Spatial mapping by serial primer extension

Abstract

The present invention provides a probe system, which comprises: a population of nucleic acid molecules, the nucleic acid molecules having an extendable end, a first group of barcode particles, each barcode particle having a nucleotide sequence, the nucleotide sequence comprising: (i) a binding sequence, which is complementary to the extendable end of the nucleic acid molecule, (ii) a unique particle tag sequence, and (iii) a first template sequence, and a second group of barcode particles, each barcode particle having a nucleotide sequence, the nucleotide sequence comprising: (i) the first template sequence and (ii) the unique particle tag sequence. In use, the nucleic acid molecules are extended using the first group of barcode particles as templates to produce extension products, and the extension products contain a complementary sequence to the unique particle tag sequence of the particle and a complementary sequence to the first template sequence. A method of mapping binding events in or on a cell sample using the probe system is also provided.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 168,132, filed on March 30, 2021, the disclosure of which is incorporated herein by reference in its entirety.

[0003] background

[0004] Sample and molecular indexing are commonly used in many genomics workflows today. Sample indexing is a commonly used method that enables samples to be sequenced and analyzed in a multiplex manner. In the sample indexing method, all nucleic acids in a specific sample are labeled with identical sequence tags, and the labeled library is merged with other libraries labeled with different barcodes, and the merged library is sequenced in parallel in a single sequencing run. Then, during analysis, the sample-specific indexing allows the software to separate the multiple sequence data into sample-specific data sets. On the other hand, the target of molecular indexing is to label each molecule in the sample with a unique sequence before pcr amplification. In these methods, each nucleic acid in the starting sample is labeled with a unique molecular index, and the sequence analysis software filters out repeated read lengths (reads) (i.e., read lengths from copies of the same molecule), and uses the index to eliminate pcr / sequencing errors.

[0005] The present disclosure employs indexing to provide spatial information, which is believed to be a new use of molecular indexing.

[0006] Overview

[0007] Among other things, the present disclosure provides a probe system comprising: (a) a population of nucleic acid molecules having extendable ends; (b) a first group of barcode particles, each barcode particle having a nucleotide sequence, the nucleotide sequence comprising: (i) a binding sequence complementary to the extendable ends of the nucleic acid molecules of (a), (ii) a unique particle tag sequence (identifier sequence), and (iii) a first template sequence; (c) a second group of barcode particles, each barcode particle having a nucleotide sequence, the nucleotide sequence comprising: (i) the first template sequence, and (ii) the unique particle tag sequence. In the probe system, the nucleic acid molecules of (a) are extended using the first group of barcode particles of (b) as templates to generate extension products, the extension products containing the complementary sequence of the unique particle tag sequence of the particles of (a)(ii) and the complementary sequence of the first template sequence.

[0008] In the following description, the nucleic acid molecule with an extendable end is a primer, and the method is implemented using primer extension. Figure 12-14In the example shown in Figure 2, gap filling / ligation or ligation-based methods are used to implement the method. Therefore, the systems and methods of the present invention should not be limited to methods based on primer extension.

[0009] In some embodiments, the method includes: hybridizing a first group of barcode particles with a first population of primer molecules, extending the hybridized primer molecules using the nucleotide sequence of the first group of barcode particles as a template to generate a first primer extension product, the first primer extension product containing a complementary sequence of a unique particle tag sequence from a barcode particle in the first group of barcode particles and a complementary sequence of a first template sequence; removing the first group of barcode particles; hybridizing the first primer extension product with a second group of barcode particles, wherein the complementary sequence of the first template sequence in the first primer extension product hybridizes with the first template sequence in the second group of barcode particles; and extending the first primer extension product using the nucleotide sequence of the second group of barcode particles as a template to generate a second primer extension product, the second primer extension product containing: a unique particle tag sequence from a barcode particle in the first group of barcode particles, the first template sequence, and a unique particle tag sequence from a barcode particle in the second group of barcode particles.

[0010] The probe system can be used in a method for mapping binding events on a cell sample. In an embodiment, the method may include: (a) obtaining: i. a sample containing primer molecules that bind to sites in or on a cell; ii. a first group of barcode particles, each barcode particle having a nucleotide sequence, the nucleotide sequence comprising: (i) a primer binding sequence that is complementary to the 3' end of the primer molecule, (ii) a unique particle label sequence, and (iii) a first template sequence; iii. a second group of barcode particles, each barcode particle having a nucleotide sequence, the nucleotide sequence comprising: (i) a first template sequence, and (ii) a unique particle label sequence; (b) specifically hybridizing the first group of barcode particles with the sample, wherein the nucleotide sequence of at least some of the barcode particles in the first group of barcode particles hybridizes with at least two primer molecules; (c) using the nucleotide sequence to which the primers hybridize as a template to extend the barcode particles in step (b); (c) hybridizing a primer to a barcode particle to produce first primer extension products, each of which comprises a first unique particle tag sequence; (d) removing a first group of barcode particles from the sample; (e) specifically hybridizing a second group of barcode particles to the first primer extension products of (c), wherein the nucleotide sequence of at least some of the barcode particles in the second group of barcode particles hybridizes to at least two molecules in the primer extension products; (f) extending the first primer extension products hybridized to the barcode particles in step (e) using the nucleotide sequence to which the primer hybridizes as a template to produce second primer extension products, the second primer extension products comprising two unique particle tag sequences; (g) determining which unique particle tag sequence or its complement is in the second primer extension product; and (h) mapping the relative positions of the primers using the unique particle tag sequences in the second primer extension products.

[0011] An alternative embodiment relates to a probe system comprising: (a) a population of primer molecules; (b) a set of barcode particles, each barcode particle having a nucleotide sequence, the nucleotide sequence comprising: (i) a primer binding sequence that is complementary to the 3' end of the primer molecule of (a), (ii) a unique particle label 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 a first template sequence; and the second oligonucleotide comprises the first template sequence and a second sequence complementary to the first sequence.

[0012] The alternative probe system can be used in a method comprising: i. hybridizing a group of barcode particles of (b) with a group 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, the first primer extension product containing: i. a complementary sequence to a unique particle tag sequence from the barcode particle and ii. a complementary sequence to a first template sequence; iii. removing the barcode particle; iv. hybridizing the first primer extension product with a ligation splint, wherein the complementary sequence to the first template sequence in two proximal first primer extension products hybridizes with a first sequence and a second sequence of the ligation splint; and ligating at least one of the first oligonucleotide or the second oligonucleotide of the hybridized ligation splint to the first primer extension product, and using the first primer extension product in the ligation product as a template to extend the 3' end of the ligated first oligonucleotide or the second oligonucleotide in the splint, thereby adding two unique particle tag sequences to the primer.

[0013] BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Those skilled in the art will appreciate that the drawings described below are for illustration purposes only. These drawings are not intended to limit the scope of the present teachings in any way.

[0015] Figure 1 A probe system of the present disclosure is schematically illustrated.

[0016] Figure 2 A method by which the probe system may be used is schematically illustrated.

[0017] Figure 3 Schematic illustration of a probe system with primer binding sites allowing amplification of extension products.

[0018] Figure 4 An embodiment of the method is schematically illustrated wherein the primer is tethered.

[0019] Figure 5 A schematic illustration of how this probe system can be used to produce an array.

[0020] Figure 6 It is also shown how the probe system can be used to produce arrays.

[0021] Figure 7 Shows how to use Figure 5 and 6 Array analysis samples indicated.

[0022] Figure 8 Schematic illustration of how the binding site can be spatially reconfigured.

[0023] Figures 9A-9CFurther details of embodiments of the method of the invention are schematically illustrated.

[0024] Fig.10 Alternative probe systems and methods of using the same are schematically illustrated.

[0025] Fig.11 A method is schematically illustrated in which a Fig.10 Alternative probe system shown.

[0026] Fig.12 The gap filling / connection method of the present invention is schematically illustrated.

[0027] Fig.13 A ligation-based format of the method of the invention is schematically illustrated, wherein nucleic acid molecules are attached to "pre-made" barcodes.

[0028] Fig.14 Schematically illustrated is a gap-filling / ligation version of the method of the invention, which can be used to add barcodes to cDNA.

[0029] Fig.15 Schematic illustration of how this approach can be implemented using a hairpin in each repeat using the rolling circle replication product.

[0030] Fig.16 then Fig.15 An example of an assay that can be designed such that the primer extension product ends at a defined nucleotide, allowing it to be used as a primer in the next step of the method is shown.

[0031] Fig.17 Schematically illustrated is an alternative example of how the assay can be designed so that the primer extension product ends at a defined nucleotide, allowing it to be used as a primer in the next step of the method.

[0032] Figures 18A-18C Correlation plots showing marker counts within each cluster are shown. The poor correlation between CD3 and HLA-DR suggests that each cluster represents a single cell.

[0033] Fig.19 Visualization of graph components (clusters) using a force-generated layout algorithm. Different antibody types can be distinguished.

[0034] definition

[0035] Before describing exemplary embodiments in greater detail, the following definitions are set forth to illustrate and define the meaning and scope of the terms used in the description.

[0036] Numerical ranges are inclusive of the numbers defining the range.Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; and amino acid sequences are written left to right in amino to carboxyl orientation, respectively.

[0037] 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 the 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 COLLINSDICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide one of the general meanings of many of the terms used herein. However, for clarity and ease of reference, certain terms are defined below.

[0038] It must 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 "a primer" refers to one or more primers, i.e., a single primer and multiple primers. It is also noted that any optional elements may be excluded when drafting the claims. Therefore, this statement is intended to serve as antecedent basis for the use of exclusive terminology such as "solely," "only," etc., or the use of a "negative" limitation when reciting claim elements.

[0039] The term "nucleotide" is intended to include those moieties that contain not only the known purine and pyrimidine bases, but also other modified heterocyclic bases. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, alkylated ribose or other heterocycles. In addition, the term "nucleotide" includes those moieties that contain haptens or fluorescent labels, and may contain not only conventional ribose and deoxyribose, but also other sugars. Modified nucleosides or nucleotides also include modifications to the sugar moiety (e.g., wherein one or more of the hydroxyl groups are substituted with halogen atoms or aliphatic groups), functionalized as ethers, amines, etc.

[0040] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to describe polymers 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 consisting of nucleotides, e.g., deoxyribonucleotides or ribonucleotides, and can be produced enzymatically or synthetically (e.g., PNAs described in U.S. Pat. No. 5,948,902 and references cited therein), which can hybridize with naturally occurring nucleic acids in a sequence-specific manner similar to two naturally occurring nucleic acids, e.g., can 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 backbones, respectively, while the backbone of PNA consists 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 acid (LNA), commonly referred to as inaccessible RNA, is a modified RNA nucleotide. The ribose moiety of the LNA nucleotide is modified by an additional bond bridge connecting the 2' oxygen and the 4' carbon. The bond bridge "locks" the ribose in the 3'-endo (north) conformation that is usually present in the A-type duplex. LNA nucleotides can be mixed with DNA or RNA residues in oligonucleotides when necessary. The term "unstructured nucleic acid" or "UNA" is a nucleic acid containing non-natural nucleotides that bind to each other with reduced stability. For example, an unstructured nucleic acid may contain G' residues and C' residues, wherein these residues correspond to non-natural forms of G and C, i.e., analogs, whose bases 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 US20050233340, which is incorporated herein by reference to disclose UNA.

[0041] The term "oligonucleotide" as used herein means a single-stranded polymer of a nucleotide of about 2 to 200 nucleotides, and the length can be up to 500 nucleotides. Oligonucleotides can be synthetic or enzymatically prepared, and in some embodiments, have a length of 30 to 150 nucleotides. Oligonucleotides can contain ribonucleotide monomers (i.e., can be oligoribonucleotides) or deoxyribonucleotide monomers. For example, the length of an oligonucleotide can be 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 80 to 100, 100 to 150 or 150 to 200 nucleotides.

[0042] The term "primer" as used herein refers to such an oligonucleotide, when placed under conditions of inducing the synthesis of a primer extension product complementary to a nucleic acid chain, i.e., in the presence of nucleotides and an inducing agent (such as DNA polymerase) and at a suitable temperature and pH, an oligonucleotide that can serve as a starting point for synthesis. The primer can be single-stranded and must be long enough to initiate the synthesis of a desired extension product in the presence of an inducing agent. The exact length of the primer will depend on many factors, including the purpose of temperature, primer source and method. For example, for diagnostic applications, depending on the complexity of the target sequence or fragment, an oligonucleotide primer generally contains 15-25 or more nucleotides, but it may also contain fewer nucleotides. The primer herein is selected to be substantially complementary to the different chains of a specific target DNA sequence. This means that the primer must have enough complementarity to hybridize with each chain. Therefore, the primer sequence does not need to reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment can be attached to the 5' end of the primer, and the remainder of the primer sequence is complementary to the chain. Alternatively, non-complementary bases or longer sequences may be interspersed into the primer, provided that the primer sequence has sufficient complementarity to that of the strand to hybridize therewith, thereby forming a template for the synthesis of extension products.

[0043] The term "primer extension product" refers to the product of primer extension 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 primer extension. The term "second primer extension product" refers to the product obtained by extending the first primer extension product. If the second primer extension product is sequenced, the entire molecule (or a large portion of the molecule) can 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.

[0044] The term "hybridization" or "hybridizes" refers to a process in which a nucleic acid chain anneals with a second complementary nucleic acid chain under normal hybridization conditions and forms a stable duplex (homoduplex or heteroduplex), 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 accomplished by annealing two complementary nucleic acid chains in a hybridization reaction. By adjusting the hybridization conditions (commonly referred to as hybridization stringency) under which a hybridization reaction occurs, the hybridization reaction can be made highly specific so that the hybridization between the two nucleic acid chains does not form a stable duplex, for example, the duplex of the double-stranded region is maintained under normal stringent conditions, unless the two nucleic acid chains contain a certain number of substantially or completely complementary specific sequences of nucleotides. For any given hybridization reaction, "normal hybridization or normal stringent conditions" are easily determined. See, for example, 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 "hybridizing" or "hybridization" refers to any process by which a nucleic acid strand binds to a complementary strand through base pairing.

[0045] A nucleic acid is considered to "selectively hybridize" to a reference nucleic acid sequence if the two sequences specifically hybridize to each other under moderate to highly stringent hybridization and washing conditions. Moderate and highly stringent 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 highly stringent conditions includes hybridization at about 42°C in 50% formamide, 5X SSC, 5X Denhardt's solution, 0.5% SDS, and 100 ug / ml denatured carrier DNA, followed by washing twice at room temperature in 2X SSC and 0.5% SDS, and washing twice more at 42°C in 0.1X SSC and 0.5% SDS.

[0046] As used herein, the term "sequencing" refers to a method by which the identity of at least 10 consecutive nucleotides (e.g., identity of at least 20, at least 50, at least 100, or at least 200 or more consecutive nucleotides) of a polynucleotide is obtained.

[0047] The term "next generation sequencing" refers to the so-called parallel synthesis sequencing or ligation sequencing platforms currently used by companies such as Illumina, Life Technologies, BGI Genomics (Complete Genomics Technology) and Roche. Next generation sequencing methods may also include nanopore sequencing methods or electronic detection-based methods, such as the Ion Torrent technology commercialized by Life Technologies.

[0048] The terms "duplex" or "duplexed" as used herein describe two complementary polynucleotides that are base paired, ie, hybridized together.

[0049] The terms "determine," "measure," "evaluate," "assess," "measure," and "analyze" are used interchangeably herein to refer to forms of measurement, including determining the presence or absence of an element. These terms include quantitative and / or qualitative determination. Assessments can be relative or absolute.

[0050] As used herein, the term "ligation" refers to the enzymatic joining of the terminal nucleotide at the 5' end of a first DNA molecule to the terminal nucleotide at the 3' end of a second DNA molecule.

[0051] The terms "plurality," "group," and "population" are used interchangeably to refer to something that contains at least 2 members. In some cases, a plurality can have at least 10, at least 100, at least 1000, at least 10,000, or at least 100,000 members.

[0052] A "primer binding site" refers to a site in a target polynucleotide or fragment to which an oligonucleotide hybridizes. If an oligonucleotide "provides" a binding site for a primer, then the primer may hybridize to the oligonucleotide or its complement.

[0053] As used herein, the term "strand" refers to a nucleic acid composed of nucleotides covalently linked together by covalent bonds, such as phosphodiester bonds.

[0054] The term "extension" as used herein refers to the extension of a nucleic acid by adding nucleotides by ligation or using a polymerase. If a nucleic acid annealed to a polynucleotide is extended, the polynucleotide serves as a template for the extension reaction. In these embodiments, the nucleic acid can be extended by a template-dependent polymerase or by ligation to an oligonucleotide complementary to the polynucleotide, wherein the polynucleotide serves as a splint.

[0055] The term "extension" includes extension of either the 3' end or the 5' end. Primer extension, ligation and gap-filling ligation reactions are types of extension.

[0056] The term "extendable 5' or 3' end" refers to the 5' phosphate group and the 3' hydroxyl group, respectively, both of which can be extended by ligation. The 3' hydroxyl group can also be extended by a polymerase.

[0057] The term "as a template" as used herein refers to: (a) a primer extension reaction in one strand acts as a template for the addition of nucleotides by a polymerase, (b) splint ligation, in which one strand acts as a template (or "splint") to connect two nucleic acid molecules together. In a ligation reaction, both molecules hybridize to the template and become connected. The connection can be at the 5' end of the nucleic acid molecule or the 3' end of the nucleic acid molecule. At the 3' end or the 5' end; (c) a gap filling / ligation reaction. In a gap filling / ligation reaction, two nucleic acids hybridize to a template with a gap in the middle. One nucleic acid molecule is extended toward another nucleic acid molecule by primer extension, and then the 3' end of the product is connected to another nucleic acid. The term "rolling circle amplification" or "RCA" for short as used herein refers to isothermal amplification using a strand displacement polymerase to produce linear concatenated copies of a circular nucleic acid template. RCA is well known in the art of molecular biology and is 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.

[0058] The term "rolling circle amplification product" as used herein refers to the concatenated product of a rolling circle amplification reaction.

[0059] As used herein, the term "surface" refers to any solid material (e.g., glass, metal, ceramic, organic polymer surface or gel) that may contain any combination of cells or biomolecules derived from cells, such as proteins, nucleic acids, lipids, oligosaccharides / polysaccharides, biomolecule complexes, organelles, cell debris or excretions (exosomes, microvesicles), etc. Tissue blots, protein blots, and glass slides are examples of solid materials with surfaces. A suspension of cells, such as mammalian cells, is another example of a surface.

[0060] As used herein, the term "splint" refers to an oligonucleotide that hybridizes to the ends of two other oligonucleotides and brings those ends together to create a ligatable junction or gap that can be filled by a gap-filling / ligation reaction.

[0061] The term "barcoded particle" as used herein is intended to refer to both barcoded RCA products and barcoded nanoparticles, wherein each particle in a population of barcoded particles is individually barcoded with a unique particle tag sequence, i.e., a sequence uniquely possessed by each particle such that the particles can be distinguished from each other by their unique tag sequence.

[0062] Additional definitions of terms may appear throughout the specification.

[0063] Description of Example Implementations

[0064] Before describing various embodiments, it should be understood that the teaching of the present disclosure is not limited to the specific embodiments described, and therefore can of course vary. It should also be understood that the terminology used herein is only used for the purpose of describing specific embodiments, and is not intended to be limiting, because the scope of the present teaching will be limited only by the appended claims.

[0065] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described in any way. Although the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to these embodiments. On the contrary, as will be appreciated by those skilled in the art, the present teachings include various alternatives, modifications, and equivalents.

[0066] 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 the present 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 now described.

[0067] 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 invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates and may need to be independently confirmed.

[0068] As will be apparent to those skilled in the art upon reading this disclosure, each individual embodiment described and illustrated herein has discrete components and features that can be readily separated or combined with the features of any other several embodiments without departing from the scope or spirit of the teachings of the present invention. Any method of recording can be performed in the order of recorded events or in any other logically possible order.

[0069] All patents and publications mentioned herein, including all sequences disclosed within such patents and publications, are expressly incorporated herein by reference.

[0070] Before describing certain aspects of the invention in more detail, it is important to note that the accompanying drawings illustrate embodiments of the probe systems and methods of the invention using RCA products. As described below, the principles illustrated in the drawings can be readily applied to barcoded nanoparticles and, therefore, the invention should not be limited to what is depicted in the drawings. In addition, only one RCA product repeat is shown in the drawings. 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 repeating molecules (which are either concatenated in the RCA product or tethered to the surface of the nanoparticle).

[0071] Furthermore, it is recognized that the probe systems and methods of the present invention need not be implemented as a primer extension assay (e.g. Figure 2 As shown in Figure 12-14 As shown, the method can be implemented using gap filling / ligation or ligation assays, both of which can involve extending the 5' end of the nucleic acid molecule using the barcoded particle as a template. Therefore, while the following description focuses on embodiments employing primer extension, it should be appreciated that the primer extension-based embodiment described below is only one example of how the method can be implemented.

[0072] exist Figure 1 Some principles of the probe system of the present invention are illustrated in FIG. Figure 1, the probe system may include: a primer molecule group 2 (as shown, it has a 3' end with sequence C-BS1'). As will be described in more detail below, the primer molecule can be a synthetic oligonucleotide or a cDNA molecule, for example, having a primer sequence at the 3' end. In some embodiments, the primer molecule can be connected to a binding agent (e.g., an oligonucleotide probe, an antibody, an aptamer, etc.), and in other embodiments, the primer molecule can be connected to a planar substrate as a lawn, wherein the 3' end of the oligonucleotide is located at the distal end of the substrate and can be extended by a polymerase. As shown, the probe system also includes a first group of barcode particles 4 and a second group of barcode particles 12. As shown, the particles in the first group of barcode particles 4 each have a nucleotide sequence, and the nucleotide sequence includes: a primer binding sequence 6 (C-BS1) complementary to the 3' end of the primer molecule 2, as well as a unique particle marker sequence 8 (UMI1) and a first template sequence 10 (C-BS2). Each particle in the second group of barcoded particles 12 has a nucleotide sequence that includes: a first template sequence 10 (i.e., the same template sequence C-BS2 as in the first group of particles) and a unique particle marker sequence 14 (UMI2). In the illustrated embodiment, the nucleotide sequence of the second group of barcoded particles does not contain a primer binding sequence 6 (CBS1). However, in other embodiments, the nucleotide sequence of the second group of barcoded particles may contain a primer binding sequence 6 (CBS1). In the probe system (e.g. Figure 2 ), primer molecules 2 are extended using the first set of barcoded particles 4 as templates, producing first primer extension products 16, which contain a complementary sequence 18 of a unique particle tag sequence of the particle sequence of the first set of barcoded particles (i.e., the complementary sequence of UMI1, or UMI1') and a complementary sequence 20 of the first template sequence (C-BS2', which is the complementary sequence of C-BS2). As will be described in more detail below, the 3' end of the primer extension product 16 is capable of hybridizing with the first template sequence 10 of the second set of particles. As shown, the 3' end of the primer extension product 16 is extended using particles from the second set of barcoded particles as templates, resulting in a second primer extension product 22, which contains a complementary sequence 18 of a unique particle tag sequence from the first set of particles, a complementary sequence 20 of the first template sequence, and a complementary sequence 24 of a unique particle tag sequence from the second set of particles. In some embodiments, there may be an internal hairpin immediately downstream of the first template sequence.

[0073] In some embodiments, the first group of barcode particles and the second group of barcode particles can be rolling circle amplification (RCA) products or barcode nanoparticles. For example, in some embodiments, the first group of barcode particles and the second group of barcode particles can be RCA products, the first group of barcode particles and the second group of barcode particles can be barcode nanoparticles, or the first group of barcode particles can be RCA products or barcode nanoparticles, and the second group of barcode particles can be another type.

[0074] In embodiments where the barcode particles are RCA products, the RCA products each contain a unique sequence in the form of a repeating sequence. In other words, if there are 1,000 RCA products, each product will have a unique sequence (referred to herein as a unique molecular marker "UMI" or a unique marker "UID"). The UID of one particle is different from the UID of other particles. The RCA products can be prepared by, for example, synthesizing an initial oligonucleotide with a degenerate sequence, circularizing the initial oligonucleotide using a splint, and amplifying the circular oligonucleotide by RCA. In some embodiments, depending on the number of unique RCA products desired, the initial oligonucleotide may contain a degenerate (e.g., random) sequence of 6-10 nucleotides or even more random nucleotides. Amplification of circular oligonucleotides with degenerate sequences should produce a population of RCA products, each of which has a unique marker (i.e., a sequence different from other RCA products in the population). For example, Wu et al (Nat. Comm. 2019 10: 3854) and US20160281134 describe methods for generating RCA products with unique labels, and are easily adapted for use herein. In some embodiments, different oligonucleotides for preparing a first group of RCA products and a second group of RCA products are made separately and then mixed together. In other embodiments, different oligonucleotides can be made in parallel on a planar support in the form of an array and then cut out from the array. For example, Cleary et al. (Nature Methods 2004 1: 241-248) and LeProust et al. (Nucleic Acids Research 2010 38: 2522-2540) describe examples of such methods. In some embodiments, one or both sets of RCA products may contain uracil of thymine, thereby allowing enzymatic degradation of the RCA products by USER (see, e.g., Bitinaite et al Nucleic Acids Res. 2007 35: 1992-2002), which contains UDG (uracil DNA glycosylase) and AP lyase, which cleaves the phosphodiester backbone at apurinic sites.

[0075] In embodiments where the barcode particles are barcode nanoparticles, the barcode nanoparticles are small beads or metal particles, etc., that are coated with oligonucleotides, wherein the surface-tethered oligonucleotide on each particle has a unique sequence that is different from the sequence in the oligonucleotides tethered to other particles in the population. In other words, if there are 1,000 barcode particles, the oligonucleotide tethered to each particle will have a unique sequence (referred to herein as a unique molecular identifier "UMI" or a unique identifier "UID". The UID of one particle is different from the UID of other particles. The particles can be of any suitable size, material, and shape. In many embodiments, the particles have a size of 10nm-200nm. Gold particles can be used (e.g., can be easily made into any diameter in the range of 1.8nm to 1500nm), but the particles can also be made of silver, silica, titanium dioxide, carbon, polymers (such as polystyrene, polyacrylates, etc.), agarose, etc. Magnetic particles of iron and various alloys can also be used (Creative Diagnostics, Shirley, NY, USA). The particles do not need to be magnetic, but in some cases magnetic nanospheres can be used (Creative Diagnostics, Shirley, NY, USA). There are several surface chemistries used to functionalize metal surfaces so that they can be linked to nucleic acids. For example, the particles can be modified to contain reactive groups including, but not limited to, N-hydroxysuccinimide esters, sulfo-N-hydroxysuccinimide esters, halo-substituted phenol esters, pentafluorophenol esters, nitro-substituted phenol esters, anhydrides, isocyanates, isothiocyanates, imidoesters, maleimides, iodoacetyl groups, hydrazides, aldehydes, or epoxides. Other suitable groups are known in the art and can be found in, for example, Hermanson, "Bioconjugate Techniques" Academic Press, 2nd Ed., 2008. The most commonly used capture agent reactive groups are NHS (which is amine reactive) and maleimide (which is sulfhydryl reactive), but many other groups can be used. These particles can also be coated with streptavidin, which can bind to the biotinylated nucleic acids. In some embodiments, the barcode particles can be made by emulsion PCR, which has been successfully used in other applications and is described in, for example, Kanagal-Shamanna et al (Methods Mol Biol 2016 1392: 33-42) and Shao et al (Plos One 2011 0024910). In some embodiments, as discussed above, the nucleic acids of the barcode particles can contain uracil, allowing them to be cleaved using USER.

[0076] In any embodiment, primer molecule 2 may contain a 5' tail that does not hybridize with any nucleic acid of the barcode particle. The 5' tail may contain a primer binding site (e.g., for a forward primer) and a target marker sequence that can be used later in the protocol. As described above, the primer molecule may be a synthetic artificial oligonucleotide having a length of 10-200 nucleotides. In some of these embodiments, the primer molecule may be connected 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 cDNA molecule having a primer sequence at the 3' end, i.e., an additional sequence that can be used as a primer. In these embodiments, cDNA can be made in the following manner, for example, by hybridizing a reverse transcription primer (e.g., a primer having a 3' end made of oligo (dT), a random sequence, or a gene-specific sequence, which optionally has a 5' end that does not hybridize with RNA, and may contain a sequence that provides a binding site for a PCR primer) with RNA, for example, RNA in situ hybridization in a sample. Reverse transcription primers can be extended in situ (in a reaction containing NTP, reverse transcriptase and any other necessary reagents) to produce a cDNA product to produce the first strand cDNA. Primer sequences can be added to the cDNA by template switching (see, for example, Zhu et al BioTechniques 2001 30:892-7), connecting 3' adapters or by tailing, for example, by terminal transferase. As mentioned above, in cDNA embodiments, cDNA can be prepared in situ, and therefore, primers can be in or on tissue sections. In other embodiments, primer molecules can be connected to a planar substrate, for example, by their 5' ends, so that they form a "lawn".

[0077] In the embodiment that primer molecule can be connected with binding agent, for example, binding agent can be oligonucleotide probe, antibody or aptamer.In these embodiments, primer molecule can additionally comprise the sequence of the target that marks binding agent.For example, if primer is connected with antibody, primer may also contain target marker sequence, and this target marker sequence marks antibody name or the target (for example, epitope) that antibody binds with it at the 5' end of primer (downstream of any primer sequence).Therefore, in some embodiments, primer can be part of capture agent-primer conjugate, wherein capture agent such as antibody or aptamer and primer are connected to single-stranded primer in non-covalent (for example, by streptavidin / biotin interaction) or covalent (for example, by click reaction etc.) mode, so that capture agent can still bind with its binding site.Oligonucleotide and capture agent can be connected by many different methods, including those using maleimide or halogen-containing group with cysteine ​​reactivity.Capture agent and oligonucleotide can be connected at 5' end proximal end or 5' end of oligonucleotide, at 3' end proximal end or 3' end of oligonucleotide or any position between the two. In some embodiments, the oligonucleotide can be connected to the capture agent by a joint separating the oligonucleotide from the capture agent. The oligonucleotide can be connected 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 primer sequence conjugated to the binding agent uniquely marks the epitope or sequence to which the binding agent is bound. For example, if 10 different antibodies are used to perform the method, each antibody is tethered to different primers, which contain the sequence of the epitope to which the labeled antibody is bound. This feature allows the method to become multiplexed, and in certain embodiments, at least 5, at least 10, at least 20 or at least 50 different antibodies bound to different markers in or on the surface of the cell can be used for the method. Each antibody is conjugated to different target marker sequences, and the antibody marker sequence allows the binding events of specific antibodies to be mapped. Such tagged antibodies are described, for example, in Wu et al (Nat. Comm. 2019 10:3854) and US20160281134, among others.

[0078] In embodiments where the primer molecules can be attached to a planar substrate, the substrate can be made by, for example, synthetically preparing the primer molecules, for example, using phosphoramidite chemistry and tethering the primer molecules via their well-known 5' ends to a planar support, for example, a glass slide, etc.

[0079] In any embodiment, the primer molecule 2 may have a 5' tail with a forward primer sequence 30, and the nucleotide sequence of the second set of barcode particles 12 may have a reverse primer sequence 32 downstream of the unique particle tag sequence 14, such as Figure 3 In this embodiment (and as Figure 4 ), the second primer extension product has forward and reverse primer sequences, thereby allowing the product to be amplified by PCR.

[0080] The lengths of the components of the nucleic acid molecules described herein can vary. In some embodiments, the primer portion that hybridizes to the 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.

[0081] In any embodiment, the first group of barcoded particles and the second group of barcoded particles can each 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 million, at least 10 million, at least 100 million, at least 1 billion, or at least 10 billion members), each of which can be uniquely labeled by its particle tag sequence. In some embodiments, the nucleic acid sequences of the barcoded particles in the first group of barcoded particles and the second group of barcoded particles can be identical to each other except for their particle tag sequences.

[0082] This article also provides a method for adding a unique particle label sequence to a primer using the probe system of the present invention. The principle of this method is as follows Figure 2As shown. In these embodiments, the method may include hybridizing a first set of barcode particles 4 with a population of primer molecules 2, extending the hybridized primer molecules using the nucleotide sequence of the first set of barcode particles as a template to produce a first extension product 16, which contains: i. a complementary sequence 18 of a unique particle tag sequence from the first set of barcode particles and ii. a complementary sequence 20 of a first template sequence. Next, the first set of barcode particles is removed, for example by denaturation and washing, or, for example, by incorporating uracil residues in place of thymidine in the first barcode particles and enzymatically treating the product, for example, using USER (NEB) containing uracil-n-glycosylase and AP lyase, which is capable of specifically cleaving phosphodiester bonds at apurinic sites, thereby degrading the first barcode particles and releasing the extended primer. In these embodiments, primer 2 (and therefore primer extension product 16) can be tethered to a support or sample (e.g., via a capture agent), and, therefore, the first set of barcode particles can be subjected to stringent washing conditions without removing the primer or primer extension product from the sample or support. In addition, the first primer extension product can be terminated at the correct position (i.e., at the end of the first template sequence) by designing a site for a restriction enzyme at that position and then digesting the primer extension product with the restriction enzyme after it is produced (which will be double-stranded), 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, which will cause the polymerase to stall. In these latter embodiments, the polymerase (which should be a non-strand-displacing polymerase) should terminate synthesis when it encounters the blocking oligonucleotide or hairpin. Alternative methods for terminating nucleic acid synthesis at a specified site are as follows Figure 15-17 shown. Fig.15 RCA products containing hairpin loops are illustrated. These hairpin loops can be cleaved by restriction enzymes to release the 5' and 3' ends as blunt ends or overhangs. Fig.16 How primer extension terminates at a specified site in the product is described. In this embodiment, the hairpin is digested to produce a blunt end or an overhang, but the RCA product is kept together as a complex. The RCA product is then hybridized with a conjugated oligonucleotide, and a strand displacement DNA polymerase (e.g., the Klenow fragment of E. coli DNA polymerase I) replicates the barcode from the RCA product, displaces the hairpin, and terminates naturally as the template ends. The primer extension product can then be used as a primer for the next set of pixels. Restriction enzyme cleavage of the hairpin can be performed before or after the RCA product is hybridized with the oligonucleotide. Fig.17 It is shown how it is possible to generate defined ends by digesting extension products after they have been made while they are still double-stranded.

[0083] Next, the method may include hybridizing the first primer extension product 16 to the second group of barcode particles 12, wherein the complementary sequence 20 of the first template sequence in the first primer extension product hybridizes to the first template sequence in the second group of barcode particles. Next, the method includes extending the first primer extension product using the nucleotide sequence of the second group of barcode particles as a template to produce a second primer extension product containing: a unique particle tag sequence 18 from a barcode particle in the first group of barcode particles, the first template sequence 20, and a unique particle tag sequence 20 from a barcode particle in the second group of barcode particles.

[0084] like Figure 3 As shown, primer molecule 2 can have a forward primer sequence, and the nucleotide sequence of the second set of barcode particles 12 can have a reverse primer sequence downstream of the unique particle tag 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 can include amplifying the second primer extension product 22 by PCR using primers that target the forward primer sequence and the reverse primer sequence. Figure 4 An embodiment of a method of tethering a primer to a support or a sample is shown.

[0085] 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 unique labels for both barcode particles, thereby marking which barcode particles hybridize with the primers in different hybridization steps. In some embodiments, and as will be described in more detail below, the method may include using unique particle label sequence pairs located in the second primer extension product to map the relative positions of the primers.

[0086] As described above, in some embodiments, the primer can be attached to the cell sample via a binding agent. In these embodiments, the unique particle label in the second primer extension product indicates the relative position of the binding agent on the cell sample. Figure 4 As shown. Figure 4 It can be seen that in these embodiments, the primers may include a target identification sequence. Figure 4In the illustrated embodiment, in steps A and B, the primer molecules contain target markers and are immobilized on the sample, such as by a binding agent. In the method, a first set of barcoded particles are hybridized to the sample, and the complementary sequences of the UMI and C-BS2 sequence in the particles to which the primers are hybridized are copied to the ends of the primer molecules using the hybridized particles as templates. In steps C and D, the first set of barcoded particles are removed and the second set of particles are hybridized. After hybridization, the primer extension products are extended to add the UMI in the second set of particles to the ends of the primer extension products. The barcode pairs copied to the ends of the primers can be analyzed to determine the relative positions of the different primers in the sample.

[0087] As described above, in alternative embodiments, the method can be implemented using gap filling / ligation and / or ligation-based methods, wherein, in these embodiments, the 5' end of the nucleic acid can be extended by gap filling / ligation or ligation reactions using the barcode particle of (b) as a template. Figure 12-14 These embodiments are schematically illustrated in FIG. Fig.12 Instead of replicating the barcode in the barcode particle by polymerization from the free 3' end of a primer coupled to a binder (as described above), a gap-filling / ligation reaction can be used to replicate the barcode onto the 5' end of an oligonucleotide conjugated to an antibody, where the oligonucleotide has a 5' phosphate. These embodiments can be accomplished using, for example, a combination of T4 DNA polymerase and T4 DNA ligase. Fig.13 Shown is how the 5' or 3' end of the oligonucleotide conjugated to the binder is attached to a "pre-made" molecule containing the particle barcode. In these embodiments, the barcode is first replicated by a gap-filling / ligation reaction. During the assay itself, the antibody's oligonucleotide is attached to the 5' or 3' end of the pre-made copy of the barcode. In this figure, the 5' end of the oligonucleotide is attached to the copy of the barcode, but this can be done the other way around, i.e. the oligonucleotide coupled to the antibody carries a free 3' end that is attached to the 5' phosphate end of the UMI copy sequence. Fig.14 It is described how to add a barcode to the 5' end of a cDNA via a gap-filling / ligation reaction. In this method, mRNA is replicated in a reverse transcription reaction (using oligo d(T) primers, random primers, or gene-specific primers), and once cDNA is generated, a barcode is added to the 5' end of the cDNA via a gap-filling ligation reaction. These and other implementations are contemplated.

[0088] As described above, in some embodiments, the primer molecules can be attached to a planar substrate, e.g., a glass slide, etc. In these embodiments, at least 10 6 , 10 7 , 10 8 or 109 Each primer molecule can be attached to a planar support and extended using the methods described above. Figure 5 The method is schematically illustrated in . In this embodiment, a first set of barcode particles and a second set of barcode particles are hybridized to sequences located in overlapping regions on a carrier 50. A first primer extension reaction adds a molecular tag to the primers hybridized to the first set of barcode particles to produce a first feature array 52 corresponding to the first primer extension products. In the example shown, there are six features 58, each corresponding to a barcode particle and each containing a different molecular tag. After the first primer extension reaction is completed, the first set of barcode particles are removed and the second set of barcode particles are hybridized to the substrate. In this step, the second set of barcode particles are hybridized to the first primer extension products, but in an overlapping manner. The extension of the hybridized second set of barcode particles adds the molecular tag from the second set of barcode particles to the first primer extension products to produce a second feature array 56 corresponding to the second primer extension products. As an example, in array 56, features 60 and 62 have the same unique tag (corresponding to feature 58) from the first barcode particle, but have different unique tags from the second barcode particle.

[0089] Still Figure 6 An embodiment of the method for obtaining the array is described in. Figure 6 , the method can start with a solid surface with an immobilized oligonucleotide. In step 1, the free 3' end of the immobilized oligonucleotide is extended into a first set of barcode particles (e.g., RCA products) with a unique molecular label. This extension step copies the complementary sequence of the unique molecular label from the first set of barcode particles to the oligonucleotide. The barcode particles are then removed. In step 2, a second set of barcode particles is added to the surface, effectively adding a second random barcode to the surface. The primer extension products are then extended, which incorporates the label of the second set of particles into the initial extension products. The second label provides information about the proximity of the first label. Since the second addition only partially overlaps the first addition, a spatial map of connected labels will be generated, such as Figure 6 The array can then be used to capture biomolecules bound in a cell or tissue sample, thereby providing spatial information about the biomolecules. The resolution may be comparable to the size of the particles, which may be 50-500 nanometers, greatly exceeding the resolution of other methods. Alternatively, biomolecules in a sample (e.g., mRNA binding probes or poly-A primers for cDNA) can be captured on a surface containing immobilized oligonucleotides (before primer extension). Examples of using such an array include Figure 7 shown.

[0090] The probe system of the present invention can be used to map binding events in or on a cell sample. Figure 8 The method is schematically illustrated in . In these embodiments, the method may include obtaining a sample containing primer molecules that bind 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 a cell, extending the primer to prepare cDNA, and attaching a second primer to the 5' end of the cDNA, wherein the cDNA becomes the primer molecule used in the present method. Other components used in the method include a first set of barcode particles as described above, i.e., particles in which each particle has a nucleotide sequence comprising: a primer binding sequence that is complementary to the 3' end of the primer molecule, a unique particle marker sequence, and a first template sequence, such as Figure 1 As shown, and a second set of barcoded particles, each particle having a nucleotide sequence, the nucleotide sequence comprising: a first template sequence and a unique particle tag sequence, such as Figure 1 In any embodiment and as Figure 1 As shown, the nucleotide sequence of the second group of barcode particles can lack the primer binding sequence in the nucleotide sequence of the first group of barcode particles. Therefore, in any embodiment, the nucleotide sequence of the second group of barcode particles can lack the primer binding sequence in the nucleotide sequence of the first group of barcode particles, that is, it can include the first template sequence and the unique particle tag sequence, but not the primer binding sequence in the nucleotide sequence of the first group of barcode particles.

[0091] As described above, the method involves specifically hybridizing a first set of barcoded particles to a sample, wherein the nucleotide sequences of at least some of the barcoded particles in the first set of barcoded particles hybridize to at least two primer molecules. Figure 8As shown, the number of primer molecules hybridized to each barcode particle may vary. Some barcode particles in the first group of barcode particles may hybridize to one primer molecule, but many should hybridize to at least two, at least 5, at least 10, or at least 20 primer molecules, depending on the density of primer molecules and the size of the particles used. As with the above method, the method includes extending the primers hybridized to the barcode particles using the nucleotide sequence to which the primers hybridize as a template to produce first primer extension products, each of which contains a first unique particle tag sequence. Next, the method may include removing the first group of barcode particles from the sample as described above, and specifically hybridizing the second group of barcode particles to the first primer extension products. In this step of the method, the nucleotide sequence of at least some of the barcode particles in the second group of barcode particles hybridizes to at least two molecules in the primer extension products. Similarly, the first primer extension product previously hybridized to the barcode particle is extended using the nucleotide sequence to which the primer hybridizes as a template to generate a second primer extension product, the second primer extension product comprising two unique particle marker sequences, one from a particle in the first group and the other from a particle in the second group. Next, the method includes determining which unique particle marker sequence or its complementary sequence is in the second primer extension product, and making a relative position map of the primers using the unique particle marker sequence in the second primer extension product.

[0092] exist Fig. 9A -C lists the steps of this method in detail. Fig. 9A -C As can be seen, the primers can contain target marker sequences, allowing the assay to become a multiplex assay. In this method, the second primer extension product group can be amplified and sequenced together. Each sequence read should contain a target marker and two particle markers, one from the first group of particles and the other from the second group of particles. Since the sites bound by the first group of particles and the second group of particles overlap in separate hybridization reactions, the primers are able to locate the sites in the sample. Specifically, a relationship map of barcode particles can be generated by determining which marker pairs are added to the primers. The target sequence can be located on the relationship map, thereby providing a map of the binding sites of the primers (or the sites to which they bind, such as epitopes or sequences).

[0093] Alternative ligation-based embodiments are described below. These embodiments avoid the use of two sets of barcode particles and require only one primer extension step. In these embodiments, label pairs can be generated by ligating nearby primer extension products by ligating splints. Fig.10 and 11These embodiments are schematically illustrated in . In these embodiments, the probe system may include: a population of primer molecules (as shown, which may contain sequences that provide primer binding sites, target markers, and primer sequences C-BS1); a set of barcode particles, the barcode particles having a nucleotide sequence, the nucleotide sequence comprising: (i) a primer binding sequence (C-BS1) that is complementary to the 3' end of the primer molecule, (ii) a unique particle marker sequence (UMI) and (iii) a first template sequence (C-BS2). The probe system does not contain a second set of barcode particles, but instead includes a connection splint. As Fig.10 As shown, the ligation splint is composed of a first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide comprises a first sequence and a first template sequence (C-BST'); and the second oligonucleotide comprises a first template sequence (C-BST') and a second sequence complementary to the first sequence. The two oligonucleotides in the splint may have the same sequence and hybridize to each other through the sequence at the 5' end.

[0094] In general, the components of the system are similar to those of the serial extension system described above, except that the alternative system has a connection splint instead of a second set of barcode particles. For example, the barcode particle can be a rolling circle amplification (RCA) product, or the barcode particle can be a barcode nanoparticle, wherein the nucleotide sequence is tethered to the surface of the barcode particle. Similarly, the primer molecule of (a) is a synthetic oligonucleotide of 10-200nt in length, which may 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.

[0095] As with the series extension embodiment, the set of barcoded particles can contain at least 10 particles, each particle having a unique particle tag sequence. In this embodiment, the set of barcoded particles can each contain at least 10 members (e.g., at least 100, at least 1,000, at least 10,000, at least 100,000, at least 1 million, at least 10 million, at least 100 million, at least 1 billion, or at least 10 billion members), each of which can be uniquely labeled by its particle tag sequence. In some embodiments, the nucleic acid sequences of the barcoded particles can be identical to each other except for their particle tag sequence.

[0096] The alternative probe system can be used in a similar manner to the serial probe system described above to map binding events in or on a cell sample. A method for adding a unique particle label sequence to a primer using an alternative probe system is provided. Fig.10 and 11The method described in may include hybridizing the group of barcode particles with a group of primer molecules (which may be immobilized on a carrier as shown in the figure), and extending the hybridized primer molecules using a nucleotide sequence as a template. This step results in the generation of a first primer extension product, which contains: i. a complementary sequence to a unique particle marker sequence (UMI) from the barcode particle and ii. a complementary sequence to a first template sequence (C-BS2). As shown in the figure, after removing the barcode particle, the method includes hybridizing the first primer extension product with a ligation splint, wherein the complementary sequence of the first template sequence in the two proximal first primer extension products hybridizes with the first sequence and the second sequence of the ligation splint. Next, the method includes ligating at least one of the first oligonucleotide or the second oligonucleotide of the hybridized ligation splint to the first extension product, and using the first extension product in the ligation product as a template to extend the 3' end of at least one of the first oligonucleotide and the second oligonucleotide in the splint, thereby adding two unique particle marker sequences to the primer. As Fig.10 As shown, the primers can be engineered into a forward primer sequence, a reverse primer sequence and a template tag sequence, thereby providing a product containing a PCR primer site, a pair of template tag sequences and two particle tags. Fig.11 As schematically illustrated, 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). Obviously, the method can include sequencing the second primer extension to mark which unique particle marker sequence pairs are in the second primer extension product. The method can also include using the unique particle marker sequence pairs in the second primer extension product to map the relative positions of the primers of i.

[0097] As with the serial 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 label in the second primer extension product can indicate the relative position of the binding agent on the cell sample. Similarly, the primer can be attached to the 3' end of a cDNA made in situ in the cell sample, wherein the unique particle label in the second primer extension product indicates the relative position of the cDNA in the cell sample.

[0098] In any embodiment, if two composite tags are added to the same molecule, the barcode particles can be mapped relative to each other. The map generated by the method can be a three-dimensional map or a two-dimensional map, depending on how the method is implemented. For example, if the complex product is fixed in the cell (e.g., generated in situ in the cell), the map generated can be three-dimensional. In other embodiments, for example, if the complex is fixed on one or more surfaces (e.g., the surface of one or more cells that can be suspended or mounted on a carrier), the map generated by the method can be two-dimensional or potentially three-dimensional, because in theory, the map can be spherical. Although the method can be applied to cells (as described below), the method can be applicable to mapping adjacent complexes fixed on any surface (e.g., a slide that may have a tissue print or a protein print, etc.). Similarly, although the complex can be anchored in a cell or on a site on a cell or on a surface by an antibody (e.g., an antibody conjugated to an oligonucleotide, the oligonucleotide having a sequence complementary to a sequence in the complex), the complex can be directly or indirectly fixed by any type of interaction (e.g., covalent or non-covalent interaction). For example, in some embodiments, the complex can be bound to the cell via a binding agent (e.g., an aptamer, an antibody, or an oligonucleotide, etc.), wherein the binding agent binds to a sequence in the complex and a site in the cell or on the surface of one or more cells. In some embodiments, the complex can be immobilized by 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 by electrostatic interactions, by streptavidin / biotin interactions, or by covalent bonds (e.g., by click coupling).

[0099] In any embodiment, the complex can be fixed in or on a cell, and the cell is a cell in a solution, a cell on a carrier (e.g., a slide), a cell in a three-dimensional tissue sample, or a cell in a tissue section. For example, a sample containing cells in a solution can be a cultured cell sample grown as a cell suspension. In other embodiments, dissociated cells can be used (the cells can be produced by dissociating cultured cells or solid tissues such as soft tissues such as the liver or spleen using trypsin, etc.). In a specific embodiment, the complex can be fixed on cells that can be found in blood, for example, cells in whole blood or their subpopulations. The subpopulation cells of whole blood include platelets, erythrocytes (erythrocytes), and leukocytes (i.e., peripheral blood leukocytes, consisting of neutrophils, lymphocytes, eosinophils, basophils, and monocytes). These five types of leukocytes can be further divided into two groups, granulocytes (also referred to 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 present in the blood circulation reservoir, rather than being isolated (sequestered) in the lymphatic system, spleen, liver or bone marrow. If cells fixed on a carrier are used, then cells can be deposited on a planar surface, for example, by culturing cells on a planar surface, such as by centrifugation, by cutting the three-dimensional object comprising the cells into slices and mounting the slices on the planar surface, i.e., producing tissue slices to prepare samples. In an alternative embodiment, a surface can be made by absorbing cellular components on the surface.

[0100] In any embodiment, the method may include affixing thousands, tens of thousands, hundreds of thousands, or at least one million barcoded particles (each with a unique label) to a population of cells (e.g., via antibodies) such that the barcoded particles effectively coat the cells on each cell. The barcoded particles can be hybridized with other oligonucleotides (these oligonucleotides are tethered to sites within or on the cells) to produce a matrix containing the barcoded particles. After hybridization, the unique label sequence of adjacent complexes can be copied from one barcoded particle to another barcoded particle. A physical map of the complexes and the sites where the barcoded particles bind to the cells can be constructed based on the copied sequences.

[0101] In addition to making a map of barcoded particles, the method may also involve performing a proximity assay between one or more binding agents (e.g., antibodies that bind to cell surface markers on cells) that bind to sites in cells or on the cell surface. In these embodiments, the product may include a pair of unique complex marker sequences and binding agent marker sequences. In some embodiments, the binding agent may be an antibody-oligonucleotide conjugate, while in other embodiments, the capture agent may be an oligonucleotide probe or cDNA. The oligonucleotide and the capture agent may be connected by many different methods, including those using maleimide or halogen-containing groups with cysteine ​​reactivity. The capture agent and the oligonucleotide may be connected at the 5' end proximal or 5' end of the oligonucleotide, at the 3' end proximal or 3' end of the oligonucleotide, or anywhere in between. In some embodiments, the oligonucleotide may be connected to the capture agent via a linker that separates the oligonucleotide from the capture agent. As described above, the oligonucleotide may be connected to the capture agent using any convenient method. In many embodiments, the primer sequence conjugated to the binding agent uniquely marks the epitope or sequence to which the binding agent binds. For example, if 10 different antibodies are used to perform the method, each antibody is tethered to a different sequence that labels the epitope to which the antibody binds. This feature allows the method to become a multiplex method, and in certain embodiments, at least 5, at least 10, at least 20, or at least 50 different antibodies that bind to different markers in or on the surface of a cell can be used for the method. Each antibody is conjugated to a different antibody tag sequence, and the antibody tag sequence allows mapping of the binding events of a specific antibody. Such tagged antibodies are described, for example, in Wu et al (Nat. Comm. 2019 10: 3854) and US20160281134 and other documents.

[0102] Implementation

[0103] Embodiment 1. A probe system, comprising: (a) a group of primer molecules; (b) a first group of barcode particles, each barcode particle having a nucleotide sequence, the nucleotide sequence comprising: (i) a primer binding sequence, which is complementary to the 3' end of the primer molecule of (a), (ii) a unique particle tag sequence, and (iii) a first template sequence; (c) a second group of barcode particles, each barcode particle having a nucleotide sequence, the nucleotide sequence comprising: (i) the first template sequence, and (ii) a unique particle tag sequence; wherein the first group of barcode particles of (b) are used as a template to extend the primer molecules of (a) to generate primer extension products, the primer extension products containing the complementary sequence of the unique particle tag sequence of the particles of (b) (ii) and the complementary sequence of the first template sequence.

[0104] Embodiment 2. The probe system according to embodiment 1, wherein the barcode particles of (b) and / or (c) are rolling circle amplification (RCA) products.

[0105] Embodiment 3. The probe system according to embodiment 1, wherein the barcode particle of (b) and / or (c) is a barcode nanoparticle, wherein the nucleotide sequence is tethered to the surface of the barcode particle.

[0106] Embodiment 4. The probe system according to any one of the preceding embodiments, wherein the primer molecule of (a) is a synthetic oligonucleotide with a length of 10-200 nt.

[0107] Embodiment 5. The probe system according to any one of the preceding embodiments, wherein the primer molecule of (a) is a cDNA molecule having a primer sequence at the 3' end.

[0108] Embodiment 6. The probe system according to any of the preceding embodiments, wherein the primer molecule of (a) has a forward primer sequence, and the nucleotide sequence of the second set of barcode particles has a reverse primer sequence located downstream of the unique particle tag sequence.

[0109] Embodiment 7. The probe system according to any of the preceding embodiments, wherein the primer molecule of (a) is linked to a binding agent (eg, an oligonucleotide probe, an antibody, an aptamer, etc.).

[0110] Embodiment 8. The probe system according to embodiment 7, wherein the primer molecules further comprise target marker sequences that indicate the binding agents to which they are attached.

[0111] Embodiment 9. The probe system according to any one of embodiments 1-6, wherein the primer molecule is attached to the planar substrate.

[0112] Embodiment 10. The probe system according to any of the preceding embodiments, wherein the first set of barcoded particles and the second set of barcoded particles independently contain at least 10,000 particles, each particle having a unique particle label sequence.

[0113] Embodiment 11. The probe system according to any of the preceding embodiments, wherein the nucleotide sequence of the second set of barcode particles lacks the primer binding sequence of (b)(i).

[0114] Embodiment 12. A method for adding a unique particle label sequence to a primer using the probe system of any one of embodiments 1-11, comprising:

[0115] i. hybridizing the first group of barcoded particles of (b) with the group of primer molecules of (a),

[0116] ii. extending the hybridized primer molecule using the nucleotide sequence of the first group of barcode particles as a template to generate a first primer extension product, the first primer extension product containing: i. a complementary sequence of a unique particle tag sequence of a barcode particle from the first group of barcode particles and ii. a complementary sequence of the first template sequence;

[0117] iii. removing the first group of barcoded particles;

[0118] iv. hybridizing the first primer extension product to the second set of barcode particles, wherein the complement of the first template sequence in the first primer extension product hybridizes to the first template sequence in the second set of barcode particles; and

[0119] v. extending the first primer extension product using the nucleotide sequence of the second set of barcode particles as a template to generate a second primer extension product, the second primer extension product comprising:

[0120] a unique particle tag sequence of a barcoded particle from the first set of barcoded particles,

[0121] The first template sequence, and

[0122] A unique particle marker sequence from a barcoded particle in the second set of barcoded particles.

[0123] Embodiment 13. A method according to embodiment 12, wherein the primer molecule has a forward primer sequence, and the nucleotide sequence of the second set of barcode particles has a reverse primer sequence located downstream of the unique particle label sequence, and the method includes amplifying the second primer extension product of v.

[0124] Embodiment 14. A method according to embodiment 12 or 13, wherein the method includes sequencing the second primer extension product of v.

[0125] Embodiment 15. A method according to embodiment 14, using a unique particle marker sequence pair in the second primer extension product to map the relative position of the primers of i.

[0126] Embodiment 16. A method according to any one of embodiments 12-15, wherein the primer is attached to a planar substrate and the method results in an array of second primer extension products located on the substrate.

[0127] Embodiment 17. A method according to any one of embodiments 12-15, wherein the primer is attached to the cell sample via a binding agent, and the unique particle label in the second primer extension product indicates the relative position of the binding agent on the cell sample.

[0128] Embodiment 18. A method according to any one of embodiments 12-15, wherein the primers produce cDNA in situ in the cell sample, and wherein the unique particle label in the second primer extension product indicates the relative position of the cDNA in the cell sample.

[0129] Embodiment 19. A method for mapping binding events in or on a cell sample, comprising: (a) obtaining: i. a sample containing primer molecules that bind to sites in or on a cell; ii. a first set of barcode particles, each barcode particle having a nucleotide sequence, the nucleotide sequence comprising: (i) a primer binding sequence that is complementary to the 3' end of the primer molecule of (a), (ii) a unique particle tag sequence, and (iii) a first template sequence; iii. a second set of barcode particles, each barcode particle having a nucleotide sequence, the nucleotide sequence comprising: (i) the first template sequence, and (ii) a unique particle tag sequence; (b) specifically hybridizing the first set of barcode particles of (a)(ii) to the sample, wherein the nucleotide sequence of at least some of the barcode particles in the first set of barcode particles hybridizes to at least two primer molecules; (c) using the nucleotide sequence to which the primers hybridize as templates to extend and step (d) removing a first group of barcode particles from the sample; (e) specifically hybridizing a second group of barcode particles from (a)(iii) to the first primer extension products from (c), wherein the nucleotide sequences of at least some of the barcode particles in the second group of barcode particles hybridize to at least two molecules in the first primer extension products; (f) extending the first primer extension products hybridized to the second barcode particles in step (e) using the nucleotide sequences to which the primers hybridize as templates to generate second primer extension products, the second primer extension products comprising two unique particle tag sequences; (g) determining which unique particle tag sequence or its complement is in the second primer extension product; and (h) mapping the relative positions of the primers of (a)(i) using the unique particle tag sequences in the second primer extension products.

[0130] Embodiment 20. A method according to embodiment 19, wherein the sample of (a) is prepared by binding a primer attached to a binding agent to a site in or on a cell.

[0131] Embodiment 21. The method of embodiment 20, wherein the binding agent is an oligonucleotide probe, an antibody or an aptamer.

[0132] Embodiment 22. A method according to embodiment 20 or 21, wherein the primers further comprise target marker sequences that indicate the binding agents to which they are attached.

[0133] Embodiment 23. The method of embodiment 19, wherein the sample of (a) is prepared by hybridizing a first primer to RNA in a cell, extending the primer to prepare cDNA, and appending a second primer to the 5' end of the cDNA.

[0134] Embodiment 24. The probe system according to any of the preceding embodiments, wherein the nucleotide sequence of the second set of barcode particles lacks the primer binding sequence of the first set of barcode particles.

[0135] Embodiment 25. A probe system comprising: (a) a population of primer molecules; (b) a set of barcode particles, each barcode particle having a nucleotide sequence, the nucleotide sequence comprising: (i) a primer binding sequence, which is complementary to the 3' end of the primer molecule of (a), (ii) a unique particle label sequence, and (iii) a first template sequence; and (c) a connecting splint, which comprises a first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide comprises a first sequence and a first template sequence; and the second oligonucleotide comprises the first template sequence and a second sequence complementary to the first sequence.

[0136] Embodiment 26. The probe system of embodiment 25, wherein the barcode particle of (b) is a rolling circle amplification (RCA) product.

[0137] Embodiment 27. The probe system according to embodiment 25, wherein the barcode particle of (b) is a barcode nanoparticle, wherein the nucleotide sequence of (b) is tethered to the surface of the barcode particle.

[0138] Embodiment 28. The probe system according to any one of embodiments 25-27, wherein the primer molecule of (a) is a synthetic oligonucleotide with a length of 10-200 nt.

[0139] Embodiment 29. The probe system according to any one of embodiments 25-27, wherein the primer molecule of (a) is a cDNA molecule having a primer sequence at the 3' end.

[0140] Embodiment 30. A probe system according to any one of embodiments 25-28, wherein the primer molecule of (a) is linked to a binding agent (e.g., an oligonucleotide probe, an antibody, an aptamer, etc.).

[0141] Embodiment 31. A probe system according to any one of embodiments 25-30, wherein a set of barcoded particles may contain at least 10,000 particles, each particle having a unique particle label sequence.

[0142] Embodiment 32. A method for adding a unique particle label sequence to a primer using the probe system of any one of embodiments 25-31, comprising:

[0143] i. hybridizing a group of barcoded particles of (b) with a group of primer molecules of (a),

[0144] ii. extending the hybridized primer molecule using the nucleotide sequence as a template to generate a first primer extension product, the first primer extension product containing: i. a complementary sequence to the unique particle tag sequence from the barcoded particle and ii. a complementary sequence to the first template sequence;

[0145] iii. Removal of barcode particles;

[0146] iv. hybridizing the first primer extension product to the ligation splint, wherein the complementary sequence of the first template sequence in the two proximal first primer extension products hybridizes to the first sequence and the second sequence of the ligation splint;

[0147] v. ligating at least one of the hybridized first oligonucleotide or second oligonucleotide of the ligation splint to the first primer extension product, and extending the 3' end of the ligated first oligonucleotide or second oligonucleotide in the splint using the first primer extension product in the ligation product as a template, thereby adding two unique particle label sequences to the primer.

[0148] Embodiment 33. A method according to embodiment 32, wherein the method includes sequencing the second primer extension product of v.

[0149] Embodiment 34. A method according to embodiment 32 or 33, further comprising mapping the relative position of the primers of i using a unique particle marker sequence pair in the second primer extension product.

[0150] Embodiment 35. A method according to any one of embodiments 32-34, wherein the primer molecule of i is attached to the cell sample via a binding agent, and the unique particle label in the second primer extension product indicates the relative position of the binding agent on the cell sample.

[0151] Embodiment 36. A method according to any one of embodiments 32-34, wherein the primer is a cDNA made in situ in the cell sample, and wherein the unique particle label in the second primer extension product indicates the relative position of the cDNA in the cell sample. Example

[0152] The following examples are put forth so as to provide those of ordinary skill in the art with additional disclosure and description on how to make and use the invention and are not intended to limit the scope of what the inventors regard as their invention nor to represent that the experiments below are all or the only experiments performed.

[0153] method

[0154] Probe Circularization and Rolling Circle Amplification: The probe oligonucleotide (1 or 2) was circularized using the template oligonucleotide (3 or 4) in a 50 μl ligation reaction containing 100 nM probe oligonucleotide, 100 nM template oligonucleotide, 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.

[0155] The non-circularized probe was digested by adding 10 U of exonuclease I and 100 U of exonuclease III to each reaction after ligation. Exonuclease digestion was performed at 37°C for 30 minutes followed by heat inactivation at 85°C for 20 minutes.

[0156] After exonuclease digestion RCA primer oligonucleotide 3 or 4 was added to each reaction to a concentration of 100 nM and allowed to hybridize to the circularized probe by incubation at 37°C for 20 minutes.

[0157] 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. Reactions were incubated at 37°C for 20 minutes and then heat inactivated at 65°C for 10 minutes.

[0158] Cell staining and fixation: Raji and Jurkat cells were aspirated from separate T75 culture flasks and spun down at 300×g for 5 minutes. The cells were counted and a cell suspension corresponding to 1 million cells was taken. The cells were washed twice in FACS buffer (2% FBS, 2mM EDTA dissolved in 1x PBS). The cells were blocked with Fc blocking agent and centrifuged to remove the supernatant. The 2TotalSeq B (Biolegend inc) antibody-oligonucleotide conjugate (oligonucleotide 8-27) library targeting various immune cell markers (CD3, CD4, etc.) was added to the cells at a concentration of 5 μg / ml for each conjugate and incubated on ice for 30 minutes. The cells stained with the conjugate were washed twice with 300 μl of FACS buffer, and then the cells were centrifuged, the supernatant was removed, and 250 μl of 1% PFA was added to fix the sample. After incubation for 10 min at room temperature, fixation was quenched by adding 12.5 μl of 2.5 M glycine followed by washing with 250 μl of 125 mM glycine in PBS. After another wash in PBS, cells were resuspended in PBS and stored at +4°C until use.

[0159] Primer extension assay: Conjugate-stained and fixed Raji and Jurkat cells were mixed in a 1:1 ratio and aliquots corresponding to a total of approximately 20 000 cells were placed in PCR tubes.

[0160] Hybridization of the RCA product with the cell-bound antibody-conjugate oligonucleotide was performed in a 40 μl reaction containing 2.5 nM RCA product (derived from oligonucleotide 1), 0.5 μM blocking oligonucleotide (5) and in a buffer containing 300 mM NaCl, 15 mM MgCl2, 20 mM Tris-HCl, pH 8. The reaction was incubated at 55°C for 15 minutes.

[0161] The sample was centrifuged at 500×g for 2 minutes to pellet the cells, the supernatant was removed and 100 μl of washing buffer (50 mM NaCl, 1 mM EDTA, 20 mM Tris-HC, pH 81) was added and the cells were resuspended. The sample was centrifuged again to pellet the cells and the supernatant was removed.

[0162] A 50 μl DNA polymerase extension reaction was performed to incorporate the pixel barcode sequence by adding a premix containing 0.2 mM dNTPs, 1 μl of Klentaq DNA polymerase in a buffer containing 50 mM Na-acetate, 10 mM Mg-acetate, 20 mM Tris-acetate, 100 μg / ml BSA. The reaction was incubated at 30°C for 15 minutes. The washing step was performed again under the same conditions as described above, followed by a USER digestion (uracil DNA glycosylase + endonuclease VII) reaction to degrade 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 at 37°C for 30 minutes.

[0163] A washing step was performed, followed by the addition of 50 μl of USER inactivation master mix consisting of 1 U of UGI protein in 50 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl, pH 8. The reaction was incubated at 37°C for 15 minutes.

[0164] After the washing step, a second RCA product hybridization reaction was performed using the RCA product derived from oligonucleotide 2 using the conditions otherwise identical to those described above for hybridization of RCA product 1. Similarly, the same extension and USER inactivation protocol was performed after the second hybridization reaction. After the second USER digestion step, a washing step was performed and the cells were then resuspended in 50 μl of washing buffer. The resulting cell suspension was quantified and diluted to a concentration of 20 cells / μl.

[0165] Following the serial extension assay, each conjugate oligonucleotide bound to the cells via antibody binding has two DNA pixel barcode sequences and one reverse PCR primer motif incorporated by the extension step of the assay.

[0166] 15 cycles of PCR were performed in a reaction containing 5 μl of diluted sample, 0.2 mM dNTPs, 0.4 μM of each forward and reverse primer containing Illumina adapter sequences (6, 7), 1 μl of Phusion DNA polymerase in 1× Phusion HF reaction buffer. The PCR reaction consisted of denaturation at 98°C for 1 minute, followed by 15 cycles of denaturation at 98°C for 10 seconds, annealing at 60°C for 30 seconds, 72°C for 40 seconds, and a final extension at 72°C for 5 minutes.

[0167] The PCR product was finally purified using Ampure XP beads according to the manufacturer's instructions but using a beads to sample ratio of 1.2. The purified PCR product was quantified using a Qubit fluorometer and sequenced on an Illumina NextSeq 2000 sequencer.

[0168]

[0169]

[0170]

[0171] result

[0172] The combination of the 2 DNA pixel barcodes incorporated onto each conjugate oligonucleotide represents two DNA pixels that were in close proximity to a molecule during the serial extension assay, since each pixel simply occupies a different region of space. This, coupled with the fact that multiple neighboring molecules will therefore share the same pixel barcode, allows for a spatial reconstruction of the relative position of each molecule using a graph theory approach by viewing the DNA pixel barcodes as nodes in a graph, and the combination of the 2 pixel barcodes for each molecule representing an edge (link) in the graph.

[0173] 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, did not contain common sequence motifs expected at a specific position, and were only observed once in the data. The average read depth was 7.99, meaning that there were an average of 7.99 copies of each unique molecule.

[0174] Of the reads remaining after filtering, a total of 3.4 million unique molecules were labeled based on their unique molecular identifier (UMI) sequences that were part of the conjugated oligonucleotide sequence. A total of 361,575 DNA pixel sequences were found from group A and 451,177 DNA pixel sequences from group B. The average number of unique conjugated oligonucleotides associated with each pixel from group A was 7.47, while the average number of unique conjugated oligonucleotides associated with each pixel from group B was 9.31.

[0175] After filtering out small graph components, the resulting graph or physical map consisted of a set of approximately 100 graph components (clusters), each of which included at least 1000 nodes. An induced subgraph was generated for each component, and the counts of each surface marker type were summarized for each cluster. The correlation of marker counts within each cluster was compared using scatter plots ( Figures 18A-18C ). If two B cell markers are plotted against each other (HLA-DR, CD19) or two T cell markers (CD3, CD7), a linear correlation of marker counts is observed. Whereas if a B cell marker (HLA-DR) is plotted against a T cell marker (CD3), no such correlation is observed. In summary, these results show that each separate cluster is generated from and represents a single cell in the experiment containing a sample with a mixture of B and T cells, Raji and Jurkat.

[0176] Representative clusters are selected and visualized using the Kamada-Kawai force-generated graph layout algorithm ( Fig.19 ). Can be represented by different colors Fig.19 Each edge (link) of the graph labels a different antibody type, where information has been decoded from the barcode sequence of each oligonucleotide conjugated to the antibody, and each node of the graph represents a DNA pixel sequence. Sequence Listing <110> pselgentechnik ag <120> Spatial mapping by serial primer extension <130> PIXL-006WO <150> 63168132 <151> 2021-03-30 <160> 27 <170> PatentIn version 3.5 <210> 1 <211> 84 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <220> <221> misc_feature <222> (23)..(42) <223> n is a, c, g, or t <400> 1 gctttaaggc cggtcctagc aannnnnnnn nnnnnnnnnn nncaacatca gtattcccag 60 gctacctgca ggttaagcgg attg 84 <210> 2 <211> 94 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <220> <221> misc_feature <222> (26)..(45) <223> n is a, c, g, or t <400> 2 caacatcagt attcccaggc taaaannnnn nnnnnnnnnn nnnnnagatc ggaagagcgt 60 cgtgtaggga aagacctgca ggttaagcgg attg 94 <210> 3 <211> 42 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 3 ttgctaggac cggccttaaa gccaatccgc ttaacctgca gg 42 <210> 4 <211> 42 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 4 tagcctggga atactgatgt tgcaatccgc ttaacctgca gg 42 <210> 5 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 5 cctgcaggtt aagcggattg 20 <210> 6 <211> 52 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 6 caagcagaag acggcatacg agatcgagta atgtgactgg agttcagacg tg 52 <210> 7 <211> 61 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 7 aatgatacgg cgaccaccga gatctacact atagcctaca ctctttccct acacgacgct 60 c 61 <210> 8 <211> 90 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <220> <221> misc_feature <222> (35)..(44) <223> n is a, c, g, or t <220> <221> misc_feature <222> (60)..(68) <223> n is a, c, g, or t <400> 8 gtgactggag ttcagacgtg tgctcttccg atctnnnnnn nnnntccctt gcgatttacn 60 nnnnnnnngc tttaaggccg gtcctagcaa 90 <210> 9 <211> 90 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <220> <221> misc_feature <222> (35)..(44) <223> n is a, c, g, or t <220> <221> misc_feature <222> (60)..(68) <223> n is a, c, g, or t <400> 9 gtgactggag ttcagacgtg tgctcttccg atctnnnnnn nnnntatccc ttgggatggn 60 nnnnnnnngc tttaaggccg gtcctagcaa 90 <210> 10 <211> 90 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <220> <221> misc_feature <222> (35)..(44) <223> n is a, c, g, or t <220> <221> misc_feature <222> (60)..(68) <223> n is a, c, g, or t <400> 10 gtgactggag ttcagacgtg tgctcttccg atctnnnnnn nnnnctgggc aattactcgn 60 nnnnnnnngc tttaaggccg gtcctagcaa 90 <210> 11 <211> 90 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <220> <221> misc_feature <222> (35)..(44) <223> n is a, c, g, or t <220> <221> misc_feature <222> (60)..(68) <223> n is a, c, g, or t <400> 11 gtgactggag ttcagacgtg tgctcttccg atctnnnnnn nnnngccgga cgacattaan 60 nnnnnnnngc tttaaggccg gtcctagcaa 90 <210> 12 <211> 90 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <220> <221> misc_feature <222> (35)..(44) <223> n is a, c, g, or t <220> <221> misc_feature <222> (60)..(68) <223> n is a, c, g, or t <400> 12 gtgactggag ttcagacgtg tgctcttccg atctnnnnnn nnnnttctgg gtccctagan 60 nnnnnnnngc tttaaggccg gtcctagcaa 90 <210> 13 <211> 90 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <220> <221> misc_feature <222> (35)..(44) <223> n is a, c, g, or t <220> <221> misc_feature <222> (60)..(68) <223> n is a, c, g, or t <400> 13 gtgactggag ttcagacgtg tgctcttccg atctnnnnnn nnnngtctct tggcttaaan 60 nnnnnnnngc tttaaggccg gtcctagcaa 90 <210> 14 <211> 90 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <220> <221> misc_feature <222> (35)..(44) <223> n is a, c, g, or t <220> <221> misc_feature <222> (60)..(68) <223> n is a, c, g, or t <400> 14 gtgactggag ttcagacgtg tgctcttccg atctnnnnnn nnnnaatagc gagcaagtan 60 nnnnnnnngc tttaaggccg gtcctagcaa 90 <210> 15 <211> 90 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <220> <221> misc_feature <222> (35)..(44) <223> n is a, c, g, or t <220> <221> misc_feature <222> (60)..(68) <223> n is a, c, g, or t <400> 15 gtgactggag ttcagacgtg tgctcttccg atctnnnnnn nnnngctgcg ctttccattn 60 nnnnnnnngc tttaaggccg gtcctagcaa 90 <210> 16 <211> 90 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <220> <221> misc_feature <222> (35)..(44) <223> n is a, c, g, or t <220> <221> misc_feature <222> (60)..(68) <223> n is a, c, g, or t <400> 16 gtgactggag ttcagacgtg tgctcttccg atctnnnnnn nnnncaatca gacctatgan 60 nnnnnnnngc tttaaggccg gtcctagcaa 90 <210> 17 <211> 90 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <220> <221> misc_feature <222> (35)..(44) <223> n is a, c, g, or t <220> <221> misc_feature <222> (60)..(68) <223> n is a, c, g, or t <400> 17 gtgactggag ttcagacgtg tgctcttccg atctnnnnnn nnnnatatgt atcacgcgan 60 nnnnnnnngc tttaaggccg gtcctagcaa 90 <210> 18 <211> 90 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <220> <221> misc_feature <222> (35)..(44) <223> n is a, c, g, or t <220> <221> misc_feature <222> (60)..(68) <223> n is a, c, g, or t <400> 18 gtgactggag ttcagacgtg tgctcttccg atctnnnnnn nnnntcaatc cttccgcttn 60 nnnnnnnngc tttaaggccg gtcctagcaa 90 <210> 19 <211> 90 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <220> <221> misc_feature <222> (35)..(44) <223> n is a, c, g, or t <220> <221> misc_feature <222> (60)..(68) <223> n is a, c, g, or t <400> 19 gtgactggag ttcagacgtg tgctcttccg atctnnnnnn nnnnctccga atcatgttgn 60 nnnnnnnngc tttaaggccg gtcctagcaa 90 <210> 20 <211> 90 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <220> <221> misc_feature <222> (35)..(44) <223> n is a, c, g, or t <220> <221> misc_feature <222> (60)..(68) <223> n is a, c, g, or t <400> 20 gtgactggag ttcagacgtg tgctcttccg atctnnnnnn nnnngtccct gcaacttgan 60 nnnnnnnngc tttaaggccg gtcctagcaa 90 <210> twenty one <211> 90 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <220> <221> misc_feature <222> (35)..(44) <223> n is a, c, g, or t <220> <221> misc_feature <222> (60)..(68) <223> n is a, c, g, or t <400> twenty one gtgactggag ttcagacgtg tgctcttccg atctnnnnnn nnnntcccac ttccgctttn 60 nnnnnnnngc tttaaggccg gtcctagcaa 90 <210> twenty two <211> 90 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <220> <221> misc_feature <222> (35)..(44) <223> n is a, c, g, or t <220> <221> misc_feature <222> (60)..(68) <223> n is a, c, g, or t <400> twenty two gtgactggag ttcagacgtg tgctcttccg atctnnnnnn nnnntggatt cccggacttn 60 nnnnnnnngc tttaaggccg gtcctagcaa 90 <210> twenty three <211> 90 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <220> <221> misc_feature <222> (35)..(44) <223> n is a, c, g, or t <220> <221> misc_feature <222> (60)..(68) <223> n is a, c, g, or t <400> twenty three gtgactggag ttcagacgtg tgctcttccg atctnnnnnn nnnncgcgaa cataagaagn 60 nnnnnnnngc tttaaggccg gtcctagcaa 90 <210> twenty four <211> 90 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <220> <221> misc_feature <222> (35)..(44) <223> n is a, c, g, or t <220> <221> misc_feature <222> (60)..(68) <223> n is a, c, g, or t <400> twenty four gtgactggag ttcagacgtg tgctcttccg atctnnnnnn nnnncagtcg tggtagatan 60 nnnnnnnngc tttaaggccg gtcctagcaa 90 <210> 25 <211> 90 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <220> <221> misc_feature <222> (35)..(44) <223> n is a, c, g, or t <220> <221> misc_feature <222> (60)..(68) <223> n is a, c, g, or t <400> 25 gtgactggag ttcagacgtg tgctcttccg atctnnnnnn nnnnatatgt cagagcaccn 60 nnnnnnnngc tttaaggccg gtcctagcaa 90 <210> 26 <211> 90 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <220> <221> misc_feature <222> (35)..(44) <223> n is a, c, g, or t <220> <221> misc_feature <222> (60)..(68) <223> n is a, c, g, or t <400> 26 gtgactggag ttcagacgtg tgctcttccg atctnnnnnn nnnnggtggc tagataatgn 60 nnnnnnnngc tttaaggccg gtcctagcaa 90 <210> 27 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <220> <221> misc_feature <222> (35)..(44) <223> n is a, c, g, or t <220> <221> misc_feature <222> (60)..(68) <223> n is a, c, g, or t <400> 27 gtgactggag ttcagacgtg tgctcttccg atctnnnnnn nnnngagatg tctgcaactn 60 nnnnnnnngc tttaaggccg gtcctagcaa 90

Claims

1. A probe system, comprising: (a) a population of nucleic acid molecules, each of which has an extendable 5' or 3' end; (b) a first set of barcoded particles, each barcoded particle having a nucleotide sequence, the nucleotide sequence comprising, in order: (i) a binding sequence which is complementary to the extendable end of the nucleic acid molecule of (a), (ii) a unique particle marker sequence, and (iii) a first template sequence; wherein each particle can be distinguished from every other particle by their unique marker sequence; (c) a second set of barcoded particles, each barcoded particle having a nucleotide sequence comprising: (i) a first template sequence, and (ii) a unique particle marker sequence, wherein the nucleotide sequence of the second set of barcoded particles does not contain the binding sequence and wherein each particle is distinguishable from every other particle by their unique marker sequence; wherein the first set of barcoded particles of (b) is used as a template, the nucleic acid molecules of (a) are extended by primer extension if the nucleic acid molecules have an extendable 3' end, or by gap-filling ligation if the nucleic acid molecules have an extendable 5' end, to generate a first extension product, the first extension product comprising a complementary sequence of the unique particle tag sequence of (ii) of (b) and a complementary sequence of the first template sequence; and wherein the second set of barcoded particles is used as a template, the first extension product is extended by primer extension if the nucleic acid molecule has an extendable 3' end, or the first extension product is extended by gap-filling ligation if the nucleic acid molecule has an extendable 5' end, to produce a second extension product, the second extension product comprising a complementary sequence to the unique particle tag sequence of (ii) of (c), a complementary sequence to the first template sequence, and a complementary sequence to the unique particle tag sequence of (ii) of (b); Wherein the barcoded particles are rolling circle amplification (RCA) products, or Wherein the barcode particle is a barcode nanoparticle wherein the nucleotide sequence is in an oligonucleotide tethered on the surface of the barcode nanoparticle.

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

4. 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 located downstream of the unique particle marker sequence. 5 . The probe system according to claim 1 , wherein the nucleic acid molecule of (a) is linked to an oligonucleotide probe, an antibody or an aptamer or a planar substrate. 6 . The probe system according to claim 5 , wherein the nucleic acid molecules of (a) further comprise target marker sequences, which indicate the oligonucleotide probes, antibodies or aptamers linked thereto.

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

8. The probe system according to claim 1, wherein the nucleic acid molecule is an oligonucleotide molecule having a 3' hydroxyl group or a 5' phosphate group.

9. The probe system of claim 1, wherein the nucleic acid molecule is cDNA.

10. A method for adding a unique particle label sequence to a nucleic acid molecule using the probe system of any one of claims 1 to 9, comprising: i. hybridizing the first set of barcoded particles of (b) with the nucleic acid molecule population of (a), ii. using the nucleotide sequence of the first set of barcode particles as a template, extending the hybridized nucleic acid molecule 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, to generate a first extension product, the first extension product containing: i. a complementary sequence of a unique particle tag sequence of a barcode particle from the first set of barcode particles and ii. a complementary sequence of the first template sequence; iii. removing the first group of barcoded particles; iv. hybridizing the first extension product to a second set of barcode particles, wherein the complement of the first template sequence in the first extension product hybridizes to the first template sequence in the second set of barcode particles; and v. using the nucleotide sequence of the second set of barcode particles as a template, extending 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, to produce a second extension product, the second extension product comprising: a complementary sequence of a unique particle marker sequence of a barcoded particle from the first set of barcoded particles, a complementary sequence to the first template sequence, and The complement of the unique particle marker sequence of a barcoded particle from the second set of barcoded particles.

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 barcode particles has a reverse primer sequence located downstream of the unique particle marker sequence, and the method comprises amplifying the second extension product of step v and / or wherein the method comprises sequencing the second extension product of step v.

12. The method of claim 11, comprising sequencing the second extension product of step v and mapping the relative positions of the nucleic acid molecules of step i using complementary sequence pairs of the unique particle tag sequence in the second extension product.

13. A method according to any one of claims 10 to 12, wherein the nucleic acid molecules are attached to a planar substrate and the method results in an array of second extension products located on the substrate, or wherein the nucleic acid molecule is attached to the cell sample via a binding agent, and the complementary sequence of the unique particle label in the second extension product indicates the relative position of the binding agent on the cell sample, wherein the binding agent is an oligonucleotide probe, an antibody or an aptamer, or The nucleic acid molecule is a cDNA made in situ in a cell sample, and the complementary sequence of the unique particle tag in the second extension product indicates the relative position of the cDNA in the cell sample.

14. A method for mapping binding events in or on a cell sample, comprising: (a) Obtain: i. A sample containing a nucleic acid molecule that binds to a site in or on a cell, the nucleic acid molecule having an extendable 5' or 3' end; ii. A first set of barcoded particles, each barcoded particle having a nucleotide sequence, the nucleotide sequence comprising, in order: (i) a binding sequence which is complementary to the extendable end of the nucleic acid molecule of (a), (ii) a unique particle marker sequence, and (iii) a first template sequence, where each particle can be distinguished from every other particle by their unique tag sequence; iii. a second set of barcoded particles, each barcoded particle having a nucleotide sequence comprising: (i) a first template sequence, and (ii) unique particle marker sequence, wherein the nucleotide sequence of the second set of barcoded particles does not contain a binding sequence and wherein each particle is distinguishable from every other particle by their unique tag sequence; (b) specifically hybridizing the first group of barcode particles of ii. of (a) with the sample, wherein the nucleotide sequences of at least some of the barcode particles in the first group of barcode particles hybridize with at least two of the nucleic acid molecules of (a); (c) using the first barcode particle as a template, extending the nucleic acid molecule hybridized to the barcode particle in step (b) by primer extension if the nucleic acid molecule has an extendable 3' end, or extending the nucleic acid molecule hybridized to the barcode particle in step (b) by gap-filling ligation if the nucleic acid molecule has an extendable 5' end, to generate first extension products, each of the first extension products comprising a complementary sequence to the first unique particle tag sequence; (d) removing the first set of barcoded particles from the sample; (e) specifically hybridizing the second group of barcode particles of iii of (a) with the first extension product of (c), wherein the nucleotide sequences of at least some of the barcode particles in the second group of barcode particles hybridize with at least two molecules in the first extension product; (f) using the second barcode particle as a template, extending the first extension product hybridized to the second barcode particle in step (e) by primer extension if the nucleic acid molecule has an extendable 3' end, or extending the first extension product hybridized to the second barcode particle in step (e) by gap-filling ligation if the nucleic acid molecule has an extendable 5' end, to generate a second extension product, the second extension product comprising complementary sequences of the two unique particle tag sequences; (g) determining which unique particle marker sequence or its complement is in the second extension product; and (h) mapping the relative position of the nucleic acid molecule of i of (a) using the unique particle marker sequence or its complement in the second extension product; Wherein the barcoded particles are rolling circle amplification (RCA) products, or Wherein the barcode particle is a barcode nanoparticle wherein the nucleotide sequence is in an oligonucleotide tethered on the surface of the barcode nanoparticle.

15. The method of claim 14, wherein the sample of (a) is prepared by binding an oligonucleotide linked to a binding agent to a site in or on a cell, wherein the binding agent is an oligonucleotide probe, an antibody or an aptamer.

16. The method of claim 15, wherein the oligonucleotides further comprise a target marker sequence that indicates the binding agent to which they are attached.

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

18. A probe system comprising: (a) a population of nucleic acid molecules, each of which has an extendable 5' or 3' end; (b) a set of barcoded particles, each barcoded particle having a nucleotide sequence, the nucleotide sequence comprising: (i) a binding sequence which is complementary to the extendable 3' or 5' end of the nucleic acid molecule of (a), (ii) a unique particle marker 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 a first template sequence; and The second oligonucleotide comprises a first template sequence and a second sequence complementary to the first sequence, wherein the first oligonucleotide and the second oligonucleotide hybridize to each other through the respective first sequence and the second sequence, Wherein, in the case where the nucleic acid molecule population has an extendable 3' end, the first template sequence of the connecting splint is located downstream of the first sequence and the second sequence of the first oligonucleotide and the second oligonucleotide; in the case where the nucleic acid molecule population has an extendable 5' end, the first template sequence of the connecting splint is located upstream of the first sequence and the second sequence of the first oligonucleotide and the second oligonucleotide; Wherein the barcoded particles in (b) are rolling circle amplification (RCA) products, or The barcode particle of (b) is a barcode nanoparticle, wherein the nucleotide sequence of (b) is in an oligonucleotide tethered on the surface of the barcode particle.

19. The probe system of claim 18, wherein the nucleic acid molecule of (a) is a synthetic oligonucleotide with a length of 10-200 nt.

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

21. The probe system of claim 18, wherein the nucleic acid molecule of (a) is linked to a binding agent, wherein the binding agent is an oligonucleotide probe, an antibody, or an aptamer.

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

23. A method for adding a unique particle label sequence to a nucleic acid molecule using the probe system of any one of claims 18 to 22, comprising: i. hybridizing a group of barcoded particles of (b) with a group of nucleic acid molecules of (a); ii. using the nucleotide sequence as a template, extending the hybridized nucleic acid molecule 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, to produce a first extension product, the first extension product containing: i. a complementary sequence to a unique particle tag sequence from a barcode particle and ii. a complementary sequence to a first template sequence; iii. Removal of barcode particles; iv. hybridizing the first extension product to the ligation splint, wherein the complementary sequence of the first template sequence in two adjacent first extension products hybridizes to the first template sequence 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 oligonucleotide or the second oligonucleotide in the splint 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, thereby adding two unique particle label sequences to the nucleic acid.

24. The method of claim 23, wherein the method comprises sequencing the second extension product of step v.

25. The method of claim 23 or 24, further comprising mapping the relative positions of the nucleic acid molecules of step i using the unique particle tag sequence pairs in the second extension product.

26. A method according to claim 23 or 24, wherein the nucleic acid molecule of step i is attached to the cell sample via a binding agent, and the unique particle label in the second extension product indicates the relative position of the binding agent on the cell sample, wherein the binding agent is an oligonucleotide probe, an antibody or an aptamer.

27. The method of claim 23 or 24, wherein the nucleic acid molecule is a cDNA made in situ in a cell sample, and wherein the unique particle label in the second extension product indicates the relative position of the cDNA in the cell sample.

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