Methods, compositions, and systems for analyte detection

Abstract

This specification provides methods and systems for identifying the proximity of analytes in a sample. The method may involve contacting one or more analytes with one or more probes. The proximity of one or more analytes may trigger a ligation event between one or more probes. An amplification reaction may be carried out involving one or more copies of a barcode or its derivatives. One or more copies of the barcode or its derivatives may be detected to identify the proximity between one or more analytes.

Description

Technical Field

[0001] Cross-reference This application claims priority to U.S. Provisional Patent Application No. 63 / 512,502, filed Jul. 7, 2023, the entire disclosure of which is hereby incorporated by reference herein.

Background Art

[0002] The proximity of an analyte within a sample has been analyzed and used to determine the state of the sample. Methods for analyzing the proximity of an analyte within a sample have been developed.

Summary of the Invention

[0003] Embodiments disclosed herein are methods for detecting an analyte in a sample, wherein the method provides a) a first probe and a second probe, the first probe comprising (i) a first binding site configured to connect to the first analyte in a first portion of the first analyte, (ii) a second binding site configured to connect to the first analyte in a second portion of the first analyte, the second binding site being adjacent to the second portion of the first analyte, (iii) a third binding site configured to connect to the second probe, (iv) a barcode, (v) a first end, and (vi) a second end, the second probe comprising (i) a fourth binding site configured to connect to the first probe, and (ii) a fifth binding site configured to connect to the second analyte. The present invention provides a method comprising: a) contacting a sample containing a first analyte and a second analyte with the first and second probes, such that (i) the first probe is connected to the first analyte, (ii) the second probe is connected to the second analyte, and (iii) the first probe is connected to the second probe; c) forming a cyclic oligonucleotide by ligating the first and second ends; d) amplifying the cyclic oligonucleotide to produce an amplified product, wherein the amplified product contains the complement of the barcode; and e) detecting the complement of the barcode or a derivative thereof using a plurality of detection probes to determine the proximity between the first analyte and the second analyte.

[0004] In some embodiments, the sample is a tissue sample. In some embodiments, the tissue sample is a fresh-frozen tissue sample. In some embodiments, the tissue sample is a formalin-fixed, paraffin-embedded tissue sample. In some embodiments, the sample is 5–250 μm thick. In some embodiments, the sample is 10–200 μm thick. In some embodiments, the sample is 25–150 μm thick.

[0005] In some embodiments, the first analyte includes nucleic acid. In some embodiments, the nucleic acid is ribonucleic acid. In some embodiments, the ribonucleic acid is messenger ribonucleic acid. In some embodiments, the ribonucleic acid is ribosomal ribonucleic acid. In some embodiments, the nucleic acid is deoxyribonucleic acid. In some embodiments, the first analyte includes polypeptide. In some embodiments, it includes ribosomal protein. In some embodiments, the first analyte includes chemical modification. In some embodiments, the second analyte includes nucleic acid. In some embodiments, the nucleic acid is ribonucleic acid. In some embodiments, the ribonucleic acid is messenger ribonucleic acid. In some embodiments, the ribonucleic acid is ribosomal ribonucleic acid. In some embodiments, the nucleic acid is deoxyribonucleic acid. In some embodiments, the second analyte includes polypeptide. In some embodiments, the polypeptide includes ribosomal protein.

[0006] In some embodiments, the first probe comprises a nucleic acid. In some embodiments, the nucleic acid comprises an oligonucleotide. In some embodiments, the oligonucleotide comprises one or more modifications. In some embodiments, one or more modifications comprises a 5'-phosphate modification. In some embodiments, one or more modifications comprises an internucleotide bond. In some embodiments, the internucleotide bond is a phosphorothioate. In some embodiments, the internucleotide bond is a phosphodiester. In some embodiments, the first probe recognizes ribonucleic acid. In some embodiments, the nucleic acid comprises a single nucleotide polymorphism. In some embodiments, if the first probe recognizes a single nucleotide polymorphism and the first analyte does not contain a single nucleotide polymorphism, ligation in c) does not occur. In some embodiments, if the first probe does not recognize a single nucleotide polymorphism and the first analyte contains a single nucleotide polymorphism, ligation in c) does not occur. In some embodiments, the nucleic acid comprises modifications. In some embodiments, the modification is selected from the group consisting of N6-methyladenosine, 5-methylcytosine, N1-methyladenosine, N7-methylguanosine, N4-acetylcytosine, pseudouridine, and N1-methylpseudridine. In some embodiments, the first probe recognizes the modification, and if the first analyte does not contain the modification, ligation in c) does not occur. In some embodiments, the first probe does not recognize the modification, and if the first analyte contains the modification, ligation in c) does not occur. In some embodiments, the first probe recognizes deoxyribonucleic acid. In some embodiments, the first probe recognizes a deoxyribonucleic acid modification. In some embodiments, the deoxyribonucleic acid modification is a methyl modification. In some embodiments, the first probe contains a first reactive chemical moiety at the first end and a second reactive chemical moiety at the second end.In some embodiments, the first reactive chemical moiety is selected from the group consisting of tetrazine, alkyne, azide, trans-cyclooctene, maleimide, N-hydroxysuccinimide ester, amine, carboxylic acid, hydroxyl, cyclopropenone, and thiol, norbornene. In some embodiments, the second reactive chemical moiety is selected from the group consisting of tetrazine, alkyne, azide, trans-cyclooctene, maleimide, N-hydroxysuccinimide ester, amine, carboxylic acid, hydroxyl, cyclopropenone, and thiol, norbornene. In some embodiments, the ligation in c) includes a reaction between the first and second reactive chemical moieties.

[0007] In some embodiments, ligation in c) includes performing a ligation reaction with a ligase. In some embodiments, the ligase is a T4 ligase. In some embodiments, the second probe comprises a nucleic acid. In some embodiments, the nucleic acid comprises an oligonucleotide. In some embodiments, the oligonucleotide comprises one or more modifications. In some embodiments, one or more modifications comprises a 5' phosphate modification. In some embodiments, one or more modifications comprises an internucleotide bond. In some embodiments, the internucleotide bond is a phosphorothioate. In some embodiments, the internucleotide bond is a phosphodiester. In some embodiments, the nucleic acid comprises an aptamer. In some embodiments, the second probe comprises a polypeptide. In some embodiments, the polypeptide comprises an antibody or antibody fragment. In some embodiments, the polypeptide comprises an affimer. In some embodiments, the polypeptide comprises a nanobody. In some embodiments, the second probe recognizes ribonucleic acid. In some embodiments, the second probe recognizes ribonucleic acid modifications. In some embodiments, the ribonucleic acid modification is selected from the group consisting of N6-methyladenosine, 5-methylcytosine, N1-methyladenosine, N7-methylguanosine, N4-acetylcytosine, pseudouridine, and N1-methylpseudridine. In some embodiments, the second probe recognizes deoxyribonucleic acid. In some embodiments, the probe recognizes a deoxyribonucleic acid modification. In some embodiments, the deoxyribonucleic acid modification is a methyl modification. In some embodiments, the second probe recognizes a polypeptide. In some embodiments, the polypeptide is a protein. In some embodiments, the protein is a transcription factor. In some embodiments, the protein is a ribosomal protein. In some embodiments, the protein is a histone. In some embodiments, the protein is a polymerase. In some embodiments, the protein is a helicase. In some embodiments, the protein is a restriction enzyme.In some embodiments, the protein is a ribonucleic acid-binding protein. In some embodiments, a second probe recognizes post-translational modifications of the protein.

[0008] In some embodiments, the barcode includes nucleic acid. In some embodiments, the nucleic acid is deoxyribonucleic acid. In some embodiments, the nucleic acid is ribonucleic acid. In some embodiments, the nucleic acid is at least 4 nucleotides long. In some embodiments, the nucleic acid is at least 6 nucleotides long. In some embodiments, the nucleic acid is at least 8 nucleotides long. In some embodiments, the nucleic acid is at least 10 nucleotides long. In some embodiments, the barcode corresponds to a first analyte. In some embodiments, the barcode corresponds to a second analyte. In some embodiments, the barcode corresponds to a first analyte adjacent to a second analyte. In some embodiments, the first probe further includes a second barcode. In some embodiments, the second barcode includes nucleic acid. In some embodiments, the nucleic acid is deoxyribonucleic acid. In some embodiments, the nucleic acid is ribonucleic acid. In some embodiments, the nucleic acid is at least 4 nucleotides long. In some embodiments, the nucleic acid is at least 6 nucleotides long. In some embodiments, the nucleic acid is at least 8 nucleotides long. In some embodiments, the nucleic acid is at least 10 nucleotides long. In some embodiments, the second barcode corresponds to the first analyte. In some embodiments, the second barcode corresponds to the second analyte. In some embodiments, the second barcode corresponds to the first analyte adjacent to the second analyte.

[0009] In some embodiments, the first binding site includes nucleic acid, which is at least 6 nucleotides long. In some embodiments, the first binding site includes nucleic acid, which is at least 10 nucleotides long. In some embodiments, the first binding site includes nucleic acid, which is at least 14 nucleotides long. In some embodiments, the first binding site includes nucleic acid, which is at least 20 nucleotides long. In some embodiments, the second binding site includes nucleic acid, which is at least 4 nucleotides long. In some embodiments, the second binding site includes nucleic acid, which is at least 10 nucleotides long. In some embodiments, the second binding site includes nucleic acid, which is at least 14 nucleotides long. In some embodiments, the second binding site includes nucleic acid, which is at least 20 nucleotides long. In some embodiments, the third binding site includes nucleic acid, which is at least 4 nucleotides long. In some embodiments, the third binding site includes nucleic acid, which is at least 8 nucleotides long. In some embodiments, the third binding site includes nucleic acid, which is at least 10 nucleotides long. In some embodiments, the third binding site includes nucleic acid, which is at least 12 nucleotides long. In some embodiments, the fourth binding site includes nucleic acid, which is at least 4 nucleotides long. In some embodiments, the fourth binding site includes nucleic acid, which is at least 8 nucleotides long. In some embodiments, the fourth binding site includes nucleic acid, which is at least 10 nucleotides long. In some embodiments, the fourth binding site includes nucleic acid, which is at least 12 nucleotides long. In some embodiments, the fifth binding site includes nucleic acid, which is at least 6 nucleotides long. In some embodiments, the fifth binding site includes nucleic acid, which is at least 8 nucleotides long. In some embodiments, the fifth binding site includes nucleic acid, which is at least 10 nucleotides long. In some embodiments, the fifth binding site includes nucleic acid, which is at least 12 nucleotides long.

[0010] In some embodiments, (c) involves performing rolling circle amplification. In some embodiments, (d) involves hybridizing a detection probe and an anchor probe from a plurality of detection probes to the amplification product. In some embodiments, the detection probe and section detection probe are ligated. In some embodiments, the detection probe includes a label. In some embodiments, the label includes a fluorescent molecule. In some embodiments, the label includes a quantum dot. In some embodiments, the label includes an enzyme. In some embodiments, the enzyme generates a signal indicating the label. In some embodiments, (e) involves detecting the label. In some embodiments, (e) involves sequencing in situ using a plurality of detection probes. In some embodiments, (e) involves imaging the sample. In some embodiments, the first probe recognizes messenger ribonucleic acid, and the second probe recognizes ribosomal proteins. In some embodiments, the second probe includes an antibody or antibody fragment. In some embodiments, the first probe recognizes messenger ribonucleic acid, and the second probe recognizes ribosomal ribonucleic acid. In some embodiments, the first probe recognizes messenger ribonucleic acid, and the second probe recognizes messenger ribonucleic acid modifications. In some embodiments, the second probe includes a reactive chemical moiety. In some embodiments, the second probe includes an antibody or antibody fragment. In some embodiments, the first probe recognizes deoxyribonucleic acid, and the second probe recognizes deoxyribonucleic acid modifications. In some embodiments, the second probe includes a reactive chemical moiety. In some embodiments, the second probe includes an antibody or antibody fragment. In some embodiments, the sample is embedded in a hydrogel. In some embodiments, ligation in c) includes ligating the first end to the second end. In some embodiments, the first and second ends are separated by at least one nucleotide when ligated after (b).In some embodiments, the method further includes performing the gap-filling reaction after (b) and before (c) such that the first and third ends are directly adjacent to each other.

[0011] Embodiments disclosed herein are methods for detecting an analyte in a sample, the method comprising: a) providing a first probe, the first probe comprising: (i) a first binding site configured to connect to the first analyte in a first portion of the first analyte; (ii) a second binding site configured to connect to the first analyte in a second portion, wherein the first portion of the first analyte is adjacent to the second portion of the first analyte; (iii) a third binding site configured to connect to the second probe; (iv) a barcode; (v) a first end; and (vi) a second end; and b) contacting, the method comprising contacting a sample comprising a plurality of analytes, including the first analyte and the second analyte, with the first probe so that the first probe connects to the first analyte. The present invention provides a method comprising: c) contacting a first and second end separated by a gap; c) performing a gap-filling reaction to fill the gap; d) forming a cyclic oligonucleotide by ligating the first and second ends; e) contacting the cyclic oligonucleotide with a second probe, wherein the second probe includes (i) a fourth binding site for linking to the first probe and (ii) a fifth binding site for linking to the second analyte; f) amplifying the cyclic oligonucleotide to produce an amplified product, wherein the amplified product includes a barcode complement; and g) detecting the barcode complement or a derivative thereof using at least one detection probe to determine the proximity between the first analyte and the second analyte.

[0012] Aspects disclosed herein are methods for detecting an analyte in a sample, wherein the method provides a) a first probe, a second probe, and a third probe, wherein the first probe includes (i) a first binding site configured to connect to a first analyte, (ii) a second binding site configured to connect to a second probe, (iii) a barcode, (iv) a first end, and (v) a second end, and the second probe includes (i) a third end configured to connect to the first probe. The third probe includes (i) a binding site configured to connect to the second probe, (ii) a fourth binding site configured to connect to the third probe, and (iii) a fifth binding site configured to connect to the second analyte, wherein the third probe includes (i) a sixth binding site configured to connect to the second probe, (ii) a seventh binding site configured to connect to the first analyte, (iii) a third end, the third end being adjacent to the first end, and (iv) a fourth end, the fourth end being adjacent to the second end a) to provide, including a fourth end, and to contact, a sample containing multiple analytes, including a first analyte and a second analyte, with the first probe, the second probe, and the third probe, such that (i) the first probe is connected to the first analyte, (ii) the second probe is connected to the second analyte, (iii) the third probe is connected to the first analyte, (iv) the first probe is connected to the second analyte, and (v) the third probe is connected to the second probe. The present invention provides a method comprising: c) forming a cyclic oligonucleotide by ligating the first and third ends and the second and fourth ends; d) amplifying the cyclic oligonucleotide to produce an amplified product, wherein the amplified product includes the complement of the barcode; and e) detecting the complement of the barcode or a derivative thereof using a plurality of detection probes to determine the proximity between a first analyte and a second analyte.

[0013] In some embodiments, the sample is a tissue sample. In some embodiments, the tissue sample is a fresh-frozen tissue sample. In some embodiments, the tissue sample is a formalin-fixed, paraffin-embedded tissue sample. In some embodiments, the sample is 5–250 μm thick. In some embodiments, the sample is 10–200 μm thick. In some embodiments, the sample is 25–150 μm thick. In some embodiments, the first analyte contains nucleic acid. In some embodiments, the nucleic acid is ribonucleic acid. In some embodiments, the messenger ribonucleic acid is messenger ribonucleic acid. In some embodiments, the messenger ribonucleic acid is ribosome messenger ribonucleic acid. In some embodiments, the nucleic acid is deoxyribonucleic acid. In some embodiments, the nucleic acid contains a single nucleotide polymorphism. In some embodiments, the first probe recognizes a single nucleotide polymorphism, and if the first analyte does not contain a single nucleotide polymorphism, ligation in c) does not occur. In some embodiments, the first probe does not recognize single nucleotide polymorphisms, and if the first analyte contains a single nucleotide polymorphism, ligation in c) does not occur. In some embodiments, the third probe recognizes single nucleotide polymorphisms, and if the first analyte does not contain a single nucleotide polymorphism, ligation in c) does not occur. In some embodiments, the third probe does not recognize single nucleotide polymorphisms, and if the first analyte contains a single nucleotide polymorphism, ligation in c) does not occur. In some embodiments, the nucleic acid contains modifications. In some embodiments, the modifications are selected from the group consisting of N6-methyladenosine, 5-methylcytosine, N1-methyladenosine, N7-methylguanosine, N4-acetylcytosine, pseudouridine, and N1-methylpseudridine. In some embodiments, the first probe recognizes modifications, and if the first analyte does not contain modifications, ligation in c) does not occur. In some embodiments, the first probe does not recognize the modification, and if the first analyte contains the modification, ligation in c) does not occur.In some embodiments, the third probe recognizes the modification, and if the first analyte does not contain the modification, ligation in c) does not occur. In some embodiments, the third probe does not recognize the modification, and if the first analyte contains the modification, ligation in c) does not occur. In some embodiments, the first analyte comprises a polypeptide. In some embodiments, the polypeptide comprises a ribosomal protein. In some embodiments, the first analyte contains chemical modifications.

[0014] In some embodiments, the second analyte includes nucleic acid. In some embodiments, the nucleic acid is ribonucleic acid. In some embodiments, the ribonucleic acid is messenger ribonucleic acid. In some embodiments, the ribonucleic acid is ribosomal ribonucleic acid. In some embodiments, the nucleic acid is deoxyribonucleic acid. In some embodiments, the second analyte includes polypeptide. In some embodiments, the polypeptide includes ribosomal protein.

[0015] In some embodiments, the first probe comprises a nucleic acid. In some embodiments, the nucleic acid comprises an oligonucleotide. In some embodiments, the oligonucleotide comprises one or more modifications. In some embodiments, one or more modifications comprises a 5'-phosphate modification. In some embodiments, one or more modifications comprises an internucleotide bond. In some embodiments, the internucleotide bond is a phosphorothioate. In some embodiments, the internucleotide bond is a phosphodiester. In some embodiments, the first probe recognizes ribonucleic acid. In some embodiments, the first probe recognizes a ribonucleic acid modification. In some embodiments, the ribonucleic acid modification is selected from the group consisting of N6-methyladenosine, 5-methylcytosine, N1-methyladenosine, N7-methylguanosine, N4-acetylcytosine, pseudouridine, and N1-methylpseudridine. In some embodiments, the first probe recognizes deoxyribonucleic acid. In some embodiments, the first probe recognizes a deoxyribonucleic acid modification. In some embodiments, the deoxyribonucleic acid modification is a methyl modification. In some embodiments, the first probe includes a first reactive chemical moiety at its first end, and the third probe includes a second reactive chemical moiety at its third end. In some embodiments, the first reactive chemical moiety is selected from the group consisting of tetrazine, alkynes, azides, trans-cyclooctene, maleimide, N-hydroxysuccinimide esters, amines, carboxylic acids, hydroxyl, cyclopropenone, and thiols, norbornene. In some embodiments, the second reactive chemical moiety is selected from the group consisting of tetrazine, alkynes, azides, trans-cyclooctene, maleimide, N-hydroxysuccinimide esters, amines, carboxylic acids, hydroxyl, cyclopropenone, and thiols, norbornene. In some embodiments, ligation in c) includes a reaction between the first and second reactive chemical moieties. In some embodiments, the first probe includes a third reactive chemical moiety at its second end, and the third probe includes a fourth reactive chemical moiety at its fourth end.In some embodiments, the third reactive chemical moiety is selected from the group consisting of tetrazine, alkyne, azide, trans-cyclooctene, maleimide, N-hydroxysuccinimide ester, amine, carboxylic acid, hydroxyl, cyclopropenone, and thiol, norbornene. In some embodiments, the fourth reactive chemical moiety is selected from the group consisting of tetrazine, alkyne, azide, trans-cyclooctene, maleimide, N-hydroxysuccinimide ester, amine, carboxylic acid, hydroxyl, cyclopropenone, and thiol, norbornene. In some embodiments, ligation in c) includes a reaction between the first and second reactive chemical moieties. In some embodiments, ligation in c) includes a ligation reaction with a ligase. In some embodiments, the ligase is a T4 ligase.

[0016] In some embodiments, the second probe comprises a nucleic acid. In some embodiments, the nucleic acid comprises an oligonucleotide. In some embodiments, the oligonucleotide comprises one or more modifications. In some embodiments, one or more modifications comprises a 5'-phosphate modification. In some embodiments, one or more modifications comprises an internucleotide bond. In some embodiments, the internucleotide bond is a phosphorothioate. In some embodiments, the internucleotide bond is a phosphodiester. In some embodiments, the nucleic acid comprises an aptamer. In some embodiments, the second probe comprises a polypeptide. In some embodiments, the polypeptide comprises an antibody or antibody fragment. In some embodiments, the polypeptide comprises an affimer. In some embodiments, the polypeptide comprises a nanobody. In some embodiments, the second probe recognizes ribonucleic acid. In some embodiments, the second probe recognizes ribonucleic acid modifications. In some embodiments, the ribonucleic acid modifications are selected from the group consisting of N6-methyladenosine, 5-methylcytosine, N1-methyladenosine, N7-methylguanosine, N4-acetylcytosine, pseudouridine, and N1-methylpseudridine. In some embodiments, the second probe recognizes deoxyribonucleic acid. In some embodiments, the second probe recognizes deoxyribonucleic acid modifications. In some embodiments, the deoxyribonucleic acid modification is a methyl modification. In some embodiments, the second probe recognizes polypeptides. In some embodiments, the polypeptide is a protein. In some embodiments, the protein is a transcription factor. In some embodiments, the protein is a ribosomal protein. In some embodiments, the protein is a histone. In some embodiments, the protein is a polymerase. In some embodiments, the protein is a helicase. In some embodiments, the protein is a restriction enzyme. In some embodiments, the protein is a ribonucleic acid-binding protein. In some embodiments, the second probe recognizes post-translational modifications of a protein. In some embodiments, the second probe contains a reactive chemical moiety.In some embodiments, the third reactive chemical moiety is selected from a list consisting of tetrazine, alkyne, azide, trans-cyclooctene, maleimide, N-hydroxysuccinimide ester, amine, carboxylic acid, hydroxyl, cyclopropenone, and thiol, norbornene. In some embodiments, the reactive chemical moiety reacts with the second analyte. In some embodiments, the reactive chemical moiety reacts with the first analyte.

[0017] In some embodiments, the third probe comprises a nucleic acid. In some embodiments, the nucleic acid comprises an oligonucleotide. In some embodiments, the oligonucleotide comprises one or more modifications. In some embodiments, one or more modifications comprises a 5'-phosphate modification. In some embodiments, one or more modifications comprises an internucleotide bond. In some embodiments, the internucleotide bond is a phosphorothioate. In some embodiments, the internucleotide bond is a phosphodiester. In some embodiments, the third probe recognizes ribonucleic acid. In some embodiments, the third probe recognizes a ribonucleic acid modification. In some embodiments, the ribonucleic acid modification is selected from the group consisting of N6-methyladenosine, 5-methylcytosine, N1-methyladenosine, N7-methylguanosine, N4-acetylcytosine, pseudouridine, and N1-methylpseudridine. In some embodiments, the third probe recognizes deoxyribonucleic acid. In some embodiments, the third probe recognizes a deoxyribonucleic acid modification. In some embodiments, the deoxyribonucleic acid modification is a methyl modification. In some embodiments, the third probe includes a reactive chemical moiety. In some embodiments, the reactive chemical moiety reacts with the third probe.

[0018] In some embodiments, one or more barcodes contain nucleic acids. In some embodiments, the nucleic acid is deoxyribonucleic acid. In some embodiments, the nucleic acid is ribonucleic acid. In some embodiments, the nucleic acid is at least 4 nucleotides long. In some embodiments, the nucleic acid is at least 6 nucleotides long. In some embodiments, the nucleic acid is at least 8 nucleotides long. In some embodiments, the nucleic acid is at least 10 nucleotides long. In some embodiments, the barcode corresponds to a first analyte. In some embodiments, the barcode corresponds to a second analyte. In some embodiments, the barcode corresponds to a first analyte adjacent to a second analyte. In some embodiments, the first probe further includes a second barcode. In some embodiments, the second barcode contains nucleic acids. In some embodiments, the nucleic acid is deoxyribonucleic acid. In some embodiments, the nucleic acid is ribonucleic acid. In some embodiments, the nucleic acid is at least 4 nucleotides long. In some embodiments, the nucleic acid is at least 6 nucleotides long. In some embodiments, the nucleic acid is at least 8 nucleotides long. In some embodiments, the nucleic acid is at least 10 nucleotides long. In some embodiments, the second barcode corresponds to the first analyte. In some embodiments, the second barcode corresponds to the second analyte. In some embodiments, the second barcode corresponds to the first analyte adjacent to the second analyte. In some embodiments, the third probe includes a third barcode. In some embodiments, the third barcode includes a nucleic acid. In some embodiments, the nucleic acid is deoxyribonucleic acid. In some embodiments, the nucleic acid is ribonucleic acid. In some embodiments, the nucleic acid is at least 4 nucleotides long. In some embodiments, the nucleic acid is at least 6 nucleotides long. In some embodiments, the nucleic acid is at least 8 nucleotides long. In some embodiments, the nucleic acid is at least 10 nucleotides long. In some embodiments, the third barcode corresponds to the first analyte. In some embodiments, the third barcode corresponds to the second analyte.In some embodiments, the third barcode corresponds to the first analyte adjacent to the second analyte. In some embodiments, the first binding site includes nucleic acid, which is at least 6 nucleotides long. In some embodiments, the first binding site includes nucleic acid, which is at least 10 nucleotides long. In some embodiments, the first binding site includes nucleic acid, which is at least 14 nucleotides long. In some embodiments, the first binding site includes nucleic acid, which is at least 20 nucleotides long. In some embodiments, the second binding site includes nucleic acid, which is at least 2 nucleotides long. In some embodiments, the second binding site includes nucleic acid, which is at least 4 nucleotides long. In some embodiments, the second binding site includes nucleic acid, which is at least 8 nucleotides long. In some embodiments, the second binding site includes nucleic acid, which is at least 10 nucleotides long. In some embodiments, the third binding site includes nucleic acid, which is at least 2 nucleotides long. In some embodiments, the third binding site includes nucleic acid, which is at least 4 nucleotides long. In some embodiments, the third binding site includes nucleic acid, which is at least 8 nucleotides long. In some embodiments, the third binding site includes nucleic acid, which is at least 12 nucleotides long. In some embodiments, the fourth binding site includes nucleic acid, which is at least 2 nucleotides long. In some embodiments, the fourth binding site includes nucleic acid, which is at least 4 nucleotides long. In some embodiments, the fourth binding site includes nucleic acid, which is at least 8 nucleotides long. In some embodiments, the fourth binding site includes nucleic acid, which is at least 12 nucleotides long. In some embodiments, the fifth binding site includes nucleic acid, which is at least 6 nucleotides long. In some embodiments, the fifth binding site includes nucleic acid, which is at least 10 nucleotides long. In some embodiments, the fifth binding site includes nucleic acid, which is at least 14 nucleotides long.In some embodiments, the fifth binding site includes nucleic acid, which is at least 20 nucleotides long. In some embodiments, the sixth binding site includes nucleic acid, which is at least 2 nucleotides long. In some embodiments, the sixth binding site includes nucleic acid, which is at least 4 nucleotides long. In some embodiments, the sixth binding site includes nucleic acid, which is at least 8 nucleotides long. In some embodiments, the sixth binding site includes nucleic acid, which is at least 12 nucleotides long. In some embodiments, the seventh binding site includes nucleic acid, which is at least 4 nucleotides long. In some embodiments, the seventh binding site includes nucleic acid, which is at least 10 nucleotides long. In some embodiments, the seventh binding site includes nucleic acid, which is at least 14 nucleotides long. In some embodiments, the seventh binding site includes nucleic acid, which is at least 20 nucleotides long.

[0019] In some embodiments, (c) involves performing rolling circle amplification. In some embodiments, (d) involves hybridizing a detection probe and an anchor probe from a plurality of detection probes to the amplification product. In some embodiments, the detection probe and the section detection probe are ligated. In some embodiments, the detection probe includes a label. In some embodiments, the label includes a fluorescent molecule. In some embodiments, the label includes a quantum dot. In some embodiments, the label includes an enzyme. In some embodiments, the enzyme generates a signal indicating the label. In some embodiments, (e) involves detecting the label. In some embodiments, (e) involves sequencing in situ using a plurality of detection probes. In some embodiments, (e) involves imaging the sample. In some embodiments, the first and third probes recognize messenger ribonucleic acid, and the second probe recognizes ribosomal ribonucleic acid. In some embodiments, the first and third probes recognize messenger ribonucleic acid, and the second probe recognizes messenger ribonucleic acid modifications. In some embodiments, the second probe includes a reactive chemical moiety. In some embodiments, the second probe includes an antibody or antibody fragment. In some embodiments, the first and third probes recognize deoxyribonucleic acid, and the second probe recognizes deoxyribonucleic acid modifications. In some embodiments, the second probe includes a reactive chemical moiety. In some embodiments, the second probe includes an antibody or antibody fragment. In some embodiments, the sample is embedded in a hydrogel. In some embodiments, ligation in c) includes ligating the first end to the third end. In some embodiments, ligation in c) includes ligating the second end to the fourth end. In some embodiments, the first and third ends are separated by at least one nucleotide when ligated after (b).In some embodiments, the method further includes performing the gap-filling reaction after (b) and before (c) such that the first and third ends are directly adjacent to each other. In some embodiments, the second and fourth ends are separated by at least one nucleotide when ligated after (b). In some embodiments, the method further includes performing the gap-filling reaction after (b) and before (c) such that the second and fourth ends are directly adjacent to each other.

[0020] Another aspect of this disclosure provides a non-temporary computer-readable medium comprising machine-executable code that implements any of the methods described above or elsewhere in this specification when executed by one or more computer processors.

[0021] Another aspect of this disclosure provides a system comprising one or more computer processors and computer memory linked thereto. The computer memory comprises machine-executable code that implements either the methods described above or elsewhere in this specification when executed by the one or more computer processors.

[0022] Further aspects and advantages of the present disclosure will be readily apparent to those skilled in the art from the following detailed description, which shows and describes only exemplary embodiments of the present disclosure. As will be understood, other different embodiments are possible, and some of their details can be modified in various obvious ways without departing from the present disclosure. Accordingly, the drawings and description should be considered illustrative and not limiting in nature.

[0023] Reference All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the incorporated publications and patents or patent applications conflict with the disclosure contained herein, this specification supersedes and / or is intended to supersede any such conflicting subject matter.

Brief Description of the Drawings

[0024] The novel features of the invention are specifically set forth in the appended claims. The features and advantages of the invention will be better understood from the following detailed description which describes exemplary embodiments in which the principles of the invention are utilized, and from the appended drawings (also referred to herein as "figures" and "FIGs"). [Figure 1A] FIG. 1A shows a two-probe system for detecting mRNA translation in situ using SplintR ligase. [Figure 1B] FIG. 1B shows a two-probe system for detecting mRNA translation in situ using SplintR ligase. [Figure 2A] FIG. 2A shows a two-probe system for detecting modified RNA or RNA / protein / DNA bound to mRNA in situ using SplintR ligase. [Figure 2B] FIG. 2B shows a two-probe system for detecting modified RNA or RNA / protein / DNA bound to mRNA in situ using SplintR ligase. [Figure 3A] FIG. 3A shows a three-probe system for detecting mRNA translation in situ using SplintR ligase and DNA ligase. [Figure 3B] FIG. 3B shows a three-probe system for detecting mRNA translation in situ using SplintR ligase and DNA ligase. [[ENDEND]] [Figure 4A]Figure 4A shows three probe systems that use SplintR ligase and DNA ligase to detect modified RNA or RNA / protein / DNA bound to mRNA in situ. [Figure 4B] Figure 4B shows three probe systems that use SplintR ligase and DNA ligase to detect modified RNA or RNA / protein / DNA bound to mRNA in situ. [Figure 5A] Figure 5A shows three probe systems for detecting mRNA translation in situ using SplintR ligase. [Figure 5B] Figure 5B shows three probe systems for detecting mRNA translation in situ using SplintR ligase. [Figure 6A] Figure 6A shows three probe systems that use SplintR ligase to detect modified RNA or RNA / protein / DNA bound to mRNA in situ. [Figure 6B] Figure 6B shows three probe systems that use SplintR ligase to detect modified RNA or RNA / protein / DNA bound to mRNA in situ. [Figure 7A] Figure 7A shows two probe systems for detecting DNA modifications, DNA-bound analytes, or analytes adjacent to DNA. [Figure 7B] Figure 7B shows two probe systems for detecting DNA modifications, DNA-bound analytes, or analytes adjacent to DNA. [Figure 8A] Figure 8A shows three probe systems for detecting DNA modifications, DNA-bound analytes, or analytes in close proximity to DNA. [Figure 8B] Figure 8B shows three probe systems for detecting DNA modifications, DNA-bound analytes, or analytes adjacent to DNA. [Figure 9] Figure 9 shows the detection of amplification product signals in a single cell. [Figure 10] Figure 10 shows a schematic diagram of the binding site within the two-probe system. [Figure 11]Figure 11 shows a schematic diagram of the binding sites within the three probe systems. [Figure 12] Figure 12 shows the process for detecting the proximity between the first analyte and the second analyte. [Figure 13] Figure 13 shows the process for detecting the proximity between the first analyte and the second analyte. [Figure 14] Figure 14 shows a computer system programmed or otherwise configured to implement the methods provided herein. [Modes for carrying out the invention]

[0025] While various embodiments of the present invention are shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided only as examples. Numerous variations, modifications, and substitutions can be conceived by those skilled in the art without departing from the present invention. It should be understood that various alternative forms to the embodiments of the present invention described herein may be used.

[0026] As used herein, the singular forms "a," "an," and "the" refer to multiple objects unless the context clearly indicates otherwise. References to "or" herein are intended to include "and / or" unless otherwise specified.

[0027] Whenever the terms “at least,” “greater than,” or “greater than or equal to” precede the first number in a sequence of two or more numbers, those terms apply to each number in that sequence. For example, 1, 2, or 3 or more is equivalent to 1 or more, 2 or more, or 3 or more.

[0028] Whenever the terms "not greater than," "less than," or "less than or equal to" precede the first number in a sequence of two or more numbers, those terms apply to each number in that sequence. For example, 3, 2, or 1 or less is equivalent to 3 or less, 2 or less, or 1 or less.

[0029] As used herein, the term “hydrogel” may refer to a network of water-insoluble polymer chains, which may also be found as a colloidal gel with water as the dispersion medium. In other words, a hydrogel can be a type of polymer material that can absorb large amounts of water without dissolving. Hydrogels can contain more than 99% water and may include natural or synthetic polymers, or combinations thereof. Hydrogels may also possess a degree of flexibility very similar to that of natural tissues, due to their significant water content. A detailed description of suitable hydrogels can be found in published U.S. Patent Application 20100055733, which is incorporated herein by reference. As used herein, the terms “hydrogel subunit” or “hydrogel precursor” may mean a hydrophilic monomer, prepolymer, or polymer that can be crosslinked or “polymerized” to form a three-dimensional (3D) hydrogel network. Without being bound by any scientific theory, it is believed that by immobilizing a biological specimen in the presence of a hydrogel subunit, the components of the specimen are crosslinked to the hydrogel subunit, thereby fixing the molecular components in place and preserving the tissue structure and cellular morphology.

[0030] As used herein, “peptide,” “oligopeptide,” or “polypeptide” may refer to two or more amino acids linked together by an amide bond (i.e., a “peptide bond”). Peptides may be linear or cyclic. Peptides may be cc, J3, γ, δ, or more, or a mixture thereof. Peptides may contain any mixture of amino acids as defined herein, such as any combination of D, L, cc, J3, γ, δ, or more amino acids.

[0031] As used herein, “protein” may refer to an amino acid sequence having multiple linked amino acids. Proteins may also be peptides having a secondary and / or tertiary structure. Histones may be a type of protein that binds to DNA and regulates its activity. Histone modifications include H3K4me1, H3K4me3, H3K36me3, H3K79me2, H3K9Ac, H3K27Ac, ​​H4K16Ac, H3K27me3, H3K9me3, Gamma H2A.X, H3S10P, or their analogues.

[0032] As used herein, “nucleotide” may comprise a nitrogen-containing heterocyclic base, a sugar, and one or more phosphate groups. A nucleotide is a monomeric unit of a nucleic acid sequence. Examples of nucleotides include, for example, ribonucleotides or deoxyribonucleotides. In ribonucleotides (RNA), the sugar is ribose, and in deoxyribonucleotides (DNA), the sugar is deoxyribose, i.e., a sugar lacking the hydroxyl group at the 2' position of ribose. The nitrogen-containing heterocyclic base can be a purine base or a pyrimidine base. Purine bases include adenine (A) and guanine (G), as well as their modified derivatives or analogs. Pyrimidine bases include cytosine (C), thymine (T), and uracil (U), as well as their modified derivatives or analogs. The C-1 atom of deoxyribose is bonded to N-1 of pyrimidine or N-9 of purine. The phosphate group can be mono-, di-, or triphosphate. While these nucleotides are natural nucleotides, it should be further understood that non-natural nucleotides, modified nucleotides, or analogues of the aforementioned nucleotides may also be used.

[0033] As used herein, “nucleic acid base” may refer to heterocyclic bases such as adenine, guanine, cytosine, thymine, uracil, inosine, xanthine, and hypoxanthine, or their heterocyclic derivatives, analogs, or tautomers. Nucleic acid bases may be spontaneously occurring or synthetic. Non-limiting examples of nucleic acid bases include adenine, guanine, thymine, cytosine, uracil, xanthine, hypoxanthine, 8-azapurine, purines substituted with methyl or bromine at the 8-position, 9-oxo-N6-methyladenine, 2-aminoadenine, 7-deazaxanthine, 7-deazaguanine, 7-deaza-adenine, N4-ethanocytosine, 2,6-diaminopurine, N6-ethano-2,6-diaminopurine, 5-methylcytosine, 5- (C3-C6)-alkynylcytosine, 5-alkynyluracil, 5-fluorouracil, 5-bromouracil, thiouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridine, isocytosine, isoguanine, inosine, 7,8-dimethylaloxazine, 6-dihydrothymine, 5,6-dihydrouracil, 4-methyl-indole, etenoadenine, and other nucleic acid bases that do not occur naturally.

[0034] The terms “nucleic acid” or “polynucleotide” may refer to deoxyribonucleotides or ribonucleotide polymers in single-stranded, double-stranded, or combination thereof, and unless otherwise specified, include known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides, such as peptide nucleic acids (PNAs) and phosphorothioate DNA. Unless otherwise indicated, a particular nucleic acid sequence includes its complementary sequence. Nucleotides include, but are not limited to, ATP, dATP, CTP, dCTP, GTP, dGTP, UTP, TTP, dUTP, 5-methyl-CTP, 5-methyl-dCTP, ITP, dITP, 2-amino-adenosine-TP, 2-amino-deoxyadenosine-TP, 2-thiothymidine tripphosphate, pyrrolo-pyrimidine tripphosphate, and 2-thiocytidine, as well as all of the above alpha-thio tripphosphates and 2'-O-methyl-ribonucleotide tripphosphates of all of the above bases. Modified bases include, but are not limited to, 5-Br-UTP, 5-Br-dUTP, 5-F-UTP, 5-F-dUTP, 5-propynyl-dCTP, and 5-propynyl-dUTP.

[0035] As used herein, the term "SEDAL" may refer to sequencing by dynamic annealing and ligation error correction (SEDAL), a method for decoding DNA sequences into imageable multicolor fluorescent signals. See Wang, Xiao et al., "Three-dimensional intact-tissue sequencing of single-cell transcriptional states," Science 361.6400 (2018).

[0036] As used herein, the term “complementary” may refer to two oligonucleotide sequences containing nucleotides that can form hydrogen bonds. Sequences may be complementary at one or more bases and / or at one or more consecutive positions.

[0037] As used herein, the terms “transcript” or “RNA transcript” may refer to the cellular output produced from RNA polymerase-catalyzed transcription of DNA. The term “mRNA transcript” may refer to an RNA transcript that has undergone further post-transcriptional processing to remove introns. mRNA transcripts can be translated into polypeptides.

[0038] As used herein, the terms “padlock” or “padlock probe” may refer to one or more oligonucleotides specific to a target nucleic acid sequence. The padlock probe may further be complementary to a secondary nucleic acid sequence directly or indirectly bound to the protein of interest, as well as additional nucleic acid sequences. The padlock probe may also contain other signaling elements, including barcode nucleic acid sequences.

[0039] As used herein, the term “amplification product” may refer to an amplified nucleic acid containing an amplified nucleotide sequence, thereby generating an amplified copy and / or a replica of the target nucleic acid. Amplification products may be generated through isothermal amplification, rolling circle amplification, or through repeated steps of denaturation, annealing, and extension for amplification of the target nucleic acid.

[0040] As used herein, the term “probe” may refer to an oligonucleotide sequence complementary to a specific sequence of DNA or RNA. A probe may contain multiple subunits, each complementary to one or more specific sequences of DNA or RNA. The shape and structure of the probe can be manipulated. By arranging typically consecutive complementary sequences at the distal end of the probe, it becomes possible for the probe to adhere in a substantially circular shape during annealing.

[0041] As used herein, “detection portion” may refer to an antibody or antibody fragment, such as a Fab, probe, aptamer, or chemical group, which can be covalently or noncovalently bonded to the analyte of interest.

[0042] The terms “comprising,” “including,” and “containing,” and their various forms, are synonymous and equally broad. Furthermore, unless explicitly stated otherwise, examples of elements or groups of elements that possess a particular characteristic, comprising, including, or having a particular characteristic, may include additional elements, regardless of whether those additional elements possess that characteristic or not.

[0043] This specification provides methods, compositions, and systems for detecting the proximity of analytes in a sample using probes. The detection methods described herein can detect the relationships between one or more analytes in a sample with high accuracy, high specificity, high sensitivity, or a combination thereof. Various types of analytes can be detected using these methods. In addition, various probe types can be used to detect one or more analytes as part of the methods described herein.

[0044] The compositions, methods, and systems provided herein may be used to measure ribosome activity. The compositions may include probes designed to detect mRNA translation, nucleic acid modification, nucleic acid interaction analytes, or combinations thereof in the presence of the probe. The probes may be conjugated to labels. The probes may function as primers and / or ligation templates.

[0045] Aspects of this disclosure provide a method for detecting an analyte in a sample. The method may include providing a first probe and / or a second probe. The first probe may include one or more binding sites. The first probe may include a first binding site (e.g., a first binding site of the first probe), a second binding site (e.g., a second binding site of the first probe), a third binding site (e.g., a third binding site of the first probe), or any combination thereof. The first binding site may be configured to connect to the first analyte in a first portion of the first analyte. The second binding site of the first probe (e.g., a second binding site of the first probe) may be configured to connect to the first analyte in a second portion of the first analyte. The third binding site (e.g., a third binding site of the first probe) may be configured to connect to the second probe. The first probe may include one or more barcodes. The first portion of the first analyte may be adjacent to the second portion of the first analyte. The second probe may include a first binding site (e.g., a fourth binding site of the second probe) and / or a second binding site (e.g., a fifth binding site of the second probe). The first binding site of the second probe (e.g., a fourth binding site of the second probe) may be configured to connect to the first probe. The second binding site of the second probe (e.g., a fifth binding site of the second probe) may be configured to connect to the second analyte. In some cases, a sample containing one or more analytes may be brought into contact with the first probe and / or the second probe. One or more analytes may include the first analyte and the second analyte. When the sample is brought into contact with the first probe and / or the second probe, the first probe may be connected to the first analyte. When the sample is brought into contact with the first probe and / or the second probe, the second probe may be connected to the second analyte. When the sample is brought into contact with the first probe and / or the second probe, the first probe may be connected to the second probe. The first probe may include a first end (e.g., the first end of the first probe) and / or a second end (e.g., the second end of the second probe).The first end of the first probe (e.g., the first end of the first probe) can be ligated to the second end of the second probe (e.g., the second end of the second probe) to form a cyclic oligonucleotide. The cyclic oligonucleotide can be amplified to produce an amplified product. The amplified product may include the complement of the barcode. In some cases, the complement of the barcode or a derivative thereof can be detected using multiple detection probes. Detection can determine the proximity between the first analyte and the second analyte.

[0046] Further aspects of the present disclosure provide a method for detecting an analyte in a sample using components shown in Figure 10. The method provides (a) a first probe (1009) and a second probe (1008), wherein the first probe (1009) has (i) a first binding site (1001) configured to connect to the first analyte (1007) in a first portion of the first analyte, and (ii) a second binding site (1002) configured to connect to the first analyte (1007) in a second portion of the first analyte, wherein the first portion of the first analyte is connected to the second portion of the first analyte The second probe (1008) includes a second binding site (1002) adjacent to the first probe (1008), a third binding site (1003) configured to connect to the second probe (1008), a barcode, a first end, and a second end, wherein the second probe (1008) includes a fourth binding site (1004) configured to connect to the first probe (1009), and a fifth binding site (1005) configured to connect to the second analyte (1006). (b) providing and (c) contacting, wherein a sample containing multiple analytes, including the first analyte (1007) and the second analyte (1006), is brought into contact with the first probe (1009) and the second probe (1008) such that (i) the first probe (1009) is connected to the first analyte (1007), (ii) the second probe (1008) is connected to the second analyte (1006), and (iii) the first probe (1009) is connected to the second probe (1008). The process includes (c) bringing the materials into contact, (d) ligating the first and second ends to form a cyclic oligonucleotide, (e) amplifying the cyclic oligonucleotide to produce an amplified product, wherein the amplified product includes the complement of the barcode, and (f) using a plurality of detection probes to detect the complement of the barcode or a derivative thereof to determine the proximity between the first analyte (1006) and the second analyte (1007).

[0047] Figure 12 schematically illustrates an example of detecting proximity between a first analyte and a second analyte using a first probe and a second probe. In this example, a first probe and a second probe are provided (1201). The first probe and the second probe may include, for example, nucleic acids. A sample containing the first analyte and the second analyte may be brought into contact with the first probe and the second probe (1202). For example, the first probe may bind to the first analyte, and the second probe may bind to the second analyte. One end of the first probe may be ligated to another end of the first probe to form a cyclic oligonucleotide (1203). The cyclic oligonucleotide may be amplified to produce one or more amplified products containing complements of the barcode sequence of the first probe (1204). The complements of the barcode sequence of the first probe may be, for example, the inverse complement of the barcode sequence. The complement of the barcode can be detected to determine the proximity between the first analyte and the second analyte (1205). For example, multiple detection probes may be added to the sample, or a subset of multiple detection probes may be coupled to one or more amplification products to reveal at least a portion of the barcode complement of the first probe.

[0048] Further aspects of the present disclosure provide a method for detecting an analyte in a sample. The method may include providing a first probe. The first probe may include one or more binding sites. The first probe may include a first binding site, a second binding site, a third binding site, or any combination thereof. The first probe may be configured to connect to a first analyte. The first probe may include a first binding site configured to connect to a first analyte in a first portion of the first analyte. (ii) A second binding site may be configured to connect to a first analyte in a second portion of the first analyte. The first portion of the first analyte may be adjacent to a second portion of the first analyte. A third binding site may be configured to connect to a second probe. The first probe may include (iv) a barcode. The first probe may include a first end. The first probe may include a second end. In some cases, a sample containing one or more analytes, including a first analyte and / or a second analyte, may come into contact with the first probe so that the first probe can be ligated to the first analyte. The first and second ends of the first probe may be separated by a gap (e.g., a single-stranded region of the first analyte that does not hybridize to the first probe and is adjacent to the first and second ends of the first probe). A gap-filling reaction may be performed to fill the gap (e.g., extending the nucleic acid sequence of the first probe from one end to the other according to the nucleic acid sequence of the single-stranded region of the first analyte that does not hybridize to the first probe). The first end may be ligated to the second end after the gap-filling reaction to form a cyclic oligonucleotide. The cyclic oligonucleotide may come into contact with the second probe. The second probe may include a fourth binding site for ligating to the first probe. The second probe may include a fifth binding site linked to the second analyte. The cyclic oligonucleotide may be amplified to produce an amplified product. The amplified product may include the complement of the barcode. The complement of the barcode or a derivative thereof may be detected using at least one detection probe, thereby allowing for the determination of the proximity between the first analyte and the second analyte.

[0049] Further embodiments disclosed herein provide a method for detecting an analyte in a sample using the components shown in Figure 10, the method comprising: a) a first probe (1009), the first probe comprising: (i) a first binding site (1001) configured to bind to the first analyte (1007) in a first portion of the first analyte; and (ii) a second binding site (1002) configured to bind to the first analyte in a second portion (1006), the first analyte a) to provide a first portion of which includes a second binding site (1002) adjacent to the second portion of the first analyte, (iii) a third binding site (1003) configured to connect to a second probe (1008), (iv) a barcode, (v) a first end, and (vi) a second end, and b) to bring into contact a plurality of analytes including a first analyte (1007) and a second analyte (1006) such that a first probe (1009) is connected to a first analyte (1007). The method includes: a) contacting a sample containing a first probe (1009) such that the first and second ends are separated by a gap; c) performing a gap-filling reaction to fill the gap; d) forming a cyclic oligonucleotide by ligating the first and second ends; e) contacting the cyclic oligonucleotide with a second probe (1006) such that the second probe includes (i) a fourth binding site (1004) that connects to the first probe, and (ii) a fifth binding site (1005) that connects to the second analyte (1006); f) amplifying the cyclic oligonucleotide to produce an amplified product such that the amplified product includes the complement of the barcode; and g) detecting the complement of the barcode or a derivative thereof using at least one detection probe to determine the proximity between the first analyte (1007) and the second analyte (1006).

[0050] The first probe may include at least one binding site configured to bind to an analyte, a probe, or a combination thereof. In some cases, at least one binding site of the first probe may be configured to bind to an analyte. The analyte may be the first analyte. In some cases, at least one binding site of the first probe may be configured to bind to a probe. In some cases, the probe to which the first probe is configured to bind may be the second probe. At least one binding site of the first probe may include a nucleic acid. The nucleic acid of at least one binding site of the first probe may have a nucleotide length of at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, or longer. The nucleic acid of at least one binding site of the first probe may have a nucleotide length of up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 6, up to about 7, up to about 8, up to about 9, up to about 10, up to about 15, up to about 20, up to about 25, up to about 30, up to about 40, up to about 50, up to about 60, up to about 70, up to about 80, up to about 90, up to about 100, or shorter. The nucleic acid may have a length of about 1 to about 100, about 2 to about 90, about 3 to about 80, about 4 to about 70, about 5 to about 60, about 6 to about 50, about 7 to about 40, about 8 to about 30, about 9 to about 25, or about 10 to 20 nucleotides. The nucleic acid of at least one binding site of the first probe may include, but not limited to, adenine (A), guanine (G), thymine (T), cytosine (C), uracil, or combinations thereof. In some cases, the nucleic acid may include modifications. Modifications of the first binding site of the first probe may include methylation, phosphate modification, or combinations thereof. Modifications may include sugar modification, sugar / skeleton modification, skeletal modification, base modification, non-native base pairing, or combinations thereof.In some cases, sugar modifications may include 2'-fluoro, 2'-O-methyl, 2'-fluoroarabinose nucleic acids, hexitol nucleic acids, 2'-O-methoxyethyl, (1'-3')-β-L-ribonucleic acid, α-L-threose nucleic acid, 3'-2'phosphonomethyl-threosyl nucleic acid, 2'-deoxyxyl nucleic acid, phosphorothioates, alkylphosphonate nucleic acids, peptide nucleic acids, or combinations thereof.

[0051] The first probe may bind to the analyte in one or more portions. In some cases, the first probe may bind to one or more portions of the analyte. In some cases, one or more portions of the analyte may overlap. For example, in some cases, the first probe may bind to a first portion of the analyte that includes at least a portion of the second portion of the first analyte to which the first probe binds. If the first analyte contains nucleic acid, the first portion and the second portion of the analyte described herein may contain portions of the same nucleic acid sequence (e.g., portions of the same nucleic acid molecule). In some cases, one or more portions of the analyte do not have to overlap. For example, if the first analyte contains nucleic acid, the first portion and the second portion of the first analyte described herein may contain two different portions of nucleic acid that are adjacent to each other or separated by one or more nucleotides. In some cases, one or more portions of the analyte may be separated from the analyte by a certain distance. For example, in some cases, the first analyte may contain nucleic acids and may be bound to a first portion and a second portion of the first analyte, where the first portion and the second portion are separated by at least one nucleotide. If the analyte contains nucleic acids, the distance between one or more portions may be at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, and at least about 100 nucleotides. If the analyte contains nucleic acids, the distance between one or more parts may be at most about 1, at most about 2, at most about 3, at most about 4, at most about 5, at most about 6, at most about 7, at most about 8, at most about 9, at most about 10, at most about 15, at most about 20, at most about 25, at most about 30, at most about 40, at most about 50, at most about 60, at most about 70, at most about 80, at most about 90, and at most about 100 nucleotides.If the analyte contains nucleic acids, the distances between one or more parts may be approximately 1 to approximately 100, approximately 2 to approximately 90, approximately 3 to approximately 80, approximately 4 to approximately 70, approximately 5 to approximately 60, approximately 6 to approximately 50, approximately 7 to approximately 40, approximately 8 to approximately 30, approximately 9 to approximately 25, or approximately 10 to approximately 20 nucleotides. In some cases, the first analyte may contain polypeptides. If the first analyte contains a polypeptide, the distance between one or more parts may be at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, about 1 to 100, about 2 to 90, about 3 to 80, about 4 to 70, about 5 to 60, about 6 to 50, about 7 to 40, about 8 to 30, about 9 to 25, or about 10 to 20 amino acids. If the first analyte contains polypeptides, the distance between one or more parts may be at most about 1, at most about 2, at most about 3, at most about 4, at most about 5, at most about 6, at most about 7, at most about 8, at most about 9, at most about 10, at most about 15, at most about 20, at most about 25, at most about 30, at most about 40, at most about 50, at most about 60, at most about 70, at most about 80, at most about 90, at most about 100, about 1 to about 100, about 2 to about 90, about 3 to about 80, about 4 to about 70, about 5 to about 60, about 6 to about 50, about 7 to about 40, about 8 to about 30, about 9 to about 25, or about 10 to about 20 amino acids.

[0052] The first probe may include a first binding site. The first binding site may be configured to bind to a first analyte. The first binding site may include nucleic acids. The nucleic acids of the first binding site may have nucleotide lengths of at least about 2, at least about 4, at least about 6, at least about 8, at least about 10, at least about 12, at least about 14, at least about 16, at least about 18, at least about 20, at least about 22, at least about 24, at least about 26, at least about 28, at least about 30, at least about 32, at least about 34, at least about 36, at least about 38, at least about 40, at least about 42, at least about 44, at least about 46, at least about 48, at least about 50, or longer. The nucleic acid of the first binding site may have a nucleotide length of up to approximately 2, up to approximately 4, up to approximately 6, up to approximately 8, up to approximately 10, up to approximately 12, up to approximately 14, up to approximately 16, up to approximately 18, up to approximately 20, up to approximately 22, up to approximately 24, up to approximately 26, up to approximately 28, up to approximately 30, up to approximately 32, up to approximately 34, up to approximately 36, up to approximately 38, up to approximately 40, up to approximately 42, up to approximately 44, up to approximately 46, up to approximately 48, up to approximately 50, or shorter. The nucleic acid of the first binding site may be approximately 2–50, 4–48, 6–46, 8–44, 10–42, 12–40, 14–38, 16–36, 18–34, 20–32, 22–30, or 24–28 nucleotides long.

[0053] The first probe may include a binding site (e.g., a third binding site of the first probe). The first binding site may be configured to bind to the first analyte. In some cases, the first analyte may include nucleic acid. In some cases, the binding site (e.g., a third binding site of the third probe) may include nucleic acid, and the nucleic acid of the binding site (e.g., a third binding site of the first probe) may hybridize to at least a portion of the nucleic acid of the first analyte. The nucleic acid of the binding site (e.g., the third binding site of the first probe) may have a nucleotide length of at least about 2, at least about 4, at least about 6, at least about 8, at least about 10, at least about 12, at least about 14, at least about 16, at least about 18, at least about 20, at least about 22, at least about 24, at least about 26, at least about 28, at least about 30, at least about 32, at least about 34, at least about 36, at least about 38, at least about 40, at least about 42, at least about 44, at least about 46, at least about 48, at least about 50, or longer. The nucleic acid at the binding site (for example, the third binding site of the first probe) may have a nucleotide length of up to approximately 2, up to approximately 4, up to approximately 6, up to approximately 8, up to approximately 10, up to approximately 12, up to approximately 14, up to approximately 16, up to approximately 18, up to approximately 20, up to approximately 22, up to approximately 24, up to approximately 26, up to approximately 28, up to approximately 30, up to approximately 32, up to approximately 34, up to approximately 36, up to approximately 38, up to approximately 40, up to approximately 42, up to approximately 44, up to approximately 46, up to approximately 48, up to approximately 50, or shorter. The nucleic acid at the binding site (for example, the third binding site of the first probe) may be approximately 2 to 50, 4 to 48, 6 to 46, 8 to 44, 10 to 42, 12 to 40, 14 to 38, 16 to 36, 18 to 34, 20 to 32, 22 to 30, or 24 to 28 nucleotides long.

[0054] The first probe may include a third binding site. The third binding site may be configured to bind to the second probe. For example, the third binding site may include nucleic acid, the second probe may include nucleic acid, and the nucleic acid of the third binding site may hybridize with the nucleic acid of the second probe. The nucleic acid of the third binding site of the first probe may have a nucleotide length of at least about 2, at least about 4, at least about 6, at least about 8, at least about 10, at least about 12, at least about 14, at least about 16, at least about 18, at least about 20, at least about 22, at least about 24, at least about 26, at least about 28, at least about 30, at least about 32, at least about 34, at least about 36, at least about 38, at least about 40, at least about 42, at least about 44, at least about 46, at least about 48, at least about 50, or longer. The nucleic acid at the third binding site of the first probe may have a nucleotide length of up to approximately 2, up to approximately 4, up to approximately 6, up to approximately 8, up to approximately 10, up to approximately 12, up to approximately 14, up to approximately 16, up to approximately 18, up to approximately 20, up to approximately 22, up to approximately 24, up to approximately 26, up to approximately 28, up to approximately 30, up to approximately 32, up to approximately 34, up to approximately 36, up to approximately 38, up to approximately 40, up to approximately 42, up to approximately 44, up to approximately 46, up to approximately 48, up to approximately 50, or shorter. The nucleic acid of the third binding site of the first probe may be approximately 2 to 50, 4 to 48, 6 to 46, 8 to 44, 10 to 42, 12 to 40, 14 to 38, 16 to 36, 18 to 34, 20 to 32, 22 to 30, or 24 to 28 nucleotides in length.

[0055] The first probe can ligate itself. For example, one end of the first probe can be connected to another end of the first probe (for example, it can be ligated). The first probe may contain nucleic acid. The nucleic acid of the first probe may contain one or more single-stranded regions, one or more double-stranded regions, or a combination thereof. The nucleic acid of the first probe may contain at least one modification to enable a ligation event. In some cases, the ligation event may include a ligation event that connects one end of the first probe to another end of the first probe. The ligation event may include a chemical reaction that forms a covalent bond. The ligation event may include the formation of a non-covalent interaction between the first end and the second end of the first probe. The first end of the first probe can be ligated to the second end of the first probe.

[0056] The chemical reaction that forms a covalent bond between one end of a first probe and another end of the first probe may be facilitated by a protein. In some cases, the protein may be an enzyme. The enzyme may be a ligase, polymerase, transposase, or a combination thereof. In some cases, the enzyme may be a ligase. If the enzyme that facilitates the chemical reaction that forms a covalent bond between one end of a first probe and another end of the first probe is a ligase, then the ligase may be one or more ligases. In some cases, the ligase may include mammalian ligases. In some cases, the ligase may be bacterial ligases. The ligase may be DNA ligase I, DNA ligase II, DNA ligase III, DNA ligase IV, or a combination thereof. In some cases, the ligase may include RNA ligases. In some cases, the ligase may ligate the 3' nucleotide of one nucleic acid to the 5' nucleotide of another nucleic acid. In some cases, the ligase may ligate the 3' end of one nucleic acid to the 5' end of the same nucleic acid. For example, a nucleic acid molecule may contain a 5' end and a 3' end, and the 5' and 3' ends of the nucleic acid molecule may be ligated to form a cyclic oligonucleotide. A ligase may ligate two nucleotides that are part of a double-stranded nucleic acid. In some cases, a double-stranded nucleic acid may contain a nick, and the nick portion may be ligated by a ligase. In some embodiments, the double-stranded nucleic acid may contain a DNA / DNA double strand. In some embodiments, the double-stranded nucleic acid may contain an RNA / DNA double strand. The ligase may include one or more of the following: T4 DNA ligase, SplintR ligase, T3 DNA ligase, T7 DNA ligase, E. coli DNA ligase, Taq ligase, RtcB ligase, or a combination thereof.

[0057] A chemical reaction that forms a covalent bond between one end of a probe and another end of the probe (for example, two different ends of the same probe) may involve a reaction between one or more chemically reactive moieties. One or more chemically reactive moieties may include chemoselective reagents. One or more chemically reactive moieties may consist of tetrazine, alkyne, azide, trans-cyclooctene, maleimide, N-hydroxysuccinimide ester, amine, carboxylic acid, hydroxyl, cyclopropenone, thiol, norbornene, or a combination thereof. A first probe (firs probe) may contain one or more chemically reactive moieties. The first probe may contain a first reactive chemical moiety at a first end and a second reactive chemical moiety at a second end. The first chemically reactive moieties may contain tetrazine, alkyne, azide, trans-cyclooctene, maleimide, N-hydroxysuccinimide ester, amine, carboxylic acid, hydroxyl, cyclopropenone, thiol, norbornene, or a combination thereof. The second chemically reactive moiety may include tetrazine, alkyne, azide, trans-cyclooctene, maleimide, N-hydroxysuccinimide ester, amine, carboxylic acid, hydroxyl, cyclopropenone, thiol, norbornene, or a combination thereof. The ligation step may include a reaction between the first and second chemically reactive moieties.

[0058] The first and second ends of the first probe may be directly adjacent to each other when the first probe is ligated to the sample (for example, the first and second ends of the first probe may be ligated without any gap-filling reaction). For example, if the sample contains a nucleic acid analyte bound to the first probe, the first end of the first probe may hybridize to one portion of the nucleic acid analyte, and the second end of the first probe may hybridize to another portion of the nucleic acid analyte, and there may be no gap (i.e., a single-stranded region on the nucleic acid analyte) between the 5' end of the first end and the 3' end of the second end, or there may be no gap between the 3' end of the first end and the 5' end of the second end. In some cases, the first and second ends may be separated by one or more nucleotides. For example, if the sample contains a nucleic acid analyte bound to a first probe, the first end of the first probe may hybridize to a portion of the nucleic acid analyte, the second end of the first probe may hybridize to another portion of the nucleic acid analyte, there may be a gap (i.e., a single-stranded region on the nucleic acid analyte) between the 5' end of the first end and the 3' end of the second end, or there may be a gap between the 3' end of the first end and the 5' end of the second end. A gap-filling reaction may be performed to allow the first and second ends of a probe to be directly adjacent to each other (e.g., the 5' end of the first end can be directly ligated to the 3' end of the second end, or the 3' end of the first end can be directly ligated to the 5' end of the second end using a ligase). The gap-filling reaction may involve extending one or two ends of the first probe by using polymerase and / or transcriptase to incorporate nucleotides according to the sequence of nucleic acids bound by the first probe.

[0059] The first probe may ligate itself to form a circular probe. The circular probe may be a circular nucleic acid. The circular nucleic acid may be single-stranded, double-stranded, or a combination thereof. The circular nucleic acid may bind to an analyte. In some cases, the analyte to which the circular nucleic acid binds may be the first analyte. The circular nucleic acid may bind to a probe. In some cases, the circular nucleic acid may bind to a second probe. The circular nucleic acid may contain at least one barcode. If the circular nucleic acid contains one or more barcodes, each of the one or more barcodes may be the same or different. For example, each barcode may contain nucleic acid, and the nucleic acid may contain the same sequence. In other cases, each barcode may contain nucleic acid, and one or more nucleotides of the nucleic acid in each barcode may differ across one or more barcodes.

[0060] The second probe may include at least one binding site configured to bind to the analyte, the probe, or a combination thereof. In some cases, at least one binding site of the second probe may be configured to bind to one or more portions of the analyte. One or more portions of the analyte may include the second analyte. In some cases, at least one binding site of the second probe may be configured to bind to one or more portions of the probe. In some cases, one or more portions of the probe may be one or more portions of the first probe. For example, the binding site of the second probe may be configured to bind to a portion of the analyte. The binding site of the second probe may include nucleic acids. At least one binding site of the second probe may include a nucleic acid sequence. The nucleic acid sequence of at least one binding site of the second probe may have a nucleotide length of at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, or longer. The nucleic acid of at least one binding site of the second probe may have a nucleotide length of up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 6, up to about 7, up to about 8, up to about 9, up to about 10, up to about 15, up to about 20, up to about 25, up to about 30, up to about 40, up to about 50, up to about 60, up to about 70, up to about 80, up to about 90, up to about 100, or shorter. The nucleic acid may have a length of about 1 to about 100, about 2 to about 90, about 3 to about 80, about 4 to about 70, about 5 to about 60, about 6 to about 50, about 7 to about 40, about 8 to about 30, about 9 to about 25, or about 10 to about 20 nucleotides. The nucleic acid of at least one binding site of the second probe may contain a variety of nucleotides, including but not limited to A, G, C, T, U, or combinations thereof. In some cases, the nucleic acid may contain modifications. Modifications may include methylation, phosphate modification, or combinations thereof.Modifications may include sugar modifications, sugar / skeleton modifications, skeleton modifications, base modifications, non-natural base pairs, or combinations thereof. In some cases, sugar modifications may include 2'-fluoro, 2'-O-methyl, 2'-fluoroarabinose nucleic acids, hexitol nucleic acids, 2'-O-methoxyethyl, (1'-3')-β-L-ribonucleic acid, α-L-threose nucleic acid, 3'-2'phosphonomethyl-threosyl nucleic acid, 2'-deoxyxyl nucleic acid, phosphorothioates, alkylphosphonate nucleic acids, peptide nucleic acids, or combinations thereof.

[0061] The second probe may include a binding site (e.g., a fourth binding site of the second probe). The binding site (e.g., a fourth binding site of the second probe) may be configured to bind to the first probe. The binding site of the second probe (e.g., a fourth binding site of the second probe) may include a nucleic acid sequence. The nucleic acid sequence of the binding site of the second probe (e.g., a fourth binding site of the second probe) may have a nucleotide length of at least about 2, at least about 4, at least about 6, at least about 8, at least about 10, at least about 12, at least about 14, at least about 16, at least about 18, at least about 20, at least about 22, at least about 24, at least about 26, at least about 28, at least about 30, at least about 32, at least about 34, at least about 36, at least about 38, at least about 40, at least about 42, at least about 44, at least about 46, at least about 48, at least about 50, or longer. The nucleic acid at the binding site of the second probe (for example, the fourth binding site of the second probe) may have a nucleotide length of up to approximately 2, up to approximately 4, up to approximately 6, up to approximately 8, up to approximately 10, up to approximately 12, up to approximately 14, up to approximately 16, up to approximately 18, up to approximately 20, up to approximately 22, up to approximately 24, up to approximately 26, up to approximately 28, up to approximately 30, up to approximately 32, up to approximately 34, up to approximately 36, up to approximately 38, up to approximately 40, up to approximately 42, up to approximately 44, up to approximately 46, up to approximately 48, up to approximately 50, or shorter. The nucleic acid at the binding site of the second probe (for example, the fourth binding site of the second probe) may be approximately 2 to 50, 4 to 48, 6 to 46, 8 to 44, 10 to 42, 12 to 40, 14 to 38, 16 to 36, 18 to 34, 20 to 32, 22 to 30, or 24 to 28 nucleotides in length.

[0062] The second probe may include a binding site (e.g., a fifth binding site of the second probe). The binding site of the second probe (e.g., a fifth binding site of the second probe) may be configured to bind to the second analyte. The binding site (e.g., a fifth binding site of the second probe) may include nucleic acid. The nucleic acid of the binding site of the second probe (e.g., a fifth binding site of the second probe) may have a nucleotide length of at least about 2, at least about 4, at least about 6, at least about 8, at least about 10, at least about 12, at least about 14, at least about 16, at least about 18, at least about 20, at least about 22, at least about 24, at least about 26, at least about 28, at least about 30, at least about 32, at least about 34, at least about 36, at least about 38, at least about 40, at least about 42, at least about 44, at least about 46, at least about 48, at least about 50, or longer. The nucleic acid at the binding site of the second probe (for example, the fifth binding site of the second probe) may have a nucleotide length of up to approximately 2, up to approximately 4, up to approximately 6, up to approximately 8, up to approximately 10, up to approximately 12, up to approximately 14, up to approximately 16, up to approximately 18, up to approximately 20, up to approximately 22, up to approximately 24, up to approximately 26, up to approximately 28, up to approximately 30, up to approximately 32, up to approximately 34, up to approximately 36, up to approximately 38, up to approximately 40, up to approximately 42, up to approximately 44, up to approximately 46, up to approximately 48, up to approximately 50, or shorter. The nucleic acid of the binding site of the second probe (for example, the fifth binding site of the second probe) may be approximately 2 to 50, 4 to 48, 6 to 46, 8 to 44, 10 to 42, 12 to 40, 14 to 38, 16 to 36, 18 to 34, 20 to 32, 22 to 30, or 24 to 28 nucleotides in length.

[0063] In some embodiments, the methods described herein may include a gap-filling reaction. The gap-filling reaction may be facilitated by a binding event between a first probe and a first analyte, where the two ends of the first probe are separated by a certain distance upon binding to the first analyte. For example, both the first probe and the first analyte may contain nucleic acids, and the first probe may hybridize to the first analyte in two parts such that when the two ends of the first probe hybridize to the first analyte, they form a double-stranded region. The double-stranded region may be separated by a single-stranded region. The single-stranded region may contain a portion of the first analyte that does not hybridize to the first probe. The single-stranded region of the first analyte may be considered a gap, and the gap-filling reaction may extend one or both ends of the first probe by adding nucleotides to the nucleic acid of the first probe according to the sequence of the first analyte that is not bound by the first probe. In a gap-filling reaction, the first analyte may serve as a template for extending and filling the first probe. The first probe may comprise a nucleic acid sequence, and the gap may comprise a nucleic acid sequence that is not bound by the first probe and comprises a single-stranded portion of the first analyte located between the first and second ends of the first probe. In some cases, the gap may comprise at least one nucleotide of the first analyte. In some cases, the gap between one end of the first probe and another end of the first probe when hybridized to the first analyte may comprise one or more nucleotides. In some cases, the gap distance between one end of the first probe and another end of the first probe when bound to the first analyte may be at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, or more nucleotides.In some cases, the gap distance between one end of the first probe and another end of the first probe when bound to the first analyte may be at most about 1, at most about 2, at most about 3, at most about 4, at most about 5, at most about 6, at most about 7, at most about 8, at most about 9, at most about 10, at most about 15, at most about 20, at most about 25, at most about 30, at most about 40, at most about 50, at most about 60, at most about 70, at most about 80, at most about 90, at most about 100, or more nucleotides. In some cases, the gap distance between one end of the first probe and the other end of the first probe when bound to the first analyte may be approximately 1 to 100, 2 to 90, 3 to 80, 4 to 70, 5 to 60, 6 to 50, 7 to 40, 8 to 30, 9 to 25, or 10 to 20 nucleotides. The gap-filling reaction may involve using an enzyme to ligate the two ends of the first probe. The enzyme may be a DNA polymerase, an RNA polymerase, or a combination thereof. Examples of DNA polymerases include Q5 High-Fidelity DNA polymerase, Q5U Hot Start High-Fidelity DNA polymerase, and Phusion High-Fidelity DNA polymerase. *Routine PCR, OneTaq DNA polymerase, Taq DNA polymerase, LongAmp Taq DNA polymerase, Hemo KlenTaq, Epimark Hot Start Taq DNA polymerase, isothermal amplification and strand substitution, Bst DNA polymerase, Bst DNA polymerase, Bst 2.0 DNA polymerase, Bst 3.0 DNA polymerase, Bsu DNA polymerase, large fragment, phi29 DNA polymerase, phi29-XT DNA polymerase, T7 DNA polymerase (unmodified), Sulfolobus DNA polymerase IV, Therminator™ DNA polymerase, DNA polymerase I (E. coli), DNA polymerase I, large (Krenow) fragment, Krenow fragment (3'→5'exo-), T4 DNA polymerase, Legacy polymerase, Vent DNA polymerase, Vent(exo-) DNA polymerase, Deep Vent DNA polymerase, Deep Vent(exo-)DNA polymerase, or a combination thereof, may be used.

[0064] Another aspect of the present disclosure provides a method for detecting an analyte in a sample. The method may include providing a first probe, a second probe, a third probe, or any combination thereof. The first probe may include a first binding site (e.g., a first binding site of the first probe) configured to bind to a first analyte. The first probe may include a second binding site (e.g., a second binding site of the first probe) configured to bind to a second probe. The first probe may include one or more barcodes. The first probe may include a first end (e.g., a first end of the first probe). The first probe may include a second end (e.g., a second end of the second probe). The second probe may include a third binding site (e.g., a third binding site of the first probe) configured to bind to the first probe. The second probe may include a first binding site (e.g., a fourth binding site of the second probe) configured to bind to a third probe. The second probe may include a second binding site (e.g., a fifth binding site of the second probe) configured to connect to the second analyte. The third probe may include a first binding site (e.g., a sixth binding site of the third probe) configured to connect to the second probe. The third probe may include a second binding site (e.g., a seventh binding site of the third probe) configured to connect to the first analyte. The third probe may include a first end (e.g., a third end of the third probe), the first end of the third probe (e.g., a third end of the third probe) may be adjacent to the first end of the first probe (e.g., a first end of the first probe). The third probe may include a second end (e.g., a fourth end of the third end). The second end of the third probe (for example, the fourth end of the third probe) may be adjacent to the second end of the first probe (for example, the second end of the second probe).A sample containing multiple analytes, including a first analyte and / or a second analyte, can be brought into contact with a first probe, a second probe, and / or a third probe, such that (i) a first probe can be coupled to a first analyte, (ii) a second probe can be coupled to a second analyte, (iii) a third probe can be coupled to a first analyte, (iv) a first probe can be coupled to a second analyte, and (v) a third probe can be coupled to a second probe. The first end of the first probe (e.g., the first end of the first probe) can be ligated to the first end of the third probe (e.g., the third end of the third probe), and the second end of the second probe (e.g., the second end of the first probe) can be ligated to the second end of the third probe (e.g., the fourth end of the third probe) to form a cyclic oligonucleotide. A cyclic oligonucleotide can be amplified to produce one or more amplification products. These amplification products may include the complement of the barcode. The complement of the barcode or its derivatives can be detected using multiple detection probes, thereby allowing for the determination of the proximity between the first analyte and the second analyte.

[0065] Another aspect of the present disclosure provides a method for detecting an analyte in a sample using the components shown in Figure 11. The method provides (a) a first probe (1111), a second probe (1110), and a third probe (1112), wherein the first probe (1111) includes (i) a first binding site (1101) configured to connect to a first analyte (1109), (ii) a second binding site (1102) configured to connect to the second probe (1110), (iii) a barcode, (iv) a first end, and (v) a second end, and the second probe (1110) includes (i) the first probe (1111) (ii) a third binding site (1103) configured to connect, (ii) a fourth binding site (1104) configured to connect to a third probe (1112), and (iii) a fifth binding site (1105) configured to connect to a second analyte (1108), wherein the third probe (1112) has (i) a sixth binding site (1106) configured to connect to a second probe (1110), (ii) a seventh binding site (1107) configured to connect to a first analyte (1109), and (iii) a third end. (i) a first probe (1111) connected to a first analyte (1109), (ii) a second probe (1110) connected to a second analyte (1108), (iii) a third probe (1112) connected to a first analyte (1109), and (iv) a first probe (1111) connected to a second analyte (1110). (v) Contacting a sample containing multiple analytes, including the first analyte (1109) and the second analyte (1108), with the first probe (1111), the second probe (1110), and the third probe (1112) such that the third probe (1112) is connected to the second probe (1110); (c) Forming a cyclic alkyl group by ligating the first and third ends and the second and fourth ends; and (d) Amplifying the cyclic alkyl group.(e) generating an amplified product, wherein the amplified product includes the complement of the barcode; and (f) using a plurality of detection probes to detect the complement of the barcode or a derivative thereof, thereby determining the proximity between the first analyte (1109) and the second analyte (1108).

[0066] Figure 13 schematically illustrates an example of detecting proximity between a first analyte and a second analyte using a first probe, a second probe, and a third probe. In this example, a first probe, a second probe, and a third probe are provided (1301). The first probe, the second probe, and the third probe may include, for example, nucleic acids. A sample containing the first analyte and the second analyte may be brought into contact with the first probe, the second probe, and the third probe (1302). For example, the first probe may bind to the first analyte, the second probe may bind to the second analyte, and the third probe may bind to the first analyte. A cyclic oligonucleotide may be formed by ligating one end of the first probe to one end of the third probe and another end of the first probe to another end of the third probe (1303). The cyclic oligonucleotide can be amplified to produce one or more amplified products containing the complement of the barcode sequence of the first probe (1304). The complement of the barcode sequence of the first probe may be, for example, the inverse complement of the barcode sequence. The barcode complement can be detected to determine the proximity between the first analyte and the second analyte (1305). For example, multiple detection probes may be added to the sample, and a subset of multiple detection probes may be conjugated to one or more amplified products to reveal at least a portion of the barcode complement of the first probe.

[0067] In some cases, the first probe may include various binding sites configured for various purposes. The first probe may include at least one binding site configured to bind to at least one analyte, at least one probe, or at least a portion of a combination thereof. For example, the first probe may include a binding site configured to bind to the first analyte. The first binding site of the first probe may be adjacent to a binding site in the third probe. The first probe may include one or more binding sites configured to bind to one or more portions of the first analyte. The first probe may include binding sites configured to bind to one or more portions of the second probe. The first binding site of the first probe may be adjacent to a binding site in the third probe. The first probe may include one or more binding sites configured to bind to the second probe.

[0068] The first probe may include a barcode. The barcode may include nucleic acids. The nucleic acids in the barcode may include, but are not limited to, combinations of nucleotides including, A, C, G, T, U, or combinations thereof. The barcode may provide information related to the first analyte, the second analyte, or combinations thereof. For example, the information related to the first analyte, the second analyte, or combinations thereof may be sequence information, positional information, expression level information, proximity information, or combinations thereof. The barcode may represent the proximity between the first analyte and the second analyte. For example, detection of a barcode may indicate that the first analyte is within 100-1000 nm of the second analyte. The barcode may be adjacent to the first binding site, the second binding site, or combinations thereof. For example, the barcode of the first probe may include nucleic acids, and the sequence of the nucleic acids may be within 0-25 nucleotides of the first binding site of the first probe. The first binding site may include the barcode or a portion thereof. For example, the barcode of the first probe may contain a nucleic acid sequence, and the first binding site of the first probe may contain another nucleic acid sequence. The nucleic acid sequence of the barcode and the nucleic acid sequence of the first binding site may be the same. For example, the nucleic acid sequence of the first binding site of the first probe may also be a barcode sequence. The second binding site may contain a barcode or a portion thereof. For example, the barcode of the first probe may contain a nucleic acid sequence, and the second binding site of the first probe may contain another nucleic acid sequence. The nucleic acid sequence of the barcode and the nucleic acid sequence of the second binding site may be the same. For example, the nucleic acid sequence of the second binding site of the first probe may also be a barcode sequence.

[0069] The first probe may include one or more modifications. These modifications may be configured to ligate the first probe to the third probe. For example, one or more modifications may be at the end of the first probe, and one or more modifications may be at the end of the third probe. The modifications of the first probe and the modifications of the third probe may react with each other. In some cases, the first probe may contain nucleic acids, and the modifications may contain one or more nucleic acid modifications. Examples of nucleic acid modifications include methylation, phosphate modification, or a combination thereof. Examples of modifications include sugar modification, sugar / skeleton modification, skeleton modification, base modification, non-native base pairing, or a combination thereof. In some cases, sugar modifications may include 2'-fluoro, 2'-O-methyl, 2'-fluoroarabinose nucleic acids, hexitol nucleic acids, 2'-O-methoxyethyl, (1'-3')-β-L-ribonucleic acid, α-L-threose nucleic acid, 3'-2'phosphonomethyl-threosyl nucleic acid, 2'-deoxyxyl nucleic acid, phosphorothioates, alkylphosphonate nucleic acids, peptide nucleic acids, or combinations thereof.

[0070] One or more modifications of the nucleic acid of the first probe may include one or more chemically reactive moieties. One or more chemically reactive moieties of the nucleic acid of the first probe may include chemoselective reagents. One or more chemically reactive moieties may consist of tetrazine, alkyne, azide, trans-cyclooctene, maleimide, N-hydroxysuccinimide ester, amine, carboxylic acid, hydroxyl, cyclopropenone, thiol, norbornene, or a combination thereof.

[0071] One or more modifications of the nucleic acid of the first probe may be located at one or both ends of the first probe. One or more modifications of the nucleic acid of the first probe may be located within the first probe. The first probe may contain nucleic acid, and the nucleic acid may contain modifications at the 5' end, the 3' end, or a combination thereof. If the first probe contains one or more modifications, each of the one or more modifications may be the same or different. In some cases, some of the one or more modifications may be the same and some may be different. For example, if the first probe contains nucleic acid and the nucleic acid contains two phosphorylation modifications, the modifications may be considered the same. If the first probe contains nucleic acid and the nucleic acid contains several, including one phosphorylation modification and one hydroxyl modification, the modifications may be considered different. One or more modifications of the nucleic acid of the first probe may include a 5' phosphate group.

[0072] A first probe can be ligated to a third probe. The first probe can be ligated to a third probe at one or more positions. For example, one end of the first probe can be ligated to one end of the third probe, and another end of the first probe can be ligated to another end of the third probe. The first probe can be ligated to a third probe as a result of the first and third probes binding to a first analyte. For example, the first and third probes can bind to a first analyte, and the end of the first probe can be directly adjacent to the end of the third probe, thereby enabling a ligation reaction between the first end of the first probe and the end of the third probe. The first probe can be ligated to a third probe as a result of the first and third probes binding to a second probe. For example, the first probe and the third probe may bind to the second probe, and the end of the first probe may be directly adjacent to the end of the third probe, thereby enabling a ligation reaction between the end of the first probe and the end of the third probe. In some cases, the first probe may contain nucleic acid, and the nucleic acid may contain a 5' end and a 3' end. In some cases, the third probe may contain nucleic acid, and the nucleic acid may contain a 5' end and a 3' end. The 3' end of the first probe and the 5' end of the third probe may be ligated to each other, the 5' end of the first probe and the 3' end of the third probe may be ligated to each other, or a combination thereof.

[0073] A first probe may be ligated to a third probe using a ligase. The ligase may be one or more ligases. In some cases, the ligase may include mammalian ligases. In some cases, the ligase may be bacterial ligases. The ligase may be DNA ligase I, DNA ligase II, DNA ligase III, DNA ligase IV, or a combination thereof. In some cases, the ligase may include RNA ligases. In some cases, the ligase may ligate the 3' nucleotide of one nucleic acid to the 5' nucleotide of another nucleic acid. In some cases, the ligase may ligate the 3' end of one nucleic acid to the 5' end of the same nucleic acid. For example, a nucleic acid may contain a 5' end and a 3' end, and the 5' and 3' ends may be ligated to each other to form a circular nucleic acid. The ligase may ligate two nucleotides that are part of a double-stranded nucleic acid. In some cases, double-stranded nucleic acids may contain nicks, and the location of the nicks may be ligated by a ligase. In some embodiments, double-stranded nucleic acids may include DNA / DNA double strands. In some embodiments, double-stranded nucleic acids may include RNA / DNA double strands. The ligase may include one or more of the following: T4 DNA ligase, SplintR ligase, T3 DNA ligase, T7 DNA ligase, E. coli DNA ligase, Taq ligase, RtcB ligase, or a combination thereof.

[0074] One or more ends of the first probe can be ligated to one or more ends of the third probe. One end of the first probe (e.g., the first end of the first probe) can be ligated to one end of the third probe (e.g., the third end of the third probe). One end can be ligated to one end of the third probe. One end can be ligated to another end of the third probe (e.g., the fourth end of the third probe). Another end of the first probe (e.g., the second end of the first probe) can be ligated to one end of the third probe. One end can be ligated to one end of the fourth probe. One end of the first probe can be ligated to one end of the second end of the first probe.

[0075] The end of the first probe may be in direct proximity to the end of the third probe when hybridizing with the analyte in the sample. In some cases, the first probe may hybridize with the analyte to form a double-stranded region containing the end of the first probe. In some cases, the third probe may hybridize with the same analyte to which it is bound by the first probe to form another double-stranded region containing the end of the third probe. The double-stranded region containing the end of the first probe may be separated from the double-stranded region containing the end of the third probe by a gap containing one or more nucleotides of the single-stranded portion of the first analyte. For example, the first probe and the third probe may each contain nucleic acids and bind to the first analyte to generate a first double-stranded region formed between the first probe and the first analyte, and a second double-stranded region formed between the first analyte and the third probe. The first double-stranded region and the second double-stranded region may be separated by a single-stranded region containing the nucleic acid sequence of the first analyte, which is not bound by either the first or third probe. In this case, the single-stranded region is considered a gap separating the first probe from the third probe by at least one nucleotide. In some cases, one or more ends of the first probe are directly adjacent to one or more ends of the third probe (for example, the first and third probes may be ligated without any gap-filling reaction). For example, the first probe and the third probe may each contain nucleic acid and bind to the first analyte to produce a first double-stranded region formed between them and the first probe, and a second double-stranded region formed between the first analyte and the third probe. The first and second double-stranded regions may not be separated by a single-stranded region containing the nucleic acid sequence of the first analyte, which is not bound by either the first or third probe. In this case, the single-stranded regions can be considered directly adjacent, without a gap separating the first probe from the third probe by at least one nucleotide (for example, the first and third probes can be ligated without any gap-filling reaction).In some cases, when bound to a sample, one or more ends of the first probe are separated from one or more ends of the third probe by one or more nucleotides in the single-stranded region of the first analyte. A gap-filling reaction may be performed to fill the gap between the ends of the first probe and the ends of the third probe with nucleotides. In some cases, when bound to a sample, the first and third ends are not separated by any nucleotides in the single-stranded region of the first analyte. In some cases, when bound to a sample, the first and third ends are separated by any one or more nucleotides in the single-stranded region of the first analyte. In some cases, when hybridizing to the first analyte, the second and fourth ends are not separated by any nucleotides in the single-stranded region of the first analyte. In some cases, when bound to a sample, the second and fourth ends are separated by any one or more nucleotides in the single-stranded region of the first analyte.

[0076] A first probe may be ligated to a third probe using one or more chemically reactive moieties. In some cases, the third probe may contain one or more chemically reactive moieties, and the first probe may contain one or more chemically reactive moieties. One or more chemically reactive moieties of the third probe may react with one or more chemically reactive moieties of the first probe, thereby ligating the third probe and the first probe. The first probe may contain a first chemically reactive moiety at its first end. The third probe may contain a second chemically reactive moiety at its third end. The first and / or second chemically reactive moieties may include tetrazine, alkyne, azide, trans-cyclooctene, maleimide, N-hydroxysuccinimide ester, amine, carboxylic acid, hydroxyl, cyclopropenone, thiol, norbornene, or a combination thereof. The ligation step may include reacting the first chemically reactive moiety with the second chemically reactive moiety. The first probe may contain a third chemically reactive moiety at its second end. The third probe may contain a fourth chemically reactive moiety at its fourth end. The third and / or fourth chemically reactive moieties may include tetrazine, alkyne, azide, trans-cyclooctene, maleimide, N-hydroxysuccinimide ester, amine, carboxylic acid, hydroxyl, cyclopropenone, thiol, norbornene, or a combination thereof. The ligation step may include reacting the third chemically reactive moiety with the fourth chemically reactive moiety.

[0077] A first probe may be ligated to a third probe to form a cyclic oligonucleotide. The cyclic oligonucleotide may contain a cyclic nucleic acid. The cyclic nucleic acid may be single-stranded, double-stranded, or a combination thereof. The cyclic nucleic acid may bind to an analyte. In some cases, the analyte may be the first analyte. The cyclic nucleic acid may bind to a second probe. The cyclic nucleic acid may contain at least one barcode. In some cases, the cyclic nucleic acid may contain one or more barcodes. Each of the one or more barcodes of the cyclic nucleic acid may be the same or different. For example, the cyclic nucleic acid may contain two barcodes, and the two barcodes may contain the same nucleic acid sequence and may be considered the same. In some cases, the cyclic nucleic acid may contain two barcodes, and the sequences of the two barcodes may differ by one or more nucleotides and may be considered different.

[0078] The first probe may include a binding site (e.g., a first binding site of the first probe). The binding site (e.g., a first binding site of the first probe) may be configured to bind to the first analyte. The binding site of the first probe (e.g., a first binding site of the first probe) may include a nucleic acid sequence. The nucleic acid sequence of the binding site of the first probe (e.g., a first binding site of the first probe) may have a nucleotide length of at least about 2, at least about 4, at least about 6, at least about 8, at least about 10, at least about 12, at least about 14, at least about 16, at least about 18, at least about 20, at least about 22, at least about 24, at least about 26, at least about 28, at least about 30, at least about 32, at least about 34, at least about 36, at least about 38, at least about 40, at least about 42, at least about 44, at least about 46, at least about 48, at least about 50, or longer. Nucleic acid sequences can have nucleotide lengths of up to approximately 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, or shorter. Nucleic acid sequences may be approximately 2 to 50, 4 to 48, 6 to 46, 8 to 44, 10 to 42, 12 to 40, 14 to 38, 16 to 36, 18 to 34, 20 to 32, 22 to 30, or 24 to 28 nucleotides long.

[0079] The first probe may include a second binding site. The second binding site may be configured to bind to the second probe. The second binding site of the first probe may include a nucleic acid. The nucleic acid of the second binding site of the first probe may have a nucleotide length of at least about 2, at least about 4, at least about 6, at least about 8, at least about 10, at least about 12, at least about 14, at least about 16, at least about 18, at least about 20, at least about 22, at least about 24, at least about 26, at least about 28, at least about 30, at least about 32, at least about 34, at least about 36, at least about 38, at least about 40, at least about 42, at least about 44, at least about 46, at least about 48, at least about 50, or longer. Nucleic acids can have a maximum nucleotide length of approximately 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, or shorter. Nucleic acids may be approximately 2–50, 4–48, 6–46, 8–44, 10–42, 12–40, 14–38, 16–36, 18–34, 20–32, 22–30, or 24–28 nucleotides long.

[0080] The second probe may include a binding site (e.g., a third binding site of the first probe). The binding site (e.g., a third binding site of the first probe) may be configured to bind to the first probe. The binding site (e.g., a third binding site of the first probe) may include a nucleic acid sequence. The nucleic acid sequence may have a nucleotide length of at least about 2, at least about 4, at least about 6, at least about 8, at least about 10, at least about 12, at least about 14, at least about 16, at least about 18, at least about 20, at least about 22, at least about 24, at least about 26, at least about 28, at least about 30, at least about 32, at least about 34, at least about 36, at least about 38, at least about 40, at least about 42, at least about 44, at least about 46, at least about 48, at least about 50, or longer. Nucleic acid sequences can have nucleotide lengths of up to approximately 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, or shorter. Nucleic acid sequences may be approximately 2 to 50, 4 to 48, 6 to 46, 8 to 44, 10 to 42, 12 to 40, 14 to 38, 16 to 36, 18 to 34, 20 to 32, 22 to 30, or 24 to 28 nucleotides long.

[0081] The second probe may include a fourth binding site. The fourth binding site may be configured to bind to the third probe. The fourth binding site may include a nucleic acid sequence. The nucleic acid sequence of the fourth binding site of the second probe may have a nucleotide length of at least about 2, at least about 4, at least about 6, at least about 8, at least about 10, at least about 12, at least about 14, at least about 16, at least about 18, at least about 20, at least about 22, at least about 24, at least about 26, at least about 28, at least about 30, at least about 32, at least about 34, at least about 36, at least about 38, at least about 40, at least about 42, at least about 44, at least about 46, at least about 48, at least about 50, or longer. Nucleic acid sequences can have nucleotide lengths of up to approximately 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, or shorter. Nucleic acid sequences may be approximately 2 to 50, 4 to 48, 6 to 46, 8 to 44, 10 to 42, 12 to 40, 14 to 38, 16 to 36, 18 to 34, 20 to 32, 22 to 30, or 24 to 28 nucleotides long.

[0082] The second probe may include a binding site (e.g., a fifth binding site of the second probe). The binding site (e.g., a fifth binding site of the second probe) may be configured to bind to the second analyte. The binding site (e.g., a fifth binding site of the second probe) may include a nucleic acid sequence. The nucleic acid sequence of the binding site of the second probe (e.g., a fifth binding site of the second probe) may have a nucleotide length of at least about 2, at least about 4, at least about 6, at least about 8, at least about 10, at least about 12, at least about 14, at least about 16, at least about 18, at least about 20, at least about 22, at least about 24, at least about 26, at least about 28, at least about 30, at least about 32, at least about 34, at least about 36, at least about 38, at least about 40, at least about 42, at least about 44, at least about 46, at least about 48, at least about 50, or longer. Nucleic acid sequences can have nucleotide lengths of up to approximately 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, or shorter. Nucleic acid sequences may be approximately 2 to 50, 4 to 48, 6 to 46, 8 to 44, 10 to 42, 12 to 40, 14 to 38, 16 to 36, 18 to 34, 20 to 32, 22 to 30, or 24 to 28 nucleotides long.

[0083] The binding site (e.g., the fifth binding site of the second probe) may contain a polypeptide. The polypeptide of the binding site (e.g., the fifth binding site of the second probe) may contain a protein or a portion thereof. In some cases, the polypeptide may contain an antibody or an antibody fragment. In some cases, the polypeptide may contain a portion or an antibody fragment of an antibody. If the binding site of the second probe (e.g., the fifth binding site of the second probe) may contain a polypeptide, the binding site (e.g., the fifth binding site of the second probe) may be linked to the fourth binding site of the second probe via a linker. The linker may contain a variety of chemical groups, including one or more ethylene groups, one or more methylene groups, one or more polyethylene glycol groups, or a combination thereof. The linker may link the binding site of the second probe (e.g., the fifth binding site of the second probe) to the fourth binding site of the second probe via one or more covalent bonds. The linker may connect the binding site of the second probe (e.g., the fifth binding site of the second probe) to the fourth binding site of the second probe via one or more non-covalent bonds. In some cases, the binding site of the second probe (e.g., the fifth binding site of the second probe) may connect to the fourth binding site of the second probe via a combination of one or more covalent interactions and one or more non-covalent interactions. For example, in some cases, the binding site of the second probe (e.g., the fifth binding site of the second probe) may contain a nucleic acid, the fourth binding site of the second probe may contain a nucleic acid, and the binding site (e.g., the fifth binding site of the second probe) and the fourth binding site may be connected by one or more phosphodiester bonds. In some cases, the second probe may contain an antibody and a nucleic acid, and the antibody may be connected to the nucleic acid via the linker. In some cases, the second probe may include an antibody configured to bind to a second analyte, an antibody configured to bind to the antibody that binds to the second analyte, and a nucleic acid linked to the antibody configured to bind to the antibody that binds to the second analyte.If the binding site of the second probe (for example, the fifth binding site of the second probe) contains a polypeptide, the polypeptide may have an amino acid length of at least about 2, at least about 4, at least about 6, at least about 8, at least about 10, at least about 12, at least about 14, at least about 16, at least about 18, at least about 20, at least about 22, at least about 24, at least about 26, at least about 28, at least about 30, at least about 32, at least about 34, at least about 36, at least about 38, at least about 40, at least about 42, at least about 44, at least about 46, at least about 48, at least about 50, or longer. Polypeptides can have amino acid lengths of up to approximately 2, up to approximately 4, up to approximately 6, up to approximately 8, up to approximately 10, up to approximately 12, up to approximately 14, up to approximately 16, up to approximately 18, up to approximately 20, up to approximately 22, up to approximately 24, up to approximately 26, up to approximately 28, up to approximately 30, up to approximately 32, up to approximately 34, up to approximately 36, up to approximately 38, up to approximately 40, up to approximately 42, up to approximately 44, up to approximately 46, up to approximately 48, up to approximately 50, or shorter. Polypeptides may have amino acid lengths of approximately 2 to 50, 4 to 48, 6 to 46, 8 to 44, 10 to 42, 12 to 40, 14 to 38, 16 to 36, 18 to 34, 20 to 32, 22 to 30, or 24 to 28 amino acids.

[0084] The third probe may be configured to bind to various components. In some cases, the third probe may bind to one or more analytes, one or more probes, or a combination thereof. The third probe may be configured to bind to the first analyte at one or more binding sites (e.g., the seventh binding site of the third probe). The third probe may be configured to bind to the second probe at one or more binding sites (e.g., the sixth binding site of the third probe).

[0085] The third probe may contain nucleic acids. The nucleic acids of the third probe may contain DNA, RNA, or a combination thereof. The nucleic acids of the third probe may contain single-stranded regions, double-stranded regions, or a combination thereof. The third probe may contain one or more modifications. One or more modifications of the third probe may be at one or more ends of the third probe, inside the third probe, or a combination thereof. If the third probe contains nucleic acids, one or more modifications may consist of one or more nucleic acid modifications. One or more modifications of the nucleic acids of the third probe may include sugar modifications, sugar / skeleton modifications, skeletal modifications, base modifications, non-native base pairs, or a combination thereof. In some cases, sugar modifications may include 2'-fluoro, 2'-O-methyl, 2'-fluoroarabinose nucleic acids, hexitol nucleic acids, 2'-O-methoxyethyl, (1'-3')-β-L-ribonucleic acid, α-L-threose nucleic acid, 3'-2'phosphonomethyl-threosyl nucleic acid, 2'-deoxyxyl nucleic acid, phosphorothioates, alkylphosphonate nucleic acids, peptide nucleic acids, or combinations thereof.

[0086] The third probe may recognize, ligate to, and / or bind to the first analyte. The first analyte may include various types of analytes, and depending on the type of analyte, the third probe may recognize specific features of the first analyte. For example, the third probe may recognize and bind to ribonucleic acid, and the specific features of ribonucleic acid may include sequence, ribonucleic acid secondary structure, ribonucleic acid tertiary structure, or a combination thereof. The first analyte may include ribonucleic acid. Ribonucleic acid may include, but is not limited to, messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), or a combination thereof. The RNA may be endogenous to the sample or exogenous to the sample. The RNA may be introduced into the sample by various means, including, but not limited to, the use of AAV or rAAV. If the second analyte includes RNA, the RNA may include one or more modifications. One or more modifications of the first analyte may include N6-methyladenosine, 5-methylcytosine, N1-methyladenosine, N7-methylguanosine, N4-acetylcytosine, pseudouridine, N1-methylpseudridine, or a combination thereof. The RNA may include single-stranded regions, double-stranded regions, or a combination thereof. The RNA may include multiple nucleotides. The multiple nucleotides may include A, C, U, G, or a combination thereof. In some cases, the RNA may include secondary structures, tertiary structures, or a combination thereof.

[0087] A third probe can recognize a first analyte. The first analyte that can be recognized by the third probe may contain nucleic acids. The nucleic acids of the first analyte may contain one or more single nucleotide polymorphisms. The third probe can bind to nucleic acids containing one or more single nucleotide polymorphisms. For example, the third probe may contain nucleic acids and can hybridize to nucleic acids containing one or more single nucleotide polymorphisms. By binding the third probe to nucleic acids containing one or more single nucleotide polymorphisms, it may be possible to ligate the third probe with the first probe. For example, by binding the third probe to nucleic acids containing one or more single nucleotide polymorphisms, the ends of the third probe may be adjacent to the ends of the first probe, and the ends of the third probe and the ends of the first probe may be ligated in the presence of a ligase. In some cases, the ligation of the third probe may include ligation of one end of the third probe with another end of the third probe. In some cases, the ligation of the third probe may include ligation between one end of the third probe and one end of the first probe.

[0088] In some cases, if the analyte contains nucleic acid and the nucleic acid may contain one or more single nucleotide polymorphisms, the third probe may not be able to recognize the analyte (e.g., it may not be able to bind to the analyte). Because the sequences of one or more single nucleotide polymorphisms are different from the complement or reverse complement of the third probe, the third probe may not be able to bind to nucleic acids containing one or more single nucleotide polymorphisms. Since the third probe may not be able to bind to nucleic acids containing one or more single nucleotide polymorphisms, the third probe may not be able to ligate in the presence of the analytes described herein. In some cases, the third probe may not need to recognize (e.g., bind to) an analyte containing one or more single nucleotide polymorphisms, and since the third probe may not be able to recognize the analyte, if the analyte contains one or more single nucleotide polymorphisms, the third probe may not need to ligate to the first probe. The analyte may be the first analyte.

[0089] In some cases, if the first analyte contains nucleic acid, and the nucleic acid may contain one or more modifications, the third probe may recognize the first analyte. The one or more modifications of the nucleic acid in the first analyte may include one or more methyl modifications. The one or more modifications of the nucleic acid in the first analyte may include N6-methyladenosine, 5-methylcytosine, N1-methyladenosine, N7-methylguanosine, N4-acetylcytosine, pseudouridine, N1-methylpseudridine, or a combination thereof. The third probe may bind to nucleic acid containing one or more modifications. Binding of the third probe to nucleic acid containing one or more modifications may enable ligation of the third probe with the first probe. For example, binding of the third probe to nucleic acid containing one or more modifications may result in the end of the third probe being adjacent to the end of the first probe, and the ends of the third probe and the first probe may be ligated in the presence of a ligase. In some cases, the ligation of the third probe may include ligation between one end of the third probe and another end of the third probe.

[0090] In some cases, if the analyte contains nucleic acid, and the nucleic acid may contain one or more modifications, the third probe may not be able to recognize the analyte. The third probe may not be able to bind to nucleic acid containing one or more modifications because the sequences of one or more modifications are different from the sequences recognized by the third probe. The third probe may not be ligated in the presence of the analytes described herein. In some cases, the third probe may not be able to recognize an analyte containing nucleic acid containing one or more modifications, and if the analyte contains one or more modifications, the third probe will not be ligated. The analyte may be the first analyte.

[0091] For example, the third probe may recognize DNA. The first probe may recognize, bind to, and / or ligate to DNA. The first analyte may contain DNA recognized by the third probe, the first probe, or a combination thereof. The first probe may recognize, bind to, and / or ligate to DNA. The DNA in the first analyte may associate with one or more histone molecules (e.g., DNA may form a complex with one or more histones). The DNA in the first analyte may associate with one or more nucleosomes (e.g., DNA may wrap around one or more histone molecules to form one or more nucleosomes). The DNA in the first analyte may associate with polymerase (e.g., DNA may interact with polymerase). DNA may associate with DNA polymerase. The DNA in the first analyte may be endogenous to the sample (e.g., DNA may be synthesized in one or more cells of the sample). The DNA of the first analyte may be exogenous to the sample (for example, DNA may be inserted into one or more cells of the sample from an external source). The DNA of the first analyte may be a combination of endogenous and exogenous DNA. The DNA of the first analyte may be single-stranded, double-stranded, or a combination thereof. The DNA of the first analyte may contain multiple nucleotides. The multiple nucleotides may contain A, C, T, G, or a combination thereof. The DNA of the first analyte may contain oligonucleotides. Oligonucleotides may be single-stranded, double-stranded, or a combination thereof. In some cases, oligonucleotides may contain secondary structures. In some cases, the DNA of the first analyte may contain secondary structures, tertiary structures, or a combination thereof. The DNA of the first analyte may contain modifications. Modifications may include methyl modifications.

[0092] The third probe may include one or more barcodes. One or more barcodes may include nucleic acids. The nucleic acids of the third probe may include, but are not limited to, combinations of nucleotides including, A, C, G, T, U, or combinations thereof.

[0093] One or more barcodes described herein may provide information relating to one or more analytes. For example, information relating to a first analyte, a second analyte, or a combination thereof may be sequence information, positional information, expression level information, proximity information, or a combination thereof. A barcode may represent the proximity between a first analyte and a second analyte. For example, detection of a barcode may indicate that the first analyte is within 100 to 1000 nm of the second analyte. A barcode may be adjacent to a binding site (e.g., a first binding site of a first probe), a second binding site, or a combination thereof. For example, the barcode of a first probe may contain nucleic acid, and the sequence of the nucleic acid may be within 0 to 25 nucleotides of the binding site of the first probe (e.g., a first binding site of a first probe). The binding site (e.g., a first binding site of a first probe) may include a barcode or a portion thereof. For example, the barcode of the first probe may include a nucleic acid sequence, the binding site of the first probe (e.g., the first binding site of the first probe) may include a nucleic acid sequence, and a portion of the nucleic acid sequence of the barcode and a portion of the nucleic acid sequence of the binding site (e.g., the first binding site of the first probe) may be the same nucleic acid sequence. The second binding site may include a barcode or a portion thereof. For example, the barcode of the first probe may include a nucleic acid sequence, the second binding site of the first probe may include a nucleic acid sequence, and a portion of the nucleic acid sequence of the barcode and a portion of the nucleic acid sequence of the second binding site may be the same nucleic acid sequence.

[0094] One or more barcodes on the third probe may provide information related to the first analyte, the second analyte, the third analyte, or a combination thereof. For example, information related to the first analyte, the second analyte, or a combination thereof may be sequence information, positional information, expression level information, proximity information, or a combination thereof. The barcode on the third probe may represent the proximity between the first analyte and the second analyte. For example, detection of a barcode may indicate that the first analyte is within 100-1000 nm of the second analyte. One or more barcodes may be adjacent to the sixth binding site, the seventh binding site, or a combination thereof. For example, the barcode on the third probe may contain nucleic acid, and the sequence of the nucleic acid in the barcode on the third probe may be within 0-25 nucleotides of the sixth binding site on the first probe. The sixth binding site on the third probe may contain a barcode or a portion thereof. For example, a portion of the nucleic acid sequence of a barcode may be the same sequence as a portion of the nucleic acid sequence of the sixth binding site. The seventh binding site may include a barcode or a portion thereof. Each of one or more barcodes may have at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least Approximately 29, at least approximately 30, at least approximately 31, at least approximately 32, at least approximately 33, at least approximately 34, at least approximately 35, at least approximately 36, at least approximately 37, at least approximately 38, at least approximately 39, at least approximately 40, at least approximately 41, at least approximately 42, at least approximately 43, at least approximately 44, at least approximately 45, at least approximately 46, at least approximately 47, at least approximately 48, at least approximately 49, at least approximately 50, at least approximately 51, at least approximately 52, at least approximately 53, at least approximately 54, at least approximately 55, at least approximately 56, at least approximately 57, at least approximately 58,At least about 59, at least about 60, at least about 61, at least about 62, at least about 63, at least about 64, at least about 65, at least about 66, at least about 67, at least about 68, at least about 69, at least about 70, at least about 71, at least about 72, at least about 73, at least about 74, at least about 75, at least about 76, at least about 77, at least about 78, at least about 79, at least about 80, at least about 81, at least about 82, at least about 83, at least about 84, at least about 85, at least about 86, at least about 87, at least about 88, at least about 89, at least about 90, at least about 91, at least about 92, at least They may contain, but are not limited to, a variety of lengths, including approximately 93, at least approximately 94, at least approximately 95, at least approximately 96, at least approximately 97, at least approximately 98, at least approximately 99, at least approximately 100, at least approximately 105, at least approximately 110, at least approximately 115, at least approximately 120, at least approximately 125, at least approximately 130, at least approximately 135, at least approximately 140, at least approximately 145, at least approximately 150, at least approximately 155, at least approximately 160, at least approximately 165, at least approximately 170, at least approximately 175, at least approximately 180, at least approximately 185, at least approximately 190, at least approximately 195, at least approximately 200, or more nucleotides. One or more barcodes can be up to approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and more At approximately 27, at a maximum of approximately 28, at a maximum of approximately 29, at a maximum of approximately 30, at a maximum of approximately 31, at a maximum of approximately 32, at a maximum of approximately 33, at a maximum of approximately 34, at a maximum of approximately 35, at a maximum of approximately 36, at a maximum of approximately 37, at a maximum of approximately 38, at a maximum of approximately 39, at a maximum of approximately 40, at a maximum of approximately 41, at a maximum of approximately 42, at a maximum of approximately 43, at a maximum of approximately 44, at a maximum of approximately 45, at a maximum of approximately 46, at a maximum of approximately 47, at a maximum of approximately 48, at a maximum of approximately 49, at a maximum of approximately 50, at a maximum of approximately 51, at a maximum of approximately 52, at a maximum of approximately 53,Up to approximately 54, up to approximately 55, up to approximately 56, up to approximately 57, up to approximately 58, up to approximately 59, up to approximately 60, up to approximately 61, up to approximately 62, up to approximately 63, up to approximately 64, up to approximately 65, up to approximately 66, up to approximately 67, up to approximately 68, up to approximately 69, up to approximately 70, up to approximately 71, up to approximately 72, up to approximately 73, up to approximately 74, up to approximately 75, up to approximately 76, up to approximately 77, up to approximately 78, up to approximately 79, up to approximately 80, up to approximately 81, up to approximately 82, up to approximately 83, up to approximately 84, up to approximately 85, up to approximately 86, up to approximately 87, up to approximately 88, up to approximately 89, up to approximately 9 nucleotides may have lengths of 0, up to approximately 91, up to approximately 92, up to approximately 93, up to approximately 94, up to approximately 95, up to approximately 96, up to approximately 97, up to approximately 98, up to approximately 99, up to approximately 100, up to approximately 105, up to approximately 110, up to approximately 115, up to approximately 120, up to approximately 125, up to approximately 130, up to approximately 135, up to approximately 140, up to approximately 145, up to approximately 150, up to approximately 155, up to approximately 160, up to approximately 165, up to approximately 170, up to approximately 175, up to approximately 180, up to approximately 185, up to approximately 190, up to approximately 195, up to approximately 200, or shorter. One or more barcodes correspond to approximately 1-200, 2-195, 3-190, 4-185, 5-180, 6-175, 7-170, 8-165, 9-160, 10-155, 11-150, 12-145, 13-140, 14-135, 15-130, 16-125, 17-120, 18-115, 19-110, 20-105, 21-100, 22-99, 23-98, 24-97, 25-96, 26-95, 27-94, Approximately 28-93, approximately 29-92, approximately 30-91, approximately 31-90, approximately 32-89, approximately 33-88, approximately 34-87, approximately 35-86, approximately 36-85, approximately 37-84, approximately 38-83, approximately 39-82, approximately 40-81, approximately 41-80, approximately 42-79, approximately 43-78, approximately 44-77, approximately 45-76, approximately 46-75, approximately 47-74, approximately 48-73, approximately 49-72, approximately 50-71, approximately 51-70, approximately 52-69, approximately 53-68, approximately 54-67, approximately 55-66, approximately 56-65, approximately 57-64,They can have lengths of approximately 58-63, 59-62, or 60-61 nucleotides.

[0095] The third probe may include a binding site (e.g., a sixth binding site of the third probe). The binding site (e.g., a sixth binding site of the third probe) may be configured to bind to (e.g., hybridize with) the second probe. The binding site of the third probe (e.g., a sixth binding site of the third probe) may include a nucleic acid sequence. The nucleic acid sequence of the binding site of the third probe (e.g., a sixth binding site of the third probe) may have a nucleotide length of at least about 2, at least about 4, at least about 6, at least about 8, at least about 10, at least about 12, at least about 14, at least about 16, at least about 18, at least about 20, at least about 22, at least about 24, at least about 26, at least about 28, at least about 30, at least about 32, at least about 34, at least about 36, at least about 38, at least about 40, at least about 42, at least about 44, at least about 46, at least about 48, at least about 50, or longer. Nucleic acid sequences can have nucleotide lengths of up to approximately 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, or shorter. Nucleic acid sequences may be approximately 2 to 50, 4 to 48, 6 to 46, 8 to 44, 10 to 42, 12 to 40, 14 to 38, 16 to 36, 18 to 34, 20 to 32, 22 to 30, or 24 to 28 nucleotides long.

[0096] The third probe may include a binding site (e.g., the seventh binding site of the third probe). The binding site of the third probe (e.g., the seventh binding site of the third probe) may be configured to bind to the first analyte or a portion thereof. The binding site of the third probe (e.g., the seventh binding site of the third probe) may include a nucleic acid sequence. The nucleic acid sequence of the binding site of the third probe (e.g., the seventh binding site of the third probe) may have a nucleotide length of at least about 2, at least about 4, at least about 6, at least about 8, at least about 10, at least about 12, at least about 14, at least about 16, at least about 18, at least about 20, at least about 22, at least about 24, at least about 26, at least about 28, at least about 30, at least about 32, at least about 34, at least about 36, at least about 38, at least about 40, at least about 42, at least about 44, at least about 46, at least about 48, at least about 50, or longer. Nucleic acid sequences can have nucleotide lengths of up to approximately 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, or shorter. Nucleic acid sequences may be approximately 2 to 50, 4 to 48, 6 to 46, 8 to 44, 10 to 42, 12 to 40, 14 to 38, 16 to 36, 18 to 34, 20 to 32, 22 to 30, or 24 to 28 nucleotides long.

[0097] Another aspect of this disclosure provides a method for detecting an analyte in a sample. The method may include providing a sample. The sample may include a first analyte and / or a second analyte. The first analyte may include a genetic anomaly. The first analyte may come into contact with a first binder. The first binder may include a barcode. The barcode's back complement can be detected with an accuracy of more than 90%. The barcode's back complement can only be generated when the first analyte is in close proximity to the second analyte.

[0098] The methods described herein may also have certain advantages that result in high detection accuracy. For example, the first probe and the second probe must be bound to each other, with the first probe bound to the first analyte and the second probe bound to the second analyte. The combination of these binding events may enable a ligation event between one end of the first probe and another end of the first probe. The ligation event may enable an amplification reaction to occur. The amplification reaction may result in the formation of an amplified product having multiple copies of the barcode or the inverse complement of the barcode that can be detected. Detecting signals associated with the proximity of one analyte to another may require multiple steps, minimizing the opportunity for spurious or nonspecific signals to be generated. As a result, the detection accuracy may be higher than that of other methods. The detection methods described herein may also have high accuracy in distinguishing modified analytes from each other, or unmodified analytes from each other, due to the specificity of the ligation reaction. The detection methods described herein may also have high accuracy in distinguishing between analytes having single nucleotide polymorphisms or between analytes not having single nucleotide polymorphisms, due to the specificity of the ligation reaction. The accuracy of detection can be measured by various metrics, including but not limited to specificity, sensitivity, detection of the correct barcode or its complement, detection of the correct barcode or its complement compared to detection of an incorrect barcode or its complement, or a combination thereof.In some cases, the detection accuracy is at least approximately 50%, at least approximately 51%, at least approximately 52%, at least approximately 53%, at least approximately 54%, at least approximately 55%, at least approximately 56%, at least approximately 57%, at least approximately 58%, at least approximately 59%, at least approximately 60%, at least approximately 61%, at least approximately 62%, at least approximately 63%, at least approximately 64%, at least approximately 65%, at least approximately 66%, at least approximately 67%, at least approximately 68%, at least approximately 69%, at least approximately 70%, at least approximately 71%, at least approximately 72%, at least approximately 73%, at least approximately 74%, and less It could be at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%. In some cases, the detection accuracy is approximately 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, and The accuracy may be approximately 75%, up to approximately 76%, up to approximately 77%, up to approximately 78%, up to approximately 79%, up to approximately 80%, up to approximately 81%, up to approximately 82%, up to approximately 83%, up to approximately 84%, up to approximately 85%, up to approximately 86%, up to approximately 87%, up to approximately 88%, up to approximately 89%, up to approximately 90%, up to approximately 91%, up to approximately 92%, up to approximately 93%, up to approximately 94%, up to approximately 95%, up to approximately 96%, up to approximately 97%, up to approximately 98%, up to approximately 99%, or up to approximately 100%.

[0099] The first analyte may contain one or more genetic abnormalities. Each of the one or more genetic abnormalities in the first analyte may be the same or different. For example, the first analyte may contain two single nucleotide polymorphisms, one of which may contain an A-to-T substitution and the other may contain an A-to-C substitution. These single nucleotide polymorphisms may be considered different. One or more genetic abnormalities may include one or more insertions, one or more deletions, one or more single nucleotide polymorphisms, one or more single nucleotide mutations, one or more copy number mutations, or a combination thereof.

[0100] The first analyte may contain nucleic acids. Nucleic acids may include RNA, DNA, or a combination thereof. In some cases, the first analyte may contain RNA, and the genetic abnormality may contain one or more RNA modifications. One or more RNA modifications may include N6-methyladenosine, 5-methylcytosine, N1-methyladenosine, N7-methylguanosine, N4-acetylcytosine, pseudouridine, N1-methylpseudridine, or a combination thereof. In some cases, the first analyte may contain DNA, and the genetic abnormality may contain one or more DNA modifications. One or more DNA modifications may include methyl modifications.

[0101] The nucleic acid of the first analyte may contain one or more single nucleotide polymorphisms. One or more single nucleotide polymorphisms of the nucleic acid of the first analyte may contain one or more variants of the reference genome. The reference genome may be a human reference genome, a mouse reference genome, a synthetic reference genome, or a combination thereof. The human reference genome may include the NCBI Build 34 sequence, NCBI Build 35 sequence, NCBI Build 36.1 sequence, GRCh37 sequence, GRCh38 sequence, T2T-CHM13 sequence, GRCh39 sequence, hg16 sequence, hg17 sequence, hg18 sequence, hg19 sequence, hs1 sequence, or a combination thereof. One or more single nucleotide polymorphisms of the nucleic acid of the first analyte may include substitutions of A to T, A to C, A to G, T to A, T to C, T to G, C to A, C to T, C to G, G to A, G to C, G to T, or combinations thereof.

[0102] The nucleic acid of the first analyte may contain one or more single-nucleotide variants. The one or more single-nucleotide variants of the nucleic acid of the first analyte may contain one or more variants relative to the reference genome. The reference genome may be a human reference genome, a mouse reference genome, a synthetic reference genome, or a combination thereof. The human reference genome may include the NCBI Build 34 sequence, NCBI Build 35 sequence, NCBI Build 36.1 sequence, GRCh37 sequence, GRCh38 sequence, T2T-CHM13 sequence, GRCh39 sequence, hg16 sequence, hg17 sequence, hg18 sequence, hg19 sequence, hs1 sequence, or a combination thereof. One or more single nucleotide variants of the nucleic acid of the first analyte may include substitutions of A to T, A to C, A to G, T to A, T to C, T to G, C to A, C to T, C to G, G to A, G to C, G to T, or combinations thereof.

[0103] The barcode's inverse complement may be formed from a barcode amplification reaction, forming an amplification product. The amplification reaction may involve generating one or more copies of the barcode or the barcode's inverse complement. The amplification reaction may include rolling circle amplification. Rolling circle amplification may be performed using a cyclic nucleic acid, which may include one or more copies of the barcode. The amplification product formed from the amplification reaction may have one or more copies of the barcode's inverse complement of a cyclic oligonucleotide. The amplification product formed from the amplification reaction is at least 1 copy, at least 2 copies, at least 3 copies, at least about 4 copies, at least about 5 copies, at least about 6 copies, at least about 7 copies, at least about 8 copies, at least about 9 copies, at least about 10 copies, at least about 20 copies, at least about 30 copies, at least about 40 copies, at least about 50 copies, at least about 60 copies, at least about 70 copies, at least about 80 copies, at least about 90 copies, at least about 100 copies, at least about 135 copies, at least about 150 copies, at least about 175 copies, at least about 200 copies, and a small number of copies of the reverse complement of one or more barcodes of the cyclic oligonucleotide. It may have at least about 300 copies, at least about 400 copies, at least about 500 copies, at least about 600 copies, at least about 700 copies, at least about 800 copies, at least about 900 copies, at least about 1000 copies, at least about 2000 copies, at least about 3000 copies, at least about 4000 copies, at least about 5000 copies, at least about 6000 copies, at least about 7000 copies, at least about 8000 copies, at least about 9000 copies, at least about 10000 copies, at least about 50000 copies, at least about 100000 copies, at least about 500000 copies, at least about 1000000 copies, or more copies. The amplification product formed from the amplification reaction may have one or more copies of the reverse complement of one or more barcodes of the cyclic oligonucleotide.The amplification product formed from the amplification reaction is a maximum of 1 copy, 2 copies, 3 copies, approximately 4 copies, approximately 5 copies, approximately 6 copies, approximately 7 copies, approximately 8 copies, approximately 9 copies, approximately 10 copies, approximately 20 copies, approximately 30 copies, approximately 40 copies, approximately 50 copies, approximately 60 copies, approximately 70 copies, approximately 80 copies, approximately 90 copies, approximately 100 copies, approximately 135 copies, approximately 150 copies, approximately 175 copies, approximately 200 copies, and most It may have approximately 300 copies, a maximum of approximately 400 copies, a maximum of approximately 500 copies, a maximum of approximately 600 copies, a maximum of approximately 700 copies, a maximum of approximately 800 copies, a maximum of approximately 900 copies, a maximum of approximately 1000 copies, a maximum of approximately 2000 copies, a maximum of approximately 3000 copies, a maximum of approximately 4000 copies, a maximum of approximately 5000 copies, a maximum of approximately 6000 copies, a maximum of approximately 7000 copies, a maximum of approximately 8000 copies, a maximum of approximately 9000 copies, a maximum of approximately 10000 copies, a maximum of approximately 500000 copies, a maximum of approximately 1000000 copies, or fewer copies. The amplification product formed from the amplification reaction is approximately 1 to approximately 1,000,000 copies, approximately 2 to approximately 500,000 copies, approximately 3 to approximately 100,000 copies, approximately 4 to approximately 50,000 copies, approximately 5 to approximately 10,000 copies, approximately 6 to approximately 9,000 copies, approximately 7 to approximately 8,000 copies, approximately 8 to approximately 7,000 copies, approximately 9 to approximately 6,000 copies, approximately 10 to It may have approximately 5000 copies, approximately 20 to 4000 copies, approximately 30 to 3000 copies, approximately 40 to 2000 copies, approximately 50 to 1000 copies, approximately 60 to 900 copies, approximately 70 to 800 copies, approximately 80 to 700 copies, approximately 90 to 600 copies, approximately 100 to 500 copies, approximately 135 to 400 copies, approximately 150 to 300 copies, and approximately 175 to 200 copies.

[0104] This disclosure relates to detecting proximity between one or more analytes. Proximity between one or more analytes may refer to the spatial proximity of one analyte to another. Proximity between one analyte and another may refer to intracellular colocalization. Proximity between one or more analytes may refer to extracellular colocalization. Proximity between one or more analytes allows a ligation event to occur between one end of a probe and another end of either the same probe or another probe. The ligation event and downstream processes may enable the detection of proximity between one analyte and another analyte. In some cases, one analyte may be too far from another analyte to cause a ligation event between one end of a probe and another end of either the same probe or another probe. The interaction proximity of the analytes described herein is approximately 1 nanometer (nm) at the maximum, approximately 2 nm, approximately 3 nm, approximately 4 nm, approximately 5 nm, approximately 6 nm, approximately 7 nm, approximately 8 nm, approximately 9 nm, approximately 10 nm, approximately 15 nm, approximately 20 nm, approximately 25 nm, approximately 30 nm, approximately 35 nm, approximately 40 nm, approximately 45 nm, approximately 50 nm, approximately 60 nm, approximately 70 nm, approximately 80 nm, and approximately 90 nm. m may refer to interactions on a scale of approximately 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, or shorter.The interaction proximity of the analytes described herein is at least about 1 nm, at least about 2 nm, at least about 3 nm, at least about 4 nm, at least about 5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, at least about 15 nm, at least about 20 nm, at least about 25 nm, at least about 30 nm, at least about 35 nm, at least about 40 nm, at least about 45 nm, at least about 50 nm, at least about 60 nm, at least about 70 nm, at least about 80 nm, and at least about 90 nm. This may refer to interactions on a scale of at least approximately 100 nm, at least approximately 110 nm, at least approximately 120 nm, at least approximately 130 nm, at least approximately 140 nm, at least approximately 150 nm, at least approximately 160 nm, at least approximately 170 nm, at least approximately 180 nm, at least approximately 190 nm, at least approximately 200 nm, at least approximately 300 nm, at least approximately 400 nm, at least approximately 500 nm, at least approximately 600 nm, at least approximately 700 nm, at least approximately 800 nm, at least approximately 900 nm, at least approximately 1000 nm, or longer.

[0105] The methods described herein also relate to detecting the proximity of analytes within a sample. Samples can be in various forms and may contain various properties and / or characteristics. Samples can be extracted from subjects. Subjects from which samples can be extracted may be human subjects. Subjects from which samples can be extracted may be non-human subjects. Non-human subjects may be rats, mice, non-human primates, fruit flies, zebrafish, or a combination thereof. Samples may contain one or more cells. In some embodiments, a sample may contain one or more cells, one or more tissue samples, one or more body fluids, or a combination thereof. Cells in a sample may be cultured cells. Cultured cells may be cultured in vivo, ex vivo, or in vitro. Samples may contain tissue samples. Tissue samples may be fresh, fresh-frozen, fixed, fixed-frozen, formalin-fixed, paraffin-embedded, or a combination thereof. In some cases, tissue samples may be fixed using cross-linking reagents. In some cases, tissue samples may be fixed using preservatives. In some cases, the crosslinking agent may include formaldehyde, formalin, glutaraldehyde, or a combination thereof.

[0106] The sample may include a tissue sample sliced ​​from a tissue block. The tissue sample may be immobilized on a substrate. The substrate may be a well plate, slide, coverslip, well, surface, flow cell, or a combination thereof. The slide may be a microscope slide. The tissue sample may have varying thicknesses. The tissue sample should have a thickness of at least approximately 1 μm, at least approximately 2 μm, at least approximately 3 μm, at least approximately 4 μm, at least approximately 5 μm, at least approximately 6 μm, at least approximately 7 μm, at least approximately 8 μm, at least approximately 9 μm, at least approximately 10 μm, at least approximately 11 μm, at least approximately 12 μm, at least approximately 13 μm, at least approximately 14 μm, at least approximately 15 μm, at least approximately 16 μm, at least approximately 17 μm, at least approximately 18 μm, at least approximately 19 μm, at least approximately 20 μm, at least approximately 21 μm, at least approximately 22 μm, at least approximately 23 μm, at least approximately 24 μm, at least approximately 25 μm, at least approximately 26 μm, at least approximately 27 μm, at least approximately 28 μm, at least approximately 29 μm, at least approximately 30 μm, and at least approximately Thickness of 31 μm, at least about 32 μm, at least about 33 μm, at least about 34 μm, at least about 35 μm, at least about 36 μm, at least about 37 μm, at least about 38 μm, at least about 39 μm, at least about 40 μm, at least about 41 μm, at least about 42 μm, at least about 43 μm, at least about 44 μm, at least about 45 μm, at least about 46 μm The thickness is at least approximately 47 μm, at least approximately 48 μm, at least approximately 49 μm, at least approximately 50 μm, at least approximately 51 μm, at least approximately 52 μm, at least approximately 53 μm, at least approximately 54 μm, at least approximately 55 μm, at least approximately 56 μm, at least approximately 57 μm, at least approximately 58 μm, at least approximately 59 μm, at least approximately 60 μm, at least approximately 61 μm,Thickness of at least approximately 62 μm, at least approximately 63 μm, at least approximately 64 μm, at least approximately 65 μm, at least approximately 66 μm, at least approximately 67 μm, at least approximately 68 μm, at least approximately 69 μm, at least approximately 70 μm, at least approximately 71 μm, at least approximately 72 μm, at least approximately 73 μm, at least approximately 74 μm, at least approximately 75 μm, at least approximately 76 μm, at least approximately 77 μm, at least approximately 78 μm, at least approximately 79 μm Thickness, at least about 80 μm, at least about 81 μm, at least about 82 μm, at least about 83 μm, at least about 84 μm, at least about 85 μm, at least about 86 μm, at least about 87 μm, at least about 88 μm, at least about 89 μm, at least about 90 μm, at least about 91 μm, at least about 92 μm, at least about 93 μm, at least about 94 μm, at least about 95 μm, at least about 96 μm, at least about 97 A thickness of μm, at least about 98 μm, at least about 99 μm, at least about 100 μm, at least about 105 μm, at least about 110 μm, at least about 115 μm, at least about 120 μm, at least about 125 μm, at least about 130 μm, at least about 135 μm, at least about 140 μm, at least about 145 μm, at least about 150 μm, at least about 155 μm, at least about 160 μm, at least about 165 μm, at least Thickness of approximately 170 μm, at least approximately 175 μm, at least approximately 180 μm, at least approximately 185 μm, at least approximately 190 μm, at least approximately 195 μm, at least approximately 200 μm, at least approximately 210 μm, at least approximately 220 μm, at least approximately 230 μm, at least approximately 240 μm, at least approximately 250 μm, at least approximately 260 μm, at least approximately 270 μm, at least approximately 280 μm, at least approximately 290 μm, at least approximately 300 μm,The thickness may be at least approximately 320 μm, at least approximately 340 μm, at least approximately 360 μm, at least approximately 380 μm, at least approximately 400 μm, at least approximately 420 μm, at least approximately 440 μm, at least approximately 460 μm, at least approximately 480 μm, at least approximately 500 μm, or greater. The tissue samples have a maximum thickness of approximately 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, and more. Thickness of approximately 18 μm, maximum thickness of approximately 19 μm, maximum thickness of approximately 20 μm, maximum thickness of approximately 21 μm, maximum thickness of approximately 22 μm, maximum thickness of approximately 23 μm, maximum thickness of approximately 24 μm, maximum thickness of approximately 25 μm, maximum thickness of approximately 26 μm, maximum thickness of approximately 27 μm, maximum thickness of approximately 28 μm, maximum thickness of approximately 29 μm, maximum thickness of approximately 30 μm, maximum thickness of approximately 31 μm, maximum thickness of approximately 32 μm, maximum thickness of approximately 33 μm, maximum thickness of approximately 34 μm, maximum thickness of approximately 35 Thickness in μm, maximum thickness of approximately 36 μm, maximum thickness of approximately 37 μm, maximum thickness of approximately 38 μm, maximum thickness of approximately 39 μm, maximum thickness of approximately 40 μm, maximum thickness of approximately 41 μm, maximum thickness of approximately 42 μm, maximum thickness of approximately 43 μm, maximum thickness of approximately 44 μm, maximum thickness of approximately 45 μm, maximum thickness of approximately 46 μm, maximum thickness of approximately 47 μm, maximum thickness of approximately 48 μm, maximum thickness of approximately 49 μm, maximum thickness of approximately 50 μm, maximum thickness of approximately 51 μm, maximum thickness of approximately 52 μm Thickness: maximum approximately 53 μm, maximum approximately 54 μm, maximum approximately 55 μm, maximum approximately 56 μm, maximum approximately 57 μm, maximum approximately 58 μm, maximum approximately 59 μm, maximum approximately 60 μm, maximum approximately 61 μm, maximum approximately 62 μm, maximum approximately 63 μm, maximum approximately 64 μm, maximum approximately 65 μm, maximum approximately 66 μm, maximum approximately 67 μm, maximum approximately 68 μm, maximum approximately 69 μm,Maximum thickness of approximately 70 μm, maximum thickness of approximately 71 μm, maximum thickness of approximately 72 μm, maximum thickness of approximately 73 μm, maximum thickness of approximately 74 μm, maximum thickness of approximately 75 μm, maximum thickness of approximately 76 μm, maximum thickness of approximately 77 μm, maximum thickness of approximately 78 μm, maximum thickness of approximately 79 μm, maximum thickness of approximately 80 μm, maximum thickness of approximately 81 μm, maximum thickness of approximately 82 μm, maximum thickness of approximately 83 μm, maximum thickness of approximately 84 μm, maximum thickness of approximately 85 μm, maximum thickness of approximately 86 μm, maximum thickness of approximately 87 μm, maximum thickness of approximately 88 μm, Maximum thickness of approximately 89 μm, maximum thickness of approximately 90 μm, maximum thickness of approximately 91 μm, maximum thickness of approximately 92 μm, maximum thickness of approximately 93 μm, maximum thickness of approximately 94 μm, maximum thickness of approximately 95 μm, maximum thickness of approximately 96 μm, maximum thickness of approximately 97 μm, maximum thickness of approximately 98 μm, maximum thickness of approximately 99 μm, maximum thickness of approximately 100 μm, maximum thickness of approximately 105 μm, maximum thickness of approximately 110 μm, maximum thickness of approximately 115 μm, maximum thickness of approximately 120 μm, maximum thickness of approximately 125 μm, maximum thickness of approximately 130 μm, maximum thickness of approximately 1 Thickness of 35 μm, maximum of approximately 140 μm, maximum of approximately 145 μm, maximum of approximately 150 μm, maximum of approximately 155 μm, maximum of approximately 160 μm, maximum of approximately 165 μm, maximum of approximately 170 μm, maximum of approximately 175 μm, maximum of approximately 180 μm, maximum of approximately 185 μm, maximum of approximately 190 μm, maximum of approximately 195 μm, maximum of approximately 200 μm, maximum of approximately 210 μm, maximum of approximately 220 μm, maximum of approximately 230 μm, maximum of approximately 240 μm The thickness can be approximately 250 μm at most, 260 μm at most, 270 μm at most, 280 μm at most, 290 μm at most, 300 μm at most, 320 μm at most, 340 μm at most, 360 μm at most, 380 μm at most, 400 μm at most, 420 μm at most, 440 μm at most, 460 μm at most, 480 μm at most, 500 μm at most, or less. The tissue sample may have a thickness of approximately 1 to 500 μm, 2 to 480 μm, 3 to 460 μm, 4 to 440 μm, 5 to 420 μm, 6 to 400 μm,Thickness of approximately 7-380 μm, approximately 8-360 μm, approximately 9-340 μm, approximately 10-320 μm, approximately 11-300 μm, approximately 12-290 μm, approximately 13-280 μm, approximately 14-270 μm, approximately 15-260 μm, approximately 16-250 μm, approximately 17-240 μm, approximately 18-230 μm, approximately 19-220 μm, approximately 20-210 μm, approximately 21-200 μm, approximately 22-195 μm Thickness: approximately 23-190 μm, approximately 24-185 μm, approximately 25-180 μm, approximately 26-175 μm, approximately 27-170 μm, approximately 28-165 μm, approximately 29-160 μm, approximately 30-155 μm, approximately 31-150 μm, approximately 32-145 μm, approximately 33-140 μm, approximately 34-135 μm, approximately 35-130 μm, approximately 36-125 μm, approximately 37-120 μm, approximately 38- Thickness of 115 μm, approximately 39 to 110 μm, approximately 40 to 105 μm, approximately 41 to 100 μm, approximately 42 to 99 μm, approximately 43 to 98 μm, approximately 44 to 97 μm, approximately 45 to 96 μm, approximately 46 to 95 μm, approximately 47 to 94 μm, approximately 48 to 93 μm, approximately 49 to 92 μm, approximately 50 to 91 μm, approximately 51 to 90 μm, approximately 52 to 89 μm, approximately 53 to 88 μm, approximately 54 to 87 μm The thickness can be approximately 55-86 μm, 56-85 μm, 57-84 μm, 58-83 μm, 59-82 μm, 60-81 μm, 61-80 μm, 62-79 μm, 63-78 μm, 64-77 μm, 65-76 μm, 66-75 μm, 67-74 μm, 68-73 μm, 69-72 μm, or 70-71 μm. In some cases, the tissue sample may be approximately 5-250 μm, 10-100 μm, or 25-150 μm thick.

[0107] The sample may be embedded in a hydrogel. The hydrogel may be formed by polymerizing monomers in the presence of the sample. The hydrogel may contain one or more polymers. One or more polymers may include poly(vinyl alcohol) (PVA), poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(acrylic acid) (PAA), and poly(acrylamide) (PAAm), or a combination thereof. The hydrogel may be formed during any step of the methods described herein. For example, the hydrogel may be formed before contacting the sample with one or more probes. The hydrogel may be formed after contacting the sample with one or more probes. In some cases, the hydrogel may be formed after contacting the sample with one or more probes, but before contacting the sample with one or more other probes. The hydrogel may be formed before the amplification step. The hydrogel may be formed after the amplification step. The hydrogel may be formed before the ligation step between one or more probes. A hydrogel may be formed after a ligation step between one or more probes. A hydrogel may be formed before a gap-filling reaction. A hydrogel may be formed after a gap-filling reaction. A hydrogel may be formed before a detection step. A hydrogel may be formed after a detection step. The sample may be embedded in a hydrogel. In some cases, the hydrogel may be formed before contacting the sample with one or more probes, and the sample may be incubated before contacting the sample with one or more probes. Before contacting the sample with one or more probes, the sample may be incubated for at least about 1 hour, at least about 12 hours, at least about 1 day, at least about 2 days, at least about 3 days, or longer. Before contacting the sample with one or more probes, the sample may be incubated for up to about 1 hour, up to about 12 hours, up to about 1 day, up to about 2 days, up to about 3 days, or shorter.

[0108] The methods described herein also relate to detecting the proximity of analytes within a sample (e.g., determining whether an analyte is associated with or close to another analyte within the sample). Proximity may be determined by analyzing one or more analytes. In some cases, various different types of analytes, including but not limited to nucleic acids, polypeptides, lipids, small molecules, cells, intracellular features, extracellular features, exogenous features, endogenous features, or combinations thereof, may be used for analysis. In some cases, one or more analytes of the same type may be analytes. For example, two different proteins may each be considered an analyte. In some cases, one or more analytes of different types may be analyzed. A protein may be analyzed for proximity to another protein. A protein may be analyzed for proximity to a nucleic acid. Nucleic acids that may be analyzed for proximity to a protein may include RNA, DNA, or combinations thereof. In some cases, a nucleic acid may be analyzed for proximity to another nucleic acid. RNA may be analyzed for proximity to another RNA. In some cases, other RNA may be different regions of the same RNA. For example, an RNA molecule may contain two distinct parts, and each of these two distinct parts can be bound by one or more probes to assess their proximity. RNA can be analyzed for its proximity to DNA. DNA can be analyzed for its proximity to other DNA. In some cases, DNA can be different regions of the same DNA. For example, a DNA molecule may contain two distinct parts, and each of these two or more parts can be bound by one or more probes to assess the proximity of two or more parts of the DNA molecule. The proximity between a first analyte and a second analyte may also be analyzed by detecting the presence of a barcode or its complement, which is amplified as a result of the spatial localization of the first and second analytes relative to each other. The first analyte can be of various types. The second analyte can be of various types.

[0109] The first analyte may contain nucleic acids. The nucleic acids of the first analyte may contain RNA, DNA, or a combination thereof. The first analyte may contain one or more genetic abnormalities. The first analyte may contain RNA. If the first analyte contains RNA, the RNA may contain various types of RNA, including but not limited to messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), or a combination thereof. The RNA may be endogenous to the sample or exogenous to the sample. The RNA may be introduced into the sample by various means, including but not limited to the use of AAV or rAAV. If the first analyte contains RNA, the RNA may contain modifications. The modifications may be N6-methyladenosine, 5-methylcytosine, N1-methyladenosine, N7-methylguanosine, N4-acetylcytosine, pseudouridine, N1-methylpseudridine, or a combination thereof. RNA may contain single-stranded regions, double-stranded regions, or combinations thereof. RNA may contain multiple nucleotides. Multiple nucleotides may contain A, C, U, G, or combinations thereof. In some cases, RNA may contain secondary structures, tertiary structures, or combinations thereof.

[0110] The first analyte may contain nucleic acids. The nucleic acids of the first analyte may contain one or more genetic abnormalities. The one or more genetic abnormalities of the first analyte may include one or more insertions, one or more deletions, one or more copy number variations, one or more single nucleotide polymorphisms, one or more single nucleotide variants, or a combination thereof. The one or more single nucleotide polymorphisms of the nucleic acids of the first analyte may include one or more variants relative to a reference genome. The reference genome may include a human reference genome, a mouse reference genome, a synthetic reference genome, or a combination thereof. The human reference genome may include the NCBI Build 34 sequence, NCBI Build 35 sequence, NCBI Build 36.1 sequence, GRCh37 sequence, GRCh38 sequence, T2T-CHM13 sequence, GRCh39 sequence, hg16 sequence, hg17 sequence, hg18 sequence, hg19 sequence, hs1 sequence, or a combination thereof. One or more single-nucleotide polymorphisms of the nucleic acid of the first analyte may include A-to-T substitutions, A-to-C substitutions, A-to-G substitutions, T-to-A substitutions, T-to-C substitutions, T-to-G substitutions, C-to-A substitutions, C-to-T substitutions, C-to-G substitutions, G-to-A substitutions, G-to-C substitutions, G-to-T substitutions, or combinations thereof. One or more single-nucleotide variants of the nucleic acid of the first analyte may include one or more variants relative to a reference genome. The reference genome may be a human reference genome, a mouse reference genome, a synthetic reference genome, or a combination thereof. The human reference genome may include the NCBI Build 34 sequence, NCBI Build 35 sequence, NCBI Build 36.1 sequence, GRCh37 sequence, GRCh38 sequence, T2T-CHM13 sequence, GRCh39 sequence, hg16 sequence, hg17 sequence, hg18 sequence, hg19 sequence, hs1 sequence, or a combination thereof. One or more single nucleotide variants may include substitutions of A to T, A to C, A to G, T to A, T to C, T to G, C to A, C to T, C to G, G to A, G to C, G to T, or combinations thereof.

[0111] The first analyte may contain polypeptides. The polypeptide of the first analyte may contain proteins, peptides, or combinations thereof. If the polypeptide of the first analyte contains proteins, the proteins may include transcription factors, ribosomal proteins, histones, polymerases, helicases, restriction enzymes, ribonucleic acid-binding proteins, enzymes, antibodies, structural proteins, protective proteins, signaling proteins, receptors, soluble proteins, transmembrane proteins, or combinations thereof. If the protein may be a signaling protein, the signaling protein may be a cytokine, a chemokine, or a combination thereof. If the polypeptide of the first analyte contains proteins, the proteins may include transcription factors, ribosomal proteins, histones, polymerases, or combinations thereof. When ribosomal proteins are listed as the first analyte, the ribosomal proteins include S3A ribosomal protein, SA ribosomal protein, S3 ribosomal protein, S9 ribosomal protein, S4(X,Y1,Y2) ribosomal protein, S2 ribosomal protein, S6 ribosomal protein, S5 ribosomal protein, S7 ribosomal protein, S15A ribosomal protein, S8 ribosomal protein, S16 ribosomal protein, S20 ribosomal protein, S10 ribosomal protein, S14 ribosomal protein, S23 ribosomal protein, S12 ribosomal protein, S18 ribosomal protein, and S29 ribosomal protein. Protein, S13 ribosomal protein, S11 ribosomal protein, S17 ribosomal protein, S15 ribosomal protein, S19 ribosomal protein, S21 ribosomal protein, S24 ribosomal protein, S25 ribosomal protein, S26 ribosomal protein, S27 ribosomal protein, S28 ribosomal protein, S30 ribosomal protein, S27A ribosomal protein, RACK1 ribosomal protein, L10A ribosomal protein, L8 ribosomal protein, L3 ribosomal protein, L4 ribosomal protein, L11 ribosomal protein, L9 ribosomal protein, L6 ribosomal protein, L7A ribosomal protein,P0 ribosomal protein, L12 ribosomal protein, L13A ribosomal protein, L13 ribosomal protein, L23 ribosomal protein, L14 ribosomal protein, L27A ribosomal protein, L15 ribosomal protein, L10 ribosomal protein, L5 ribosomal protein, L18 ribosomal protein, L19 ribosomal protein, L18A ribosomal protein, L21 ribosomal protein, L17 ribosomal protein, L22 ribosomal protein, L23A ribosomal protein, L26 ribosomal protein, L24 ribosomal protein, L27 ribosomal protein Possible examples include L28 ribosomal protein, L35 ribosomal protein, L29 ribosomal protein, L7 ribosomal protein, L30 ribosomal protein, L31 ribosomal protein, L32 ribosomal protein, L35A ribosomal protein, L34 ribosomal protein, L36 ribosomal protein, L37 ribosomal protein, L38 ribosomal protein, L39 ribosomal protein, L40 ribosomal protein, L41 ribosomal protein, L36A ribosomal protein, L37A ribosomal protein, P1 / P2(αβ) ribosomal protein, or combinations thereof.

[0112] The first analyte may include a protein, which may contain one or more post-translational modifications. Examples of one or more post-translational modifications of the protein in the first analyte include myristoylation, palmitoylation, farnesylation, geranylgeranylation, glypiation, glycosylphosphatidylinositol, lipoylation, flavin moiety attachment, heme C attachment, phosphopantetheinylation, retinilidensch base formation, translation factor modification, diphthamide formation, ethanolamine phosphoglycerol, hypsin formation, β-lysine addition to lysine, acylation (e.g., O-acylation, N-acylation, and S-acylation), acetylation, formylation, alkylation, amidation, arginylation, polyglutamylation, polyglycylation, and butyrylation. Possible modifications include γ-carboxylation, glycosylation, polysialylation, malonylation, hydroxylation, nucleotide addition, phosphate esterification (O bond), phosphoramidate (N bond) formation, phosphorylation, adenylation, uridilylation, propionylation, pyroglutamate formation, S-glutathionylation, S-nitrosylation, S-sulfenylation, S-sulfinylation, S-sulfonylation, succinylation, sulfation, glycation, carbamylation, carbonylation, spontaneous isopeptide bond formation, biotinylation, carbamylation, oxidation, pegylation, ubiquitination, SUMOylation, nedylation, ISGation, citrullination, deamidation, eliminylation, or combinations thereof. The first analyte may include chemical modifications. One or more post-translational modifications of the protein in the first analyte may include alkylation, phosphorylation, or combinations thereof.

[0113] The second analyte may contain nucleic acids. The nucleic acids of the second analyte may contain RNA, DNA, or a combination thereof. The second analyte may contain one or more genetic abnormalities. The second analyte may contain RNA. If the second analyte contains RNA, the RNA may contain various types of RNA, including but not limited to messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), or a combination thereof. The RNA of the second analyte may be endogenous to the sample or exogenous to the sample. The RNA may be introduced into the sample by various means, including but not limited to the use of AAV or rAAV. If the second analyte contains RNA, the RNA may contain modifications. The modifications may be N6-methyladenosine, 5-methylcytosine, N1-methyladenosine, N7-methylguanosine, N4-acetylcytosine, pseudouridine, N1-methylpseudridine, or a combination thereof. RNA may contain single-stranded regions, double-stranded regions, or combinations thereof. RNA may contain multiple nucleotides. Multiple nucleotides may contain A, C, U, G, or combinations thereof. In some cases, RNA may contain secondary structures, tertiary structures, or combinations thereof.

[0114] The second analyte may contain DNA. The DNA in the second analyte may associate with one or more histone molecules (e.g., DNA may form a complex with one or more histones). The DNA in the second analyte may associate with one or more nucleosomes (e.g., DNA may wrap around one or more histone molecules to form one or more nucleosomes). The DNA in the second analyte may associate with polymerase (e.g., DNA may interact with polymerase). The DNA in the second analyte may associate with DNA polymerase. The DNA in the second analyte may be endogenous to the sample (e.g., DNA may be synthesized in one or more cells of the sample). The DNA in the second analyte may be exogenous to the sample (e.g., DNA may be inserted into one or more cells of the sample from an external source). The DNA in the second analyte may be a combination of endogenous and exogenous DNA. The DNA in the second analyte may be single-stranded, double-stranded, or a combination thereof. The DNA of the second analyte may contain multiple nucleotides. These multiple nucleotides may include A, C, T, G, or combinations thereof. The DNA of the second analyte may contain oligonucleotides. Oligonucleotides may be single-stranded, double-stranded, or combinations thereof. In some cases, oligonucleotides may contain secondary structures. In some cases, the DNA of the second analyte may contain secondary structures, tertiary structures, or combinations thereof. The DNA of the second analyte may contain modifications. These modifications may include methyl modifications.

[0115] If the second analyte contains nucleic acid, the nucleic acid may contain one or more genetic abnormalities. One or more genetic abnormalities in the second analyte may include one or more insertions, one or more deletions, one or more copy number variations, one or more single nucleotide polymorphisms, one or more single nucleotide variants, or a combination thereof. One or more single nucleotide polymorphisms in the second analyte may include one or more variants relative to the reference genome. The reference genome may be a human reference genome, a mouse reference genome, a synthetic reference genome, or a combination thereof. The human reference genome may include the NCBI Build 34 sequence, NCBI Build 35 sequence, NCBI Build 36.1 sequence, GRCh37 sequence, GRCh38 sequence, T2T-CHM13 sequence, GRCh39 sequence, hg16 sequence, hg17 sequence, hg18 sequence, hg19 sequence, hs1 sequence, or a combination thereof. One or more single-nucleotide polymorphisms of the second analyte may include A-to-T substitutions, A-to-C substitutions, A-to-G substitutions, T-to-A substitutions, T-to-C substitutions, T-to-G substitutions, C-to-A substitutions, C-to-T substitutions, C-to-G substitutions, G-to-A substitutions, G-to-C substitutions, G-to-T substitutions, or combinations thereof. One or more single-nucleotide variants of the second analyte may include one or more variants relative to a reference genome. The reference genome may be a human reference genome, a mouse reference genome, a synthetic reference genome, or a combination thereof. The human reference genome may include the NCBI Build 34 sequence, NCBI Build 35 sequence, NCBI Build 36.1 sequence, GRCh37 sequence, GRCh38 sequence, T2T-CHM13 sequence, GRCh39 sequence, hg16 sequence, hg17 sequence, hg18 sequence, hg19 sequence, hs1 sequence, or combinations thereof. One or more single-nucleotide variants of the second analyte may include substitutions of A to T, A to C, A to G, T to A, T to C, T to G, C to A, C to T, C to G, G to A, G to C, G to T, or combinations thereof.

[0116] The second analyte may contain polypeptides. The polypeptides of the second analyte may contain proteins, peptides, or combinations thereof. If the polypeptides of the second analyte contain proteins, the proteins may include transcription factors, ribosomal proteins, histones, polymerases, helicases, restriction enzymes, ribonucleic acid-binding proteins, enzymes, antibodies, structural proteins, protective proteins, signaling proteins, receptors, soluble proteins, transmembrane proteins, or combinations thereof. If the protein may be a signaling protein, the signaling protein may be a cytokine, a chemokine, or a combination thereof. If the polypeptides of the second analyte contain proteins, the proteins may include transcription factors, ribosomal proteins, histones, polymerases, or combinations thereof. When ribosomal proteins are listed as the second analyte, the ribosomal proteins include S3A ribosomal protein, SA ribosomal protein, S3 ribosomal protein, S9 ribosomal protein, S4(X,Y1,Y2) ribosomal protein, S2 ribosomal protein, S6 ribosomal protein, S5 ribosomal protein, S7 ribosomal protein, S15A ribosomal protein, S8 ribosomal protein, S16 ribosomal protein, S20 ribosomal protein, S10 ribosomal protein, S14 ribosomal protein, S23 ribosomal protein, S12 ribosomal protein, S18 ribosomal protein, and S29 ribosomal protein. Protein, S13 ribosomal protein, S11 ribosomal protein, S17 ribosomal protein, S15 ribosomal protein, S19 ribosomal protein, S21 ribosomal protein, S24 ribosomal protein, S25 ribosomal protein, S26 ribosomal protein, S27 ribosomal protein, S28 ribosomal protein, S30 ribosomal protein, S27A ribosomal protein, RACK1 ribosomal protein, L10A ribosomal protein, L8 ribosomal protein, L3 ribosomal protein, L4 ribosomal protein, L11 ribosomal protein, L9 ribosomal protein, L6 ribosomal protein, L7A ribosomal protein,P0 ribosomal protein, L12 ribosomal protein, L13A ribosomal protein, L13 ribosomal protein, L23 ribosomal protein, L14 ribosomal protein, L27A ribosomal protein, L15 ribosomal protein, L10 ribosomal protein, L5 ribosomal protein, L18 ribosomal protein, L19 ribosomal protein, L18A ribosomal protein, L21 ribosomal protein, L17 ribosomal protein, L22 ribosomal protein, L23A ribosomal protein, L26 ribosomal protein, L24 ribosomal protein, L27 ribosomal protein Possible examples include L28 ribosomal protein, L35 ribosomal protein, L29 ribosomal protein, L7 ribosomal protein, L30 ribosomal protein, L31 ribosomal protein, L32 ribosomal protein, L35A ribosomal protein, L34 ribosomal protein, L36 ribosomal protein, L37 ribosomal protein, L38 ribosomal protein, L39 ribosomal protein, L40 ribosomal protein, L41 ribosomal protein, L36A ribosomal protein, L37A ribosomal protein, P1 / P2(αβ) ribosomal protein, or combinations thereof.

[0117] The second analyte may include a protein, which may contain one or more post-translational modifications. Examples of one or more post-translational modifications of the protein in the second analyte include myristoylation, palmitoylation, farnesylation, geranylgeranylation, glypiation, glycosylphosphatidylinositol, lipoylation, flavin moiety attachment, heme C attachment, phosphopantetheinylation, retinilidenschiff base formation, translation factor modification, diphthamide formation, ethanolamine phosphoglycerol, hypsin formation, β-lysine addition to lysine, acylation (e.g., O-acylation, N-acylation, and S-acylation), acetylation, formylation, alkylation, amidation, arginylation, polyglutamylation, polyglycylation, and butyrylation. Possible modifications include γ-carboxylation, glycosylation, polysialylation, malonylation, hydroxylation, nucleotide addition, phosphate esterification (O bond), phosphoramide (N bond) formation, phosphorylation, adenylation, uridilylation, propionylation, pyroglutamate formation, S-glutathionylation, S-nitrosylation, S-sulfenylation, S-sulfinylation, S-sulfonylation, succinylation, sulfation, glycation, carbamylation, carbonylation, spontaneous isopeptide bond formation, biotinylation, carbamylation, oxidation, pegylation, ubiquitination, SUMOylation, nedylation, ISGation, citrullination, deamidation, eliminylation, or combinations thereof. The second analyte may include chemical modifications. One or more post-translational modifications of the protein in the second analyte may include alkylation, phosphorylation, or combinations thereof.

[0118] The first probe may contain various components. In some cases, the first probe may contain nucleic acids. The nucleic acids of the first probe may contain one or more deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a combination thereof. The nucleic acids of the first probe may contain one or more modifications. In some cases, one or more modifications of the nucleic acids of the first probe may include DNA modifications. In some cases, one or more modifications may be RNA modifications. One or more modifications may be located at one or both ends of the first probe, inside the first probe, or a combination thereof. One or more modifications of the first probe may be used to ligate the first probe to itself, to ligate the first probe to another probe (e.g., a third probe), to directly or indirectly detect the first probe, or a combination thereof. One or more modifications may include phosphate modifications. In some cases, the phosphate modifications may include 5'-phosphate modifications. In some cases, the phosphate modifications may include 3'-phosphate modifications. The first probe may contain nucleic acids. The nucleic acid of the first probe may be an oligonucleotide. The oligonucleotide may include modifications. In some cases, the oligonucleotide may include a 5' phosphate modification. One or more modifications of the nucleic acid may include an internucleotide bond. The internucleotide bond of the nucleic acid of the first probe may include a phosphorothioate, a phosphodiester, or a combination thereof. The internucleotide bond may include locked nucleic acid (LNA). The internucleotide bond may provide advantages to the method described herein by increasing the melting temperature of the oligonucleotide interaction, increasing the stability of the probe interaction, improving the specificity of the probe prerecognition, or a combination thereof.

[0119] The first probe may include a variety of lengths. If the first probe contains nucleic acids, the nucleic acids may be at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 2 6, at least about 27, at least about 28, at least about 29, at least about 30, at least about 31, at least about 32, at least about 33, at least about 34, at least about 35, at least about 36, at least about 37, at least about 38, at least about 39, at least about 40, at least about 41, at least about 42, at least about 43, at least about 44, at least about 45, at least about 46, at least about 47, at least about 48, at least about 49, at least about 50, at least about 51, at least about 52, at least about 5 3, at least about 54, at least about 55, at least about 56, at least about 57, at least about 58, at least about 59, at least about 60, at least about 61, at least about 62, at least about 63, at least about 64, at least about 65, at least about 66, at least about 67, at least about 68, at least about 69, at least about 70, at least about 71, at least about 72, at least about 73, at least about 74, at least about 75, at least about 76, at least about 77, at least about 78, at least about 79, at least about 8 0, at least about 81, at least about 82, at least about 83, at least about 84, at least about 85, at least about 86, at least about 87, at least about 88, at least about 89, at least about 90, at least about 91, at least about 92, at least about 93, at least about 94, at least about 95, at least about 96, at least about 97, at least about 98, at least about 99, at least about 100, at least about 105, at least about 110, at least about 115, at least about 120, at least about 125, at least about 130,The nucleotide length may be at least approximately 135, at least approximately 140, at least approximately 145, at least approximately 150, at least approximately 155, at least approximately 160, at least approximately 165, at least approximately 170, at least approximately 175, at least approximately 180, at least approximately 185, at least approximately 190, at least approximately 195, at least approximately 200, or longer. If the first probe contains nucleic acids, the nucleic acids are at most approximately 1, at most approximately 2, at most approximately 3, at most approximately 4, at most approximately 5, at most approximately 6, at most approximately 7, at most approximately 8, at most approximately 9, at most approximately 10, at most approximately 11, at most approximately 12, at most approximately 13, at most approximately 14, at most approximately 15, at most approximately 16, at most approximately 17, at most approximately 18, at most approximately 19, at most approximately 20, at most approximately 21, at most approximately 22, at most approximately 23, at most approximately 24, at most approximately 25, at most approximately 26, at most approximately 27, at most approximately 28, at most Large: approximately 29, Maximum: approximately 30, Maximum: approximately 31, Maximum: approximately 32, Maximum: approximately 33, Maximum: approximately 34, Maximum: approximately 35, Maximum: approximately 36, Maximum: approximately 37, Maximum: approximately 38, Maximum: approximately 39, Maximum: approximately 40, Maximum: approximately 41, Maximum: approximately 42, Maximum: approximately 43, Maximum: approximately 44, Maximum: approximately 45, Maximum: approximately 46, Maximum: approximately 47, Maximum: approximately 48, Maximum: approximately 49, Maximum: approximately 50, Maximum: approximately 51, Maximum: approximately 52, Maximum: approximately 53, Maximum: approximately 54, Maximum: approximately 55, Maximum: approximately 56, Maximum: approximately 57, Maximum: approximately 58 , up to approximately 59, up to approximately 60, up to approximately 61, up to approximately 62, up to approximately 63, up to approximately 64, up to approximately 65, up to approximately 66, up to approximately 67, up to approximately 68, up to approximately 69, up to approximately 70, up to approximately 71, up to approximately 72, up to approximately 73, up to approximately 74, up to approximately 75, up to approximately 76, up to approximately 77, up to approximately 78, up to approximately 79, up to approximately 80, up to approximately 81, up to approximately 82, up to approximately 83, up to approximately 84, up to approximately 85, up to approximately 86, up to approximately 87, up to approximately 88, maximum approximately 89, maximum approximately 90, maximum approximately 91, maximum approximately 92, maximum approximately 93, maximum approximately 94, maximum approximately 95, maximum approximately 96, maximum approximately 97, maximum approximately 98, maximum approximately 99, maximum approximately 100, maximum approximately 105, maximum approximately 110, maximum approximately 115, maximum approximately 120, maximum approximately 125, maximum approximately 130, maximum approximately 135, maximum approximately 140, maximum approximately 145, maximum approximately 150, maximum approximately 155, maximum approximately 160, maximum approximately 165, maximum approximately 170, maximum approximately 175,The nucleotide lengths may be approximately 180, 185, 190, 195, 200, or shorter. If the first probe contains nucleic acid, the nucleic acid may be approximately 1-200, 2-195, 3-190, 4-185, 5-180, 6-175, 7-170, 8-165, 9-160, 10-155, 11-150, 12-145, 13-140, and 14 ~135, 15~130, 16~125, 17~120, 18~115, 19~110, 20~105, 21~100, 22~99, 23~98, 24~97, 25~96, 26~95, 27~94, 28~93, 29~92 , approximately 30-91, approximately 31-90, approximately 32-89, approximately 33-88, approximately 34-87, approximately 35-86, approximately 36-85, approximately 37-84, approximately 38-83, approximately 39-82, approximately 40-81, approximately 41-80, approximately 42-79, approximately 43-78, approximately 44-77, approximately 45-76, approximately 46 The nucleotide lengths may be approximately 75, 47-74, 48-73, 49-72, 50-71, 51-70, 52-69, 53-68, 54-67, 55-66, 56-65, 57-64, 58-63, 59-62, or 60-61.

[0120] The first probe may include one or more binding sites. One or more binding sites of the first probe may bind to one or more other probes, one or more analytes, one or more features of a sample, or a combination thereof. In some cases, the first probe may include at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, or more binding sites. In some cases, the first probe may contain up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 6, up to about 7, up to about 8, up to about 9, up to about 10, up to about 15, up to about 20, up to about 25, up to about 30, up to about 40, up to about 50, up to about 60, up to about 70, up to about 80, up to about 90, up to about 100, or fewer binding sites. In some cases, the first probe may contain about 1 to about 100, about 2 to about 90, about 3 to about 80, about 4 to about 70, about 5 to about 60, about 6 to about 50, about 7 to about 40, about 8 to about 30, about 9 to about 25, or about 10 to about 20 binding sites.

[0121] The first probe may recognize, bind to, and / or ligate to at least a portion of the first analyte. The first analyte may include various types of analytes, and depending on the type of analyte, the first probe may recognize, bind to, and / or ligate to specific features. The first probe may recognize ribonucleic acid. The first analyte may include ribonucleic acid. The ribonucleic acid (RNA) of the first analyte may include, but not limited to, messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), or combinations thereof. The RNA may be endogenous to the sample or exogenous to the sample. The RNA may be introduced into the sample by various means, including, but not limited to, the use of AAV or rAAV. If the second analyte includes RNA, the RNA may include modifications. The RNA modifications of the first analyte may be N6-methyladenosine, 5-methylcytosine, N1-methyladenosine, N7-methylguanosine, N4-acetylcytosine, pseudouridine, N1-methylpseudridine, or a combination thereof. The RNA of the first analyte may contain single-stranded regions, double-stranded regions, or a combination thereof. The RNA may contain multiple nucleotides. The multiple nucleotides may contain A, C, U, G, or a combination thereof. In some cases, the RNA may contain secondary structures, tertiary structures, or a combination thereof.

[0122] The first probe can recognize the first analyte. If the first probe can recognize the first analyte, the first analyte may contain nucleic acids, which may contain one or more single nucleotide polymorphisms. The first probe can bind to nucleic acids containing one or more single nucleotide polymorphisms. By binding the first probe to nucleic acids containing one or more single nucleotide polymorphisms, it may be possible to ligate one end of the first probe with another end of the first probe or with another end of another probe. In some cases, the ligation of the first probe may include ligation of one end of the first probe with another end of the first probe. In some cases, the ligation of the first probe may include ligation of one end of the first probe with a third probe.

[0123] The first probe cannot recognize the analyte. In some cases in which the first probe recognizes the analyte, the analyte may contain nucleic acids, and the nucleic acids may contain one or more single nucleotide polymorphisms. The first probe does not need to bind to nucleic acids containing one or more single nucleotide polymorphisms because the sequences of the one or more single nucleotide polymorphisms are different from the sequences recognized by the first probe. The first probe does not need to be ligated in the presence of the analytes described herein. In some cases, the first probe does not need to recognize analytes containing nucleic acids containing one or more single nucleotide polymorphisms, and if the analyte contains one or more single nucleotide polymorphisms, the first probe is not ligated. The analyte may be the first analyte.

[0124] A first probe may recognize a first analyte. In some cases in which the first probe may recognize a first analyte, the first analyte may include a nucleic acid, which may include one or more modifications. The one or more modifications of the first analyte may include N6-methyladenosine, 5-methylcytosine, N1-methyladenosine, N7-methylguanosine, N4-acetylcytosine, pseudouridine, N1-methylpseudridine, or a combination thereof. The first probe may bind to a nucleic acid containing one or more modifications. Binding of the first probe to the nucleic acid of the first analyte containing one or more modifications may allow ligation of one end of the first probe to another end of the first probe and / or another end of another probe. In some cases, ligation of the first probe may include ligation of one end of the first probe to another end of the first probe. In some cases, the ligation of the first probe may include ligation between one end of the first probe and one end of the third probe.

[0125] The first probe cannot recognize the analyte. In some cases where the first probe cannot recognize the analyte, the analyte may contain nucleic acids, which may contain one or more modifications. The first probe does not need to bind to nucleic acids containing one or more modifications because the sequences of the one or more modifications are different from the sequences recognized by the first probe. The first probe does not need to be ligated in the presence of the analytes described herein. In some cases, the first probe does not need to recognize analytes containing nucleic acids containing one or more modifications, and if the analyte contains one or more modifications, the first probe is not ligated. The analyte may be the first analyte.

[0126] The second probe may contain various components. In some cases, the second probe may contain nucleic acids. The nucleic acids of the second probe may contain one or more deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a combination thereof. The nucleic acids of the second probe may contain oligonucleotides. The oligonucleotides of the nucleic acids of the second probe may contain one or more modifications. The nucleic acids of the second probe may contain one or more modifications. In some cases, one or more modifications of the nucleic acids of the second probe may be DNA modifications. In some cases, one or more modifications of the nucleic acids of the second probe may be RNA modifications. One or more modifications of the nucleic acids of the second probe may contain phosphate modifications. In some cases, the phosphate modifications may include 5'-phosphate modifications. In some cases, the phosphate modifications of the nucleic acids of the second probe may include 3'-phosphate modifications. In some cases, the oligonucleotides of the nucleic acids of the second probe may contain 5'-phosphate modifications. One or more modifications of the nucleic acids of the second probe may include internucleotide bonds. The nucleotide-nucleotide bond of the nucleic acid of the second probe may include a phosphorothioate, a phosphodiester, or a combination thereof. The nucleotide-nucleotide bond may include locked nucleic acid (LNA). The nucleotide-nucleotide bond of the nucleic acid of the second probe may provide advantages to the method described herein by increasing the melting temperature of the oligonucleotide interaction, increasing the stability of the probe interaction, improving the specificity of the probe's pre-recognition, or a combination thereof.

[0127] The second probe may include a variety of lengths. If the first probe contains nucleic acids, the nucleic acids may be at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 2 6, at least about 27, at least about 28, at least about 29, at least about 30, at least about 31, at least about 32, at least about 33, at least about 34, at least about 35, at least about 36, at least about 37, at least about 38, at least about 39, at least about 40, at least about 41, at least about 42, at least about 43, at least about 44, at least about 45, at least about 46, at least about 47, at least about 48, at least about 49, at least about 50, at least about 51, at least about 52, at least about 5 3, at least about 54, at least about 55, at least about 56, at least about 57, at least about 58, at least about 59, at least about 60, at least about 61, at least about 62, at least about 63, at least about 64, at least about 65, at least about 66, at least about 67, at least about 68, at least about 69, at least about 70, at least about 71, at least about 72, at least about 73, at least about 74, at least about 75, at least about 76, at least about 77, at least about 78, at least about 79, at least about 8 0, at least about 81, at least about 82, at least about 83, at least about 84, at least about 85, at least about 86, at least about 87, at least about 88, at least about 89, at least about 90, at least about 91, at least about 92, at least about 93, at least about 94, at least about 95, at least about 96, at least about 97, at least about 98, at least about 99, at least about 100, at least about 105, at least about 110, at least about 115, at least about 120, at least about 125, at least about 130,The nucleotide length may be at least approximately 135, at least approximately 140, at least approximately 145, at least approximately 150, at least approximately 155, at least approximately 160, at least approximately 165, at least approximately 170, at least approximately 175, at least approximately 180, at least approximately 185, at least approximately 190, at least approximately 195, at least approximately 200, or longer. If the second probe contains nucleic acids, the nucleic acids are at most approximately 1, at most approximately 2, at most approximately 3, at most approximately 4, at most approximately 5, at most approximately 6, at most approximately 7, at most approximately 8, at most approximately 9, at most approximately 10, at most approximately 11, at most approximately 12, at most approximately 13, at most approximately 14, at most approximately 15, at most approximately 16, at most approximately 17, at most approximately 18, at most approximately 19, at most approximately 20, at most approximately 21, at most approximately 22, at most approximately 23, at most approximately 24, at most approximately 25, at most approximately 26, at most approximately 27, at most approximately 28, at most Large: approximately 29, Maximum: approximately 30, Maximum: approximately 31, Maximum: approximately 32, Maximum: approximately 33, Maximum: approximately 34, Maximum: approximately 35, Maximum: approximately 36, Maximum: approximately 37, Maximum: approximately 38, Maximum: approximately 39, Maximum: approximately 40, Maximum: approximately 41, Maximum: approximately 42, Maximum: approximately 43, Maximum: approximately 44, Maximum: approximately 45, Maximum: approximately 46, Maximum: approximately 47, Maximum: approximately 48, Maximum: approximately 49, Maximum: approximately 50, Maximum: approximately 51, Maximum: approximately 52, Maximum: approximately 53, Maximum: approximately 54, Maximum: approximately 55, Maximum: approximately 56, Maximum: approximately 57, Maximum: approximately 58 , up to approximately 59, up to approximately 60, up to approximately 61, up to approximately 62, up to approximately 63, up to approximately 64, up to approximately 65, up to approximately 66, up to approximately 67, up to approximately 68, up to approximately 69, up to approximately 70, up to approximately 71, up to approximately 72, up to approximately 73, up to approximately 74, up to approximately 75, up to approximately 76, up to approximately 77, up to approximately 78, up to approximately 79, up to approximately 80, up to approximately 81, up to approximately 82, up to approximately 83, up to approximately 84, up to approximately 85, up to approximately 86, up to approximately 87, up to approximately 88, maximum approximately 89, maximum approximately 90, maximum approximately 91, maximum approximately 92, maximum approximately 93, maximum approximately 94, maximum approximately 95, maximum approximately 96, maximum approximately 97, maximum approximately 98, maximum approximately 99, maximum approximately 100, maximum approximately 105, maximum approximately 110, maximum approximately 115, maximum approximately 120, maximum approximately 125, maximum approximately 130, maximum approximately 135, maximum approximately 140, maximum approximately 145, maximum approximately 150, maximum approximately 155, maximum approximately 160, maximum approximately 165, maximum approximately 170, maximum approximately 175,The nucleotide lengths may be approximately 180, 185, 190, 195, 200, or shorter. If the second probe contains nucleic acid, the nucleic acid may be approximately 1-200, 2-195, 3-190, 4-185, 5-180, 6-175, 7-170, 8-165, 9-160, 10-155, 11-150, 12-145, 13-140, and 14 ~135, 15~130, 16~125, 17~120, 18~115, 19~110, 20~105, 21~100, 22~99, 23~98, 24~97, 25~96, 26~95, 27~94, 28~93, 29~92 , approximately 30-91, approximately 31-90, approximately 32-89, approximately 33-88, approximately 34-87, approximately 35-86, approximately 36-85, approximately 37-84, approximately 38-83, approximately 39-82, approximately 40-81, approximately 41-80, approximately 42-79, approximately 43-78, approximately 44-77, approximately 45-76, approximately 46 The nucleotide lengths may be approximately 75, 47-74, 48-73, 49-72, 50-71, 51-70, 52-69, 53-68, 54-67, 55-66, 56-65, 57-64, 58-63, 59-62, or 60-61.

[0128] The second probe may include one or more binding sites. One or more binding sites may bind to one or more other probes, one or more analytes, one or more features of the sample, or a combination thereof. In some cases, the second probe may include at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, or more binding sites. In some cases, the second probe may contain up to approximately 1, up to approximately 2, up to approximately 3, up to approximately 4, up to approximately 5, up to approximately 6, up to approximately 7, up to approximately 8, up to approximately 9, up to approximately 10, up to approximately 15, up to approximately 20, up to approximately 25, up to approximately 30, up to approximately 40, up to approximately 50, up to approximately 60, up to approximately 70, up to approximately 80, up to approximately 90, up to approximately 100, or fewer. In some cases, the second probe may contain approximately 1 to approximately 100, approximately 2 to approximately 90, approximately 3 to approximately 80, approximately 4 to approximately 70, approximately 5 to approximately 60, approximately 6 to approximately 50, approximately 7 to approximately 40, approximately 8 to approximately 30, approximately 9 to approximately 25, or approximately 10 to approximately 20 binding sites.

[0129] The second probe may include an aptamer. The aptamer of the second probe may include a nucleic acid. The aptamer of the second probe may include a secondary structure, a tertiary structure, or a combination thereof. The aptamer may be configured to recognize, bind to, and / or link to an analyte. For example, the aptamer may include a sequence that recognizes and binds to a protein of interest, and an associated tertiary structure. The analyte recognized by the aptamer, bound to, and / or linked to it may be the second analyte. The analyte recognized by the aptamer, bound to, and / or linked to it may include a nucleic acid, a polypeptide, or a combination thereof. For example, the analyte may include a DNA-binding protein containing an epitope recognized by the aptamer. The aptamer may include RNA, DNA, polypeptide, xenonucleic acid (XNA), or a combination thereof.Aptamers are at least about 1 kilodalton (kDa), at least about 2kDa, at least about 3kDa, at least about 4kDa, at least about 5kDa, at least about 6kDa, at least about 7kDa, at least about 8kDa, at least about 9kDa, at least about 10kDa, at least about 11kDa, at least about 12kDa, at least about 13kDa, at least about 14kDa, at least about 15kDa, at least about 16kDa, at least about 17kDa, at least about 18kDa, at least about 19kDa, at least about 20kDa, at least about 21kDa, at least about 22kDa, at least about 23kDa, at least about 24kDa, at least about 25kDa, and less Each may have a molecular weight of approximately 26 kDa, at least approximately 27 kDa, at least approximately 28 kDa, at least approximately 29 kDa, at least approximately 30 kDa, at least approximately 31 kDa, at least approximately 32 kDa, at least approximately 33 kDa, at least approximately 34 kDa, at least approximately 35 kDa, at least approximately 36 kDa, at least approximately 37 kDa, at least approximately 38 kDa, at least approximately 39 kDa, at least approximately 40 kDa, at least approximately 41 kDa, at least approximately 42 kDa, at least approximately 43 kDa, at least approximately 44 kDa, at least approximately 45 kDa, at least approximately 46 kDa, at least approximately 47 kDa, at least approximately 48 kDa, at least approximately 49 kDa, or at least approximately 50 kDa.Aptamers have a maximum of approximately 1kDa, 2kDa, 3kDa, 4kDa, 5kDa, 6kDa, 7kDa, 8kDa, 9kDa, 10kDa, 11kDa, 12kDa, 13kDa, 14kDa, 15kDa, 16kDa, 17kDa, 18kDa, 19kDa, 20kDa, 21kDa, 22kDa, 23kDa, 24kDa, 25kDa, and 26kDa. Da may have a molecular weight of up to approximately 27kDa, up to approximately 28kDa, up to approximately 29kDa, up to approximately 30kDa, up to approximately 31kDa, up to approximately 32kDa, up to approximately 33kDa, up to approximately 34kDa, up to approximately 35kDa, up to approximately 36kDa, up to approximately 37kDa, up to approximately 38kDa, up to approximately 39kDa, up to approximately 40kDa, up to approximately 41kDa, up to approximately 42kDa, up to approximately 43kDa, up to approximately 44kDa, up to approximately 45kDa, up to approximately 46kDa, up to approximately 47kDa, up to approximately 48kDa, up to approximately 49kDa, or up to approximately 50kDa. Aptamers are approximately 1-50kDa, 2-49kDa, 3-48kDa, 4-47kDa, 5-46kDa, 6-45kDa, 7-44kDa, 8-43kDa, 9-42kDa, 10-41kDa, 11-40kDa, 12-39kDa, 13-38kDa, and It may have a molecular weight of approximately 14-37 kDa, approximately 15-36 kDa, approximately 16-35 kDa, approximately 17-34 kDa, approximately 18-33 kDa, approximately 19-32 kDa, approximately 20-31 kDa, approximately 21-30 kDa, approximately 22-29 kDa, approximately 23-28 kDa, or approximately 24-27 kDa, approximately 25-26 kDa.

[0130] The second probe may include a polypeptide. The polypeptide of the second probe may include a protein, a peptide, or a combination thereof. The protein of the polypeptide of the second probe may include a protein-binding protein, a DNA-binding protein, or a combination thereof. The protein of the polypeptide of the second probe may be a nucleic acid-binding protein. The protein may include an antibody, an antibody fragment, an affimer, a nanobody, or a combination thereof. The antibody or antibody fragment of the second probe may include, but not limited to, various isotypes, including IgG, IgM, IgA, IgD, IgE, or a combination thereof. The antibody or antibody fragment of the second probe may include an fc domain that recognizes an analyte (e.g., the second analyte). The second probe may include one or more antibodies or antibody fragments. In some cases, the second probe may include an antibody that recognizes, binds to, and / or ligates to the second analyte, and an antibody that recognizes, binds to, and / or ligates to the antibody that recognizes, binds to, and / or ligates to the second analyte. One or more antibodies of the second probe may contain one or more modifications. One or more modifications of one or more antibodies of the second probe may contain nucleic acid modifications directly or indirectly conjugated to one or more antibodies or antibody fragments of the second probe. The nucleic acid modifications of the second probe may contain a binding site (e.g., a fourth binding site of the second probe). The binding site of the nucleic acid modification of the second probe may function as a primer to initiate amplification. The binding site of the nucleic acid modification of the second probe may function as a binding site to one or more portions of the first probe and / or the third probe, and upon binding of one or more portions of the first probe, it may initiate ligation of one end of the first probe to another end of the first probe and / or another end of the third probe.

[0131] A second probe may recognize a second analyte. The second analyte may include various types of analytes, and depending on the type of analyte, the second probe may recognize specific features. For example, the second probe may recognize and bind to ribosomal proteins, and the specific features of ribosomal proteins may include epitopes that are recognized and bound by anti-ribosomal antibodies. The second analyte may include ribonucleic acid. The ribonucleic acid (RNA) of the second analyte may include, but not limited to, messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), or combinations thereof. The RNA may be endogenous to the sample or exogenous to the sample. The RNA of the nucleic acid of the second analyte may be introduced into the sample by various means, including, but not limited to, the use of AAV or rAAV. If the second analyte includes RNA, the RNA may include modifications. Modifications may include N6-methyladenosine, 5-methylcytosine, N1-methyladenosine, N7-methylguanosine, N4-acetylcytosine, pseudouridine, N1-methylpseudridine, or combinations thereof. The RNA of the nucleic acid of the second analyte may contain single-stranded regions, double-stranded regions, or combinations thereof. The RNA may contain multiple nucleotides. The multiple nucleotides may contain A, C, U, G, or combinations thereof. In some cases, the RNA may contain secondary structures, tertiary structures, or combinations thereof.

[0132] The second probe may recognize the second analyte. If the second probe may recognize the second analyte, the second analyte may contain nucleic acids, which may contain one or more modifications. The nucleic acids of the second analyte may contain RNA. The one or more modifications of the nucleic acids of the second analyte may include N6-methyladenosine, 5-methylcytosine, N1-methyladenosine, N7-methylguanosine, N4-acetylcytosine, pseudouridine, N1-methylpseudridine, or a combination thereof. The second probe may bind to nucleic acids containing one or more modifications.

[0133] The second probe may recognize polypeptides. The polypeptide of the second analyte may include proteins, peptides, or combinations thereof. If the polypeptide of the second analyte includes proteins, the proteins may include transcription factors, ribosomal proteins, histones, polymerases, helicases, restriction enzymes, ribonucleic acid-binding proteins, enzymes, antibodies, structural proteins, protective proteins, signaling proteins, receptors, soluble proteins, transmembrane proteins, or combinations thereof. If the protein may be a signaling protein, the signaling protein may be a cytokine, a chemokine, or a combination thereof. If the polypeptide of the second analyte includes proteins, the proteins may include transcription factors, ribosomal proteins, histones, polymerases, or combinations thereof. When ribosomal proteins are listed as the second analyte, the ribosomal proteins include S3A ribosomal protein, SA ribosomal protein, S3 ribosomal protein, S9 ribosomal protein, S4(X,Y1,Y2) ribosomal protein, S2 ribosomal protein, S6 ribosomal protein, S5 ribosomal protein, S7 ribosomal protein, S15A ribosomal protein, S8 ribosomal protein, S16 ribosomal protein, S20 ribosomal protein, S10 ribosomal protein, S14 ribosomal protein, S23 ribosomal protein, S12 ribosomal protein, S18 ribosomal protein, and S29 ribosomal protein. Protein, S13 ribosomal protein, S11 ribosomal protein, S17 ribosomal protein, S15 ribosomal protein, S19 ribosomal protein, S21 ribosomal protein, S24 ribosomal protein, S25 ribosomal protein, S26 ribosomal protein, S27 ribosomal protein, S28 ribosomal protein, S30 ribosomal protein, S27A ribosomal protein, RACK1 ribosomal protein, L10A ribosomal protein, L8 ribosomal protein, L3 ribosomal protein, L4 ribosomal protein, L11 ribosomal protein, L9 ribosomal protein, L6 ribosomal protein, L7A ribosomal protein,P0 ribosomal protein, L12 ribosomal protein, L13A ribosomal protein, L13 ribosomal protein, L23 ribosomal protein, L14 ribosomal protein, L27A ribosomal protein, L15 ribosomal protein, L10 ribosomal protein, L5 ribosomal protein, L18 ribosomal protein, L19 ribosomal protein, L18A ribosomal protein, L21 ribosomal protein, L17 ribosomal protein, L22 ribosomal protein, L23A ribosomal protein, L26 ribosomal protein, L24 ribosomal protein, L27 ribosomal protein Possible examples include L28 ribosomal protein, L35 ribosomal protein, L29 ribosomal protein, L7 ribosomal protein, L30 ribosomal protein, L31 ribosomal protein, L32 ribosomal protein, L35A ribosomal protein, L34 ribosomal protein, L36 ribosomal protein, L37 ribosomal protein, L38 ribosomal protein, L39 ribosomal protein, L40 ribosomal protein, L41 ribosomal protein, L36A ribosomal protein, L37A ribosomal protein, P1 / P2(αβ) ribosomal protein, or combinations thereof.

[0134] The second probe can recognize one or more post-translational modifications of a protein. Examples of one or more post-translational modifications of the protein in the second analyte include myristoylation, palmitoylation, farnesylation, geranylgeranylation, glypiation, glycosylphosphatidylinositol, lipoylation, flavin moiety attachment, heme C attachment, phosphopantetheinylation, retinilidenschiff base formation, translation factor modification, diphthamide formation, ethanolamine phosphoglycerol, hypsin formation, β-lysine addition to lysine, acylation (e.g., O-acylation, N-acylation, and S-acylation), acetylation, formylation, alkylation, amidation, arginylation, polyglutamylation, polyglycylation, and butyrylation. Possible modifications include γ-carboxylation, glycosylation, polysialylation, malonylation, hydroxylation, nucleotide addition, phosphate esterification (O bond), phosphoramide (N bond) formation, phosphorylation, adenylation, uridilylation, propionylation, pyroglutamate formation, S-glutathionylation, S-nitrosylation, S-sulfenylation, S-sulfinylation, S-sulfonylation, succinylation, sulfation, glycation, carbamylation, carbonylation, spontaneous isopeptide bond formation, biotinylation, carbamylation, oxidation, pegylation, ubiquitination, SUMOylation, nedylation, ISGation, citrullination, deamidation, eliminylation, or combinations thereof. The second analyte may include chemical modifications. One or more post-translational modifications of the protein in the second analyte may include alkylation, phosphorylation, or combinations thereof.

[0135] The methods described herein also relate to analyzing the proximity of one or more analytes using one or more barcodes or derivatives thereof. A barcode or derivative thereof may provide information relating to one or more analytes, the proximity of one or more analytes, or a combination thereof. A barcode or derivative thereof may indicate the presence of an analyte. For example, detection of a barcode or derivative thereof in a tissue sample may indicate the presence of an analyte recognized and bound to one or more probes containing a barcode within the sample. A barcode or derivative thereof may indicate the proximity of one analyte to another. For example, detection of a barcode or derivative thereof in a tissue sample may indicate that a first analyte and a second analyte are complexed with each other within the tissue sample. A barcode or derivative thereof may be used in combination with other information to indicate the presence and / or spatial localization of an analyte. For example, a barcode or derivative thereof may be detected as a result of a first probe and a second probe binding to a messenger RNA sequence containing a specific single nucleotide polymorphism. In conjunction with this, the sample may be stained with nuclear stain, which may be detected using an imaging system. Detection of barcodes or their derivatives, and detection through nuclear staining, can provide information related to the presence of messenger RNA containing single nucleotide polymorphisms and its location within the nucleus and / or extranucleus of the sample. Barcodes or their derivatives can be used in combination with other information to indicate the proximity of one analyte to another. For example, barcodes or their derivatives may be detected to determine whether a particular messenger RNA sequence is in close proximity to ribosomal RNA, and dye-labeled antibodies may be used to detect the presence of ribosomal proteins. In conjunction, detection of barcodes or their derivatives, and detection of dye-labeled antibodies, can provide information related to the translational state of messenger RNA. Barcodes or their derivatives can be detected in situ. Barcodes or their derivatives can be detected using one or more detection rounds.For example, a barcode or its derivative may contain 8 nucleotides, and sequence information associated with 2 of these 8 nucleotides can be obtained over 7 detection rounds using detection probes and anchor probes, and by removing the corresponding detection probes and anchor probes. Figure 9 shows how data associated with a barcode sequence can be collected over multiple rounds. The amplified products can be quantified under a fluorescence microscope using in situ sequencing. The number of amplified products or the fluorescence signal intensity of the amplified products represents the translation efficiency of specific mRNA in specific cells. Amplified products from different mRNA species can be distinguished by different barcode A sequences.

[0136] The probes described herein, including the first probe, the second probe, the third probe, and other probes, may each contain one or more barcodes. In some cases, the probes of the method described herein may contain at least about 1 barcode, at least about 2 barcodes, at least about 3 barcodes, at least about 4 barcodes, at least about 5 barcodes, at least about 6 barcodes, at least about 7 barcodes, at least about 8 barcodes, at least about 9 barcodes, at least about 10 barcodes, at least about 11 barcodes, at least about 12 barcodes, at least about 13 barcodes, at least about 14 barcodes, at least about 15 barcodes, at least about 16 barcodes, at least about 17 barcodes, at least about 18 barcodes, at least about 19 barcodes, at least about 20 barcodes, at least about 21 barcodes, at least about 22 barcodes, at least about 23 barcodes, at least about 24 barcodes, and at least about 25 barcodes. It may include at least approximately 26 barcodes, at least approximately 27 barcodes, at least approximately 28 barcodes, at least approximately 29 barcodes, at least approximately 30 barcodes, at least approximately 31 barcodes, at least approximately 32 barcodes, at least approximately 33 barcodes, at least approximately 34 barcodes, at least approximately 35 barcodes, at least approximately 36 barcodes, at least approximately 37 barcodes, at least approximately 38 barcodes, at least approximately 39 barcodes, at least approximately 40 barcodes, at least approximately 41 barcodes, at least approximately 42 barcodes, at least approximately 43 barcodes, at least approximately 44 barcodes, at least approximately 45 barcodes, at least approximately 46 barcodes, at least approximately 47 barcodes, at least approximately 48 barcodes, at least approximately 49 barcodes, at least approximately 50 or more barcodes.In some cases, the probe of the method described herein may have up to approximately 1 barcode, up to approximately 2 barcodes, up to approximately 3 barcodes, up to approximately 4 barcodes, up to approximately 5 barcodes, up to approximately 6 barcodes, up to approximately 7 barcodes, up to approximately 8 barcodes, up to approximately 9 barcodes, up to approximately 10 barcodes, up to approximately 11 barcodes, up to approximately 12 barcodes, up to approximately 13 barcodes, up to approximately 14 barcodes, up to approximately 15 barcodes, up to approximately 16 barcodes, up to approximately 17 barcodes, up to approximately 18 barcodes, up to approximately 19 barcodes, up to approximately 20 barcodes, up to approximately 21 barcodes, up to approximately 22 barcodes, up to approximately 23 barcodes, up to approximately 24 barcodes, and up to approximately 25 barcodes. It may contain up to approximately 26 barcodes, up to approximately 27 barcodes, up to approximately 28 barcodes, up to approximately 29 barcodes, up to approximately 30 barcodes, up to approximately 31 barcodes, up to approximately 32 barcodes, up to approximately 33 barcodes, up to approximately 34 barcodes, up to approximately 35 barcodes, up to approximately 36 barcodes, up to approximately 37 barcodes, up to approximately 38 barcodes, up to approximately 39 barcodes, up to approximately 40 barcodes, up to approximately 41 barcodes, up to approximately 42 barcodes, up to approximately 43 barcodes, up to approximately 44 barcodes, up to approximately 45 barcodes, up to approximately 46 barcodes, up to approximately 47 barcodes, up to approximately 48 barcodes, up to approximately 49 barcodes, up to approximately 50 barcodes, or more.In some cases, the probes of the methods described herein may have approximately 1 to approximately 50 barcodes, approximately 2 to approximately 49 barcodes, approximately 3 to approximately 48 barcodes, approximately 4 to approximately 47 barcodes, approximately 5 to approximately 46 barcodes, approximately 6 to approximately 45 barcodes, approximately 7 to approximately 44 barcodes, approximately 8 to approximately 43 barcodes, approximately 9 to approximately 42 barcodes, approximately 10 to approximately 41 barcodes, approximately 11 to approximately 40 barcodes, approximately 12 to approximately 39 barcodes, approximately 13 to The probe may contain approximately 38 barcodes, approximately 14 to 37 barcodes, approximately 15 to 36 barcodes, approximately 16 to 35 barcodes, approximately 17 to 34 barcodes, approximately 18 to 33 barcodes, approximately 19 to 32 barcodes, approximately 20 to 31 barcodes, approximately 21 to 30 barcodes, approximately 22 to 29 barcodes, approximately 23 to 28 barcodes, or approximately 24 to 27 barcodes, or approximately 25 to 26 barcodes. The first probe may contain 1 barcode, 2 barcodes, 3 barcodes, 4 barcodes, or more barcodes. The first probe may contain 2 barcodes, with the second barcode of the 2 barcodes corresponding to the first analyte. The first probe may contain 2 barcodes, with the second barcode of the 2 barcodes corresponding to the second analyte. The first probe may include two barcodes. The second barcode of the two barcodes corresponds to the first analyte that is in close proximity to the second analyte.

[0137] A barcode containing one or more barcodes as described herein may contain nucleic acids. Nucleic acids may contain DNA, RNA, or a combination thereof. Nucleic acids may contain multiple nucleotides, including but not limited to A, T, C, U, G, or a combination thereof. Barcode nucleic acids may contain one or more non-natural nucleotides. Nucleic acids may contain one or more nucleic acid modifications. Nucleic acids are at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 31, at least about 32, at least about 33, at least about 34, at least about 35, at least about 36, at least about 37, at least about 38, at least about 39, at least about 40, at least about 41, at least about 42, at least about 43, at least about 44, at least about 45, at least about 46, at least about 4 7, at least about 48, at least about 49, at least about 50, at least about 51, at least about 52, at least about 53, at least about 54, at least about 55, at least about 56, at least about 57, at least about 58, at least about 59, at least about 60, at least about 61, at least about 62, at least about 63, at least about 64, at least about 65, at least about 66, at least about 67, at least about 68, at least about 69, at least about 70, at least about 71, at least about 72, at least about 73, at least about 74, at least about 75, at least about 76, at least about 77, at least about 78, at least about 79, at least about 80, at least about 81, at least about 82, at least about 83, at least about 84, at least about 85, at least about 86, at least about 87, at least about 88, at least about 89, at least about 90, at least about 91, at least about 92, at least about 93,The nucleotides may include, but are not limited to, a variety of lengths, including at least about 94, at least about 95, at least about 96, at least about 97, at least about 98, at least about 99, at least about 100, at least about 105, at least about 110, at least about 115, at least about 120, at least about 125, at least about 130, at least about 135, at least about 140, at least about 145, at least about 150, at least about 155, at least about 160, at least about 165, at least about 170, at least about 175, at least about 180, at least about 185, at least about 190, at least about 195, at least about 200, or longer. Nucleic acids are at most approximately 1, at most approximately 2, at most approximately 3, at most approximately 4, at most approximately 5, at most approximately 6, at most approximately 7, at most approximately 8, at most approximately 9, at most approximately 10, at most approximately 11, at most approximately 12, at most approximately 13, at most approximately 14, at most approximately 15, at most approximately 16, at most approximately 17, at most approximately 18, at most approximately 19, at most approximately 20, at most approximately 21, at most approximately 22, at most approximately 23, at most approximately 24, at most approximately 25, at most Approximately 26 at the beginning, approximately 27 at the end, approximately 28 at the end, approximately 29 at the end, approximately 30 at the end, approximately 31 at the end, approximately 32 at the end, approximately 33 at the end, approximately 34 at the end, approximately 35 at the end, approximately 36 at the end, approximately 37 at the end, approximately 38 at the end, approximately 39 at the end, approximately 40 at the end, approximately 41 at the end, approximately 42 at the end, approximately 43 at the end, approximately 44 at the end, approximately 45 at the end, approximately 46 at the end, approximately 47 at the end, approximately 48 at the end, approximately 49 at the end, and approximately 50 at the end. , up to approximately 51, up to approximately 52, up to approximately 53, up to approximately 54, up to approximately 55, up to approximately 56, up to approximately 57, up to approximately 58, up to approximately 59, up to approximately 60, up to approximately 61, up to approximately 62, up to approximately 63, up to approximately 64, up to approximately 65, up to approximately 66, up to approximately 67, up to approximately 68, up to approximately 69, up to approximately 70, up to approximately 71, up to approximately 72, up to approximately 73, up to approximately 74, up to Approximately 75, maximum approximately 76, maximum approximately 77, maximum approximately 78, maximum approximately 79, maximum approximately 80, maximum approximately 81, maximum approximately 82, maximum approximately 83, maximum approximately 84, maximum approximately 85, maximum approximately 86, maximum approximately 87, maximum approximately 88, maximum approximately 89, maximum approximately 90, maximum approximately 91, maximum approximately 92, maximum approximately 93, maximum approximately 94, maximum approximately 95, maximum approximately 96, maximum approximately 97, maximum approximately 98, maximum approximately 99,They may have nucleotide lengths of up to approximately 100, up to approximately 105, up to approximately 110, up to approximately 115, up to approximately 120, up to approximately 125, up to approximately 130, up to approximately 135, up to approximately 140, up to approximately 145, up to approximately 150, up to approximately 155, up to approximately 160, up to approximately 165, up to approximately 170, up to approximately 175, up to approximately 180, up to approximately 185, up to approximately 190, up to approximately 195, up to approximately 200, or shorter. Nucleic acids range from approximately 1 to 200, 2 to 195, 3 to 190, 4 to 185, 5 to 180, 6 to 175, 7 to 170, 8 to 165, 9 to 160, 10 to 155, 11 to 150, 12 to 145, 13 to 140, 14 to 135, and 15 to 15. 130, approximately 16-125, approximately 17-120, approximately 18-115, approximately 19-110, approximately 20-105, approximately 21-100, approximately 22-99, approximately 23-98, approximately 24-97, approximately 25-96, approximately 26-95, approximately 27-94, approximately 28-93, approximately 29-92, approximately 30-91 , approximately 31-90, approximately 32-89, approximately 33-88, approximately 34-87, approximately 35-86, approximately 36-85, approximately 37-84, approximately 38-83, approximately 39-82, approximately 40-81, approximately 41-80, approximately 42-79, approximately 43-78, approximately 44-77, approximately 45-76, approximately 46-75, They may have lengths of approximately 47-74, 48-73, 49-72, 50-71, 51-70, 52-69, 53-68, 54-67, 55-66, 56-65, 57-64, 58-63, 59-62, or 60-61 nucleotides.

[0138] One or more barcodes described herein may also correspond to a first analyte. For example, the presence of a first analyte may be indicated by the detection of a barcode or a derivative thereof in a sample. One or more barcodes described herein may also correspond to a second analyte. For example, the presence of a second analyte may be indicated by the detection of a barcode or a derivative thereof in a sample. One or more barcodes described herein may also correspond to the proximity of one analyte to another. For example, the detection of a barcode or a derivative thereof in a sample may indicate that the first analyte is complexed with or otherwise in close proximity to the second analyte in the sample. In some cases, a barcode may correspond to a first analyte in proximity to a second analyte. A barcode or a derivative of a barcode may be detected in a manner corresponding to the proximity of a first analyte to a second analyte.

[0139] Barcode derivatives may include the inverse complement of the barcode. The inverse complement of the barcode may include sequences that can hybridize to the barcode. Barcode derivatives may be formed from amplification reactions. Amplification reactions may include rolling circle amplification reactions in which an amplification product is produced, and the amplification product contains multiple copies of the barcode derivative. Multiple copies of the barcode derivative may be linked to one another within the amplification product.

[0140] The methods described herein may also include amplifying a cyclic oligonucleotide by performing an amplification reaction. A cyclic oligonucleotide may be formed by ligating two ends of a first probe. A cyclic oligonucleotide may also be formed by ligating one end of a first probe to one end of a third probe, and ligating the second end of a first probe to the second end of a third probe. The amplification reaction may include performing a rolling circle amplification reaction using the cyclic oligonucleotide. The amplification reaction may include the use of a primer that binds to the cyclic oligonucleotide and initiates amplification. The primer that binds to the cyclic oligonucleotide may include a portion of one or more probes described herein. For example, a second probe may bind to a cyclic oligonucleotide and initiate a rolling circle amplification reaction. The rolling circle amplification reaction may produce one or more amplification products. The one or more amplification products produced by the rolling circle amplification reaction may include multiple copies of the cyclic oligonucleotide. Multiple copies of the cyclic oligonucleotide may be linked together to form a long chain of nucleic acid. The cyclic oligonucleotide may include one or more copies of a barcode or its derivatives. One or more copies of a barcode may include one or more copies of the same barcode (e.g., the same nucleic acid sequence). One or more copies of a barcode may include one or more copies of different barcodes.Each of the one or more amplification products produced by rolling circle amplification is one or more barcodes of a cyclic oligonucleotide, or at least one copy, at least two copies, at least three copies, at least about four copies, at least about five copies, at least about six copies, at least about seven copies, at least about eight copies, at least about nine copies, at least about ten copies, at least about 20 copies, at least about 30 copies, at least about 40 copies, at least about 50 copies, at least about 60 copies, at least about 70 copies, at least about 80 copies, at least about 90 copies, at least about 100 copies, at least about 135 copies, at least about 150 copies, at least about 175 copies. It may include copies, at least approximately 200 copies, at least approximately 300 copies, at least approximately 400 copies, at least approximately 500 copies, at least approximately 600 copies, at least approximately 700 copies, at least approximately 800 copies, at least approximately 900 copies, at least approximately 1000 copies, at least approximately 2000 copies, at least approximately 3000 copies, at least approximately 4000 copies, at least approximately 5000 copies, at least approximately 6000 copies, at least approximately 7000 copies, at least approximately 8000 copies, at least approximately 9000 copies, at least approximately 10000 copies, at least approximately 500000 copies, at least approximately 500000 copies, at least approximately 1000000 copies, or more copies.Each of the one or more amplification products generated by rolling circle amplification is a cyclic oligonucleotide with one or more barcodes, or a maximum of 1 copy, 2 copies, 3 copies, approximately 4 copies, approximately 5 copies, approximately 6 copies, approximately 7 copies, approximately 8 copies, approximately 9 copies, approximately 10 copies, approximately 20 copies, approximately 30 copies, approximately 40 copies, approximately 50 copies, approximately 60 copies, approximately 70 copies, approximately 80 copies, approximately 90 copies, approximately 100 copies, approximately 135 copies, approximately 150 copies, and approximately 175 copies. This may include copies, up to approximately 200 copies, up to approximately 300 copies, up to approximately 400 copies, up to approximately 500 copies, up to approximately 600 copies, up to approximately 700 copies, up to approximately 800 copies, up to approximately 900 copies, up to approximately 1000 copies, up to approximately 2000 copies, up to approximately 3000 copies, up to approximately 4000 copies, up to approximately 5000 copies, up to approximately 6000 copies, up to approximately 7000 copies, up to approximately 8000 copies, up to approximately 9000 copies, up to approximately 10000 copies, up to approximately 500000 copies, up to approximately 500000 copies, up to approximately 1000000 copies, or fewer copies. Each of the one or more amplification products produced by rolling circle amplification is approximately 1 to approximately 1,000,000 copies, approximately 2 to approximately 500,000 copies, approximately 3 to approximately 100,000 copies, approximately 4 to approximately 50,000 copies, approximately 5 to approximately 10,000 copies, approximately 6 to approximately 9,000 copies, approximately 7 to approximately 8,000 copies, approximately 8 to approximately 7,000 copies, It may have approximately 9 to 6000 copies, approximately 10 to 5000 copies, approximately 20 to 4000 copies, approximately 30 to 3000 copies, approximately 40 to 2000 copies, approximately 50 to 1000 copies, approximately 60 to 900 copies, approximately 70 to 800 copies, approximately 80 to 700 copies, approximately 90 to 600 copies, approximately 100 to 500 copies, approximately 135 to 400 copies, approximately 150 to 300 copies, and approximately 175 to 200 copies.

[0141] Rolling circle amplification reactions can be carried out using polymerase. The polymerase can be DNA polymerase. Examples of DNA polymerases include Q5 High-Fidelity DNA polymerase, Q5U Hot Start High-Fidelity DNA polymerase, and Phusion High-Fidelity DNA polymerase. * Routine PCR, OneTaq DNA polymerase, Taq DNA polymerase, LongAmp Taq DNA polymerase, Hemo KlenTaq, Epimark Hot Start Taq DNA polymerase, isothermal amplification and strand substitution, Bst DNA polymerase, Bst DNA polymerase, Bst 2.0 DNA polymerase, Bst 3.0 DNA polymerase, Bsu DNA polymerase, large fragment, phi29 DNA polymerase, phi29-XT DNA polymerase, T7 DNA polymerase (unmodified), Sulfolobus DNA polymerase IV, Therminator™ DNA polymerase, DNA polymerase I (E. coli), DNA polymerase I, large (Krenow) fragment, Krenow fragment (3'→5'exo-), T4 DNA polymerase, Legacy polymerase, Vent DNA polymerase, Vent(exo-) DNA polymerase, Deep Vent DNA polymerase, Deep Possible polymerases include Vent(exo-)DNA polymerase, or combinations thereof. Examples of polymerases include phi29 DNA polymerase, phi29-XT DNA polymerase, or combinations thereof.

[0142] The amplification reaction may be carried out using a buffer. The buffer may contain a variety of components, including but not limited to MgCl2, NaCl, CaCl2, ethylenediaminetetraacetic acid (EDTA), 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (Triton X-100), polysorbate 20 (Tween 20), sodium lauryl sulfate (SDS), 2-amino-2-hydroxymethylpropane-1,3-diol (Tris), shear DNA, water, or combinations thereof.

[0143] Amplification reactions can occur at certain temperatures. The temperatures are approximately 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, and 51°C. The temperature could be approximately 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, or higher. The temperatures range from approximately 4 to 95°C, 5 to 94°C, 6 to 93°C, 7 to 92°C, 8 to 91°C, 9 to 90°C, 10 to 89°C, 11 to 88°C, 12 to 87°C, 13 to 86°C, 14 to 85°C, 15 to 84°C, 16 to 83°C, 17 to 82°C, 18 to 81°C, 19 to 80°C, 20 to 79°C, 21 to 78°C, 22 to 77°C, 23 to 76°C, 24 to 75°C, 25 to 74°C, 26 to 73°C, and 27 to 75°C. It could be 72°C, approximately 28-71°C, approximately 29-70°C, approximately 30-69°C, approximately 31-68°C, approximately 32-67°C, approximately 33-66°C, approximately 34-65°C, approximately 35-64°C, approximately 36-63°C, approximately 37-62°C, approximately 38-61°C, approximately 39-60°C, approximately 40-59°C, approximately 41-58°C, approximately 42-57°C, approximately 43-56°C, approximately 44-55°C, approximately 45-54°C, approximately 46-53°C, approximately 47-52°C, approximately 48-51°C, or approximately 49-50°C.

[0144] The amplification reaction may take place over a certain period of time. The length of time may be at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, at least about 60 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, or longer. The duration of time is approximately 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, and 10 hours. It can be up to approximately 11 hours, up to approximately 12 hours, up to approximately 13 hours, up to approximately 14 hours, up to approximately 15 hours, up to approximately 16 hours, up to approximately 17 hours, up to approximately 18 hours, up to approximately 19 hours, up to approximately 20 hours, up to approximately 21 hours, up to approximately 22 hours, up to approximately 23 hours, up to approximately 24 hours, up to approximately 1 day, up to approximately 2 days, up to approximately 3 days, up to approximately 4 days, or shorter.The length of time can range from approximately 5 minutes to 24 hours, 10 minutes to 23 hours, 15 minutes to 22 hours, 20 minutes to 21 hours, 25 minutes to 20 hours, 30 minutes to 19 hours, 40 minutes to 18 hours, 45 minutes to 17 hours, 50 minutes to 16 hours, 55 minutes to 15 hours, 60 minutes to 14 hours, 1 hour to 13 hours, 2 hours to 12 hours, 3 hours to 11 hours, 4 hours to 10 hours, 5 hours to 9 hours, or 6 hours to 8 hours.

[0145] The second probe may be a primer for the amplification reaction. The second probe may bind to a cyclic oligonucleotide to initiate amplification. For example, the second probe may bind to a cyclic oligonucleotide (e.g., hybridize) and then bind to a second analyte. The second probe may bind to a cyclic oligonucleotide to form a double-stranded nucleic acid having an overhang region. The double-stranded nucleic acid formed between the second probe and the circular oligonucleotide may contain at least about 1 base pair, at least about 2 base pairs, at least about 3 base pairs, at least about 4 base pairs, at least about 5 base pairs, at least about 6 base pairs, at least about 7 base pairs, at least about 8 base pairs, at least about 9 base pairs, at least about 10 base pairs, at least about 11 base pairs, at least about 12 base pairs, at least about 13 base pairs, at least about 14 base pairs, at least about 15 base pairs, at least about 16 base pairs, at least about 17 base pairs, at least about 18 base pairs, at least about 19 base pairs, at least about 20 base pairs, at least about 21 base pairs, at least about 22 base pairs, at least about 23 base pairs, at least about 24 base pairs, at least about 25 base pairs, at least about 26 base pairs, at least about 27 base pairs, at least about 28 base pairs, at least about 29 base pairs, at least about 30 base pairs, or more base pairs. Double-stranded nucleic acids can contain up to approximately 1 base pair, up to approximately 2 base pairs, up to approximately 3 base pairs, up to approximately 4 base pairs, up to approximately 5 base pairs, up to approximately 6 base pairs, up to approximately 7 base pairs, up to approximately 8 base pairs, up to approximately 9 base pairs, up to approximately 10 base pairs, up to approximately 11 base pairs, up to approximately 12 base pairs, up to approximately 13 base pairs, up to approximately 14 base pairs, up to approximately 15 base pairs, up to approximately 16 base pairs, up to approximately 17 base pairs, up to approximately 18 base pairs, up to approximately 19 base pairs, up to approximately 20 base pairs, up to approximately 21 base pairs, up to approximately 22 base pairs, up to approximately 23 base pairs, up to approximately 24 base pairs, up to approximately 25 base pairs, up to approximately 26 base pairs, up to approximately 27 base pairs, up to approximately 28 base pairs, up to approximately 29 base pairs, up to approximately 30 base pairs, or fewer base pairs.Double-stranded nucleic acids can have lengths of approximately 1 to 30 base pairs, 2 to 29 base pairs, 3 to 28 base pairs, 4 to 27 base pairs, 5 to 26 base pairs, 6 to 25 base pairs, 7 to 24 base pairs, 8 to 23 base pairs, 9 to 22 base pairs, 10 to 21 base pairs, 11 to 20 base pairs, 12 to 19 base pairs, 13 to 18 base pairs, 14 to 17 base pairs, and 15 to 16 base pairs.

[0146] Barcodes or their derivatives can be detected. Detection of barcodes or their derivatives can provide information relating to the proximity of one analyte to another. Detection of barcodes or their derivatives can provide information relating to the sequence of the barcode or its derivative. Information relating to the sequence of the barcode or its derivative can be obtained over one or more cycles of detection. Each detection round can provide information relating to a portion of the barcode or its derivative. For example, a single round of detection may reveal sequence information for two nucleotides of the barcode or its derivative. Subsequent detection rounds may reveal information about other nucleotides in the barcode or its derivative. Other nucleotides in the barcode or its derivative may overlap with the two nucleotides of the barcode or its derivative revealed in one round, or they may not overlap with the two nucleotides of the barcode or its derivative revealed in one round. In some cases, detection methods based on hybridization can be used to detect barcodes or their derivatives. For example, a dye-labeled probe may be bound to the barcode or its derivative, and sequence information of the barcode or its derivative may be revealed based on the sequence of the dye-labeled probe. In some cases, detection methods based on sequencing can be used to detect barcodes or their derivatives.

[0147] Detecting a barcode or a derivative thereof may involve contacting a sample with a material and performing a reaction based on in situ sequencing. Various in situ sequencing-based reactions may be performed, including but not limited to synthesis sequencing, sequencing by oligonucleotide ligation and detection (SOLiD), sequencing with error-reduction by dynamic annealing and ligation (SEDAL), or combinations thereof. Details of performing SEDAL sequencing can be found in its entirety in PCT / US2019 / 025835, which is referenced by means of this document. In some cases, detection of a barcode or its complement may involve in situ sequencing using multiple detection probes. In some embodiments, the barcode complement may include the barcode's inverse complement.

[0148] After amplification, the sample may come into contact with multiple probes. These probes may include anchor probes and detection probes. An anchor probe may be used for the detection of a barcode or its derivative sequence by hybridizing to a portion of the amplified product sequence adjacent to the binding site of the detection probe, thereby enabling a ligation event between one or more ends of one or more probes. Ligation between the anchor probe and the detection probe may increase the melting temperature associated with the ligated product compared to the melting temperature of the detection probe alone. The increased melting temperature leads to improved stability of the double-strand formed between the detection probe and the amplified product, thereby enabling more specific detection of the detection probe bound to the amplified product. An anchor probe may bind to the amplified product formed during amplification. One of the multiple anchor probes may bind to all or part of the barcode or its derivative. The detection probe may bind to all or part of the barcode or its derivative. That particular anchor probe may bind to a sequence adjacent to the barcode or its derivative. The detection probe may bind to a sequence adjacent to the barcode or its derivative.

[0149] The detection probe may contain nucleic acids. Nucleic acids may include DNA, RNA, or a combination thereof. The nucleic acids in the detection probe may be single-stranded, double-stranded, or a combination thereof. The nucleic acids in the detection probe may include LNA. The detection probe may contain a label. The label may be a detectable label, a linker, or a combination thereof. The detectable label may contain a fluorescent molecule. The fluorescent molecule may be included.

[0150] Detecting a barcode or its inverse complement may involve reading the signal associated with the barcode or its inverse complement. Various methods, including hybridization-based detection, sequencing-based detection, or a combination thereof, can be used to read the signal associated with the barcode's inverse complement. Reading the signal associated with a barcode or its inverse complement may be performed in situ. One or more rounds of reading may be performed to collect one or more signals associated with a barcode or its inverse complement. In cases where one or more rounds of reading are performed, signals may be removed between rounds.

[0151] In some cases, a detection probe that binds to a barcode or its inverse complement may be added to the sample. The detection probe may contain nucleic acids. The detection probe may contain one or more labels. The labels may contain one or more fluorescent molecules, one or more quantum dots, one or more proteins, one or more mass tags, one or more chromophores, or a combination thereof. In some cases, one or more proteins may contain enzymes, such as horseradish peroxidase. The enzyme may produce a signal indicating the label. Examples of fluorescent molecules include AlexaFluo Texas Red, Rhodamine B, Rhodamine 6G, Rhodamine 10, TMR-iodoacetamide, Lisamin Rhodamine B sulfonyl chloride, Lisamin Rhodamine B sulfonyl hydrazine, Texas Red sulfonyl chloride, Texas Red hydrazide, coumarins and coumarin derivatives such as AMCA, AMCA-NHS, AMCA-sulfo-NHS, AMCA-HPDP, DCIA, AMCE-hydrazide, BODIPY FL C3-SE, BODIPY 530 / 550 C3, BODIPY 530 / 550 C3-SE, BODIPY 530 / 550 C3 hydrazide, BODIPY 493 / 503 C3 hydrazide, BODIPY FL C3 hydrazide, BODIPY FL IA, BODIPY 530 / 551 IA, and Br-BODIPY BODIPY and derivatives such as 493 / 503, Cascade Blue acetylazide, Cascade Blue cadaverine, Cascade Blue ethylenediamine, Cascade Blue hydrazide, Lucifer Yellow, Lucifer Yellow CH and other Cascade Blue and derivatives, indolium cyanine dyes, benzoindolium cyanine dyes, pyridium cyanine dyes, thiozolium cyanine dyes, quinolinium cyanine dyes, imidazolium cyanine dyes, cyanines and derivatives such as C y3, C y5, BCPDA, TBP, TMT, BHHCT, BCOT, europium chelate, terbium chelate, Alexa Fluor dye, DyLight dye, Atto dye, LightCycler Red dye, CAL Flour dye and other lanthanide chelates and derivatives, OregonGreen dye, WellRED dye, IRD dye, phycoerythrin and phycobilin dyes, malachite green, stilbene, DEG dye, JOE and its derivatives such as C y3, C y5 and C y7, carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxynaptofluorescein, fluorescein isothiocyanate, NHS-fluorescein, iodoacetamidefluorescein, fluorescein maleimide, SAMSA-fluorescein, fluorescein thiosemicarbazide, carbohydrazinomethylthioacetyl-aminofluorescein, and other fluoresceins. Luorethine and fluorescein derivatives, TRITC, TMR, Lisamin Rhodamine, TEX615, TYE(trademark)665, TYE705, SUN, ATTO(trademark)425, ATTO(trademark)488, ATTO(trademark)532, ATTO(trademark)550, ATTO(trademark)565, ATTO(trademark)Rho101, ATTO(trademark)590, ATTO(trademark)633, ATTO(trademark)647, ATTO(trademark)700, Alexa Examples of labels include, but are not limited to, Fluor® 488 (NHS ester), Alexa Fluor® 532 (NHS ester), Alexa Fluor® 546 (NHS ester), Alexa Fluor® 594 (NHS ester), Alexa Fluor® 647 (NHS ester), Alexa Fluor® 660 (NHS ester), Alexa Fluor® 750 (NHS ester), IRDye® 700, IRDye® 800, Rhodamine Red®, 5-TAMRA®, Texas Red®-X, Lightcycler® 640, Dy 750, or combinations thereof. One or more labels may be attached to the detection probe at one or more ends, within the detection probe, or in combination thereof. If the detection probe contains nucleic acid, one or more labels may be attached to the nucleic acid at the 5' end, at the 3' end, within the nucleic acid, or in combination thereof.

[0152] The label of the detection probe may be attached to the nucleic acid using a linker. The linker may contain a variety of chemical groups, including one or more ethylene groups, one or more methylene groups, one or more polyethylene glycol groups, or a combination thereof. The detection probe may be reversibly bound to the barcode or the barcode's reverse complement. The detection probe may be irreversibly bound to the barcode or the barcode's reverse complement. The detection probe may be irreversibly bound to the barcode or the barcode's reverse complement via nucleic acid hybridization.

[0153] In some cases, one or more detection probes may be added to the sample. Each of the one or more detection probes may bind to a barcode or its inverse complement. In some cases, each of the one or more detection probes may bind to one or more barcodes or one or more inverse complements of barcodes in the sample. Each of the barcodes or its inverse complements may be associated with a different analyte or set of analytes. In some cases, each of the one or more detection probes may contain a different label. In some cases, at least two of the detection probes may contain different labels. In some cases, all of the one or more detection probes may contain different labels. In some cases, one or more detection probes may contain the same label (e.g., the same fluorescent dye and / or the same linker).

[0154] In some cases, one or more detection probes may be added to a sample and bound to one or more barcodes or the inverse complements of one or more barcodes. The signal associated with the detection probe can be detected. Detection of the signal may be performed using an imaging system, for example, an imaging system described herein. The imaging system may comprise a microscope, camera, stage, sample holder, computer, or a combination thereof. The imaging system may collect the signal associated with the detection probe by illuminating the sample with light. The light used to illuminate the sample may include one or more wavelengths. The imaging system may collect the light emitted by the sample at one or more wavelengths. One or more wavelengths of illumination or collection may include, but are not limited to, light having the following wavelengths: at least about 260 nm, at least about 265 nm, at least about 270 nm, at least about 280 nm, at least about 285 nm, at least about 290 nm, at least about 295 nm, at least about 300 nm, at least about 305 nm, at least about 310 nm, at least about 315 nm, at least about 320 nm, at least about 325 nm, at least about 330 nm, at least about 335 nm, at least about 340 nm, at least about 345 nm, at least about 350 nm, at least about 355 nm, at least about 360 nm, at least about 365 nm, at least about 370 nm, at least about 375 nm, at least about 380 nm, and less At least approximately 385nm, at least approximately 390nm, at least approximately 395nm, at least approximately 400nm, at least approximately 405nm, at least approximately 410nm, at least approximately 415nm, at least approximately 420nm, at least approximately 425nm, at least approximately 430nm, at least approximately 435nm, at least approximately 440nm, at least approximately 445nm, at least approximately 450nm, at least approximately 455nm, at least approximately 460nm, at least approximately 465nm, at least approximately 470nm, at least approximately 475nm, at least approximately 480nm, at least approximately 485nm, at least approximately 490nm, at least approximately 495nm, at least approximately 500nm, at least approximately 505nm, at least approximately 510nm, at least approximately 515nm, at least approximately 520nm,At least approximately 525nm, at least approximately 530nm, at least approximately 535nm, at least approximately 540nm, at least approximately 545nm, at least approximately 550nm, at least approximately 555nm, at least approximately 560nm, at least approximately 565nm, at least approximately 570nm, at least approximately 575nm, at least approximately 580nm, at least approximately 585nm, at least approximately 590nm, at least approximately 595nm, at least approximately 600nm, at least approximately 605nm, at least approximately 610nm, at least approximately 615nm, at least approximately 620nm, at least approximately 625nm, at least approximately 630nm, at least approximately 635nm, at least approximately 640nm, at least approximately 645nm, at least approximately 650nm, at least approximately 655nm, at least approximately 660nm, at least approximately 665nm, at least about 670nm, at least about 675nm, at least about 680nm, at least about 685nm, at least about 690nm, at least about 695nm, at least about 700nm, at least about 705nm, at least about 710nm, at least about 715nm, at least about 720nm, at least about 725nm, at least about 730nm, at least about 735nm, at least about 740nm, at least about 745nm, at least about 750nm, at least about 755nm, at least about 760nm, at least about 765nm, at least about 770nm, at least about 775nm, at least about 780nm, at least about 785nm, at least about 790nm, at least about 795nm, at least about 800nm, or longer. One or more wavelengths of illumination or collection may include light having the following wavelengths: up to approximately 260 nm, up to approximately 265 nm, up to approximately 270 nm, up to approximately 280 nm, up to approximately 285 nm, up to approximately 290 nm, up to approximately 295 nm, up to approximately 300 nm, up to approximately 305 nm, up to approximately 310 nm, up to approximately 315 nm, up to approximately 320 nm, up to approximately 325 nm, up to approximately 330 nm, up to approximately 335 nm, up to approximately 340 nm, up to approximately 345 nm, up to approximately 350 nm, up to approximately 355 nm, up to approximately 360 nm, up to approximately 365 nm, up to approximately 370 nm, up to approximately 375 nm, up to approximately 380 nm, up to approximately 385 nm, up to approximately 390 nm, up to approximately 395 nm, up to approximately 400 nm.Up to approximately 405nm, up to approximately 410nm, up to approximately 415nm, up to approximately 420nm, up to approximately 425nm, up to approximately 430nm, up to approximately 435nm, up to approximately 440nm, up to approximately 445nm, up to approximately 450nm, up to approximately 455nm, up to approximately 460nm, up to approximately 465nm, up to approximately 470nm, up to approximately 475nm, up to approximately 480nm, up to approximately 485nm, up to approximately 490nm, up to approximately 495nm, up to approximately 500nm, up to Approximately 505nm, up to approximately 510nm, up to approximately 515nm, up to approximately 520nm, up to approximately 525nm, up to approximately 530nm, up to approximately 535nm, up to approximately 540nm, up to approximately 545nm, up to approximately 550nm, up to approximately 555nm, up to approximately 560nm, up to approximately 565nm, up to approximately 570nm, up to approximately 575nm, up to approximately 580nm, up to approximately 585nm, up to approximately 590nm, up to approximately 595nm, up to approximately 600nm, up to approximately 60 5nm, up to approximately 610nm, up to approximately 615nm, up to approximately 620nm, up to approximately 625nm, up to approximately 630nm, up to approximately 635nm, up to approximately 640nm, up to approximately 645nm, up to approximately 650nm, up to approximately 655nm, up to approximately 660nm, up to approximately 665nm, up to approximately 670nm, up to approximately 675nm, up to approximately 680nm, up to approximately 685nm, up to approximately 690nm, up to approximately 695nm, up to approximately 700nm, up to approximately 705nm , up to approximately 710nm, up to approximately 715nm, up to approximately 720nm, up to approximately 725nm, up to approximately 730nm, up to approximately 735nm, up to approximately 740nm, up to approximately 745nm, up to approximately 750nm, up to approximately 755nm, up to approximately 760nm, up to approximately 765nm, up to approximately 770nm, up to approximately 775nm, up to approximately 780nm, up to approximately 785nm, up to approximately 790nm, up to approximately 795nm, up to approximately 800nm, or shorter nm. One or more wavelengths of illumination or collection may include light having the following wavelengths: approximately 260 to approximately 800 nm, approximately 265 to approximately 795 nm, approximately 270 to approximately 790 nm, approximately 280 to approximately 785 nm, approximately 285 to approximately 780 nm, approximately 290 to approximately 775 nm, approximately 295 to approximately 770 nm, approximately 300 to approximately 765 nm, approximately 305 to approximately 760 nm, approximately 310 to approximately 755 nm, approximately 315 to approximately 750 nm, approximately 320 to approximately 745 nm.Approximately 325 to approximately 740nm, approximately 330 to approximately 735nm, approximately 335 to approximately 730nm, approximately 340 to approximately 725nm, approximately 345 to approximately 720nm, approximately 35 0 to about 715 nm, about 355 to about 710 nm, about 360 to about 705 nm, about 365 to about 700 nm, about 370 to about 695 nm, about 375 to about 690nm, about 380 to about 685nm, about 385 to about 680nm, about 390 to about 675nm, about 395 to about 670nm, about 400 to about 665 nm, approximately 405 to approximately 660 nm, approximately 410 to approximately 655 nm, approximately 415 to approximately 650 nm, approximately 420 to approximately 645 nm, approximately 425 to approximately 640 nm, approximately 430-635nm, 435-630nm, 440-625nm, 445-620nm, 450-615nm, 455-610nm, 460-605nm, 465-600nm, 470-595nm, 475-590nm, 480-585nm, 485-580nm, 490-575nm, 495-570nm, 500-565nm, 505-560nm, 510-555nm, 515-550nm, 520-545nm, 525-540nm, or 530-535nm.

[0155] One or more detection probes may bind to a sample and detect sequences associated with a barcode or its inverse complement. In some cases, the sequences associated with a barcode or its inverse complement may include the entire barcode sequence or its inverse complement. In some cases, the sequences associated with a barcode or its inverse complement may include a portion of the barcode sequence or its inverse complement. In some cases, the detection probe may detect at least about 1 nucleotide, at least about 2 nucleotides, at least about 3 nucleotides, at least about 4 nucleotides, at least about 5 nucleotides, at least about 6 nucleotides, at least about 7 nucleotides, at least about 8 nucleotides, at least about 9 nucleotides, at least about 10 nucleotides, at least about 11 nucleotides, at least about 12 nucleotides, at least about 13 nucleotides, at least about 14 nucleotides, at least about 15 nucleotides, at least about 16 nucleotides, at least about 17 nucleotides, at least about 18 nucleotides, at least about 19 nucleotides, at least about 20 nucleotides, at least about 21 nucleotides, at least Detect approximately 22 nucleotides, at least approximately 23 nucleotides, at least approximately 24 nucleotides, at least approximately 25 nucleotides, at least approximately 26 nucleotides, at least approximately 27 nucleotides, at least approximately 28 nucleotides, at least approximately 29 nucleotides, at least approximately 30 nucleotides, at least approximately 31 nucleotides, at least approximately 32 nucleotides, at least approximately 33 nucleotides, at least approximately 34 nucleotides, at least approximately 35 nucleotides, at least approximately 36 nucleotides, at least approximately 37 nucleotides, at least approximately 38 nucleotides, at least approximately 39 nucleotides, at least approximately 40 nucleotides, or more, associated with the barcode or its reverse complement.In some cases, the detection probe may contain up to approximately 1 nucleotide, up to approximately 2 nucleotides, up to approximately 3 nucleotides, up to approximately 4 nucleotides, up to approximately 5 nucleotides, up to approximately 6 nucleotides, up to approximately 7 nucleotides, up to approximately 8 nucleotides, up to approximately 9 nucleotides, up to approximately 10 nucleotides, up to approximately 11 nucleotides, up to approximately 12 nucleotides, up to approximately 13 nucleotides, up to approximately 14 nucleotides, up to approximately 15 nucleotides, up to approximately 16 nucleotides, up to approximately 17 nucleotides, up to approximately 18 nucleotides, up to approximately 19 nucleotides, up to approximately 20 nucleotides, up to approximately 21 nucleotides, and up to approximately 22 nucleotides. The rectoid detects nucleotides associated with the barcode or its reverse complement, up to approximately 23 nucleotides, up to approximately 24 nucleotides, up to approximately 25 nucleotides, up to approximately 26 nucleotides, up to approximately 27 nucleotides, up to approximately 28 nucleotides, up to approximately 29 nucleotides, up to approximately 30 nucleotides, up to approximately 31 nucleotides, up to approximately 32 nucleotides, up to approximately 33 nucleotides, up to approximately 34 nucleotides, up to approximately 35 nucleotides, up to approximately 36 nucleotides, up to approximately 37 nucleotides, up to approximately 38 nucleotides, up to approximately 39 nucleotides, up to approximately 40 nucleotides, or fewer. In some cases, the detection probe detects nucleotides associated with the barcode or its reverse complement, approximately 1 to 40 nucleotides, 2 to 39 nucleotides, 3 to 38 nucleotides, 4 to 37 nucleotides, 5 to 36 nucleotides, 6 to 35 nucleotides, 7 to 34 nucleotides, 8 to 33 nucleotides, 9 to 32 nucleotides, 10 to 31 nucleotides, 11 to 30 nucleotides, 12 to 29 nucleotides, 13 to 28 nucleotides, 14 to 27 nucleotides, 15 to 26 nucleotides, 16 to 25 nucleotides, 17 to 24 nucleotides, 18 to 23 nucleotides, 19 to 22 nucleotides, or 20 to 21 nucleotides.

[0156] Additional probes may be added to the sample during the detection process. In some cases, one or more anchor probes may be added to the sample. One or more anchor probes may bind to all or part of the barcode or its inverse complement. One or more anchor probes may bind to a region adjacent to the barcode or its inverse complement. The region adjacent to the barcode or its inverse complement may be part of a nucleic acid. The nucleic acid may be an amplification product. In some cases, one or more anchor probes may bind to both the barcode and the sequence adjacent to the barcode. In some cases, one or more anchor probes may bind to both the inverse complement of the barcode and the sequence adjacent to its inverse complement. One or more anchor probes may contain nucleic acid. The nucleic acid may include RNA, DNA, or a combination thereof. The nucleic acid may contain one or more modifications. One or more modifications may include phosphorylation modifications, LNA, or a combination thereof. Phosphorylation modifications may include 5' phosphorylation modifications. In some cases, the detection probe and anchor probe are hybridized to the amplification product generated from rolling circle amplification. The detection probe and anchor probe may be ligated after hybridization to the amplification product.

[0157] One or more anchor probes may bind to a barcode or its reverse complement, or a sequence adjacent to a barcode or its reverse complement, such that one or more anchor probes are adjacent to one or more detection probes. One or more anchor probes may be directly adjacent to one or more detection probes in the absence of intervening nucleotides. In some cases, one or more anchor probes may be adjacent to one or more detection probes in the presence of one or more intervening nucleotides. Intervening nucleotides may be filled between one or more anchor probes and one or more detection probes by performing a gap-filling reaction. One or more anchor probes may be ligated to one or more detection probes. Ligation between anchor probes and detection probes may be performed using enzymatic ligation, chemical ligation, or a combination thereof. Enzymatic ligation may be performed by ligase. Ligases may include one or more of the following: T4 DNA ligase, SplintR ligase, T3 DNA ligase, T7 DNA ligase, E. coli DNA ligase, Taq ligase, RtcB ligase, or a combination thereof.

[0158] Detection of signals associated with one or more detection probes may be performed after ligating one or more detection probes with one or more anchor probes. In some cases, one or more detection probes may transiently bind to the sample. In some cases, one or more detection probes may bind to the sample for a certain duration without noticeable dissociation. One or more detection probes ligated to one or more anchor probes may bind more strongly to the sample than one or more detection probes alone.

[0159] In some cases, one or more labels associated with the sample may be removed after signal detection. Labels may be removed from the sample by chemical or enzymatic means. In some cases, labels may be cleaved from the detection probe using enzymes, chemical cleavage reagents, or a combination thereof. Enzymes may include restriction enzymes, polymerases, ligases, transposons, or a combination thereof. Chemical cleavage reagents may include reducing agents, reactive oxygen species, or a combination thereof. Labels may be removed from the sample by removing, digesting, or destroying one or more detection probes from the sample. In some cases, one or more detection probes may be ligated to one or more anchor probes. One or more detection probes may be removed from the sample by using temperature and incubating the sample at a high temperature for a period of time. One or more detection probes may be removed from the sample by incubating the sample with a chemical that interferes with nucleic acid hybridization, such as one or more chaotropic agents. In some cases, one or more chaotropic agents may include glycine, arginine, histidine hydrochloride, sodium hydroxide, formamide, dimethyl sulfoxide (DMSO), guanidinium chloride, or a combination thereof. One or more detection probes may be removed from the sample by incubating the sample with one or more chaotropic reagents and incubating the sample at a high temperature. One or more detection probes may also be removed from the sample using enzymes, including but not limited to DNAse, RNAse, restriction enzymes, or a combination thereof.

[0160] In some cases, after signal detection, one or more labels associated with the sample may be altered so that the labels can no longer emit a signal. Labels may be denatured, for example, if the labels may contain polypeptides. Denaturation may be carried out using heat, salts, solvents (e.g., methanol, ethanol, or a combination thereof), acids, bases, or a combination thereof. Labels may be photobleached, for example, if the labels may contain fluorescent moieties. Photobleaching may be carried out by exposing the sample to light, including ambient light, reactive chemical species, reducing reagents, bases, acids, or a combination thereof.

[0161] One or more detection rounds may be performed. Each detection round may involve contacting the sample with one or more detection probes, one or more anchor probes, or a combination thereof. For example, the sample may contain an amplification product containing the reverse complement of an 8-nucleotide barcode sequence. A detection round may involve adding multiple anchor probes and multiple detection probes to the sample to enable binding of the anchor probes and detection probes to the amplification product, where the sequence of the detection probe provides information related to the first two nucleotides of the 8-nucleotide reverse complement of the barcode sequence. After binding, the anchor probes and detection probes may be ligated, and the sample may be washed to remove all unbound detection probes. The sample may be imaged to detect signals associated with the detection probes. After imaging, detection probes ligated to the anchor probes may be removed by incubating the sample with a chaotropic reagent. The combination of detection probe binding, detection probe ligation, sample imaging, and detection probe removal constitutes a detection round in this case. In some cases, one or more detection probes may be the same across one or more rounds. For example, detection probes having the same sequence may be added in the first and second cycles. In some cases, one or more detection probes may differ over one or more rounds. For example, detection probes having different sequences may be added in the first round compared to the second round. In some cases, one or more anchor probes may be the same over one or more rounds. For example, anchor probes having the same sequence may be added in the first and second cycles. In some cases, one or more anchor probes may differ over one or more rounds. For example, anchor probes having different sequences may be added in the first round compared to the second round. Each detection round may include adding probes to the sample, imaging the sample using an imaging system to generate one or more images, and optionally removing labels from the sample.

[0162] One or more images generated by imaging a sample during a described detection round may be analyzed. The images may contain information related to a sequence of barcodes or the inverse complements of barcodes. In some cases, one or more images from one round of detection may contain information related to a portion of a sequence of one or more barcodes or the inverse complements of one or more barcodes. In some cases, one or more images from another detection round may contain information related to another portion of a sequence or the inverse complements of one or more barcodes or barcodes. By analyzing images generated during one or more detection rounds, the identification of one or more barcodes or the inverse complements of one or more barcodes may be obtained. The identification of one or more barcodes or the inverse complements of one or more barcodes may enable the identification of one or more targets, as described herein. Spatial maps, images, displays, summaries, tables, or combinations thereof may be generated based on the analysis described herein. Information related to both target identification and spatial location may be determined based on the analysis described herein. Spatial location may include information related to the location of the target in the x, y, z directions, or combinations thereof.

[0163] In some cases, the first probe may recognize messenger RNA, and the second probe may recognize ribosomal proteins. The proximity between messenger RNA and ribosomal proteins can be determined. The proximity between messenger RNA and ribosomal proteins can provide information related to protein translation. For example, barcodes or their derivatives may be detected as a result of the messenger RNA and ribosomal proteins being in close proximity to each other. The close proximity of messenger RNA and ribosomal proteins may cause a ligation event at one end of a nucleic acid probe to another end of the nucleic acid probe, which may result in the generation of multiple copies of the barcode or its derivatives. Detection of barcodes or their derivatives may indicate that the messenger RNA is part of a complex with ribosomal proteins, which may occur during the translation of the protein encoded by the messenger RNA. An example of this type of detection is shown in Figures 1A and 1B, which illustrate a two-probe system for detecting messenger RNA (mRNA) translation. The first detection scheme shown in Figure 1A includes a first probe (101) that recognizes messenger RNA (102) and a second probe (103) containing an antibody (104) that recognizes ribosomes (107). In this case, the second probe includes a primary antibody (104) and a secondary antibody (105) conjugated to a nucleic acid (106) (e.g., a primer). The primary antibody may be an anti-ribosomal protein antibody. The nucleic acid conjugated to the secondary antibody may include a fourth binding site that binds to a binding site on the first probe (e.g., a third binding site on the first probe). The first probe may include a 5'-phosphorylated base and a 3'-free deoxyribose that may be complementary to adjacent positions on the target oligo (mRNA) and can be annealed to the nucleic acid conjugated to the antibody. The first probe may include two optional barcodes, shown here as barcode A and barcode B. The second probe may contain barcode B. If there is a gap between the two ends, that gap may be filled with DNA bases using reverse transcriptase-mediated polymerization.The nick between the 5' and 3' ends of the first probe can be ligated in the presence of mRNA using a ligase capable of generating a cyclic oligonucleotide (e.g., SplintR ligase). The cyclic oligonucleotide can serve as a template for a rolling circle amplification (RCA) reaction using a DNA polymerase (e.g., Phi29 polymerase). In this case, the nucleic acid conjugated to the secondary antibody can serve as a primer to initiate the RCA reaction. The antibody can recognize ribosomal components directly (e.g., when the nucleic acid is conjugated to a primary antibody that recognizes ribosomal proteins) or indirectly (e.g., when the nucleic acid is conjugated to a secondary antibody that recognizes ribosomal proteins). The scheme shown in Figure 1B involves the use of a nucleic acid probe (108) that recognizes ribosomal RNA. In each case shown in Figures 1A and 1B, the RCA product can be embedded in a hydrogel, thereby preserving the spatial arrangement of the nucleic acids within the sample. An optional barcode A in the first probe can be specific to each target nucleic acid. Barcode B, of any choice in the first and second probes, may be specific to the protein to which the antibody binds. Barcode A or barcode B or its complement may be identified by ligation-based, hybridization-based, or sequence-based in situ sequencing using a fluorescently labeled probe that may be visible under a microscope using a laser.

[0164] Another example of a set for detecting mRNA translation is shown in Figures 3A and 3B. In this case, mRNA translation is detected by detecting the presence of a ribosome adjacent to the mRNA using three probes. The detection of ribosomes and mRNA in Figure 3A is similar to that in Figure 1A, with one difference being the inclusion of an additional probe (e.g., a third probe) (301). In this case, the additional probe may bind to mRNA and nucleic acids conjugated to a secondary probe (302) that can bind to a primary antibody (303) that can bind to a ribosome (304). The primary antibody may be conjugated to a secondary antibody (308) that is conjugated to an oligonucleotide (309) that binds to the first and third probes. This additional probe may be ligated to the first probe (305) at two positions. The additional probe may include a 5' phosphorylation modification and a 3' hydroxyl group. The 5' phosphorylation modification is the binding site to the nucleic acid conjugated to the secondary antibody and may be ligated to the 3' hydroxyl group of the first probe. An additional probe (e.g., a third probe) may be ligated to the first probe (305) at a second site, e.g., a binding site on mRNA. In some cases, a gap may exist between the ends of the first and third probes, and a gap-filling reaction may be performed. The first probe may contain barcode A of any choice. The additional probe (e.g., a third probe) may contain barcode B of any choice. The nucleic acid conjugated to the secondary antibody may contain barcode B of any choice. In some cases, a gap may exist between both ends of the probe. The gap between the two ends of the probe may be filled with DNA bases via DNA polymerase-mediated polymerization. The nick between the 5' end of the first probe and the 3' end of the third probe may bind to a target (e.g., mRNA) or a detection portion conjugate nucleotide primer (e.g., a second probe) and be ligated. Ligation between the third probe and the first probe can be performed using SplintR ligase, T4 DNA ligase, or a combination thereof.Ligation can generate a cyclic oligonucleotide that can function as a template for the RCA reaction by DNA polymerase. A detection portion conjugate nucleotide primer (e.g., a second probe) can function as a primer to initiate the RCA reaction. If the detection portion is an antibody, the antibody can recognize ribosomal components directly (when the oligo is conjugated to a primary antibody that recognizes ribosomal proteins) or indirectly (when the oligo is conjugated to a secondary antibody that recognizes ribosomal proteins). The ribosomal component may be a ribosomal protein. If the detection portion is a nucleic acid probe, e.g., ribosomal RNA (306), the nucleic acid probe (e.g., a second probe) (307) can bind to the ribosomal RNA as shown in Figure 3B. The RCA product can be embedded in a hydrogel. An optional barcode A in the first probe may be specific to each target nucleic acid (308). An optional barcode B in the first and second probes may be specific to the protein to which the antibody binds. Barcode A can be specifically identified by ligation-based or hybridization-based in situ sequencing using a fluorescently labeled oligo that is visible under a microscope using a laser.

[0165] Examples of other sets for detecting mRNA translation are shown in Figures 5A and 5B. In this case, primer probes containing unmodified or modified RNA chimeric sequences can serve as templates for SplintR-mediated DNA ligation between a first probe (501) and a second probe (502). Both the first and second probes may contain a 5'-phosphorylated base and a 3'-free deoxyribose. The 5' of the first probe and the 3' of the second probe may be complementary to the adjacent positions on the target mRNA (503) (e.g., mRNA, "the nucleotide of interest") and can be annealed to the target mRNA (503). The 5' of the second probe (501) and the 3' of the first probe (501) may be complementary to the adjacent positions on a detection partial conjugate nucleotide primer (e.g., the second probe). If there is a gap between the two ends, that gap may be filled by DNA bases via DNA polymerase-mediated polymerization. The nick between the 5' and 3' ends of the first and second probes can be annealed on a target oligo or antibody-conjugated nucleotide primer and ligated by SplintR ligase and / or T4 DNA ligase. Ligation may generate a cyclic oligonucleotide. The cyclic oligonucleotide can serve as a template for the RCA reaction by DNA polymerase. The detection portion conjugated nucleotide primer (e.g., the second probe) can serve as a primer to initiate the RCA reaction. If the detection portion is an antibody, the antibody can recognize ribosomal components directly (if the oligo is conjugated with a primary antibody that recognizes ribosomal proteins) or indirectly (if the oligo is conjugated with a secondary antibody that recognizes ribosomal proteins). If the detection portion is a nucleic acid probe, the nucleic acid probe can recognize ribosomal RNA. The RCA product can be embedded in a hydrogel. An optional barcode A in the first probe may be specific to each target nucleic acid. The barcode B of the first probe and an optional antibody-conjugated nucleotide primer may be specific to the protein to which the antibody binds.Barcode A can be specifically identified by ligation-based or hybridization-based in situ sequencing using a fluorescently labeled oligo that is visible under a microscope using a laser. Figure 5B shows a similar workflow, but includes an additional gap-filling reaction (505) to connect a second probe to an additional probe (504) that binds to the first probe.

[0166] In some cases, the first probe may recognize messenger RNA, and the second probe may recognize ribosomal RNA. The proximity of the messenger RNA and ribosomal RNA can be determined. For example, a barcode or its derivatives, which can be amplified as a result of a ligation reaction between the two ends of the first probe based on the binding of the first probe to the messenger RNA, the binding of the second probe to the ribosomal RNA, and the binding of the first probe to the second probe, can be detected (e.g., using an imaging system). The proximity of the messenger RNA and ribosomal RNA can provide information related to protein translation. For example, the detection of the presence of a barcode or its derivatives may indicate that the messenger RNA is complexed with the ribosomal RNA, which may indicate that the protein encoded by the messenger RNA is being translated.

[0167] In some cases, the first probe may recognize messenger RNA, and the second probe may recognize, ligate to, and / or bind to messenger RNA modifications. RNA modifications may be incorporated, for example, by metabolic labeling. The proximity of messenger RNA and messenger RNA modifications may be determined. The proximity of messenger RNA and messenger RNA modifications may provide information related to protein translation, mRNA transcription, or a combination thereof....

Claims

1. A method for detecting analytes in a sample, a) To provide a first probe and a second probe, The first probe, (i) A first bonding site configured to connect to a first analyte in the first part, (ii) A second bonding site configured to connect to the first analyte in the second portion, wherein the first portion is adjacent to the second bonding site, (iii) A third coupling portion configured to be connected to the second probe, (iv) barcode, (v) the first terminal, and (vi) The second end Includes, The second probe, (i) A fourth coupling site configured to be connected to the first probe, and (ii) A fifth binding site configured to connect to the second analyte Including providing, b) To make contact, (i) The first probe is connected to the first analyte, (ii) The second probe is connected to the second analyte, (iii) The first probe is connected to the second probe, A sample containing a plurality of analytes, including the first analyte and the second analyte, is brought into contact with the first probe and the second probe. c) Forming a cyclic oligonucleotide by ligating the first and second ends, d) Amplifying the cyclic oligonucleotide to produce an amplified product, wherein the amplified product includes the complement of the barcode. e) Using multiple detection probes to detect the complement or derivative of the barcode, thereby determining the proximity between the first analyte and the second analyte. Methods that include...

2. The method according to claim 1, wherein the sample is a tissue sample.

3. The method according to claim 1, wherein the tissue sample is a fresh frozen tissue sample.

4. The method according to claim 1, wherein the tissue sample is a formalin-fixed paraffin-embedded tissue sample.

5. The method according to any one of claims 1 to 4, wherein the sample has a thickness of 5 to 250 μm.

6. The method according to any one of claims 1 to 4, wherein the sample has a thickness of 10 to 200 μm.

7. The method according to any one of claims 1 to 4, wherein the sample has a thickness of 25 to 150 μm.

8. The method according to any one of claims 1 to 4, wherein the first analyte comprises nucleic acid.

9. The method according to claim 8, wherein the nucleic acid is ribonucleic acid.

10. The method according to claim 9, wherein the ribonucleic acid is messenger ribonucleic acid.

11. The method according to claim 9, wherein the ribonucleic acid is ribosomal ribonucleic acid.

12. The method according to claim 8, wherein the nucleic acid is deoxyribonucleic acid.

13. The method according to any one of claims 1 to 8, wherein the first analyte comprises a polypeptide.

14. The method according to claim 13, wherein the polypeptide comprises a ribosomal protein.

15. The method according to any one of claims 1 to 14, wherein the first analyte includes chemical modification.

16. The method according to any one of claims 1 to 15, wherein the second analyte comprises nucleic acid.

17. The method according to claim 16, wherein the nucleic acid is ribonucleic acid.

18. The method according to claim 17, wherein the ribonucleic acid is messenger ribonucleic acid.

19. The method according to claim 17, wherein the ribonucleic acid is ribosomal ribonucleic acid.

20. The method according to claim 16, wherein the nucleic acid is deoxyribonucleic acid.

21. The method according to any one of claims 1 to 20, wherein the second analyte comprises a polypeptide.

22. The method according to claim 21, wherein the polypeptide comprises a ribosomal protein.

23. The method according to any one of claims 1 to 22, wherein the first probe comprises nucleic acid.

24. The method according to claim 23, wherein the nucleic acid includes an oligonucleotide.

25. The method according to claim 24, wherein the oligonucleotide includes one or more modifications.

26. The method according to claim 25, wherein one or more of the modifications include a 5'-phosphate modification.

27. The method according to any one of claims 25 to 26, wherein one or more of the modifications include internucleotide bonding.

28. The method according to claim 27, wherein the nucleotide bond is a phosphorothioate.

29. The method according to claim 27, wherein the nucleotide bond is a phosphodiester.

30. The method according to any one of claims 1 to 29, wherein the first probe recognizes ribonucleic acid.

31. The method according to any one of claims 8 to 12, wherein the nucleic acid comprises a single nucleotide polymorphism.

32. The method according to claim 31, wherein the first probe recognizes the single nucleotide polymorphism, and if the first analyte does not contain the single nucleotide polymorphism, the ligation in c) does not occur.

33. The method according to claim 31, wherein the first probe does not recognize the single nucleotide polymorphism, and if the first analyte contains the single nucleotide polymorphism, the ligation in c) does not occur.

34. The method according to any one of claims 8 to 12, wherein the nucleic acid includes modifications.

35. The above modification is N ６ - Methyladenosine, 5-methylcytosine, N １ - Methyladenosine, N ７ - Methylguanosine, N ４ - Acetylcytosine, pseudouridine, and N １ - The method according to claim 34, selected from the group consisting of methylpseudridine.

36. The method according to any one of claims 34 to 35, wherein if the first probe recognizes the modification and the first analyte does not contain the modification, the ligation in c) does not occur.

37. The method according to any one of claims 34 to 35, wherein the first probe does not recognize the modification, and if the first analyte contains the modification, the ligation in c) does not occur.

38. The method according to any one of claims 1 to 37, wherein the first probe recognizes deoxyribonucleic acid.

39. The method according to any one of claims 1 to 37, wherein the first probe recognizes deoxyribonucleic acid modification.

40. The method according to claim 39, wherein the deoxyribonucleic acid modification is a methyl modification.

41. The method according to any one of claims 1 to 40, wherein the first probe includes a first reactive chemical moiety at its first end and a second reactive chemical moiety at its second end.

42. The method according to claim 41, wherein the first reactive chemical moiety is selected from the group consisting of tetrazine, alkyne, azide, trans-cyclooctene, maleimide, N-hydroxysuccinimide ester, amine, carboxylic acid, hydroxyl, cyclopropenone, and thiol, norbornene.

43. The method according to claim 41, wherein the second reactive chemical moiety is selected from the group consisting of tetrazine, alkyne, azide, trans-cyclooctene, maleimide, N-hydroxysuccinimide ester, amine, carboxylic acid, hydroxyl, cyclopropenone, and thiol, norbornene.

44. The method according to any one of claims 41 to 43, wherein the ligation in c) includes a reaction between the first reactive chemical portion and the second reactive chemical portion.

45. The method according to any one of claims 1 to 40, wherein the ligation in c) includes carrying out a ligation reaction with a ligase.

46. The method according to claim 45, wherein the ligase is T4 ligase.

47. The method according to any one of claims 1 to 46, wherein the second probe comprises nucleic acid.

48. The method according to claim 47, wherein the nucleic acid includes an oligonucleotide.

49. The method according to claim 48, wherein the oligonucleotide includes one or more modifications.

50. The method according to claim 49, wherein one or more of the modifications include a 5'-phosphate modification.

51. The method according to any one of claims 48 to 49, wherein one or more of the modifications include internucleotide bonds.

52. The method according to claim 51, wherein the nucleotide bond is a phosphorothioate.

53. The method according to claim 51, wherein the nucleotide bond is a phosphodiester.

54. The method according to any one of claims 29 to 53, wherein the nucleic acid comprises an aptamer.

55. The method according to any one of claims 1 to 54, wherein the second probe comprises a polypeptide.

56. The method according to claim 55, wherein the polypeptide comprises an antibody or an antibody fragment.

57. The method according to claim 56, wherein the polypeptide comprises an adfimer.

58. The method according to claim 56, wherein the polypeptide comprises a nanobody.

59. The method according to any one of claims 1 to 58, wherein the second probe recognizes ribonucleic acid.

60. The method according to any one of claims 1 to 58, wherein the second probe recognizes ribonucleic acid modification.

61. wherein the ribonucleic acid modification is N ６ -methyladenosine, 5-methylcytosine, N １ -methyladenosine, N ７ -methylguanosine, N ４ -acetylcytosine, pseudouridine, and N １ -methylpseudouridine, the method according to claim 60.

62. The method according to any one of claims 1 to 58, wherein the second probe recognizes deoxyribonucleic acid.

63. The method according to any one of claims 1 to 58, wherein the second probe recognizes deoxyribonucleic acid modification.

64. The method according to claim 63, wherein the deoxyribonucleic acid modification is a methyl modification.

65. The method according to any one of claims 1 to 58, wherein the second probe recognizes a polypeptide.

66. The method according to claim 65, wherein the polypeptide is a protein.

67. The method according to claim 66, wherein the protein is a transcription factor.

68. The method according to claim 66, wherein the protein is a ribosomal protein.

69. The method according to claim 66, wherein the protein is a histone.

70. The method according to claim 66, wherein the protein is polymerase.

71. The method according to claim 66, wherein the protein is a helicase.

72. The method according to claim 66, wherein the protein is a restriction enzyme.

73. The method according to claim 66, wherein the protein is a ribonucleic acid-binding protein.

74. The method according to any one of claims 66 to 73, wherein the second probe recognizes the post-translational modification of the protein.

75. The method according to any one of claims 1 to 74, wherein the barcode comprises nucleic acid.

76. The method according to claim 75, wherein the nucleic acid is deoxyribonucleic acid.

77. The method according to claim 75, wherein the nucleic acid is ribonucleic acid.

78. The method according to any one of claims 75 to 77, wherein the nucleic acid is at least 4 nucleotides long.

79. The method according to any one of claims 75 to 77, wherein the nucleic acid is at least 6 nucleotides long.

80. The method according to any one of claims 75 to 77, wherein the nucleic acid is at least 8 nucleotides long.

81. The method according to any one of claims 75 to 77, wherein the nucleic acid is at least 10 nucleotides long.

82. The method according to any one of claims 1 to 81, wherein the barcode corresponds to the first analyte.

83. The method according to any one of claims 1 to 81, wherein the barcode corresponds to the second analyte.

84. The method according to any one of claims 1 to 81, wherein the barcode corresponds to the first analyte that is adjacent to the second analyte.

85. The method according to any one of claims 1 to 84, wherein the first probe further comprises a second barcode.

86. The method according to claim 85, wherein the second barcode includes nucleic acid.

87. The method according to claim 86, wherein the nucleic acid is deoxyribonucleic acid.

88. The method according to claim 86, wherein the nucleic acid is ribonucleic acid.

89. The method according to any one of claims 85 to 88, wherein the nucleic acid is at least 4 nucleotides long.

90. The method according to any one of claims 85 to 88, wherein the nucleic acid is at least 6 nucleotides long.

91. The method according to any one of claims 85 to 88, wherein the nucleic acid is at least 8 nucleotides long.

92. The method according to any one of claims 85 to 88, wherein the nucleic acid is at least 10 nucleotides long.

93. The method according to any one of claims 85 to 92, wherein the second barcode corresponds to the first analyte.

94. The method according to any one of claims 85 to 92, wherein the second barcode corresponds to the second analyte.

95. The method according to any one of claims 85 to 92, wherein the second barcode corresponds to the first analyte that is adjacent to the second analyte.

96. The method according to any one of claims 1 to 95, wherein the first binding site comprises a nucleic acid, and the nucleic acid is at least 6 nucleotides long.

97. The method according to any one of claims 1 to 95, wherein the first binding site comprises a nucleic acid, and the nucleic acid is at least 10 nucleotides long.

98. The method according to any one of claims 1 to 95, wherein the first binding site comprises a nucleic acid, and the nucleic acid is at least 14 nucleotides long.

99. The method according to any one of claims 1 to 95, wherein the first binding site comprises a nucleic acid, and the nucleic acid is at least 20 nucleotides long.

100. The method according to any one of claims 1 to 99, wherein the second binding site includes a nucleic acid, and the nucleic acid is at least 4 nucleotides long.

101. The method according to any one of claims 1 to 99, wherein the second binding site comprises a nucleic acid, and the nucleic acid is at least 10 nucleotides long.

102. The method according to any one of claims 1 to 99, wherein the second binding site comprises a nucleic acid, and the nucleic acid is at least 14 nucleotides long.

103. The method according to any one of claims 1 to 99, wherein the second binding site comprises a nucleic acid, and the nucleic acid is at least 20 nucleotides long.

104. The method according to any one of claims 1 to 103, wherein the third binding site comprises a nucleic acid, and the nucleic acid is at least 4 nucleotides long.

105. The method according to any one of claims 1 to 103, wherein the third binding site comprises a nucleic acid, and the nucleic acid is at least 8 nucleotides long.

106. The method according to any one of claims 1 to 103, wherein the third binding site comprises a nucleic acid, and the nucleic acid is at least 10 nucleotides long.

107. The method according to any one of claims 1 to 103, wherein the third binding site comprises a nucleic acid, and the nucleic acid is at least 12 nucleotides long.

108. The method according to any one of claims 1 to 107, wherein the fourth binding site includes a nucleic acid, and the nucleic acid is at least 4 nucleotides long.

109. The method according to any one of claims 1 to 107, wherein the fourth binding site includes a nucleic acid, and the nucleic acid is at least 8 nucleotides long.

110. The method according to any one of claims 1 to 107, wherein the fourth binding site comprises a nucleic acid, and the nucleic acid is at least 10 nucleotides long.

111. The method according to any one of claims 1 to 107, wherein the fourth binding site comprises a nucleic acid, and the nucleic acid is at least 12 nucleotides long.

112. The method according to any one of claims 1 to 111, wherein the fifth binding site comprises a nucleic acid, and the nucleic acid is at least 6 nucleotides long.

113. The method according to any one of claims 1 to 111, wherein the fifth binding site comprises a nucleic acid, and the nucleic acid is at least 8 nucleotides long.

114. The method according to any one of claims 1 to 111, wherein the fifth binding site comprises a nucleic acid, and the nucleic acid is at least 10 nucleotides long.

115. The method according to any one of claims 1 to 111, wherein the fifth binding site comprises a nucleic acid, and the nucleic acid is at least 12 nucleotides long.

116. (c) The method according to any one of claims 1 to 115, wherein (c) involves performing rolling circle amplification.

117. The method according to any one of claims 1 to 116, wherein (e) comprises hybridizing a detection probe and an anchor probe among the plurality of detection probes to the amplification product.

118. The method according to claim 117, wherein the detection probe and the section detection probe are ligated.

119. The method according to any one of claims 117 to 118, wherein the detection probe includes a label.

120. The method according to claim 119, wherein the label includes a fluorescent molecule.

121. The method according to claim 119, wherein the label includes a quantum dot.

122. The method according to claim 119, wherein the label comprises an enzyme.

123. The method according to claim 122, wherein the enzyme generates a signal indicating the label.

124. The method according to any one of claims 117 to 123, wherein (e) is the detection of the sign.

125. The method according to any one of claims 1 to 124, wherein (e) sequence determination is performed in situ using the plurality of detection probes.

126. The method according to any one of claims 1 to 125, wherein (d) is imaging the sample.

127. The method according to claim 1, wherein the first probe recognizes messenger ribonucleic acid and the second probe recognizes ribosomal proteins.

128. The method according to claim 127, wherein the second probe comprises an antibody or an antibody fragment.

129. The method according to claim 1, wherein the first probe recognizes messenger ribonucleic acid and the second probe recognizes ribosomal ribonucleic acid.

130. The method according to claim 1, wherein the first probe recognizes messenger ribonucleic acid and the second probe recognizes messenger ribonucleic acid modifications.

131. The method according to claim 130, wherein the second probe includes a reactive chemical portion.

132. The method according to claim 130, wherein the second probe comprises an antibody or an antibody fragment.

133. The method according to claim 1, wherein the first probe recognizes deoxyribonucleic acid and the second probe recognizes deoxyribonucleic acid modifications.

134. The method according to claim 133, wherein the second probe includes a reactive chemical portion.

135. The method according to claim 133, wherein the second probe comprises an antibody or an antibody fragment.

136. The method according to any one of claims 1 to 135, wherein the sample is embedded in a hydrogel.

137. The method according to any one of claims 1 to 136, wherein the first and second ends are separated by at least one nucleotide when ligated after (b).

138. The method according to claim 137, further comprising carrying out a gap-filling reaction after (b) and before (c) such that the first end and the second end are directly adjacent to each other.

139. A method for detecting analytes in a sample, a) To provide a first probe, The first probe, (i) A first bonding site configured to connect to a first analyte in the first part, (ii) A second bonding site configured to connect to the first analyte in the second portion, wherein the first portion is adjacent to the second bonding site, (iii) A third connection site configured to connect to a second probe, (iv) barcode, (v) the first terminal, and (vi) The second end Including providing, b) Bringing a sample containing a plurality of analytes, including the first analyte and the second analyte, into contact with the first probe, such that the first end and the second end are separated by a gap, c) Filling the gap by performing a gap-filling reaction, d) Forming a cyclic oligonucleotide by ligating the first and second ends, e) Contacting the cyclic oligonucleotide with the second probe, wherein the second probe (i) A fourth connection site connected to the first probe, and (ii) A fifth bonding site connected to the second analyte Including contact, f) Amplifying the cyclic oligonucleotide to produce an amplification product, wherein the amplification product includes the complement of the barcode. g) Using at least one detection probe to detect the complement or derivative of the barcode, thereby determining the proximity between the first analyte and the second analyte. Methods that include...

140. A method for detecting analytes in a sample, a) To provide a first probe, a second probe, and a third probe, The first probe, (i) A first binding site configured to connect to the first analyte, (ii) A second coupling portion configured to be connected to the second probe, (iii) barcode, (iv) the first terminal, and (v) Second terminal Includes, The second probe, (i) A third coupling portion configured to be connected to the first probe, (ii) A fourth coupling site configured to be connected to the third probe, and (iii) A fifth binding site configured to connect to the second analyte Includes, The third probe, (i) A sixth coupling portion configured to be connected to the second probe, (ii) A seventh bonding site configured to connect to the first analyte, (iii) A third end, wherein the third end is adjacent to the first end, and (iv) A fourth end, wherein the fourth end is adjacent to the second end. Including providing, b) To make contact, (i) The first probe is connected to the first analyte, (ii) The second probe is connected to the second analyte, (iii) The third probe is connected to the first analyte, (iv) The first probe is connected to the second analyte, (v) The third probe is connected to the second probe, A sample containing a plurality of analytes, including the first analyte and the second analyte, is brought into contact with the first probe, the second probe, and the third probe. c) Forming a cyclic oligonucleotide by ligating the first and third ends and the second and fourth ends, d) Amplifying the cyclic oligonucleotide to produce an amplified product, wherein the amplified product includes the complement of the barcode. e) Using multiple detection probes to detect the complement or derivative of the barcode, thereby determining the proximity between the first analyte and the second analyte. Methods that include...

141. The method according to claim 140, wherein the sample is a tissue sample.

142. The method according to claim 141, wherein the tissue sample is a fresh frozen tissue sample.

143. The method according to claim 141, wherein the tissue sample is a formalin-fixed paraffin-embedded tissue sample.

144. The method according to any one of claims 140 to 143, wherein the sample has a thickness of 5 to 250 μm.

145. The method according to any one of claims 140 to 143, wherein the sample has a thickness of 10 to 200 μm.

146. The method according to any one of claims 140 to 143, wherein the sample has a thickness of 25 to 150 μm.

147. The method according to any one of claims 140 to 143, wherein the first analyte comprises nucleic acid.

148. The method according to claim 147, wherein the nucleic acid is ribonucleic acid.

149. The method according to claim 148, wherein the messenger ribonucleic acid is a messenger ribonucleic acid.

150. The method according to claim 148, wherein the messenger ribonucleic acid is a ribosome messenger ribonucleic acid.

151. The method according to claim 147, wherein the nucleic acid is deoxyribonucleic acid.

152. The method according to any one of claims 147 to 151, wherein the nucleic acid comprises a single nucleotide polymorphism.

153. The method according to claim 152, wherein the first probe recognizes the single nucleotide polymorphism, and if the first analyte does not contain the single nucleotide polymorphism, the ligation in c) does not occur.

154. The method according to claim 152, wherein the first probe does not recognize the single nucleotide polymorphism, and if the first analyte contains the single nucleotide polymorphism, the ligation in c) does not occur.

155. The method according to claim 152, wherein the third probe recognizes the single nucleotide polymorphism, and if the first analyte does not contain the single nucleotide polymorphism, the ligation in c) does not occur.

156. The method according to claim 152, wherein the third probe does not recognize the single nucleotide polymorphism, and if the first analyte contains the single nucleotide polymorphism, the ligation in c) does not occur.

157. The method according to any one of claims 147 to 151, wherein the nucleic acid includes modifications.

158. The above modification is N ６ - Methyladenosine, 5-methylcytosine, N １ - Methyladenosine, N ７ - Methylguanosine, N ４ - Acetylcytosine, pseudouridine, and N １ - The method according to claim 157, selected from the group consisting of methylpseudridine.

159. The method according to any one of claims 157 to 158, wherein the first probe recognizes the modification, and if the first analyte does not contain the modification, the ligation in c) does not occur.

160. The method according to any one of claims 157 to 158, wherein the first probe does not recognize the modification, and if the first analyte contains the modification, the ligation in c) does not occur.

161. The method according to any one of claims 157 to 158, wherein the third probe recognizes the modification, and if the first analyte does not contain the modification, the ligation in c) does not occur.

162. The method according to any one of claims 157 to 158, wherein the third probe does not recognize the modification, and if the first analyte contains the modification, the ligation in c) does not occur.

163. The method according to any one of claims 140 to 151, wherein the first analyte comprises a polypeptide.

164. The method according to claim 163, wherein the polypeptide comprises a ribosomal protein.

165. The method according to any one of claims 140 to 164, wherein the first analyte includes chemical modification.

166. The method according to any one of claims 140 to 165, wherein the second analyte comprises nucleic acid.

167. The method according to claim 166, wherein the nucleic acid is ribonucleic acid.

168. The method according to claim 167, wherein the ribonucleic acid is messenger ribonucleic acid.

169. The method according to claim 168, wherein the ribonucleic acid is ribosomal ribonucleic acid.

170. The method according to claim 166, wherein the nucleic acid is deoxyribonucleic acid.

171. The method according to any one of claims 140 to 170, wherein the second analyte comprises a polypeptide.

172. The method according to claim 171, wherein the polypeptide comprises a ribosomal protein.

173. The method according to any one of claims 140 to 172, wherein the first probe comprises nucleic acid.

174. The method according to claim 173, wherein the nucleic acid includes an oligonucleotide.

175. The method according to claim 174, wherein the oligonucleotide includes one or more modifications.

176. The method according to claim 175, wherein one or more of the modifications include a 5'-phosphate modification.

177. The method according to any one of claims 175 to 176, wherein the one or more modifications include internucleotide bonding.

178. The method according to claim 177, wherein the nucleotide bond is a phosphorothioate.

179. The method according to claim 177, wherein the nucleotide bond is a phosphodiester.

180. The method according to any one of claims 140 to 179, wherein the first probe recognizes ribonucleic acid.

181. The method according to any one of claims 140 to 179, wherein the first probe recognizes ribonucleic acid modification.

182. The ribonucleic acid modification is N ６ - Methyladenosine, 5-methylcytosine, N １ - Methyladenosine, N ７ - Methylguanosine, N ４ - Acetylcytosine, pseudouridine, and N １ - The method according to claim 181, selected from the group consisting of methylpseudridine.

183. The method according to any one of claims 140 to 179, wherein the first probe recognizes deoxyribonucleic acid.

184. The method according to any one of claims 140 to 179, wherein the first probe recognizes deoxyribonucleic acid modification.

185. The method according to claim 184, wherein the deoxyribonucleic acid modification is a methyl modification.

186. The method according to any one of claims 140 to 185, wherein the first probe includes a first reactive chemical moiety at its first end, and the third probe includes a second reactive chemical moiety at its third end.

187. The method according to claim 186, wherein the first reactive chemical moiety is selected from the group consisting of tetrazine, alkyne, azide, trans-cyclooctene, maleimide, N-hydroxysuccinimide ester, amine, carboxylic acid, hydroxyl, cyclopropenone, and thiol, norbornene.

188. The method according to claim 186, wherein the second reactive chemical moiety is selected from the group consisting of tetrazine, alkyne, azide, trans-cyclooctene, maleimide, N-hydroxysuccinimide ester, amine, carboxylic acid, hydroxyl, cyclopropenone, and thiol, norbornene.

189. The method according to any one of claims 186 to 188, wherein the ligation in c) includes a reaction between the first reactive chemical portion and the second reactive chemical portion.

190. The method according to any one of claims 140 to 185, wherein the first probe includes a third reactive chemical moiety at the second end, and the third probe includes a fourth reactive chemical moiety at the fourth end.

191. The method according to claim 190, wherein the third reactive chemical moiety is selected from the group consisting of tetrazine, alkyne, azide, trans-cyclooctene, maleimide, N-hydroxysuccinimide ester, amine, carboxylic acid, hydroxyl, cyclopropenone, and thiol, norbornene.

192. The method according to claim 190, wherein the fourth reactive chemical moiety is selected from the group consisting of tetrazine, alkyne, azide, trans-cyclooctene, maleimide, N-hydroxysuccinimide ester, amine, carboxylic acid, hydroxyl, cyclopropenone, and thiol, norbornene.

193. The method according to any one of claims 190 to 192, wherein the ligation in c) includes a reaction between the first reactive chemical portion and the second reactive chemical portion.

194. The method according to any one of claims 140 to 185, wherein the ligation in c) includes carrying out a ligation reaction with a ligase.

195. The method according to claim 194, wherein the ligase is T4 ligase.

196. The method according to any one of claims 140 to 195, wherein the second probe comprises nucleic acid.

197. The method according to claim 196, wherein the nucleic acid includes an oligonucleotide.

198. The method according to claim 197, wherein the oligonucleotide comprises one or more modifications.

199. The method according to claim 198, wherein one or more of the modifications include a 5'-phosphate modification.

200. The method according to any one of claims 198 to 199, wherein the one or more modifications include internucleotide bonds.

201. The method according to claim 200, wherein the nucleotide bond is a phosphorothioate.

202. The method according to claim 201, wherein the nucleotide bond is a phosphodiester.

203. The method according to any one of claims 196 to 202, wherein the nucleic acid comprises an aptamer.

204. The method according to any one of claims 140 to 203, wherein the second probe comprises a polypeptide.

205. The method according to claim 204, wherein the polypeptide comprises an antibody or an antibody fragment.

206. The method according to claim 205, wherein the polypeptide comprises an adfimer.

207. The method according to claim 205, wherein the polypeptide comprises a nanobody.

208. The method according to any one of claims 140 to 207, wherein the second probe recognizes ribonucleic acid.

209. The method according to any one of claims 140 to 207, wherein the second probe recognizes ribonucleic acid modification.

210. The ribonucleic acid modification is N ６ - Methyladenosine, 5-methylcytosine, N １ - Methyladenosine, N ７ - Methylguanosine, N ４ - Acetylcytosine, pseudouridine, and N １ - The method according to claim 209, selected from the group consisting of methylpseudridine.

211. The method according to any one of claims 140 to 207, wherein the second probe recognizes deoxyribonucleic acid.

212. The method according to any one of claims 140 to 207, wherein the second probe recognizes deoxyribonucleic acid modification.

213. The method according to claim 212, wherein the deoxyribonucleic acid modification is a methyl modification.

214. The method according to any one of claims 140 to 207, wherein the second probe recognizes a polypeptide.

215. The method according to claim 214, wherein the polypeptide is a protein.

216. The method according to claim 215, wherein the protein is a transcription factor.

217. The method according to claim 215, wherein the protein is a ribosomal protein.

218. The method according to claim 215, wherein the protein is a histone.

219. The method according to claim 215, wherein the protein is a polymerase.

220. The method according to claim 215, wherein the protein is a helicase.

221. The method according to claim 215, wherein the protein is a restriction enzyme.

222. The method according to claim 215, wherein the protein is a ribonucleic acid-binding protein.

223. The method according to any one of claims 140 to 222, wherein the second probe recognizes the post-translational modification of the protein.

224. The method according to any one of claims 140 to 223, wherein the second probe comprises a reactive chemical portion.

225. The method according to claim 224, wherein the third reactive chemical moiety is selected from the list consisting of tetrazine, alkyne, azide, trans-cyclooctene, maleimide, N-hydroxysuccinimide ester, amine, carboxylic acid, hydroxyl, cyclopropenone, and thiol, norbornene.

226. The method according to claim 224, wherein the reactive chemical portion reacts with the second analyte.

227. The method according to any one of claims 140 to 226, wherein the third probe comprises nucleic acid.

228. The method according to claim 227, wherein the nucleic acid includes an oligonucleotide.

229. The method according to claim 228, wherein the oligonucleotide comprises one or more modifications.

230. The method according to claim 229, wherein one or more of the modifications include a 5'-phosphate modification.

231. The method according to any one of claims 229 to 230, wherein one or more of the modifications include internucleotide bonding.

232. The method according to claim 231, wherein the nucleotide bond is a phosphorothioate.

233. The method according to claim 232, wherein the nucleotide bond is a phosphodiester.

234. The method according to any one of claims 140 to 233, wherein the third probe recognizes ribonucleic acid.

235. The method according to any one of claims 140 to 233, wherein the third probe recognizes ribonucleic acid modification.

236. The ribonucleic acid modification is N ６ - Methyladenosine, 5-methylcytosine, N １ - Methyladenosine, N ７ - Methylguanosine, N ４ - Acetylcytosine, pseudouridine, and N １ - The method according to claim 235, selected from the group consisting of methylpseudridine.

237. The method according to any one of claims 140 to 233, wherein the third probe recognizes deoxyribonucleic acid.

238. The method according to any one of claims 140 to 233, wherein the third probe recognizes deoxyribonucleic acid modification.

239. The method according to claim 238, wherein the deoxyribonucleic acid modification is a methyl modification.

240. The method according to any one of claims 140 to 239, wherein the third probe comprises a reactive chemical portion.

241. The method according to claim 240, wherein the reactive chemical portion reacts with the third probe.

242. The method according to any one of claims 140 to 241, wherein the barcode includes nucleic acid.

243. The method according to claim 242, wherein the nucleic acid is deoxyribonucleic acid.

244. The method according to claim 242, wherein the nucleic acid is ribonucleic acid.

245. The method according to any one of claims 242 to 244, wherein the nucleic acid is at least 4 nucleotides long.

246. The method according to any one of claims 242 to 244, wherein the nucleic acid is at least 6 nucleotides long.

247. The method according to any one of claims 242 to 244, wherein the nucleic acid is at least 8 nucleotides long.

248. The method according to any one of claims 242 to 244, wherein the nucleic acid is at least 10 nucleotides long.

249. The method according to any one of claims 140 to 248, wherein the barcode corresponds to the first analyte.

250. The method according to any one of claims 140 to 248, wherein the barcode corresponds to the second analyte.

251. The method according to any one of claims 140 to 248, wherein the barcode corresponds to the first analyte that is adjacent to the second analyte.

252. The method according to any one of claims 140 to 251, wherein the first probe further includes a second barcode.

253. The method according to claim 252, wherein the second barcode contains nucleic acid.

254. The method according to claim 253, wherein the nucleic acid is deoxyribonucleic acid.

255. The method according to claim 253, wherein the nucleic acid is ribonucleic acid.

256. The method according to any one of claims 252 to 255, wherein the nucleic acid is at least 4 nucleotides long.

257. The method according to any one of claims 252 to 255, wherein the nucleic acid is at least 6 nucleotides long.

258. The method according to any one of claims 252 to 255, wherein the nucleic acid is at least 8 nucleotides long.

259. The method according to any one of claims 252 to 255, wherein the nucleic acid is at least 10 nucleotides long.

260. The method according to any one of claims 252 to 259, wherein the second barcode corresponds to the first analyte.

261. The method according to any one of claims 252 to 259, wherein the second barcode corresponds to the second analyte.

262. The method according to any one of claims 252 to 259, wherein the second barcode corresponds to the first analyte that is adjacent to the second analyte.

263. The method according to any one of claims 140 to 262, wherein the third probe includes a third barcode.

264. The method according to claim 263, wherein the third barcode includes nucleic acid.

265. The method according to claim 264, wherein the nucleic acid is deoxyribonucleic acid.

266. The method according to claim 264, wherein the nucleic acid is ribonucleic acid.

267. The method according to any one of claims 264 to 266, wherein the nucleic acid is at least 4 nucleotides long.

268. The method according to any one of claims 264 to 266, wherein the nucleic acid is at least 6 nucleotides long.

269. The method according to any one of claims 264 to 266, wherein the nucleic acid is at least 8 nucleotides long.

270. The method according to any one of claims 264 to 266, wherein the nucleic acid is at least 10 nucleotides long.

271. The method according to any one of claims 263 to 270, wherein the third barcode corresponds to the first analyte.

272. The method according to any one of claims 263 to 270, wherein the third barcode corresponds to the second analyte.

273. The method according to any one of claims 252 to 270, wherein the third barcode corresponds to the first analyte that is adjacent to the second analyte.

274. The method according to any one of claims 140 to 273, wherein the first binding site comprises a nucleic acid, and the nucleic acid is at least 6 nucleotides long.

275. The method according to any one of claims 140 to 273, wherein the first binding site comprises a nucleic acid, and the nucleic acid is at least 10 nucleotides long.

276. The method according to any one of claims 140 to 273, wherein the first binding site comprises a nucleic acid, and the nucleic acid is at least 14 nucleotides long.

277. The method according to any one of claims 140 to 273, wherein the first binding site comprises a nucleic acid, and the nucleic acid is at least 20 nucleotides long.

278. The method according to any one of claims 140 to 277, wherein the second binding site includes a nucleic acid, and the nucleic acid is at least two nucleotides long.

279. The method according to any one of claims 140 to 277, wherein the second binding site includes a nucleic acid, and the nucleic acid is at least 4 nucleotides long.

280. The method according to any one of claims 140 to 277, wherein the second binding site includes a nucleic acid, and the nucleic acid is at least 8 nucleotides long.

281. The method according to any one of claims 140 to 277, wherein the second binding site comprises a nucleic acid, and the nucleic acid is at least 10 nucleotides long.

282. The method according to any one of claims 140 to 281, wherein the third binding site includes a nucleic acid, and the nucleic acid is at least two nucleotides long.

283. The method according to any one of claims 140 to 281, wherein the third binding site comprises a nucleic acid, and the nucleic acid is at least 4 nucleotides long.

284. The method according to any one of claims 140 to 281, wherein the third binding site comprises a nucleic acid, and the nucleic acid is at least 8 nucleotides long.

285. The method according to any one of claims 140 to 281, wherein the third binding site comprises a nucleic acid, and the nucleic acid is at least 12 nucleotides long.

286. The method according to any one of claims 140 to 285, wherein the fourth binding site includes a nucleic acid, and the nucleic acid is at least two nucleotides long.

287. The method according to any one of claims 140 to 285, wherein the fourth binding site includes a nucleic acid, and the nucleic acid is at least 4 nucleotides long.

288. The method according to any one of claims 140 to 285, wherein the fourth binding site includes a nucleic acid, and the nucleic acid is at least 8 nucleotides long.

289. The method according to any one of claims 140 to 285, wherein the fourth binding site includes a nucleic acid, and the nucleic acid is at least 12 nucleotides long.

290. The method according to any one of claims 140 to 289, wherein the fifth binding site includes a nucleic acid, and the nucleic acid is at least 6 nucleotides long.

291. The method according to any one of claims 140 to 289, wherein the fifth binding site includes a nucleic acid, and the nucleic acid is at least 10 nucleotides long.

292. The method according to any one of claims 140 to 289, wherein the fifth binding site includes a nucleic acid, and the nucleic acid is at least 14 nucleotides long.

293. The method according to any one of claims 140 to 289, wherein the fifth binding site includes a nucleic acid, and the nucleic acid is at least 20 nucleotides long.

294. The method according to any one of claims 140 to 293, wherein the sixth binding site includes a nucleic acid, and the nucleic acid is at least two nucleotides long.

295. The method according to any one of claims 140 to 293, wherein the sixth binding site includes a nucleic acid, and the nucleic acid is at least 4 nucleotides long.

296. The method according to any one of claims 140 to 293, wherein the sixth binding site includes a nucleic acid, and the nucleic acid is at least 8 nucleotides long.

297. The method according to any one of claims 140 to 293, wherein the sixth binding site includes a nucleic acid, and the nucleic acid is at least 12 nucleotides long.

298. The method according to any one of claims 140 to 297, wherein the seventh binding site comprises a nucleic acid, and the nucleic acid is at least four nucleotides long.

299. The method according to any one of claims 140 to 297, wherein the seventh binding site comprises a nucleic acid, and the nucleic acid is at least 10 nucleotides long.

300. The method according to any one of claims 140 to 297, wherein the seventh binding site comprises a nucleic acid, and the nucleic acid is at least 14 nucleotides long.

301. The method according to any one of claims 140 to 297, wherein the seventh binding site comprises a nucleic acid, and the nucleic acid is at least 20 nucleotides long.

302. (c) The method according to any one of claims 140 to 301, wherein rolling circle amplification is performed.

303. The method according to any one of claims 140 to 302, wherein (d) comprises hybridizing a detection probe and an anchor probe among the plurality of detection probes to the amplification product.

304. The method according to claim 303, wherein the detection probe and the section detection probe are ligated.

305. The method according to any one of claims 303 to 304, wherein the detection probe includes a label.

306. The method according to claim 305, wherein the label includes a fluorescent molecule.

307. The method according to claim 305, wherein the label includes a quantum dot.

308. The method according to claim 305, wherein the label comprises an enzyme.

309. The method according to claim 308, wherein the enzyme generates a signal indicating the label.

310. The method according to any one of claims 305 to 309, wherein (e) is the detection of the sign.

311. The method according to any one of claims 140 to 310, wherein (e) sequence determination is performed in situ using the plurality of detection probes.

312. The method according to any one of claims 140 to 311, wherein (e) is imaging the sample.

313. The method according to claim 140, wherein the first probe and the third probe recognize messenger ribonucleic acid, and the second probe recognizes ribosomal ribonucleic acid.

314. The method according to claim 140, wherein the first probe and the third probe recognize messenger ribonucleic acid, and the second probe recognizes messenger ribonucleic acid modifications.

315. The method according to claim 314, wherein the second probe includes a reactive chemical portion.

316. The method according to claim 314, wherein the second probe comprises an antibody or an antibody fragment.

317. The method according to claim 140, wherein the first probe and the third probe recognize deoxyribonucleic acid, and the second probe recognizes deoxyribonucleic acid modification.

318. The method according to claim 317, wherein the second probe includes a reactive chemical portion.

319. The method according to claim 317, wherein the second probe comprises an antibody or an antibody fragment.

320. The method according to any one of claims 140 to 319, wherein the sample is embedded in a hydrogel.

321. The method according to any one of claims 144 to 320, wherein the ligation in (c) includes ligating the first end to the third end.

322. The method according to any one of claims 144 to 320, wherein the ligation in (c) includes ligating the second end to the fourth end.

323. The method according to any one of claims 140 to 320, wherein the first and third ends are separated by at least one nucleotide when ligated after (b).

324. The method according to claim 323, further comprising carrying out a gap-filling reaction after (b) and before (c) such that the first end and the third end are directly adjacent to each other.

325. The method according to any one of claims 140 to 324, wherein the second and fourth ends are separated by at least one nucleotide when ligated after (b).

326. The method according to claim 323, further comprising carrying out a gap-filling reaction after (b) and before (c) such that the second end and the fourth end are directly adjacent to each other.

327. A method for detecting analytes in a sample, (a) To provide the sample, wherein the sample comprises a first analyte and a second analyte, and the first analyte contains a genetic abnormality. (b) Contacting the first analyte with a first binder, wherein the first binder includes a barcode, (c) Detecting the inverse complement of the barcode with an accuracy of more than 90%, wherein the inverse complement of the barcode is generated only when the first analyte is in close proximity to the second analyte. Methods that include...

328. The method according to claim 327, wherein the first analyte comprises nucleic acid.

329. The method according to claim 327, wherein the nucleic acid includes ribonucleic acid.

330. The method according to claim 329, wherein the gene abnormality includes ribonucleic acid modification.

331. The method according to claim 330, wherein the ribonucleic acid modification is selected from the group consisting of N6-methyladenosine, 5-methylcytosine, N1-methyladenosine, N7-methylguanosine, N4-acetylcytosine, pseudouridine, and N1-methylpseudridine.

332. The method according to claim 327, wherein the nucleic acid comprises deoxyribonucleic acid.

333. The method according to claim 332, wherein the gene abnormality includes deoxyribonucleic acid modification.

334. The method according to claim 332, wherein the deoxyribonucleic acid modification includes a methyl group.

335. The method according to any one of claims 327 to 334, wherein the gene abnormality includes a single nucleotide polymorphism.

336. The method according to any one of claims 327 to 329 and 332, wherein the gene abnormality includes an insertion.

337. The method according to any one of claims 327 to 329 and 332, wherein the gene abnormality includes a deletion.

338. The method according to any one of claims 327 to 329 and 332, wherein the gene abnormality includes a single nucleotide polymorphism.

339. The method according to any one of claims 327 to 329 and 332, wherein the gene abnormality comprises a single nucleotide mutation.

340. The method according to any one of claims 323 to 329 and 332, wherein the gene abnormality includes copy number variation.

Images