relative positions of the primers using pairs of unique particle identifier sequences in the second primer extension products.

[0080] As mentioned above, in some embodiments, the primers may be attached to the cell sample via a binder. In these embodiments, the unique particle identifier in the second primer extension product indicates the relative location of the binder on the cell sample. This embodiment is shown in FIG. 4. As can be seen in FIG. 4, in these embodiments, the primers may include a target identification sequence. In the embodiment shown in FIG. 4, in steps A and B, the primer molecules contain a target identifier and are immobilized to the sample, for example, via a binder. In this method, a first set of barcoded particles is hybridized to the sample, and the UMI and complement of the C-BS2 sequence from the primer-hybridized particles are copied onto the end of the primer molecule using the hybridized particles as a template. In steps C and D, the first set of barcoded particles is removed and a second set of particles is hybridized. After hybridization, the primer extension products are extended to add the UMI from the second set of particles to the end of the primer extension products. The pairs of barcodes copied onto the ends of the primers can be analyzed to determine the relative positions of the different primers in a sample.

[0081] As indicated above, in alternative embodiments, the method may be carried out using a gap-filling / ligation and / or ligation-based approach, in which the 5' end of the nucleic acid may be extended by a gap-filling / ligation or ligation reaction using the barcoded particle of (b) as a template. These embodiments are shown diagrammatically in Figures 12-14. With respect to Figure 12, instead of copying the barcode from the barcoded particle by polymerizing from the free 3' end of the binder-conjugated primer (as described above), a gap-filling / ligation reaction may be used to copy the barcode onto the 5' end of an antibody-conjugated oligonucleotide bearing a 5' phosphate. These embodiments may be carried out, for example, using a combination of T4 DNA polymerase and T4 DNA ligase. Figure 13 shows how the 5' or 3' end of a binder-conjugated oligonucleotide may be ligated to a "pre-prepared" molecule containing the barcode of the particle. In these embodiments, the barcode is first copied by a gap-filling / ligation reaction. During the assay itself, the antibody oligonucleotide is ligated to the 5' or 3' end of a pre-prepared copy of the barcode. In this figure, the 5' end of the oligonucleotide is ligated to the copy of the barcode, but this can be done in reverse, i.e., the oligonucleotide linked to the antibody has a free 3' end that is ligated to the 5' phosphate of the UMI copy sequence. Figure 14 shows how the 5' end of the cDNA can be added to the barcode via a gap-filling / ligation reaction. In this method, the mRNA is copied in a reverse transcription reaction (using an oligo nd(T) primer, random primer or gene-specific primer) and once the cDNA is produced, the barcode is added to the 5' end of the cDNA via a gap-filling ligation reaction. These and other implementations can be envisioned.

[0082] As mentioned above, in some embodiments, the primer molecules may be attached to a flat substrate, such as a glass slide. In these embodiments, at least 10 6 , 10 7 , 10 8 or 10 9 The primer molecules can be attached to a flat support and extended using the method described above. This method is shown diagrammatically in FIG. 5. In this embodiment, the first and second sets of barcoded particles hybridize to sequences in overlapping sections on the support 50. A first primer extension reaction adds molecular identifiers to the primers hybridized to the first set of barcoded particles, resulting in a first array of features 52 corresponding to the first primer extension products. In the example shown, there are six features 58, each of which corresponds to a barcoded particle and contains a different molecular identifier. After the first primer extension reaction is completed, the first set of barcoded particles is removed and the second set of barcoded particles is hybridized to the substrate. In this step, the second set of barcoded particles hybridize to the first primer extension products, but in an overlapping manner to the first set of barcoded particles. Extension of the hybridized second set of barcoded particles adds molecular identifiers from the second set of barcoded particles to the first primer extension products to produce a second array of features 56 that correspond to the second primer extension products. As an example, in array 56, features 60 and 62 have the same unique identifier from the first barcoded particle (corresponding to feature 58), but different unique identifiers from the second barcoded particle.

[0083] This embodiment of the method, resulting in an array, is also shown in FIG. 6. With reference to FIG. 6, the method may begin with a solid surface with immobilized oligonucleotides. In step 1, the free 3' ends of the immobilized oligonucleotides are extended to a first set of barcoded particles (e.g., RCA products) with unique molecular identifiers. This extension step copies the complements of the unique molecular identifiers from the first set of barcoded particles onto the oligonucleotides. The barcoded particles are then removed. In step 2, a second set of barcoded particles is added to the surface, effectively adding a second random barcode to the surface. The primer extension products are then extended, incorporating the identifiers of the second set of particles into the initial extension products. The second identifiers provide information about which first identifiers are in close proximity. The second addition has only a partial overlap with the first, resulting in a spatial map of linked identifiers, shown at the bottom of FIG. 6. The array can then be used to capture bound biomolecules in a cell or tissue sample, thereby providing spatial information of the biomolecules. Resolution will likely be at the size of the particle, which may be 50-500 nm, greatly exceeding the resolution of other methods. Alternatively, capture of biomolecules in a sample (e.g., mRNA binding probes or polyA primers for cDNA) can be performed on a surface containing immobilized oligonucleotides (prior to primer extension). An example of the use of such an array is shown in Figure 7.

[0084] The probe system can be used to map binding events in or on a cell sample. This method is shown diagrammatically in FIG. 8. In these embodiments, the method can include obtaining a sample containing a primer molecule bound to a site in or on a cell. For example, as described above, the sample can be prepared by binding a primer attached to a binding agent (e.g., an oligonucleotide probe, an antibody, or an aptamer) to a site in or on a cell, or by hybridizing a first primer to RNA in the cell, extending the primer to prepare cDNA, and adding a second primer to the 5' end of the cDNA, such that the cDNA becomes the primer molecule used in the method. Other components used in the method include the first set of barcoded particles discussed above, i.e., particles each having a nucleotide sequence including a primer binding sequence complementary to the 3' end of a primer molecule, a unique particle identifier sequence, and a first template sequence, as shown in Figure 1, as well as a second set of barcoded particles each having a nucleotide sequence including a first template sequence and a unique particle identifier sequence, as shown in Figure 1. In any embodiment, and as shown in Figure 1, the nucleotide sequences of the second set of barcoded particles may be devoid of the primer binding sequences present in the nucleotide sequences of the first set of barcoded particles. Thus, in any embodiment, the nucleotide sequences of the second set of barcoded particles may be devoid of the primer binding sequences present in the nucleotide sequences of the first set of barcoded particles, and may include the first template sequence and the unique particle identifier sequence, but may not include the primer binding sequences present in the nucleotide sequences of the first set of barcoded particles.

[0085] As described above, the method involves specifically hybridizing a first set of barcoded particles to a sample, where the nucleotide sequence of at least some of the first set of barcoded particles hybridizes to at least two primer molecules. As shown in FIG. 8, the number of primer molecules hybridized to each barcoded particle can vary. Some of the first set of barcoded particles may hybridize to one primer molecule, but many should hybridize to at least 2, at least 5, at least 10, or at least 20 primer molecules, depending on the density of the primer molecules and the size of the particles used. As with the method described above, the method includes extending the primers hybridized to the barcoded particles using the nucleotide sequence to which the primers hybridize as a template to produce first primer extension products, each of which includes a first unique particle identifier sequence. The method may then include removing the first set of barcoded particles from the sample, as described above, and specifically hybridizing the second set of barcoded particles with the first primer extension product. In this step of the method, at least some nucleotide sequences of the second set of barcoded particles hybridize to at least two molecules of the primer extension product. Similarly, the first primer extension product previously hybridized to the barcoded particles is extended using the nucleotide sequence to which the primer hybridized as a template to produce a second primer extension product containing two unique particle identifier sequences, one from the particles in the first set and the other from the particles in the second set. The method then includes determining which unique particle identifier sequence or its complement is in the second primer extension product, and using the unique particle identifier sequence in the second primer extension product to map the relative positions of the primers.

[0086] The steps of this method are shown in detail in Figures 9A-C. As can be seen from Figures 9A-C, the primers may contain target identifier sequences, which may allow the assay to be multiplexed. In this method, the population of second primer extension products may be amplified and sequenced in unison. Each sequence read should contain two particle identifiers, one from the first set of particles and the other from the second set of particles, in addition to the target identifier. Because the sites where the first and second sets of particles bind overlap in separate hybridization reactions, the primers may be mapped to sites in the sample. In particular, a relational map of barcoded particles may be produced by unravelling which pairs of identifiers are attached to the primers. The target sequences may be mapped to the relational map, which may provide a map of the binding sites of the primers (or the sites to which they bind, e.g., epitopes or sequences).

[0087] Alternative ligation-based embodiments are described below. These embodiments avoid the use of two sets of barcoded particles, and instead only one primer extension step is required. In these embodiments, identifier pairs can be created by connecting nearby primer extension products via a ligation splint. These embodiments are shown diagrammatically in Figures 10 and 11. In these embodiments, the probe system can include a population of primer molecules (which may contain sequences providing a primer binding site, a target identifier, and a primer sequence C-BS1, as shown); a set of barcoded particles each having a nucleotide sequence that includes (i) a primer binding sequence (C-BS1) complementary to the 3' end of the primer molecule, (ii) a unique particle identifier sequence (UMI), and (iii) a first template sequence (C-BS2). Instead of a second set of barcoded particles, the probe system includes a ligation splint. As shown in Figure 10, a ligation splint is composed of a first oligonucleotide and a second oligonucleotide, where the first oligonucleotide comprises a first sequence and a first template sequence (C-BST'); and the second oligonucleotide comprises a second sequence complementary to the first sequence and the first template sequence (C-BST'). The two oligonucleotides in the splint have the same sequence and can hybridize to each other via sequences at their 5' ends.

[0088] In general, the components of this system are similar to those of the serial extension system described above, except that the alternative system has a ligation splint instead of a second set of barcoded particles. For example, the barcoded particles can be rolling circle amplification (RCA) products, or the barcoded particles can be barcoded nanoparticles, and the nucleotide sequence is tethered to the surface of the barcoded particles. Similarly, the primer molecule of (a) is a synthetic oligonucleotide of 10-200 nt in length that can be linked to a binding agent (e.g., an oligonucleotide probe, an antibody, an aptamer, etc.). In some embodiments, the primer molecule can be a cDNA molecule having a primer sequence at the 3' end.

[0089] Similar to the sequential extension embodiment, the set of barcoded particles may contain at least 10 particles, each having a unique particle identifier sequence. In this embodiment, the set of barcoded particles may include at least 10 members (e.g., at least 100, at least 1,000, at least 10,000, at least 100,000, at least 1M, at least 10M, at least 100M, at least 1B, or at least 10B members), each uniquely identifiable by their particle identifier sequence. In some embodiments, the nucleic acid sequences of the barcoded particles may be identical to one another except for their particle identifier sequences.

[0090] Alternative probe systems can be used to map binding events in or on cell samples in a manner similar to the sequential probe system described above. A method is provided for attaching unique particle identifier sequences to primers using alternative probe systems. This method, shown in Figures 10 and 11, can include hybridizing a set of barcoded particles with a population of primer molecules (which can be immobilized on a support as shown) and extending the hybridized primer molecules using a nucleotide sequence as a template. This step results in the production of a first primer extension product that contains i. the complement of the unique particle identifier sequence (UMI) from the barcoded particle and ii. the complement of the first template sequence (C-BS2). After the barcoded particles are removed, the method includes hybridizing the first primer extension product with a ligation splint, in which the complement of the first template sequence in the two proximal first primer extension products hybridizes to the first and second sequences of the ligation splint, as shown.The method then includes ligating at least one of the first and second oligonucleotides of the hybridized ligation splint to the first primer extension product, and extending the 3' end of at least one of the first and second oligonucleotides in the splint using the first primer extension product in the ligation product as a template, thereby adding two unique particle identifier sequences to the primer.As shown in Figure 10, the primer can be operated on the forward and reverse primer sequences and the target identification sequence, thereby providing a product containing a PCR primer site, a pair of target identification sequences, and two particle identifiers. These sequences can be analyzed to determine the relative positions of the barcoded particles and primers (or the binders, probes or cDNA molecules to which they are tethered), as shown diagrammatically in Figure 11. As will be apparent, the method can include sequencing the second primer extension, thereby identifying which unique particle identifier sequence pairs are in the second primer extension product.The method may further comprise mapping the relative positions of the primers of i. using the pair of unique particle identifier sequences present in the second primer extension product.

[0091] Similar to the sequential extension embodiment described above, the primer molecule can be attached to the cell sample via a binding agent (e.g., an antibody, aptamer, or probe), and the unique particle identifier in the second primer extension product can indicate the relative location of the binding agent on the cell sample. Similarly, the primer can be the 3' end of a cDNA prepared in situ in the cell sample, and the unique particle identifier in the second primer extension product can indicate the relative location of the cDNA in the cell sample.

[0092] In any embodiment, barcoded particles can be mapped relative to each other when two complex identifiers are attached to the same molecule. The map produced by the method can be a three-dimensional map or a two-dimensional map, depending on how the method is performed. For example, when the complex products are immobilized within cells (e.g., produced in situ within cells), the map produced can be three-dimensional. In other embodiments, for example, when the complexes are immobilized on one or more surfaces (e.g., the surface of one or more cells that may be in suspension or attached to a support), the map produced by the method can be two-dimensional, or potentially three-dimensional, since theoretically the map can be spherical. Although the method can be applied to cells (as described below), the method can be adapted to map adjacent complexes immobilized on any surface, such as a glass slide, for example, a tissue blot or a Western blot. Similarly, the complex can be immobilized at a site in or on a cell or on the surface via an antibody (e.g., an antibody conjugated to an oligonucleotide having a sequence complementary to a sequence in the complex), but the complex can be immobilized directly or indirectly via any type of interaction, e.g., covalent or non-covalent interactions. For example, in some embodiments, the complex can be bound to a cell via a binding agent (e.g., an aptamer, an antibody, or an oligonucleotide, etc.) that binds to a sequence in the complex and a site within the cell or on the surface of one or more cells. In some embodiments, the complex can be immobilized via hybridization to an oligonucleotide that also hybridizes to a nucleic acid (e.g., to cellular RNA), or the RCA product can be non-covalently immobilized to a site via electrostatic interactions, via streptavidin / biotin interactions, or by covalent binding (e.g., via click coupling).

[0093] In any embodiment, the complex may be immobilized in or on cells that are in solution, on a support (e.g., a slide), in a three-dimensional sample of tissue, or in a tissue section. A sample containing cells in solution may be, for example, a sample of cultured cells grown as a cell suspension. In other embodiments, dissociated cells (which may be produced by dissociating cells in culture or in a solid tissue, e.g., soft tissue such as the liver of the spleen, using trypsin, etc.) may be used. In certain embodiments, the complex may be immobilized on cells that can be found in blood, e.g., cells in whole blood or subpopulations of cells thereof. Subpopulations of cells in whole blood include platelets, red blood cells (erythrocytes), and white blood cells (i.e., peripheral blood white blood cells, which are composed of neutrophils, lymphocytes, eosinophils, basophils, and monocytes). These five types of leukocytes can be further divided into two groups: granulocytes (also known as polymorphonuclear leukocytes, including neutrophils, eosinophils, and basophils) and mononuclear leukocytes (including monocytes and lymphocytes). Lymphocytes can be further divided into T cells, B cells, and NK cells. Peripheral blood cells are found in the circulating pool of blood and are not sequestered within the lymphatic system, spleen, liver, or bone marrow. When cells immobilized on a support are used, the sample can be prepared, for example, by growing the cells on a flat surface, depositing the cells on a flat plane, cutting the three-dimensional object containing the cells into sections, for example by centrifugation, and mounting the sections on the flat surface, i.e., producing a tissue section. In an alternative embodiment, the surface can be prepared by absorbing cellular components onto the surface.

[0094] In any embodiment, the method may include immobilizing thousands, tens of thousands, hundreds of thousands, or at least a million barcoded particles (each with a unique identifier) ​​to a population of cells (e.g., via antibodies) such that the barcoded particles on each cell effectively coat the cell. The barcoded particles may hybridize to other oligonucleotides tethered to sites in or on the cells to produce a matrix containing the barcoded particles. After hybridization, the unique identifier sequences of adjacent complexes may be copied from one barcoded particle to another. In addition to the complexes, a physical map of the sites where the barcoded particles bind to the cells may be constructed based on the copied sequences.

[0095] In addition to creating a map of barcoded particles, the method may involve performing a proximity assay between one or more binding agents (e.g., antibodies that bind cell surface markers on cells) that are bound to sites within a cell or on the surface of a cell. In these embodiments, the product may contain a pair of binding agent identifier sequences in addition to a unique complex identifier sequence. In some embodiments, the binding agent may be an antibody-oligonucleotide conjugate, and in other embodiments, the capture agent may be an oligonucleotide probe or cDNA. The oligonucleotide and capture agent may be linked via several different methods, including using maleimides or halogen-containing groups that are cysteine ​​reactive. The capture agent and oligonucleotide may be linked proximal or at the 5' end of the oligonucleotide, proximal or at the 3' end of the oligonucleotide, or anywhere in between. In some embodiments, the oligonucleotide may be linked to the capture agent by a linker that spaces the oligonucleotide from the capture agent. The oligonucleotide may be linked to the capture agent using any convenient method, as described above. In many embodiments, the sequence of the oligonucleotide conjugated to the binding agent uniquely identifies the epitope or sequence to which the binding agent binds. For example, if the method is performed using 10 different antibodies, each antibody is tethered to a different sequence that identifies the epitope to which the antibody binds. This feature allows the method to be multiplexed, and in some embodiments, at least 5, at least 10, at least 20, or at least 50 different antibodies that bind to different markers within or on the surface of a cell can be used in the method. Each antibody is conjugated to a different antibody identifier sequence, which allows the binding event of a particular antibody to be mapped. Such tagged antibodies are described, for example, in Wu et al. (Nat.Comm.2019 10:3854) and US Patent Publication No. 20160281134.

[0096] Embodiment Embodiment 1. A probe system comprising: (a) a population of primer molecules; (b) (i) a first set of barcoded particles, each having a nucleotide sequence comprising a primer binding sequence complementary to a 3' end of the primer molecules of (a), (ii) a unique particle identifier sequence, and (iii) a first template sequence; and (c) a second set of barcoded particles, each having a nucleotide sequence comprising (i) the first template sequence, and (ii) a unique particle identifier sequence, wherein extension of the primer molecules of (a) using the first set of barcoded particles of (b) as templates produces primer extension products containing a complement of the unique particle identifier sequence of the particle of (b)(ii) and a complement of the first template sequence.

[0097] Embodiment 2. The probe system of embodiment 1, wherein the barcoded particles of (b) and / or (c) are rolling circle amplification (RCA) products.

[0098] A probe system as described in embodiment 1, wherein the barcoded particles of embodiment 3 (b) and / or (c) are barcoded nanoparticles and the nucleotide sequence is tethered to the surface of the barcoded particles.

[0099] A probe system according to any one of embodiments 1 to 3, wherein the primer molecule of embodiment 4 (a) is a synthetic oligonucleotide having a length of 10 to 200 nt.

[0100] A probe system according to any one of the preceding embodiments, wherein the primer molecule of embodiment 5 (a) is a cDNA molecule having a primer sequence at the 3' end.

[0101] Embodiment 6. A probe system described in any one of embodiments 1 to 5, wherein the primer molecule of (a) has a forward primer sequence and the nucleotide sequence of the second set of barcoded particles has a reverse primer sequence downstream of the unique particle identifier sequence.

[0102] Embodiment 7. A probe system described in any one of embodiments 1 to 6, wherein the primer molecule of (a) is linked to a binding agent (e.g., an oligonucleotide probe, an antibody, an aptamer, etc.).

[0103] Embodiment 8. The probe system of embodiment 7, wherein the primer molecules further comprise target identifier sequences that indicate the binding agents to which they are linked.

[0104] Embodiment 9. A probe system according to any one of embodiments 1 to 6, wherein the primer molecules are linked to a flat substrate.

[0105] Embodiment 10. A probe system as described in any of embodiments 1 to 9, wherein the first and second sets of barcoded particles independently contain at least 10,000 particles each having a unique particle identifier sequence.

[0106] Embodiment 11. A probe system according to any one of embodiments 1 to 10, wherein the nucleotide sequence of the second set of barcoded particles lacks the primer binding sequence of (b)(i).

[0107] Embodiment 12. A method of adding a unique particle identifier sequence to a primer using a probe system according to any one of embodiments 1 to 11, comprising: i. hybridizing said population of primer molecules of (a) with said first set of barcoded particles of (b); ii. extending the hybridized primer molecule using the nucleotide sequence of the first set of barcoded particles as a template to produce a first primer extension product that contains i. the complement of a unique particle identifier sequence from a barcoded particle in the first set of barcoded particles and ii. the complement of the first template sequence; iii. removing the first set of barcoded particles; iv. hybridizing the second set of barcoded particles with the first primer extension products, wherein the complement of the first template sequence in the first primer extension products hybridizes to the first template sequence in the second set of barcoded particles; v. extending the first primer extension product using the nucleotide sequences of the second set of barcoded particles as templates; a unique particle identifier sequence from a barcoded particle in said first set of barcoded particles; the first template sequence, and a unique particle identifier sequence from a barcoded particle in said second set of barcoded particles. producing a second primer extension product containing A method comprising:

[0108] Embodiment 13. The method of embodiment 12, wherein the primer molecule has a forward primer sequence and the nucleotide sequence of the second set of barcoded particles has a reverse primer sequence downstream of the unique particle identifier sequence, and the method comprises amplifying the second primer extension product of v.

[0109] The method of embodiment 12 or 13, comprising sequencing the second primer extension product of embodiment 14.v.

[0110] Embodiment 15. The method of embodiment 14, wherein a pair of unique particle identifier sequences present in the second primer extension product is used to map the relative positions of the primers of i.

[0111] Embodiment 16 The method of any of embodiments 12 to 15, wherein the primer is attached to a flat substrate and the method results in an array of the second primer extension products on the substrate.

[0112] Embodiment 17. The method of any of embodiments 12 to 15, wherein the primer is attached to the cell sample via a binding agent, and the unique particle identifier in the second primer extension product indicates the relative location of the binding agent on the cell sample.

[0113] Embodiment 18. The method of any one of embodiments 12 to 15, wherein the primer is a cDNA prepared in situ in a cell sample, and the unique particle identifier in the second primer extension product indicates the relative location of the cDNA in the cell sample.

[0114] Embodiment 19. A method of mapping binding events in or on a cell sample, comprising: (a) i. a sample containing primer molecules bound to sites in or on a cell; ii. a first set of barcoded particles, each having a nucleotide sequence comprising: (i) a primer binding sequence complementary to a 3' end of the primer molecules of (a), (ii) a unique particle identifier sequence, and (iii) a first template sequence; and iii. a second set of barcoded particles, each having a nucleotide sequence comprising: (i) the first template sequence, and (ii) a unique particle identifier sequence. (b) specifically hybridizing the first set of barcoded particles of (a)(ii) to the sample, wherein at least a portion of the nucleotide sequences of the first set of barcoded particles hybridize to at least two primer molecules; and (c) extending the primers hybridized to the barcoded particles in step (b) using the nucleotide sequences to which the primers hybridize as templates to obtain first unique nucleotide sequences. (d) producing first primer extension products each comprising a particle identifier sequence; (e) removing the first set of barcoded particles from the sample; (f) specifically hybridizing the second set of barcoded particles of (a)(iii) to the first primer extension products of (c), wherein a nucleotide sequence of at least a portion of the second set of barcoded particles hybridizes to at least two molecules of the primer extension products; and (g) removing the barcoded particles of step (e). (g) extending the first primer extension product hybridized to the added particle using the nucleotide sequence to which the primer hybridized as a template to produce a second primer extension product comprising the two unique particle identifier sequences; (g) determining which of the unique particle identifier sequences or its complement is present in the second primer extension product; and (h) using the unique particle identifier sequences present in the second primer extension product to map the relative positions of the primers of (a)(i).

[0115] Embodiment 20. The method of embodiment 19, wherein the sample of (a) is prepared by binding a primer attached to a binding agent to a site in or on the cell.

[0116] Embodiment 21 The method of embodiment 20, wherein the binding agent is an oligonucleotide probe, an antibody or an aptamer.

[0117] Embodiment 22 The method of embodiment 20 or 21, wherein the primers further comprise target identifier sequences that indicate the binding agents to which they are linked.

[0118] Embodiment 23. The method of embodiment 19, wherein the sample of (a) is prepared by hybridizing a first primer to RNA in the cell, extending the primer to prepare cDNA, and adding a second primer to the 5' end of the cDNA.

[0119] Embodiment 24. The probe system of any preceding embodiment, wherein the nucleotide sequences of the second set of barcoded particles lack the primer binding sequences of the first set of barcoded particles.

[0120] Embodiment 25. A probe system comprising: (a) a population of primer molecules; (b) a set of barcoded particles, each having a nucleotide sequence comprising: (i) a primer binding sequence complementary to the 3' end of the primer molecule of (a), (ii) a unique particle identifier sequence, and (iii) a first template sequence; and (c) a ligation splint comprising a first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide comprises a first sequence and the first template sequence; and the second oligonucleotide comprises a second sequence complementary to the first sequence and the first template sequence.

[0121] The probe system of embodiment 25, wherein the barcoded particles of embodiment 26 (b) are rolling circle amplification (RCA) products.

[0122] Embodiment 27. The probe system of embodiment 25, wherein the barcoded particle of (b) is a barcoded nanoparticle, and the nucleotide sequence of (b) is tethered to the surface of the barcoded particle.

[0123] Embodiment 28. A probe system according to any one of embodiments 25 to 27, wherein the primer molecule (a) is a synthetic oligonucleotide having a length of 10 to 200 nt.

[0124] Embodiment 29. A probe system described in any one of embodiments 25 to 27, wherein the primer molecule of (a) is a cDNA molecule having a primer sequence at the 3' end.

[0125] Embodiment 30. A probe system described in any of embodiments 25 to 28, wherein the primer molecule of (a) is linked to a binding agent (e.g., an oligonucleotide probe, an antibody, an aptamer, etc.).

[0126] Embodiment 31. A probe system described in any of embodiments 25 to 30, wherein the set of barcoded particles may contain at least 10,000 particles each having a unique particle identifier sequence.

[0127] Embodiment 32. A method for adding a unique particle identifier sequence to a primer using a probe system according to any one of embodiments 25 to 31, comprising: i. hybridizing said set of barcoded particles of (b) with said population of primer molecules of (a); ii. extending the hybridized primer molecule using the nucleotide sequence as a template to produce a first primer extension product that contains i. the complement of the unique particle identifier sequence from the barcoded particle and ii. the complement of the first template sequence; iii. removing the barcoded particles; iv. hybridizing the first primer extension products with the ligation splint, wherein the complements of the first template sequence in two proximal first primer extension products hybridize to the first and second sequences of the ligation splint; v. ligating at least one of the first or second oligonucleotides of the hybridized ligation splint to the first primer extension product and extending the 3' end of the ligated first or second oligonucleotide in the splint using the first primer extension product in the ligation product as a template, thereby adding two unique particle identifier sequences to the primer; A method comprising:

[0128] The method of embodiment 32, comprising sequencing the second primer extension product of embodiment 33.v.

[0129] Embodiment 34. The method of embodiment 32 or 33, further comprising mapping the relative positions of the primers of i. using the pair of unique particle identifier sequences present in the second primer extension product.

[0130] The method of any of embodiments 32 to 34, wherein the primer molecule of embodiment 35.i. is attached to a cell sample via a binding agent, and the unique particle identifier in the second primer extension product indicates the relative position of the binding agent on the cell sample.

[0131] Embodiment 36. The method of any of embodiments 32 to 34, wherein the primer is a cDNA prepared in situ in a cell sample, and the unique particle identifier in the second primer extension product indicates the relative location of the cDNA in the cell sample. EXAMPLES

[0132] The following examples are put forth to provide those of ordinary skill in the art with further disclosure and description of how to make and use the invention, and are not intended to limit the scope of what the inventors regard as the invention, nor are they intended to represent that the following experiments are all or the only experiments performed.

[0133] method Probe circularization and rolling circle amplification: Probe oligo (1 or 2) was circularized with template oligo (3 or 4) in a 50 μl ligation reaction containing 100 nM probe oligo, 100 nM template oligo, 1 mM ATP, 200 U T4 DNA ligase, and reaction buffer containing 33 mM Tris-acetate, 10 mM Mg-acetate, 66 mM K-acetate, 1 mM DTT, and 0.1% Tween 20. The reaction was incubated at 37° C. for 30 minutes.

[0134] 10 U Exonuclease I and 100 U Exonuclease III were added to each reaction after ligation to digest the non-circularized probe. Exonuclease digestion was carried out at 37° C. for 30 min, followed by heat inactivation at 85° C. for 20 min.

[0135] RCA primer oligo 3 or 4 was added to each reaction solution after exonuclease digestion to a concentration of 100 nM, and hybridized by incubation at 37° C. for 20 minutes to circularize the probe.

[0136] Rolling circle amplification was performed in 75 μl reactions containing 2.5 nM circularized probe, 0.75 mM of each d(AUGC)TP, and 7.5 U phi29 DNA polymerase in the same reaction buffer used for probe circularization. The reactions were incubated at 37° C. for 20 min, followed by heat inactivation at 65° C. for 10 min.

[0137] Cell staining and fixation: Raji and Jurkat cells were aspirated from separate T75 flasks and spun down at 300×g for 5 min. Cells were counted and a cell suspension corresponding to 1M cells was collected. Cells were washed twice in FACS buffer (2% FBS, 2mM EDTA in 1x PBS). Cells were blocked with Fc blocking agent, spun down and the supernatant removed. A pool of 2 TotalSeq B (Biolegend inc) antibody-oligonucleotide conjugates (oligo 8-27) targeting various immune cell markers (CD3, CD4, etc.) at a concentration of 5μg / ml of each conjugate was added to the cells and incubated on ice for 30 min. Conjugate stained cells were washed twice with 300μl FACS buffer, after which the cells were spun down, the supernatant removed and 250μl of 1% PFA was added to fix the samples. After 10 min incubation at RT, fixation was quenched by addition of 12.5 μl of 2.5 M glycine, followed by a wash with 250 μl of 125 mM glycine in PBS. After a further wash in PBS, cells were resuspended in PBS and stored at +4° C. until use.

[0138] Primer extension assay: Conjugate stained and fixed Raji and Jurkat cells were mixed in a 1:1 ratio and aliquots corresponding to approximately 20,000 cells in total were placed into PCR tubes.

[0139] Hybridization of the RCA products to cell-bound antibody-conjugated oligos was performed in 300 mM NaCl, 15 mM MgCl 2The reaction was performed in a 40 μl reaction containing 2.5 nM RCA product (originating from Oligo 1) and 0.5 μM blocking oligo (5) in a buffer containing 20 mM Tris-HCl (pH 8). The reaction was incubated at 55° C. for 15 min.

[0140] The samples were centrifuged at 500×g for 2 minutes to pellet the cells, the supernatant was removed, and 100 μl of wash buffer (50 mM NaCl, 1 mM EDTA, 20 mM Tris-HC, pH 8) was added and the cells were resuspended. The samples were centrifuged again to pellet the cells and the supernatant was removed.

[0141] A 50 μl DNA polymerase extension reaction was performed to incorporate pixel barcode sequences by adding a master mix containing 1 μl Klentaq DNA polymerase, 0.2 mM dNTPs in a buffer containing 50 mM Na-acetate, 10 mM Mg-acetate, 20 mM Tris-acetate, 100 μg / ml BSA. The reaction was incubated for 15 min at 30°C. After a washing step was performed again under the same conditions as previously described, a USER digestion (uracil DNA glycosylase + endonuclease VII) reaction was performed to degrade the DNA pixels by adding 50 μl of a buffer containing 50 mM NaCl, 1 mM EDTA, 20 mM Tris-HC and 1 U of USER enzyme. The reaction was incubated for 30 min at 37°C.

[0142] After a washing step, 50 μl of USER inactivation master mix was added, consisting of 1 U UGI protein in 50 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl, pH 8. The reaction was incubated at 37°C for 15 min.

[0143] After a washing step, a second RCA product hybridization reaction was performed using the RCA product originating from oligo 2, but using otherwise the same conditions as previously described for the hybridization of RCA product 1. Similarly, the same protocol for extension and USER inactivation was performed after the second hybridization reaction. After the second USER digestion step, a washing step was performed, after which the cells were resuspended in 50 μl of washing buffer. The resulting cell suspension was quantified and diluted to a concentration of 20 cells / μl.

[0144] After the sequential extension assay, each conjugated oligonucleotide bound to a cell via antibody binding had two DNA pixel barcode sequences and a reverse PCR primer motif incorporated throughout the extension step of the assay.

[0145] PCR was performed for 15 cycles in a reaction containing 5 μl of diluted sample, 0.2 mM dNTPs, 0.4 μM each of forward and reverse primers containing Illumina adapter sequences (6, 7), and 1 μl of Phusion DNA polymerase in 1× Phusion HF reaction buffer. The PCR reaction consisted of denaturation at 98°C for 1 min, followed by 15 cycles of denaturation at 98°C for 10 s, annealing at 60°C for 30 s, and 72°C for 40 s, followed by a final extension at 72°C for 5 min.

[0146] The PCR products were finally purified using Ampure XP beads according to the manufacturer's instructions but using a bead-to-sample ratio of 1.2. The purified PCR products were quantified using a Qubit fluorometer and sequenced on an Illumina NextSeq 2000 sequencer. [Table 1-1] [Table 1-2]

[0147] result The combination of the two DNA pixel barcodes incorporated into each conjugated oligo represents the two DNA pixels that were in close proximity to the molecule during the successive extension assays, since each pixel occupies only a distinct region of space. This, together with the fact that multiple adjacent molecules therefore share the same pixel barcode, allows the spatial reconstruction of the relative position of each molecule using a graph theory approach by considering the DNA pixel barcodes as nodes in a graph, and the combination of the two pixel barcodes of each molecule representing the edges (links) in the graph.

[0148] A series of data filtering steps were performed on the 40 million reads generated. Briefly, the filtering steps consisted of removing reads that were shorter than expected length, did not contain the expected common sequence motif at a certain position, and were observed only once in the data. The average read depth was 7.99, i.e., there were an average of 7.99 copies of each unique molecule.

[0149] Of the reads remaining after filtering, a total of 3.4M unique molecules were identified based on the unique molecular identifier (UMI) sequence, which is part of the conjugation oligo sequence. A total of 361575 DNA pixel sequences from Set A and 451177 DNA pixel sequences from Set B were found. The average number of unique conjugated oligos associated with each pixel from Set A was 7.47 and for pixel Set B it was 9.31.

[0150] After filtering out small graph components, the generated graph, or physical map, consisted of a set of approximately 100 graph components (clusters), each containing at least 1000 nodes. Induced subgraphs were generated for each of the components, and counts of each surface marker type were summarized for each cluster. Correlations of marker counts within each cluster were compared using scatter plots (Figures 18A-18C). Linear correlations in marker counts were observed when the two B cell markers were plotted against each other (HLA-DR, CD19) or the two T cell markers (CD3, CD7). No such correlation was observed when the B cell marker (HLA-DR) was instead plotted against the T cell marker (CD3). Taken together, these results indicate that each separate graph cluster was generated that represents a single cell in this experiment containing a sample with a mixture of B and T cells, Raji and Jurkat.

[0151] Representative clusters were selected and visualized using the Kamada-Kawai force-generated graph layout algorithm (Figure 19). Each edge (link) of the graph in Figure 19 can be represented by a different color that identifies a different antibody type, with information being decoded from the barcode sequence of each oligonucleotide conjugated to the antibody, and each node of the graph represents a DNA pixel sequence.

Claims

1. (a) a population of nucleic acid molecules having an extendable 5' or 3' end; (b) a binding sequence complementary to the extendable end of the nucleic acid molecule of (i)(a); (ii) a unique particle identifier sequence, and (iii) a first set of barcoded particles, each having a nucleotide sequence that includes, in order, a first template sequence, wherein each particle can be distinguished from one another by their unique identifier sequence; and (c) (i) the first template sequence, and (ii) a unique particle identifier sequence a second set of barcoded particles each having a nucleotide sequence comprising: extension of the nucleic acid molecules of (a) by primer extension if the nucleic acid molecules have an extendable 3' end, or gap-filling ligation if the nucleic acid molecules have an extendable 5' end, using the first set of barcoded particles of (b) as templates produces a first extension product that contains a complement of a unique particle identifier sequence of a particle of (b)(ii) and a complement of the first template sequence of the first and second sets; extension of the first extension product by primer extension if the nucleic acid molecule has an extendable 3' end, or by gap-filling ligation if the nucleic acid molecule has an extendable 5' end, using the second set of barcoded particles as templates, produces a second extension product that contains the complement of the unique particle identifier sequence of the particle in (c)(ii) and the complement of the unique particle identifier sequence of the particle in (b)(ii). Probe system.

2. 2. The probe system of claim 1, wherein the barcoded particles of (b) and / or (c) are rolling circle amplification (RCA) products.

3. 2. The probe system of claim 1, wherein the barcoded particles of (b) and / or (c) are barcoded nanoparticles, and the nucleotide sequence is tethered to a surface of the barcoded nanoparticle.

4. the nucleic acid molecule of (a) is a synthetic oligonucleotide of 10-200 nt in length; and / or The probe system according to claim 1 , wherein the nucleic acid molecule of (a) is a cDNA molecule having an extendable 3′ end.

5. 5. The probe system of claim 1, wherein the nucleic acid molecule of (a) comprises a forward primer sequence and the nucleotide sequence of the second set of barcoded particles comprises a reverse primer sequence downstream of the unique particle identifier sequence.

6. The probe system of claim 1 , wherein the nucleic acid molecule of (a) is linked to a binding agent or a flat substrate.

7. The probe system of claim 6 , wherein the nucleic acid molecules of (a) further comprise target identifier sequences that indicate the binding agents to which they are linked.

8. 8. The probe system of claim 1, wherein the first and second sets of barcoded particles independently contain at least 10,000 particles each having a unique particle identifier sequence.

9. The probe system according to any one of claims 1 to 8, wherein the nucleic acid molecule is an oligonucleotide molecule or a cDNA having a 3' hydroxyl or a 5' phosphate.

10. 8. A method of adding a unique particle identifier sequence to a nucleic acid molecule using a probe system according to any one of claims 1 to 7, comprising the steps of: i. hybridizing said population of nucleic acid molecules of (a) with said first set of barcoded particles of (b); ii. extending the hybridized nucleic acid molecule by primer extension if the nucleic acid molecule has an extendable 3' end, or by gap-fill ligation if the nucleic acid molecule has an extendable 5' end, using the nucleotide sequence of the first set of barcoded particles as a template to produce a first extension product that contains i. the complement of a unique particle identifier sequence from a barcoded particle in the first set of barcoded particles, and ii. the complement of the first template sequence; iii. removing the first set of bar-coded particles; iv. hybridizing the second set of barcoded particles with the first extension products, wherein the complement of the first template sequence in the first extension products hybridizes to the first template sequence in the second set of barcoded particles; v. using the nucleotide sequences of the second set of barcoded particles as templates to extend the first extension products by primer extension if the nucleic acid molecules have extendable 3' ends, or by gap-filling ligation if the nucleic acid molecules have extendable 5' ends; the complement of a unique particle identifier sequence from a barcoded particle in said first set of barcoded particles; the complement of the first template sequence, and the complement of a unique particle identifier sequence from a barcoded particle in said second set of barcoded particles. and producing a second extension product comprising: A method comprising:

11. 11. The method of claim 10, wherein the nucleic acid molecule has a forward primer sequence and the nucleotide sequence of the second set of barcoded particles has a reverse primer sequence downstream of the unique particle identifier sequence, and the method comprises amplifying the second extension product of v. and / or sequencing the second extension product of v.

12. v. sequencing the second extension product of using pairs of unique particle identifier sequences present in the second extension product to map the relative positions of the nucleic acid molecules of i.; The method of claim 11 , comprising:

13. the nucleic acid molecule is attached to a planar substrate and the method results in an array of the second extension products on the substrate; or the nucleic acid molecule is attached to a cell sample via a binding agent and the complement of the unique particle identifier in the second extension product indicates the relative location of the binding agent on the cell sample; or A method according to any of claims 10 to 12, wherein the nucleic acid molecule is a cDNA prepared in situ in a cell sample, and the complement of the unique particle identifier in the second extension product indicates the relative position of the cDNA in the cell sample.

14. 1. A method for mapping binding events in or on a cell sample, comprising: (a) i. a sample containing a nucleic acid molecule bound to a site in or on a cell, the nucleic acid molecule having an extendable 5' or 3' end; ii. a binding sequence complementary to the extendable end of the nucleic acid molecule of (i)(a); (ii) a unique particle identifier sequence, and (iii) a first template sequence a first set of barcoded particles each having a nucleotide sequence including, in order, iii. (i) the first template sequence, and (ii) a unique particle identifier sequence a second set of barcoded particles each having a nucleotide sequence comprising: and obtaining (b) specifically hybridizing the first set of barcoded particles of (a)(ii) to the sample, wherein nucleotide sequences of at least some of the first set of barcoded particles hybridize to at least two of the nucleic acid molecules of (a); (c) using the first barcoded particle as a template, extending the nucleic acid molecule hybridized to the barcoded particle in step (b) by primer extension if the nucleic acid molecule has an extendable 3' end, or by gap-fill ligation if the nucleic acid molecule has an extendable 5' end to produce first extension products, each of which comprises the complement of a first unique particle identifier sequence; (d) removing the first set of barcoded particles from the sample; (e) specifically hybridizing the second set of barcoded particles of (a)(iii) to the first extension products of (c), wherein at least a nucleotide sequence of at least a portion of the second set of barcoded particles hybridizes to at least two molecules of the first extension products; (f) using the second barcoded particle as a template to extend the first extension product hybridized to the second barcoded particle in step (e) by primer extension if the nucleic acid molecule has an extendable 3' end, or by gap-fill ligation if the nucleic acid molecule has an extendable 5' end to produce a second extension product comprising the complement of the two unique particle identifier sequences; (g) determining which unique particle identifier sequences or their complements are present in the second extension product; (h) using the unique particle identifier sequence, or its complement, present in the second extension product to map the relative positions of the nucleic acids of (a)(i); A method comprising:

15. (a) the sample is prepared by binding an oligonucleotide linked to a binding agent to a site in or on the cell; Optionally, the binding agent is an oligonucleotide probe, an antibody or an aptamer.

16. 16. The method of claim 14 or 15, wherein the oligonucleotides further comprise target identifier sequences that indicate the binding agents to which they are linked.

17. 15. The method of claim 14, wherein the nucleic acid molecule of (a) is a cDNA molecule.

18. (a) a population of nucleic acid molecules having an extendable 5' or 3' end; (b) a binding sequence complementary to the 3' or 5' end of the nucleic acid molecule of (i)(a); (ii) a unique particle identifier sequence, and (iii) a first template sequence a set of barcoded particles, each having a nucleotide sequence comprising: (c) a ligation splint comprising a first oligonucleotide and a second oligonucleotide, the first oligonucleotide comprises a first sequence and, downstream of the first sequence, the first template sequence; and the second oligonucleotide comprises a second sequence complementary to the first sequence and the first template sequence downstream of the second sequence, and the first oligonucleotide and the second oligonucleotide hybridize to each other via their respective first and second sequences; The ligation splint; Including, the population of nucleic acid molecules contains an extendable 3' end, and the first template sequence of the ligation splint is downstream of the first and second sequences of the first and second oligonucleotides; the population of nucleic acid molecules contains an extendable 5' end, and the first template sequence of the ligation splint is upstream of the first and second sequences of the first and second oligonucleotides. Probe system.

19. 20. The probe system of claim 18, wherein the barcoded particles of (b) are rolling circle amplification (RCA) products.

20. 20. The probe system of claim 18, wherein the barcoded particle of (b) is a barcoded nanoparticle, and the nucleotide sequence of (b) is tethered to a surface of the barcoded particle.

21. The probe system according to any one of claims 18 to 20, wherein the nucleic acid molecule (a) is a synthetic oligonucleotide having a length of 10 to 200 nt.

22. 21. The probe system of claim 18, wherein the nucleic acid molecule of (a) is a cDNA molecule.

23. 22. A probe system according to any one of claims 18 to 21, wherein the nucleic acid molecule of (a) is linked to a binding agent.

24. 24. The probe system of claim 18, wherein the set of barcoded particles can contain at least 10,000 particles each having a unique particle identifier sequence.

25. 25. A method of adding a unique particle identifier sequence to a nucleic acid using a probe system according to any one of claims 18 to 24, comprising the steps of: i. hybridizing said set of barcoded particles of (b) to said population of nucleic acid molecules of (a); ii. extending the hybridized nucleic acid molecule by primer extension if the nucleic acid molecule has an extendable 3' end, or by gap-fill ligation if the nucleic acid molecule has an extendable 5' end, using the nucleotide sequence as a template to produce a first extension product that contains i. the complement of a unique particle identifier sequence from the barcoded particle and ii. the complement of the first template sequence; iii. Removing the barcoded particles; iv. hybridizing the first extension products with the ligation splint, wherein the complements of the first template sequence in two proximal first extension products hybridize to the first and second sequences of the ligation splint; v. ligating at least one of the first oligonucleotide or the second oligonucleotide of the hybridized ligation splint to the first extension product and extending the 3' or 5' end of the ligated first or second oligonucleotide in the splint by primer extension if the nucleic acid molecule has an extendable 3' end, or by gap-fill ligation if the nucleic acid molecule has an extendable 5' end, using the first extension product in the ligation product as a template to add two unique particle identifier sequences to the nucleic acid; A method comprising:

26. v. sequencing the second extension product.

27. 27. The method of claim 25 or 26, further comprising mapping the relative positions of the nucleic acid molecules of i. using the pairs of unique particle identifier sequences present in the second extension product.

28. 28. The method of any of claims 25-27, wherein the nucleic acid molecule of i. is attached to a cell sample via a binding agent, and the unique particle identifier in the second extension product indicates the relative location of the binding agent on the cell sample.

29. 28. The method of any one of claims 25 to 27, wherein the nucleic acid molecule is a cDNA prepared in situ in a cell sample, and the unique particle identifier in the second extension product indicates the relative location of the cDNA in the cell sample.

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